*3.1. Isolation and Identification of Antibiotic-Degrading Bacteria*

Twelve and eight bacterial strains with the ability to use CTC, AMO and SMX as carbon sources under aerobic and anaerobic conditions were isolated from the sludge samples. The five aerobic isolates showing the greatest degrading capability were strains SF1, SF2, SF3, SF4 and A12. When the bacterial counts increased, the level of CTC, AMO and SMX decreased. The order of CTC, AMO and SMX degradation was strain SF1 > strain A12 > strain SF2 > strain SF3 > strain SF4. Strains SF1 and A12 exhibited the greatest aerobic degradation capability. The degradation of CTC, AMO and SMX by strain SF1 after 8 days of incubation was equal to 81.6, 89.1, and 95.9%, respectively (Table 1). The degradation of CTC, AMO and SMX by strain A12 after 8 days of incubation was equal to 69.4, 81.4, and 89.6%, respectively. The degradation of CTC, AMO and SMX by strains SF1 and A12 after 8 days of incubation was equal to 89.4, 93.4, and 99.3%, respectively. The order of the efficiency of the aerobic degradation of antibiotics was as follows: strains SF1 and A12 > strain SF1 > strain A12.

**Table 1.** Remaining percentages (%) of chlortetracycline (CTC), amoxicillin (AMO) and sulfamethoxazole (SMX) after incubation with antibiotic-degrading bacteria.


Note(s): <sup>a</sup> aerobic conditions for 8 days. <sup>b</sup> anaerobic conditions for 15 days. Data from three independent experiments are presented as the means ± SE.

Five anaerobic isolates exhibited the greatest antibiotic-degradation capability were strains B, SANA, SANB, SANC, SANC, and SAND. When the bacterial counts increased, the level of CTC, AMO and SMX decreased. The order of CTC, AMO and SMX degradation is strain B > strain SANA > strain SANB > strain SANC > strain SAND. Strains B and SANA exhibited the greatest antibiotic degradation capability under anaerobic conditions among the five tested isolates (B, SANA, SANB, SANC, SANC, and SAND). The degradation of CTC, AMO and SMX by strain B after 15 days of incubation was equal to 81.6, 89.1, and 95.1%, respectively (Table 1). The degradation of CTC, AMO and SMX by strain SANA after 15 days of incubation was equal to 59.4, 81.4, and 89.6%, respectively. The degradation of CTC, AMO and SMX by strain B and SANA after 15 days of incubation was equal to 89.6, 93.4, and 99.5%, respectively. The order of the efficiency of the anaerobic degradation of antibiotics was as follows: strains B and SANA > strain B > strain SANA. Yang et al. (2019) reported that the cocultures of strains M10 and M12 can enhance malachite green degradation in milkfish pond sediments. The addition of both bacterial strains M10 and M12 produced better results than each of the single cultures [31]. The aerobic stains SF1 and A12 and the anaerobic strains B and SANA were used in subsequent studies.

The colony morphology and scanning electron micrographs of the four bacterial strains are shown in Figure 1. All of them were Gram-negative, rod-shaped bacteria.

Phylogenetic analysis of strains A12, SF1, B and SANA, based on the 16S rRNA gene, is shown in Figure 2. The strains A12, SF1, B and SANA are closely related to *Pseudmonas pseudoalcaligenes* (99%), *Pseudmonas taiwanensis* (96%), *Bacillus flexus* (99%) and *Clostridium butyricum* (99%), respectively. *Pseudomonas* bacteria are widespread in various natural environments, such as soil, plants, animals, air and water. *Pseudmonas taiwanensis* is an aerobic Gram-negative, rod-shaped, motile, nonspore-forming bacterial strain isolated from soil [33]. *Pseudomonas pseudoalcaligenes* is an aerobic Gram-negative bacterium. It is able to use cyanide as a nitrogen source, and can be used for bioremediation [34]. *Bacillus flexus* is a Gram-positive, rod-shaped, endospore-forming bacterium. This bacterium may be isolated from feces and soil. *Bacillus flexus* has been shown to exert activity mainly against polyvinyl chloride additives and exhibits a low biodegradation rate of polyvinyl chloride polymers [35]. *Clostridium butyricum is* a strictly anaerobic endospore-forming Gram-positive bacillus. This bacterium has been studied for its efficiency in decolorizing various remazol reactive dyes [36].

**Figure 1.** Colony morphology and scanning electron micrographs of isolated bacterial strains SF1 (Pseudmonas sp.) (**A**,**B**), A12 (Pseudmonas sp.) (**C**,**D**), SANA (Clostridium sp.) (**E**,**F**), and B (Bacillus sp.) (**G**,**H**).

**Figure 2.** Phylogenetic analysis of the 16S rRNA genes of the four bacterial strains (A12, B, SF1, SANA). Bootstrapping values at branch points indicate the number of times that the same branch was observed out of 1000 repeats of the phylogenetic reconstruction.

#### *3.2. Degradation of Antibiotics in Sludge with Isolated Bacteria*

The antibiotic concentrations in the sterile controls were first examined at the end of the 10th or 15th day incubation periods, under aerobic or anaerobic conditions, respectively. The remaining percentages of antibiotics ranged from 91.7 to 95.3%. Therefore, it was concluded that the antibiotic degradation that occurred in all of the following experiments was due to microbial action.

The aerobic and anaerobic degradation of the antibiotics in sludge containing the isolated bacterial strains is shown in Figures 3–5. The degradation of the antibiotics was increased in sludge containing the isolated bacterial strains. As shown in Figures 3A, 4A and 5A, the aerobic degradation half-lives of OTC, TC, CTC, SDM, SMX, SMZ and AMO were 3.0, 4.6, 2.3, 2.5, 3.1, 1.7 and 2.0 days, respectively. The anaerobic degradation half-lives of OTC, TC, CTC, SDM, SMX, SMZ and AMO were 4.4, 7.0, 3.4, 4.1, 6.8, 2.4 and 3.0 days, respectively (Figures 3B, 4B and 5B). The order of the degradation rates of the antibiotics was CTC > OTC > TC> AMO > SMX > SDM > SMZ.

The order of SA degradation rates in sludge was SMX > SDM > SMZ. Similar trends were observed in a study by Yang et al. (2016) in which fungal enzymes were used to increase the degradation of SAs in sludge [37]. The order of the TC degradation rates in sludge was CTC > OTC > TC. Similar results were reported by Suda et al. (2012) from a study in which TC antibiotics were treated with the laccase enzyme in the presence of 1-hydroxybenzotriazole [38]. The order of the antibiotic degradation rates was TCs > AMO > SAs. Antibiotics with lower molecular weights are easier to degrade than those with higher molecular weights. Larger functional groups may hinder degradation by affecting the interactions between target compounds and the bacteria or enzymes [39]. Moreover, the degradation of compounds with complex structures may require more reaction steps.

**Figure 3.** Degradation of tetracyclines with and without degrading bacteria in sludge. Aerobic (**A**) and anaerobic (**B**) degradation of TCs. TC: tetracycline, CTC: chlortetracycline, OTC: oxytetracycline. B: isolated antibiotic-degrading bacteria. Data from three independent experiments are presented as the means ± SE.

**Figure 4.** Degradation of sulfonamides with and without degrading bacteria in sludge. Aerobic (**A**) and anaerobic (**B**) degradation of SAs. SMZ: sulfamethazine, SMX: sulfamethoxazole, SDM: sulfadimethoxine. B: isolated antibiotic-degrading bacteria. Data from three independent experiments are presented as the means ± SE.

**Figure 5.** Degradation of amoxicillin with and without degrading bacteria in sludge. Aerobic (**A**) and anaerobic (**B**) degradation of AMO. AMO: amoxicillin. B: isolated antibiotic-degrading bacteria. Data from three independent experiments are presented as the means ± SE.
