*3.4. Microbial Communities in the Repeated Addition Experiments*

To obtain deeper insight into the conditions in the repeated addition experiments, the microbial communities in the repeated addition experiments were analyzed. As shown in Figure 6, the five experimental conditions (original sludge, sludge in aerobic conditions after the 3rd addition, sludge and bacteria in aerobic conditions after the 3rd addition, sludge in anaerobic conditions after the 3rd addition, sludge and bacteria in anaerobic conditions after the 3rd addition) exhibited differences in the composition of the major microbial communities (46, 45, 24, 33 and 26 microbial genera). The lists of the five groups of major microbial genera in the five experimental conditions are listed in Table S2.

**Figure 6.** Major microbial communities (genus level) in sludge after the 3rd repeated addition experiment. Control: original sludge. A, S: aerobic conditions, sludge only. A, S + B: aerobic conditions, sludge + bacteria. An, S: anaerobic conditions, sludge only. An, S + B: anaerobic conditions, sludge + bacteria. The major microbial genera (red color in heatmap) under the five experimental conditions are listed in Table S2.

For validation, a list of xenobiotic biodegradation-associated bacteria/archaea in the KEGG database was used to identify bacteria/archaea with xenobiotic degradation ability in the repeated addition experiments. The identified bacterial/archaeal genera were used as key words to perform text mining using the NCBI PubMed database. Under aerobic conditions, 25 of the 63 identified bacterial/archaeal genera have been reported to be antibiotic-degrading bacteria/archaea (Figure 7A). Under anaerobic conditions, 25 of the 63 identified bacterial/archaeal genera have been reported to be antibiotic-degrading bacteria/archaea (Figure 7B).

In total, 24 reported antibiotic-degrading bacterial genera (*Achromobacter*, *Acidovorax*, *Acinetobacter*, *Alcaligenes*, *Bacillus*, *Burkholderia*, *Castellaniella*, *Comamonas*, *Corynebacterium*, *Cupriavidus*, *Dechloromonas*, *Geobacter*, *Gordonia*, *Klebsiella*, *Mycobacterium*, *Novosphingobium*, *Pandoraea*, *Pseudomonas*, *Rhodococcus*, *Sphingomonas*, *Thauera*, *Treponema*, *Vibrio and Xanthobacter*) are common to the aerobic and anaerobic groups. These results suggest that the 24 bacterial genera may be the major antibiotic-degrading bacteria in sludge. The PubMed ID, title and abstract of these reports are collected and summarized in Table S3.

The distribution of the number of major microbial genera exhibiting different aromatic compound degradation pathways is shown in Figure 8. Most of the microbial genera were associated with six reaction modules: M00548 (benzene degradation, benzene ≥ catechol), M00551 (benzoate degradation, benzoate ≥ catechol/methylbenzoate ≥ methylcatechol), M00568 (catechol ortho-cleavage, catechol ≥ 3-oxoadipate), M00569 (catechol meta-cleavage, catechol ≥ acetyl-CoA/4-methylcatechol ≥ propanoyl-CoA), M00623 (phthalate degradation, phthalate ≥ protocatechuate) and M00638 (salicylate degradation, salicylate ≥ gentisate). Moreover, more microbial genera associated with aromatic compound degradation were identified in the sludge with isolated antibiotic-degrading bacteria than in the sludge without the bacteria. These results provide explanations for the increased antibiotic-degrading effects associated with the addition of isolated antibiotic-degrading bacteria in sludge observed in Figures 3–5 and Table 2.

Yin et al. (2020) identified a TC-degrading strain, TR5, that could degrade TC quickly (~90% within 36 h) when the initial TC concentration was 200 mg/L [40]. Its efficiency is much higher than those of the TC-degrading bacteria identified in this study. However, TR5 was identified as *Klebsiella* pneumoniae according to 16S rRNA gene sequencing and biochemical properties. Great care must be taken in the application of pathogenic bacteria in wastewater treatment. Wu et al. (2020) identified two TC-degrading strains, *Raoultella* sp. XY-1 and *Pandoraea* sp. XY-2, which degraded 81.72% TC within 12 days in lysogeny broth (LB) medium [41]. The bacterial strains identified in this study exhibit a higher TC-degrading efficiency than *Raoultella* sp. XY-1 and *Pandoraea* sp. XY-2. Wen et al. reported that 95% of the doxycycline (50 mg/L) was degraded by the recombinant strain *Escherichia coli* ETD-1, with tetX, within 48 h [42]. Although the doxycycline-degrading ability of recombinant *Escherichia coli* is very significant, great care must be taken in the application of microorganisms carrying recombinant genes in the environment. Sodhi et al. (2020) identified the AMO-degrading bacterium, *Alcaligenes* sp. MMA, which was able to remove up to 84% of amoxicillin in 14 days in M9 minimal media [43]. The bacterial strains identified in this study exhibit a higher AMO-degrading efficiency than *Alcaligenes* sp. MMA. Liang et al. (2019) reported that *Achromobacter* sp. JL9 was able to utilize SMX as its sole nitrogen source for growth, with an SMX biodegradation efficiency of 63.10% [44]. Moreover, Nguyen et al. (2019) showed that SMX and sodium acetate could be cometabolized as carbon sources, with the highest removal efficiencies of 82.44%, 80.2%, and 79.45% for NH4 <sup>+</sup>-N, NO3 - -N, and SMX, respectively [45]. The bacterial strains identified in this study exhibit a higher SMX-degrading efficiency than *Achromobacter* sp. Wang et al. (2018) demonstrated that *Acinetobacter* sp. could mineralize 98.8% of SMX, but only 17.5% and 20.5% of sulfadiazine and SMZ, respectively [46]. The bacterial strains identified in this study could degrade 98%, 91% and 88% SMX, SDM and SMZ, respectively. Overall, the antibiotic-degrading efficiency of the bacterial strains identified in this study is very good. Moreover, these bacterial strains exhibit the ability to degrade multiple antibiotics.

**Figure 7.** Xenobiotic biodegradation-associated bacteria after the 3rd repeated addition experiment under aerobic (**A**) and anaerobic (**B**) conditions. Control: original sludge. Sludge and B: sludge containing the isolated bacterial strains. a: amoxicillin-degrading bacteria. s: sulfonamide-degrading bacteria. t: tetracycline-degrading bacteria.

**Figure 8.** Xenobiotic biodegradation-associated microbial genera after the 3rd repeated addition experiment under aerobic and anaerobic conditions. Mxxxxx are Kyoto Encyclopedia of Genes and Genomes (KEGG) database module IDs. aero: sludge under aerobic conditions. anaero: sludge under anaerobic conditions. aero\_B: sludge with antibiotic-degrading bacteria under aerobic conditions. anaero: sludge with antibiotic-degrading bacteria under anaerobic conditions.
