*2.2. Characterization of Cd2+ Resistance*

The ST11 strain was assessed for its ability to grow in the presence of Cd2+. As shown in Figure 2, no difference was found from the control samples when ST11 was incubated for 48 h in a culture medium containing <0.3 mmol/L Cd2+. The growth of ST11 was slightly suppressed in the presence of 0.5–1.0 mmol/L Cd2+ and significantly suppressed in the presence of 1.5 mmol/L Cd2+. The effective concentration-25 (EC25) and effective concentration-50 (EC50) were defined as the Cd2+ concentration required to obtain 25% and 50% cell growth inhibition effects, respectively. The EC<sup>25</sup> and EC<sup>50</sup> of ST11 toward Cd2+ were 1.21 and 2.01 mmol/L, respectively.

*Catalysts* **2022**, *12*, x FOR PEER REVIEW 3 of 13

**Figure 1.** Phylogenetic analysis of strain ST11 and related species by the Neighbor‐Joining method based on 16S rRNA gene sequences. Bootstrap values (%) are indicated at the nodes, and the scale bars represent 0.001 substitutions per site. *2.2. Characterization of Cd2+ Resistance* **Figure 1.** Phylogenetic analysis of strain ST11 and related species by the Neighbor-Joining method based on 16S rRNA gene sequences. Bootstrap values (%) are indicated at the nodes, and the scale bars represent 0.001 substitutions per site. *Catalysts* **2022**, *12*, x FOR PEER REVIEW 4 of 13

The ST11 strain was assessed for its ability to grow in the presence of Cd2+. As shown

**Figure 2.** Growth curves (**A**) and inhibition fitting curve (**B**) of ST11 in the presence of Cd2+. The cells were cultured with the Luria‐Bertani (LB) medium in the presence of Cd2+ (0–1.5 mmol/L) at 30 °C at 150 rpm in darkness for 48 h. At the timed interval, flasks were taken out and 1 mL culture was withdrawn for OD600 measurements. **Figure 2.** Growth curves (**A**) and inhibition fitting curve (**B**) of ST11 in the presence of Cd2+. The cells were cultured with the Luria-Bertani (LB) medium in the presence of Cd2+ (0–1.5 mmol/L) at 30 ◦C at 150 rpm in darkness for 48 h. At the timed interval, flasks were taken out and 1 mL culture was withdrawn for OD<sup>600</sup> measurements.

The adsorption capacities of ST11 for Cd2+ were shown in Figure 3. Before and after cell adsorption, the difference of Cd2+ detected was negligible. The result shows that ST11

**Figure 3.** Adsorption isotherm of Cd2+ on ST11 cells as a function of Cd2+ concentration. The cells were harvested at 6380× *g* centrifugation for 10 min after ST11 was cultured in LB for 48 h. The cells were washed three times with deionized water to remove the residual medium. The obtained wet cells were used directly for Cd2+ adsorption without further treatment. The adsorption of Cd2+ by

*Arthrobacter* is prevalent in the agricultural soil environment, and it could degrade many kinds of environmental pollutants, but evidence of its resistance to heavy metals is limited [10]. For instance, a quinaldine‐degrading *Arthrobacter* sp. Rue61a was suggested to have the potential to tolerate heavy metals, such as Zn2+, Pb2+, and Co2+ [31]. However, for atrazine‐degrading *Arthrobacter*, a strain with the ability to resist heavy metals has not been reported. In this study, *Arthrobacter* sp. ST11 was shown to survive in the presence

cells have limit adsorption capacity for Cd2+.

ST11 cells was carried out at 25 °C and pH 7.

The adsorption capacities of ST11 for Cd2+ were shown in Figure 3. Before and after cell adsorption, the difference of Cd2+ detected was negligible. The result shows that ST11 cells have limit adsorption capacity for Cd2+ . The adsorption capacities of ST11 for Cd2+ were shown in Figure 3. Before and after cell adsorption, the difference of Cd2+ detected was negligible. The result shows that ST11 cells have limit adsorption capacity for Cd2+.

**Figure 2.** Growth curves (**A**) and inhibition fitting curve (**B**) of ST11 in the presence of Cd2+. The cells were cultured with the Luria‐Bertani (LB) medium in the presence of Cd2+ (0–1.5 mmol/L) at 30 °C at 150 rpm in darkness for 48 h. At the timed interval, flasks were taken out and 1 mL culture was

withdrawn for OD600 measurements.

*Catalysts* **2022**, *12*, x FOR PEER REVIEW 4 of 13

A B

**Figure 3.** Adsorption isotherm of Cd2+ on ST11 cells as a function of Cd2+ concentration. The cells were harvested at 6380× *g* centrifugation for 10 min after ST11 was cultured in LB for 48 h. The cells were washed three times with deionized water to remove the residual medium. The obtained wet cells were used directly for Cd2+ adsorption without further treatment. The adsorption of Cd2+ by ST11 cells was carried out at 25 °C and pH 7. **Figure 3.** Adsorption isotherm of Cd2+ on ST11 cells as a function of Cd2+ concentration. The cells were harvested at 6380× *g* centrifugation for 10 min after ST11 was cultured in LB for 48 h. The cells were washed three times with deionized water to remove the residual medium. The obtained wet cells were used directly for Cd2+ adsorption without further treatment. The adsorption of Cd2+ by ST11 cells was carried out at 25 ◦C and pH 7.

*Arthrobacter* is prevalent in the agricultural soil environment, and it could degrade many kinds of environmental pollutants, but evidence of its resistance to heavy metals is limited [10]. For instance, a quinaldine‐degrading *Arthrobacter* sp. Rue61a was suggested to have the potential to tolerate heavy metals, such as Zn2+, Pb2+, and Co2+ [31]. However, for atrazine‐degrading *Arthrobacter*, a strain with the ability to resist heavy metals has not been reported. In this study, *Arthrobacter* sp. ST11 was shown to survive in the presence *Arthrobacter* is prevalent in the agricultural soil environment, and it could degrade many kinds of environmental pollutants, but evidence of its resistance to heavy metals is limited [10]. For instance, a quinaldine-degrading *Arthrobacter* sp. Rue61a was suggested to have the potential to tolerate heavy metals, such as Zn2+, Pb2+, and Co2+ [31]. However, for atrazine-degrading *Arthrobacter*, a strain with the ability to resist heavy metals has not been reported. In this study, *Arthrobacter* sp. ST11 was shown to survive in the presence of up to 1.5 mmol/L Cd2+. The microbial metabolism of heavy metals includes extracellular complexation, extracellular precipitation, cation outflow, in-vivo detoxification, and in vivo complexation [32]. Living ST11 cells do not adsorb Cd2+, so the resistance of ST11 to heavy metals may be based on cation outflow or the absence of a Cd2+ binding site on the cell membrane of ST11.

#### *2.3. Biodegradation of Crystalline and NAPL-Dissolved Atrazine*

The cell growth and biodegradation of crystalline and NAPL-dissolved atrazine by ST11 are shown in Figure 4A,B, respectively. Di(2-ethylhexyl) phthalate (DEHP) is a commonly used typical NAPL [34] that is biocompatible and could not be used by ST11. Meanwhile, atrazine could be degraded by physical, chemical, and biological methods [35]. In the absence of microorganisms, they could attenuate naturally under some physical and chemical factors [18]. A non-inoculated control experiment was set up in the present study to exclude the effects of temperature, pH, dissolved oxygen, ionic strength, and other physical and chemical factors. For the whole culture cycle, significant bacterial growth of non-investigated samples was not detected under these conditions. For inoculated samples, during the first 16 h, the numbers of cells produced at the two systems were not statistically (Student's *t*-test) significant. The bacterial growth rates during this phase were 0.0171 ± 0.0013 and 0.0185 ± 0.0005 OD600/h with 1.85 mmol/L crystalline and DEHPdissolved atrazine, respectively. Subsequent bacterial growth led to an increased number

of cells until growth stopped after 40 h. At that time, the average final OD<sup>600</sup> values of cells were 0.61 ± 0.01 and 0.44 ± 0.03 with crystalline and DEHP-dissolved atrazine, respectively. In the following 8 h, the cells almost stopped growing. Most of the bioavailable atrazine at that point was assumed to be consumed. Therefore, residual atrazine was detected after 48 h of culture (Figure 4B). Physical and chemical factors were found to degrade atrazine by no more than 15%. With crystalline atrazine as the substrate, the degradation ratio of atrazine increased significantly and reached 68% after 48 h. When atrazine was dissolved in DEHP, the degradation ratio of atrazine decreased to less than 55%, which is consistent with the low number of cells. studied [38], little is known about the mode of its acquisition when it is present in an unavailable phase, such as soil and sediment solids or NAPL. For a naphthalene‐degrad‐ ing *Arthrobacter* sp., the attachment of this strain to the NAPL‐water interface is necessary, and the cells at the interface could degrade organic compounds at rates higher than those of abiotic partitioning [39]. However, for ST11 cells that could not adhere to the oil‐water interface (Figure 5), the biodegradation of atrazine in DEHP was limited by the mass trans‐ fer of the substrate between the two oil‐water phases. Therefore, the findings of this study indicated that when atrazine is dissolved in NAPL in soil, its biodegradation is likely to be restricted, which may be one of the reasons why atrazine is a persistent contaminant in

For other hydrophobic organic compounds in soil, such as polycyclic aromatic hy‐ drocarbons, the bioavailability mechanism of substrates in the non‐aqueous phase has been deeply studied [34,37]. Although the bioremediation of atrazine has been extensively

bioavailability. The mass transfer of atrazine is not limited by the mass transfer rate, and ST11 cells could only use the substrate dissolved in water. When atrazine is a crystal, the mass transfer between the solid and water is only limited by the saturated solubility of atrazine. At this time, the concentration of atrazine in the aqueous phase could be approx‐ imately regarded as the saturated solubility (0.153 mmol/L). When atrazine is dissolved in a NALP, the mass transfer of atrazine between the NAPL and water is affected by the octanol‐water partition coefficient (log *P*ow, 2.28) of atrazine. In the experiments, the addi‐ tional amount of atrazine in DEHP was 11.13 mmol/L. If the difference between DEHP and octanol was not considered, the concentration of atrazine in the aqueous phase was only 0.058 mmol/L. Therefore, when atrazine was used as a substrate in crystal form or dissolved in DEHP, the bioavailability of the former was 2.8 times that of the latter. The low bioavailability of atrazine in DEHP delayed its degradation by ST11 (Figure 4B).

arable soil [4].

*Catalysts* **2022**, *12*, x FOR PEER REVIEW 6 of 13

**Figure 4.** Cell growth (**A**) and biodegradation of atrazine (**B**) in crystalline form or in DEHP by ST11. The initial concentration of atrazine was 1.85 mmol/L. For biological samples, 1 mL of bacterial sus‐ pension (OD600 1.0) was inoculated. For blank tests, no inoculation was prepared to control the non‐ biological effects. (**A**) The growth was monitored spectrophotometrically by measuring OD600 at an interval of 4–8 h. (**B**) At the end of culture (48 h), the biodegradation ratio of atrazine was detected. Data points represent the mean of three replicates and error bars show the standard deviation. Dif‐ ferent lower‐case letters (a, b, and c) over the bars indicate significant differences at *p* < 0.05. **Figure 4.** Cell growth (**A**) and biodegradation of atrazine (**B**) in crystalline form or in DEHP by ST11. The initial concentration of atrazine was 1.85 mmol/L. For biological samples, 1 mL of bacterial suspension (OD<sup>600</sup> 1.0) was inoculated. For blank tests, no inoculation was prepared to control the non-biological effects. (**A**) The growth was monitored spectrophotometrically by measuring OD<sup>600</sup> at an interval of 4–8 h. (**B**) At the end of culture (48 h), the biodegradation ratio of atrazine was detected. Data points represent the mean of three replicates and error bars show the standard deviation. Different lower-case letters (a, b, and c) over the bars indicate significant differences at *p* < 0.05.

The atrazine degradation pathway includes hydrolysis, deamination, dealkylation, and ring cleavage [2]. Liquid Chromatography-Ultraviolet (LC-UV) analysis was performed at 225 nm to determine whether a new metabolite was generated after atrazine degradation (Figure S2). A new absorption peak (Figure S2c) appeared at the retention time of 4.145 min, which was not found in the atrazine standard (Figure S2a) and the culture medium before atrazine was degraded (Figure S2b). In the process of atrazine biodegradation by the *Bacillus licheniformis* ATLJ-5 strain, intermediate metabolites hydroxyatrazine and n-isopropylammelide were detected by LC-UV [8]. Similarly, during the process of ST11 using atrazine, a UV detectable metabolite was found. This finding suggested that the new compound was a metabolite of the degraded atrazine.

Microscopic observation explained that atrazine dissolved in DEHP had decreased bioavailability (Figure 5). For the microbial adhesion to hydrocarbons (MATH) experiment of ST11, the solution was vigorously homogenized and then allowed to stand for 2 h to ensure complete phase separation. The OD<sup>600</sup> of cells in the water phase increased from 0.6 to 0.68 (Figure 5B), possibly because some DEHP micro-droplets suspended in the water phase hindered the transmission of light. The DEHP oil phase (Figure 5B) was as clear and transparent as the sterile sample (Figure 5A), and no obvious emulsification was observed. The liquid at the oil-water interface was sampled for microscopic observation (Figure 5C). The results showed that the ST11 cells were evenly scattered in the water phase and not observed on the oil-water interface. Therefore, ST11 is a water-soluble bacterium that could only use atrazine dissolved in the water phase in the oil-water two-phase system.

**Figure 5.** Visual aspect of two‐phase systems with DEHP as a NAPL phase. (**A**) 5 mL of DEHP, 20 mL of MSM; (**B**) 5 mL of DEHP, 20 mL of cells suspension in MSM with OD600 0.6; (**C**) Microscopic images of oil‐water interface. The arrow indicates ST11 cells. **Figure 5.** Visual aspect of two-phase systems with DEHP as a NAPL phase. (**A**) 5 mL of DEHP, 20 mL of MSM; (**B**) 5 mL of DEHP, 20 mL of cells suspension in MSM with OD<sup>600</sup> 0.6; (**C**) Microscopic images of oil-water interface. The arrow indicates ST11 cells.

*2.4. Effect of Cd2+ on the Growth of Strain ST11 and Atrazine Biodegradation* The effects of Cd2+ on ST11 cell density and degradation of atrazine in crystal form or dissolved in DEHP were determined (Figure 6). At Cd2+ concentrations below 0.3 mmol/L, cell growth and atrazine degradation were stimulated with Cd2+ concentration. The high‐ est removal of atrazine (almost 100% at 48 h) in crystal form or dissolved in DEHP oc‐ curred in the case of Cd2+ concentration of 0.1 and 0.2 mmol/L, respectively. The DEHP‐ dissolved atrazine with decreased bioavailability, which may cause more Cd2+, is needed to obtain high degradation ratio. When the concentration of Cd2+ was increased from 0.5 to 1.5 mmol/L, the effect of stimulating cell growth and atrazine degradation gradually weakened. However, in this Cd2+ concentration range, the growth was partially inhibited when ST11 was used soluble substrates (Figure 2A). A notable detail that at the same Cd2+ concentration (such as 0.1 mmol/L), the biodegradation enhancement effect of Cd2+ on crystalline atrazine was stronger than that of the DEHP‐dissolved atrazine. As mentioned, the main reason could be the lower bioavailability of atrazine in DEHP. Correspondingly, the promotion of atrazine biodegradation in the presence of the Cd2+ concentration of 0.05– Atrazine has low bioavailability because of its low aqueous solubility (log *P*ow, 2.28; water solubility, 0.153 mmol/L at 20 ◦C) [36]. However, when atrazine is used as a substrate in crystalline and DEHP-soluble forms, it could be speculated to have different bioavailability. The mass transfer of atrazine is not limited by the mass transfer rate, and ST11 cells could only use the substrate dissolved in water. When atrazine is a crystal, the mass transfer between the solid and water is only limited by the saturated solubility of atrazine. At this time, the concentration of atrazine in the aqueous phase could be approximately regarded as the saturated solubility (0.153 mmol/L). When atrazine is dissolved in a NALP, the mass transfer of atrazine between the NAPL and water is affected by the octanol-water partition coefficient (log *P*ow, 2.28) of atrazine. In the experiments, the additional amount of atrazine in DEHP was 11.13 mmol/L. If the difference between DEHP and octanol was not considered, the concentration of atrazine in the aqueous phase was only 0.058 mmol/L. Therefore, when atrazine was used as a substrate in crystal form or dissolved in DEHP, the bioavailability of the former was 2.8 times that of the latter. The low bioavailability of atrazine in DEHP delayed its degradation by ST11 (Figure 4B).

1.5 mmol/L could be attributed to the enhanced atrazine catabolism (Figure 6). Low con‐ centrations of heavy metal ions could stimulate the catabolism of organic substances [40]. Cu2+ concentrations of 15 and 2 mg/L stimulated the degradation of decabromodiphenyl ether and benzo[a]pyrene, respectively [41,42]. An assessment of Cd pollution in arable soil in China showed that the average and maximum Cd concentrations were 0.0024 and 1.36 mmol/kg, respectively [6], both lower than the 1.5 mmol/L shown in the present study. Therefore, ST11 is not affected by soil Cd when it degrades atrazine in arable soils. As is well known, Cd, similar to other heavy metals, such as copper, zinc, and lead, has biological toxicity [5]. Cd could attach to proteins with sulfhydryl functional groups or glutathione, thus interfering with the synthesis of cysteine or directly damaging DNA [43]. In addition, Cd could easily penetrate the cell membrane of bacteria, causing the emission of substances in cells and affecting the metabolic process of cells [44]. The results For other hydrophobic organic compounds in soil, such as polycyclic aromatic hydrocarbons, the bioavailability mechanism of substrates in the non-aqueous phase has been deeply studied [34,37]. Although the bioremediation of atrazine has been extensively studied [38], little is known about the mode of its acquisition when it is present in an unavailable phase, such as soil and sediment solids or NAPL. For a naphthalene-degrading *Arthrobacter* sp., the attachment of this strain to the NAPL-water interface is necessary, and the cells at the interface could degrade organic compounds at rates higher than those of abiotic partitioning [39]. However, for ST11 cells that could not adhere to the oil-water interface (Figure 5), the biodegradation of atrazine in DEHP was limited by the mass transfer of the substrate between the two oil-water phases. Therefore, the findings of this study indicated that when atrazine is dissolved in NAPL in soil, its biodegradation is likely to be restricted, which may be one of the reasons why atrazine is a persistent contaminant in arable soil [4].

#### soluble substrates (Cd2+ concentration of 0.5–1.5 mmol/L) and hydrophobic substrate at‐ *2.4. Effect of Cd2+ on the Growth of Strain ST11 and Atrazine Biodegradation*

of the present study showed that Cd inhibited and stimulated the growth of ST11 using

razine (Cd2+ concentration of ≤1.5 mmol/L), respectively. ST11 cells have negligible Cd adsorption capacity, which may be one of the reasons why they could resist high Cd con‐ centration. In addition, the antagonistic effect of hydrophobic organics on Cd could be used to mitigate Cd effect on soil living organisms [45]. The results of the present study are consistent with that of the previous study. The biodegradation of crystalline atrazine and NAPL‐solubilized atrazine by ST11 could not be affected by Cd2+ with concentrations up to 1.5 mmol/L. The effects of Cd2+ on ST11 cell density and degradation of atrazine in crystal form or dissolved in DEHP were determined (Figure 6). At Cd2+ concentrations below 0.3 mmol/L, cell growth and atrazine degradation were stimulated with Cd2+ concentration. The highest removal of atrazine (almost 100% at 48 h) in crystal form or dissolved in DEHP occurred in the case of Cd2+ concentration of 0.1 and 0.2 mmol/L, respectively. The DEHPdissolved atrazine with decreased bioavailability, which may cause more Cd2+, is needed to obtain high degradation ratio. When the concentration of Cd2+ was increased from 0.5 to 1.5 mmol/L, the effect of stimulating cell growth and atrazine degradation gradually weakened. However, in this Cd2+ concentration range, the growth was partially inhibited when ST11 was used soluble substrates (Figure 2A). A notable detail that at the same Cd2+

concentration (such as 0.1 mmol/L), the biodegradation enhancement effect of Cd2+ on crystalline atrazine was stronger than that of the DEHP-dissolved atrazine. As mentioned, the main reason could be the lower bioavailability of atrazine in DEHP. Correspondingly, the promotion of atrazine biodegradation in the presence of the Cd2+ concentration of 0.05–1.5 mmol/L could be attributed to the enhanced atrazine catabolism (Figure 6). Low concentrations of heavy metal ions could stimulate the catabolism of organic substances [40]. Cu2+ concentrations of 15 and 2 mg/L stimulated the degradation of decabromodiphenyl ether and benzo[a]pyrene, respectively [41,42]. An assessment of Cd pollution in arable soil in China showed that the average and maximum Cd concentrations were 0.0024 and 1.36 mmol/kg, respectively [6], both lower than the 1.5 mmol/L shown in the present study. Therefore, ST11 is not affected by soil Cd when it degrades atrazine in arable soils. *Catalysts* **2022**, *12*, x FOR PEER REVIEW 8 of 13

**Figure 6.** Effect of Cd2+ of different concentrations on bacterial growth and atrazine biodegradation. (**A**) crystalline atrazine; (**B**) DEHP dissolved atrazine. The initial concentration of atrazine was 1.85 mmol/L. Ctr: control samples with no cells inoculated. For each biological flask, 1 mL of bacterial suspension (OD600 1.0) was inoculated. At an interval of 24 h, the growth was monitored spectro‐ photometrically by measuring OD600 and the residual atrazine was detected. Data points represent the mean of three replicates and error bars show the standard deviation. **Figure 6.** Effect of Cd2+ of different concentrations on bacterial growth and atrazine biodegradation. (**A**) crystalline atrazine; (**B**) DEHP dissolved atrazine. The initial concentration of atrazine was 1.85 mmol/L. Ctr: control samples with no cells inoculated. For each biological flask, 1 mL of bacterial suspension (OD<sup>600</sup> 1.0) was inoculated. At an interval of 24 h, the growth was monitored spectrophotometrically by measuring OD<sup>600</sup> and the residual atrazine was detected. Data points represent the mean of three replicates and error bars show the standard deviation.

**3. Materials and Methods** *3.1. Chemicals* Atrazine (product number TCI‐A1650, 97.0%), CdCl2∙2.5H2O (≥99.0%), ethyl acetate (>99%), and methanol (>99%) were purchased from Sinopharm (Shanghai, China). DEHP (≥99.5%) was obtained from Aladdin (Shanghai, China). 2‐(5‐Bromo‐2‐pyridylazo)‐5‐(di‐ ethylamino) phenol (5‐Br‐PADAP, 97%) and Triton X‐114 (laboratory grade) were re‐ ceived from Sigma‐Aldrich (Shanghai, China). The other reagents and solvents were of analytical grade and used directly. The stock solution of atrazine was prepared by dis‐ solving atrazine in dichloromethane (92.73 mmol/L). *3.2. Microorganism and Culture Conditions* Soil samples were collected by hand‐picking in a 5–10 cm soil layer with a modified sampling technique [46]. Each sample was placed in a portable icebox and then trans‐ ferred to the laboratory. An efficient atrazine‐degrading bacterium *Arthrobacter* sp. ST11 As is well known, Cd, similar to other heavy metals, such as copper, zinc, and lead, has biological toxicity [5]. Cd could attach to proteins with sulfhydryl functional groups or glutathione, thus interfering with the synthesis of cysteine or directly damaging DNA [43]. In addition, Cd could easily penetrate the cell membrane of bacteria, causing the emission of substances in cells and affecting the metabolic process of cells [44]. The results of the present study showed that Cd inhibited and stimulated the growth of ST11 using soluble substrates (Cd2+ concentration of 0.5–1.5 mmol/L) and hydrophobic substrate atrazine (Cd2+ concentration of <sup>≤</sup>1.5 mmol/L), respectively. ST11 cells have negligible Cd adsorption capacity, which may be one of the reasons why they could resist high Cd concentration. In addition, the antagonistic effect of hydrophobic organics on Cd could be used to mitigate Cd effect on soil living organisms [45]. The results of the present study are consistent with that of the previous study. The biodegradation of crystalline atrazine and NAPL-solubilized atrazine by ST11 could not be affected by Cd2+ with concentrations up to 1.5 mmol/L.

#### was isolated from herbicide‐polluted soil. The bacterium was cultivated on LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter of Milli‐Q deionized water; pH **3. Materials and Methods**

#### *3.1. Chemicals*

6.8; stored at room temperature after sterilization) at 30 °C, with shaking at 150 rpm for 18 h. The cells were harvested by centrifugation at 6380× *g* for 10 min at 4 °C and washed twice with mineral salt medium (MSM, 5.8 g Na2HPO4, 0.9 g KH2PO4, 0.2 g MgSO4∙7H2O, and 1 mL of trace element solution per liter of Milli‐Q deionized water; pH 6.5). The com‐ position of the trace element solution was as follows: 0.4 g Na2B4O7∙10H2O, 0.5 g Atrazine (product number TCI-A1650, 97.0%), CdCl2·2.5H2O (≥99.0%), ethyl acetate (>99%), and methanol (>99%) were purchased from Sinopharm (Shanghai, China). DEHP (≥99.5%) was obtained from Aladdin (Shanghai, China). 2-(5-Bromo-2-pyridylazo)- 5-(diethylamino) phenol (5-Br-PADAP, 97%) and Triton X-114 (laboratory grade) were

Na2MoO4∙2H2O, 0.8 g CuSO4∙5H2O, 2 g FeSO4∙7H2O, 2 g MnSO4∙H2O, 10 g ZnSO4∙7H2O, and 5 g EDTA disodium per liter of Milli‐Q deionized water; pH 6.5. The cells were resus‐

The resistance of ST11 to Cd2+ was investigated. The Cd2+ concentrations ranged from 0 to 1.5 mmol/L for ST11. The cells were cultured with an LB medium without Cd2+ at 30 °C at 150 rpm in darkness for 24 h. After the cells were washed three times and resus‐ pended with Milli‐Q deionized water, 1 mL of cell culture (OD600 = 1.0) was transferred into 150 mL sterilized flasks containing 30 mL of LB medium with various Cd2+ concen‐ trations. The flasks were incubated at 150 rpm in darkness at 30 °C for 48 h. Cell growth

*3.3. Cd2+ Resistance and Growth Curve of ST11*

received from Sigma-Aldrich (Shanghai, China). The other reagents and solvents were of analytical grade and used directly. The stock solution of atrazine was prepared by dissolving atrazine in dichloromethane (92.73 mmol/L).

#### *3.2. Microorganism and Culture Conditions*

Soil samples were collected by hand-picking in a 5–10 cm soil layer with a modified sampling technique [46]. Each sample was placed in a portable icebox and then transferred to the laboratory. An efficient atrazine-degrading bacterium *Arthrobacter* sp. ST11 was isolated from herbicide-polluted soil. The bacterium was cultivated on LB medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter of Milli-Q deionized water; pH 6.8; stored at room temperature after sterilization) at 30 ◦C, with shaking at 150 rpm for 18 h. The cells were harvested by centrifugation at 6380× *g* for 10 min at 4 ◦C and washed twice with mineral salt medium (MSM, 5.8 g Na2HPO4, 0.9 g KH2PO4, 0.2 g MgSO4·7H2O, and 1 mL of trace element solution per liter of Milli-Q deionized water; pH 6.5). The composition of the trace element solution was as follows: 0.4 g Na2B4O7·10H2O, 0.5 g Na2MoO4·2H2O, 0.8 g CuSO4·5H2O, 2 g FeSO4·7H2O, 2 g MnSO4·H2O, 10 g ZnSO4·7H2O, and 5 g EDTA disodium per liter of Milli-Q deionized water; pH 6.5. The cells were resuspended with MSM, and the optical density at 600 nm (OD600) was adjusted to 1.0.
