*Article* **Biodegradation of Crystalline and Nonaqueous Phase Liquid-Dissolved ATRAZINE by** *Arthrobacter* **sp. ST11 with Cd2+ Resistance**

**Jiameng Zhang, Zhiliang Yu, Yaling Gao, Meini Wang, Kai Wang and Tao Pan \***

Jiangxi Province Key Laboratory of Mining and Metallurgy Environmental Pollution Control, School of Resource and Environmental Engineering, Jiangxi University of Science and Technology, Ganzhou 341000, China **\*** Correspondence: t.pan@jxust.edu.cn; Tel.: +86-13698057756

**Abstract:** A newly isolated cadmium (Cd)-resistant bacterial strain from herbicides-polluted soil in China could use atrazine as the sole carbon, nitrogen, and energy source for growth in a mineral salt medium (MSM). Based on 16S rRNA gene sequence analysis and physiochemical tests, the bacterium was identified as *Arthrobacter* sp. and named ST11. The biodegradation of atrazine by ST11 was investigated in experiments, with the compound present either as crystals or dissolved in di(2-ethylhexyl) phthalate (DEHP) as a non-aqueous phase liquid (NAPL). After 48 h, ST11 consumed 68% of the crystalline atrazine in MSM. After being dissolved in DEHP, the degradation ratio of atrazine was reduced to 55% under the same conditions. Obviously, the NAPL-dissolved atrazine has lower bioavailability than the crystalline atrazine. Cd2+ at concentrations of 0.05–1.5 mmol/L either had no effect (<0.3 mmol/L), slight effects (0.5–1.0 mmol/L), or significantly (1.5 mmol/L) inhibited the growth of ST11 in Luria-Bertani medium. Correspondingly, in the whole concentration range (0.05–1.5 mmol/L), Cd2+ promoted ST11 to degrade atrazine, whether crystalline or dissolved in DEHP. Refusal to adsorb Cd2+ may be the main mechanism of high Cd resistance in ST11 cells. These results may provide valuable insights for the microbial treatment of arable soil co-polluted by atrazine and Cd.

**Keywords:** atrazine; *Arthrobacter* sp. ST11; biodegradation; nonaqueous-phase liquid; cadmium

## **1. Introduction**

Arable soil is often polluted with herbicides [1]. Atrazine (6-chloro-4-N-ethyl-2-Npropan-2-yl-1,3,5-triazine-2,4-diamine) is one of the most widely-used persistent chlorine herbicides that often remain in agricultural fields and water bodies for several years at concentrations of hundreds of µg/kg [2]. Atrazine has lower bio-accessibility when present in an unavailable phase, such as crystalline form, soil and sediment solids, or non-aqueous phase liquid (NAPL). The environmental persistence of atrazine has been shown to be a vastly significant problem [2]. No strong evidence could prove that atrazine causes cancer; however, it affects the endocrine response and thus has a potential effect on human reproduction and development [3]. Although it was banned for use by the European Union in 2004, atrazine remains legal in China [2]. Atrazine is expected to persist in arable soil sources for decades [4], thereby calling for appropriate remediation measures.

Cadmium (Cd), a potentially toxic heavy metal with no known biological function, occurs widely in nature [5]. A national-scale study of soil Cd pollution in China reported that the average and maximum concentrations of Cd in arable soil were 0.0024 and 1.36 mmol/kg, respectively [6]. As one of the most toxic trace elements in the environment, Cd could cause serious health problems to microorganisms, plants, animals, and humans [5,7].

Bioremediation is cost-effective and environmentally friendly and, thus, has become one of the most popular approaches for removing atrazine [2]. Biodegradation by indigenous

**Citation:** Zhang, J.; Yu, Z.; Gao, Y.; Wang, M.; Wang, K.; Pan, T. Biodegradation of Crystalline and Nonaqueous Phase Liquid-Dissolved ATRAZINE by *Arthrobacter* sp. ST11 with Cd2+ Resistance. *Catalysts* **2022**, *12*, 1653. https://doi.org/10.3390/ catal12121653

Academic Editor: Jasmina Nikodinovi´c-Runi´c

Received: 25 September 2022 Accepted: 14 December 2022 Published: 15 December 2022

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microbial populations is considered an important process that affects the fate of atrazine in contaminated sites [2]. Since the 1990s, different atrazine-degrading bacteria and fungi have been isolated from contaminated sites [8,9], including *Arthrobacter* [10], *Nocardioides* [11], *Shewanella* [12], *Rhodococcus* [13], *Stenotrophomonas* [14], *Pseudomonas* [15], *Paenarthrobacter* [16], and *Trametes* [17]. Bacteria are the foundation of microbial bioremediation, which can outperform fungi in the potential for atrazine-specific bioaugmentation [18]. However, the low bioavailability of atrazine in the environment hindered the degradation efficiency of these strains. Although many enhancement methods have been applied [19–21], little is known about the mode of acquisition of atrazine when it is present in an unavailable phase. Heavy metals and synthetic pesticides often co-occur in soil, although their hazards are usually evaluated separately and in bulk soil [22]. Coexisting heavy metals may stimulate or inhibit the biodegradation of herbicides [23,24]. When Cd pollution coexists, the fate of atrazine in the soil will be difficult to predict. The potential ecological risk of combined pollution of atrazine and Cd in waters and soils still exists, and it cannot be ignored, even when said risk is lower than that of atrazine or Cd alone [25,26]. Overall, the search and isolation of specific bacterial strains that could degrade atrazine efficiently with Cd resistance are of great interest.

*Arthrobacter* is prevalent in the agricultural soil environment, and it could degrade many kinds of environmental pollutants [10,27]. Many *Arthrobacter* strains have been isolated to degrade atrazine, such as *Arthrobacter* Sp. DNS10, *Arthrobacter* sp. LY-1, and *Arthrobacter Aurescens* TC1 [28–30]. In addition, some *Arthrobacter* strains were reported to have metal resistance [31,32]. However, atrazine-degrading *Arthrobacter* strains with Cd resistance have not yet been found.

The three primary goals of this study were designed to address gaps that currently exist in the research. The first goal was to isolate high-efficiency atrazine-degrading bacteria with Cd resistance and characterize them through 16S rRNA gene sequencing analysis and physiochemical tests. The second goal was to investigate the bioavailability of crystalline and NAPL-dissolved atrazine to the strain. The third goal was to explore the mechanisms of the effect of Cd2+ on atrazine biodegradation. Finally, some valuable insights into the treatment of atrazine in soil with Cd pollution were provided.

#### **2. Results and Discussion**

#### *2.1. Identification and Characterization of Test Strain*

An efficient atrazine-degrading bacterium ST11 was isolated from herbicide-polluted soil. Under scanning electron microscopy (SEM), ST11 appeared as rods when rapidly dividing and cocci when in the stationary phase (Figure S1), showing the typical characteristics of *Arthrobacter* cells [33]. The strain was further identified as *Arthrobacter* sp. by physiochemical tests (Table S1) and 16S rDNA analysis (GenBank OP435654). A phylogenetic tree was constructed using an approximate maximum-likelihood analysis (Figure 1).
