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 土壤学报  2021, Vol. 58 Issue (4): 957-967  DOI: 10.11766/trxb202002200514 0

### 引用本文

CHEN Xinyao, XIAO Zufei, ZHU Yanhua, et al. Impacts of Combined Pollution of Copper and Doxycycline on Activities of Soil Microbes and Enzymes. Acta Pedologica Sinica, 2021, 58(4): 957-967.

### 作者简介

1. 苏州科技大学环境科学与工程学院, 江苏苏州 215009;
2. 苏州工业园区环境监测站, 江苏苏州 215027;
3. 苏州高新区环境监测大队, 江苏苏州 215000

Impacts of Combined Pollution of Copper and Doxycycline on Activities of Soil Microbes and Enzymes
CHEN Xinyao1,2, XIAO Zufei1, ZHU Yanhua3, WU Jian1, HUA Qianwen1, ZHANG Yuan1
1. School of Environmental Science and Engineering, Suzhou University of Science and Technology, Suzhou, Jiangsu 215009, China;
2. Environmental Monitoring Station of Suzhou Industrial Part, Suzhou, Jiangsu 215027, China;
3. Environmental Monitoring Brigade of Suzhou High-tech District, Suzhou, Jiangsu 215000, China
Abstract: 【Objective】Nowadays, more attention should be paid to the situation of agricultural environment and the quality and safety issues of agricultural produces because more and more various pollutants are entering into the agricultural soil. Among the pollutants, heavy metals and antibiotics are two of the most important types of pollutants in the farmland soil. In this study, soil microorganisms are used as indicators to illustrate impacts of combined pollution of heavy metals and antibiotics on soil environment.【Method】An in-lab analogue incubation experiment was conducted in this study, with the soil spiked with doxycycline (DOX, 0, 8 and 15 mg·kg-1) and copper (Cu, 0, 100 and 400 mg·kg-1), singly or combinedly, at a varying rate as the representative pollutants of antibiotic and heavy metal, respectively. Effects of the pollutants on respiration of soil microbes, activities of soil enzymes, like urease, sucrase and catalase, and abundance of tet ARGs (tetracycline-resistance genes) were investigated.【Result】Results show that throughout the whole incubation period (30 days), the pollution of DOX and Cu, either singly or combinedly, inhibited significantly respiration of soil microbes in intensity, and activities of sucrase and catalase, but stimulated that of urease, and the effect was apparently catalase than on sucrase. To sum up, the effects of combined pollutions is much higher than that of singe pollutions and the addition of DOX stimulated the initial effect of Cu on respiration of soil microbes or activities of soil enzymes. Correlation analysis shows that the activity of catalase/urease was significantly and negatively related to Cu concentration and urease activity significantly and negatively correlated with sucrase activity. Besides, it was also found that the total relative abundance of four types of antibiotic resistance genes (ARGs) decreased first and then turned backwards, which was probably owing to the tie lag of about 7-15 days the soil system needs to adapt and age after the addition of the pollutants. Compared with other treatments, the addition of high concentrations of Cu and DOX significantly increased the abundance of ARGs at the mid-and late-stages of the incubation, especially the relative abundance of tetA and tetW. The addition of a high concentration (400 mg·kg-1) of Cu improved the ability of DOX to induce high relative abundance of tet ARGs.【Conclusion】All the findings in this study indicate that combined pollution of Cu and DOX has dramatic impacts on functions of the soil microbial system.
Key words: Doxycycline    Copper    Soil microbial respiration    Enzyme activity    Antibiotic resistance genes

1 材料与方法 1.1 供试材料

1.2 实验设计

1.3 微生物呼吸分析

 $微生物呼吸强度\rm{=}\frac{\times }{\times \times }$ (1)

1.4 土壤酶活性分析

1.5 DNA提取和定量实时PCR（qPCR）分析

 ${C_1} = \frac{{L{\rm{ \times }}C}}{{N{\rm{ \times }}M{\rm{ \times }}{{10}^9}}}$ (2)

 $耐药基因拷贝数=\frac{{{C_1} \times V}}{{{M_1}}}$ (3)

1.6 数据处理

2 结果 2.1 铜和强力霉素污染对土壤微生物呼吸的影响

 图 1 铜和强力霉素单一及复合污染对土壤微生物呼吸的影响 Fig. 1 Impacts of Cu and/or DOX pollution on respiration of soil microbes

DOX单一污染在培养期会始终抑制对土壤微生物呼吸，抑制率在培养1 d最大。对于Cu单一污染，结果表明Cu400处理下微生物呼吸始终保持在较低水平，而Cu100处理下微生物呼吸强度在培养期内虽均低于CK，但随培养时间不断恢复增强。

2.2 铜和强力霉素污染对土壤酶活性的影响

 注：不同小写字母表示同一时间节点不同污染处理方式之间的差异性显著（P < 0.05）。   Note: Different lowercase letters represent significant differences between different treatments at the same time point(P < 0.05). 图 2 铜和强力霉素单一及复合污染对土壤脲酶、蔗糖酶及过氧化氢酶活性的影响 Fig. 2 Impacts of Cu and/or DOX pollution on activities of urease, sucrase and catalase in the soil

DOX、Cu单一及复合污染对土壤蔗糖酶活性的影响见图 2。Cu、DOX单一及复合污染对蔗糖酶在培养期内主要为抑制作用，抑制强度随时间推移逐渐减弱，30 d基本恢复至初始水平。DOX单一处理对蔗糖酶活性整体呈抑制作用，DOX8对蔗糖酶活性的抑制率随时间逐渐增大；DOX15前期抑制酶活性，终期（30 d）激活蔗糖酶活性。单一Cu胁迫及其与DOX复合胁迫对蔗糖酶活性均呈现“抑制-激活”趋势。研究还发现Cu100+DOX8/15复合作用的修复转折点是22 d，而Cu400+DOX8/15复合作用修复转折点提前，为15 d。

2.3 铜和强力霉素污染对抗生素抗性基因相对丰度的影响

 图 3 铜和强力霉素单一及复合污染对土壤抗生素抗性基因相对丰度的影响 Fig. 3 Impacts of Cu and/or DOX pollution on relative abundance of tetracycline resistance genes

 图 4 铜和强力霉素单一及复合污染对土壤抗生素抗性基因相对丰度总和的影响 Fig. 4 Impacts of Cu and/or DOX pollution on relative abundance of total tetracycline resistance genes
3 讨论 3.1 铜和强力霉素对土壤微生物呼吸的作用

3.2 铜和强力霉素对土壤酶活性的作用

3.3 铜和强力霉素污染条件下土壤微生物对污染物的适应性

4 结论