随着我国工矿企业的发展，土壤汞污染问题日益突现，部分受汞污染的土壤仍然用于农作物种植，对人类健康和生态环境造成了极大的危害。旬阳汞资源富集，汞矿开发历史悠久，前期炼汞排放的废气、废水和废渣已对周围的大气、水体以及土壤造成污染，部分农作物出现汞超标现象。汞在农作物中的累积给当地人民群众饮食安全带来了严重威胁，因此土壤汞污染治理势在必行。微生物修复因具有经济和安全特点，受到众多学者的关注，成为土壤汞污染治理的潜在技术。本文以旬阳汞锑矿周边土壤为采样区，通过野外调查、室内试验和表征手段，在筛选出具有抗汞特性的土著菌株的基础上，进一步研究分离菌的培养条件、生长特性和去汞效果与机理，旨在为该区土壤汞污染的微生物修复提供菌种资源和理论依据，主要得出以下研究成果：

（1）试验区土壤汞的浓度分布范围为6.28~46.5 mg/kg，平均值为17.4 mg/kg。pH值范围为5.63~8.69，平均值为7.54，总体呈中性偏酸性。汞污染土壤中分离出两种耐汞菌株，微生物菌剂中分离出一种耐汞菌株，16S rRNA鉴定分别为东京芽孢杆菌（K型）、肺炎克雷伯氏菌（N型）和枯草芽孢杆菌（W型）。三种菌株的耐汞浓度分别为30 mg/L、30 mg/L和15 mg/L，分离时间一般为12~30 h，且汞含量越高，分离时间越长。

（2）在30℃、室内震荡培养条件下，三种菌株均经历适应期、对数期、稳定期和衰亡期四个生长阶段，时间分别为3~20 h、20~40 h、45~50 h和20~27 h，生长周期约为120 h，其中菌在含汞环境中适应期较长，表现为N型和复合型菌适应期最短，W型菌适应期最长，适应期以后，三种菌株的生长规律基本与无汞时相一致。

（3）菌株种类，培养方式、汞浓度和pH值对菌株的生长均有一定影响。复合菌优于单菌生长，在pH值为6~8、汞浓度在5~15 mg/L、震荡培养时均有利于菌的生长，OD₆₀₀值达到1.6。静态培养时生长周期要延至5~7 d，OD₆₀₀值下降0.5左右。

（4）共存重金属离子对KNW型菌对汞的去除具有明显的胁迫作用。Zn²⁺、Cu²⁺、Cd²⁺、Cr⁶⁺、Ni²⁺或多种重金属离子共存时，对复合菌在去汞的过程中起到抑制作用，其中Zn²⁺和多种金属离子共存胁迫作用最为明显，Cu²⁺和Cd²⁺次之，Cr⁶⁺和Ni²⁺作用最小；而Pb²⁺与其它离子表现不同，在复合菌去汞的过程中起到了促进作用，汞的吸附率提高了10%。此外，复合菌对其它重金属也表现出一定的吸附作用，表明该菌对汞是非选择性吸附。

（5）单菌、两种菌复合以及三种菌复合在培养72 h后，对10 mg/L汞的去除率分别为30~40%、50~60%和84%。复合菌的去汞效果明显优于单菌，表明菌种之间为共生关系，可以协同作用来提高汞的去除效果。SEM、EDS、FTIR表征与Zeta电位分析结果表明，分离菌去汞机制是生物吸附、沉淀和挥发共同作用的结果，其中菌体表面的氨基和磷酸基官能团发挥了重要作用。

关 键 词：汞土壤污染；生物修复；复合菌；重金属

研究类型：基础研究

With the development of industrial and mining enterprises in China, the problem of soil mercury pollution has become increasingly prominent. Some mercury contaminated soils are still used for crop planting, which has caused great harm to human health and ecological environment. Xunyang is rich in mercury resources and has a long history of mercury mine development. The waste gas, wastewater and waste residue discharged from Mercury Smelting in the early stage have polluted the surrounding atmosphere, water and soil, and some crops have exceeded the standard of mercury. The accumulation of mercury in crops poses a serious threat to the food safety of local people, so the treatment of soil mercury pollution is imperative. Microbial remediation has attracted the attention of many scholars because of its economic and safety characteristics, and has become a potential technology for the treatment of soil mercury pollution. In this paper, the soil around the Xunyang mercury-antimony mine was taken as the sampling area, and the culture conditions, growth characteristics and mercury removal of the isolated bacteria were further studied on the basis of screening out indigenous strains with mercury-resistant properties through field investigations, laboratory tests and characterization methods. The purpose of this study is to provide bacterial resources and theoretical basis for the microbial remediation of soil mercury pollution in this area. The main research results are as follows:

(1) The distribution range of soil mercury concentration in the experimental area was 6.28~46.5 mg/kg, with an average value of 17.4 mg/kg. The pH range is 5.63~8.69, the average is 7.54, and the overall is neutral to acid. Two mercury-resistant strains were isolated from the mercury-contaminated soil, and one mercury-resistant strain was isolated from the microbial inoculum. 16S rRNA was identified as Bacillus tokyo (type K), Klebsiella pneumoniae (type N) and Bacillus subtilis (type W). The mercury-tolerant concentrations of the three strains were 30 mg/L, 30 mg/L and 15 mg/L, respectively, and the separation time was generally 12~30 h, and the higher the mercury content, the longer the separation time.

(2) Under the condition of 30℃ and indoor shaking culture, the three strains all experienced four growth stages: adaptation period, logarithmic period, stable period and decay period, and the time was 3~20 h, 20~40 h, 45~50 h and 20~27 h, the growth period is about 120 h, among which the bacteria have a longer adaptation period in the mercury-containing environment, and the N type and compound type bacteria have the shortest adaptation period, and the W type bacteria have the longest adaptation period. The growth laws of the strains were basically the same as those without mercury.

(3) Bacterial species, cultivation method, mercury concentration and pH value all have certain influence on the growth of strains. Compound bacteria are better than single bacteria growth. When the pH value is 6~8, the mercury concentration is 5~15 mg/L, and the shaking culture is conducive to the growth of bacteria, the OD₆₀₀ value can reach 1.6. In static culture, the growth cycle was extended to 5~7 days, and the OD₆₀₀ value decreased by about 0.5.

(4) The coexistence of heavy metal ions has obvious stress on the removal of mercury by KNW bacteria. When Zn²⁺, Cu²⁺, Cd²⁺, Cr⁶⁺, Ni²⁺ or a variety of heavy metal ions coexist, it can inhibit the complex bacteria in the process of mercury removal. Among them, the coexistence of Zn²⁺ and various metal ions has the most obvious stress effect, followed by Cu²⁺ and Cd²⁺. Cr⁶⁺ and Ni²⁺ have the least effect; while Pb²⁺ is different from other ions, it plays a promoting role in the process of mercury removal by compound bacteria, and the adsorption rate of mercury is increased by 10%. In addition, the composite bacteria also showed a certain adsorption effect on other heavy metals, indicating that the bacteria were non-selective adsorption of mercury.

(5) The removal rates of 10 mg/L mercury for single bacteria, two bacteria compound and three bacteria compound were 30~40%, 50~60% and 84% respectively after culturing for 72 h. The mercury removal effect of the compound bacteria was significantly better than that of the single bacteria, indicating that the bacteria were in a symbiotic relationship and could synergize to improve the mercury removal effect. The results of SEM, EDS, FTIR characterization and Zeta potential analysis showed that the mercury removal mechanism of the isolated bacteria was the result of the combined action of biosorption, precipitation and volatilization, and the amino and phosphate functional groups on the surface of the bacteria played an important role.

Key words: Mercury soil pollution; Bioremediation; Compound bacteria; Heavy metal

Thesis type: Fundamental research

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