The Inhibitory Effect of Cadmium And/Or Mercury on Soil Enzyme Activity, Basal Respiration, and Microbial Community Structure in Coal Mine–Affected Agricultural Soil
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Annals of Microbiology (2019) 69:849–859 https://doi.org/10.1007/s13213-019-01478-3 ORIGINAL ARTICLE The inhibitory effect of cadmium and/or mercury on soil enzyme activity, basal respiration, and microbial community structure in coal mine–affected agricultural soil Liugen Zheng1 & Yang Li1 & Wenqin Shang1 & Xianglin Dong2 & Quan Tang3 & Hua Cheng1 Received: 19 October 2018 /Accepted: 26 April 2019 /Published online: 6 May 2019 # Università degli studi di Milano 2019 Abstract Purpose The Cd and Hg contents in soils can be elevated due to coal mining. To estimate the effects of these two heavy metals on soil enzymes and the key microbial groups, coal mine–affected agricultural soils were cultured for 30 days with Cd and/or Hg. Methods Soil enzyme activities were measured by a colorimetric method, and microbial abundance was assessed according to real-time quantitative PCR analysis of the 16S rRNA and 18S rRNA genes. In addition, the microbial communities were analyzed by Illumina sequencing. Results Heavy metals inhibited soil enzyme activities. For example, both Cd and Hg decreased 25.52–34.89% of the soil catalase activity; the highest level of Hg (30 mg kg−1) decreased 76.50–89.88% of the soil urease activity and 85.60–92.92% of the soil dehydrogenase activity; and the soil acid phosphatase activity significantly decreased by 15.18–32.64% under all the levels of Cd and decreased 17.09–30.32% under the high levels of the Cd–Hg combination (> 3 mg kg−1). In addition, increased Cd levels affected bacterial number more than fungal abundance; however, addition of Hg alone decreased the bacterial number but increased the fungal abundance. Furthermore, the bacterial communities but not fungal communities were altered by heavy metals. A total of 23 highly sensitive genera and 16 highly resistant genera were identified. The sensitive genera were assigned to Actinobacteria, Acidobacteria, Candidate division WS3, Chloroflexi, Gemmatimonadetes, Proteobacteria, and Thermotogae, while the resistant genera were affiliated to Bacteroidetes and Proteobacteria. Conclusion Soils containing the highest level of the combination of Cd and Hg exhibited the lowest soil enzyme activities; bacterial communities were more sensitive to heavy metal contamination than fungi. Keywords Coal mine . Agricultural soil . Soil enzymes . Bacterial community . Fungal communities Introduction heavy metals occurs mainly during natural soil formation (dos Santos et al. 2017), and anthropogenic deposits originate from Cd and Hg are two of the most toxic heavy metals and pose a sewage irrigation, sludge application, solid waste treatment, serious threat to soil ecosystems. Accumulation of these toxic automobile exhausts, industrial activities (Jan et al. 2010), and mining (Šajn et al. 2013;Lietal.2014). It is well known that these heavy metals have negative effects on biological func- Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-019-01478-3) contains supplementary tions in soil, including soil enzyme activity (Khan et al. 2010; material, which is available to authorized users. Pan and Yu 2011), microbial abundance (Chen et al. 2014), microbial activity (Chodak et al. 2013;Chenetal.2015), and * Liugen Zheng microbial community structure (Khan et al. 2010;Freyand [email protected] Rieder 2013). Microorganisms in soils exhibit a certain adaptability to 1 School of Resources and Environmental Engineering, Anhui heavy metals. However, identification of an indicator of heavy University, Hefei, China metal pollution in soils remains challenging. Previous studies 2 Geological Survey Division, Huaibei Coal Mining Group have found that soil enzymes can facilitate soil chemical reac- Corporation, Huaibei 235001, China tions and metabolic processes, such as biogeochemical nutri- 3 School of Life Sciences, Anhui University, Hefei, China ent cycling (Moreno et al. 2003). These soil enzymes are 850 Ann Microbiol (2019) 69:849–859 −1 −1 −1 sensitive to heavy metal contamination (Xian et al. 2015)and CuT, 25.66 mg kg ;NiT, 42.67 mg kg ;PbT, 34.83 mg kg ; −1 −1 are often used to estimate the adverse effects of pollutants on ZnT, 69.20 mg kg ;andHgT,0.0633mgkg . soil quality (Wang et al. 2007; Gao et al. 2010). For example, inhibition of urease or phosphatase activity by heavy metals Experimental setup has been reported (Pan and Yu 2011). Meanwhile, heavy metals also have considerable effects on the abundance and The samples were adjusted to 40% water-holding capacity composition of microbial communities (Pan and Yu 2011; with deionized water and preincubated at 25 °C for 7 days. Frey and Rieder 2013), which ultimately lead to changes in Cd and Hg were added to the soil as an aqueous mixture of soil microbial activities, including soil basal respiration and CdCl2 and HgCl2. A control group (ck, sterile water micro- enzyme activity (Pan and Yu 2011;Xianetal.2015). cosms) and nine test groups (each test group with three re- The Cd and Hg levels in soils are particularly elevated due peats) were examined. In the Cd test, concentration gradients to coal mining in the Linhuan subsidence of Huaibei were set up as follows: 0.3 mg kg−1 (Cd0.3), 3 mg kg−1 (Cd3), Coalfield, Anhui Province, China. The mean concentrations and 30 mg kg−1 (Cd30); in the Hg test, the concentration of Cd and Hg were 0.26 mg kg−1 and 0.04 mg kg-1,respec- gradients were as follows: 0.3 mg kg−1 (Hg0.3), 3 mg kg−1 tively, which were as high as 3.2 and 2.9 times the background (Hg3), and 30 mg kg−1 (Hg30); and in the Cd and Hg test, the values of the surface soil of Huaibei City according to our concentration gradients were as follows: 0.3 mg of Cd and previous research (Shang et al. 2016). Therefore, in this re- 0.3mgofHgkg−1 (CdHg0.3),3mgofCdand3mgof gion, Cd and Hg are likely to affect both soil microbial diver- Hg kg−1 (CdHg3),30mgofCdand30mgofHgkg−1 sity and soil microbial processes because heavy metals can (CdHg30). A total of 30 independent incubations were per- have long-term hazardous effects on soil ecosystems and ad- formed. The treated soils were placed in plastic bags and verse effects on soil biological processes (Lee et al. 2002; mixed thoroughly, and then, the soils were kept at 25 °C for Pérez-de-Mora et al. 2006). Although several studies found 24 h to obtain a uniform distribution of the metal and an that polymetal co-contamination could result the decrease of equilibrium between Cd, Hg, and soil binding sites. Then, actinomycetes (Xu et al. 2019), and Proteobacteria, the soils (500 g) were transferred into 1000-mL plastic jars Firmicutes, and Acidobacteria were most altered by heavy and covered with the supplied air-permeable caps throughout metal contents (Frey and Rieder 2013;Dengetal.2015; the experiment to minimize evaporation but permit aeration. Jiang et al. 2019). Few studies have identified microorganisms The microcosms were incubated in the dark at 25 °C and 60% that exhibit resistance or sensitivity to these two metals and external humidity in climatic chambers. Water loss was deter- could be used as indicators of heavy metal pollution in soils. mined every other day by weight and was compensated by Therefore, in the present study, we determined the effects of addition of sterile water. Destructive sampling was performed increasing Cd and Hg concentrations on the soil microbial in triplicate after incubation of the microcosms for 30 days. communities in coal mining–affected agricultural soil and aimed to screen groups that were resistant or sensitive to Cd Enzyme activities and basal respiration assays and Hg contaminations. Soil urease activity was determined by the indophenol-based + colorimetric method, and the NH4 released by urease- mediated enzymatic hydrolysis of urea was determined color- Materials and methods imetrically at 578 nm (Guan 1986). Acid phosphatase activity was measured by the buffer method, and the p-nitrophenol Soil characteristics (PNP) levels in the filtrate were determined colorimetrically at 410 nm (Guan 1986). Dehydrogenase activity was tested by Soil samples were collected from the top 0–20 cm of the reduction of 2,3,5-triphenyltetrazolium chloride (TTC), and Linhuan subsidence of Huaibei Coalfield, Anhui Province, the triphenyl formazan (TPF) released was extracted with China (latitude N 33° 36′, longitude E 116° 36′), which is methanol and assayed at 485 nm(Guan 1986). Catalase activ- located in the warm-temperate, semihumid, monsoon climate ity was measured by the titration method (Guan 1986), and zone. The collected soil samples were stored in a polyethylene soil basal respiration (SBR) (CO2 production by incubation bag and transported in ice to the laboratory, where the samples for 24 h at 25 °C) was measured by the sodium hydroxide were sieved through a 2-mm mesh and stored at − 20 °C. The absorption method (Xu et al. 2019). soil properties were as follows: pH, 6.82; soil organic matter content, 20.64 g kg−1; total nitrogen content, 4.5 g kg−1;avail- Illumina sequencing able potassium content, 140.5 mg kg−1; available phosphorus −1 −1 content, 4.28 mg kg ; EC, 185.5 μscm ;CdT (the subscript Total DNA was extracted from each sample with a soil −1 −1 T indicates total content), 0.30 mg kg ;CrT, 90.11 mg kg ; extraction kit (E.Z.N.A. Soil DNA Kit, Omega) Ann Microbiol (2019) 69:849–859 851 according to the manufacturer’s instructions. The V3– Data analysis V4 region of the 16S rRNA gene of each sample was amplified by the 338F/806R primer pair (338F: 5′-ACT Data are expressed as the mean value ± SE. Basic data calcu- CCT ACG GGA GGC AGC A-3′ and 806R: 5′-GGA lation was performed by using Excel 2010, and graphs were CTA CHV GGG TWT CTA AT-3′).