2.1 Soil sample source
In this experiment, mercury-contaminated soils were collected from agricultural fields in Luanchuan County, Luoyang City, Henan Province (33°N, 111°E) using the five-point sampling method, and the samples were primarily from the protoplasmic soil layer within the top 20 cm of the soil. The mercury content of the soil was determined by atomic fluorescence with a detection limit of 0.002 mg/kg.
2.2 Screening mercury-resistant strains
Soil samples were collected from the heavy metal-contaminated soil in the mining area, 10 g of soil was placed in a 250 mL beaker, 90 mL of sterile water was added to create a bacterial suspension and the suspension was magnetically stirred for 30 min and left to stand. Then, 1 mL of supernatant was inoculated with a pipette gun in LB medium containing 10 mg/L Hg2+ in a 30°C, the samples were shaken at 150 r/min in an incubator and the samples incubated for 2 h for 10×series (10− 2, 10− 3, 10− 4 and 10− 5) gradient dilutions (Naguib et al., 2019). A dilution of the sample was spread on solid LB medium containing 20 mg/L Hg2+ and incubated in a biochemical incubator at 37°C for 48 hours. After the colonies grew on the medium, the concentration of mercuric chloride in the LB solid medium was increased continuously. A high concentration of mercury-tolerant strains was screened by picking and culturing individual colonies on an LB solid medium containing a high mercury concentration.
The selected mercury-resistant strains were cultured in line on LB solid medium without mercury, and the strains were repeatedly purified and screened. The morphological characteristics of the colonies on the medium were observed and recorded. Inoculations of the mercury-resistant strain were placed into LB liquid medium and incubated at 30°C and 150 rpm in a constant temperature shaker until the solution became cloudy. The solution was preserved using glycerol. With the aid of a pipette on an ultraclean table, 0.2 mL of glycerol was pipetted into a 1.5 mL centrifuge tube, and 0.2 mL of the bacterial seed solution was mixed with gentle shaking. The 1.5 mL centrifuge tube containing the bacterial solution was placed in a sealed bag and placed in a refrigerator at -40°C for use in subsequent experiments.
2.3 Strain identification
Identification of bacteria by colony morphology and biochemical tests, including Gram staining (Oliveira et al., 2015), starch hydrolysis test, gelatin hydrolysis test, oil hydrolysis test, indole test, sugar fermentation test, methyl red test, volt-pop (VP) test, H2S test, and strain motility test.
2.4 Molecular biology identification
The genomic DNA of the strain was extracted, and the 16S rDNA gene (Pushkar, Sevak, & Sounderajan, 2018) was amplified by polymerase chain reaction (PCR) using bacterial universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-ACGGCTACCTTGTTACGA-3'). The PCR (Aatif, Arslan, Mushtaq, Zakia, & Stanley, 2019) products were sent to Shanghai Bioengineering Co. Ltd. for sequencing, and the sequencing results were compared with the GenBank database of the National Center for Biotechnology Information (NCBI) to identify the genus of the strain. The phylogenetic tree was constructed by the neighbor-joining method of MEGA X software.
2.5 Exploration of optimal growth conditions for bacteria strains
To investigate the effect of different media on the growth of experimental strains (Bravo, Vega-Celedón, Gentina, & Seeger, 2020), 50 mL of each of 8 liquid media, including LB medium, beef paste peptone medium, high type 1 medium, PDA medium, Choi medium, sand medium, glycerol medium, and PSA medium, were prepared in a 250 mL conical flask, and standard liquid medium containing 10 mg/L Hg2+ and no Hg2+ was prepared. Inoculation was performed after the medium was adjusted to pH 7.0, sterilized at 121°C for 30 minutes, and cooled (fresh bacterial seed solution to medium = 2:100). The culture was incubated for 36 h at 37°C and 150 r/min in a constant temperature shaking incubator. The cultured bacterial solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the centrifuge tube was placed in a vacuum drying oven and dried at 70°C to a constant weight. Each group was divided into three replicates. The dry weight of the strain was calculated as follows:
$$\text{m}=\left({\text{M}}_{1}-{\text{m}}_{1}\right)-({\text{M}}_{2}-{\text{m}}_{2})$$
where M1 is the initial mass of the experimental group, M2 is the initial mass of the control group, m1 is the mass of the experimental group after drying, and m2 is the mass of the control group after drying.
Under the optimum conditions, LB liquid medium containing 10 mg/L Hg2+ and LB liquid medium without Hg2+ were used. The following range of inoculums were used for the test strains: 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%, and the OD600nm values were determined after 36 hours of incubation at 150 rpm and 30°C.
The pH of liquid LB medium was adjusted to 4, 5, 6, 7, 8, 9, and 10 with hydrochloric acid (HCl) or sodium hydroxide (NaOH), and the OD600nm values were measured after inoculating fresh bacterial solutions into LB liquid medium and incubating the solutions at 37°C for 36 hours at 150 rpm to obtain the proper pH range for strain growth.
Based on the same medium preparation method, pH, and inoculation method, the OD600 nm values were determined after 36 h of incubation (Pepi et al., 2011) at 20°C, 25, 30, 35°C, and 40°C in a constant temperature shaking incubator. Under the above optimal conditions, the bacteria were inoculated in liquid LB (Abu-Dieyeh, Alduroobi, & Al-Ghouti, 2019) media containing 0, 0.5%, 1%, 2%, 4%, 6%, and 8% sodium chloride, and the OD600 nm values were measured after incubation to obtain the optimal salt concentration. Each group of experiments was repeated three times.
2.6 Determination of the tolerance of strains to mercury
Under optimal growth conditions, LB liquid medium was prepared with Hg2+ concentrations of 0, 1, 2, 4, 8, 16, 32, 64, and 128 mg/L, sterilized at 121°C for 30 min, inoculated with 2% fresh bacterial broth into LB liquid medium, and incubated with shaking for 120 h. To determine the minimum inhibitory concentration of heavy metal mercury on the experimental strains, the optical density (OD600nm) values of each medium were measured regularly. The minimum inhibitory concentration (MIC) of Hg2+ on the experimental strains was determined at regular intervals by measuring the OD600 nm values of each medium for 120 h.
According to the minimum inhibitory concentration (MIC) of Hg2+ on the strains, 2% fresh bacterial solution was inoculated into liquid LB medium with 5, 10, and 20 mg/L Hg2+, LB medium without mercury was used as a blank control, and the optical density was measured regularly in a UV spectrophotometer to study the changes in the growth pattern of the strains under mercury stress.
2.7 Determination of mercury volatilization and adsorption capacity of the strain
As part of the experiment, three groups were established. The positive control group contained no mercury, the negative control group was not inoculated with the strain. The experimental group was inoculated with 2% fresh bacterial broth in liquid LB medium containing 10 mg/L Hg2+ and under optimal growth conditions, and three parallel treatments were conducted for each group. The samples were removed at regular intervals and centrifuged at 8000 rpm for 10 minutes. The process of digesting the supernatant and bacterium was performed as follows. A total of 1 mL supernatant from the centrifuged culture solution was mixed with 10 mL of HCl in a 50 mL Teflon crucible, the sample was heated continuously on an electric furnace, and the sample was digested until the volume was reduced to 3 mL. A total of 5 mL nitric acid was added to the solution, and the solution was heated for 1 h. The steps were repeated until the digestion was complete and the white fumes were exhausted. Then, the digestion solution was transferred to a 50 mL volumetric flask after it had cooled to room temperature and was shaken well with a fixed volume.
A cold atomic absorption spectrophotometer was used to measure the mercury concentration in the supernatant and the mercury content in the bacteria. The efficiency of total mercury removal was the initial mercury content of the culture medium minus the mercury content of the supernatant, divided by the initial mercury content of the culture medium; the efficiency of mercury adsorption by the bacteria was determined by the mercury content of the bacteria divided by the initial mercury content of the culture medium.
The mercury concentration of the supernatant and the mercury content of the bacteria were measured using a cold atomic absorption spectrophotometer. The efficiency of total mercury removal is the initial mercury content of the culture medium minus the mercury content of the supernatant, divided by the initial mercury content of the culture medium, and the efficiency of mercury adsorption by the bacteria is the mercury content of the bacteria divided by the initial mercury content of the culture medium. The total removal rate of mercury minus the adsorption efficiency of the bacteria on mercury is the volatilization efficiency of mercury (Ghosh, Sadhukhan, Ghosh, Chaudhuri, & Mandal, 1996). The heavy metal concentrations in the digestion solution and the blank control were measured three times. The calculation was performed as follows:
$${\text{q}}_{1}=\frac{({\text{a}}_{1}-{\text{b}}_{1})}{{\text{a}}_{1}}$$
$${\text{q}}_{2}=\frac{{c}_{1}}{{a}_{1}}$$
$${\text{q}}_{3}={\text{q}}_{1}-{\text{q}}_{2}$$
where a1 is the initial mercury content of the culture solution (mg/L); b1 is the mercury content of the supernatant (mg/L); c1 is the mercury content of the bacteria (mg/L); q1 is the total removal rate (%); q2 is the adsorption efficiency (%); and q3 is the volatilization efficiency (%).
2.8 Transformation infrared spectroscopy
To investigate the response mechanism of the LBA119 strain to mercury stress, Fourier transform infrared (FTIR) spectroscopy (Pepi et al., 2011) was used to analyze the changes in the adsorption functional groups of bacteria before and after treatment with the heavy metal Hg2+. The bacteria were incubated in LB medium with mercury (10 mg/L) and without mercury for 36 h. The cultures were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the organisms were washed three times with phosphate buffer solution. The strains were freeze-dried under a vacuum, and the dried samples were thoroughly mixed with potassium bromide (KBr) powder in an agate mortar (bacterial mass: KBr mass = 1:100), pressed into thin slices using a solid press, and measured using a Fourier infrared spectrometer to record the infrared spectra in the region of 4000 − 400 cm− 1. The obtained spectral data were processed using Omnic 9.0 software.
2.9 Testing the strains for resistance to different types of heavy metal ions
LB liquid medium containing single heavy metal ions (Mn2+, Zn2+, Pb2+, Cd2+, and Cr6+) was prepared and inoculated with 2% fresh bacterial broth and incubated on a shaker at 30°C for approximately 36 h. LB medium with different concentration gradients (1, 100, 500, 1000, 1500, 2000, 3000, 4000, and 5000 mg/L) of heavy metals was first set medium to determine the crude tolerance of the strain to heavy metal concentrations (Ruiz-Díez et al., 2012). The concentration interval was then narrowed to determine the maximum concentration of heavy metals that could be tolerated. Bacterial concentrations were determined using the 600 nm optical density method (OD600nm) to determine the minimum inhibitory concentration (MIC). Five heavy metal resistance assays were performed independently (Zhang, Chen, & Liu, 2012), with three parallel groups for each group.
2.10 Study on the bioremediation effect of strains of bacteria on mercury-contaminated soil
In this experiment, mercury-contaminated soil was collected in Luanchuan County, Henan Province, from farmland in the molybdenum mining area. The soil was air-dried and sieved, and then a specific quantity of HgCl2 solution was added to it. The soil samples were prepared with mercury concentrations of 5, 50, and 100 mg/L, stirred well, and left to stand for several days before being stirred again; then, the mercury soil was aged for 60 days.
2.11 Strain for remediation of mercury-contaminated soil
A logarithmic growth stage was achieved by incubating the bacteria. An adjustment of pH 4.6 was made to the seed solution used for inoculating LBA119 bacteria. Blank LB medium was added to the control group at the same concentration. After inoculation, the soil and inoculum were gently mixed on a shaker at 30 rpm for 20 minutes to conduct an exploratory experiment on the biological removal of mercury from the soil (Koçberber & Dönmez, 2007).
The study involved several experimental groups, which were prepared as follows. (1) Restoration group: 100 g of mercury-aged soil samples, 95 mL of distilled water, and 5 mL of bacterial seed solution were added and shaken well to ensure that all the soil was suspended in the liquid. Sterile distilled water was replenished quantitatively every day to maintain the water content of the soil, and the new bacterial solution was added every 7 d, with an additional amount of 5 mL each time. (2) Control group: 100 g of aged soil samples containing mercury, 95 mL of distilled water, and 5 mL of LB medium were added and shaken well, and 5 mL of fresh medium was added every 7 d.
In each experimental group, three replicates were conducted. Incubation for 7, 15, and 30 days was conducted in an incubator (temperature 30°C, light interval 8 h light, 16 h dark). Each group was provided 0.5 g of air-dried soil that was ground and digested. Using cold atomic fluorescence spectrometry (Ji, Zhang, Bararunyeretse, & Li, 2018), the mercury content was determined, and the mercury removal efficiency was calculated.
Mercury remediation efficiency was calculated by subtracting the initial mercury content of the soil from the mercury content at the time of sampling. The results are the mean ± standard deviation of three independent replicates. The efficiency of the strains for remediating mercury in the soil was calculated as follows:
$$\text{q}\left(\text{%}\right)=\frac{\text{a}-\text{b}}{\text{a}}\times 100\text{%}$$
where q is the removal efficiency (%) of mercury in soil by the strain; a is the initial mercury content of soil; and b is the mercury content of soil at different sampling times.