Properties of sediment
The sediment sample selected in this study was from Puti Lake, Jiaxing, Zhejiang Province, China (E 120.73°, N 30.95°). The grab dredger was used to collect the sediment sample from the bottom layer within 0-30 cm. The coarse suspended matter was removed using a 20 mesh screen. The physical and chemical properties of the sediment were measured as follows: pH 7.29, Eh -159.83 mv, total nitrogen 3.04 g/kg, total carbon 31.11 g/kg, acid volatile sulfur (AVS) 2.20 mg/kg and total solid 22.10%. The concentrations of various heavy metals were as follows: Ni 84 mg/kg, Cu 284 mg/kg, Zn 394 mg/kg. The heavy metal concentrations of the sediment were analyzed using the method mentioned in Analysis of heavy metal contents after the acid digestion of the sediment.
Properties of sulfur substrate
The sulfur substrate used in the research included sulfur-covered biochar particles, sulfur powder and sulfur powder mixed with surfactant rhamnolipid (see Table 1 for details). The sulfur-covered biochar particles were prepared by the laboratory: The bamboo biochar was purchased from Lin'an Yaoshi Biochar Industry Co., Ltd. Bamboo sawdust was anaerobic-burned at 500 °C for 8h, and the bamboo biochar particles were sieved with a mortar mill to obtain the particle size required for the research. After that, the bamboo biochar particles were washed three times with distilled water, and dried at 105 °C for 6h. The surface area of the prepared bamboo biochar particles was 332.10 m2·g-1. The main components included C (56.05 %), H (1.32 %), N (0.23 %), O (2.62 %), ash (39.78 %), P (0.29 %) and Na (0.01 %).
And the sulfur-covered biochar particles were produced by solidifying melted elemental sulfur on the surface of the bamboo biochar at 120 °C. After cooling down, the weight of the attached melted sulfur on the bamboo biochar particles was measured in order to control the same sulfur content in each experiment group.
Table 1 The properties of the different sulfur substrates
Code
|
Sulfur substrate
|
Sulfur content
|
Particle size
|
Source
|
Note
|
|
S
|
sulfur powder
|
≥ 99.5%
|
200-300 μm
|
Shijiazhuang Jiyanzhengnong corporation
|
/
|
|
|
|
SC
|
sulfur-covered biochar particles
|
70%
|
irregular particles with size of about 5 mm
|
prepared by the laboratory
|
prepared by solidifying melted elemental sulfur on the surface of the bamboo biochar
|
|
|
|
R
|
sulfur powder mixed with rhamnolipid
|
≥ 99.5%
|
200-300 μm
|
VICTEX corporation
|
rhamnolipid surfactant is secreted by Pseudomonas. The added concentration in the research was 0.3 g/L [10]
|
|
|
|
C
|
none
|
/
|
/
|
/
|
control check group, no sulfur substrate added
|
|
|
|
N
|
none
|
/
|
/
|
/
|
sterilization treatment group, added 200 mg/L NaN3
|
|
Preparation of the sludge-enriched inoculum
The inoculum used in this experiment was obtained from the acclimation of the sludge indigenous bacterium in Hangzhou Qige Wastewater Treatment Plant. And the preparation methods were consistent with the previous research [18].
Bioleaching experiment
Three experiment groups were set up according to the sulfur substrate added: sulfur powder group (experimental code: S-A), sulfur-covered biochar particles group (experimental code: SC-A), sulfur powder mixed with rhamnolipid (experimental code: R-A). The bioleaching experiments were conducted in a 250 mL conical flask containing 2.5 g dry weight of sediment, 150 mL of distilled water (the sediment concentration was decided by pre-experiment), 3 g/L of sulfur substrate (calculated by sulfur content) and 3 mL of sludge-enriched inoculum (-A represented the addition of the inoculum). Control groups without sulfur substrate and sterilization groups without sulfur substrate but with 200 mg/L NaN3 were set at the same time (the experimental code: C/N).
The conical flasks were placed in a shaking incubator at 28 °C and 180 r/min. Each treatment consisted of nine parallel groups, three of which were used to measure pH, concentration of SO42- and heavy metal on a daily basis. Distilled water was added daily to compensate for the loss of vaporization. The supernatant was withdrawn daily and the concentration of SO42- and heavy metals was analyzed. The other six parallel groups were tested for microbiological analysis on Day 4 and Day 9, respectively.
When it came to Day 9, the Tessier sequential extraction method was employed to determine the content of different heavy metal forms in the solid phase after bioleaching [19]. The forms of heavy metals were represented as follows in Fig. 2: Res=residual state; Org=organic state; Fe-Mn=iron-manganese oxidation state; Car=carbonate-bound state; Exc=exchangeable state.
After the first round, the sulfur-covered biochar particles of the SC-A group were recovered by filtration and washed with sterile physiological saline 3 times before being used for the second round of bioleaching. The experimental conditions of the second round were the same as those of the first round, but no bioleaching functional bacteria or sulfur substrate were added in all experimental groups. The third round of bioleaching experiment was carried out by the same experimental method. The pH value, concentration of SO42- and heavy metals of the samples were measured every day to investigate the bioleaching effect of the sulfur-covered biochar particles in the multiple recycling rounds.
Analysis method
The pH value was measured using pH meter (PB-10) using the National Standard Method HJ 962-2018. The concentration of SO42- was detected by ion chromatography (ICS-1100); total nitrogen, total carbon, and total phosphorus of the sediment sample were determined using an elemental analyzer (Elementar vario MAX CNS). The specific surface area of the biochar samples were measured using the American Tristar III3020 automatic specific surface area. The BET (Brunauer–Emmerr–Teller) equation was used to calculate the surface area of the bamboo biochar particles [20]. The EA110 elemental analyzer was utilized to determine the percentages of elements C, H, and N in the biochar samples [21]. Field emission scanning electron microscope (FEI SIRION-100) was utilized to observe the surface structure and the colonization of the microorganisms on the sulfur-covered biochar particles.
Analysis of heavy metal content
The heavy metal concentration of the sample was detected by an inductively coupled plasma mass spectrometer (ICP-MS) (PQMS 10-5000S-AR091). The limit of detection (LOD) of the ICP-MS is 1ppb, the accuracy was < 5%, and the relative standard deviation (RSD) was < 5%. To control the analytical quality of the analytical procedure, a certified reference material (GBW-07405) was applied and analyzed following the same procedure. The recoveries of Ni, Cu and Zn were in the range of 74.3%~113.3% (n=3). The recoveries of heavy metals during the sequential extraction process were in the ranges of 79.08~117.92%, 76.16~107.94% and 74.93~113.62%, respectively. The method to calculate the recoveries of heavy metal during the sequential extraction process was written in the Supplementary Material. These results indicated that our methods were reliable and precise enough for the purposes of this study.
Microbiological analysis
Using the iHAAQ methodology proposed by Lou et al., the qPCR analyses were performed to quantify the specific genes of the extracted DNA with three replicates using a StepOnePlus TM Real-Time PCR System instrument (Applied Biosystems, Foster City, CA, USA). The high-throughput sequencing was performed by using Illumina Miseq platform following standard protocols. The quality control of raw sequencing reads was performed using QIIME software (version 1.7.0). The absolute abundance of each genus level of the top 30 most abundant microorganisms in the sediment was calculated by multiplying the total abundance of bacteria (the copy number of the 16S rRNA gene in the V4 region measured by qPCR) and the corresponding relative abundance obtained by high-throughput sequencing [17].
Data analysis
The mathematical calculations involved in the study were done using Matlab 2017 software. The chart making and function curve fitting involved in the research were completed using Origin 8.0 software. The correlation analysis was performed using SPSS V22.0 software.
Heavy metal bioleaching curves were fitted using logistic equations [22]: (see Equation 1 in the Supplementary Files)
In the formula, M is the concentration of heavy metal (mg/kg) in the liquid phase; Mlimit is the upper limit bioleaching concentration of heavy metal (mg/kg); t is the bioleaching time; x, B, and p are constants.
We assumed that when M = 95%Mlimit, the bioleaching is finished. Then based on equation (1), T95% could be calculated using equation (2): (see Equation 2 in the Supplementary Files)
Using equation (1) to further determine the derivative of the time t and the bioleaching time TVmax (day), at which the maximum bioleaching rate Vmax (mg·kg-1·day- 1) is reached, can be obtained.