An Experimental Study to Improve the Nutrients and the Mechanical Properties of Copper Tailings Sand in China’s Arid Zone by Biomineralization of Locally Isolated Urease-Producing Bacteria

Abstract

Microbially induced carbonate precipitation (MICP) technology is an environmentally friendly technique that can contribute to tailings consolidation and ecosystem restoration. Our study found that local MICP bacteria, Lysinibacillus fusiformis, could remediate copper tailings pollution at different slope positions (K1, K2, and K3) in arid areas. We analyzed the effect of MICP treatment on the slag from macro- to microscopic levels with a dissolution test, soil physical and chemical tests, wind tunnel testing experiments, and scanning electron microscopy (SEM). The results demonstrated that the MICP bacteria, L. fusiformis, effectively remodeled the slag structure, thereby preventing the diffusion of tailing slag caused by wind erosion. This structural remodeling resulted in a significant increase in slag strength (maximum strength: 2707 KPa) and an increased content of CaCO₃. Furthermore, it led to a significant reduction in total salinity content (36.4–43.6%), pH (4.1–4.4%), and improvement in nutritional status (total potassium content (16–31.4%) and the available phosphorus content (2.1–2.3 times) of the tailings slag (p < 0.05). There was also a 15% increase in urease and catalase activity in K1, a 7% increase in invertase activity in K3 (p < 0.05), and a significant increase in carbon and nitrogen microbial biomass in K1, K2, and K3 (p < 0.05).

1. Introduction

The mineral industry makes considerable fortunes annually from valuable metals, such as copper, lead, zinc, silver, and gold, producing billions of tons of tailings near mine sites [1]. These metal tailings contribute to the accumulation of potentially toxic levels of metals in surface soils, leading to soil erosion and nutrient depletion in the topsoil, which hinder vegetation recovery in the affected regions [2]. The vast mine wastelands covering extensive areas are responsible for soil contamination with heavy metals, including cadmium (Cd), nickel (Ni), and copper (Cu), which pose a significant threat to biodiversity [3]. These heavy metals alter the physicochemical properties of soil, reduce agricultural productivity, and endanger human health through bioaccumulation in the food chain [4]. Currently, approximately 3.33 million hectares of farmland in China are unsuitable for growing crops, primarily due to heavy metal contamination [5]. In northern China, the metal smelting industry serves as an economic asset; however, it results in the accumulation of heavy metals in farmland and sewage irrigation water [6]. Agriculture in the arid northwest region of China heavily relies on irrigation, and the metal smelting industry poses a significant risk to farmland and water resources in this area. As a result, there is an urgent need to establish environmental laws and implement pollution control measures.

Microbially induced carbonate precipitation (MICP) is a promising technique for heavy metal containment. However, the harsh local environment in sand tailings, characterized by significant temperature variations between day and night [7] and limited water availability [8], poses challenges to the activity of urease bacteria responsible for bacterial enzymatic hydrolysis [9]. It could reduce the effectiveness of environmental regulations and the remediation of copper mining waste using MICP technology. Thus, we need to screen the highly adaptive habitat urease strains for these extreme environmental factors on site (e.g., drought, strong wind) using the general method with some modifications [10,11]. In the MICP method, Sporosarcina pasteurii and Bacillus megaterium are the most active urease-producing bacteria [12]. In this study, we used Lysinibacillus fusiformis (a patent of our laboratory) as the urease strain (MICP bacteria) for application on the copper tailings sands of the experimental sites. This strain exhibited greater adaptability to the environment and higher urease activity.

The sands from the tailings slope (37.39° N, 105.17° E) were classified based on their location, and we ensured that the tailings particles used in the experiment corresponded to three size standards (K1, K2, and K3). To compare the anti-shearing strength of tailings sand and the effect of nutrient modification of the sand by MICP technology, we used different reactor volumes, including the fermented liquids of Lysinibacillus fusiformis and MICP reaction solution, to obtain a similar height of the whole effector system in the box. The box contained L. fusiformis bacteria with a urea and calcium chloride concentration of 1 M in varying volumes ranging from 900 mL to 1150 mL. The overburdened sand completely solidified after an 18-day treatment period. After treatment of the sand samples, SEM, calcite content, nutrient content, copper content, unconfined compressive strength (UCS), and their relationships were investigated. Finally, based on the experimental results, we could determine the most appropriate reaction fluid volumes of MICP technology for use in field applications.

2. Materials and Methods

2.1. Tailing Sand Sample

The tailing sand used in this study was sourced from the copper tailings sand located in the arid zone of Ningxia Province, China (Figure 1 shows the location of the copper tailings and the sampling site). To classify the sands based on their dominant particle diameter at different slope locations of the tailing, we followed the method outlined in HJ-T166—2004, the Chinese soil standard. This resulted in the division of the sands into three size categories: small-sized sand (d < 5 mm) from the top slope of the tailing (referred to as K1), medium-sized sand (d: 5~15 mm) from the middle slope of the tailing (referred to as K2), and large-sized sand (d > 15 mm) from the bottom slope of the tailing (referred to as K3). Further details regarding the characteristics of the tailings sand are provided in

Table 1. The physical and chemical properties of the copper tailings sand.

pH	Water Content (%)	Carbonate Ion (mg/kg)	Water-Soluble Salt (mg/kg)	Available Phosphorus (mg/kg)	Alkali-Hydrolyzed Nitrogen (mg/kg)	Available Potassium (mg/kg)
9.23	2.24	0	26,101	12.35	11.97	25.47

.

2.2. Strain Screening and Identification

2.2.1. Strain Screening and Observation

A 1 g tailing slag sample was placed into a 10 mL sterile centrifuge tube with 9 mL of sterile water. The sample was then placed on a shaker (150 rpm, 30 °C) for enrichment and culture. The plate streaking method was then used to screen and purify the strains in the enriched bacterial solution on the urease-producing screening medium (KH₂PO₄ 2 g/L, peptone 10 g/L, glucose 10 g/L, NaCl 5 g/L, urea 20 g/L, phenol red 0.01 g/L, pH 7 ± 0.2) [13]. After 3–5 times of purification, strains with a fast growth rate, a large growth volume, and strong urease activity were screened. We used the LB Broth Medium (pH = 7) with safranin O staining of 80 cm³ to observe the bacillus morphology and its biofilm (Figure 2A,B.) by optical microscope (Leica DM3000). The growth curve of bacillus (Figure 2D) was measured and the colony variety calculated on the LB solid and liquid mediums.

2.2.2. Gene Sequencing and Phylogenetic Tree Construction

The gene sequencing for the strains was completed by Shanghai Bioengineering Co., Ltd. (Shanghai, China). The bacteria were collected by centrifugation at 12,000 rpm at room temperature for 1 min. The DNA of the target strains was extracted using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0. The universal primers for bacterial species identification (27F, AGTTTGATCMTGGCTCAG, and 1492R, GGTTACCTTGTTACGACTT) were used to perform PCR amplification on 16 s rDNA (pre-denatured at 94 °C for 5 min; circulated 27 times a cycle, including at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; subsequently repaired and extended at 72 °C for 10 min; reaction terminated at 4 °C). A 1% agarose was used to perform electrophoresis (200 V, 100 mA) for 15 min to observe the PCR products and DNA bands. At last, PCR primers were employed to directly sequence the PCR products.

The BLAST program was used to search for matching sequences in the NCBI GenBank database; similarity of the gene sequence of not less than 99% was used as the search criteria. MEGA 7.0 was used to construct a phylogenetic tree. According to the principles of molecular biology, a sequence similarity higher than 96% can be regarded as the same species (Figure 2C).

2.3. Cementation Media

In our study, the cementation solution employed consisted of urea and calcium chloride in equimolar concentrations of 1 mol/L. The ratio between the bacteria solution and the cementation solution was maintained at a 1:1 ratio, constituting our MICP treatment solution system [14].

2.4. Molds

There were two types of sand molds used in this study. One is the disposable sterilized syringe described in Yongming’s research with little change as the sample for the strength testing [15]. However, the difference in our research was that the total volume of the syringe was 80 cm³. The molds we used were rectangle sterilized culture boxes, which had a transverse diameter of 20 cm, a vertical diameter of 35 cm, a total height of 10 cm, and a total volume of 7000 cm³. All boxes were filled with iron tailings sand to the same thickness (3.5 cm). During the MICP process, the volume ratio of the bacterial solution and cementation solution used for potting was 1:1.

The weight of different slopes of copper tailing (K1, K2, and K3) filling the boxes was 3.5 kg. MICP treated the different sizes of the tailing sands (K1 + M, K2 + M, and K3 + M) for immobilization to reach the same height (7 cm), which consumed the time and dosage of cementation fluid seen in

Table 2. Consumption of bacteria solutions and reaction solutions.

Sample	Number	Mass (kg)	Thick (cm)	Cementation Fluid ① (mL)	Cementation Fluid ② (mL)	Cementation Fluid ③ (mL)	Treat Period (d)
K1	5	6.5	3.5	0	0	0	15
K2	5	6.5	3.5	0	0	0	15
K3	5	6.5	3.5	0	0	0	15
K1 + M	5	6.5	3.5	850	300	200	15
K2 + M	5	6.5	3.5	600	500	200	15
K3 + M	5	6.5	3.5	300	600	200	15

Note: The experiment had suffered three times MICP treatment named ①, ② and ③, which had been 15 days in each treatment.

. Furthermore, the treatment process is shown in Figure 3.

It took 18 days to finish the MICP treatment to the standard of total desiccation of treated copper tailings, as seen in “After MICP” in Figure 3. All the experiment sample materials achieved from the tailings in the “After MICP” situation. This experimental study was conducted in the summer with a daytime temperature of about 37.6 °C and a nighttime temperature of about 21.4 °C.

2.5. Experimental Ways for the Strength Test

The syringe mold was selected for the strength test, and the way for testing was following Yongming’s research [15]. We used the Model TSZ-3 Strain Controlled Triaxial Test Apparatus for strength testing, which apparatus was produced by Nanjing Soil Instrument Factory Co., Ltd. (Nanjing, China), a load sensor with a range of 0~200 N, a probe with a diameter of 3 mm, and a displacement sensor with a range of −25~25 mm. The probe was just above the surface of the sample before the test. The strain speed ratio was set to 3 mm/min.

2.6. Physical and Chemical Properties Tests

All the samples were sifted with a 2 mm griddle to prepare 10 g for physical and chemical property testing. We used 5 g, adding 25 mL of deionized water for pH testing by the method (ISO 10396: 2005). Other index tests consumed 0.5 g of the sifted sample. The content of CaCO₃ was tested in Pan et al.’s research [16]. Total salinity content was measured by bulk electrical conductivity and permittivity [17]. The total carbon and nitrogen determinations in soils were tested according to the research of Wright and Bailey [18]. Other indexes were organic matter determined by ignition method (pp: 34), total phosphorus content determined by Sodium hydroxide melting—molybdenum-antimony resistance colorimetric method (pp: 76), and total potassium determined by sodium hydroxide melt with flame photometer detection method (pp: 101) [19]. The soil enzyme activities were based on the methods in Jiang’s research, including urease activity, sucrase activity, and catalase activity [20].

2.7. Scanning Electron Microscopy (SEM)

We used a field emission scanning electron microscope (SIGMA 500) produced by Zeiss Corporation in Germany to observe the surface microstructure of the tailing sand specimens. The SEM was operated in high-vacuum mode at a voltage of 5 kV. All sand specimens were sputter-coated with gold prior to examination.

2.8. Statistical Analysis

All biochemical analyses had at least three biological replicates. Data were statistically analyzed with R 3.3.0 (SPSS Inc., Chicago, IL, USA) using a two-factorial completely randomized design. Two-way analyses of variance (ANOVA) were employed to test the effects of tailing sand size (K1, K2, and K3), MICP treatment, and their interaction, and means were separated using Duncan’s multiple range tests. The data were to meet assumptions of normality and homogeneity of variance. Differences were considered significant at p < 0.05.

3. Results

3.1. MICP Role on Different Copper Tailings Sand Sizes by Strength and CaCO₃ Content Test

The compressive strength of the materials was evaluated through strength testing to assess their performance. Following the treatment with MICP technology, there was an overall improvement in the maximum force exhibited by each size of tailing sand, along with an increase in displacement. Among the treated samples, K3 + M exhibited the highest force, although its displacement was comparatively lower than that of K1 + M and K2 + M. The force-displacement curve for K1 showed two peaks, whereas K1 + M exhibited a single peak that was higher than the peaks observed in K1. Notably, K2 + M demonstrated higher force and displacement values compared to K2 (Figure 4A). Additionally, the MICP treatment resulted in increased CaCO₃ content in K1, K2, and K3, although the differences observed were not statistically significant (Figure 4B).

3.2. Effects of MICP Treatment on the Microstructure of Different-Sized Tailing Sands

Based on the SEM images, it is clear that crystal growth changes and lamellar structure generation occur as tailing sand size changes. The sand of ≤5 mm treated by MICP (Figure 5D) showed more lamellar structure compared to the non-MICP sample (Figure 5A). The size of 5~15 mm copper tailings with MICP (Figure 5E) generated more pore space between sands. The size of ≥15 mm copper tailings treated by MICP (Figure 5F) showed more pore space and lamellar structure than samples with non-MICP treatment (Figure 5C). Furthermore, all the changes and forms of bonding in all samples are due to the formation of calcite precipitation.

3.3. Effects of MICP Treatment on the Salinity and pH of Different-Sized Tailing Sand

Pretreatment with MICP had a significant reduction in the total salinity content and pH of the tailing sand at different sand levels (small, middle, and large) (Figure 6). The effect of MICP on salinity reduction was extreme, with p < 0.001 in each size of tailing sand. However, the effect of MICP on pH was either much more obvious with p < 0.005 in small- or large-sized tailing sand or p < 0.01 in middle-sized tailing sand. Only MICP treatment (T) had a significant role, and tailing sand size (S) and their relationship (S × T) were not relevant to salinity and pH.

3.4. Effects of MICP Treatment on the Nutrition of Different-Sized Tailing Sands

MICP treatment had significant effects on total potassium content, the available phosphorus content, and the available potassium content of the tailing sand at different sand levels (small, middle, and large) (Figure 7), but caused no change in total phosphorus content. The MICP method increased the total potassium content and the available phosphorus content in different sizes of tailing sand, obviously. The available potassium content was only decreased in all sizes of tailing sand by MICP treatment (at least p < 0.005). MICP treatment (T) and tailing sand size (S) had a significant role in total potassium content, the available phosphorus content, and the available potassium content. The relationship between tailing sand size and MICP treatment (S × T) was the only obvious role in the available potassium content (S × P: 0.0240).

3.5. Effects of MICP Treatment on the Nitrogen and Carbon of Different-Sized Tailing Sands

The MICP technique could improve the total nitrogen content and the carbon content of the tailing sand, whose effects were significant except for the effect on the carbon content in small- and large-sized tailing sands (Figure 8). However, the ratio of carbon and nitrogen was obviously decreased by MICP treatment. The tailing sand size treatment (S) and the MICP treatment (T) both played significant roles in the total nitrogen content, the carbon content, and their ratios. The relationship between S and T (S × P) just had an obvious effect on the total nitrogen content (p = 0.0034) and the total nitrogen content (p = 0.0153).

3.6. Effects of MICP Treatment on the Microbial Biomass Nitrogen and Carbon of Different-Sized Tailing Sands

The MICP technique increased the microbial biomass nitrogen and carbon content significantly in the small, middle, and large sizes of the tailings sand. However, the size of tailing sand (S) had no obvious influence on these two indices (p > 0.05). The relationship between S and T (S × T) had the same role as S. The only remarkable effect was caused by MICP treatment (T) in the microbial biomass nitrogen content (T < 0.0001) and the microbial biomass carbon content (T < 0.0001) (Figure 9).

3.7. Effects of MICP Treatment on the Enzyme Activity of Different-Sized Tailing Sands

The MICP technique improved the urease activity remarkably in the small, middle, and large sizes of the tailing sand (p < 0.01 and p < 0.005). The catalase activity and invertase activity of tailing sand also improved by MICP treatment, but the obvious effect only appeared in small-sized sand (catalase activity, p < 0.01) and in large-sized sand (invertase activity, p < 0.05). Only the remarkable effect was the reason for MICP treatment (T) in the urease activity (T < 0.0001), catalase activity (T = 0.0072), and invertase activity (T = 0.0044). The tailing sand size treatment (S) and the relationship between S and T (S × T) did not play a significant role in the enzyme activity in the tailing sand (Figure 10).

3.8. The Correlation Analysis of Tailing Sand Physicochemical Properties with MICP Treatment

Before the MICP treatment, the total nitrogen content (Total N) had a significantly negative relationship with the total carbon content (Total C) (p < 0.05) and the ratio of carbon and nitrogen (C:N) (p < 0.01). Total C had an extremely positive relationship with C:N (p < 0.01) and an obviously negative relationship with total potassium content (Total K) (p < 0.05) and invertase activity (p < 0.05). Total phosphorus content (Total P) only had a positive relationship with catalase activity (p < 0.05). Total K had an extremely positive correlation with available potassium content (Available K) (p < 0.01). Available phosphorus content (Available P) showed a highly negative connection with soil microbial biomass carbon content (M−carbon) (p < 0.01). Total C had no relationship with Total P. Total K was not related to invertase activity (Figure 11A).

After the MICP treatment, the correlation among those indexes showed some variety. Total N had either a significant relation with Total C or C:N, but the significance levels were both up to p < 0.01. Meanwhile, Total N showed an extremely positive relationship with Total K (p < 0.01). C:N showed an obvious correlation with Total K (p < 0.05). MICP treatment showed a significant positive relationship (p < 0.05) between Available P and Available K (Figure 11B). The correlation between Total C and C:N showed no variation with tailing sand treated by MICP compared to the original tailing sand (Figure 11A,B).

4. Discussion

MICP technology is widely used in soil remediation to address various environmental concerns, including wind erosion, acid rain erosion, and soil metal contamination. In this study, we utilized MICP-capable strains, namely Acinetobacter guillouiae from coal mines [10], Sporosarcina pasteurii from limestone caves [11], and our target strain (Lysinibacillus fusiformis) from copper tailing sand. These strains have the ability to produce urease, which facilitates the formation of cementitious mineral crystals. These crystals effectively prevent the diffusion of heavy metal ions into the environment and improve the structural stability of the sand. However, the application of MICP technology in arid areas has been relatively less explored, primarily due to limited studies on nutrient diversity analysis.

In our research, the arid zone MICP strain Lysinibacillus fusiformis improved the compression strength of tailing sand, demonstrating a 2.4-fold increase in compression strength (K2 + M compared to K2) and a nearly 110% increase in movement length (displacement in K1 + M compared to K1), as evidenced by the strength test. SEM analysis revealed a marginal increase in the CaCO3 content of the various tailing sand sizes (K1, K2, and K3), better crystal growth, and more pores after MICP treatment. We observed similar results for another urease-producing bacteria, Sporosarcina. Sporosarcina pasteurii increased the mineralization of coal dust by 49.8% while simultaneously increasing the CaCO3 content and the mineralized products generated [21]. Our object of study was the sand with high salinity (>2%) and pH (>9), which was not adaptable for ordinary urease-producing strains’ work in biomineralizing the tailing sand. We used the native MICP functional bacteria to manage the arid tailing sand, which showed MICP application reduced the total salinity and pH of the sand and certified the highly alkaline salinity fitness of Lysinibacillus fusiformis. The MICP technique application in coal gangue generated two distinct morphologies of CaCO₃ named vaterite and calcite and some traces of microbial growth (2–3 μm in length), which also accompanied the improvement in CaCO₃ content [22].

The mine operation activities could change the groundwater dynamics in the surrounding area, and the saline groundwater could move toward the mining area under the mine drainage conditions [23]. The salinity of the tailing sand in our study area was higher, which is likely to limit nutrient recovery from the tailing sand. Another regional study in Baia Mare, Romania (an area heavily polluted by mine tailings), showed that mining activities caused lower total nitrogen content in the soil and higher levels of copper, manganese, zinc, and other metals [24]. The MICP technique stabilized the mechanical properties of the gangue and added much exogenous nitrogen to the tailing sand system as well, which is better for the nutrition circulation of the soil. In this study, MICP treatment of the original stockpile sand increased the total nitrogen content by 3.29 to 4.23 times, the carbon content by less than 50%, the microcarbon content by 1.17 times, and the micronitrogen content by 1.08 times. In our research, MICP technology was more effective at accumulating nitrogen elements than converting carbon elements, as indicated by the ratio of carbon and nitrogen content and the increased rate of micro-carbon and micro-nitrogen content. The above findings could explain the high activity of Lysinibacillus fusiformis in urea decomposition, which could be the nitrogen source for the tailings sand. Furthermore, a recent study highlighted the positive effects of bamboo biochar, rich in available nitrogen (AN), available phosphorus (AP), available potassium (AK), and organic carbon (OC), on mine rehabilitation. Tailing sand treated with bamboo biochar exhibited higher nutrient content (AN, AP, AK, and OC) and promoted better growth of cabbage plants [25].

Enzyme activity plays a crucial role in the mineralization of soil organic carbon, providing insights into soil nutrition and property restoration. When an additional 5% biochar was applied, it significantly increased peroxidase activity as well as the levels of freely oxidizable carbon and nitrogen [26]. Among soil enzymes, catalase activity is highly sensitive to contamination, while invertase and urease activities are less sensitive [27]. In our study, MICP technology significantly improved urease activity, catalase activity, and invertase activity in tailing sand, but enzyme activity was unrelated to other indices. As a result, we conclude that the MICP treatment could directly and significantly impact the enzyme activities in copper tailing. The data proved that a pH > 7 promotes urease activity in urease-producing bacteria [28]. MICP technology always uses increased amounts of urea as a nitrogen source, promoting the growth and regeneration of urease-producing bacteria. A recent study demonstrated the highest urease activity at the initial 0.5 M urea concentration and a slightly alkaline environment, producing the spherical structure of calcium carbonate [29]. In our study, the MICP application with a 0.5 M urea concentration resulted in lamellar-structured calcium carbonate with a 10–20% calcium carbonate enhancement, which can resist wind erosion and increase tailings nutrition in a high-salinity tailings environment (pH > 9).

Mine tailings pose significant environmental challenges due to their toxic nature, high concentration of pollutants, and interactions with environmental factors [30]. In arid or semi-arid climates, wind dispersion and erosion processes result in the widespread dispersal of tailings, hampering the growth of local vegetation. The application of the MICP technique presents a promising approach to partially transforming tailing sands into land with favorable soil properties and biostability. Our research provides a more precise and efficient method for remediating copper tailings in arid areas by harnessing the power of bacteria and urea under field conditions. However, further investigations are necessary to fully understand the MICP regulation mechanism, particularly regarding the functional analysis of the microbiome involved.

5. Conclusions

In conclusion, our results show that copper tailings sand has poor soil nutrition and a soil structure that is prone to erosion. This paper presented how the local MICP bacteria, Lysinibacillus fusiformis, could remediate copper tailings pollution at different slope positions (K1, K2, and K3) in arid areas through the solidification and nutrient regulation of tailings sand. The results showed the habitat MICP bacteria treatment could prevent diffusion of tailing slag caused by wind erosion (maximum strength: 2707 KPa) and increase the content of CaCO₃. The salinity and nutrition of the tailing sand were modified by the MICP technique with our bacteria. This indicated that this method is especially suitable for the solidification and ecological restoration of copper tailing sand to reinforce tailing ponds and increase the nutrition of tailing sands.