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Article

Feasibility Analysis of Bacterial-Treated Coal Gangue for Soil Improvement: Growth-Promoting Effects of Alfalfa

by
Yaya Wang
*,
Mingwu Liu
,
Zhiting Di
,
Weilong Cao
and
Shihua He
College of Chemistry and Chemical Engineering, Xi’an University of Science and Technology, Xi’an 710001, China
*
Author to whom correspondence should be addressed.
Minerals 2024, 14(7), 676; https://doi.org/10.3390/min14070676
Submission received: 16 May 2024 / Revised: 24 June 2024 / Accepted: 25 June 2024 / Published: 28 June 2024
(This article belongs to the Section Biomineralization and Biominerals)
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Abstract

:
The long-term storage of coal gangue (CG) mountains causes serious environmental problems such as water and air pollution. Thus, sustainable reclamation practices are urgently needed to minimize the environmental impacts brought by CG mountains. Pikovskaya medium was employed to screen microorganisms, which were subsequently utilized to promote the solubilization of CG. XRF, SEM, XRD, and HPLC techniques were employed to characterize the CG before and after bacterial treatment. In this study, we have successfully isolated and purified a bacterial strain, identified as Stenotrophomonas bentonitica BII-R7, which possesses the ability to facilitate the solubilization of nutrient elements from CG. Factors including initial inoculation ratio, incubation time, CG particle size, CG concentration, pH, and temperature were examined to investigate their effects on the biosolubilization of CG. Furthermore, the mechanism underlying the CG solubilization was also probed. Our data demonstrated that low-molecular-weight organic acids, such as acetic acid and formic acid, may harbor a crucial role in promoting the solubilization of CG. Lastly, we found that Stenotrophomonas bentonitica BII-R7, in conjugation with CG, can increase the alfalfa seed germination percentage and promote the growth of alfalfa. Together, these data provide evidence that bacterial-treated CG can be utilized for soil improvement and land reclamation.

1. Introduction

Given China’s energy structure, which is rich in coal, poor in oil, and low in gas, coal has become the primary energy source in the country, accounting for more than 50% of total energy consumption annually in recent years [1,2]. Coal gangue (CG) is a major solid waste generated during coal mining and processing [3,4]. Currently, it is estimated that around 750 million tons of CG are generated annually, resulting in the formation of more than nineteen hundred gangue mountains [5,6]. The large number of CG mountains has led to many serious environmental issues [7,8,9]. For instance, the long-term stacking of CG mountains can lead to the leakage of heavy metals into surrounding underground water and soils, posing great risks to human health [10,11,12]. Additionally, weathered CG can release toxic gases such as sulfur dioxide, hydrogen sulfide, and nitrogen oxides, which could seriously pollute the surrounding atmospheric environment [13,14].
The clean utilization of CG has become an urgent issue that requires immediate resolution [15,16]. Consequently, a series of studies have been conducted to address the environmental problems caused by CG dumps by producing high-value-added materials [17]. For instance, some researchers demonstrated that CG can be employed as coarse aggregates in structural concrete [18,19,20]. Furthermore, studies have shown that CG can also serve as backfill materials in backfill mining technology, enabling efficient utilization of CG [21,22]. In addition to these investigations, a novel CG microsphere/geopolymer composite foam can be synthesized by incorporating CG microspheres into geopolymer foams, potentially applicable in membrane supports, catalysis, and filtration fields [23]. Valuable critical metals including aluminum (Al), lithium (Li), gallium (Ga), and rare earth elements can also be extracted from CG [24,25].
Recently, a number of studies have verified that CG can be utilized to improve soil quality and crop growth. For instance, waste CG was shown to restrain the dispersivity and enhance the water stability of dispersive soil, which results in altering the dispersive soil into nondispersive soil [26]. Additionally, one study indicated that a Si-K-based amendment by CG and plant ash is effective in facilitating the growth of maize plants in saline soils compared to equal amounts of Si/K fertilizer without CG supplement [27]. CG can also be utilized to prepare silicon fertilizer due to the fact that most types of CG are shown to contain high contents of silicon elements, implying that CG-based fertilizer could meet the agricultural needs of China [28,29,30]. Similarly, a slow-release fertilizer prepared by ferric/phosphorus composite coating on CG was developed and shows great promise for application in agricultural activities [31]. Selenium-enriched CG was also reported to have great potential to produce selenium fertilizer using CG [29]. Despite these findings demonstrating that CG has the potential to serve as fertilizer and improve soil quality, studies on the utilization of CG in combination with microorganisms to enhance plant growth have been rare.
In this study, we have successfully isolated and purified a bacterium, identified as Stenotrophomonas bentonitica BII-R7, which can facilitate the solubilization of available phosphorus (AP) and available potassium (AK) in CG. Factors such as initial inoculation ratio, incubation time, CG particle size, CG concentration, temperature, and pH were examined to investigate their impacts on the solubilization efficiency of CG by this bacterial strain. Furthermore, the mechanism underlying the solubilization behavior and the growth-promoting effects of alfalfa by CG were also investigated.

2. Materials and Methods

2.1. Sample Collection

CG samples were collected from Dahaize coal mine, located in Shenmu County, Shaanxi Province, West China. The samples were stored in ziplock bags before transporting them to the laboratory. The soil samples for bacterial screening were collected from Xi’an University of Science and Technology and were immediately transported to the lab, where they were stored at −80 °C until analysis. Sandy soils for pot experiments were collected from Yulin City, situated at an altitude and longitude of 38.29° N, 109.73° E, in the northern part of Shaanxi province, China. Yulin City experiences a continental semi-arid climate with limited precipitation, making it a challenging environment for agriculture [32,33].

2.2. Bacterial Culture

The bacteria were cultured in Pikovskaya (PVK) medium containing glucose (10 g), (NH4)2SO4 (0.5 g), yeast extract (0.5 g), MgSO4 (0.3 g), FeSO4 (0.03 g), MnSO4 (0.03 g), NaCl (0.3 g), KCl (0.3 g), Ca3(PO4)2 (5 g), and agar (15 g) in 1 L of deionized water [34]. The PVK medium was employed to screen bacteria from soil samples. The National Botanical Research Institute’s phosphate growth medium (NBRIP) contained L−1: glucose (10 g), Ca3(PO4)2 (5 g), MgCl2 (5 g), MgSO4·7H2O (0.25 g), KCl (0.2 g), and (NH4)2SO4 (0.1 g); it was employed to analyze the ability of phosphate solubilizing microorganisms in dissolving the calcium phosphate [35]. Luria-Bertani (LB) medium containing tryptone (10 g), NaCl (10 g), and yeast extract (5 g) in one liter of deionized water was utilized to purify the screened bacteria. The pH of the prepared medium was adjusted to 7.2 and sterilized at 121 °C for 15 min.

2.3. Bacteria Isolation and Purification

The bacteria were isolated using the serial dilution method by spreading the diluted samples on the PVK medium. Briefly, 5 g of sieved soil were added to 45 mL of sterilized water and shaken at 160 rpm for 30 min. Then, the samples were serially diluted from 10−1 to 10−6, of which 100 µL of serially diluted soil samples were plated on the PVK medium. The Petri plates were subsequently incubated at 30 °C incubator for one week. The colonies that exhibited a clear zone on agar plates were individually picked and further purified using the LB medium. To obtain purified bacteria, the isolated colonies were purified and cultured for at least three generations.

2.4. Bacteria Identification

The morphology of the bacterial colonies was analyzed on an LB agar plate and an optical microscope (EX30, Ningbo, China) by using the gram staining method. Bergey’s Manual of Systematic Bacteriology was employed to analyze the cell morphology. The genomic DNA of the isolated bacteria was purified using the Bacterial Genomic DNA Extraction Kit (Solarbio, China) according to the manufacturer’s protocol. After extraction, the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-AAGGAGGTGATCCAGCCGCA-3′) were employed to amplify the 16S rRNA gene according to the following PCR conditions: 95 °C, 5 min; 94 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min; and 72 °C, 10 min, of which Step 2 to Step 4 were repeated for 30 cycles. The PCR reaction included 2 µL of forward primer, 2 µL of reverse primer, 4 µL of genomic DNA (68 ng/µL), 25 µL of 2xTaq PCR Master Mix (Shenggong, China), and 2 µL of nuclease-free water. The amplified PCR samples were then sequenced by Shenggong Biotechnology (Shanghai, China) and analyzed using the GenBank BLAST function on the National Center for Biotechnology Information (NCBI) website. The phylogenetic tree was constructed using the neighbor-joining method (MEGA6.0). The reliability of interior branches in the phylogenetic trees was assessed by the bootstrap method with 1000 resamplings.

2.5. Available P and Available K Measurements

The concentration of available P in the sample was measured by the molybdenum-antimony resistance colorimetry method. Briefly, 10 mL of the centrifuged supernatant sample was mixed with 20 mL of distilled water and one drop of 2,4-dinitrophenol. Then, sulfuric acid (2 mol/L) was added to the mixture until the solution became colorless. A total of 0.75 mL of ascorbic acid and 5mL of molybdate were then added to the above solution. The mixture was diluted to 50 mL volume using distilled water and subsequently incubated at room temperature for 30 min. The absorbance of the solution was determined at 700 nm with a UV–Vis spectrophotometer (Puxi TU-1900, Beijing, China). The concentration of available K was analyzed by the Flame atomic absorption spectrophotometry (FP640, Shanghai, China) following the manufacturer’s procedures. In brief, a standard curve was generated by measuring the intensity of emitted light, which is proportional to the potassium concentration in the sample, of a series of standard solutions containing known concentrations of KCl. A total of 2 mL of the centrifuged supernatant was taken to measure the emission intensity of light and the concentration of AK was determined according to the standard curve.

2.6. Solubilization of Nutrient Elements from CG

The screened and purified bacteria were employed to promote the solubilization of P and K elements from CG samples. In brief, 10 mL of overnight bacterial culture, with OD600 = 1.6, was inoculated into 90 mL of modified NBRIP medium, of which calcium phosphate was replaced with 2 g of CG. Factors including incubation time, initial inoculation ratio, CG concentration, CG particle size, incubation temperature, and pH were analyzed to assess their effects on the CG nutrient solubilization. The cultures were then incubated in a constant shaking incubator at 180 rpm and 30 °C, and the supernatant of the culture was collected daily to determine the concentrations of AP and AK. The control experiments were carried out without adding bacteria into the culture medium and each experiment was conducted in triplicate.

2.7. Coal Gangue Analysis

The major components of raw CG samples were analyzed by X-ray fluorescence (XRF). X-ray diffraction (XRD), equipped with a Bruker D8 Advance instrument with Cu-Kα radiation (λ = 0.15472 nm), was employed to characterize the CG before and after bacterial treatment. Diffraction patterns were obtained at 2θ angle of 5 degrees to 90 degrees at a scan rate of 10 degrees⋅min−1. Scanning Electron Microscopy (ZEISS Sigma 500, Göttingen, Germany), combined with a Bruker XFlash 6130 detector, was used to characterize the surface morphology and composition of raw CGs and CGs treated with bacteria.

2.8. High-Performance Liquid Chromatography (HPLC) Analysis

The content of organic acids in the supernatant sample was analyzed by HPLC (LC-20AD, Shimadzu, Kyoto, Japan). In brief, the sample was filtered by a Millipore filter and subsequently concentrated with a rotary vacuum evaporator. A total of 10 µL of the filtrate was injected into the HPLC system equipped with an Ultimate AQ-C18 column (4.6 mm × 50 mm) using an autosampler injector. Disodium hydrogenphosphate, filtered through a 0.2 µm membrane filter to remove particulates and degassed to remove dissolved gases, was used as the mobile phase at a flow rate of 0.7 mL/min. The retention time of the signal was measured at a wavelength of 210 nm.

2.9. Pot Experiment

The alfalfa seeds were soaked in sterile water for 30 min for washing. The isolated bacteria were cultured in LB medium overnight until reaching an OD600 of ~1.0. A total of 480 g sandy soils and 480 g CG were added to each plastic pot (15.5 cm diameter × 10.9 cm depth) and 3 mL of overnight bacterial culture was subsequently mixed with the CG and sandy soils. The sterile water was added for control experiments. In the pot experiment, 30 alfalfa seeds were sown in each pot and each treatment was conducted in triplicate. The alfalfa was grown for almost one month in the laboratory and the pots were watered regularly. To evaluate the growth-promoting effects of alfalfa treated with bacteria-infused CG, growth parameters including seed germination percentage, main root length, fresh weight, and above-ground plant height were assessed. The seed germination percentage of alfalfa was determined after one week of growth, while main root length, fresh weight, and above-ground plant height were measured after almost twenty days of growth.

2.10. Statistical Analysis

All statistical analyses were conducted using IBM SPSS 26.0 software. The data presented show the mean ± standard error from experiments conducted in triplicate, where applicable. Duncan’s Multiple Range Test (DMRT) was used at a significance level of 5% to examine the effects of bacteria-treated CG.

3. Results

3.1. Components of Raw CG

Our data reveal that oxides of silica and aluminum (59.13%) are the major chemical elements of raw CG (Table 1), which is consistent with the findings reported by others [22,36]. Apart from silica and aluminum, iron oxide and potassium oxide account for approximately 5% of the raw CG. Furthermore, raw CG samples also contain elements such as phosphorus, calcium, and magnesium, which are proven to be critical for plant growth.

3.2. Bacterial Isolation and Identification

PVK agar plates were employed to isolate bacteria, which can potentially facilitate the conversion of insoluble phosphorus into rapidly available phosphorus for direct uptake by plants or crops. A total of eighteen bacterial strains were isolated from the soil samples and subsequently used to promote the solubilization of nutrient elements from CG. One bacterial strain, referred to as W21 in this study, showed a superior ability to solubilize CG and was therefore chosen for further investigation. The colony and microscopic morphology of the screened W21 strain were examined.
The W21 colonies are rod-shaped, smooth, and have a slightly yellowish color (Figure 1A). Additionally, they exhibit an earthy odor and belong to Gram-negative bacteria. The genomic DNA of the W21 bacterial strain was isolated and employed for 16S rRNA sequencing. Next, primers were used to amplify the 16S rRNA gene from the bacterial DNA sample. The amplified 16S rRNA gene fragments were further sequenced. The sequenced 16S rRNA gene fragments were analyzed using the BLAST tool in the NCBI GenBank database. The phylogenetic tree of the W21 bacterial strain was constructed with MEGA 6.0 software. As shown in Figure 1B, the W21 strain showed 99.9% percentage sequence similarity with Stenotrophomonas bentonitica BII-R7 and was identified as Stenotrophomonas bentonitica BII-R7.

3.3. Study of Factors Affecting CG Solubilization

The W21 bacterial strain was employed to dissolve the nutrients, especially phosphorus and potassium, from CG. Factors including incubation time, initial inoculation ratio, CG particle size, and CG concentration were investigated to examine their impacts on CG solubilization.
The overnight culture of W21 bacteria was mixed with CG and then incubated at 30 °C in a constant shaker at a speed of 180 rpm. The supernatant of the culture was collected every day for AP and AK measurements. Our data showed that the concentrations of AP and AK increased with the incubation time, reaching 105.3 mg/kg and 731 mg/kg, respectively, when the culture was incubated for 5 days, indicating that W21 bacteria could promote the solubilization of insoluble P and K into AP and AK (Figure 2A). However, their concentrations declined after incubating for more than five days, implying that the nutrients present in the culture medium may have been consumed by W21 bacteria, thus making it unsuitable for bacterial growth.
To examine the effect of initial inoculation ratio (IIR) on CG nutrient solubilization, different initial inoculation ratios such as 1%, 3%, 5%, 10% and 20% were analyzed. As exhibited in Figure 2B, the data showed that the concentration of AP and AK increased with the rise in IIR. However, the increase was not significant once the IIR exceeded 10%, indicating that a 10% initial inoculation ratio was sufficient for CG solubilization.
The effect of CG particle size on CG nutrient solubilization was also investigated. Briefly, various CG particle sizes ranging from 20 to 100 mesh screens, corresponding to sizes from 0.85 mm to 0.15 mm, were used in this study. Our results demonstrated that the concentrations of AP and AK reached maximum when the CG particle size was 40-mesh, implying that a 40-mesh CG particle size can be utilized for CG nutrient solubilization (Figure 2C).
Different amounts of CG, ranging from 0.5 g to 10 g, were also utilized in the current study to examine their effects on CG nutrient solubilization. The data exhibited that the content of AP and AK increased with the higher concentration of CG. However, the increase was not significant once the concentration of CG exceeded 5 g in 100 mL of culture medium (Figure 2D). Collectively, the above data indicate that the optimal culture conditions for CG solubilization include a 40-mesh CG particle size, 10% initial inoculation ratio, 5 days of incubation time, and 5g of CG in 100 mL of modified NBRIP medium.

3.4. Optimization of Incubation Conditions on CG Solubilization

The impacts of incubation conditions such as pH and temperature on CG nutrient solubilization were also investigated. Various pH conditions ranging from 4 to 10 of the culture medium were analyzed to investigate their effects on AP and AK solubilization of CG. The data in Figure 3A showed that the content of AP and AK reached maximum when the pH ~was close to 7.0. However, it seems that the pH of the culture medium did not exhibit a remarkable influence on CG solubilization, suggesting that W21 bacteria could promote the nutrient solubilization of CG under a wide range of pH conditions. Likewise, various temperatures, ranging from 10 °C to 50 °C, were also investigated to examine their effects on the solubilization of CG. Our data revealed that the concentration of AP and AK reaches the maximum when the temperature is 40 °C (Figure 3B). However, the contents of AP and AK were relatively low when the temperature was below 30 °C or above 40 °C, suggesting that temperature has a remarkable impact on the solubilization efficiency of CG.

3.5. XRD and SEM Analysis of CG before and after Bacterial Treatment

As shown in Figure 4, peaks representing the main elements of phosphorus and potassium present in monetite and annite, respectively, were hardly observable after W21 bacteria treatment, implying that these two elements in CG are possibly transformed into soluble forms by W21 bacteria. Furthermore, the peak representing muscovite almost disappeared, indicating that the potassium element existing in muscovite may also be transformed into a soluble form. The SEM data showed that W21 bacteria were absorbed on the surface of the CG (Figure 5). Furthermore, it seemed that the CG became more porous compared to the raw CG after bacterial treatment. Taken together, this evidence verifies that the physical and chemical properties of CG have altered after bacterial treatment.

3.6. The Mechanism of CG Nutrient Solubilization

To examine the mechanism underlying the CG nutrient solubilization by bacterial treatment, an HPLC experiment was conducted to investigate the low-molecular-weight organic acids secreted by the W21 bacterial strain. The data exhibited that W21 bacteria mainly facilitate the solubilization of P and K elements from CG via secreting acetic acid and formic acid (Figure 6 and Table 2). Additionally, a small amount of 2,3-dihydroxybernsteinsaeure, oxalic acid, lactic acid, and malic acid were also secreted by W21 bacteria to promote the solubilization of CG. The biochemical characteristics of the low-molecular-weight organic acids secreted by W21 were displayed in the Supplementary Materials (Table S1). In summary, our data indicate that low-molecular-weight organic acids secreted by bacteria promote the solubilization of CG nutrient elements.

3.7. Growth-Promoting Effects of Alfalfa by Bacterial-Treated CG

To further verify the feasibility of bacterial-treated CG in soil improvement, we mixed W21 bacteria, CG, and sandy soils together to grow alfalfa in plastic pots. Our results exhibited that the addition of W21 bacteria and CG into sandy soils increased the seed germination percentage of alfalfa compared to the control experiments (Figure 7A and Table 3). Furthermore, the data exhibited that the roots were also longer in W21 bacteria and CG supplement experiment groups compared to the control groups (Figure 7B). The fresh weight and plant height of alfalfa is also larger in experimental groups with W21 and CG addition compared to the control groups (Table 3). In conclusion, these data support the notion that W21 bacteria, in conjugation with CG, could be employed as fertilizers to improve soil quality and promote plant growth.

4. Discussion

Large piles of coal gangue can not only release harmful chemicals such as heavy metals, sulfur compounds, and acid mine drainage but also cause soil erosion and loss of vegetation cover. These environmental problems may severely disrupt local ecosystems and habitats, leading to a reduction in biodiversity and serious health risks to nearby communities. Thus, there is an urgent need to find sustainable management strategies to address these environmental issues caused by coal gangue. In the present study, we attempted to isolate and purify bacteria capable of promoting the nutrient solubilization of CG, which could be utilized for soil improvement to promote plant growth.
We have successfully isolated a bacterial strain, referred to as Stenotrophomonas bentonitica BII-R7, which could promote the release of AP and AK from CG. Although a similar study demonstrated that a bacterial strain isolated from a dump yard of CG is capable of solubilizing nutrient elements such as available phosphorus, potassium, and silicon from CG, it did not validate the growth-promoting effects of CG on plants [37]. In the present study, we have further verified that Stenotrophomonas bentonitica BII-R7, isolated using PVK medium, could increase the alfalfa seed germination percentage and stimulate the growth of alfalfa compared to the control experiments. Consistently, one recent study reveals that Stenotrophomonas maltophilia could be a potential biocontrol agent and bio-fertilizer to enhance wheat seed germination percentage and promote wheat production [38]. Moreover, other studies also verify that several Stenotrophomonas species possess genes involved in plant growth promotion [39,40]. How bacterial-treated CG could facilitate plant growth will be investigated in the near future. Taken together, this evidence suggests that Stenotrophomonas bentonitica BII-R7 combined with CG has great potential to be utilized as bioinoculants for agricultural biotechnology.
Several key issues must be addressed if large amounts of CG and Stenotrophomonas bentonitica bacteria are utilized to improve soil quality. First, a series of investigations have demonstrated that long-term stacking of CG may cause the leakage of heavy metals into the soils around the mining area [41,42]. Furthermore, weathered CG may release harmful gases such as sulfur dioxide (SO2) and nitric oxide (NO), which can contribute to air pollution and acid rain formation [43]. Second, some studies have shown that polycyclic aromatic compounds (PACs) present in CG may contaminate the soils and thus pose potential health risks to nearby villages [44,45]. Lastly, one recent study indicated that the addition of CG into soils may reduce the diversity of bacteria in soil [46]. These negative effects of CG on the soil microbial community and toxic metal release and PAC contaminants in soil must be considered when applying CG for soil improvement.
Key factors including initial inoculation ratio, CG concentration, incubation time, CG particle size, pH, and temperature were examined to investigate their impacts on CG nutrient solubilization. Our data revealed that these factors play a key role in CG biosolubilization. These factors should be taken into consideration when CG and W21 bacteria are utilized for soil improvement. Additionally, both XRD and SEM data revealed that the chemical and physical properties of CG had changed after bacterial treatment. Lastly, low-molecular-weight organic acids, especially acetic acid (51.5%) and formic acid (37.2%), of which relative contents reach approximately 88.7%, are primarily responsible for CG nutrient solubilization. This finding contrasts with the data reported by others, which indicated that tartaric acid showed the highest solubilization of CG [37]. To sum up, the evidence presented in this study indicates that W21 bacteria, in conjugation with CG, could be utilized as fertilizers to stimulate plant growth, offering a sustainable reclamation strategy to mitigate the environmental impacts of CG.

5. Conclusions

In this study, we have isolated and purified a bacterial strain that is capable of facilitating the solubilization of AP and AK from CG. Crucial factors affecting the solubilization ability of CG were also examined. Furthermore, the results indicate that W21 bacteria, in conjugation with CG, possess growth-promoting effects on alfalfa. It is also intriguing to explore whether CG and W21 bacteria would have similar growth-promoting effects on other types of crops. Moreover, CG combined with W21 bacteria may also be used to reclaim barren or degraded land, which benefits land stabilization, erosion prevention, and landscape restoration for agricultural purposes. Clean utilization of CG can mitigate environmental problems such as water and air pollution caused by long-term stacking of CG. In summary, the findings presented in this study hold great promise for the sustainable utilization of CG in agriculture and the environment.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/min14070676/s1, Table S1: Biochemical characteristics of organic acids (T: Toxic; Xn: Harmful; Xi: Irritant; C: Corrosive).

Author Contributions

Conceptualization, Y.W. and M.L.; methodology, Y.W., M.L. and Z.D.; software, M.L.; validation, Y.W. and M.L.; formal analysis, Y.W., M.L., Z.D. and W.C.; investigation, Y.W., M.L., Z.D., W.C. and S.H.; resources, Y.W. and S.H.; data curation, Y.W. and M.L.; writing-original draft preparation, Y.W., M.L. and Z.D.; writing-review and editing Y.W., M.L., Z.D. and S.H.; visualization, Y.W., M.L., Z.D., W.C. and S.H.; supervision, Y.W.; project administration, Y.W. and M.L.; funding acquisition, Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This project was supported by the National Natural Science Foundation of China (Grant NO. 42202206).

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Acknowledgments

We thank the lab member Xinkai Mu from Xiangrong Liu’s research group for his assistance with lab equipment.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Minerals 14 00676 g001
Figure 1. Bacteria purification and identification. (A) The colony morphology and microscopic morphology of screened W21 bacterial strain; (B) phylogenetic trees of bacterial strains.
Figure 1. Bacteria purification and identification. (A) The colony morphology and microscopic morphology of screened W21 bacterial strain; (B) phylogenetic trees of bacterial strains.
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Figure 2. Factors affecting the solubilization of CG nutrients by W21 bacteria. (A) Incubation time; (B) initial inoculation ratio; (C) particle size; and (D) coal gangue amount in 100 mL culture medium. (***, p < 0.001; **, p < 0.01).
Figure 2. Factors affecting the solubilization of CG nutrients by W21 bacteria. (A) Incubation time; (B) initial inoculation ratio; (C) particle size; and (D) coal gangue amount in 100 mL culture medium. (***, p < 0.001; **, p < 0.01).
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Figure 3. Optimization of incubation conditions on CG solubilization (A) pH; (B) temperature. (***, p < 0.001; **, p < 0.01).
Figure 3. Optimization of incubation conditions on CG solubilization (A) pH; (B) temperature. (***, p < 0.001; **, p < 0.01).
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Figure 4. XRD patterns of raw CG and bacterial-treated CG.
Figure 4. XRD patterns of raw CG and bacterial-treated CG.
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Figure 5. SEM pictures of CG. (A) raw CG; (B) bacterial-treatment CG.
Figure 5. SEM pictures of CG. (A) raw CG; (B) bacterial-treatment CG.
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Figure 6. LC-MC spectrum of the organic acids in the liquid products.
Figure 6. LC-MC spectrum of the organic acids in the liquid products.
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Figure 7. Growth-promoting effects on alfalfa by W21 bacteria and CG. (A) seed germination of alfalfa without (left) and with W21 bacteria addition (right); (B) root of the alfalfa from control group (left) and W21 bacteria addition group (right).
Figure 7. Growth-promoting effects on alfalfa by W21 bacteria and CG. (A) seed germination of alfalfa without (left) and with W21 bacteria addition (right); (B) root of the alfalfa from control group (left) and W21 bacteria addition group (right).
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Table 1. The main chemical elements of raw coal gangue.
Table 1. The main chemical elements of raw coal gangue.
ComponentSiO2Al2O3Fe2O3CaOK2OSO3MgOTiO2
Content (wt. %)43.8215.312.953.152.091.250.960.73
Table 2. The relative content of organic acids in the liquid products.
Table 2. The relative content of organic acids in the liquid products.
NO.Retention Time
(min)		CompoundsContent
(%)
14.405Oxalic acid2.612
25.052(2R,3R)-2,3-Dihydroxybernsteinsaeure4.012
35.397Formic acid37.155
46.573Malonic acid0.819
56.986DL-Malic acid1.516
68.045DL-Lactic acid2.363
78.476Acetic acid51.523
Table 3. Effects of W21 bacteria on different growth parameters of alfalfa. (***, p < 0.001).
Table 3. Effects of W21 bacteria on different growth parameters of alfalfa. (***, p < 0.001).
Sample NameSeed Germination Percentage (%)Root Length (mm)Plant Height (mm)Fresh Weight (mg)
Sandy soil and CG70.0 ± 3.3335.56 ± 3.1842.5 ± 2.1333.0 ± 2.69
Sandy soil, CG
and W21 bacteria		81.7 ± 5.16 ***46.4 ± 4.22 ***60.3 ± 3.66 ***42.0 ± 3.52 ***
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Wang, Y.; Liu, M.; Di, Z.; Cao, W.; He, S. Feasibility Analysis of Bacterial-Treated Coal Gangue for Soil Improvement: Growth-Promoting Effects of Alfalfa. Minerals 2024, 14, 676. https://doi.org/10.3390/min14070676

AMA Style

Wang Y, Liu M, Di Z, Cao W, He S. Feasibility Analysis of Bacterial-Treated Coal Gangue for Soil Improvement: Growth-Promoting Effects of Alfalfa. Minerals. 2024; 14(7):676. https://doi.org/10.3390/min14070676

Chicago/Turabian Style

Wang, Yaya, Mingwu Liu, Zhiting Di, Weilong Cao, and Shihua He. 2024. "Feasibility Analysis of Bacterial-Treated Coal Gangue for Soil Improvement: Growth-Promoting Effects of Alfalfa" Minerals 14, no. 7: 676. https://doi.org/10.3390/min14070676

APA Style

Wang, Y., Liu, M., Di, Z., Cao, W., & He, S. (2024). Feasibility Analysis of Bacterial-Treated Coal Gangue for Soil Improvement: Growth-Promoting Effects of Alfalfa. Minerals, 14(7), 676. https://doi.org/10.3390/min14070676

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