CN116903421A - Method for passivating heavy metals and activating fertilizer by utilizing microorganisms - Google Patents

Abstract

A method for passivating heavy metals and activating fertilizer by utilizing microorganisms comprises the steps of coal gangue pretreatment, microorganism breeding and mixed fermentation operation, wherein the coal gangue is treated by indigenous microorganism SK1 bacteria, and the metabolic products (organic acids) of the SK1 bacteria can selectively destroy mineral compositions in the coal gangue and convert insoluble phosphorus, potassium and silicon into plant absorbable effective phosphorus, quick-acting potassium and effective silicon. The three extracellular polymers and cellular structures of SK1 bacteria are well-tolerated by lead and form lead phosphate precipitates that are passivated (passivation > 91%). Three extracellular polymers of SK1 bacteria have remarkable adsorption effect on chromium (VI), and the three components of the internal structure of the bacteria, metabolic products and pyrite in coal gangue are combined to effectively reduce heavy metal chromium (VI) and achieve passivation effect (the removal rate is more than 86%). The invention has the characteristics of low medicament consumption and low energy consumption, the content of the activated effective silicon, the activated effective phosphorus and the activated quick-acting potassium of the gangue is obviously increased, and the passivation effect of heavy metals lead and chromium (VI) is obvious.

Description

Method for passivating heavy metals and activating fertilizer by utilizing microorganisms

Technical Field

The invention relates to the technical field of coal gangue recycling, in particular to a method for passivating heavy metals and activating fertilizers by utilizing microorganisms.

Background

Gangue is solid waste with the largest discharge amount in the coal industry, and the accumulation of the gangue occupies a large amount of land resources to pollute the surrounding environment, so that the efficient treatment and resource utilization of the gangue are urgent. At present, the main utilization approaches of the coal gangue include: (1) The clay gangue can be used as a silicon-aluminum material to burn ordinary silicate cement, special cement, clinker-free cement, sintered brick and the like to provide SiO for the building material ₂ 、Al ₂ O ₃ Fe in a small amount ₂ O ₃ (Shi Mingjun, she Haijun. Comprehensive utilization of gangue in building [ J)]Shanxi construction, 2007, 30:188-189; su Lei, shao Wei, ma Baoguo analysis of the performance of sintered bricks based on sludge-gangue-shale ternary System [ J ]]Tile, 2018, 4:23-27). (2) Valuable elements are extracted from the coal gangue, and the valuable elements extracted from the coal gangue have the characteristics of wide raw material sources, abundant reserves and low cost. The contents of Si, al and Fe in gangue are relatively high, while the contents of rare noble metals such as V, ga are also considerable (Gu Min, yang Lei. The technical research on extracting alumina by calcining and activating gangue [ J)]Mineral comprehensive utilization 2020, 2:140-144; li Lingyue, su Jingcheng, xue Fangming Experimental study on the recovery of rare noble metals from gangue [ J]Shandong chemical industry, 2017, 46 (3): 38-40. (3) The use of coal gangue as road embankment filler for road construction has the obvious advantages of large consumption and no need of special treatment, and the underground backfill is an effective way for consuming a great amount of solid waste (Cheng Gongguang. The coal gangue should be used in road construction)By study [ D]University of changan, 2009; liu Dong the properties of gangue and its comprehensive utilization shallow analysis [ J ]]Inner Mongolia science and economy 2010, 8:91-92.). (4) The application of the coal gangue in agriculture comprises a plurality of nutrient elements and 15% -25% of organic matters which are necessary for plant growth, and the soil conditioner prepared by scientifically and reasonably adding the coal gangue has the potential of reducing the soil adhesion and improving the soil porosity, so that the soil is compacted and suitable for cultivation, and therefore, the coal gangue can also be used for preparing agricultural fertilizers and soil conditioners (Feng Anda. The research on the comprehensive effect of microorganism combination on solid wastes in coal mine areas [ D)]Northwest university of agriculture and forestry science and technology, 2008).

At present, two methods for preparing fertilizer and soil conditioner by using coal gangue mainly exist: the first is chemical method and the second is microbiological method. Zhu Fuying and the like are used for producing the gangue silicon fertilizer through a mechanical activation-high temperature roasting process, and have obvious promotion effect on plant growth and soil improvement (Zhu Fuying, liu Sheng, xu Hui and the like. A process for preparing the water-soluble silicon fertilizer by using the gangue, CN108821801A [ P/OL ]. 2018, 11-16). The bioremediation technology for improving the physicochemical properties of the coal gangue by utilizing microorganisms is a hot spot emerging technology (Gu Qianqian, cheng Fan, xie Chengwei) for utilizing the coal gangue in recent years, a study [ J ]. The comprehensive utilization of fly ash, 2012, 2:28-31) for preparing the coal gangue fertilizer by utilizing silicate bacteria (GY 03). The preparation of the soil conditioner by utilizing the microorganism to treat the coal gangue becomes one of the hot spots of the comprehensive treatment research of the coal gangue due to the advantages of economy, environmental protection, large treatment capacity, in-situ treatment and the like. However, the heavy metals Pb (II) and Cr (VI) with higher content in the gangue cause ecological hidden trouble, so how to treat the gangue by utilizing microorganisms to prepare the soil conditioner and promote the dissolution of nutrient elements in the gangue and the passivation and removal of the heavy metals are key to realizing the utilization of the gangue in a 'reduction, recycling and harmless' way.

Disclosure of Invention

The invention aims at overcoming the defects in the prior art, and provides a method for passivating heavy metals and activated fertilizers by utilizing microorganisms, which has the advantages of low medicament consumption and low energy consumption, obviously increased contents of effective silicon, effective phosphorus and quick-acting potassium after the coal gangue is activated, obvious passivation effect of heavy metals Pb (II) and Cr (VI), and capability of preparing the coal gangue into soil conditioner and being applied to the improvement of barren soil.

The aim of the invention can be achieved by the following technical measures:

the method for passivating heavy metals and activating fertilizers by utilizing microorganisms comprises the operations of coal gangue pretreatment, microorganism breeding, mixed fermentation and the like, and comprises the following steps:

a. pretreatment of coal gangue and microorganism breeding: firstly, processing coal gangue into powder with the particle size smaller than 115 mu m through crushing and ore grinding procedures, and preserving for later use; placing coal gangue powder which is naturally weathered for 60-90 days into a sterilized container, wherein the liquid-solid mass ratio is 10-20:1 adding sterile water, soaking for 4-10 days at 30-45deg.C, dipping supernatant with an inoculating loop in LB solid medium under ultra-clean environment, culturing under streak culture, purifying for 3-5 generations, and storing in a refrigerator at 0-4deg.C for use; preparing bacterial plates on glass slides of the purified bacterial strains of 3-5 generations, gram-staining the bacterial plates, observing the forms of bacteria through a high-power microscope, and screening the purified bacterial strains with strong propagation speed and propagation stability through a plate streak culture mode to obtain SK1 bacteria;

b. and (3) mixed fermentation: SK1 bacteria are scraped into sterile physiological saline by an inoculating loop in an ultra-clean workbench after being cultured for 2-4 days at 25-35 ℃ in LB solid culture medium, and uniformly shaken, and the concentration of bacteria liquid is changed by controlling the scraped bacteria amount. Adding gangue powder into a container, and adding the gangue powder into the container according to the liquid-solid mass ratio of 4-6:1 adding distilled water for soaking to determine the pH value, pouring the prepared bacterial liquid into gangue powder, vibrating and uniformly mixing, standing and fermenting for 4-10 days at 25-35 ℃ in a constant temperature incubator, and then drying at 40-60 ℃ in an oven to obtain the soil conditioner.

The coal gangue is taken from medium-grain-size coal gangue with grain size of 5-20 mm after coal is washed and selected, then the medium-grain-size coal gangue is crushed into small grains with diameter of less than 3 mm, and then the small grains are ground into powder with grain size of less than 115 mu m.

Each 1000 parts of mL distilled water in the LB solid culture medium contains 10-20 g parts of peptone, 3-9 parts of beef extract powder g parts of sodium chloride 10-30 parts of g parts of agar 15-45 parts of g parts of distilled water, and is heated to dissolve, the pH value is regulated to 7.2-7.5, and then the mixture is packaged in a container and sterilized in a sterilizing pot at 121 ℃ for 20 min.

The SK1 bacteria are gram-negative bacteria, have small colony volume, 0.4-1.0 mu m wide and 1.2-3.0 mu m long, are mucilaginous and slightly convex, are light yellow, have smooth and round surfaces and are not transparent, and have short rod shapes, smooth tail ends and no endophytic spores.

The soil conditioner is applied to soil to be improved (including barren soil such as saline-alkali soil, sandy land and the like), wherein available phosphorus and quick-acting potassium are measured according to the Chinese forestry industry standards LY/T1233-1999 and LY/T1236-1999, available silicon is measured according to the Chinese agriculture industry standards NY/T1121.15-2006, soluble lead and soluble chromium (VI) are measured respectively according to GB/T8152.15-2021 and GB 31893-2015, a coomassie brilliant blue staining method is adopted for measuring protein, and an anthrone method taking glucose as a standard is adopted for measuring polysaccharide. The method comprises the steps of performing microscopic feature monitoring on a soil conditioner by adopting X-ray diffraction analysis (XRD), X-ray fluorescence spectrometry (XRF), inductively coupled plasma mass spectrometry (ICP-MS), scanning electron microscope energy spectrum analysis (SEM-EDS), performing analysis on microscopic features of the soil conditioner and SK1 bacteria by utilizing Fourier infrared spectroscopy analysis (FT-IR), three-dimensional fluorescence spectrometry (3D-EEM) and X-ray photoelectron spectroscopy analysis (XPS), wherein the effective silicon content in the soil conditioner is 500-1000 mg/kg, the effective phosphorus content is 200-400 mg/kg, the quick-acting potassium content is 400-700 mg/kg, the content of soluble lead is reduced to more than 91%, and the removal rate of soluble chromium (VI) is more than 86%.

The beneficial effects of the invention are as follows:

because the invention adopts indigenous microorganism-SK 1 bacteria to treat the coal gangue, the invention can activate the fertility of available phosphorus (200-400 mg/kg), quick-acting potassium (400-700 mg/kg) and available silicon (500-1000 mg/kg) in the coal gangue, and can also deactivate heavy metal lead and chromium (VI). The metabolites of SK1 are various organic acids that selectively destroy the mineral composition of phosphorus, silicon, and potassium in gangue, thereby converting insoluble phosphorus, potassium, and silicon into plant absorbable effective phosphorus, quick-acting potassium, and effective silicon. The dissolved phosphate forms a lead phosphate precipitate with lead under the action of SK1 bacteria, while the three extracellular polymers and cellular structures of SK1 bacteria are well-tolerated by lead, which can be present in large amounts in the bacterial structure for passivation (passivation rate greater than 91%). The three SK1 extracellular polymers have remarkable adsorption effect on chromium (VI), and the combination of the internal structure of bacteria, metabolic products and pyrite in coal gangue effectively reduces heavy metal chromium (VI) and achieves passivation effect (the removal rate is more than 86%).

Therefore, the invention has the characteristics of low medicament consumption and low energy consumption, the content of the activated effective silicon, the activated effective phosphorus and the activated quick-acting potassium of the gangue is obviously increased, the passivation effect of heavy metal lead and chromium (VI) is obvious, and the gangue can be prepared into soil conditioner and applied to the improvement of barren soil.

Drawings

Figure 1 XRD pattern analysis chart of gangue raw ore.

FIG. 2 is a graph showing the effect of the amount of bacteria on nutrient elution.

FIG. 3 is a scanning electron microscope observation chart of the adhesion of the SK1 strain on the surface of the mineral phase.

Figure 4 XRD spectra of gangue before and after SK1 bacteria treatment.

FIG. 5 shows Pb (II) elution amount chart in gangue soak solution under bacteria and bacteria free condition.

FIG. 6 shows the variation of extracellular polymer components during Pb (II) passivation by SK1 strain.

FIG. 7 shows the relative change of Pb (II) content of the extracellular polymer during Pb (II) passivation by the SK1 strain.

FIG. 8 shows the variation of bacterial cell composition during Pb (II) inactivation by SK1 strain.

FIG. 9 is a graph showing the variation of the relative content of Pb (II) in the cell fraction of the Pb (II) passivating process by the SK1 strain.

FIG. 10 is an infrared analysis chart of solid precipitate after centrifugation of Pb (II) -containing and Pb (II) -free culture solutions.

FIG. 11 shows a three-dimensional fluorescence analysis of the cell fraction of the SK1 strain.

FIG. 12 is a three-dimensional fluorescence analysis of extracellular polymer of SK1 strain.

FIG. 13 is a graph showing the Cr (VI) ion content of the gangue soak solution under the bacteria-free and aseptic conditions.

FIG. 14 is a graph showing the reduction effect of Cr (VI) by various organic acids.

FIG. 15 shows the change pattern of the extracellular polymer removal Cr (VI) of the SK1 strain.

FIG. 16 is a chart showing an infrared spectrum analysis of the strain SK1 before and after Cr (VI) removal.

FIG. 17 is a chart of three-dimensional fluorescence spectroscopy of SK1 strain.

FIG. 18 XPS analysis of Cr valence before and after Cr (VI) removal by SK1 strain.

Fig. 19 is a process flow diagram of the present invention.

Detailed Description

The invention will be further described with reference to the following examples (drawings):

the gangue contains phosphorus, potassium, silicon and other nutrient elements, and usually only the parts of the elements existing in the forms of available phosphorus, quick-acting potassium and available silicon can be directly absorbed and utilized by plants, and the analysis of the selected gangue nutrient elements and heavy metals is shown in tables 1 and 2.

As can be seen from Table 1, although the content of the nutrient elements in the gangue is high, most of the nutrient elements are difficult to be absorbed and utilized by plants, and the preparation of the soil conditioner by converting the nutrient elements into the effective state can realize the recycling of the gangue, so that the gangue needs to be activated to improve the content of the effective state nutrient elements.

As can be seen from table 2, the heavy metal content in the gangue is as follows from high to low: cr > Pb > Zn > Cu > Ni. In life, cr (VI) and Pb (II) have great harm to organisms, and the Pb and Cr content in the gangue exceeds the standard, so that the removal effect of the strain on soluble Cr (VI) and Pb (II) in the gangue soil conditioner in the process of preparing the soil conditioner by the SK1 strain is very necessary to be explored.

The gangue contains phosphorus, potassium, silicon and other nutrient elements, and X-ray diffraction analysis is carried out on the gangue sample to explore the occurrence forms of various nutrient elements, and the result is shown in figure 1.

The main minerals in the gangue are Quartz (Quartz), pyrite (pyrrite) and Kaolinite (Kaolinite), and in addition, very small amounts of Monetite (Monetite), muscovite (Muscovite), iron mica (anite ferrian) and chlorocone (Cronstedtite), and in combination with the chemical composition analysis of the gangue, it can be determined that the nutrient element phosphorus exists in the gangue in the form of Monetite, while potassium is present in the mica, and that the silicon element exists mainly in the form of Quartz and Kaolinite, and that a small amount of silicon exists in the gangue in the form of mica and chlorocone.

The influence of the bacterial concentration on the activating effect of the coal gangue is examined, and the content of the nutrient elements is measured, and the result is shown in figure 2.

The contents of available phosphorus, quick-acting potassium and available silicon in the gangue are increased and then decreased along with the increase of the concentration of bacterial liquid. When the bacterial liquid concentration is 2.28X10 respectively ¹² CFU/mL、1.14×10 ¹² CFU/mL and 2.85X10 ¹¹ At CFU/mL, the available phosphorus, quick-acting potassium and available silicon contents reach 264.2 mg/kg, 572.53 mg/kg and 522.7 mg/kg respectively. The bacteria and minerals can be contacted with each other through electrostatic attraction and hydrophobic force, and the adsorption condition of SK1 bacteria on the surface of gangue particles is shown in figure 3.

The surface of the mineral sample treated with bacteria had adsorbed a large amount of SK1 strain, and the bacteria were not uniformly distributed on the surface of the coal gangue, and the bacteria density was very high in some areas, and little or no bacteria adsorption in some areas. Bacteria adsorbed on the mineral surface may secrete extracellular polymers and produce a large amount of pili, and thus, the bacteria may adhere closely to the mineral surface. The outer membrane of bacteria is composed of a number of proteins, such as lipopolysaccharides, phospholipids and lipoproteins, some of the protein components of the membrane play an important role in the adsorption of bacteria on mineral surfaces.

XRD analysis was performed on SK1 bacteria treated gangue slag and compared with raw ore, and the results are shown in FIG. 4.

Characteristic peaks of quartz and kaolinite in the gangue slag are not significantly changed, whereas in the residues treated with the SK1 strain, P-containing triclinic calcium phosphate (CaHPO) ₄ ) And muscovite (KAl) containing K and Si ₂ (AlSi ₃ O ₁₀ ) (OH) ₂ ) The diffraction peak intensities of (c) were all reduced or even disappeared, indicating that these minerals containing phosphorus and potassium were dissolved during the treatment of SK1 bacteria. Iron mica (KFe) ₃ FeSi ₃ O ₁₀ (OH) ₂ ) The diffraction peak of (2) is weakened, which also shows that SK1 bacteria can dissolve ferrous iron to obtain energy, and organic acid generated by metabolism can promote solubilization of K and Si at the same time, and the solubilization effect of bacteria on muscovite and iron mica is better than that of quartz.

The leaching condition of Pb (II) in the process of treating coal gangue by SK1 strain is explored, a sterile LB liquid culture medium is taken as a control group, and the sterile LB liquid culture medium and the bacteria content are respectively taken to be 2.85 multiplied by 10 ¹² The CFU/mL bacterial liquid is used for treating coal gangue, and the concentration change condition of Pb (II) in the system is respectively measured, and the result is shown in figure 5.

On the 1 st day of the reaction, the experimental group inoculated with the SK1 strain had a higher Pb (II) elution amount than the control group, and it was presumed that the SK1 strain eluted the nutrient element with Pb (II) being eluted. The concentration of Pb (II) in the experimental group gradually decreases, and the concentration of Pb (II) in the control group gradually increases, which proves that the SK1 strain can promote the fixation of Pb (II) in the system.

The change in polysaccharide and protein in the extracellular polymer was measured, and the relative content of Pb (II) in the extracellular polymer was measured, and the results are shown in FIGS. 6 and 7, respectively.

The protein content of the three extracellular polymers is greatly different, and the size relationship is as follows: S-EPS > TB-EPS > LB-EPS, the process of the inside-out excretion of bacterially produced proteins is illustrated. Although the protein content in LB-EPS is the lowest, it is notable that the LB-EPS protein content is always higher than that of the control group (0 mg/L Pb (II)) in 1-5 days of treatment, and the relative Pb (II) concentration in LB-EPS is continuously increased in combination with the change of Pb (II) content of the three extracellular polymers in FIG. 7, which indicates that Pb (II) is more sensitive to Pb (II) after entering LB-EPS, and a large amount of protein binding and Pb (II) fixation are called. Within 1-5 days, the contents of LB-EPS and TB-EPS proteins obviously increase and decrease, and the relative Pb (II) content is always increasing, which indicates the slow diffusion process of Pb (II) from the solution to bacterial cells. For polysaccharide, comparing S-EPS, LB-EPS and TB-EPS, the polysaccharide content in the three extracellular polymers can be found to be greatly different, and the size relationship is as follows: S-EPS > TB-EPS > LB-EPS, illustrating the process of excretion of bacterially produced polysaccharide from the inside to the outside. The change trend of the polysaccharide of the three extracellular polymers is observed, the increase of the polysaccharide in the S-EPS is obviously faster than that of the TB-EPS and the LB-EPS on the 1 st day, the polysaccharide content reaches the maximum value on the 2 nd day, the content in the S-EPS is maintained in a relatively stable state, the TB-EPS and the LB-EPS start to decline, the strong affinity of the polysaccharide to Pb (II) is demonstrated, a large amount of polysaccharide is secreted by bacteria at the beginning, and the polysaccharide is transported to the outermost layer to block the invasion of Pb (II) to the polysaccharide. The polysaccharide content of LB-EPS was significantly higher than that of the control group for the first 3 days, indicating that bacteria store a higher concentration of polysaccharide in the second loosely bound extracellular polymer to block Pb (II) entry after sensing the presence of Pb (II) in addition to the outermost layer to formulate a large amount of polysaccharide. In connection with the change of Pb (II) in FIG. 7, it was found that there was a significant decrease in the levels of LB-EPS and TB-EPS polysaccharides starting on day 2, whereas the increase in Pb (II) concentration in these two components was more pronounced, indicating that Pb (II) diffused into the LB-EPS and TB-EPS layers after 2 days, in order to protect the cells from binding of a large amount of polysaccharides to Pb (II).

In conclusion, the extracellular polymeric substances secreted by bacteria have a significantly altered composition of polysaccharides and proteins in the presence of Pb (II) compared to those in the absence of Pb (II). Compared with polysaccharide, the change of protein is slightly delayed, and compared with the change of control group, no polysaccharide is obvious, so that both components are favorable for protecting bacterial cells from toxic action of Pb (II), and the polysaccharide has the characteristics of quick response and quick diffusion compared with the protein. However, analysis of the content of each component found that the protein content was higher than that of the polysaccharide in several polymers, it was presumed that the bacteria first secreted a large amount of polysaccharide to adsorb and fix Pb (II) and to block it as far as possible in the outermost layer, and then as the stress time of Pb (II) by the bacteria became longer, the bacteria produced a large amount of protein to bind to Pb (II) and lost the ability of Pb (II) to continue to cause damage to bacterial cells. Changes in the contents of polysaccharide and protein in the cytoplasm and the cell membrane were measured, and the relative contents of Pb (II) in the cytoplasm and the cell membrane were also measured, and the results are shown in FIGS. 8 and 9.

As can be seen from FIG. 8, for the protein, the cytoplasmic protein content was significantly reduced at a Pb (II) concentration of 600 mg/L compared to the control group (Pb (II) concentration of 0 mg/L), and was maintained at a lower concentration throughout 8 days of culture, while the protein content in the cell membrane was also lower in the first 4 days of culture, and the protein content in the cell membrane was increased after the 4 th day, and the relative Pb (II) content in the cell membrane was increased and maintained at a higher level (87%), indicating that the protein in the cell membrane has an accelerating effect on the SK1 strain against the effect of Pb (II). Regarding the polysaccharide, the content of the polysaccharide in cytoplasm was decreased compared with that of the control group Pb (II) at 600 mg/L, but the change trend of the polysaccharide in cytoplasm of the experimental group and the control group was about the same, indicating that the polysaccharide in cytoplasm has no obvious effect in the influence of bacteria against Pb (II). In the cell membrane, the polysaccharide content increased significantly after 4 days, combined with the rapid increase in the Pb (II) content of the cell membrane after 4 days in fig. 9, demonstrated that the polysaccharide also contributed to the immobilization of Pb (II) in the cell membrane.

In the presence of Pb (II), the protein and polysaccharide contents in the bacterial cells were significantly changed from those in the control group. Wherein the protein content in the cytoplasm varies more than the polysaccharide. The content change of protein and polysaccharide in the cell membrane has strong correlation with the Pb (II) content change: the protein in the cell membrane increases with an increase in its Pb (II) content, which begins to decrease as the Pb (II) content remains relatively stable; the polysaccharide content in the cell membrane increases with an increase in its Pb (II) content, which continues to increase as the Pb (II) content remains relatively stable. Infrared (FT-IR) analysis has been widely used for qualitative identification of functional groups and therefore it has also been used to determine the bioadsorption in which the functional groups are involved. The infrared analysis of the solid precipitate after centrifugation of the bacterial liquid with Pb (II) and Pb (II) -free culture is shown in FIG. 10.

As can be seen from FIG. 10, inIn the absence of Pb (II) culture, at 3290.59 cm ^-1 (-OH or-NH) ₂ ），2925.69 cm ^-1 （C-H），1645.42 cm ^-1 (c=o of protein), 1538.24 cm ^-1 (N-H and C-N of an amide group), 1400.08 cm ^-1 (c=o of amide I), 1239.29 cm ^-1 (C-N in amino group), 986.78 cm ^-1 (C-O-P and-PO in polysaccharide) ₄ ^3- The telescopic vibration absorption band of C-N in the amino group) forms a distinct absorption peak. Comparing the infrared spectra of Pb (II) and Pb (II) -free, it was found that after Pb (II) treatment, raw material 1645.42 cm ^-1 The c=o corresponding peak of the protein at this point shifted to 1659.82 cm ^-1 Here, it is illustrated that Pb (II) causes asymmetric stretching vibration of c=o in proteins secreted by bacteria. 1400.08 cm ^-1 The absorption peak is red shifted, by 1400.08 cm ^-1 Move to 1397.07 cm ^-1 1453.57 cm after Pb (II) treatment ^-1 The peak intensity enhancement at (C-H and COO-) suggests that both carboxylic acid or carboxylate and amide are involved in Pb (II) removal. 986.78 to 1077.82 cm in the presence of Pb (II) ^-1 The position of the C-O-P stretching vibration peak in the polysaccharide is obviously blue shifted, which indicates that the active group-PO on bacterial cells ₄ ^3- Amine groups and the C-O-P sugar rings in polysaccharides are the main contributors to bacterial immobilization and Pb (II) removal. Substance Pb generated in XRD analysis result in combination with FIG. 4 ₅ (PO ₄ ) ₃ (OH) describes-PO ₄ ^3- Mainly participate in the precipitation reaction of Pb (II), and other substances, proteins and polysaccharides play an important role in the adsorption of Pb (II) by bacteria. The effect of Cr (VI) on bacterial cell and composition of three extracellular products was studied using 3D-EEM and the results are shown in FIGS. 11 and 12.

A peak was identified at 275/335 nm, which was considered the protein tryptophan, while a peak was identified at 230/325 nm in the cell membrane fraction, which could be considered tyrosine. Comparing the changes of cytoplasmic peaks before and after Pb (II) addition (FIGS. 12 (a) and (b)), it can be found that the peak intensity of tryptophan is significantly reduced after Pb (II) addition, which means that the protein tryptophan in the cell membrane contributes more to the adsorption of Pb (II) in the process of immobilization, whereas comparing the change of the cell membrane can find that the tryptophan changes less in the cell membrane, which means that the tryptophan content in the cell membrane is higher and 600 mg/L Pb (II) does not affect the activity of tryptophan in the bacterial cell, and tyrosine is reduced after Pb (II) addition, which means that tyrosine also participates in the adsorption and immobilization of Pb (II), but the influence of Pb (II) on the activity of tyrosine is greater. Taken together, it was determined that tryptophan is mainly involved in Pb (II) fixation in cell membranes and cytoplasm after Pb (II) enters cells, and tyrosine is also involved but contributes less.

Peaks were identified in the soluble extracellular polymer at 290/370 nm and 380/425 nm, which were considered as soluble microbial by-products and humic acid-like organics, respectively, and the peak intensities were reduced both before and after the addition of Pb (II), and the higher fixed amount of Pb (II) combined with S-EPS could speculate that these organics adsorbed and fixed a large amount of Pb (II), and the presence of Pb (II) also affected its activity, resulting in reduced peak intensities. The loosely bound and tightly bound extracellular polymers identify peaks identical to those of cell membranes, and the changes of peaks before and after Pb (II) treatment are identical, thus indicating the same mechanism of adsorption and fixation of LB-EPS and TB-EPS to Pb (II) as that of cell membranes.

Exploring the dissolution condition of Cr (VI) in coal gangue when the coal gangue is treated by bacterial liquid, taking an LB liquid culture medium as a control group, and respectively taking the sterile LB liquid culture medium and the bacterial content of 2.85 multiplied by 10 ¹¹ The concentration change of Cr (VI) in each of the systems was measured in CFU/mL of the bacterial solutions, and the results are shown in FIG. 13.

Along with the extension of the treatment time, the Cr (VI) content in the coal gangue leachate is reduced by 86% compared with the aseptic environment, and the Cr (VI) content in the coal gangue leachate can be stabilized near 0.012 mg/L after the SK1 strain is treated. This well demonstrates that bacteria play a role in Cr (VI) removal.

The SK1 strain metabolites contained a large amount of organic acids, and for this purpose the organic acid reducing property against Cr (VI) was investigated in the range of organic acid concentration that the SK1 strain was capable of producing, and the results are shown in FIG. 14.

The relation of the organic acid to the reduction of Cr (VI) shows that the oxalic acid removal effect is most obvious, and the Cr (VI) removal rate in acetic acid is not relevant with the time extension, so that the acetic acid is not provided with the capability of reducing Cr (VI), tartaric acid and formic acid have certain reducing capability, but the final reducing effect is not provided with oxalic acid. Although oxalic acid, tartaric acid and formic acid all have certain reducing capability, the strain has limited acid production capability, and the removal rate of Cr (VI) is lower than 10 percent.

The great reason for the tolerance of bacterial cells to Cr (VI) is that the adsorption and reduction of Cr (VI) in the external environment by the extracellular polymer is effective means for resisting the toxicity of Cr (VI) by the SK1 strain. Thus the bacterial removal of Cr (VI) needs to be analysed both for extracellular components and for the bacterial cell itself. Bacterial extracellular products were extracted daily in experiments of removing Cr (VI) from SK1 strain, cr (VI) content in each layer of extracellular products was measured, and total removal rate of Cr (VI) was calculated, and experimental results are shown in fig. 15.

Along with the increase of the culture time of the SK1 strain, the Cr (VI) content in the three-layer extracellular polymer is in a decreasing trend, and the Cr (VI) content tends to be gentle in 7-8 days. The higher Cr (VI) content in the three-layer extracellular polymer than in the loosely bound and tightly bound extracellular polymers indicates that the adsorption of Cr (VI) by the extracellular polymer is mainly completed by the soluble extracellular polymer, while Cr (VI) is not increased in the loosely bound and tightly bound extracellular polymers with the extension of the treatment time, and the three-layer extracellular polymer has Cr (VI) removing capability, wherein the soluble extracellular polymer has the strongest removing capability and plays a main role in the process of resisting Cr (VI) by bacteria. Along with the reduction of the Cr (VI) content in the bacterial extracellular polymer, the Cr (VI) removal rate in the system is always increased, and the Cr (VI) removal rate is stable from the 7 th day, and finally can reach 95%. The IR spectra of the SK1 strain before and after Cr (VI) removal are shown in FIG. 16.

After Cr (VI) removal, the cells were 3290.59 cm ^-1 The absorption band at the site blue shifted, indicating-OH in the carbohydrate and-NH in the protein ₂ Stretching vibration occurs. 1652 cm ^-1 The absorption bands at this point are usually produced by C=O and C-N stretching vibrations of the protein, which in the presence of Cr (VI) slightly blue-shift the absorption bands, which is possibleBoth groups are bound to metal ions. 1538 cm ^-1 The nearby absorption peak is generated by stretching of protein amide II band C-N and N-H deformation vibration, and after Cr (VI) is treated, the protein amide II band C-N is subjected to 1452 and 1452 cm ^-1 The absorption band is enhanced where the change in absorption band is usually due to CH in proteins and lipids ₂ Bending vibrations are generated. After removal of Cr (VI) source 1400 cm ^-1 The nearby absorption band has shifted red, and the absorption band at this position is believed to be related to the C-O and COO-stretching movements of amide III in the protein. At 1239.29 cm ^-1 The absorption band is formed by symmetrically stretching the phosphoric acid diester, 1071.72 cm ^-1 986.78 cm ^-1 The absorption bands are generated by the vibration of sugar ring (C-O-C) of uronic acid and phosphate group (C-O-P) of nucleic acid functional group, and the absorption peak changes at the two positions are most obvious, 986.78 cm after Cr (VI) is removed ^-1 The absorbent band disappears and 1071.72 cm ^-1 The absorption band is obviously enhanced, which shows that Cr (VI) has influence on polysaccharide and intracellular metabolism of bacteria. In summary, it was determined that the polysaccharides and proteins metabolized by the SK1 strain during the treatment of Cr (VI) by the SK1 strain are the main participants, which adsorb and reduce Cr (VI). In addition, the presence of Cr (VI) also has an effect on the nucleic acid of the SK1 strain, presumably due to the entry of part of Cr (VI/III) into the cell interior.

Since the production of bacterial extracellular polymers is controlled by bacterial metabolism, when Cr (VI) is present in the environment, it is often possible to influence the metabolic processes of the bacteria, and it is necessary to study the removal of Cr (VI) by bacterial extracellular polymers alone. Three extracellular polymers were characterized using three-dimensional fluorescence spectroscopy (3D-EEM) and the results are shown in FIG. 17.

The organic composition of the soluble extracellular polymer is greatly different from the other two. The soluble extracellular polymerization contains microbial byproducts and humic acid-like organic matters (Ex/Em=290/370 nm and 380/425 nm), and the main components of the loosely-combined and tightly-combined extracellular polymers are tryptophan (Ex/Em=275/335 nm) and tyrosine (Ex/Em=230/325 nm). In combination with the existing studies, tryptophan and tyrosine play an increasing role in the reduction of Cr (IV) by bacteria, so we can infer that the same process exists in the reduction of Cr (VI) by SK1 strain. The soluble extracellular polymer does not contain tryptophan and tyrosine, but the removal rate of Cr (IV) is the highest, which shows that SK1 strain has other ways to remove Cr (VI). The SK1 strain is a gram-negative bacterium, polysaccharide in extracellular polymers of the gram-negative bacterium is anionic, humic acid in the extracellular polymers is electronegative, both substances have adsorption effect on metal ions, and the content of polysaccharide in the soluble extracellular polymers is far higher than that of the combined extracellular polymers, so that the adsorption of Cr (III/VI) by the soluble extracellular polymers is also helpful for Cr removal. In summary, it can be inferred that in the process of removing Cr (VI) from the extracellular polymeric substance, the soluble extracellular polymeric substance adsorbs and reduces Cr (VI), and a small amount of Cr (VI) is reduced by two amino acids contained in the soluble extracellular polymeric substance after entering the loosely bound extracellular polymeric substance and the tightly bound extracellular polymeric substance.

To determine the valence change of chromium before and after SK1 treatment, the chromium in solution before and after SK1 strain treatment was characterized by XPS, and the results are shown in fig. 18.

The XPS results of FIG. 18 show that Cr (III) is present in the system prior to SK1 strain treatment, and that Cr2p3/2 Binding Energy (BE) is approximately 577.32 eV and 586.22 eV, which is the phenomenon that part of the components in LB liquid medium have a reducing effect on Cr (VI). The peak at the Binding Energy (BE) of about 579.02 eV is a characteristic peak of Cr (VI). Cr (III) occurs at lower binding energies (574.28 eV and 571.48 eV) after bacterial treatment, and generally the lower the valence of most elements, the lower the binding energy. Thus, we can consider that these peaks are caused by reduction of Cr (VI) to a lower valence state. XPS results show that when the liquid medium containing Cr (VI) is treated by the SK1 strain, the characteristic peak of Cr (III) is obviously enhanced, and the peak of the corresponding Cr (VI) is weakened or even disappeared, which shows that under the reduction action of the SK1 strain, the Cr (VI) in the system is reduced to Cr (III) for removal.

As shown in fig. 19, to avoid repetitive description, technical parameters according to the embodiment of the present invention will be described as follows: the coal gangue is medium-grain-size coal gangue with grain size of 5-20 mm after coal is washed and selected; each 1000 mL distilled water in the LB solid culture medium contains 10-20 g peptone, 3-9 g beef extract powder, 10-30 g sodium chloride and 15-45 g agar, and is packaged in a container after heating and dissolving, regulating the pH value to 7.2-7.5 and sterilizing for 20min at 121 ℃ in a sterilizing pot; the SK1 bacteria are gram-negative bacteria, have small colony volume, 0.4-1.0 mu m wide and 1.2-3.0 mu m long, are mucilaginous and slightly convex, are light yellow, have smooth and round surfaces and are not transparent, and have short rod shapes and smooth tail ends and are free of endospores; the soil to be improved comprises barren soil such as saline-alkali soil, sand and the like; the soil improvement monitoring method comprises the steps of measuring available phosphorus and available potassium according to the Chinese forestry industry standards LY/T1233-1999 and LY/T1236-1999, measuring available silicon according to the Chinese agriculture industry standards NY/T1121.15-2006, measuring soluble lead and soluble chromium (VI) respectively according to the GB/T8152.15-2021 and the GB 31893-2015, measuring protein by adopting a Coomassie brilliant blue staining method, measuring polysaccharide by adopting an anthrone method taking glucose as a standard, adopting X-ray diffraction analysis (XRD), X-ray fluorescence spectrum analysis (XRF), inductively coupled plasma mass spectrometry (ICP-MS), carrying out microscopic feature monitoring on the soil improvement agent by using a scanning electron microscope energy spectrum analysis (SEM-EDS), carrying out analysis on microscopic features of the soil improvement agent and SK1 bacteria by utilizing a Fourier infrared spectrum analysis (FT-IR), and carrying out three-dimensional fluorescence spectrum analysis (3D-EEM) and X-ray photoelectron energy spectrum analysis (XPS), and not being described in the specific embodiments.

Example 1

Crushing the coal gangue with the medium particle size of 5-20 mm into small particles with the diameter of less than 3 mm, grinding into powder with the particle size of less than 115 mu m, and preserving for later use. Placing coal gangue powder which is naturally weathered for 60-70 days into a sterilized conical flask, wherein the liquid-solid mass ratio is 10-13:1 adding sterile water, soaking for 4-6 days at 30-45deg.C, dipping supernatant in an ultra-clean workbench with an inoculating loop, culturing in LB solid medium under streak culture, purifying for 3-5 generations, and storing in a refrigerator at 0-4deg.C for use. Preparing bacterial plates from the purified bacterial strains of 3-4 generations on a glass slide, gram staining the bacterial plates, observing the form of bacteria by a high-power microscope, and screening the purified bacterial strains with strong propagation speed and propagation stability by a plate streak culture mode to obtain SK1 bacteria. SK1 bacteria are scraped into sterile physiological saline by an inoculating loop in an ultra-clean workbench after being cultured for 2-4 days at 25-35 ℃ in LB solid culture medium, and uniformly shaken, and the concentration of bacteria liquid is changed by controlling the scraped bacteria amount. Adding gangue powder into a conical flask, and adding the gangue powder into the conical flask according to the liquid-solid mass ratio of 4-5:1 adding distilled water to soak, measuring the pH value of the bacterial liquid, pouring the bacterial liquid into gangue powder, vibrating and uniformly mixing, standing and fermenting for 4-6 days at 25-35 ℃ in a constant temperature incubator, and then drying at 40-5 0 ℃ in an oven to obtain the soil conditioner. The soil conditioner is applied to soil to be improved, wherein the effective silicon content in the soil conditioner is 500-700 mg/kg, the effective phosphorus content is 200-280 mg/kg, the quick-acting potassium content is 400-530mg/kg, the content of soluble lead is reduced to more than 91%, and the removal rate of soluble chromium (VI) is more than 86%.

Example 2

Crushing the coal gangue with the medium particle size of 5-20 mm into small particles with the diameter of less than 3 mm, grinding into powder with the particle size of less than 115 mu m, and preserving for later use. Placing coal gangue powder which is naturally weathered for 70-80 days into a sterilized conical flask, wherein the mass ratio of liquid to solid is 13-17:1 adding sterile water, soaking at 30-45deg.C for 6-8 days, dipping supernatant in an ultra-clean workbench, culturing in LB solid medium under streak culture, purifying for 3-5 generations, and storing in a refrigerator at 0-4deg.C for use. Preparing bacterial plates from the purified bacterial strains of 3-4 generations on a glass slide, gram staining the bacterial plates, observing the form of bacteria by a high-power microscope, and screening the purified bacterial strains with strong propagation speed and propagation stability by a plate streak culture mode to obtain SK1 bacteria. SK1 bacteria are scraped into sterile physiological saline by an inoculating loop in an ultra-clean workbench after being cultured for 2-4 days at 25-35 ℃ in LB solid culture medium, and uniformly shaken, and the concentration of bacteria liquid is changed by controlling the scraped bacteria amount. Adding coal gangue into conical bottle powder according to the mass ratio of liquid to solid of 5-6:1 adding distilled water for soaking to determine the pH value, pouring the prepared bacterial liquid into gangue powder, vibrating and uniformly mixing, standing and fermenting for 6-8 days at 25-35 ℃ in a constant temperature incubator, and then drying at 50-60 ℃ in an oven to obtain the soil conditioner. The soil conditioner is applied to soil to be improved, wherein the effective silicon content in the soil conditioner is 600-800 mg/kg, the effective phosphorus content is 240-320 mg/kg, the quick-acting potassium content is 450-610 mg/kg, the content of soluble lead is reduced to more than 92%, and the removal rate of soluble chromium (VI) is more than 88%.

Example 3

Crushing the coal gangue with the medium particle size of 5-20 mm into small particles with the diameter of less than 3 mm, grinding into powder with the particle size of less than 115 mu m, and preserving for later use. Placing coal gangue powder which is naturally weathered for 80-90 days into a sterilized conical flask, wherein the mass ratio of liquid to solid is 17-20:1 adding sterile water, soaking at 30-45deg.C for 8-10 days, dipping supernatant in an ultra-clean workbench, culturing in LB solid medium under streak culture, purifying for 3-5 generations, and storing in a refrigerator at 0-4deg.C for use. Preparing bacterial plates from the purified 4-5 generation bacterial strains on a glass slide, gram staining the bacterial plates, observing the form of bacteria by a high-power microscope, and screening the purified bacteria to obtain the bacterial strains with strong propagation speed and propagation stability, namely SK1 bacteria by a plate streak culture mode. SK1 bacteria are scraped into sterile physiological saline by an inoculating loop in an ultra-clean workbench after being cultured for 2-4 days at 25-35 ℃ in LB solid culture medium, and uniformly shaken, and the concentration of bacteria liquid is changed by controlling the scraped bacteria amount. Adding gangue powder into a conical flask, and adding the gangue powder into the conical flask according to the liquid-solid mass ratio of 4-6:1 adding distilled water for soaking to determine the pH value, pouring the prepared bacterial liquid into gangue powder, vibrating and uniformly mixing, standing and fermenting for 8-10 days at 25-35 ℃ in a constant temperature incubator, and then drying at 40-60 ℃ in an oven to obtain the soil conditioner. The soil conditioner is applied to soil to be improved, wherein the effective silicon content in the soil conditioner is 700-1000 mg/kg, the effective phosphorus content is 280-400 mg/kg, the quick-acting potassium content is 510-700 mg/kg, the content of soluble lead is reduced to more than 93%, and the removal rate of soluble chromium (VI) is more than 90%.

Claims (4)

1. A method for passivating heavy metals and activating fertilizers by utilizing microorganisms is characterized by comprising the following steps: the method comprises the following steps:

a. pretreatment of coal gangue and microorganism breeding: firstly, processing coal gangue into powder with the particle size smaller than 115 mu m through crushing and ore grinding procedures, and preserving for later use; placing coal gangue powder which is naturally weathered for 60-90 days into a sterilized conical flask, wherein the liquid-solid mass ratio is 10-20:1 adding sterile water, soaking for 4-10 days at 30-45deg.C, dipping supernatant with an inoculating loop in LB solid medium under ultra-clean environment, culturing under streak culture, purifying for 3-5 generations, and storing in a refrigerator at 0-4deg.C for use; preparing bacterial plates on glass slides of the purified bacterial strains of 3-5 generations, gram-staining the bacterial plates, observing the forms of bacteria through a high-power microscope, and screening the purified bacterial strains with strong propagation speed and propagation stability through a plate streak culture mode to obtain SK1 bacteria;

b. and (3) mixed fermentation: the SK1 bacteria are scraped into sterile physiological saline by an inoculating loop in an ultra-clean workbench after being cultured for 2-4 days at 25-35 ℃ in an LB solid culture medium, and uniformly shaken, and the concentration of bacteria liquid is changed by controlling the scraped bacteria amount; adding gangue powder into a container, and adding the gangue powder into the container according to the liquid-solid mass ratio of 4-6:1 adding distilled water for soaking to determine the pH value, pouring the prepared bacterial liquid into gangue powder, vibrating and uniformly mixing, standing and fermenting for 4-10 days at 25-35 ℃ in a constant temperature incubator, and then drying at 40-60 ℃ in an oven to obtain the soil conditioner.

2. The method for passivating heavy metals and activating fertilizer by utilizing microorganisms according to claim 1, wherein the method comprises the following steps: the coal gangue is selected from medium-grain-size coal gangue with grain size of 5-20 mm after coal is washed, then the medium-grain-size coal gangue is crushed into small grains with diameter of less than 3 mm, and then the small grains are ground into powder with grain size of less than 115 mu m.

3. The method for passivating heavy metals and activating fertilizer by utilizing microorganisms according to claim 1, wherein the method comprises the following steps: each 1000 parts of mL distilled water in the LB solid culture medium contains 10-20 g parts of peptone, 3-9 parts of beef extract powder g parts of sodium chloride 10-30 parts of g parts of agar 15-45 parts of g parts of distilled water, and is heated to dissolve, the pH value is regulated to 7.2-7.5, and then the mixture is packaged in a container and sterilized in a sterilizing pot at 121 ℃ for 20 min.

4. The method for passivating heavy metals and activating fertilizer by utilizing microorganisms according to claim 1, wherein the method comprises the following steps: the SK1 bacteria are gram-negative bacteria, have small colony volume, 0.4-1.0 mu m wide and 1.2-3.0 mu m long, are mucilaginous and slightly convex, are light yellow, have smooth and round surfaces and are not transparent, and have short rod shapes, smooth tail ends and no endophytic spores.