Study on Metabolites and Desiliconization of Bacillus licheniformis JXF

Bacillus licheniformis belongs to silicate bacteria, which is a facultative energy-producing bacterium capable of decomposing silicate minerals and releasing elements such as potassium and silicon. In the mineral processing field, mainly for desilication silicate bacteria silicate mineral bauxite, etc., in order to improve the quality of the mineral purposes, but such use is experimental research stage. Studies on the desiliconization mechanism of silicate bacteria have not been reported in the system. The leaching mechanism of silicate bacteria is currently different. Some people think that it is caused by organic acids produced by silicate bacteria, others are thought to be due to the extracellular polysaccharide produced during the growth process, or both. The result of synergy, but there is no experimental argument. To this end, this paper made a comprehensive experimental study on this problem, analyzed various metabolites that may be produced during the growth and reproduction of Bacillus licheniformis, and studied the leaching of various metabolites on silicate minerals.

First, the test materials and methods

(1) Materials

The Bacillus licheniformis JXF strain was isolated and screened from the silicon fertilizer by the laboratory. Tested mineral quartz 85% purity (isolated from the Zhengzhou gibbsite), kaolinite, potassium feldspar (92% purity). Medium: Based on the Ashbe substrate medium, three different mediums containing nitrogen, nitrogen and quartz powder were prepared.

(2) Analysis content and method

Three 500ml cone bottles were filled with 150ml medium I, II, III, and sterilized at 120 °C for 2h. After cooling, the Bacillus licheniformis suspension was inserted into the cone bottle at 2% inoculum and shaken at 36 °C. 200 r / min), cultured 0, 6, 12, 24, 36, 48, 72, 96h after analysis.

Determination of organic acids: The content of organic acids in various fermentation broths was determined by high performance liquid chromatography using sulfuric acid extraction.

Determination of amino acid: The fermentation broth was first treated with trichloroacetic acid, diluted with 0.015 mol/L hydrochloric acid, and then determined by an amino acid analyzer.

Determination of polysaccharides in the fermentation broth of the three kinds of culture medium: ethanol was added to the fermentation broth after extracting the organic acid, centrifuged at 60 ° C, and weighed to obtain a crude polysaccharide.

(III) Analysis of silicate metabolites leaching potassium feldspar

Potassium feldspar was leached by shake flask leaching using various Bacillus licheniformis metabolites. Determination of silicon in the leachate by silicon molybdenum blue spectrophotometry.

Second, the test results analysis

(1) Metabolites in the fermentation broth of JXF strains

1. Determination of organic acid content in organic acid fermentation broth of fermentation strain showed that the content of organic acid in the nitrogen-free quartz-containing powder medium was the largest. Through qualitative detection, it was found that the following four organic acids were detected in the fermentation broth of different periods: oxalic acid, tartaric acid, citric acid and malic acid. The quantitative test results are shown in Table 1. It can be seen from the results in Table 1 that the bacteria are not the most in the initial stage of fermentation (6 to 48 h), but the organic acid is more synthetic due to the strong metabolic activity of bacteria. With the prolongation of bacterial fermentation time, although the number of cells increased greatly, the amount of organic acids synthesized by bacteria decreased to a large extent, which indicates that in the process of bacterial growth and reproduction, due to the continuous consumption of nutrients in the fermentation broth, The organic acid synthesized by the cells and secreted into the fermentation broth is used as a nutrient by the bacteria.

Table 1 Test results of components in the fermentation broth

2. The amino acid test in the fermentation broth of JXF-1 strain found that the amount and type of various amino acids produced by JXF strains were basically the same in three different media, so the test only determined the nitrogen-free medium containing quartz powder. The amino acid species and their content. Through qualitative and quantitative analysis, it was found that silicate bacteria can synthesize various types of amino acids, and there are mainly 15 kinds of amino acids that can be detected. The results are shown in Table 2. It can be seen from the results in Table 2 that in different fermentation periods, the amount of amino acids synthesized by the strains is quite different. Several amino acids such as asparagine, semi-leucine, methionine, leucine and valine are mainly In the early stage of fermentation, with the prolongation of fermentation time, the concentration of these amino acids in the fermentation broth gradually decreased, indicating that the strains fully utilized them as nutrients for their own growth and reproduction. With the prolongation of fermentation time, the concentrations of several amino acids such as glycine, methionine, arginine and serine in the fermentation broth gradually increased, indicating that the bacteria can continuously synthesize these amino acids. At the same time, the types of amino acids synthesized by bacteria are different in different fermentation stages, such as glycine, alanine, leucine and isoleucine. Hemi-amino acid is mainly synthesized in the early stage of bacterial fermentation, and several amino acids such as proline, phenylalanine, aspartic acid and serine are mainly synthesized in the late fermentation stage. In the fermentation process of bacteria, the highest content of synthetic amino acids is alanine, followed by glycine, and the least amount of synthetic amino acids is aspartic acid.

Table 2 Amino acid types and contents of bacterial synthesis

Note: “—” means that no data has been detected.

3. The polysaccharides in the fermentation broth of the strains were found in the separation of JXF strains, fermentation culture and leaching of silicate minerals. The strains were found in nitrogen-containing medium, nitrogen-free medium, and nitrogen-free quartz-containing silicate minerals. There is a large difference in the content of capsular polysaccharide produced in the medium. Therefore, the above three kinds of mediums were selected for the test, and the dynamic changes of the capsules in each medium were qualitatively and quantitatively analyzed. The measurement results are shown in Fig. 1. The test results show that JXF-1 bacteria can synthesize several kinds of sugar alcohols such as glucose, arabinose, xylose, mannitol, sucrose, fructose, lactose, maltose, raffinose, sorbitol and inositol. In the nitrogen-free quartz medium, the strain has the strongest ability to produce capsular polysaccharide, followed by the nitrogen-free medium. In the nitrogen medium, the strain produced the least capsular polysaccharide, which was only 3.76 g/L, and only about 25% of the capsular polysaccharide in the nitrogen-free aluminous medium. At the same time, it can be seen from Fig. 1 that with the prolongation of fermentation time of the strain, the content of capsular polysaccharide has a great change. Within 0~48h, the yield of polysaccharide increases with time, and the fermentation is 48h. The yield of capsular polysaccharides reached the highest in three different media. After 48h, the content of capsular polysaccharide decreased with the prolongation of fermentation time, indicating that the capsular polysaccharide in the culture solution was partially used by bacteria. The growth and reproduction of nutrients is utilized to reduce the content of polysaccharides in the bacterial liquid. The above results indicate that the JXF-1 strain has a lower ability to form capsular polysaccharide in the presence of an effective multi-nitrogen source, and more synthetic pods in the case of less nitrogen or no nitrogen. Membrane polysaccharide; in the nitrogen-free bauxite medium, due to the presence of a large amount of silica, the strain can mass-produce the capsular polysaccharide.

Fig.1 Effect of different media on the production of capsular polysaccharides from JXF-1 strains

(2) Solubilization of silicate minerals by JXF bacterial metabolites

Various metabolites of silicate bacteria have certain solubility in potassium in potassium-containing minerals, but whether they can activate the silicon, the current mineral processing scholars have not done systematic research, most of them are speculation. In this paper, the soaking effect of JXF bacterial metabolites on silicate minerals was studied experimentally.

1. Dissolution of potassium feldspar by organic acid According to the range of four organic acids detected in the fermentation broth of silicate bacteria, oxalic acid, tartaric acid, citric acid and malic acid solution with a maximum concentration of 200 mg/L were prepared. At the same time, the EDTA disodium with a pH of 2.5 was prepared and compared with distilled water. 10 g of potassium feldspar powder was separately added to 100 mL of the above organic acid and EDTA solution, and shaken at 25 ° C for 7 days, and then filtered, and the contents of aluminum and silicon in the filtrate were measured. The results are shown in Table 3. It can be seen from the results in Table 3 that the dissolution of potassium feldspar by organic acid under acidic conditions is better than the dissolution of potassium feldspar by organic acid under neutral conditions, indicating that the action of organic acid to dissolve silicate minerals has acid dissolution. It also contains the complexing action of organic acids, and plays a major role in the coordination of organic acids. As can be seen from Table 3 while the results, since EDTA has a strong effect of metal ions with the silicate minerals of silicon, aluminum or the like, thereby increasing the dissolution effect of these ions, silicon EDTA solution, the more the amount of aluminum The solution containing each organic acid is much higher. However, if 25% hydrogen peroxide is used to destroy the structure of the above organic acid, it is found that their ability to dissolve potassium feldspar is greatly reduced, indicating that the above organic acid dissolves potassium feldspar and releases the elements such as silicon and aluminum to its unique structure. That is, it is closely related to the content of a group such as -COOH.

Table 3 Solubilization of potassium feldspar by organic acids

The results of the dissolution test of potassium feldspar at different concentrations of the four organic acids detected in the fermentation broth are shown in Fig. 2. As a control, potassium feldspar was leached by using four kinds of mixed organic acids of 25, 50, 100, and 150 mg/L in the relative proportions of various organic acids, and the leaching results are shown in Fig. 3.

Figure 2 Dissolution of potassium feldspar by four organic acids

Figure 3: Dissolution of potassium feldspar by mixed organic acids

It can be seen from Fig. 2 and Fig. 3 that the dissolution of silicon in potassium feldspar by various organic acids is oxalic acid> mixed organic acid (oxalic acid + citric acid + malic acid + tartaric acid) > citric acid > tartaric acid > malic acid . The content of silicon in the leachate did not change much with the change of concentration of tartaric acid and malic acid.

2, amino acid on the potassium feldspar, kaolinite soaking in a 500mL cone type bottle with 100mL distilled water and 10g potassium feldspar or kaolinite and mixed amino acids (take the test measured silicate bacteria fermentation broth maximum The concentration of each amino acid was mixed. After 7 days of shaking at 25 ° C, the contents of aluminum and silicon in the supernatant were measured. The results are shown in Table 4. It can be seen from the results in Table 4 that the amino acid has the ability to leaching aluminum and silicon in the silicate mineral. When the structure of the mixed amino acid is destroyed by the action of hydrogen peroxide, the leaching ability is greatly reduced. This indicates that the -COOH and -NH ₂ groups in the amino acid have the ability to cooperate with silicon in kaolinite and potassium feldspar.

Table 4 Amino acid dissolution of potassium feldspar

3. Solubilization of potassium feldspar by polysaccharides A certain amount of polysaccharide was added to the cone bottle and formulated into various concentrations as shown in Table 5. After 7 days of shaking at 25 ° C, the content of aluminum and silicon in the solution was determined. The test results are shown in Table 5. It can be seen from the data in Table 5 that the polysaccharide produced by the fermentation of silicate bacteria has a certain leaching effect on aluminum and silicon in potassium feldspar. With the increase of polysaccharide content, the content of aluminum and silicon in the supernatant is obvious. increase. The polysaccharide is degraded by 25% hydrogen peroxide, and the organic structure of the polysaccharide is destroyed due to its oxidation, so that the leaching ability is lowered. This is because the polysaccharide contains various organic groups having a complexing action, and forms a complex with various metal ions in the mineral, thereby increasing the content of aluminum and silicon in the solution.

Table 5 Polysaccharides secreted by silicate bacteria soaked in silicon and aluminum in potassium feldspar

4, organic acid, amino acid, polysaccharide synergistic effect on the dissolution of potassium feldspar in a 500mL cone type of each metabolite and the composition of the test minerals - mixed organic acid composition: each concentration of 200mg / L 4 kinds of organic acids (oxalic acid, tartaric acid, citric acid, malic acid); mixed amino acid composition: the maximum concentration of each amino acid mixed composition; polysaccharide concentration of 8.5g / L; potassium feldspar 15g / L.

Organic acids, amino acids and polysaccharides have obvious leaching effects on silicon and aluminum in silicate minerals. The test results are shown in Fig. 4. It can be seen from Fig. 4 that the ability of each metabolite to decompose potassium feldspar is: mixed organic acid > mixed amino acid > polysaccharide. The ability of a mixture of amino acids, organic acids, and polysaccharides to dissolve potassium feldspar is greatly increased compared to the solubility of a single metabolite. In addition, the ability of bacterial metabolites to leach potassium and feldspar in silicon and aluminum is related to its structure. The structure of metabolites is destroyed by hydrogen peroxide, and their ability to activate potassium feldspar is greatly reduced. At the same time, the activation of potassium feldspar by the metabolites of silicate bacteria is mainly through the cooperation of organic substances with complex groups, and each metabolite has an organic group which forms a complex with various metals in the mineral, especially aluminum and silicon. Groups such as -COOH, -NH ₂ or -COO ^- , -NH ₄ ⁺ , all have a certain ability to cooperate with metal ions.

Figure 4 Dissolution of potassium feldspar by different metabolites

1-capsular polysaccharide; 2-mixed amino acid + capsular polysaccharide; 3-mixed organic acid;

4-mixed organic acid + polysaccharide; 5-mixed organic acid + mixed amino acid;

6-mixed amino acid + mixed organic acid + capsular polysaccharide

Scanning electron micrographs of different substances treated with K-feldspar are shown in Fig. 5. It can be seen from Fig. 5 that the ability of the metabolites of the JXF strain to destroy the potassium feldspar character structure is greater than the destruction of the potassium feldspar lattice structure by 0.01 mol of nitric acid, and the various metabolism of the strains by the action of H ₂ O ₂ . After the product, its ability to destroy the K-feldspar lattice structure decreases.

Figure 5 Scanning electron microscopy of different materials after treatment with potassium feldspar

(a) organic acid + amino acid + capsular polysaccharide; (b) organic acid + amino acid + capsular polysaccharide + H ₂ O ₂ ;

(c) 0.01 mol HNO ₃ ; (d) K-feldspar ore; (e) distilled water

Third, the conclusion

(1) It was verified that silicate bacteria can be metabolized to produce four kinds of organic acids, various amino acids and extracellular polysaccharides in common Ashbee matrix liquid medium. Although there is no significant difference in the types and amounts of amino acids produced by JXF strains in different media, there is a large difference in the amount of organic acids and extracellular polysaccharides produced by metabolism. The results of this study have not yet been found. Report. The leaching effect of a single metabolite produced by the strain on silicon is significantly lower than the synergistic effect of various metabolites.

(2) The JXF strain can synthesize and secrete four kinds of organic acids and various amino acids such as oxalic acid, citric acid, tartaric acid and malic acid into the fermentation broth. With the prolongation of fermentation time, the content of four organic acids gradually decreased, indicating that with the gradual consumption of nutrients in the culture solution, bacteria began to use the organic acids secreted by themselves as the material basis for reproductive growth.

(C) JXF strain can synthesize 15 of 20 kinds of amino acids in the fermentation process, including 2 kinds of basic amino acids, 2 kinds of acidic amino acids and 2 kinds of amino acids with hydroxyl groups. Among them, several amino acids such as glycine, methionine, serine and lysine are mainly synthesized in the early stage of fermentation. Different types of synthetic amino acids are different in different fermentation stages.

(4) Among the three different fermentation media containing nitrogen, nitrogen and quartz powder, the ability of the strain to synthesize polysaccharides is quite different. In the nitrogen medium, the bacteria produce the least polysaccharide, but In the nitrogen-free quartz medium, the strain produced the largest amount of polysaccharide.

(5) Shake flask leaching and electron microscopy experiments show that organic acids, amino acids and polysaccharides have the ability to destroy the crystal structure of K-feldspar and release aluminum and silicon, because these organic compounds have various metal ions in the minerals. Organic groups with a certain acid solubility. Various metabolites have synergistic effects in leaching silicate minerals. The mixture of the three is the most obvious damage to the K-feldspar lattice structure, and its leaching effect is also significantly better than their respective effects on minerals.