Method for repairing heavy metals by inducing phosphate precipitation through microorganisms

Abstract

The invention discloses a method for repairing heavy metal by inducing phosphate precipitation through microorganisms, and relates to the technical field of water body heavy metal repairing, the method comprises the following steps: step (1): bacillus subtilis is subjected to plate activation and inoculated into a culture medium A for overnight culture until OD600 is 1; (2) re-inoculating the bacillus subtilis liquid obtained in the step (1) into the culture medium A, culturing for 24 hours, sampling every 2 hours, and measuring OD600; (3) re-inoculating the bacillus subtilis liquid obtained in the step (1) into a culture medium B, culturing for 6 days, and sampling every day to measure the content of phosphate radicals; according to the method, the MIPP technology is applied to repairing of heavy metals Zn < 2 + >, Co < 2 + > and Mn < 2 + > which are still insufficient in research at present, reference value is provided for research in the field, and the method has obvious advantages in the aspects of Mn < 2 + > tolerance and repairing effect.

Description

Technical Field

[0001] The invention relates to the technical field of heavy metal restoration in water bodies, and in particular to a method for restoring heavy metals by microbial induced phosphate precipitation. Background Art

[0002] In recent years, with the rapid development of the economy, the types and quantities of heavy metals discharged into the environment have continued to increase due to industrialization and some unsafe agricultural activities (such as mining, smelting, electroplating, chemical production, and excessive use of pesticides and fertilizers). These heavy metals include zinc, cadmium, copper, lead, mercury, manganese, cobalt and other elements. They show strong toxicity even at extremely low concentrations, posing a serious threat to the environment and human health. For example, excessive exposure to zinc may cause anemia and damage the kidney and nervous system. At present, a variety of physical, chemical and biological methods have been applied to the problem of heavy metal pollution, such as adsorption, ion exchange, chemical precipitation, membrane filtration, plant extraction and transport, etc. Compared with these methods, biomineralization has received widespread attention due to its environmental friendliness, simple operation and low cost, and has good application prospects.

[0003] According to the mineralization products, biomineralization can be divided into microbially induced carbonate precipitation (abbreviated as MICP), phosphate precipitation (abbreviated as MIPP) and sulfide precipitation (abbreviated as MISP). Among them, MICP is more common in nature, so it has received more research; MISP is mainly used in anaerobic environments. The phosphate product of MIPP has better stability in nature, and no harmful gas ammonia is produced during the MIPP process, which is more environmentally friendly. Its main mechanism is that microorganisms produce phosphatase or phytase during their growth. These enzymes can decompose organic or inorganic phosphorus sources to produce phosphate ions. Phosphate ions combine with heavy metal ions to form phosphate precipitates, thereby achieving the purpose of removing heavy metals.

[0004] MIPP has been widely used in many fields, including biocement production, soil quality improvement, preparation of nano-functional materials, safe treatment of heavy metals in waste incineration fly ash, soil remediation, and removal of heavy metals in wastewater. However, there are relatively few studies on MIPP, especially in the remediation of heavy metals. Most of the research focuses on the remediation of lead, while the remediation research on other heavy metals is still insufficient. For example, in the literature "Han LJ, Li JS, Chen Z, et al. Stabilization of Pb(II)in wastewater and tailings by commercial bacteria through microbially induced phosphate precipitation(MIPP)" (Science of the Total Environment, 2023, 868:161628.) and the literature "Xue ZF, Cheng WC, Wang L, et al. Immobilizing lead in aqueous solution and loess soil using microbially induced carbonate / phosphate precipitation(MICP / MIPP)under harsh pHenvironments" (Journal of Hazardous Materials, 2024, 480: 135884.), Han et al. found that commercial Bacillus subtilis not only has higher phosphatase activity than isolated phosphatase-producing microorganisms, but also has obvious advantages in lead tolerance and removal rate; Xue et al. studied the removal of lead by Bacillus subtilis and Bacillus megacephala under different pH conditions, and the phosphate precipitation induced by Bacillus megacephala has greater advantages in the removal of lead. It can be seen that microbial induced phosphate precipitation has great potential in the removal of lead, but for other heavy metals, its research is still relatively small.

[0005] Therefore, the present invention proposes a method for repairing heavy metals by microbial-induced phosphate precipitation. Summary of the invention

[0006] The purpose of the present invention is to solve the defects in the prior art and to propose a method for repairing heavy metals by microbial induced phosphate precipitation.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for repairing heavy metals by microbial induced phosphate precipitation, more specifically, using Bacillus subtilis to repair heavy metal Zn in water 2+ 、Co 2+ and Mn 2+ , the method comprises the following steps:

[0009] Step (1): Bacillus subtilis was activated on a plate and inoculated into medium A and cultured overnight until OD 600 is 1;

[0010] Step (2): The Bacillus subtilis solution obtained in step (1) was re-inoculated into medium A and cultured for 24 hours, and samples were taken every 2 hours to measure OD 600 ;

[0011] Step (3): re-inoculating the Bacillus subtilis solution obtained in step (1) into medium B and culturing for 6 days, taking samples every day to measure the phosphate content;

[0012] Step (4): The Bacillus subtilis solution obtained in step (1) is re-inoculated into the culture medium containing different Zn 2+ 、Co 2+ and Mn 2+ The OD was measured after culturing for 24 hours. 600 ;

[0013] Step (5): The Bacillus subtilis solution obtained in step (1) is re-inoculated into the same Zn 2+ 、Co 2+ and Mn 2+ The cells were cultured in medium B with different pH values ​​for 6 days and the Zn content in the supernatant was measured. 2+ 、Co 2+ and Mn 2+ concentration.

[0014] Furthermore, the components of the culture medium A in step (1), step (2) and step (4) include beef extract, trypsin and sodium chloride, and the components of the culture medium B in step (3) and step (5) include beef extract, trypsin, sodium chloride, sodium β-glycerophosphate pentahydrate and magnesium chloride.

[0015] Furthermore, the measurement of phosphate in step (3) adopts the molybdenum blue method.

[0016] Furthermore, in step (4), Zn 2+ The concentration is 0-1.5mmol / L, Co 2+ The concentration is 0-1.2mmol / L, Mn 2+ The concentration is 0-12mmol / L.

[0017] Furthermore, the Zn in step (5) 2+ The concentration is 1mmol / L, Co 2+ The concentration is 0.5mmol / L, Mn 2+ The concentration is 6mmol / L and the pH is 5-9.

[0018] Furthermore, the culture conditions in the above steps (1) to (5) are 30° C. and the shaking speed is 220 rpm.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention applies MIPP technology to the heavy metal Zn, which is currently understudied. 2+ 、Co 2+ and Mn 2+ The repair of Mn 2+ It has obvious advantages in terms of tolerance and repair effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0022] Figure 1 is a growth curve diagram of Bacillus subtilis in an embodiment of the present invention;

[0023] Figure 2 Schematic diagram of the phosphatase activity of Bacillus subtilis in an embodiment of the present invention;

[0024] Figure 3 The Bacillus subtilis for Zn in the embodiment of the present invention 2+ Schematic diagram of tolerance;

[0025] Figure 4 is the activity of Bacillus subtilis for Co 2+ Schematic diagram of tolerance;

[0026] Figure 5 is the activity of Bacillus subtilis for Mn in the embodiment of the present invention. 2+ Schematic diagram of tolerance;

[0027] Figure 6 The results of the experiment on the effect of Bacillus subtilis on Zn at different initial pH values ​​are shown in Fig. 2+ 、Co 2+ and Mn 2 + Schematic diagram of the removal rate. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention;

[0029] Reference Figure 1-6 , a method for remediating heavy metals by microbial induced phosphate precipitation, the method targeting Bacillus subtilis (collection number CCTCC AB 98002);

[0030] The method comprises the following steps:

[0031] Step (1): Bacillus subtilis was activated on a plate and inoculated into medium A and cultured overnight until OD 600 The components of culture medium A are 10 g / L beef extract, 10 g / L tryptone, and 5 g / L sodium chloride.

[0032] Step (2): The Bacillus subtilis solution obtained in step (1) was re-inoculated into medium A and cultured for 24 hours, and samples were taken every 2 hours to measure OD 600 ;

[0033] Step (3): The Bacillus subtilis liquid obtained in step (1) is re-inoculated into culture medium B and cultured for 6 days, and samples are taken every day to measure the phosphate content; the phosphate content is determined by the molybdenum blue method, the principle is that phosphate forms phosphomolybdenum yellow with ammonium molybdate, and phosphomolybdenum yellow can be reduced to a blue complex molybdenum blue, and the concentration of molybdenum blue can be reflected according to the absorbance at 660 nanometers, thereby indicating the concentration of phosphate. The components of culture medium B are 10g / L beef extract, 10g / L tryptone, 5g / L sodium chloride, 10mmol / L magnesium chloride, and 50mmol / L sodium β-glycerophosphate pentahydrate.

[0034] Step (4): The Bacillus subtilis solution obtained in step (1) is re-inoculated into the culture medium containing different Zn 2+ 、Co 2+ and Mn 2+ The OD was measured after culturing for 24 hours. 600 ;

[0035] Specific: Zn used 2+ The concentrations were 0, 0.4, 0.8, 1, 1.2, and 1.5 mmol / L; the Co 2+ The concentrations were 0, 0.4, 0.8, 1, 1.1, and 1.2 mmol / L; the Mn 2+ The concentrations are 0, 4, 8, 10, 11, and 12 mmol / L.

[0036] Step (5): The Bacillus subtilis solution obtained in step (1) is re-inoculated into the same Zn 2+ 、Co 2+and Mn 2+ The cells were cultured in medium B with different pH values ​​for 6 days and the Zn content in the supernatant was measured. 2+ 、Co 2+ and Mn 2+ concentration.

[0037] Specifically: Centrifuge the bacterial solution at 5000 rpm for 10 minutes and measure the Zn content of the supernatant. 2+ 、Co 2+ and Mn 2+ Content, according to the Zn 2+ 、Co 2+ and Mn 2+ Calculation of Zn content 2+ 、Co 2+ and Mn 2+ Removal rate. Zn used 2+ The concentration was 1mmol / L, and the Co 2+ The concentration is 0.5mmol / L, and the Mn 2+ The concentration was 6 mmol / L. The initial pH value was adjusted to 5, 6, 7, 8, and 9 respectively using 15% sulfuric acid or sodium hydroxide particles.

[0038] Furthermore, the culture conditions in the above steps (1) to (5) are 30° C. and the shaking speed is 220 rpm.

[0039] The results are as follows:

[0040] Reference Figure 1 The figure is a growth curve of Bacillus subtilis in the embodiment of the present invention; it can be seen that in the first 4 hours, the growth curve of Bacillus subtilis remains basically flat, the number of bacteria does not change much, and it is in a slow period at this time, and the bacterial metabolism is slow; from 4 to 20 hours, the growth curve of Bacillus subtilis has an obvious rapid increase, and the bacteria are in the logarithmic period at this time, the bacterial metabolism is vigorous and begins to multiply in large quantities, and the number of bacteria increases exponentially; after 20 hours, due to the consumption of nutrients and the increase of metabolites, the bacterial growth is affected, and the bacterial growth rate slows down.

[0041] Since phosphatase can hydrolyze sodium β-glycerophosphate pentahydrate in the culture medium into phosphate, the phosphatase activity of bacteria can be reflected by measuring the phosphate content of the system.

[0042] Reference Figure 2 Schematic diagram of the phosphatase activity of Bacillus subtilis in an embodiment of the present invention; it can be seen that as time goes by, Bacillus subtilis metabolizes and produces more and more phosphatase to decompose sodium β-glycerophosphate pentahydrate, and the phosphate content in the solution increases accordingly. On about the fourth day, due to insufficient bacterial activity, the metabolic phosphatase has reached its limit, no phosphate is produced, and the phosphate produced by hydrolysis reaches a maximum value.

[0043] Reference Figure 3-6 are respectively the Zn 2+ 、Co 2+ and Mn 2+ Schematic diagram of the tolerance of these three heavy metal ions;

[0044] For Zn 2+ , the tolerance of Bacillus subtilis is 1.2mmol / L. When the concentration exceeds 1.2mmol / L, the growth of bacteria is inhibited and cannot grow normally. 2+ , which is significantly more toxic to Bacillus subtilis than Zn 2+ and Mn 2+ Even at very low concentrations, the growth of Bacillus subtilis was affected, and the inhibition became more severe as the concentration increased. 2+ , Bacillus subtilis showed significantly higher tolerance. 2+ The growth of Bacillus subtilis was inhibited only when the concentration reached 11mmol / L, showing a high tolerance.

[0045] Reference Figure 6 The results of the experiment on the effect of Bacillus subtilis on Zn at different initial pH values ​​are shown in Fig. 2+ 、Co 2+ and Mn 2+ Schematic diagram of removal rate;

[0046] For Zn 2+ Regardless of the initial pH value, the removal rate of Bacillus subtilis was not much different and was very low, all around 5%, with almost no removal and a very poor removal effect. 2+ and Mn 2+ , the removal rate under acidic conditions is significantly lower than that under neutral and alkaline conditions. This can be attributed to the fact that phosphate precipitates have a certain solubility under acidic conditions, and the removal rate increases with the increase of alkalinity. 2+ , the removal rate can reach up to about 32%; for Mn 2+ , the removal rate can reach up to 92%. Obviously, for these three heavy metal ions, Bacillus subtilis has the highest removal rate for Mn 2+ The removal rate of Co 2+ Next, Zn 2+ lowest.

[0047] Therefore, considering the comprehensive effect of Bacillus subtilis on Zn 2+ 、Co 2+ and Mn 2+ The MIPP technology used in this invention has a high tolerance and removal rate for heavy metal Mn2+ It shows remarkable effect in the restoration of , and has good research and application value.

Claims

1. A method for repairing heavy metals by microbial induced phosphate precipitation, characterized in that: The method comprises the following steps: Step (1): Bacillus subtilis was activated on a plate and inoculated into medium A and cultured overnight until OD 600 is 1; Step (2): The Bacillus subtilis solution obtained in step (1) was re-inoculated into medium A and cultured for 24 hours, and samples were taken every 2 hours to measure OD 600 ; Step (3): re-inoculating the Bacillus subtilis solution obtained in step (1) into medium B and culturing for 6 days, taking samples every day to measure the phosphate content; Step (4): The Bacillus subtilis solution obtained in step (1) is re-inoculated into the culture medium containing different Zn 2+ 、Co 2+ and Mn 2+ The OD was measured after culturing for 24 hours. 600 ; Step (5): The Bacillus subtilis solution obtained in step (1) is re-inoculated into the same Zn 2+ 、Co 2+ and Mn 2+ The cells were cultured in medium B with different pH values ​​for 6 days and the Zn content in the supernatant was measured. 2+ 、Co 2+ and Mn 2+ concentration.

2. The method for repairing heavy metals by microbial induced phosphate precipitation according to claim 1, characterized in that: The components of the culture medium A in the steps (1), (2) and (4) include beef extract, trypsin and sodium chloride, and the components of the culture medium B in the steps (3) and (5) include beef extract, trypsin, sodium chloride, sodium β-glycerophosphate pentahydrate and magnesium chloride.

3. The method for repairing heavy metals by microbial induced phosphate precipitation according to claim 1, characterized in that: The measurement of phosphate in step (3) adopts the molybdenum blue method.

4. The method for repairing heavy metals by microbial induced phosphate precipitation according to claim 1, characterized in that: In the step (4), Zn 2+ The concentration is 0-1.5mmol / L, Co 2+ The concentration is 0-1.2mmol / L, Mn 2+ The concentration is 0-12mmol / L.

5. The method for repairing heavy metals by microbial induced phosphate precipitation according to claim 1, characterized in that: The Zn in step (5) 2+ The concentration is 1mmol / L, Co 2+ The concentration is 0.5mmol / L, Mn 2+ The concentration is 6mmol / L and the pH is 5-9.

6. The method for repairing heavy metals by microbial induced phosphate precipitation according to claim 1, characterized in that: The culture conditions in steps (1) to (5) are 30° C. and the shaking speed is 220 rpm.

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