CN115805233A - Method for removing Cr (VI) in soil by sulfate reducing bacteria unsaturated biomembrane - Google Patents
Method for removing Cr (VI) in soil by sulfate reducing bacteria unsaturated biomembrane Download PDFInfo
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Abstract
The invention belongs to the technical field of soil heavy metals, and discloses a method for removing Cr (VI) in soil by using a sulfate reducing bacteria unsaturated biomembrane. The method uses different environmental pressures as influencing factors, cultures the sulfate reducing bacteria unsaturated biomembrane, and carries out the adsorption reduction experiment on Cr (VI). The method is simple to operate and environment-friendly, and by analyzing the EPS components, the yield and the composition proportion of extracellular polymers of the unsaturated biomembrane of the sulfate-reducing bacteria are correspondingly changed under the influence of different environmental pressures, so that the adsorption and reduction performance of Cr (VI) is remarkably changed, and a new thought is provided for solving the influence of the environmental pressure and pollutants on the EPS secretion of the bacteria and the treatment effect of the pollutants in the unsaturated environment.
Description
Technical Field
The invention belongs to the technical field of soil heavy metal remediation, and particularly relates to a method for removing Cr (VI) in soil by using a sulfate reducing bacteria unsaturated biomembrane.
Background
Cr is considered to be a carcinogen harmful to human health. The chemical form of Cr determines its toxicity, bioavailability and solubility. In the environment, cr is mainly present in the form of Cr (vi) and Cr (iii), which are generally sparingly soluble and less toxic than the high solubility and strong oxidizing properties of Cr (vi) compounds. In industrial processes such as leather tanning, alloy making, plating and pigments, etc., improper handling and uncontrolled release thereof results in large amounts of Cr-containing contaminants entering the soil, causing soil contamination and thus causing widespread concern. The microorganism can convert Cr (VI) into Cr (III), and has the characteristics of economic feasibility, no secondary pollution and the like, so that the microorganism is considered to be a treatment strategy with prospect in repairing soil heavy metal pollution.
In the biofilm formation process, a large amount of EPS is secreted by microorganisms, and the EPS is used as a structural scaffold to connect the microorganisms together to form a biofilm. Microbial cells and EPS tightly associate to form a biofilm, which is also the most common form of microbial presence in nature. Sharma et al reported in the Phosphatase medial bioprecipitation of lead as a pyrophorite by Achromobacter xylosoxidans that extracellular precipitation in biofilms is the major mechanism for lead removal by Achromobacter xylosoxidans. Hou et al, paper excellular polymeric substructures from coater-top carbon Sinorhizobium meliliti immobiize Cu 2+ It was also confirmed that EPS plays a key role in Cu (II) immobilization.
The unsaturated biofilm is formed by connecting cells with EPS secreted by microorganisms attached to the surface of soil particles, and the EPS can be used as a medium for conveying unsaturated biofilm substances. Therefore, in Cr-contaminated soils, EPS plays a great role as a mediator of the interaction of unsaturated biofilms with heavy metals.
In recent years, it has been found that the problem of Micro Plastics (MPs) contamination in soil is also very severe compared to heavy metal contamination of soil. It is reported in the literature that MPs can provide special niches due to their potential metabolic adaptation to specific biological groups, but while providing a living space for microorganisms, MPs can also negatively affect biofilms. MPs have an effect on the growth and development of microorganisms, EPS secretion, reactive oxygen species production and enzyme activity. However, at present, little information is available about the influence of MPs on the biofilm community, and research on natural biofilm community in water environment is mainly focused. In the actual soil environment, the condition that two pollutants of Cr (VI) and micro-plastic exist simultaneously can occur, and the influence of the composite system on the EPS secretion of the unsaturated biomembrane of the microorganism and the influence on the process of adsorbing and reducing the Cr (VI) by the microorganism are unknown.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a culture method of a sulfate reducing bacteria unsaturated biomembrane, which is characterized in that Cr (VI) and micro-plastic are simultaneously introduced into a culture system in different modes under an unsaturated environment to induce and culture the sulfate reducing bacteria unsaturated biomembrane, so that the secretion of EPS of sulfate reducing bacteria is improved, EPS components are obviously changed, and the adsorption reduction effect of the sulfate reducing bacteria unsaturated biomembrane on the Cr (VI) is improved.
In order to realize the purpose, the invention is realized by the following technical scheme:
a method for removing Cr (VI) in soil by sulfate reducing bacteria unsaturated biomembrane comprises the following steps:
(1) Culture of unsaturated biomembrane of sulfur Desulfovibriosp
Inoculating Desulfosibrio sp CMX bacteria stored in a refrigerator at the temperature of-80 ℃ to LB culture medium with the pH value of 7.0 for activation culture for 10 hours, centrifuging activated Desulibrio sp CMX bacteria liquid for 5 minutes at the temperature of 4 ℃ and 5000g, separating the Desulibrio sp CMX bacteria from the culture medium, washing the Desulibrio sp CMX bacteria twice by phosphate buffer solution with the pH value of 7.2 to remove residual culture medium, and then re-suspending the washed Desulibrio sp CMX bacteria in 0.9wt NaCl solution containing 100-200mg/L polystyrene micro plastic PS-MPs to obtain Desulibrio sp CMX bacteria inoculation liquid; transferring the polycarbonate membrane to the surface of a solid culture medium containing Cr (VI) by using sterile forceps, inoculating a Desulfovibrio sp.CMX bacterium inoculation solution to the center of the polycarbonate membrane, and performing inverted culture in an anaerobic box to obtain a sulfur Desulfovibrio sp.CMX bacterium unsaturated biological membrane;
the solid culture medium containing Cr (VI) comprises 3g/L NaCl and K 2 HPO 4 ·3H 2 O 0.66g/L，NH 4 Cl 1g/L，NaSO 4 1g/L, 6.315g/L of sodium lactate, 15mg/L of Cr (VI) and the pH value of 7.8-8.2.
(2) Sampling the cultured Desulovibrosp.CMX unsaturated biomembrane at intervals of 24h, extracting colloidal and capsule EPS of the Desulovibrosp.CMX bacteria, analyzing components of the colloidal and capsule EPS, and determining the adsorption reduction amount of the sulfur Desulavibrosp.CMX unsaturated biomembrane on Cr (VI);
CMX (Desulfovirosp) colloidal and capsular EPS (Expandable polystyrene) extraction method: resuspending the cultured desalfovibrosp.CMX bacteria unsaturated biomembrane in 0.9wt% NaCl solution in turn, centrifuging at 10000g for 30min, collecting supernatant, and filtering with 0.22 μm cellulose acetate filter membrane to obtain colloidal EPS; then, the remaining precipitated cells were resuspended in a 0.9% by weight NaCl solution containing 10mmol/LEDTA, and after standing at room temperature for 2 hours, centrifuged at 12000g for 30 minutes, and the supernatant was collected and filtered with a 0.22 μm cellulose acetate filter to obtain capsule-shaped EPS; the residual thallus is intracellular component.
Measurement of adsorbed Cr (VI) content of unsaturated biomembrane of Desulfovibrio sp. Respectively putting the Desulfovibrio sp CMX bacteria unsaturated biomembranes cultured in different time periods into a polytetrafluoroethylene digestion tank, digesting for 2 hours at 200 ℃, diluting the digested solution to a certain volume by using ultrapure water, and measuring the total Cr content by using an inductively coupled plasma emission spectrometer.
Cmx itself has some adsorption and reduction effects on heavy metals, but in an unsaturated environment, different concentrations of micro-plastics have a great influence on the same. Under the influence of a certain concentration of micro-plastic, the component proportion of the Desulfosibriosp CMX bacteria EPS is changed while the bacterial activity is maintained, the bacteria are promoted to generate more EPS, and the adsorption reduction effect of the Desulibriosp CMX bacteria unsaturated biomembrane on Cr (VI) is improved.
Compared with the prior art, the invention has the beneficial effects that:
the invention relates to a method for culturing a Desulfovibrio sp.CMX bacteria unsaturated biomembrane under the stress of specific environmental pressure (culture temperature, pH value, osmotic pressure and Cr (VI) concentration) in an unsaturated environment. On the basis, the micro plastic with a certain concentration is introduced into a culture system containing Cr (VI), so that the activity of Desulfovibrio sp CMX bacteria is ensured, more EPS is secreted, the component proportion of EPS is correspondingly changed, and the adsorption reduction effect of the sulfate reducing bacteria unsaturated biomembrane on Cr (VI) is improved to a certain extent. The method provides a new method for repairing Cr (VI) pollution in soil containing micro-plastics by adopting a microbial remediation technology in an actual environment.
Drawings
FIG. 1 shows the growth of the unsaturated biofilm cells of Desulfovibrio sp.
Fig. 2 shows the time-dependent change of cell EPS composition of desalfovibrio sp. (A: colloidal EPS high molecular weight per milligram of biofilm; B: capsule EPS high molecular weight per milligram of biofilm).
Fig. 3 shows the effect of Cr (vi) on the desalfovibrosp. (A: the influence of Cr (VI) on colloidal EPS protein components; B: the influence of Cr (VI) on colloidal EPS polysaccharide components; C: the influence of Cr (VI) on capsular EPS protein components; D: the influence of Cr (VI) on capsular EPS polysaccharide components).
CMX bacteria unsaturated biofilm the adsorption reduction of Cr (VI) and the spatiotemporal distribution of Cr (VI) in the biofilm are shown in FIG. 4. ( A: CMX bacteria have the adsorption and reduction effects on Cr (VI); b: spatial and temporal distribution of Cr (VI) in Desulfovibriosp )
Fig. 5 shows the effect of polystyrene micro-plastic on the desalfovibrosp. (A: the influence of polystyrene microplastics on the colloidal EPS protein component; B: the influence of polystyrene microplastics on the colloidal EPS polysaccharide component; C: the influence of polystyrene microplastics on the capsular EPS protein component; D: the influence of polystyrene microplastics on the capsular EPS polysaccharide component).
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
EXAMPLE 1Desulfovibrio sp. CMX non-saturated biofilm culture and EPS extraction and compositional determination
1. Culture of unsaturated biofilms
The sulfate reducing bacteria (Desulfovibrio sp. CMX, obtained by screening anaerobic sludge from a spring willow sewage treatment plant in Dalian city in this laboratory) selected in this example were cultured in the following specific manner:
(1) Desulfoviro sp.CMX preserved at-80 ℃ with 70% glycerol and 30% LB was incubated with LB medium (10 g/L peptone, 10g/L NaCl,5g/L yeast extract powder adjusted to pH 7.0 with 1mol/L NaOH and 1mol/L HCl) at constant temperature (30 ℃) for 10h with shaking (150 rpm) until the end of the logarithmic phase growth as an inoculum.
(2) Sterilizing the polycarbonate membrane in a sterilizing pot at 121 deg.C for 20min before inoculation, transferring the cooled polycarbonate membrane (diameter 13mm, pore diameter 0.1 μm, thickness 6 μm, whatman, clifton, NJ) to solid inorganic salt culture medium (NaCl 3g/L, K 2 HPO 4 ·3H 2 O 0.66g/L，NH 4 Cl 1g/L，NaSO 4 1g/L, 6.315g/L sodium lactate, 10g/L agar, pH 8.0), inoculating 10 μ L of the inoculum with a sterile pipette to the center of the polycarbonate membrane, sealing the culture medium with parafilm sealing membrane to prevent the dish from drying, and culturing in an inverted manner in an anaerobic incubator at 30 ℃ to obtain Desulfovibrio sp.CMX unsaturated biofilm.
2. Extraction of gel-like and capsule-like EPS
The polycarbonate membrane together with the Desulovibrio sp. CMX unsaturated biofilm was removed from the surface of the solid medium with sterile forceps, suspended in 2mL of sterile 0.9% NaCl solution, centrifuged at 10000g at 4 ℃ for 30min and the supernatant taken as the gel EPS fraction, and the sample was stored at-20 ℃ until analysis. The remaining pellet was resuspended in 2mL of 10mmol/LEDTA in 0.9% NaCl, allowed to stand at room temperature for 2h, and centrifuged at 12000g at 4 ℃ for 30min to give a supernatant, termed "capsule EPS", which was stored at-20 ℃ until analysis.
3. Quantitative analysis of EPS component
The main components of EPS were determined using the following method: the protein content is determined by Lowry method, and bovine serum albumin is used as standard substance; measuring polysaccharide by phenol-sulfuric acid method, and using glucose as standard substance; the composition of EPS is characterized by both polysaccharide and protein.
As can be seen from FIG. 2, the activity of Desulfovibrio sp.CMX cells cultured at pH 8.0 and NaCl concentration 51mmol/L at 30 ℃ was high, while the EPS secretion was high. At 24h, the colloidal EPS and encapsulated EPS secreted per mg biofilm fell to 148.5ug and 392.2ug, respectively, whereas at 96h, the colloidal EPS and encapsulated EPS secreted per mg biofilm fell to 85ug and 206ug, respectively, which is likely because the pre-secreted EPS provided a suitable environment for Desulfovibrio sp.
Example 2 Change of Desulfovibrio sp. CMX unsaturated biofilm EPS under Cr (VI) stress and adsorptive reduction of Cr (VI)
1. Culture of Desulfovibrio sp.CMX unsaturated biomembrane in Cr (VI) -containing environment
The components of the solid inorganic salt culture medium are unchanged. A certain volume of K 2 Cr 2 O 7 Filtering the solution by using a 0.22 mu m filter membrane, adding the filtered solution into a sterilized inorganic salt culture medium to ensure that the concentration of Cr (VI) in the solid inorganic salt culture medium is 15mg/L, shaking up the solution, after the inorganic salt culture medium is cooled and solidified, flatly paving a polycarbonate membrane for growing a Desulfovibrio sp.CMX unsaturated biomembrane on the surface of the solid culture medium containing Cr (VI), wherein the culture temperature is 30 ℃.
2. Determination of Cr adsorption reduction by sulfate reducing bacteria unsaturated biomembrane
The total Cr concentration was measured by inductively coupled plasma emission spectrometer (ICP-OES, AVIO 500) as follows:
and (3) putting the cultured unsaturated biomembrane into a polytetrafluoroethylene digestion tank for digestion, adding 5mL of nitric acid at intervals of 30min, and digesting for 2h at 200 ℃. The digested solution was diluted with 5% HCl to a final volume of 5mL. The samples were subjected to measurement of total Cr concentration on an inductively coupled plasma emission spectrometer (ICP-OES, AVIO 500).
As can be seen from fig. 3, when Cr (VI) is introduced into the above environmental conditions at a certain concentration, the secretion of the Desulfovibrio sp. At 72h, the protein content in the colloidal EPS and the capsular EPS secreted by each mg of the biomembrane was increased from 23ug and 215ug to 28ug and 253ug, respectively, indicating that the biomembrane cultured with Cr secreted more extracellular protein. Also at 72h, the polysaccharide content in both gel-like EPS and capsule-like EPS increased from 57ug and 95ug to 93ug and 114ug, respectively, per mg of biofilm. This also suggests that Cr (VI) promotes the biofilm to secrete more exopolysaccharides.
As can be seen from fig. 5, the Desulfovibrio sp. From 24h to 120h, the adsorption of the biomembrane to Cr is increased, which shows that the activity of the microorganism is high and the EPS secretion capability is stronger; about 120h, the adsorption reduction effect of each mg of the biomembrane on the Cr (VI) reaches the maximum value, which is about 6.5 ug. Relevant researches show that EPS plays an important role in maintaining the stability of microbial activity and can maintain the stable structure of a biomembrane of a repair system, which is probably the reason why the biomembrane secretes more EPS to maintain self activity when being poisoned by external heavy metals, and indirectly proves why Desulovibrio sp.CMX secretes more EPS under the influence of certain concentration of Cr (VI).
Example 3 Change of polystyrene micro-Plastic (PS-MPs) under stress in Desulfovibrio sp.CMX unsaturated biofilm EPS and Effect on Cr (VI) adsorptive reduction
10h activated Desulovibrio sp.CMX was centrifuged at 5000g for 5min at 4 ℃ to separate the bacterial cells from the medium, washed twice with phosphate buffered saline (PBS; pH 7.2) to remove the residual medium, and finally resuspended in 0.9% saline.
A certain amount of polystyrene microsphere stock solution (milky white suspension, 10 g/L) with the particle size of 1 micron is respectively added into the resuspended bacterial solution, so that the concentration of the polystyrene microspheres is 150mg/L. Inoculating 10 μ L of the inoculum containing polystyrene microspheres with different concentrations to the center of polycarbonate membrane by using a sterile pipette, sealing the culture medium with a sealing membrane, and culturing in an anaerobic incubator at 30 deg.C in an inverted manner. Samples were taken at 24h intervals and Desulfovibrio sp. Cmx gum-like, capsule-like EPS was extracted and analyzed as in example 1. And the adsorption reduction amount of Cr by the Desulfovibrio sp.cmx unsaturated biofilm was measured according to the method in example 2.
As can be seen from fig. 4, the polystyrene micro-plastic (PS-MPs) at a specific concentration also has a corresponding effect on the secretion of the Desulfovibrio sp. Under the influence of polystyrene micro plastics (PS-MPs), at 96h, the protein content in the colloidal EPS and the capsule EPS secreted by the biomembrane is reduced from 25ug and 277ug to 21ug and 256ug respectively, and the polysaccharide content in the colloidal EPS and the capsule EPS in the biomembrane is increased from 137ug and 152ug to 153ug and 164ug respectively.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (4)
1. A method for removing Cr (VI) in soil by using a sulfate reducing bacteria unsaturated biomembrane is characterized by comprising the following steps:
(1) Culture of unsaturated biomembrane of sulfur Desulfovibriosp
Inoculating Desulfosibrio sp CMX bacteria stored in a refrigerator at the temperature of-80 ℃ to LB culture medium with the pH value of 7.0 for activation culture for 10 hours, centrifuging activated Desulibrio sp CMX bacteria liquid for 5 minutes at the temperature of 4 ℃ and 5000g, separating the Desulibrio sp CMX bacteria from the culture medium, washing the Desulibrio sp CMX bacteria twice by phosphate buffer solution with the pH value of 7.2 to remove residual culture medium, and then respectively re-suspending the washed Desulibrio sp CMX bacteria in 0.9wt NaCl solution containing 50-150mg/L polystyrene micro plastic PS-MPs to obtain Desulibrio sp CMX bacteria inoculation liquid; transferring the polycarbonate membrane to the surface of a solid culture medium containing Cr (VI) by using sterile forceps, inoculating a Desulfovibrio sp.CMX bacterium inoculation solution to the center of the polycarbonate membrane, and performing inverted culture in an anaerobic box to obtain a sulfur Desulfovibrio sp.CMX bacterium unsaturated biological membrane;
(2) Sampling the cultured Desulovibrio sp.CMX unsaturated biomembrane at intervals of 24h, extracting colloidal and capsular EPS of the Desulovibrio sp.CMX bacteria, analyzing components of the colloidal and capsular EPS, and determining the adsorption reduction amount of Cr (VI) by the sulfur Desulivibrio sp.CMX unsaturated biomembrane.
2. The method for removing Cr (VI) in soil by using the sulfate-reducing bacteria unsaturated biofilm as claimed in claim 1, wherein the Cr (VI) -containing solid culture medium comprises 3g/L NaCl and K 2 HPO 4 ·3H 2 O0.66g/L，NH 4 Cl 1g/L，NaSO 4 1g/L, 6.315g/L of sodium lactate, 15mg/L of Cr (VI) and the pH value of 7.8-8.2.
3. The method for removing Cr (VI) in soil by using the sulfate-reducing bacteria unsaturated biomembrane as claimed in claim 1, wherein the method for extracting colloidal and capsular EPS of Desulfovibriosp. CMX comprises the following steps: resuspending the cultured Desulovibrosp.CMX bacterial non-saturated biomembrane in turn in 0.9wt% NaCl solution, centrifuging at 10000g for 30min, collecting supernatant, and filtering with 0.22 μm cellulose acetate filter membrane to obtain colloidal EPS; then, the remaining precipitated cells were resuspended in a 0.9% by weight NaCl solution containing 10mmol/LEDTA, and after standing at room temperature for 2 hours, centrifuged at 12000g for 30 minutes, and the supernatant was collected and filtered with a 0.22 μm cellulose acetate filter to obtain capsular EPS; the residual thallus is intracellular component.
4. The method for removing Cr (VI) in soil by using the sulfate-reducing bacteria unsaturated biofilm as claimed in claim 1, wherein the content of Cr (VI) adsorbed by the desulfovibrio sp CMX unsaturated biofilm is determined by: respectively putting the Desulfovibrio sp CMX bacteria unsaturated biomembranes cultured in different time periods into a polytetrafluoroethylene digestion tank, digesting for 2 hours at 200 ℃, diluting the digested solution to a certain volume by using ultrapure water, and measuring the total Cr content by using an inductively coupled plasma emission spectrometer.
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