CN114410527B - Broad-spectrum soil heavy metal passivation microbial inoculum and preparation method and application thereof - Google Patents
Broad-spectrum soil heavy metal passivation microbial inoculum and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a broad-spectrum soil heavy metal passivation microbial inoculum, a preparation method and application thereof, which contains Geobacter sulfurreducens HMP-1 and auxiliary materials. The broad-spectrum heavy metal passivating bacterial agent directly forms metal sulfide minerals with heavy metal ions through sulfydryl (-HS) rich in the surface of Geobacter sulfurreducens HMP-1 bacterial body, thereby reducing the toxicity of heavy metals in the environment. The microbial agent can effectively solve the problems of limited heavy metal species, indirect passivation effect and large economic investment of the existing biological microbial agent, has the heavy metal removal rate of more than 70% on soil and rice, reduces the toxicity of broad-spectrum heavy metal under the condition of not affecting the physicochemical property of the soil, and promotes the development of the technology of repairing heavy metal pollution by microorganisms.
Description
Technical Field
The invention belongs to the technical field of pollution treatment engineering, and particularly relates to a broad-spectrum soil heavy metal passivation microbial inoculum, and a preparation method and application thereof.
Background
Heavy metal (Cd, cu, pb, zn, hg, etc.) pollution has been investigated as one of the most prominent inorganic pollutants in farmland soil worldwide. Because rice, wheat and various vegetables have strong absorption capacity to heavy metals, heavy metals in soil are easy to absorb and remain in plants, if the heavy metal exceeding the standard is taken for a long time, serious injury is caused to human bodies, and therefore, the repair of heavy metal pollution of the soil is an important difficult problem to be solved in order to ensure the grain safety and the human health.
At present, in reports and practice of soil heavy metal restoration, the chemical passivating agent can well fix heavy metals in soil. The chemical passivating agent repairing technology is to react with heavy metals in soil by using a chemical passivating agent, so that the damage of the heavy metals to crops is reduced. Although the method can reduce the plant effectiveness of heavy metals to a certain extent, the method has high economic investment and complex operation, and can change the physicochemical property of soil and influence the quality of soil environment. Compared with the traditional chemical passivating agent, the microbial agent can reduce the effectiveness of heavy metals through the actions of biological adsorption, precipitation, transformation, complexation, alkalization and the like, can maintain the soil fertility, and is a repair technology with great potential in the technical field of pollution treatment engineering in recent years.
However, the microbial agents for soil heavy metal remediation in the related art have more defects or limitations. The Chinese patent with application publication number of CN 110713959A discloses a microbial agent for repairing heavy metal pollution, which comprises the following components in parts by weight: 1-2 parts of water producing alcaligenes, 1-2 parts of bacillus subtilis, 90-100 parts of peptone, 15-20 parts of yeast extract, 0.5-1 part of ferric phosphate, 30-40 parts of urea and 0.2-0.5 part of sodium hydroxide. The principle of the microbial inoculum for passivating the heavy metals in soil is as follows: the microorganism secretes urease to hydrolyze urea, so that the pH of soil is improved, and heavy metal ions in the soil form carbonate precipitates, thereby having the effect of passivating heavy metals. However, the pH rise caused by urea hydrolysis belongs to an indirect passivation effect, which not only has the problem of high production cost of the microbial inoculum, but also has the use defect of unstable passivation effect of heavy metals, and is difficult to be used in actual production and use. The Chinese patent with application publication number of CN 07384433A discloses a soil heavy metal passivator, which comprises the following components in parts by weight: 30 parts of peanut shell, 15 parts of pine needles of Korean pine, 10 parts of sepiolite, 10 parts of fly ash, 5 parts of chitin, 3 parts of sodium metasilicate pentahydrate, 0.4 part of sodium tripolyphosphate, 0.1 part of choline chloride, 1.2 parts of microorganism and 0.05 part of quicklime. The passivating agent has small microorganism component ratio, and is mainly used for passivating heavy metals through a series of abiotic reactions such as the exclusive adsorption, surface complexation, ion exchange, coprecipitation and the like of various chemical substances, so that the inherent defects of the traditional chemical passivating agent, such as the influence on the soil environment quality, and the like, can not be overcome. The Chinese patent with application publication number of CN 110951642A discloses a microbial heavy metal passivation microbial inoculum which is prepared from equal amounts of evenly mixed bacillus cereus and rhodococcus ruber fixed by gel microspheres. The principle of the microbial inoculum for passivating heavy metals is as follows: the microorganism reduces iron oxide to ferrous ions, which reduce Gr (VI) to less toxic Gr (III). However, the microbial inoculum also belongs to an indirect passivation effect, is only suitable for repairing chromium pollution, has no efficacy on other heavy metal pollution, and has no universality.
Therefore, the development of the broad-spectrum, efficient and stable heavy metal passivation microbial inoculum has great significance for future agriculture and environmental management.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a broad-spectrum soil heavy metal passivation microbial inoculum, and a preparation method and application thereof. The broad-spectrum heavy metal passivation microbial inoculum contains Geobacter sulfurreducens HMP-1 and auxiliary materials, and is directly formed into metal sulfide minerals with heavy metal ions through sulfydryl (-HS) rich in Geobacter sulfurreducens HMP-1 microbial surfaces, so that the toxicity of heavy metals in the environment is reduced. The microbial agent can effectively solve the problems of limited heavy metal species, indirect passivation and large economic investment of the conventional biological microbial agent, thereby realizing high-efficiency reduction of the toxicity of broad-spectrum heavy metals, achieving the removal rate of more than 70 percent on the heavy metals in soil and rice, and promoting the development of the technology for repairing heavy metal pollution by microorganisms while realizing the reduction of the toxicity of broad-spectrum heavy metals under the condition of not affecting the physicochemical properties of the soil.
In a first aspect of the invention, a broad spectrum heavy metal passivating microbial inoculant is provided.
According to a first aspect of the present invention, in some embodiments of the present invention, the broad-spectrum heavy metal-inactivating bacteria agent comprises Geobacter sulfurreducens HMP-1 and an adjuvant.
According to a first aspect of the invention, in some embodiments of the invention, the Geobacter sulfurreducens HMP-1 strain is geobacillus thioreductase HMP-1, classified under the name Geobacter sulfurreducens, deposited with the collection of microorganism strains in the cantonese province, accession number: GDMCC No.62189.
In some preferred embodiments of the invention, the Geobacter sulfurreducens HMP-1 strain is enriched in sulfhydryl groups (-HS), and the heavy metal ions are bound by the-HS to form metal sulfide precipitate rapidly, so that the heavy metal ions are removed without attaching any substrate or highly enriched heavy metal plants. Meanwhile, geobacter sulfurreducens HMP-1 has strong heavy metal tolerance and can be suitable for the treatment of high-concentration metal pollution environments.
In some preferred embodiments of the present invention, the construction method of the heavy metal passivated geobacillus engineering strain comprises the following steps:
(1) Inserting a cysteine gene into the empty vector pUC19 to obtain a recombinant plasmid pUC-HMP;
(2) The recombinant plasmid pUC-HMP is transformed into competent Geobacter sulfurreducens to obtain the recombinant plasmid.
According to a second aspect of the present invention, in some embodiments of the present invention, the recombinant plasmid pUC-HMP further comprises a marker gene, a promoter gene and a sequence upstream and downstream of the target insertion site.
The sequence upstream and downstream of the target insertion site is used for achieving gene insertion at a specific location and for subsequent amplification detection.
In some preferred embodiments of the invention, the marker genes include a selection gene and a reporter gene.
In some more preferred embodiments of the invention, the marker gene is a selection gene.
In some embodiments of the invention, the selection gene is a gentamicin resistance gene.
Of course, the person skilled in the art can reasonably select other marker genes to replace gentamicin resistance genes according to actual use requirements, so as to achieve the screening purpose.
In some embodiments of the invention, the promoter is a PompJ strong promoter gene.
Of course, the person skilled in the art can reasonably select other promoter genes to replace the PompJ strong promoter gene according to the actual use requirement, so as to achieve the same purpose.
In some embodiments of the invention, the Geobacter sulfurreducens is Geobacter sulfurreducens PCA.
In some preferred embodiments of the invention, the nucleotide sequence of the recombinant plasmid pUC-HMP is shown in SEQ ID NO. 1.
In some preferred embodiments of the present invention, the construction method of the heavy metal passivated geobacillus engineering strain specifically comprises: the empty vector pUC19 is taken as a vector plasmid, and is mixed with 5 XIn-Fusion HD Enzyme Premix, cysteine gene, gentamicin resistance gene, pompJ strong promoter gene and DNA sequences (the length of the upstream and downstream DNA sequences is 500 bp) at the upstream and downstream insertion sites of a target, and then the mixture is reacted for 15min at 50 ℃ In a PCR instrument, so that the recombinant plasmid pUC-HMP is obtained. pUC-HMP was linearized using the restriction endonuclease ScaI. Linearized pUC-HMP was transformed into Geobacter sulfurreducens PCA competent cells. And (3) screening by using an NBAF solid plate containing 50g/mL gentamicin.
According to the first aspect of the present invention, in some preferred embodiments of the present invention, the mass ratio of Geobacter sulfurreducens HMP-1 to the auxiliary material in the broad-spectrum heavy metal-inactivating bacteria agent is 1: (30-45).
In some more preferred embodiments of the present invention, the mass ratio of Geobacter sulfurreducens HMP-1 to the auxiliary materials in the broad-spectrum heavy metal-inactivating bacteria agent is 1: (35-40).
According to a first aspect of the present invention, in some preferred embodiments of the present invention, the adjunct comprises rice straw charcoal, bran coat and bran coat.
In some preferred embodiments of the present invention, the adjunct is rice straw biochar, chaff, and bran.
The preparation method of the rice straw biochar comprises the following steps: and (3) placing the dried rice straw in a muffle furnace, and carrying out anaerobic firing at 200 ℃ for 3 hours to obtain the rice straw.
In some preferred embodiments of the present invention, the supplementary material comprises 5 to 20 parts of rice straw biochar, 25 to 40 parts of bran and 15 to 30 parts of bran according to parts by mass.
In some preferred embodiments of the invention, the auxiliary materials are 10 to 15 parts of rice straw biochar, 20 to 35 parts of bran and 20 to 25 parts of bran according to parts by mass.
According to the first aspect of the present invention, in some preferred embodiments of the present invention, the viable count of Geobacter sulfurreducens HMP-1 in the broad-spectrum heavy metal-inactivated microbial agent is 2×10 or more 8 Each/g.
In some more preferred embodiments of the present invention, the number of viable bacteria of Geobacter sulfurreducens HMP-1 in the broad-spectrum heavy metal-inactivated microbial inoculum is 2.9X10 or more 8 Each/g.
In a second aspect, the invention provides a preparation method of the broad-spectrum heavy metal passivation microbial inoculum according to the first aspect, which comprises the following steps:
inoculating Geobacter sulfurreducens HMP-1 bacterial liquid into a culture medium according to the volume ratio of 2-3%, performing anaerobic fermentation at 28-32 ℃ for 6-7 days, drying into powder, and then performing anaerobic fermentation according to the following steps of 1: mixing the components (30-45) with auxiliary materials uniformly.
According to a second aspect of the present invention, in some preferred embodiments of the present invention, the inoculum size of the Geobacter sulfurreducens HMP-1 bacterial liquid is 3% (by volume).
According to a second aspect of the invention, in some preferred embodiments of the invention, the Geobacter sulfurreducens HMP-1 fermentation temperature is 30℃and the fermentation time is 7 days.
According to a second aspect of the invention, in some preferred embodiments of the invention, the mass ratio of Geobacter sulfurreducens HMP-1 to adjuvant is 1: (35-40).
According to a second aspect of the present invention, in some preferred embodiments of the present invention, the adjunct comprises rice straw charcoal, bran coat and bran coat.
In some preferred embodiments of the present invention, the adjunct is rice straw biochar, chaff, and bran.
The preparation method of the rice straw biochar comprises the following steps: and (3) placing the dried rice straw in a muffle furnace, and carrying out anaerobic firing at 200 ℃ for 3 hours to obtain the rice straw.
In some preferred embodiments of the present invention, the supplementary material comprises 5 to 20 parts of rice straw biochar, 25 to 40 parts of bran and 15 to 30 parts of bran according to parts by mass.
In some preferred embodiments of the invention, the auxiliary materials are 10 to 15 parts of rice straw biochar, 20 to 35 parts of bran and 20 to 25 parts of bran according to parts by mass.
According to the second aspect of the present invention, in some preferred embodiments of the present invention, the number of viable Geobacter sulfurreducens HMP-1 bacteria in the broad-spectrum heavy metal-inactivated microbial inoculum prepared by the method is 2×10 or more 8 Each/g.
In some more preferred embodiments of the present invention, the number of viable bacteria of Geobacter sulfurreducens HMP-1 in the broad-spectrum heavy metal-inactivated microbial inoculum prepared by the method is required to be 2.9X10 or more 8 Each/g.
According to a second aspect of the invention, in some preferred embodiments of the invention, the preparation method is specifically:
(1) Activating the strain: geobacter sulfurreducens HMP-1 was inoculated into NBAF liquid medium for activation.
(2) Preparing bacterial liquid: inoculating the activated Geobacter sulfurreducens HMP-1 bacterial liquid into a new NBAF liquid culture medium according to an inoculum size of 3% (v/v), and carrying out anaerobic fermentation at 30 ℃ for 7 days;
(3) Preparing a solid microbial inoculum: drying the fermented bacterial liquid at a low temperature in vacuum to obtain Geobacter sulfurreducens HMP-1 bacterial powder, and then carrying out mass 1: and (35-40) fully and uniformly mixing Geobacter sulfurreducens HMP-1 bacterial powder, rice straw biochar, chaff and bran according to the proportion.
The third aspect of the invention provides the broad-spectrum heavy metal passivation microbial inoculum according to the first aspect of the invention or the application of the broad-spectrum heavy metal passivation microbial inoculum prepared by the preparation method according to the second aspect of the invention in soil heavy metal passivation.
In the invention, the inventor proves through experiments that the content of various effective state heavy metals in the soil can be greatly influenced by the use and non-use of the broad-spectrum soil heavy metal passivation microbial inoculum disclosed by the invention, and the content of the effective state heavy metals in the soil after the use of the broad-spectrum soil heavy metal passivation microbial inoculum disclosed by the invention is reduced by at least 70% compared with the soil without the use of the broad-spectrum soil heavy metal passivation microbial inoculum, namely, the removal rate of the broad-spectrum soil heavy metal passivation microbial inoculum disclosed by the invention on various heavy metals in the soil is higher than 70%, so that the broad-spectrum soil heavy metal passivation microbial inoculum disclosed by the invention can be suitable for passivation of broad-spectrum heavy metals.
In a fourth aspect, the invention provides an application of the broad-spectrum heavy metal passivation microbial inoculum prepared by the first aspect or the preparation method of the second aspect in preparation of a crop heavy metal absorption resistant preparation.
According to a fourth aspect of the invention, in some preferred embodiments of the invention, the crop plants include rice, wheat and vegetables of various types.
In the invention, the inventor finds that the total effective state heavy metal content of the paddy rice treated by the broad-spectrum soil heavy metal passivation microbial inoculum is reduced by more than 70% by comparing the total effective state heavy metal content of the paddy rice treated by the broad-spectrum soil heavy metal passivation microbial inoculum with the total effective state heavy metal content of the paddy rice treated by the broad-spectrum soil heavy metal passivation microbial inoculum, namely the broad-spectrum soil heavy metal passivation microbial inoculum has remarkable inhibition effect on the effective accumulation amount of various heavy metals in crops, which indicates that the broad-spectrum soil heavy metal passivation microbial inoculum can be suitable for reducing the absorption of various heavy metals by general crops.
The beneficial effects of the invention are as follows:
1. the broad-spectrum soil heavy metal passivation microbial inoculum disclosed by the invention contains an effective amount of Geobacter sulfurreducens HMP-1, so that the metal passivation microbial inoculum can directly react with broad-spectrum heavy metals to form metal sulfides to achieve the effect of passivating the heavy metals, and the Geobacter sulfurreducens HMP-1 has better tolerance to a high-concentration metal environment, so that the application range is wide.
2. The broad-spectrum soil heavy metal passivation microbial inoculum provided by the invention has the advantages that the materials are low in price and easy to obtain, and the physical and chemical properties of soil cannot be influenced after application, so that the broad-spectrum soil heavy metal passivation microbial inoculum can be used for effectively removing heavy metals, simultaneously, the planting of crops is not damaged, and better soil fertility is maintained.
3. The broad-spectrum soil heavy metal passivation microbial inoculum has great influence on the content of effective heavy metal in soil, and compared with the soil without the broad-spectrum soil heavy metal passivation microbial inoculum, the broad-spectrum soil heavy metal passivation microbial inoculum has at least 70 percent lower effect and has wider heavy metal passivation effect compared with the traditional passivation microbial inoculum.
4. The broad-spectrum soil heavy metal passivating microbial inoculum disclosed by the invention can effectively reduce the absorption of common crops to various heavy metals, so that the generation probability of heavy metal polluted crops is reduced, and the food safety problem is avoided.
Drawings
FIG. 1 is a plasmid map of a recombinant plasmid pUC-HMP in an example of the present invention.
FIG. 2 is a preparation flow of a broad-spectrum soil heavy metal passivating microbial inoculum in an embodiment of the invention.
FIG. 3 is a scanning electron microscope image of the broad-spectrum soil heavy metal passivating agent combined with the active state Cd.
Fig. 4 is a scanning electron microscope image of the broad-spectrum soil heavy metal passivating agent combined with the effective Cu.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to the following specific embodiments. It should be understood that the detailed description is presented herein for purposes of illustration only and is not intended to limit the invention.
The experimental materials and reagents used, unless otherwise specified, are those conventionally available commercially.
Experimental materials
In the following examples, the microorganism used was Geobacillus thioreductase HMP-1 (Geobacter sulfurreducens HMP-1, classified under the name Geobacter sulfurreducens) deposited at the Guangdong province microorganism strain collection (GDMCC for short), address: building 5 of Guangzhou city, first, china, no. 100, college of China, no. 59, with a preservation date of 2022, 1 month and 5 days, and a preservation number: GDMCC No.62189. The bacteria are characterized in that: the bacillus is a rod-shaped gram-negative bacterium, the most suitable growth temperature is 30 ℃, and the anaerobic and obligate heterotrophic bacterium has a suitable pH of about 7, and is characterized in that the surface of the bacterium has rich sulfhydryl (-HS), can form metal sulfide minerals with heavy metal ions, and has strong heavy metal tolerance.
The Geobacter sulfurreducens HMP-1 is an engineering strain of geobacillus for passivating heavy metals, which is artificially constructed, and the construction method comprises the following steps:
(1) Construction of pUC-HMP:
the recombinant plasmid pUC-HMP was obtained by mixing pUC19 (TaKaRa In Japan) as a vector plasmid with 5 XIn-Fusion HD Enzyme Premix (TaKaRa In Japan), a cysteine gene, a gentamicin resistance gene, a PompJ strong promoter gene and DNA sequences upstream and downstream of the target insertion site (both of which were 500bp In length) and then reacting at 50℃for 15 minutes In a PCR instrument.
Wherein, the cysteine gene, the gentamicin resistance gene, the PompJ strong promoter gene and the DNA sequence at the upstream and downstream of the target insertion site are synthesized by biological companies.
The nucleotide sequence of the constructed recombinant plasmid pUC-HMP is shown as SEQ ID NO. 1, and the plasmid map is shown as figure 1.
(2) pUC-HMP was linearized with restriction enzyme ScaI, and then the linearized pUC-HMP was taken in a total amount of 10-15. Mu.g and concentrated to 10. Mu.L.
The concentration method comprises the following steps: 1) 15 μg of the linearized pUC-HMP plasmid is taken in a 1.5mL centrifuge tube, 10 μl of 3M sodium acetate (pH=5.2) is added, and the mixture is mixed by shaking; 2) Adding 250 μL of glacial ethanol (pre-cooling at 4deg.C), shaking, mixing, standing at-20deg.C for at least 30min, and centrifuging at 15000g at 4deg.C for 30min; 3) Discarding the supernatant, adding 1mL 70% glacial ethanol (pre-cooling at 4deg.C), centrifuging at 15000g at 4deg.C for 30min; 4) Discarding the supernatant, adding 500 μl of glacial ethanol (pre-cooling at 4deg.C), centrifuging at 15000g at 4deg.C for 30min; 5) The supernatant was discarded, left at room temperature for 10min, ethanol was evaporated, 10. Mu.L of sterile water was added to dissolve the plasmid, and the concentration was determined and deemed optimal when the concentration was greater than 1. Mu.g/. Mu.L.
(3) mu.L of the concentrated linearized pUC-HMP was transformed into Geobacter sulfurreducens PCA (purchased from American type culture Collection, strain ATCC-51573) competent cells by the transient electric shock method using an electrotransfer apparatus (Micropulser, bio-Rad, USA) and cultured in NBAF liquid medium for 16-18 hours (in this step, 1% final concentration of yeast was added in advance to the NBAF medium). After 18h of cultivation, the bacterial liquid was concentrated to 2mL in an anaerobic tank. 200. Mu.L of the mixture was plated on NBAF solid plates containing 50g/mL gentamicin, and cultured anaerobically. After the single colony grows out of the flat plate, a single colony is picked up, and is verified, and Geobacter sulfurreducens HMP-1 is obtained after verification is correct.
The conversion parameters of the instantaneous electric shock method are as follows: the shock parameter was 1.47Kv/cm for 5ms.
The NBAF media formulations for the strains cultured in this example are shown in Table 1.
TABLE 1NBAF media formulation
The pH of the NBAF medium was adjusted to 6.5-7.0.
The NB salts in the NBAF medium are specifically: 25g NH/liter 100 XNB salt solution 4 Cl，42g KH 2 PO 4 ，22g K 2 HPO 4 ，38g KCl，36g NaCl。
The NB minerals in NBAF medium were specifically: each liter of NB mineral solution contains 2.14g nitrilotriacetic acid (NTA), 0.1g MnCl 2 ·4H 2 O，0.3g FeSO 4 ·7H 2 O，0.17g CoCl 2 ·6H 2 O，0.2g ZnSO 4 ·7H 2 O，0.03g CuCl 2 ·2H 2 O，0.005g AlK(SO 4 ) 2 ·2H 2 O，0.005g H 3 BO 3 ，0.09g Na 2 MnO 4 ·2H 2 O，0.11g NiSO 4 ·6H 2 O，0.02g Na 2 WO 4 ·2H 2 O。
The DL vitamins in the NBAF medium are specifically: the DL vitamin solution contains 0.005g vitamin B5,0.0001g vitamin B12,0.005g p-aminobenzoic acid, 0.005g alpha-lipoic acid, 0.005g nicotinic acid, 0.005g vitamin B1,0.005g riboflavin, 0.01g vitamin B6 and 0.002g folic acid.
The prepared NBAF medium was treated with a gas mixture (CO) 2 ：N 2 =80%/20%) was aerated to remove oxygen from the medium.
The solid NBAF is prepared by adding 1.5% agar into the NBAF liquid culture medium, and performing aeration treatment to remove oxygen in the culture medium.
The preparation method of the Geobacter sulfurreducens PCA competent cells comprises the following steps: geobacter sulfurreducens PCA cultured to logarithmic phase was centrifuged (4500×g 12 min) after precooling at 4℃and the cells were collected, and washed twice (4500×g 12 min) with 4℃precooled electrotransfer buffer shaking the tube clockwise, and finally the cells were resuspended with 1mL electrotransfer solution and transferred to a 2mL anaerobic centrifuge tube, centrifuged at 8000×g for 5min, the supernatant was discarded, 20. Mu.L of electrotransfer solution was added to resuspend the cells, 40. Mu.L was aspirated and placed in a 2mL new anaerobic centrifuge tube, and used to transform linearized plasmids, all under anaerobic conditions.
The formula of the electrotransport buffer solution is as follows: final concentration of 175mM sucrose, 1mM MgCl 2 And 1mM Heps (ph=7).
The preparation method of the rice straw biochar in the following embodiment comprises the following steps: and (3) placing the dried rice straw in a muffle furnace, and carrying out anaerobic firing at 200 ℃ for 3 hours to obtain the rice straw.
Example 1A broad-spectrum soil heavy metal passivating microbial agent
In the embodiment, the preparation method of the broad-spectrum soil heavy metal passivation microbial inoculum comprises the following steps:
(1) Activating the strain: the above Geobacter sulfurreducens HMP-1 was inoculated into the above NBAF liquid medium for activation.
(2) Preparing bacterial liquid: inoculating the activated Geobacter sulfurreducens HMP-1 bacterial liquid into a new NBAF liquid culture medium according to an inoculum size of 3% (v/v), and carrying out anaerobic fermentation at 30 ℃ for 7 days;
(3) Preparing a solid microbial inoculum: drying the fermented bacterial liquid at a low temperature in vacuum to obtain Geobacter sulfurreducens HMP-1 bacterial powder, and then carrying out mass 1:35, fully and uniformly mixing the Geobacter sulfurreducens HMP-1 bacterial powder and auxiliary materials (ensuring that the number of viable bacteria in the final product meets the standard of GB 20287-2006).
Wherein, the composition of the auxiliary materials of the microbial inoculum is specifically as follows: 15 parts of rice straw charcoal, 30 parts of bran coat and 20 parts of bran coat by weight.
The preparation flow is shown in figure 2.
The method for detecting the viable count of Geobacter sulfurreducens HMP-1 in the prepared broad-spectrum soil heavy metal passivation microbial agent refers to national standard GB20287-2006 microbial agent.
The result shows that the number of Geobacter sulfurreducens HMP-1 viable bacteria in the broad-spectrum soil heavy metal passivation microbial inoculum in the embodiment is 3.6X10 8 The number of the active bacteria per gram accords with the regulations in the national standard GB20287-2006 microbial inoculum (the number of the active bacteria is required to be more than or equal to 2 multiplied by 10) 8 And/g).
Example 2A broad-spectrum soil heavy metal passivating microbial agent
In the embodiment, the preparation method of the broad-spectrum soil heavy metal passivation microbial inoculum comprises the following steps:
(1) Activating the strain: the above Geobacter sulfurreducens HMP-1 was inoculated into the above NBAF liquid medium for activation.
(2) Preparing bacterial liquid: inoculating the activated Geobacter sulfurreducens HMP-1 bacterial liquid into a new NBAF liquid culture medium according to an inoculum size of 3% (v/v), and carrying out anaerobic fermentation at 30 ℃ for 7 days;
(3) Preparing a solid microbial inoculum: drying the fermented bacterial liquid at a low temperature in vacuum to obtain Geobacter sulfurreducens HMP-1 bacterial powder, and then carrying out mass 1:40, and fully and uniformly mixing the Geobacter sulfurreducens HMP-1 bacterial powder and auxiliary materials (ensuring that the number of viable bacteria in the final product meets the standard of GB 20287-2006).
Wherein, the composition of the auxiliary materials of the microbial inoculum is specifically as follows: 10 parts of rice straw charcoal, 35 parts of bran coat and 25 parts of bran coat by weight.
The method for detecting the viable count of Geobacter sulfurreducens HMP-1 in the prepared broad-spectrum soil heavy metal passivation microbial agent refers to national standard GB20287-2006 microbial agent.
The result shows that the number of Geobacter sulfurreducens HMP-1 viable bacteria in the broad-spectrum soil heavy metal passivation microbial inoculum in the embodiment is 2.9X10 8 The number of the active bacteria per gram accords with the regulations in the national standard GB20287-2006 microbial inoculum (the number of the active bacteria is required to be more than or equal to 2 multiplied by 10) 8 And/g).
Practical use effect of broad-spectrum soil heavy metal passivation microbial inoculum
(1) Application of broad-spectrum soil heavy metal passivation microbial inoculum in removing soil heavy metal residues:
in this example, the soil sample is from a paddy field in Fuzhou, fujian.
Sample treatment: and naturally air-drying and grinding the collected soil sample, and sieving the ground soil sample with a 2mm sieve for later use.
The specific experimental steps are as follows:
the broad-spectrum soil heavy metal passivating microbial inoculum in example 1 was taken and mixed with a soil sample according to an addition amount of 3% (mass ratio), and a microcosm experiment (model ecological system, experimental method reference to related laboratory manual in the field) was performed, and a soil sample without the broad-spectrum soil heavy metal passivating microbial inoculum was used as a control. The test period was 30 days.
And after the test is finished, measuring the content of the heavy metal in the soil effective state.
The content of each heavy metal is measured by inductively coupled plasma mass spectrometry (ICP-MS), and specific test methods are performed by referring to related laboratory manuals in the field.
The results are shown in Table 2.
TABLE 2 removal of effective State heavy metals from soil after 30 days of microcosm experiment
| Experimental grouping | Cd removal rate | Pb removal Rate | Hg removal Rate | Zn removal rate | Cu removal rate |
| Non-passivation microbial inoculum | 0 | 0 | 0 | 0 | 0 |
| 3% of passivating microbial inoculum | 72％ | 77％ | 89％ | 76％ | 80％ |
It can be found that the use and non-use of the broad-spectrum soil heavy metal passivation microbial inoculum in example 1 brings great influence to the content of various effective heavy metals in the soil, and the content of the effective heavy metals in the soil after the use of the broad-spectrum soil heavy metal passivation microbial inoculum in example 1 is reduced by at least 70% compared with the soil without the use of the broad-spectrum soil heavy metal passivation microbial inoculum in example 1, namely, the removal rate of the broad-spectrum soil heavy metal passivation microbial inoculum in example 1 to various heavy metals in the soil is higher than 70%, which indicates that the broad-spectrum soil heavy metal passivation microbial inoculum in example 1 can be suitable for passivation of broad-spectrum heavy metals.
To further verify the effective heavy metal removal effect of the broad-spectrum soil heavy metal-passivating microbial agents of example 1, the inventors respectively put an equal amount of the broad-spectrum soil heavy metal-passivating microbial agents into the bacteria containing Cd 2+ And Cu 2+ Incubation was performed in NBAF liquid medium (20 mg/L concentration) and scanning was performed using a Scanning Electron Microscope (SEM). As a control, a broad-spectrum soil heavy metal-inactivating bacteria group was not used.
The results are shown in FIGS. 3 to 4.
As a result, it was found that Cd was able to be removed from the soil by using a broad-spectrum soil heavy metal-inactivating agent 2+ And Cu 2+ The particles are relatively concentrated and distributed on the surface of Geobacter sulfurreducens HMP-1 (FIGS. 3-4, cd) 2+ And Cu 2+ Binding to-HS on the surface of Geobacter sulfurreducens HMP-1 to form visible particles (e.g., cdS, cuS) precipitated and attached to Geobacter sulfurreducens HMP-1, whereas no particles were observed in the medium without the broad-spectrum soil heavy metal-inactivating agent, indicating Cd 2+ 、Cu 2+ No active state conversion process occurs.
(2) Application of broad-spectrum soil heavy metal passivation microbial inoculum in reducing crop heavy metal accumulation:
in this example, the soil sample was from a heavy metal test field from the university of farm and forestry, fujian.
The specific experimental steps are as follows:
the broad-spectrum soil heavy metal passivation microbial inoculum in the example 2 is taken and mixed with nitrogen, phosphorus and potassium fertilizers according to the addition amount of 2 percent (mass ratio) to be applied to a test field. And (5) rice transplanting is carried out in the test field. And (5) taking out the rice in the test field 50 days after transplanting, and detecting the content of the effective heavy metal. Rice in a test field without using the broad-spectrum soil heavy metal inactivating agent of example 2 was used as a control.
The quantitative detection of the content of the effective heavy metal mainly detects Cd, pb, hg, zn, cu accumulated in the rice.
The detection standards and methods refer to the determination of cadmium in food safety national standard food (GB/T5009.15-2014), the determination of lead in food safety national standard food (GB/5009.12-2017), the determination of total mercury and organic arsenic in food safety national standard food (GB/5009.17-2014), the determination of zinc in food safety national standard food (GB/5009.14-2017) and the determination of copper in food safety national standard food (GB/5009.13-2017) respectively.
The results are shown in Table 3.
| Experimental grouping | Cd removal rate | Pb removal Rate | Hg removal Rate | Zn removal rate | Cu removal rate |
| Non-passivation microbial inoculum | 0 | 0 | 0 | 0 | 0 |
| 2% of passivating microbial inoculum | 75％ | 86％ | 90％ | 83％ | 81％ |
As a result, the total effective state heavy metal content of the rice treated by the broad-spectrum soil heavy metal passivation microbial inoculum in the embodiment 2 is reduced by more than 70% by comparing the total effective state heavy metal content of the rice treated by the broad-spectrum soil heavy metal passivation microbial inoculum in the embodiment 2 with the total effective state heavy metal content of the rice treated by the broad-spectrum soil heavy metal passivation microbial inoculum in the embodiment 2, namely the effective accumulation amount of various heavy metals in crops is obviously inhibited by the broad-spectrum soil heavy metal passivation microbial inoculum in the embodiment 2, so that the broad-spectrum soil heavy metal passivation microbial inoculum in the embodiment of the invention can be suitable for reducing the absorption of various heavy metals by general crops.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A broad-spectrum heavy metal passivating bacterial agent is characterized by comprisingGeobacter sulfurreducensHMP-1 and auxiliary materials; the saidGeobacter sulfurreducensHMP-1 is Geobacillus thioreductase HMP-1, and is classified and named asGeobacter sulfurreducensDeposited in the collection of microorganism strains of Guangdong province, accession number: GDMCC No.62189.
2. The broad-spectrum heavy metal passivating bacterial agent according to claim 1, wherein in the broad-spectrum heavy metal passivating bacterial agent,Geobacter sulfurreducensthe mass ratio of HMP-1 to auxiliary materials is 1: (30-45).
3. The broad-spectrum heavy metal passivating microbial inoculum according to claim 1, wherein the auxiliary materials comprise rice straw biochar, chaff and bran.
4. The broad-spectrum heavy metal passivating microbial inoculum according to claim 1, wherein the auxiliary materials comprise, by mass, 5-20 parts of rice straw biochar, 25-40 parts of bran and 15-30 parts of bran.
5. The broad-spectrum heavy metal-inactivating bacteria agent according to claim 1, wherein in the broad-spectrum heavy metal-inactivating bacteria agentGeobacter sulfurreducensThe number of viable bacteria of HMP-1 is 2×10 or more 8 Each/g.
6. The broad-spectrum heavy metal-inactivating bacteria agent according to claim 1, wherein in the broad-spectrum heavy metal-inactivating bacteria agentGeobacter sulfurreducensThe number of viable bacteria of HMP-1 is 2.9X10 or more 8 Each/g.
7. The preparation method of the broad-spectrum heavy metal passivation microbial inoculum according to any one of claims 1-6, comprising the following steps:
will beGeobacter sulfurreducensThe HMP-1 bacterial liquid is inoculated into a culture medium according to the volume ratio of 2-3%, anaerobic fermentation is carried out for 6-7 days at the temperature of 28-32 ℃, and after the bacterial liquid is dried into powder, the bacterial liquid is subjected to anaerobic fermentation according to the following steps of 1: mixing the mass ratio of (30-45) with auxiliary materials uniformly to obtain the compound;
the saidGeobacter sulfurreducensHMP-1 is Geobacillus thioreductase HMP-1, and is classified and named asGeobacter sulfurreducensDeposited in the collection of microorganism strains of Guangdong province, accession number: GDMCC No.62189.
8. The broad-spectrum heavy metal passivation microbial inoculum according to any one of claims 1-6 or the application of the broad-spectrum heavy metal passivation microbial inoculum prepared by claim 7 in soil heavy metal passivation.
9. The broad-spectrum heavy metal passivating microbial inoculum according to any one of claims 1-6 or the broad-spectrum heavy metal passivating microbial inoculum prepared by claim 7, and application thereof in preparing crop heavy metal absorption resistant preparations.
10. The use according to claim 9, wherein the crop plants include rice, wheat and vegetables of various types.
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