Rhodococcus-containing bioremediation fungicide and application thereof

Abstract

The invention relates to the technical field of application of bioremediation microbial agents to environmental remediation, and particularly provides a bioremediation microbial agent containing rhodococcus and application of the bioremediation microbial agent. The bioremediation microbial agent comprises rhodococcus fermentation liquor and a stabilizer. The bioremediation microbial agent is used for degrading cyanide, can be particularly used for degrading cyanide in sewage and soil containing various cyanide and / or high-concentration cyanide, has the degradation efficiency obviously higher than that of a traditional chemical remediation agent, does not cause secondary environmental pollution, is more environment-friendly and has stable performance.

Description

Technical Field

[0001] The present application relates to the technical field of using bioremediation agents for environmental remediation, and further to a bioremediation agent containing Rhodococcus and its application. Background Art

[0002] In the last century, the rapid development of chemical synthesis has brought about environmental pollution caused by synthetic products, which has become one of the urgent problems to be solved worldwide. This includes the discharge and leakage of industrial wastewater during industrial synthesis. Cyanide with cyanide (CN) in industrial wastewater is a highly toxic product. The national comprehensive sewage discharge standard (GB 8978-1996) stipulates that the discharge standard of total cyanide compounds is 0.300 mg / L. Cyanide, COD, etc. in the wastewater discharge process will diffuse and enrich in the soil and groundwater in large quantities. Therefore, it is very necessary to develop an environmentally friendly and efficient bioremediation agent.

[0003] Common chemical degradation agents include water-soluble molecular polymers, ozone, activated carbon, etc. Most chemical degradation agents do not fundamentally remove pollutants when degrading cyanide and are prone to secondary pollution. However, there are few reports on bioremediation agents, but among many environmental remediation methods, bioremediation agents are undoubtedly a low-cost, side-effect-free and effective method. Most Rhodococci are screened from contaminated soil or water bodies, so they can achieve the degradation of pollutant compounds. In addition, Rhodococci have many advantages, such as: they can adapt to a variety of extreme environments, are not limited by oxygen and nutrient deficiencies, and most importantly, genetically modified Rhodococci can efficiently express a variety of enzyme systems, such as hydratase, hydrolase, etc. Rhodococci contain a hydrophobic surface that can fully contact with organic matter, which can improve the degradation efficiency of cyanide.

[0004] However, existing biodegradation methods can only degrade cyanide in wastewater at low concentrations, and there is no precedent for using Rhodococcus for the remediation of cyanide-containing environments. Summary of the invention

[0005] In view of the difficulties and problems faced by the prior art, the present application provides a bioremediation agent containing Rhodococcus and its application. After artificial modification, the Rhodococcus in the biological agent cooperates with a stabilizer to efficiently degrade a variety of cyanides of different concentrations. In addition, based on the obvious advantages of the biological agent, it is successfully applied to the degradation of cyanide in complex cyanide-containing wastewater and cyanide-containing soil. The bioremediation agent is environmentally friendly, efficient and stable.

[0006] This application involves the following contents:

[0007] 1. A bioremediation agent containing Rhodococcus, wherein the bioremediation agent comprises Rhodococcus fermentation broth and a stabilizer.

[0008] 2. According to the bioremediation agent described in item 1, the stabilizer is selected from any one or more of polyols, polyaldehydes and polymers.

[0009] 3. According to the bioremediation agent of item 2, the polyol is selected from any one or more of glycerol, sorbitol and mannitol;

[0010] The polymer is selected from any one or more of polyethylene glycol and polyethylene glycol lactone;

[0011] The polyaldehyde is selected from any one or more of glutaraldehyde and glyoxal.

[0012] 4. The bioremediation agent according to any one of items 2-3, wherein the stabilizer is selected from any one or more of glycerol, polyethylene glycol and glutaraldehyde;

[0013] Preferably, the stabilizer is a combination of glycerol, polyethylene glycol and glutaraldehyde;

[0014] More preferably, the stabilizer is prepared from 20-35wt% glycerol, 0.03-0.10wt% glutaraldehyde, and 4-10M polyethylene glycol.

[0015] 5. According to the bioremediation bacterial agent of item 1, the mass ratio of the Rhodococcus fermentation broth to the stabilizer is (1-90):(10-99);

[0016] Preferably, the OD600 of the Rhodococcus fermentation broth is higher than 0.2;

[0017] Preferably, the mass ratio of the Rhodococcus fermentation broth to the stabilizer is (10-50):(10-90);

[0018] More preferably, the mass ratio of the Rhodococcus fermentation broth to the stabilizer is (30-50):(50-70).

[0019] 6. The bioremediation agent according to any one of items 1 to 5, wherein the Rhodococcus in the Rhodococcus fermentation broth is obtained by genetic recombination; an exogenous recombinant plasmid containing a nitrile metabolic enzyme-related gene sequence is introduced into the wild-type Rhodococcus ruber to achieve the recombinant construction of the Rhodococcus;

[0020] Preferably, the recombinantly constructed Rhodococcus expresses nitrile metabolic enzyme-related enzymes and mutants thereof;

[0021] More preferably, the recombinant Rhodococcus expresses a protein having any one or more amino acid sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 5;

[0022] Further preferably, the recombinant Rhodococcus expresses a protein having any one or more amino acid sequences as shown in SEQ ID NO:3 to SEQ ID NO:5.

[0023] 7. The bioremediation agent according to any one of items 1 to 6, wherein the Rhodococcus has any one or more of the following enzyme activities:

[0024] (1) Cyanide hydrolase activity;

[0025] (2) Cyanide hydratase activity.

[0026] 8. Use of the bioremediation agent described in any one of items 1 to 7, wherein the bioremediation agent is used to degrade cyanide;

[0027] Preferably, the application includes degradation of cyanide in water and / or degradation of cyanide in soil.

[0028] 9. The use according to item 8, wherein the cyanide is an organic cyanide and / or an inorganic cyanide;

[0029] The organic cyanide is selected from any one or more of acrylonitrile and methacrylonitrile;

[0030] The inorganic cyanide is selected from any one or more of sodium cyanide, potassium cyanide and ammonium cyanate.

[0031] 10. The use according to any one of items 8-9, wherein when the bioremediation agent is used to degrade cyanide in water and / or soil, the concentration of cyanide in the water and / or soil is higher than 0.3 mg / L;

[0032] Preferably, the cyanide concentration is 1-350 mg / L.

[0033] 11. According to any one of items 8-10, when the bioremediation agent is used to degrade cyanide in water, the mass ratio of the bioremediation agent to water is (0.5-10):(1-50);

[0034] Preferably, the mass ratio of the bioremediation bacterial agent to water is (1-5):(1-40).

[0035] 12. According to any one of items 8-9, when the bioremediation agent is used to degrade cyanide in soil, the volume mass ratio of the bioremediation agent to soil is (10-60) mL: (0.1-20) g;

[0036] Preferably, the volume mass ratio of the bioremediation bacterial agent to soil is (10-40) mL: (2-15) g.

[0037] 13. According to any one of items 8-12, when the bioremediation agent is used for cyanide degradation, the degradation temperature is 20-70°C;

[0038] Preferably, the degradation temperature is 20-50°C;

[0039] More preferably, the degradation temperature is 35-45°C.

[0040] 14. The use according to any one of items 8 to 12, wherein when the bioremediation agent is used for the degradation of cyanide, the degradation pH is 3-12;

[0041] Preferably, the degradation pH is 5-9;

[0042] More preferably, the degradation pH is 7-9.

[0043] Effects of the Invention

[0044] The present application provides a bioremediation agent containing engineered Rhodococcus and its application in environmental remediation. The engineered Rhodococcus of the present application contains a rich cyanide metabolism-related enzyme system, and the enzyme system has high activity and strong tolerance. The reaction degradation rate reaches 70-99% in the process of degrading cyanide. It can have enzymatic activity for various different types of cyanide. Therefore, for sewage or soil with complex cyanide components, the bioremediation agent has the characteristics of high degradation efficiency and fast degradation rate for various cyanides; in addition, the engineered Rhodococcus of the present application and the stabilizer work together to ensure that the bioremediation agent can quickly degrade high-concentration cyanide in the polluted environment and achieve efficient environmental remediation. Therefore, the bioremediation agent provided by the present invention can be used to repair cyanide pollution sources, and has the advantages of high repair efficiency and short repair time in repairing cyanide pollution, which provides technical support for the use of bioprocesses in subsequent industrial treatment of such environmental problems.

[0045] In addition, compared with traditional chemical degradation agents, this bioremediation agent has a high degradation rate for cyanide, low operating costs, no secondary pollution, and can well ensure that industrial wastewater and soil meet the standards continuously and stably. It has high social and economic benefits and is of great significance to environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 .Infrared spectrum of functional group analysis of the reaction liquid after degradation of engineered Rhodococcus 2.

[0047] Figure 2 .Comparison of the effects of Rhodococcus before and after modification and the degradation of cyanide by chemical degradation agents for 3 days; among them, "Rhodococcus (not)" refers to the fermentation broth of Rhodococcus that has not been recombinantly constructed, "Rhodococcus 1" refers to the fermentation broth of engineered Rhodococcus 1, and "Rhodococcus 2" refers to the fermentation broth of engineered Rhodococcus 2.

[0048] Figure 3 .The effect of stabilizers with different component configurations on the performance of bioremediation agents; the stabilizer and Rhodococcus fermentation broth are added in a mass ratio; for example, Figure 3 The 5% glycerol on the horizontal axis in a means that the mass percentage of glycerol in the biological bacterial agent is 5%.

[0049] Figure 4 .The cyanide degradation effect of the bioremediation agent prepared by different ratios of engineered Rhodococcus 2 fermentation broth and stabilizer.

[0050] Figure 5 .Comparison of the cyanide degradation effects of bioremediation agents made from different strains (strain 1 is Bacillus subtilis 168, strain 2 is Bacillus megaterium, strain 3 is Escherichia coli, and strain 4 is engineered Rhodococcus 2).

[0051] Figure 6 .Effects of temperature (6a) and pH (6b) on cyanide degradation by bioremediation agents. Specific implementation methods

[0053] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in components' functions as the criterion for distinction.

[0054] As mentioned throughout the specification and claims, "including" or "comprising" is an open term and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the present disclosure belongs.

[0055] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments. References to "about" a value or parameter herein include (and describe) variations to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0056] As used herein, reference to a "not" value or parameter generally means and describes an "except" value or parameter. For example, the method is not used to treat cancer type X, meaning that the method is used to treat cancers other than type X.

[0057] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0058] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. This statement is therefore intended to serve as antecedent basis for the use of exclusive terminology such as "only", "only" and the like in connection with the recitation of claim elements, or the use of a limitation of "no".

[0059] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0060] As used herein, "Rhodococcus" refers to unmodified, natural Rhodococcus. In the present application, the selection of Rhodococcus is not specifically limited; the specific selection of Rhodococcus can be any one or more of Rhodococcus ruber TH, Rhodococcus rhodochrous J1, Rhodococcusrhodochrous M8, Rhodococcus pyridinivorans and Rhodococcus sp.

[0061] In one embodiment of the present application, the Rhodococcus is Rhodococcus ruber.

[0062] The terms "glycerol" and "glycerin" are used interchangeably.

[0063] "Engineered Rhodococcus", "Rhodococcus" and "recombinantly modified Rhodococcus" can be used interchangeably in this application, and refer to Rhodococcus obtained by genetic modification at the genetic level through gene recombination, editing and other means.

[0064] "Recombinant Rhodococcus fermentation broth" and "Rhodococcus fermentation broth" can be used interchangeably in this application, and refer to the fermentation broth obtained by fermenting Rhodococcus obtained by genetically modifying Rhodococcus through gene recombination, editing, etc.

[0065] “Non-recombinant Rhodococcus fermentation broth” and “natural Rhodococcus fermentation broth” are used interchangeably in this application, and refer to the fermentation broth obtained by fermenting natural, non-artificially modified Rhodococcus.

[0066] The present application provides a bioremediation bacterial agent containing Rhodococcus, wherein the bioremediation bacterial agent comprises Rhodococcus fermentation broth and a stabilizer.

[0067] The present application also provides a bioremediation bacterial agent containing Rhodococcus, wherein the bioremediation bacterial agent consists of Rhodococcus fermentation broth and a stabilizer.

[0068] In the present application, the Rhodococcus fermentation broth includes a certain amount of viable Rhodococcus and active proteins, enzymes, etc.; and the addition of stabilizers ensures that the active cells and / or molecules such as the Rhodococcus, proteins and enzymes can survive stably and / or remain active for a long time.

[0069] The Rhodococcus fermentation broth described in the present application is obtained by fermenting and culturing Rhodococcus. The Rhodococcus fermentation broth includes recombinant Rhodococcus cells, components in the culture broth that are not consumed by the recombinant Rhodococcus, and primary and / or secondary metabolites of the recombinant Rhodococcus. The culture medium used for fermenting and culturing Rhodococcus is conventionally selected in the art and is not otherwise limited in the present application. The culture medium can achieve enrichment of the Rhodococcus cells and obtain the Rhodococcus fermentation broth.

[0070] In some embodiments, the Rhodococcus in the Rhodococcus fermentation broth is obtained by genetic recombination; an exogenous recombinant plasmid containing a nitrilase gene sequence is introduced into a wild-type Rhodococcus (Rhodococcus ruber) to realize the recombinant construction of Rhodococcus. The expression product of the nitrilase gene sequence is a nitrilase-related enzyme system. In some embodiments, the nitrilase-related enzyme system is a wild-type nitrile metabolic enzyme and its mutants. In some specific embodiments, the amino acid sequence of the wild-type nitrile metabolic enzyme β subunit is shown in SEQ ID NO: 1; in some specific embodiments, the amino acid sequence of the wild-type nitrile metabolic enzyme α subunit is shown in SEQ ID NO: 2; in some specific embodiments, the amino acid sequence of the mutant nitrile metabolic enzyme β subunit mutant is shown in SEQ ID NO: 4; in some specific embodiments, the amino acid sequence of the mutant nitrile metabolic enzyme α subunit mutant is shown in SEQ ID NO: 5.

[0071] In some embodiments, the exogenous recombinant plasmid is obtained by introducing an exogenous gene, i.e., a nitrile metabolism-related enzyme, into the basic plasmid, so that it expresses the cyanide-related enzyme system gene in the rhodococcus, so that the recombinant rhodococcus has degradation activity for a variety of cyanides and high degradation activity. In the present application, there is no special restriction on the basic plasmid, which can realize the insertion of exogenous genes, and the available basic plasmids can be, for example, pNV-18.1 plasmids, PET plasmids, PET-28a plasmids; the basic plasmids used in the present application can be commercially available, or can be obtained by natural extraction, or can be obtained by genetic modification (such as gene knockout, insertion, recombination, etc.). The present application does not limit the method of introducing an exogenous promoter on the basic plasmid. In some embodiments, an exogenous promoter is introduced on the basic plasmid by connecting after double enzyme digestion. In some embodiments, the restriction endonuclease used for enzyme digestion is selected from BamH I, EcoR I, Hind II and BgLII. In some embodiments, after the basic plasmid is digested by enzymes, the digested fragments are purified and recovered, and the target gene fragments are obtained and connected to the digested basic plasmid to obtain an exogenous recombinant plasmid.

[0072] In the present application, the obtained exogenous recombinant plasmid is introduced into erythrocytes to obtain the recombinantly constructed Rhodococcus. The present application does not limit the method of introducing the exogenous recombinant plasmid into erythrocytes; for example, the exogenous recombinant plasmid is transformed into erythrocytes by electrotransformation, or by chemical reagent method.

[0073] The stabilizer is water-soluble and is selected from any one or more of polyols, polyaldehydes and polymers.

[0074] In the present application, "polyol" refers to a polyol having 2-6 carbon atoms and containing at least two hydroxyl groups. In some embodiments, the carbon number of the polyol is 2-4, 3-5 or 4-6; in a specific embodiment, the carbon number of the polyol is 2, 3, 4, 5 or 6. In some embodiments, the polyol contains at least three hydroxyl groups. In some embodiments, the number of hydroxyl groups on the polyol is 2-6; in some embodiments, the number of hydroxyl groups on the polyol is 2-4; in some embodiments, the number of hydroxyl groups on the polyol is 5-6. In a specific embodiment, the number of hydroxyl groups on the polyol is 3, 5, or 6. In some embodiments, the polyol is selected from any one or more of glycerol, sorbitol and mannitol. In some preferred embodiments, the polyol is glycerol.

[0075] In the present application, "polyaldehyde" refers to a polyaldehyde having 2-4 carbon atoms and containing at least two carbonyl groups. In some embodiments, the carbon atoms of the polyaldehyde are 2, 3 or 4. In some embodiments, the polyaldehyde is selected from any one or more of glutaraldehyde and glyoxal. In some preferred embodiments, the polyaldehyde is glutaraldehyde.

[0076] In some embodiments, the polymer is selected from any one or more of polyethylene glycol and polyethylene glycol lactone. In some preferred embodiments, the polymer is polyethylene glycol; further, the polyethylene glycol is polyethylene glycol 4000.

[0077] In this application, the understanding of "water-soluble" is consistent with the general understanding of those skilled in the art. The stabilizer does not react chemically with water and is only physically dissolved in water. "Water-soluble" means that the solubility (wt%) in water at room temperature is at least 25%, 30%, 45%, 50%, 65%, 80%, 95% or 100%.

[0078] In some embodiments, the stabilizer is a combination of a polyol, a polyaldehyde and a polymer. In some embodiments, the stabilizer is a combination of a polyol and a polymer. In some embodiments, the stabilizer is a combination of a polyol and a polyaldehyde. In some embodiments, the stabilizer is a combination of a polymer and a polyaldehyde. In some embodiments, the stabilizer is a polyol. In some embodiments, the stabilizer is a polymer. In some embodiments, the stabilizer is a polyaldehyde.

[0079] In some embodiments, the stabilizer is a combination of polyethylene glycol, glycerol and glutaraldehyde; in some embodiments, the stabilizer is a combination of polyethylene glycol and glycerol; in some embodiments, the stabilizer is a combination of polyethylene glycol and glutaraldehyde; in some embodiments, the stabilizer is a combination of glycerol and glutaraldehyde; in some embodiments, the stabilizer is polyethylene glycol; in some embodiments, the stabilizer is glycerol; in some embodiments, the stabilizer is glutaraldehyde. In a preferred embodiment, the stabilizer is glycerol.

[0080] In some embodiments, the mass ratio of the Rhodococcus fermentation broth to the stabilizer is (1-90):(10-99). In some embodiments, the mass ratio of the Rhodococcus fermentation broth to the stabilizer is (1-10):(10-99), (5-18):(10-99), (15-24):(10-99), (22-37):(10-99), (31-49):(10-99), (45-68):(10-99), (62-84):(10-99), (77-95):(10-99) or (86-90):(10-99). In some embodiments, the mass ratio of the Rhodococcus fermentation broth to the stabilizer is (1-90):(10-18), (1-90):(15-28), (1-90):(22-46), (1-90):(36-57), (1-90):(55-71), (1-90):(64-82), (1-90):(79-93) or (1-90):(88-99).

[0081] In the present application, when the mass ratio of the Rhodococcus fermentation broth and the stabilizer is described above, it is generally believed that the OD600 of the Rhodococcus fermentation broth is higher than 0.2, for example, the OD600 of the Rhodococcus fermentation broth is in the range of 0.25-1.5; specifically, the OD600 of the Rhodococcus fermentation broth can be, for example, 0.25-0.35, 0.3-0.55, 0.45-0.65, 0.6-0.85, 0.7-0.95, 0.9-1.25, 1.18-1.3 or 1.25-1.45; further, the OD600 of the Rhodococcus fermentation broth is in the range of 0.6-1.3. Of course, it is fully understood by those skilled in the art that it can also be converted based on experience, or the OD600 of the Rhodococcus fermentation broth can be controlled to the above range by dilution or concentration, and then mixed.

[0082] The present application is constructed by genetic recombination of Rhodococcus, so that the enzyme system related to cyanide metabolism is more efficiently expressed, and then the Rhodococcus with high recombinant construction for multiple cyanide hydrolysis efficiency is obtained. And the bioremediation bacterial agent prepared with the Rhodococcus fermentation liquid and stabilizer as the main raw materials, its efficiency for multiple cyanide hydrolysis is further significantly improved. When the Rhodococcus fermentation liquid and stabilizer (such as glutaraldehyde) are mixed in a mass ratio of 1:9, its cyanide degradation rate is only 70%, but when the Rhodococcus fermentation liquid and stabilizer are mixed in a mass ratio of 4:6, its cyanide degradation rate is at least 99%, even reaching 100%, and the cyanide degradation rate is improved by at least 29%. When the Rhodococcus fermentation liquid and glycerol are mixed in a mass ratio of 1:9, its cyanide degradation rate is only 40%, but when the Rhodococcus fermentation liquid and glycerol are mixed in a mass ratio of 4:6, its cyanide degradation rate is at least 79%, even reaching 82% or higher, and the cyanide degradation rate is improved by at least 39%. When the Rhodococcus fermentation broth and polyethylene glycol 4000 are mixed at a mass ratio of 1:9, the cyanide degradation rate is only 37%, but when the Rhodococcus fermentation broth and polyethylene glycol 4000 are mixed at a mass ratio of 4:6, the cyanide degradation rate is at least 59%, even reaching 63% or higher, and the cyanide degradation rate is increased by at least 22%. Therefore, the bioremediation bacterial agent of the present application is a remarkable and excellent cyanide degradation effect exerted by the combined action of the engineered Rhodococcus and the stabilizer.

[0083] It is further found in the present application that when the stabilizer is selected from a plurality of glycerol, glutaraldehyde and polyethylene glycol, a higher cyanide degradation effect is further obtained.

[0084] In some embodiments, the stabilizer is a combination of glycerol, glutaraldehyde and polyethylene glycol. In some embodiments, the stabilizer is a combination of glycerol, glutaraldehyde and polyethylene glycol. In some embodiments, the stabilizer is prepared by 20-35wt% glycerol, 0.03-0.10wt% glutaraldehyde, and 4-10M polyethylene glycol. In some embodiments, the amount of glycerol added to the stabilizer is 20-26wt%, 25-28wt%, 27-31wt%, 30-33wt% or 32-35wt%; in a specific embodiment, the amount of glycerol added to the stabilizer is 20wt%, 23wt%, 25wt%, 26wt%, 27wt%, 29wt%, 31wt%, 32wt%, 33wt% or 35wt%. In some embodiments, the amount of glutaraldehyde added to the stabilizer is 0.03-0.05wt%, 0.04-0.07wt%, 0.06-0.08wt% or 0.07-0.10wt%; in a specific embodiment, the amount of glutaraldehyde added to the stabilizer is 0.035wt%, 0.048wt%, 0.052wt%, 0.067wt%, 0.072wt%, 0.081wt%, 0.088wt%, 0.094wt% or 0.10wt%. In some embodiments, the amount of polyethylene glycol added to the stabilizer is 4-6M, 5-8M or 7-10M; in a specific embodiment, the amount of glutaraldehyde added to the stabilizer is 4M, 5.5M, 6M, 7.5M, 8.5M, 9M or 10M.

[0085] The present application found that when a stabilizer is used alone, 30wt% glycerol, 0.07wt% glutaraldehyde or 8M polyethylene glycol 4000, the cyanide degradation rate of the prepared bioremediation bacterial agent is better; in a specific embodiment, the stabilizer component is 30wt% glycerol, 0.07wt% glutaraldehyde and 8M polyethylene glycol 4000; the cyanide degradation efficiency of the bioremediation bacterial agent prepared by the stabilizer of this component is better, at least up to 97.82%, or even up to 99%.

[0086] The present application also provides an application of a bioremediation bacterial agent, wherein the bioremediation bacterial agent is used for degrading cyanide; specifically, it is used for degrading cyanide in water and / or degrading cyanide in soil.

[0087] In some embodiments, when the bioremediation agent is used to degrade cyanide in water, the degradation temperature of the bioremediation agent is 20-70°C, for example, 20-30°C, 28-41°C, 33-50°C, 46-58°C, 52-67°C or 65-70°C. In a specific embodiment, when the bioremediation agent is used to degrade cyanide in water, the degradation temperature of the bioremediation agent is 20°C, 25°C, 28°C, 30°C, 32°C, 35°C, 37°C, 39°C, 40°C, 43°C, 45°C, 49°C, 50°C, 55°C, 59°C, 62°C, 65°C, 68°C or 70°C.

[0088] In some preferred embodiments, the degradation temperature of the bioremediation agent is 20-50° C., for example, 20-31° C., 27-36° C., 32-41° C., 36-45° C., 42-47° C. or 45-50° C. In a specific preferred embodiment, when the bioremediation agent is used to degrade cyanide in water, the degradation temperature of the bioremediation agent is 20° C., 24° C., 29° C., 33° C., 38° C., 43° C., 46° C., 48° C. or 50° C.

[0089] In some more preferred embodiments, the degradation temperature of the bioremediation agent is 35-45°C; for example, 35-38°C, 36-42°C, 37-44°C or 40-45°C. In a specific more preferred embodiment, when the bioremediation agent is used to degrade cyanide in water, the degradation temperature of the bioremediation agent is 35°C, 37°C, 39°C, 42°C, 43°C or 45°C. In some specific embodiments, the bioremediation agent of the present application is used to degrade cyanide in sewage; when the cyanide concentration is as high as 300 mg / L, the degradation temperature is 35-45°C, and the degradation rate of cyanide by the bioremediation agent is as high as 99%, or even 100%.

[0090] In some embodiments, when the bioremediation agent is used for the degradation of cyanide, the degradation pH is 3-12; for example, 3-6, 5-8, 6-9 or 8-12. In some preferred embodiments, when the bioremediation agent is used for the degradation of cyanide, the degradation pH is 5-9; for example, 5-6.4, 6.2-7, 7-8.3 or 8.2-9. In some more preferred embodiments, when the bioremediation agent is used for the degradation of cyanide, the degradation pH is 7-9; for example, 7-7.6, 7.4-8.2, 8.0-8.7 or 8.5-9. In some specific embodiments, the bioremediation agent of the present application is used for the degradation of cyanide in sewage; when the cyanide concentration is as high as 300 mg / L, the degradation pH is 7-9, and the degradation rate of cyanide by the bioremediation agent is as high as 99%, or even 100%.

[0091] In some embodiments, when the bioremediation agent is used for the degradation of cyanide, the degradation time is 1-5 days; for example, 1-3, 2-4 or 3-5. In some preferred embodiments, when the bioremediation agent is used for the degradation of cyanide, the degradation time is 3-5; for example, 3-4, 4 or 4.5-5 days. In some specific embodiments, the bioremediation agent of the present application is used for the degradation of cyanide in sewage; when the cyanide concentration is as high as 300 mg / L, it is degraded for 5 days, and the degradation rate of cyanide by the bioremediation agent is as high as 99%, or even 100%.

[0092] In some embodiments, the bioremediation agent is used to degrade cyanide in water. In some further embodiments, the mass ratio of the bioremediation agent to water is (0.5-10):(1-50). In some embodiments, the mass ratio of the bioremediation agent to water is (0.5-10):(1-10), (0.5-10):(9-22), (0.5-10):(20-35), (0.5-10):(33-48), (0.5-4):(1-50), (3-6):(1-50), (5-8):(1-50) or (7-10):(1-50). In some preferred embodiments, the mass ratio of the bioremediation agent to water is (1-5):(1-40), such as (1-5):(1-10), (1-5):(9-22), (1-5):(20-35), (1-5):(32-40), (1-3):(1-40), (2-4):(1-40) or (3-5):(1-40). In some embodiments, the bioremediation agent is used for the degradation of cyanide in soil. In some further embodiments, the volume mass ratio of the bioremediation bacteria agent to the soil is (10-60) mL: (0.1-20) g; further can be (10-60) mL: (0.1-5) g, (10-60) mL: (4-9) g, (10-60) mL: (8-15) g, (10-60) mL: (14-20) g, (10-18) mL: (0.1-20) g, (17-24) mL: (0.1-20) g, (22-36) mL: (0.1-20) g, (34-48) mL: (0.1-20) g, (45-51) mL: (0.1-20) g or (50-60) mL: (0.1-20) g. In some preferred embodiments, the volume mass ratio of the bioremediation agent to the soil is (10-40) mL:(2-15) g; further, it can be (10-40) mL:(2-8) g, (10-40) mL:(7-12) g, (10-40) mL:(11-15) g, (10-19) mL:(2-15) g, (18-31) mL:(2-15) g or (28-40) mL:(2-15) g. Example

[0093] The specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0094] The calculation formula of cyanide degradation rate in this application is:

[0095] Cyanide degradation rate (%) = (cyanide concentration in the sample before degradation (mg / L) - cyanide concentration in the sample after degradation (mg / L)) / cyanide concentration in the sample before degradation (mg / L) × 100%; the sample is selected from any one of water samples and soil samples.

[0096] Example 1. Recombinant construction of Rhodococcus

[0097] The natural Rhodococcus ruber was used as the host, the artificially modified pNV plasmid was selected as the expression vector, and a recombinant expression plasmid containing nitrile metabolic enzymes was constructed. The recombinant plasmid was transformed into the competent cells of the natural Rhodococcus by electroporation. Subsequently, a single colony was selected for colony PCR verification to determine whether the target gene was successfully transferred, and the successfully transformed Rhodococcus was stored for later use.

[0098] The specific method is as follows:

[0099] The natural Rhodococcus was used as a host, the artificially modified pNV plasmid was selected as an expression vector, and a recombinant expression plasmid containing nitrile metabolic enzymes was constructed.

[0100] The method for constructing the artificially modified pNV plasmid is as follows: using plasmid pNV-18.1 as an expression vector, the plasmid is digested with endonucleases BamH I and EcoR I, and then the wild-type target gene 1, wild-type target gene 2, and mutant target gene 3 after digestion are respectively connected to the plasmid vector to obtain recombinant pNV plasmid 1, recombinant pNV plasmid 2, and recombinant pNV plasmid 3.

[0101] The recombinant pNV plasmid 1 and the recombinant pNV plasmid 2 were simultaneously transformed into the competent cells of Rhodococcus from natural sources by electrotransformation. Then, a single colony was picked for colony PCR verification to determine whether the target gene was successfully transferred, and the successfully transformed engineered Rhodococcus 1 was preserved for future use; the recombinant pNV plasmid 2 and the recombinant pNV plasmid 3 were simultaneously transformed into the competent cells of Rhodococcus from natural sources by electrotransformation, and then a single colony was picked for colony PCR verification, and the successfully transformed engineered Rhodococcus 2 was preserved for future use.

[0102] Among them, the wild-type target gene 1 expresses the β subunit (amino acid sequence as shown in SEQ ID NO: 1) and α subunit (amino acid sequence as shown in SEQ ID NO: 2) of the nitrile metabolizing enzyme respectively; the amino acid sequence of the expression product of the wild-type target gene 2 is shown in SEQ ID NO: 3; the mutant target gene 3 expresses the β subunit mutant (amino acid sequence as shown in SEQ ID NO: 4) and the α subunit mutant (amino acid sequence as shown in SEQ ID NO: 5).

[0103] The amino acid sequences of the β subunit and α subunit of the wild-type nitrile metabolizing enzyme are as follows:

[0104] The β subunit has an amino acid sequence as shown in SEQ ID NO: 1, which is as follows:

[0105] MDGIHDTGGMTGYGPVPYQKDEPFFHYEWEGRTLSILTWMHLKGMSWWDKSRFFRESMGNENYVNEIRNSYYTHWLSAAERILVADKIITEEERKHRVQEILEGRYTDRNPSRKF DPAEIEKAIERLHEPHSLALPGAEPSFSLGDKVKVKNMNPLGHTRCPKYVRNKIGEIVTSHGCQIYPESSSAGLGDDPRPLYTVAFSAQELWGDDGNGKDVVCVDLWEPYLISA;

[0106] The α subunit has an amino acid sequence as shown in SEQ ID NO: 2, which is as follows:

[0107] MSEHVNKYTEYEARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKSWVDPEYRKWLEEDATAAMASLGYAGEQAHQISAVFNDSQTHHVVVCTLCSCYPWPVLGLPPAWYKSMEYRSRVVADCRGVLKRDFGFDIPDEVEVRVWDSSSEIRYIVIPERPAGTDGWSEDELAKLVSRDSMIGVSNALTPQEVIV.

[0108] The amino acid sequence of the wild-type nitrile metabolizing enzyme is shown in SEQ ID NO: 3, and is as follows:

[0109] MVEYTNTFKVAAVQAQPVWFDAAKTVDKTVSIIAEAARNGCELVAFPEVFIPGYPYHIWVDSPLAGMAKFAVRYHENSLTMDSPHVQRLLDAARDHNIAVVVGISERDGGSLYMTQLIIDADGQLVARRRKLKPTHVERSVYGEGNGSDISVYDMPFARLGALNCWEHFQTLTKYAMYSMHEQ VHVASWPGMSLYQPEVPAFGVDAQLTATRMYALEGQTFVVCTTQVVTPEAHEFFCENEEQRKLIGRGGGFARIIGPDGRDLATPLAEDEEGILYADIDLSAITLAKQAADPVGHYSRPDVLSLNFNQRRTTPVNTPLSTIHATHTFVPQFGALDGVRELNGADEQRALPSTHSDETDRATASI.

[0110] The amino acid sequences of the β subunit and α subunit of the mutant nitrile metabolizing enzyme are as follows:

[0111] The β subunit mutant, whose amino acid sequence is shown in SEQ ID NO:4, is as follows:

[0112] MDGIHDTGGMTGYGPVPYQKDEPFFHYEWEGRTLSILTWMHLKGMSWWDKSRFFRESMGNENYVNEIRNSYYTHWLSAAERILVADKIITEEERKHRVQEILEGRYTDRNPSRKF DPAEIEKAIERLHEPHSLALPGAEPKFKEGDKVKVKNMNPLGHTRCPKYVRSKIGEIVTSHGCQIYPESSSAGLGDDPRPLYTVAFSAQELWGDDGNGKCVVCVDLWEPYLISA;

[0113] The α subunit mutant, whose amino acid sequence is shown in SEQ ID NO:5, is as follows: MSEHVNKYTEYEARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKSWVDPEYRKWLEEDATAAMASLGYAGEQAHQISAVFNDSQTHHVVVCTLCSCYPWPVLGLPPAWYKSMEYRSRVVADCRGVLKRDFGFDIPDEVEVRVWDSSSEIRYIVIPERPAGTDGWSEDELAKLVSRDSMIGVSNALTPQEVIV.

[0114] Example 2: Rhodococcus fermentation culture

[0115] The fermentation culture method of Rhodococcus is as follows:

[0116] (1) The recombinant strain (recombinant Rhodococcus) was picked up with a 10 μL inoculation loop from the streaked plate and placed in 15 mL of liquid seed culture medium, and cultured in a shaking incubator at 30° C. and 200 rpm for 3 days.

[0117] (2) Determine the absorbance value OD600 of the seed solution, calculate the inoculation volume according to the initial OD600=1, the inoculation volume shall not exceed 10% of the fermentation medium, inoculate into the fermentation medium containing antibiotics, and culture in a shaking incubator at 30°C and 200 rpm for 2 days, and then collect the Rhodococcus fermentation stock solution (i.e., the recombinant Rhodococcus fermentation solution or the non-recombinant Rhodococcus fermentation solution; OD600 is about 0.2-1.6).

[0118] Example 3. Degradation activity verification

[0119] This experiment mainly examines the expression of hydrolase and hydratase activities of the engineered Rhodococcus 2 after successful transformation. The specific experimental steps are as follows.

[0120] The engineered Rhodococcus 2 (obtained by fermentation culture using the method of Example 2; OD600 is 1.2) was mixed with 50 mg / L of different cyanide solutions (sodium cyanide, potassium cyanide, acrylonitrile) at a mass ratio of 1:20 in a conical flask, and the mixture was sealed with a film in a full-temperature shaking incubator for reaction for 3 days. The reaction conditions were: temperature of 30°C, pH of 7, and post-processing detection was performed after the reaction.

[0121] The results showed that the residual amounts of sodium cyanide, potassium cyanide and acrylonitrile corresponding to the engineered Rhodococcus 2 were 26.7 mg / L, 30.2 mg / L and 20.4 mg / L, respectively. Then, the components in the reaction solution were detected by infrared spectroscopy. Figure 1 As shown, there are characteristic peaks of carboxyl and amine groups in the solution, indicating that the Rhodococcus after successful transformation can express hydrolases and hydratases.

[0122] Example 4. Cyanide degradation by engineered and natural Rhodococcus

[0123] This experiment mainly investigates the degradation effects of bioremediation agents and chemical degradation agents prepared by engineered Rhodococcus 1, engineered Rhodococcus 2 and non-recombinant Rhodococcus on cyanide. The fermentation broth of the above-mentioned different Rhodococcus (OD600 is about 1) was selected and mixed with a stabilizer (specific components of the stabilizer are: 20wt% glycerol, 4M PEG 4000, 0.03wt% glutaraldehyde) at a mass ratio of 1:1 to prepare bioremediation agents. The above-mentioned bioremediation agents and chemical degradation agents (chemical degradation agents are Fe 2+ The mixture was prepared with hydrogen peroxide in a mass ratio of 1:1) and acrylonitrile (350 mg / L) in a mass ratio of 5:40 and added into a 500 mL conical flask. The reaction was carried out in a full temperature shaking incubator at a temperature of 30°C, a pH of 7 and a stirring speed of 200 rpm / min for 3 days.

[0124] After the experiment, the test Figure 2 As shown, after the bioremediation agent prepared by the fermentation broth of engineered Rhodococcus 2 degraded the cyanide solution, the cyanide content in the solution dropped from 350 mg / L to 98 mg / L, and the degradation rate was 72.00%, which was higher than the cyanide degradation rate of the bioremediation agent prepared by other Rhodococcus stock solutions; and after three days of degradation, the degradation rate of the bioremediation agent prepared by the engineered Rhodococcus stock solution was much higher than that of the chemical degradation agent.

[0125] Example 5. Effect of stabilizer on cyanide degradation by bioremediation bacteria

[0126] This experiment mainly investigates the effect of stabilizers prepared with different addition amounts of ingredients on the degradation effect of bioremediation agents. By optimizing the addition of different concentrations of glycerol, polyethylene glycol 4000, and glutaraldehyde to form a stabilizer (using single-factor experimental optimization, optimizing glycerol, polyethylene glycol 4000, and glutaraldehyde in turn), the stabilizer and the engineered Rhodococcus 2 fermentation broth (OD600 is about 1) are used in a mass ratio of 3:7 to form a bioremediation agent (engineered Rhodococcus 2 fermentation broth (OD600 is about 1) as a control), and a biodegradation reaction is carried out to obtain a biodegraded solution. Among them, the bioremediation agent: acrylonitrile aqueous solution (280 mg / L) are mixed at a mass ratio of 1:10, and the reaction is carried out in a full-temperature shaking incubator sealed with a film for 3 days. The biodegradation reaction conditions are: temperature of 30°C and pH value of 7. The biodegraded solution is centrifuged for solid-liquid separation to obtain a filtrate. The results are as follows Figure 3 As shown in the figure (the original solution in the figure is the fermentation solution), the degradation effect of the stabilizer prepared with different ingredients is different. When the components are 30wt% glycerol, 0.07wt% glutaraldehyde, and 8M polyethylene glycol 4000, the decyanation rate of the filtrate is 97.82%.

[0127] Example 6. Effect of stabilizer on cyanide degradation by bioremediation bacteria

[0128] This experiment mainly investigates the effect of different addition ratios of engineered Rhodococcus 2 fermentation broth (OD600 is about 1) and stabilizer (configured according to the ratio of Example 5, 30wt% glycerol, 0.07wt% glutaraldehyde, 8M polyethylene glycol 4000) on the cyanide degradation effect. A cyanide solution (i.e., acrylonitrile aqueous solution) with a concentration of 280 mg / L is prepared, and the fermentation broth of engineered Rhodococcus 2 (OD600 is about 1) is mixed with the bioremediation bacteria agent and cyanide solution prepared with different stabilizer ratios to carry out biodegradation reaction to obtain a biodegraded solution. Among them, the two are mixed at a mass ratio of bioremediation bacteria agent: acrylonitrile aqueous solution = 1:10, and the reaction is carried out in a film-sealed full-temperature shaking incubator for 3 days. The biodegradation reaction conditions are: temperature of 30°C and pH of 7. The biodegraded solution is centrifuged for solid-liquid separation to obtain a filtrate. Figure 4 As shown, after the fermentation liquid of engineered Rhodococcus 2 reacts with different stabilizer ratios, the cyanide degradation rate of the obtained filtrate is about 70% to 99%. When the mass ratio of the fermentation liquid of engineered Rhodococcus 2 to the stabilizer is 40wt%:60wt%, the decyanation rate of the filtrate is 98.93%.

[0129] Example 7. Effect of different strains on cyanide degradation

[0130] This experiment mainly investigated the effects of different strains on cyanide degradation.

[0131] Preparation of strains: Referring to the method of Example 1, the recombinant expression vector was transferred into Bacillus subtilis 168, Bacillus magaterium, and Escherichia coli, respectively, to obtain engineered Bacillus subtilis, engineered Bacillus magaterium, and engineered Escherichia coli, respectively. The fermentation broths of different strains were obtained by conventional methods.

[0132] Select bioremediation agents containing different strains (fermentation broths of different strains (OD600 is about 1) and stabilizers are configured in a mass ratio of 4:6, and the stabilizer is configured according to the ratio described in Example 5, specifically 30wt% glycerol, 0.07wt% glutaraldehyde, 8M polyethylene glycol 4000) and 200mg / L acrylonitrile aqueous solution are mixed in a mass ratio of 1:8 and added to a 500mL conical flask. The temperature is 30°C, pH is 7, and the stirring speed is 200rpm / min. The reaction is carried out in a full-temperature shaking incubator for 3 days. After the experiment, it was detected that the cyanide content in the bioremediation agent containing Escherichia coli dropped from 235mg / L to 120mg / L, and the degradation rate was 48.93%. The cyanide content in the degradation system containing Rhodococcus was reduced from 235mg / L to 48mg / L, and the degradation rate was 79.57%. The degradation rates of other strains are as follows Figure 5 shown.

[0133] Example 8. Effect of temperature and pH on cyanide degradation rate

[0134] This experiment mainly investigates the effects of the temperature and pH of the degradation environment on the efficiency of bioremediation agents in degrading cyanide. Under the condition of pH = 7, 40 mL of 300 mg / L acrylonitrile aqueous solution was added to a dry conical flask, and then 2 mL of bioremediation agents (the stabilizer was obtained by the configuration according to the ratio of Example 5, and the specific components were: 30 wt% glycerol, 0.07 wt% glutaraldehyde, 8 M polyethylene glycol 4000; the bioremediation agents were configured according to the mass ratio of engineered red coccus 2 fermentation broth (OD600 is about 1) and stabilizer 4:6), and the reaction was carried out in a full-temperature shaking incubator at a stirring speed of 200 rpm / min for 1-5 days. After the experiment, the cyanide content at different temperatures was detected. The results are as follows: Figure 6 (a) As shown. At a temperature of 40°C, 40 mL of 300 mg / L acrylonitrile aqueous solution was added to a dry conical flask, and 2 mL of the above-configured bioremediation agent was added. The reaction was carried out in a full-temperature shaking incubator at a stirring speed of 200 rpm / min for 1-5 days. After the experiment, the cyanide content at different pH values ​​was tested. The results are shown in FIG. Figure 6 (b) as shown. Figure 5 The results show that the optimum temperature for cyanide degradation is 40°C and the optimum pH is 7. Under the optimum conditions, 300 mg / L cyanide can be degraded by about 99% within 5 days.

[0135] Application Examples

[0136] Application Example 1. Degradation of cyanide in actual wastewater by bioremediation agents

[0137] This experiment mainly investigates the degradation effect of cyanide in actual wastewater by bioremediation agents containing engineered Rhodococcus 2.

[0138] Take 500mL of chemical wastewater and mix it evenly with 10mL of bioremediation agent (stabilizer prepared according to the method of Example 5, the specific components of each stabilizer are shown in Table 1; bioremediation agent is prepared according to the mass ratio of engineered red coccus 2 fermentation broth to stabilizer of 4:6, and the OD600 value of each fermentation broth is shown in Table 1) for biodegradation reaction. The degradation temperature is 40°C, pH is 7, and the shaker speed is 200rpm / min. The reaction is sealed in a full-temperature shaking incubator. Three replicates are set for each experiment. Sampling was carried out on days 0, 2, 4, and 5 of the culture, and the initial concentration of cyanide in the wastewater was measured to be 324mg / L on day 0 (with the detection of cyanide functional groups as the target). The sampling method is to weigh 2 mL of the sample in the conical flask and centrifuge it, take the supernatant to remove water with anhydrous sodium sulfate, pass it through a 0.22 μm organic filter membrane and put it into the injection bottle for testing. After 5 days of cultivation, the degradation rate of cyanide in each experimental group is more than 96%, and the cyanide degradation rate of each experimental group is shown in Table 1. The results show that the bioremediation agent containing engineered Rhodococcus 2 can be directly added to high-concentration chemical cyanide wastewater for degradation reaction.

[0139] Table 1 Stabilizer composition

[0140]

[0141] Application Example 2. Degradation of cyanide in soil by bioremediation agents

[0142] This experiment mainly investigates the degradation effect of bioremediation agents containing Rhodococcus (stabilizers are prepared according to the method of Example 5, and the specific components of each stabilizer are shown in Table 2; bioremediation agents are prepared according to the mass ratio of engineered Rhodococcus 2 fermentation broth to stabilizer of 4:6, and the OD600 values ​​of each fermentation broth are shown in Table 2) on cyanide in actual soil. Take 10g of sludge from the chemical industry and mix it evenly with 30mL of bioremediation agent for biodegradation reaction. The degradation temperature is 40°C, the pH is 7, and the shaker speed is 200rpm / min. The reaction is sealed in a full-temperature shaking incubator. Three replicates are set for each experiment. Sampling was carried out on days 0, 2, 4, and 5 of the culture, and the initial concentration of cyanide in the sludge was measured on day 0 to be 108mg / L (with the detection of cyanide functional groups as the target). The sampling method is to weigh 5 mL of the sample suspension in the conical flask, centrifuge and filter, remove water from the supernatant with anhydrous sodium sulfate, pass through a 0.22 μm organic filter membrane into a sampling bottle, and conduct on-machine detection. After 5 days of cultivation, the degradation rate of each experimental group reached more than 96%. The cyanide degradation rate of each experimental group is shown in Table 2, indicating that the bioremediation agent containing engineered Rhodococcus 2 can be directly added to the cyanide-containing soil for degradation reaction.

[0143] Table 2 Stabilizer composition

[0144]

Claims

1. A bioremediation agent containing Rhodococcus, wherein the bioremediation agent comprises Rhodococcus fermentation broth and a stabilizer.

2. The bioremediation agent according to claim 1, wherein the stabilizer is selected from any one or more of polyols, polyaldehydes and polymers.

3. The bioremediation agent according to claim 2, wherein the polyol is selected from any one or more of glycerol, sorbitol and mannitol; The polymer is selected from any one or more of polyethylene glycol and polyethylene glycol lactone; the polyaldehyde is selected from any one or more of glutaraldehyde and glyoxal.

4. The bioremediation agent according to any one of claims 2 to 3, wherein the stabilizer is selected from any one or more of glycerol, polyethylene glycol and glutaraldehyde; Preferably, the stabilizer is a combination of glycerol, polyethylene glycol and glutaraldehyde; More preferably, the stabilizer is prepared from 20-35wt% glycerol, 0.03-0.10wt% glutaraldehyde, and 4-10M polyethylene glycol.

5. The bioremediation bacterial agent according to claim 1, wherein the mass ratio of the Rhodococcus fermentation broth to the stabilizer is (1-90): (10-99); Preferably, the OD600 of the Rhodococcus fermentation broth is higher than 0.2; Preferably, the mass ratio of the Rhodococcus fermentation broth to the stabilizer is (10-50):(10-90); More preferably, the mass ratio of the Rhodococcus fermentation broth to the stabilizer is (30-50):(50-70).

6. The bioremediation agent according to any one of claims 1 to 5, wherein the Rhodococcus in the Rhodococcus fermentation broth is obtained by genetic recombination; an exogenous recombinant plasmid containing a nitrile metabolic enzyme-related gene sequence is introduced into the wild-type Rhodococcus (Rhodococcus ruber) to achieve the recombinant construction of the Rhodococcus; Preferably, the recombinantly constructed Rhodococcus expresses nitrile metabolic enzyme-related enzymes and mutants thereof; More preferably, the recombinant Rhodococcus expresses a protein having any one or more amino acid sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 5; Further preferably, the recombinant Rhodococcus expresses a protein having any one or more amino acid sequences as shown in SEQ ID NO:3 to SEQ ID NO:

5.

7. The bioremediation agent according to any one of claims 1 to 6, wherein the Rhodococcus has any one or more of the following enzyme activities: (1) Cyanide hydrolase activity; (2) Cyanide hydratase activity.

8. Use of the bioremediation agent according to any one of claims 1 to 7, wherein the bioremediation agent is used to degrade cyanide; Preferably, the application includes degradation of cyanide in water and / or degradation of cyanide in soil.

9. The use according to claim 8, wherein the cyanide is an organic cyanide and / or an inorganic cyanide; The organic cyanide is selected from any one or more of acrylonitrile and methacrylonitrile; The inorganic cyanide is selected from any one or more of sodium cyanide, potassium cyanide and ammonium cyanate.

10. The use according to any one of claims 8 to 9, wherein when the bioremediation agent is used for the degradation of cyanide in water and / or soil, the concentration of cyanide in the water and / or soil is higher than 0.3 mg / L; Preferably, the cyanide concentration is 1-350 mg / L.

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