Rhodococcus-containing bioremediation microbial agent and application thereof

Abstract

The application relates to the technical field of biological remediation bacteria for environmental remediation, and specifically provides a biological remediation bacteria containing rhodococcus and application thereof. The biological remediation bacteria comprises a rhodococcus fermentation liquor and a stabilizer. The biological remediation bacteria is used for degradation of cyanide, and can be used for degradation of cyanide in sewage containing various cyanides and / or high-concentration cyanides and soil. The degradation efficiency is significantly higher than that of a traditional chemical remediation agent, the biological remediation bacteria does not cause secondary environmental pollution, is more environmentally friendly, and is stable in performance.

Description

Technical Field

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

[0002] The environmental pollution caused by synthetic products resulting from the rapid development of chemical synthesis in the last century has become one of the most pressing global problems. This includes the discharge and leakage of industrial wastewater during industrial synthesis processes. Cyanide, containing the cyanide group (CN), is a highly toxic substance. The national standard for total cyanide compounds (GB 8978-1996) stipulates a discharge standard of 0.300 mg / L. Cyanide and COD generated during waste discharge can diffuse and accumulate in large quantities in soil and groundwater. Therefore, developing an environmentally friendly and efficient bioremediation agent is essential.

[0003] Common chemical degradation agents include water-soluble polymers, ozone, and activated carbon. However, most chemical degradation agents do not fundamentally remove pollutants when degrading cyanide and are prone to causing secondary pollution. While there are fewer reports on bioremediation agents, among various environmental remediation methods, bioremediation is undoubtedly a low-cost, side-effect-free, and effective approach. Rhodococcus bacteria are mostly screened from contaminated soil or water bodies, thus enabling the degradation of pollutants. In addition, Rhodococcus bacteria have many advantages, such as adaptability to various extreme environments, not being limited by oxygen and nutrient deficiencies, and most importantly, genetically modified Rhodococcus bacteria can efficiently express multiple enzyme systems, such as hydratases and hydrolases. Rhodococcus bacteria contain hydrophobic surfaces that allow for sufficient contact with organic matter, improving the degradation efficiency of cyanide-containing compounds.

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

[0005] To address the difficulties and problems encountered in existing technologies, this application provides a bioremediation agent containing Rhodococcus bacteria and its application. The Rhodococcus bacteria in this bioremediation agent, after artificial modification, work in conjunction with a stabilizer to efficiently degrade various cyanides at different concentrations. Furthermore, based on the significant advantages of this bioremediation agent, it has been successfully applied to the degradation of cyanide in complex cyanide-containing wastewater and cyanide-containing soil. This bioremediation agent is environmentally friendly, highly efficient, and stable.

[0006] This application involves the following:

[0007] 1. A bioremediation agent containing Rhodococcus, said bioremediation agent comprising Rhodococcus ferment broth and a stabilizer.

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

[0009] 3. The bioremediation agent according to item 2, wherein 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 20-35 wt% glycerol, 0.03-0.10 wt% glutaraldehyde, and 4-10 M polyethylene glycol.

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

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

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

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

[0019] 6. The bioremediation agent according to any one of items 1-5, wherein the Rhodococcus fermentation broth contains Rhodococcus bacteria obtained through gene recombination; the Rhodococcus recombination is constructed by introducing an exogenous recombinant plasmid containing a gene sequence related to nitrile metabolic enzymes into wild-type Rhodococcus ruber.

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

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

[0022] More preferably, the recombinant Rhodococcus expresses a protein with 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 1-6, 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. The application of the bioremediation agent described in any one of items 1-7, wherein the bioremediation agent is used to degrade cyanide;

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

[0028] 9. In the application described in item 8, 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. In any of the applications described in items 8-9, 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;

[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 for the degradation of 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 agent to water is (1-5):(1-40).

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

[0036] Preferably, the volume-to-mass ratio of the bioremediation agent to the 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℃;

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

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

[0040] 14. According to any one of items 8-12, 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] Invention Effects

[0044] This application provides a bioremediation agent containing engineered Rhodococcus bacteria and its application in environmental remediation. The engineered Rhodococcus bacteria in this application contain abundant cyanide metabolism-related enzymes with high activity and strong tolerance, achieving a degradation rate of 70-99% in the cyanide degradation process. It exhibits enzymatic activity against various types of cyanide, thus providing a high-efficiency and rapid degradation rate for wastewater or soil with complex cyanide compositions. Furthermore, the engineered Rhodococcus bacteria and stabilizer work together to ensure the rapid degradation of high concentrations of cyanide in the polluted environment, achieving efficient environmental remediation. Therefore, the bioremediation agent provided by this invention can be used to remediate cyanide pollution sources, offering advantages of high remediation efficiency and short remediation time, providing technical support for subsequent industrial treatment of such environmental problems using biological processes.

[0045] In addition, compared with traditional chemical degradation agents, this bioremediation agent has a high degradation rate of cyanide, low operating cost, no secondary pollution, and effectively ensures that industrial wastewater and soil meet standards continuously and stably. It has high social and economic benefits and is of great significance to environmental protection. Attached Figure Description

[0046] Figure 1 Infrared spectrum of functional groups in the reaction solution after degradation of Rhodococcus 2 by engineering modification.

[0047] Figure 2 Comparison of the effects of Rhodococcus modification before and after, and the degradation of cyanide by chemical degradation agent after 3 days; where "Rhodococcus (unreconstructed)" refers to the fermentation broth of Rhodococcus without recombination, "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 formulations on the performance of bioremediation agents; wherein the stabilizer and Rhodococcus ferment broth are added in a mass ratio; for example, Figure 3 In section a, the 5% glycerol on the horizontal axis means that the mass percentage of glycerol in the biological agent is 5%.

[0049] Figure 4 The cyanide degradation effect of bioremediation agents prepared by combining Rhodococcus 2 fermentation broth and stabilizer in different ratios was investigated.

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

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

[0052] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in their functions.

[0053] As used throughout the specification and claims, the terms "comprising" or "including" are open-ended and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0054] It should be understood that the embodiments of this application described herein include embodiments that are "composed of" and / or "substantially composed of". References to values ​​or parameters of "about" herein include (and describe) variations of that value or parameter itself. For example, a reference to "about X" includes a description of "X".

[0055] As used herein, references to “not” values ​​or parameters generally refer to and describe “except” values ​​or parameters. For example, “The method is not used to treat type X cancer” means that the method is used to treat cancers other than type X.

[0056] As used in this article, the term “approximately XY” has the same meaning as “approximately X to approximately Y”.

[0057] As used herein and in the appended claims, the singular forms “a / an” and “the” include the plural objects unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a preliminary basis for the use of exclusive terms such as “only” or “merely” in conjunction with the description of the elements of the claim, or for the use of the limitation of “no”.

[0058] As used herein, the term "and / or" in words 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 words 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).

[0059] As used herein, "Rhodococcus" refers to unmodified, naturally occurring Rhodococcus. In this application, no specific restrictions are placed on the selection of Rhodococcus; the specific selection may be any one or more of Rhodococcus ruber TH, Rhodococcus rhodochrous J1, Rhodococcus rhodochrous M8, Rhodococcus pyridinivorans, and the genus Rhodococcus sp.

[0060] In one embodiment of this application, the rubococcus is Rhodococcus ruber.

[0061] The terms "glycerol" and "glycerin" are used interchangeably.

[0062] The terms “engineered Rhodococcus”, “Rhodococcus”, and “recombinant Rhodococcus” can be used interchangeably in this application, referring to Rhodococcus obtained by modifying it at the gene level through gene recombination, editing, or other means.

[0063] In this application, "recombinant Rhodococcus ferment broth" and "Rhodococcus ferment broth" can be used interchangeably, referring to the fermentation broth obtained by fermenting Rhodococcus obtained by genetically modifying it through gene recombination, editing or other means at the gene level.

[0064] The terms "unreconstituted Rhodococcus ferment broth" and "natural Rhodococcus ferment broth" are used interchangeably in this application and refer to ferment broth obtained by fermenting natural, unmodified Rhodococcus.

[0065] This application provides a bioremediation agent containing Rhodococcus, the bioremediation agent comprising Rhodococcus ferment broth and a stabilizer.

[0066] This application also provides a bioremediation agent containing Rhodococcus, which is composed of Rhodococcus ferment broth and a stabilizer.

[0067] In this application, the Rhodococcus fermentation broth includes a certain amount of surviving Rhodococcus bacteria as well as active proteins, enzymes, etc.; and the addition of stabilizers ensures that the active cells and / or molecules such as Rhodococcus bacteria, proteins, and enzymes can survive stably for a long time and / or maintain their activity.

[0068] The Rhodococcus fermentation broth described in this application is obtained by fermenting Rhodococcus. The Rhodococcus fermentation broth includes recombinant Rhodococcus cells, components in the culture medium not consumed by the recombinant Rhodococcus, and primary and / or secondary metabolites of the recombinant Rhodococcus. The culture medium used for fermenting the Rhodococcus is conventionally chosen in the art, and there are no other limitations in this application; the culture medium is sufficient to achieve cell enrichment of Rhodococcus and obtain the Rhodococcus fermentation broth.

[0069] In some embodiments, the Rhodococcus fermentation broth contains Rhodococcus bacteria obtained through gene recombination; the recombinant construction of Rhodococcus is achieved by introducing an exogenous recombinant plasmid containing a nitrile hydrolase gene sequence into wild-type Rhodococcus ruber. The expression product of the nitrile hydrolase gene sequence is a nitrile hydrolase-related enzyme system. In some embodiments, the nitrile hydrolase-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 is shown in SEQ ID NO:4; in some specific embodiments, the amino acid sequence of the mutant nitrile metabolic enzyme α subunit is shown in SEQ ID NO:5.

[0070] In some embodiments, the exogenous recombinant plasmid is obtained by introducing an exogenous gene, i.e., a nitrile metabolism-related enzyme, into a base plasmid, thereby enabling it to express cyanide-related enzyme genes from Rhodococcus, resulting in the recombinant Rhodococcus possessing high degradation activity against multiple cyanides. In this application, no special restrictions are placed on the base plasmid; it can be used to allow for the insertion of exogenous genes. Suitable base plasmids include, for example, pNV-18.1 plasmid, PET plasmid, and PET-28a plasmid. The base plasmid used in this application can be commercially available, naturally extracted, or obtained through gene modification (e.g., gene knockout, insertion, recombination, etc.). This application does not restrict the method of introducing the exogenous promoter into the base plasmid. In some embodiments, the exogenous promoter is introduced into the base plasmid through double enzyme digestion followed by ligation. In some embodiments, the restriction endonucleases used for digestion are selected from BamHI, EcoRI, HindII, and BgLII. In some implementations, after digesting the basic plasmid with enzymes, the digested fragments are purified and recovered to obtain the target gene fragment, which is then ligated into the digested basic plasmid to obtain an exogenous recombinant plasmid.

[0071] In this application, the obtained exogenous recombinant plasmid is introduced into erythrocytes to obtain recombinant erythrococci. This application does not limit the method of introducing the exogenous recombinant plasmid into erythrocytes; for example, the exogenous recombinant plasmid can be transformed into erythrocytes by electroporation, or by using chemical reagents.

[0072] The stabilizer is water-soluble and is selected from any one or more of polyols, polyaldehydes, and polymers.

[0073] In this application, "polyol" refers to a polyol having 2-6 carbon atoms and containing at least two hydroxyl groups. In some embodiments, the polyol has 2-4, 3-5, or 4-6 carbon atoms; in one specific embodiment, the polyol has 2, 3, 4, 5, or 6 carbon atoms. In some embodiments, the polyol contains at least three hydroxyl groups. In some embodiments, the polyol has 2-6 hydroxyl groups; in some embodiments, the polyol has 2-4 hydroxyl groups; in some embodiments, the polyol has 5-6 hydroxyl groups. In one specific embodiment, the polyol has 3, 5, or 6 hydroxyl groups. 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.

[0074] In this application, "polyaldehyde" refers to a polyaldehyde having 2-4 carbon atoms and containing at least two carbonyl groups. In some embodiments, the polyaldehyde has 2, 3, or 4 carbon atoms. In some embodiments, the polyaldehyde is selected from any one or more of glutaraldehyde and glyoxal. In some preferred embodiments, the polyaldehyde is glutaraldehyde.

[0075] In some embodiments, the polymer is selected from 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.

[0076] In this application, "water solubility" is understood in accordance with the general understanding of those skilled in the art; the stabilizer does not chemically react with water and only physically dissolves in water. "Water solubility" refers to a solubility (wt%) in water at room temperature of at least 25%, 30%, 45%, 50%, 65%, 80%, 95%, or 100%.

[0077] In some embodiments, the stabilizer is a combination of polyols, polyaldehydes, and polymers. In some embodiments, the stabilizer is a combination of polyols and polymers. In some embodiments, the stabilizer is a combination of polyols and polyaldehydes. In some embodiments, the stabilizer is a combination of polymers and polyaldehydes. In some embodiments, the stabilizer is a polyol. In some embodiments, the stabilizer is a polymer. In some embodiments, the stabilizer is a polyaldehyde.

[0078] 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.

[0079] 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).

[0080] In this application, when describing the mass ratio of Rhodococcus ferment broth to stabilizer, it is generally assumed that the OD600 of the Rhodococcus ferment broth is higher than 0.2, for example, the OD600 of the Rhodococcus ferment broth is in the range of 0.25-1.5. Specifically, the OD600 of the Rhodococcus ferment 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; furthermore, the OD600 of the Rhodococcus ferment broth is in the range of 0.6-1.3. Of course, those skilled in the art can fully understand that conversion can be made based on experience, or the OD600 of the Rhodococcus ferment broth can be controlled to the above range by dilution or concentration before mixing.

[0081] This application involves recombinantly constructing *Rhodococcus* to more efficiently express its cyanide metabolism-related enzyme systems, thereby obtaining a recombinant *Rhodococcus* strain with high hydrolytic efficiency against multiple cyanides. The bioremediation agent prepared using this *Rhodococcus* fermentation broth and stabilizer as the main raw materials further significantly improves the efficiency of hydrolytic degradation against multiple cyanides. When the *Rhodococcus* fermentation broth and stabilizer (e.g., glutaraldehyde) are mixed at a mass ratio of 1:9, the cyanide degradation rate is only 70%. However, when the *Rhodococcus* fermentation broth and stabilizer are mixed at a mass ratio of 4:6, the cyanide degradation rate is at least 99%, even reaching 100%, an increase of at least 29%. When the *Rhodococcus* fermentation broth and glycerol are mixed at a mass ratio of 1:9, the cyanide degradation rate is only 40%. However, when the *Rhodococcus* fermentation broth and glycerol are mixed at a mass ratio of 4:6, the cyanide degradation rate is at least 79%, even reaching 82% or higher, an increase of at least 39%. When Rhodococcus ferment broth and polyethylene glycol 4000 are mixed at a mass ratio of 1:9, the cyanide degradation rate is only 37%. However, when the Rhodococcus ferment broth and polyethylene glycol 4000 are mixed at a mass ratio of 4:6, the cyanide degradation rate is at least 59%, and even reaches 63% or higher, representing an increase of at least 22%. Therefore, the bioremediation agent of this application achieves its significantly superior cyanide degradation effect through the combined action of engineered Rhodococcus and stabilizers.

[0082] This application further discovers that when the stabilizer is selected from multiple of glycerol, glutaraldehyde, and polyethylene glycol, an even higher cyanide degradation effect is obtained.

[0083] 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 formulated from 20-35 wt% glycerol, 0.03-0.10 wt% glutaraldehyde, and 4-10M polyethylene glycol. In some embodiments, the amount of glycerol added to the stabilizer is 20-26 wt%, 25-28 wt%, 27-31 wt%, 30-33 wt%, or 32-35 wt%; in one specific embodiment, the amount of glycerol added to the stabilizer is 20 wt%, 23 wt%, 25 wt%, 26 wt%, 27 wt%, 29 wt%, 31 wt%, 32 wt%, 33 wt%, or 35 wt%. In some embodiments, the amount of glutaraldehyde added to the stabilizer is 0.03-0.05 wt%, 0.04-0.07 wt%, 0.06-0.08 wt%, or 0.07-0.10 wt%; in one specific embodiment, the amount of glutaraldehyde added to the stabilizer is 0.035 wt%, 0.048 wt%, 0.052 wt%, 0.067 wt%, 0.072 wt%, 0.081 wt%, 0.088 wt%, 0.094 wt%, or 0.10 wt%. In some embodiments, the amount of polyethylene glycol added to the stabilizer is 4-6 M, 5-8 M, or 7-10 M; in one specific embodiment, the amount of glutaraldehyde added to the stabilizer is 4 M, 5.5 M, 6 M, 7.5 M, 8.5 M, 9 M, or 10 M.

[0084] This application has found that when a single stabilizer is used, the bioremediation bacterial agent prepared with 30 wt% glycerol, 0.07 wt% glutaraldehyde, or 8M polyethylene glycol 4000 exhibits a better cyanide degradation rate. In one specific embodiment, the stabilizer components are 30 wt% glycerol, 0.07 wt% glutaraldehyde, and 8M polyethylene glycol 4000. The bioremediation bacterial agent prepared with this stabilizer component has an even better cyanide degradation efficiency, at least as high as 97.82%, and even as high as 99%.

[0085] This application also provides an application of a bioremediation agent, which is used to degrade cyanide; specifically, it includes the degradation of cyanide in water and / or the degradation of cyanide in soil.

[0086] 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.

[0087] 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 for the degradation of 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.

[0088] In some 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 for the degradation of 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 this application is used for the degradation of cyanide in wastewater; 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%.

[0089] In some embodiments, when the bioremediation agent is used to degrade 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 to degrade 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 to degrade 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, when the bioremediation agent of this application is used to degrade cyanide in wastewater, when the cyanide concentration is as high as 300 mg / L and the degradation pH is 7-9, 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 to degrade cyanide, the degradation time is 1-5 days; for example, 1-3, 2-4, or 3-5 days. In some preferred embodiments, when the bioremediation agent is used to degrade cyanide, the degradation time is 3-5 days; for example, 3-4, 4, or 4.5-5 days. In some specific embodiments, the bioremediation agent of this application is used to degrade cyanide in wastewater; when the cyanide concentration is as high as 300 mg / L, the degradation takes 5 days, and the degradation rate of cyanide by the bioremediation agent is as high as 99%, or even 100%.

[0091] In some embodiments, the bioremediation agent is used for the degradation of 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), for example (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-to-mass ratio of the bioremediation agent to the soil is (10-60) mL:(0.1-20) g; further, it 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-to-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

[0092] Specific embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided 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.

[0093] The formula for calculating the cyanide degradation rate in this application is as follows:

[0094] 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.

[0095] Example 1. Recombinant construction of Rhodococcus

[0096] Using Rhodococcus ruber, a naturally derived strain, as the host, and an artificially modified pNV plasmid as the expression vector, a recombinant expression plasmid containing a nitrile metabolic enzyme was constructed. The recombinant plasmid was transformed into competent Rhodococcus ruber cells using electroporation. Single colonies were then selected for colony PCR verification to confirm successful gene transfer. Successfully transformed Rhodococcus ruber cells were then preserved for future use.

[0097] The specific method is as follows:

[0098] Using Rhodococcus purpureus of natural origin as the host, and selecting an artificially modified pNV plasmid as the expression vector, a recombinant expression plasmid containing nitrile metabolic enzymes was constructed.

[0099] The artificially modified pNV plasmid was constructed as follows: using plasmid pNV-18.1 as the expression vector, the plasmid was digested with restriction enzymes BamHI and EcoRI. Then, the digested wild-type target gene 1, wild-type target gene 2, and mutant target gene 3 were respectively ligated into the plasmid vector to obtain recombinant pNV plasmid 1, recombinant pNV plasmid 2, and recombinant pNV plasmid 3.

[0100] Recombinant pNV plasmid 1 and recombinant pNV plasmid 2 were simultaneously transformed into naturally derived Rhodococcus competent cells using electroporation. Single colonies were then selected for colony PCR verification to confirm successful gene transfer. The successfully transformed engineered Rhodococcus 1 was preserved for future use. Similarly, recombinant pNV plasmid 2 and recombinant pNV plasmid 3 were simultaneously transformed into naturally derived Rhodococcus competent cells using electroporation. Single colonies were then selected for colony PCR verification. The successfully transformed engineered Rhodococcus 2 was preserved for future use.

[0101] 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 metabolic enzyme, respectively; the amino acid sequence of the expression product of the wild-type target gene 2 is shown in SEQ ID NO:3; and the mutant target gene 3 expresses the β subunit mutant (amino acid sequence as shown in SEQ ID NO:4) and α subunit mutant (amino acid sequence as shown in SEQ ID NO:5), respectively.

[0102] The amino acid sequences of the β and α subunits of the wild-type nitrile metabolic enzyme are as follows:

[0103] The β subunit, whose amino acid sequence is shown in SEQ ID NO:1, is as follows:

[0104] MDGIHDTGGMTGYGPVPYQKDEPFFHYEWEGRTLSILTWMHLKGMSWWDKSRFFRESMGNENYVNEIRNSYYTHWLSAAERILVADKIITEEERKHRVQEILEGRYTDRNPSRKF DPAEIEKAIERLHEPHSLALPGAEPSFSLGDKVKVKNMNPLGHTRCPKYVRNKIGEIVTSHGCQIYPESSSAGLGDDPRPLYTVAFSAQELWGDDGNGKDVVCVDLWEPYLISA;

[0105] The α subunit, whose amino acid sequence is shown in SEQ ID NO:2, is as follows:

[0106] MSEHVNKYTEYEARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKSWVDPEYRKWLEEDATAAMASLGYAGEQAHQISAVFNDSQTHHVVVCTLCSCYPWPVLGLPPAWYKSMEYRSRVVADCRGVLKRDFGFDIPDEVEVRVWDSSSEIRYIVIPERPAGTDGWSEDELAKLVSRDSMIGVSNALTPQEVIV.

[0107] The amino acid sequence of the wild-type nitrile metabolic enzyme is shown in SEQ ID NO:3, and is as follows:

[0108] MVEYTNTFKVAAVQAQPVWFDAAKTVDKTVSIIAEAARNGCELVAFPEVFIPGYPYHIWVDSPLAGMAKFAVRYHENSLTMDSPHVQRLLDAARDHNIAVVVGISERDGGSLYMTQLIIDADGQLVARRRKLKPTHVERSVYGEGNGSDISVYDMPFARLGALNCWEHFQTLTKYAMYSMHEQ VHVASWPGMSLYQPEVPAFGVDAQLTATRMYALEGQTFVVCTTQVVTPEAHEFFCENEEQRKLIGRGGGFARIIGPDGRDLATPLAEDEEGILYADIDLSAITLAKQAADPVGHYSRPDVLSLNFNQRRTTPVNTPLSTIHATHTFVPQFGALDGVRELNGADEQRALPSTHSDETDRATASI.

[0109] The amino acid sequences of the β and α subunits of the mutant nitrile metabolic enzyme are as follows:

[0110] The β subunit mutant, whose amino acid sequence is shown in SEQ ID NO:4, is as follows:

[0111] MDGIHDTGGMTGYGPVPYQKDEPFFHYEWEGRTLSILTWMHLKGMSWWDKSRFFRESMGNENYVNEIRNSYYTHWLSAAERILVADKIITEEERKHRVQEILEGRYTDRNPSRKF DPAEIEKAIERLHEPHSLALPGAEPKFKEGDKVKVKNMNPLGHTRCPKYVRSKIGEIVTSHGCQIYPESSSAGLGDDPRPLYTVAFSAQELWGDDGNGKCVVCVDLWEPYLISA;

[0112] The α-subunit mutant has the amino acid sequence shown in SEQ ID NO:5, specifically as follows: MSEHVNKYTEYEARTKAIETLLYERGLITPAAVDRVVSYYENEIGPMGGAKVVAKSWVDPEYRKWLEEDATAAMASLGYAGEQAHQISAVFNDSQTHHVVVCTLCSCYPWPVLGLPPAWYKSMEYRSRVVADCRGVLKRDFGFDIPDEVEVRVWDSSSEIRYIVIPERPAGTDGWSEDELAKLVSRDSMIGVSNALTPQEVIV.

[0113] Example 2: Rhodococcus fermentation culture

[0114] The fermentation culture method for Rhodococcus is as follows:

[0115] (1) Use a 10 μL inoculation loop to pick the correct colony from the streak plate and place it into 15 mL of liquid seed culture medium. Incubate at 30 °C and 200 rpm for 3 days in a shaking incubator.

[0116] (2) Measure the absorbance value OD600 of the seed liquid, calculate the inoculation volume according to the initial OD600 = 1, and 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℃ and 200rpm for 2 days. Then collect the original fermentation broth of Rhodococcus (i.e., recombinant Rhodococcus fermentation broth or non-recombinant Rhodococcus fermentation broth; OD600 is about 0.2-1.6).

[0117] Example 3. Degradation Activity Verification

[0118] This experiment primarily investigates and verifies the expression of hydrolytic and hydratase activities in the successfully transformed engineered Rhodococcus 2. The specific experimental steps are as follows.

[0119] Engineered Rhodococcus 2 (obtained by fermentation culture using the method in Example 2; OD600 1.2) was mixed with 50 mg / L different cyanide solutions (sodium cyanide, potassium cyanide, and acrylonitrile) at a mass ratio of 1:20 in Erlenmeyer flasks, and the mixtures were reacted for 3 days in a sealed, full-temperature shaking incubator. The reaction conditions were: temperature 30℃, pH 7. Post-reaction analysis was performed after the reaction.

[0120] 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. Furthermore, infrared spectroscopy was used to detect the components in the reaction solution, such as... Figure 1 As shown, the presence of carboxyl and amine characteristic peaks in the solution indicates that the successfully transformed Rhodococcus can express hydrolases and hydratases.

[0121] Example 4. Cyanide degradation of engineered Rhodococcus and natural Rhodococcus

[0122] This experiment mainly investigated the effects of bioremediation agents and chemical degradation agents prepared using engineered Rhodococcus 1, engineered Rhodococcus 2, and unreconstituted Rhodococcus, respectively, on cyanide degradation. Different Rhodococcus fermentation broths (OD600 around 1) were selected and mixed with a stabilizer (the stabilizer components were: 20wt% glycerol, 4M PEG 4000, and 0.03wt% glutaraldehyde) at a 1:1 mass ratio to prepare bioremediation agents. The bioremediation agents and chemical degradation agents (the chemical degradation agent is Fe...) were then used... 2+ The mixture (prepared with hydrogen peroxide at a mass ratio of 1:1) was mixed with acrylonitrile (350 mg / L) at a mass ratio of 5:40 and added to a 500 mL Erlenmeyer flask. The mixture was then reacted for 3 days in a full-temperature shaking incubator at 30 °C, pH 7, and a stirring speed of 200 rpm / min.

[0123] After the experiment, the results were tested, such as... Figure 2 As shown, after the bioremediation agent prepared from the fermentation broth of engineered Rhodococcus 2 degraded the cyanide solution, the cyanide content in the solution decreased from 350 mg / L to 98 mg / L, with a degradation rate of 72.00%, which was higher than the cyanide degradation rate of other bioremediation agents prepared from Rhodococcus stock solutions. Moreover, after three days of degradation, the degradation rate of the cyanide solution prepared from the bioremediation agent prepared from the engineered Rhodococcus stock solution was much higher than that of chemical degradation agents.

[0124] Example 5. Effect of stabilizers on the degradation of cyanide by bioremediation agents

[0125] This experiment mainly investigated the effect of stabilizers prepared with different amounts of added components on the degradation effect of bioremediation agents. Stabilizers were prepared by optimizing the addition of different concentrations of glycerol, polyethylene glycol 4000, and glutaraldehyde (single-factor optimization was used, optimizing glycerol, polyethylene glycol 4000, and glutaraldehyde sequentially). The stabilizers were then mixed with engineered Rhodococcus 2 fermentation broth (OD600 approximately 1) at a mass ratio of 3:7 to form the bioremediation agent (engineered Rhodococcus 2 fermentation broth (OD600 approximately 1) served as a control). The biodegradation reaction was then carried out to obtain the biodegraded solution. In another experiment, the bioremediation agent and acrylonitrile aqueous solution (280 mg / L) were mixed at a mass ratio of 1:10 and reacted for 3 days in a sealed, full-temperature shaking incubator. The biodegradation reaction conditions were: temperature 30℃ and pH 7. The biodegraded solution was then centrifuged to separate the solid and liquid phases, obtaining the filtrate. The results are as follows: Figure 3 (The original liquid in the attached figure is the fermentation broth) As shown, the degradation effect varies depending on the stabilizer prepared with different components. When the stabilizer is prepared with 30wt% glycerol, 0.07wt% glutaraldehyde, and 8M polyethylene glycol 4000, the decyanation rate of the filtrate is 97.82%.

[0126] Example 6. Effect of stabilizers on the degradation of cyanide by bioremediation agents

[0127] This experiment mainly investigated the effect of different addition ratios of engineered Rhodococcus 2 fermentation broth (OD600 around 1) and stabilizer (prepared according to the ratio in Example 5, 30wt% glycerol, 0.07wt% glutaraldehyde, and 8M polyethylene glycol 4000) on cyanide degradation. A cyanide solution (i.e., acrylonitrile aqueous solution) with a concentration of 280 mg / L was prepared. The engineered Rhodococcus 2 fermentation broth (OD600 around 1) was mixed with bioremediation agents prepared in different stabilizer ratios and the cyanide solution, and a biodegradation reaction was carried out to obtain a biodegraded solution. Specifically, the bioremediation agent and acrylonitrile aqueous solution were mixed at a mass ratio of 1:10 and reacted for 3 days in a sealed, full-temperature shaking incubator. The biodegradation reaction conditions were: temperature 30℃ and pH 7. The biodegraded solution was centrifuged to separate the solid and liquid phases, and the filtrate was obtained. Figure 4 As shown, after the engineered Rhodococcus 2 fermentation broth was reacted with different stabilizer ratios, the cyanide degradation rate of the resulting filtrate was approximately 70%–99%. When the mass ratio of the engineered Rhodococcus 2 fermentation broth to stabilizer was 40wt%:60wt%, the cyanide removal rate of the filtrate was 98.93%.

[0128] Example 7. Effects of different strains on cyanide degradation

[0129] This experiment mainly investigates the effects of different bacterial strains on the degradation of cyanide.

[0130] Preparation of strains: Following the method in Example 1, the recombinant expression vector was transformed into Bacillus subtilis 168, Bacillus magaterium, and Escherichia coli, respectively, to obtain engineered Bacillus subtilis, engineered Bacillus magaterium, and engineered Escherichia coli. Fermentation broths of different strains were obtained using conventional methods.

[0131] Bioremediation agents containing different strains (fermentation broths of different strains (OD600 around 1) and stabilizers were prepared at a mass ratio of 4:6; the stabilizers were prepared according to the ratio described in Example 5, specifically 30wt% glycerol, 0.07wt% glutaraldehyde, and 8M polyethylene glycol 4000) and 200mg / L acrylonitrile aqueous solution were mixed at a mass ratio of 1:8 and added to a 500mL Erlenmeyer flask. The reaction was carried out for 3 days in a full-temperature shaking incubator at 30℃, pH 7, and a stirring speed of 200rpm / min. After the experiment, the cyanide content in the bioremediation agent containing Escherichia coli decreased from 235mg / L to 120mg / L, with a degradation rate of 48.93%. In the degradation system containing Rhodococcus, the cyanide content decreased from 235mg / L to 48mg / L, with a degradation rate of 79.57%. The degradation rates of other strains were as follows: Figure 5 As shown.

[0132] Example 8. Effects of temperature and pH on cyanide degradation rate

[0133] This experiment mainly investigated the effects of temperature and pH on the degradation efficiency of cyanide by bioremediation agents. Under pH 7 conditions, 40 mL of 300 mg / L acrylonitrile aqueous solution was added to a dry conical flask, followed by 2 mL of bioremediation agent (prepared according to the stabilizer ratio in Example 5, specifically consisting of 30 wt% glycerol, 0.07 wt% glutaraldehyde, and 8M polyethylene glycol 4000; the bioremediation agent was prepared at a mass ratio of 4:6 of the fermentation broth of *Rhodococcus spp.* (OD600 approximately 1) to the stabilizer). The reaction was carried out in a full-temperature shaking incubator at a stirring speed of 200 rpm for 1-5 days. After the experiment, the cyanide content at different temperatures was measured, and the results are as follows: Figure 6 As shown in (a). At 40℃, 40 mL of a 300 mg / L acrylonitrile aqueous solution was added to a dry conical flask, followed by 2 mL of the prepared bioremediation bacterial agent. The mixture was stirred at 200 rpm / min and incubated in a full-temperature shaking incubator for 1-5 days. After the experiment, the cyanide content at different pH levels was measured, and the results are shown below. Figure 6 As shown in (b). Figure 5 The results showed that the optimal temperature for cyanide degradation was 40℃, the optimal pH was 7, and under the optimal conditions, approximately 99% of 300 mg / L cyanide could be degraded within 5 days.

[0134] Application examples

[0135] Application Example 1. Degradation of cyanide in actual wastewater by bioremediation agents

[0136] This experiment mainly investigates the degradation effect of a bioremediation agent containing engineered Rhodococcus 2 on cyanide in actual wastewater.

[0137] 500 mL of chemical wastewater was directly mixed with 10 mL of bioremediation agent (the stabilizer was prepared according to the method in Example 5; the specific components of each stabilizer are shown in Table 1; the bioremediation agent was prepared according to a mass ratio of 4:6 of the fermentation broth of *Rhodococcus 2* for engineering modification to the stabilizer; the OD600 values ​​of each fermentation broth are shown in Table 1). The biodegradation reaction was carried out in a sealed, full-temperature shaking incubator at a degradation temperature of 40℃, pH of 7, and a shaking speed of 200 rpm / min. Each experiment was performed in triplicate. Samples were taken on days 0, 2, 4, and 5 of the culture. The initial concentration of cyanide in the wastewater was measured to be 324 mg / L on day 0 (targeting the cyano functional group). The sampling method involved weighing 2 mL of sample from each conical flask, centrifuging, and then passing the supernatant through anhydrous sodium sulfate to remove water, followed by filtering through a 0.22 μm organic filter membrane into a sample vial for analysis. After 5 days of incubation, the cyanide degradation rate in each experimental group exceeded 96%. The cyanide degradation rates for each experimental group are shown in Table 1. This result indicates that the bioremediation agent containing engineered Rhodococcus 2 can be directly added to high-concentration chemical wastewater containing cyanide for degradation.

[0138] Table 1. Stabilizer Composition

[0139]

[0140] Application Example 2. Degradation of Cyanide in Soil by Bioremediation Agents

[0141] This experiment mainly investigated the degradation effect of a bioremediation agent containing Rhodococcus bacteria (the stabilizer was prepared according to the method in Example 5, and the specific components of each stabilizer are shown in Table 2; the bioremediation agent was prepared according to the mass ratio of Rhodococcus 2 fermentation broth to stabilizer of engineering modification at 4:6, and the OD600 values ​​of each fermentation broth are shown in Table 2) on cyanide in actual soil. 10g of sludge from the chemical plant periphery was directly mixed with 30mL of the bioremediation agent for biodegradation. The degradation reaction was carried out in a sealed, full-temperature shaking incubator at a degradation temperature of 40℃, pH of 7, and a shaking speed of 200rpm / min. Each experiment was repeated in triplicate. Samples were taken on days 0, 2, 4, and 5 of the incubation period. The initial concentration of cyanide in the sludge was measured to be 108mg / L on day 0 (using cyano functional groups as the target analyte). The sampling method involved weighing 5 mL of the sample suspension from the conical flask, centrifuging, filtering, and then passing the supernatant through anhydrous sodium sulfate to remove water before filtering it through a 0.22 μm organic filter membrane into a sample bottle for instrumental testing. After 5 days of incubation, the degradation rate of each experimental group reached over 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 cyanide-containing soil for degradation reaction.

[0142] Table 2. Stabilizer Composition

[0143]

Claims

1. A bioremediation agent containing Rhodococcus, said bioremediation agent comprising Rhodococcus ferment broth and a stabilizer; The stabilizer includes glycerol, polyethylene glycol, and glutaraldehyde; wherein... The content of glycerol is at least 20 wt%, glutaraldehyde is at least 0.03 wt%, and polyethylene glycol is at least 4 M. The Rhodococcus bacteria in the fermentation broth were obtained through gene recombination. The recombinant Rhodococcus expresses proteins with amino acid sequences as shown in SEQ ID NO:1 to SEQ ID NO:3, or the recombinant Rhodococcus expresses proteins with amino acid sequences as shown in SEQ ID NO:3 to SEQ ID NO:5; The polyethylene glycol is polyethylene glycol 4000.

2. The bioremediation agent according to claim 1, wherein the stabilizer is prepared from 20-35 wt% glycerol, 0.03-0.10 wt% glutaraldehyde, and 4-10 M polyethylene glycol.

3. The bioremediation agent according to claim 1, wherein the mass ratio of the Rhodococcus ferment broth to the stabilizer is (1-90):(10-99).

4. The bioremediation agent according to claim 1, wherein the OD600 of the Rhodococcus ferment broth is higher than 0.

2.

5. The bioremediation agent according to claim 1, wherein the mass ratio of the Rhodococcus ferment broth to the stabilizer is (10-50): (10-90).

6. The bioremediation agent according to claim 1, wherein the mass ratio of the Rhodococcus ferment broth to the stabilizer is (30-50): (50-70).

7. The bioremediation agent according to claim 1, in wild-type Rhodococcus (… Rhodococcus ruber The recombinant plasmid containing a gene sequence related to nitrile metabolism enzymes was introduced into the Rhodococcus recombinant structure; the recombinant Rhodococcus expressed the nitrile metabolism enzyme system and its mutants.

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

9. The application of the bioremediation agent according to any one of claims 1-8, wherein the bioremediation agent is used to degrade cyanide.

10. The application according to claim 9, wherein the application includes degradation of cyanide in water and / or degradation of cyanide in soil.

11. The application according to claim 9, 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.

12. In the application according to claim 9, 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.

13. In the application according to claim 9, when the bioremediation agent is used for the degradation of cyanide in water and / or soil, the cyanide concentration is 1-350 mg / L.

14. In the application according to claim 9, when the bioremediation agent is used for the degradation of cyanide in water, the mass ratio of the bioremediation agent to water is (0.5-10):(1-50).

15. In the application according to claim 9, when the bioremediation agent is used for the degradation of cyanide in water, the mass ratio of the bioremediation agent to water is (1-5):(1-40).

16. In the application according to claim 9, when the bioremediation agent is used for the degradation of cyanide in soil, the volume-to-mass ratio of the bioremediation agent to the soil is (10-60) mL:(0.1-20) g.

17. In the application according to claim 9, when the bioremediation agent is used for the degradation of cyanide in soil, the volume-to-mass ratio of the bioremediation agent to the soil is (10-40) mL:(2-15) g.

18. In the application according to claim 9, when the bioremediation agent is used for cyanide degradation, the degradation temperature is 20-70℃.

19. In the application according to claim 9, when the bioremediation agent is used for cyanide degradation, the degradation temperature is 20-50℃.

20. In the application according to claim 9, when the bioremediation agent is used for cyanide degradation, the degradation temperature is 35-45℃.

21. In the application according to claim 9, when the bioremediation agent is used for the degradation of cyanide, the degradation pH is 3-12.

22. In the application according to claim 9, when the bioremediation agent is used for the degradation of cyanide, the degradation pH is 5-9.

23. In the application according to claim 9, when the bioremediation agent is used for the degradation of cyanide, the degradation pH is 7-9.

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