CN109576187B

CN109576187B - Cyanide degradation strain and method for degrading cyanide by using same

Info

Publication number: CN109576187B
Application number: CN201811617188.9A
Authority: CN
Inventors: 刘元元; 杨传伦; 徐泽平; 吴文雷; 苗青; 王春; 张心青
Original assignee: Chambroad Chemical Industry Research Institute Co Ltd
Current assignee: Chambroad Chemical Industry Research Institute Co Ltd
Priority date: 2018-12-27
Filing date: 2018-12-27
Publication date: 2022-04-15
Anticipated expiration: 2038-12-27
Also published as: CN109576187A

Abstract

The invention belongs to the technical field of biological engineering, and provides a cyanide degradation strain and a method for degrading cyanide by using the same, wherein the provided cyanide degradation production strain is bacillus, 16S rDNA identifies the bacillus cereus, the strain code is YJY18-02, and the preservation number of the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC No. 16151. The strain can be applied to degrading cyanide, and the degradation rate of the strain for degrading cyanide with the concentration of 300mg/L can reach more than 90%.

Description

Cyanide degradation strain and method for degrading cyanide by using same

Technical Field

The invention belongs to the technical field of bioengineering, and provides a cyanide degradation strain and a method for degrading cyanide by using the same.

Background

Cyanide refers to a compound containing a cyano group. Cyanide belongs to a highly toxic substance, and the toxic effect of the cyanide on human bodies is mainly to combine with blood ferricytochrome oxidase to generate cyanide ferricytochrome oxidase, so that the blood loses the effect of oxygen transfer, the tissue is anoxic to cause asphyxia, and the hydrocyanic acid which touches 50mg/L of oral mucosa can die immediately. It discharges a large amount of cyanide-containing waste water in the production processes of non-ferrous metal mineral extraction of gold, silver and copper, cyaniding electroplating, chemical industry, coking, heat treatment and other industries.

The cyanide-containing waste water treatment method comprises a chemical method, a chemical physical method, a biological method, a natural degradation method and a combined process method. The biological method is to decompose cyanide into CO by using cyanide-degrading bacteria₂Ammonia, etc. In recent years, the biological method for degrading cyanide has been developed greatly, and is considered to be the cyanide degradation technology with the most potential and prospect due to low cost, small environmental pollution, good quality of discharged water and small amount of slag.

In recent years, researchers at home and abroad do a lot of work on screening and separating cyanide degradation microbial agents, wherein various strains such as pseudomonas, pseudomonas fluorescens, nocardia and the like are separated. The Agrobacterium tumefaciens TQH6 screened from Chinese patent CN20150829703 degrades cyanide, and when the cyanide concentration is 1-10mg/L, the degradation effect is good, and the cyanide degradation rate is 90-100% in 18-78h, but the degradation rate is rapidly reduced when the cyanide concentration is increased. The key of the biological method for treating cyanide-containing wastewater is to find a microbial strain with high cyanide concentration resistance and good degradation effect, so that screening and degrading a high-concentration cyanide strain from the nature becomes one of the important tasks of researchers.

Disclosure of Invention

Aiming at the defects of the technology, the invention provides a cyanide degradation strain and a method for degrading cyanide by using the same, the provided cyanide degradation production strain is bacillus, is identified as bacillus cereus by 16S rDNA, has a strain code of YJY18-02, and has a preservation number of CGMCC No. 16151; the strain can be applied to degrading cyanide, and the degradation rate of the strain for degrading cyanide with the concentration of 300mg/L can reach more than 90%.

The specific technical scheme provided by the invention is as follows:

firstly, a new Bacillus cereus strain is obtained, the preservation number of the strain is CGMCC No.16151, and the specific obtaining method is as follows:

(1) separating 20 strains capable of degrading cyanide from oil refinery soil in places of Shandong Binzhou, and separating and purifying;

(2) 2 strains with high degradation capability are screened from 20 strains capable of degrading cyanide;

(3) 2 to-be-selected bacteria are subjected to relatively deep researches on physiological and biochemical characteristics, genetic stability, degradation rate and the like, a strain which is strong in degradation capability, easy to culture and stable in passage characteristic is finally screened out, and the strain is initially named as YJY-103;

(4) the strain is taken as an original strain, and a conventional mutagenesis method, such as repeated mutagenesis and secondary screening of UV and chemical mutagens, is adopted to finally obtain the strain YJY18-02 capable of efficiently degrading cyanide;

the inventor carries out biological preservation on the strain in China general microbiological culture Collection center with the preservation number of CGMCC No.16151, and the strain is detected to be in a survival state.

Morphological characteristics of the above cyanide-degrading strains: gram staining positive, rod shape, large thallus, two ends blunt round, short-chain rod arrangement, no capsule. Circular spores smaller than the thallus can be formed after 6h of culture.

The bacterial strain forms a circular and convex bacterial colony on common nutrient agar, and the surface of the bacterial colony is rough, has wax light, is opaque and is similar to ground glass.

The physiological and biochemical reactions of the strain are as follows:

carbohydrate test: the strain can decompose glucose, maltose, dextrin, sucrose and salicin, can utilize citrate, produces acid without producing gas, does not decompose lactose, xylose and mannitol, does not produce indole, and has positive VP test; nitrogen-containing compound test: on lead acetate gelatin medium, the bacterium H₂Negative S, can liquefy gelatin; and (3) carrying out an opalescent reaction: 10 percent ofCulturing on a yolk agar plate for 3h to grow aseptic colonies, but forming turbid rings at the seeding positions, and expanding the diameter of the turbid rings to 5-6mm after 6 h; positive in catalase test.

The biochemical reaction of the strain is consistent with that of Bacillus cereus. Therefore, the strain obtained by screening can be preliminarily determined to be the bacillus cereus.

The inventor simultaneously carries out 16S rDNA sequencing on the strain, the nucleotide sequence of the sequence is shown as Seq ID No:1, the sequence is that the nucleotide sequence of the 16S rDNA of the strain YJY18-02 has more than 99 percent of homology with the nucleotide sequence of different strains of Bacillus (Bacillus sp.), and has 100 percent of homology with 5 strains specifically marked as Bacillus cereus, thereby further determining that the strain provided by the invention is the Bacillus cereus strain.

The invention also aims to provide a method for degrading cyanide by using the strain, which can be more particularly applied to a treatment process for treating cyanide-containing wastewater and comprises the following specific steps:

A. activating strains:

B. preparing liquid seeds:

C. and (3) fermentation control: propagating seed liquid through liquid fermentation, producing fermentation liquor through liquid fermentation amplification, and obtaining thalli after centrifugation;

D. preparing a solid microbial inoculum: centrifuging the fermented liquid to remove supernatant, adding diatomite and bran into the precipitate, mixing, air drying, and pulverizing;

wherein, the fermentation liquor and the solid microbial inoculum prepared by the steps can be used for degrading cyanide.

Furthermore, the specific production steps are as follows:

(1) activating strains: transferring the test tube slant strains stored on the nutrient agar culture medium at 4 ℃ to room temperature (20-25 ℃) for activation for 4-8 h;

(2) preparing liquid seeds: preparing activated test tube slant strains into a strain suspension by using 10mL of sterilized distilled water on an aseptic operation table, washing the strain suspension into triangular flasks filled with a sterilized liquid seed culture medium, inoculating 1 triangular flask with 1 test tube strain, and performing shake culture for 10-15h to prepare a seed solution;

(3) and (3) fermentation process: inoculating the prepared liquid seeds into a sterilized fermentation culture medium in an inoculation amount of 5-10% (v/v), and performing liquid fermentation to obtain the bacillus cereus thallus;

(4) preparing a solid microbial inoculum: and (3) performing solid-liquid separation on the fermented mycelia, adding assistants such as diatomite and the like into the mycelia, drying at the temperature lower than 60 ℃, crushing, and sieving with a 80-mesh sieve to obtain the bacillus cereus microbial inoculum.

The method for determining the conditions of the strain for producing the bacillus cereus comprises the following steps:

(1) determining the optimal culture conditions of liquid seeds and the optimal culture conditions of liquid fermentation by adopting a single-factor test and an orthogonal test and changing the temperature, the initial pH value, the inoculation amount and the culture time;

(2) the optimal culture medium composition of the liquid seed and the optimal culture medium composition of the liquid fermentation of the strain are determined by changing the carbon-nitrogen source and the inorganic salt composition of the culture medium by adopting a single factor test and an orthogonal test.

The compositions of the strains, the liquid seed culture medium and the fermentation conditions obtained by the single factor test and the orthogonal test are as follows:

the liquid seed culture medium comprises the following components: 1-2g/L glucose, 1-4g/L yeast powder, 0.5-1.0g/L sodium chloride, 0.2-0.5g/L magnesium sulfate and pH 7.2;

liquid seed culture conditions: the inoculation amount is that each triangular flask is inoculated with a test tube, the culture temperature is 30-35 ℃, the rotating speed of a shaking table is 160-200rpm, and the culture time is 10-15 h;

the compositions and fermentation conditions of the liquid fermentation culture medium of the strain obtained by the single factor test and the orthogonal test are as follows:

liquid medium composition: corn flour 5-20g/L, soybean meal 5-25g/L, ammonium nitrate 1-5g/L, manganese sulfate 0.1-0.5g/L, magnesium sulfate 0.3-0.8g/L, sodium chloride 0.1-0.8g/L, ferrous sulfate 0.01-0.05g/L, calcium chloride 0.1-0.5g/L, and pH 7.0-7.5;

liquid fermentation conditions: the inoculation amount is 5-10% (v/v), the culture temperature is 30-35 ℃, the rotating speed of a shaking table is 160-200rpm, and the culture time is 44-48 h;

in order to improve the degradation efficiency of cyanide, the inventor prepares the thalli in the fermentation liquor into a solid microbial inoculum, and the specific steps are as above, the thalli are adsorbed on diatomite and bran, and are ventilated and dried at 20-50 ℃ to prepare the solid cyanide degradation microbial inoculum, the survival rate of the thalli in the degradation microbial inoculum is above 70-80%, and compared with the treatment effect of directly adding the same amount of mycelia, the degradation rate is improved by above 10%. The prepared solid microbial inoculum is convenient to store and transport, and the diatomite in the solid carrier can promote the functions of neutralization, flocculation, adsorption, sedimentation and filter aid of sewage suspended matters in sewage treatment.

The microbial inoculum obtained by the invention can effectively treat wastewater with cyanide concentration of less than 350mg/L, and only needs to add the solid microbial inoculum prepared by the method into a sewage treatment aerobic system according to one thousandth of the total amount of the wastewater during treatment, and then the treatment is carried out according to an aerobic treatment process.

The bacterial strain obtained by the invention has the following advantages of degrading cyanide:

1. the cost is low. The main raw materials in the culture medium used for fermentation are corn flour and bean pulp which are agricultural products, and the culture medium has the advantages of huge yield, low price and wide sources.

2. Good application effect and high degradation efficiency. According to the invention, the diatomite is used as a carrier, so that the survival rate of thalli is high, the effect is good, and the bran is added into the microbial inoculum, so that the microbial inoculum can be utilized by microorganisms after the effect is exerted, and the microbial inoculum has important significance for protecting the ecological environment and reducing the content of organic pollutants polluting the natural environment, and particularly has a good degradation effect on cyanides. When the method is implemented in water polluted by cyanide, the cyanide can be effectively removed, and the removal rate of the pollutants in the water can reach 90% or more.

In conclusion, the invention provides a bacillus cereus strain and a method for degrading cyanide by using the strain for liquid fermentation, the strain can be applied to degrading cyanide, and the degradation rate of the microbial inoculum obtained by fermentation of the method is more than 90% at the concentration of 300mg/L cyanide.

Preservation information

Preservation time: 7 month and 25 days 2018

The name of the depository: china general microbiological culture Collection center

The preservation number is: CGMCC No.16151

The address of the depository: microbial research institute of Hospital's institute of Zhongkou No.1 Xilu, Beijing, Chaoyang, and Hokkaido

Classification nomenclature-Bacillus cereus

Drawings

FIG. 1 shows cyanide degradation by Bacillus cereus according to the present invention at various temperatures.

Detailed Description

The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the above subject matter is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention, except for the specific description, the following examples all adopt the conventional prior art to complete the strains adopted in the following examples are the strains with the preservation number of CGMCC No. 16151.

Example 1

(1) Activating strains: transferring the test tube slant strains stored on the nutrient agar culture medium at the temperature of 4 ℃ to the room temperature (20-25 ℃) for activation for 4 hours;

(2) preparing liquid seeds: preparing activated test tube slant strains into a strain suspension by using 10mL of sterilized distilled water on an aseptic operation table, washing the strain suspension into triangular flasks filled with a sterilized seed culture medium under an aseptic condition, inoculating 1 test tube strain into 1 triangular flask, and culturing at a natural pH value and a culture temperature of 32 ℃ and a shaking table rotating speed of 200r/min for 15 h;

seed culture medium: 1g/L glucose, 1g/L yeast powder, 0.5g/L sodium chloride, 0.2g/L magnesium sulfate, pH7.2, sterilization conditions of 121 ℃, 0.15Mpa and 20 min;

(3) fermentation and regulation processes: inoculating liquid seeds into a fermentation culture medium in an inoculation amount of 10% (v/v), stirring at a rotation speed of 175r/min, and culturing at 32 ℃ for 48 h;

the fermentation medium comprises 12g/L of corn flour, 15g/L of soybean meal, 1.5g/L of ammonium nitrate, 0.2g/L of manganese sulfate, 0.4g/L of magnesium sulfate, 0.3g/L of sodium chloride, 0.05g/L of ferrous sulfate, 0.2g/L of calcium chloride, pH7.2, sterilization conditions of 121 ℃, 0.15Mpa and sterilization for 20 min; (ii) a

And after the fermentation is finished, centrifuging the fermentation liquor at 4000rpm for 20min to obtain thalli, adding 2% (w/w) of diatomite and 1% (w/w) of bran to adsorb to obtain a solid microbial inoculum, and determining the survival rate of spores of the solid microbial inoculum to be 78%.

Example 2

(1) Activating strains: transferring the test tube slant strains stored on an LB culture medium at the temperature of 4 ℃ to the room temperature (20-25 ℃) for activation for 6 hours;

(2) preparing liquid seeds: preparing activated test tube slant strains into a strain suspension by using 10mL of sterilized distilled water on an aseptic operation platform, washing the strain suspension into triangular flasks filled with a sterilized seed culture medium, inoculating 1 test tube strain into 1 triangular flask, and culturing at the temperature of 30 ℃ and the rotating speed of a shaking table of 200r/min for 12h, wherein the pH value is natural;

the seed culture medium comprises the following components: 1.2g/L glucose, 1.2g/L yeast powder, 0.5g/L sodium chloride, 0.2g/L magnesium sulfate, pH7.2, sterilization conditions of 121 ℃, 0.15Mpa and 20 min; (ii) a

(3) Fermentation and regulation processes: inoculating liquid seeds into 2L fermentation medium at an inoculum size of 10% (v/v), and culturing at 32 deg.C for 48 h;

the fermentation medium comprises the following components: 15g/L of corn flour, 15g/L of soybean meal, 2g/L of ammonium nitrate, 0.2g/L of manganese sulfate, 0.4g/L of magnesium sulfate, 0.2g/L of sodium chloride, 0.05g/L of ferrous sulfate, 0.2g/L of calcium chloride, pH7.3, sterilization conditions of 121 ℃, 0.15Mpa and sterilization for 20 min;

and after the fermentation is finished, centrifuging the fermentation liquor at 4000rpm for 20min to obtain thalli, adding 1.5% (w/w) of diatomite and 1.2% (w/w) of bran to adsorb to obtain the solid microbial inoculum, and determining the survival rate of spores of the solid microbial inoculum to be 80%.

Example 3

Effect of temperature on cyanide degradation efficiency

In an inorganic salt culture medium containing cyanide, the formula is as follows: per liter contains100mg/L cyanide, 0.5g K₂HPO₄、0.5g KH₂PO₄、0.01g FeSO₄、0.1g CaCl₂、0.1g MgSO₄、0.1gMnSO₄、0.2g NaCl、1g NH₄NO₃The method comprises the steps of supplementing 1L of distilled water, keeping the pH natural, placing the mixture in shaking tables for culturing at the temperatures of 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃, respectively, wherein the addition amount of the solid microbial inoculum obtained in example 1 is 0.1% (w/w), sampling for 24 hours to determine the cyanide concentration, and obtaining a result shown in figure 1. as can be seen from figure 1, the optimum temperature of the bacterial strain for degrading the cyanide reaches 100 percent at 30 ℃, and the cyanide degradation rate of the bacterial strain can exceed 90 percent at each temperature point.

Example 4

1g of the solid microbial inoculum prepared in the example 1 is weighed and inoculated into 1000mL of coking wastewater with the cyanide content of 290mg/L, the wastewater indexes of COD are 200mg/L and ammonia nitrogen is 2000mg/L, aeration treatment is carried out for 24h at 30 ℃, residues are determined, and the removal rate of the cyanide is 95%. The method for measuring the cyanide content is an isonicotinic acid-pyrazolone spectrophotometry in national standard HJ 484-2009.

Example 5

The fermentation broth from example 2 was used as the experimental broth. 2L of waste water containing 150mg/L of cyanide per liter, 0.5 percent of liquid microbial inoculum addition, taking 10mL of fermentation liquor, centrifuging for 20min at 4000rpm to obtain thalli, adding the thalli into the waste water, carrying out aeration treatment for 24h at 30 ℃, and measuring the residue, wherein the removal rate of the cyanide is 97 percent. The method for measuring the cyanide content is an isonicotinic acid-pyrazolone spectrophotometry in national standard HJ 484-2009.

Example 6

A certain petrochemical company coking device produces cyanide-containing waste water 10m per day³Detection and analysis show that COD is about 900mg/L, ammonia nitrogen is 110mg/L, and cyanide is 301 mg/L. Utilizing a certain sewage treatment plant 100m³The waste water is treated by the comprehensive aerobic tank, the dosage of the solid bactericide for initially degrading cyanide (example 1) is one thousandth, the temperature of the aerobic tank is controlled to be 25-35 ℃, the dissolved oxygen is 2-6mg/L, water is continuously fed in and discharged, and 10m of water is fed in every day³The water inlet flow is 0.42m³The residence time is 10 days,the microbial inoculum is replenished once every ten days, the effluent index is tracked and detected every day, the device continuously operates for two months, the effluent COD is less than 50mg/L, the ammonia nitrogen is less than 5mg/L, the cyanide is less than 5mg/L, the removal rate of the cyanide is more than 95%, and the low-cost effective treatment of the high cyanide wastewater is realized. The method for measuring the cyanide content is an isonicotinic acid-pyrazolone spectrophotometry in national standard HJ 484-2009.

Comparative example

1g of the solid microbial inoculum prepared in example 1 was weighed and inoculated into 1000mL of coking wastewater with a cyanide content of 100mg/L, and a commercially available solid preparation was added in an amount of 0.1% of the microbial inoculum to prepare 1000mL of coking wastewater with a cyanide content of 100mg/L, aeration treatment was started at 30 ℃ and cyanide residues were measured at regular intervals until the cyanide content was not changed. This experiment was repeated 3 times. The method for measuring the cyanide content is an isonicotinic acid-pyrazolone spectrophotometry in national standard HJ 484-2009. The cyanide removal rate of the commercial solid preparation for 24 hours reaches 60%, the cyanide removal rate of the commercial solid preparation for 48 hours reaches 65%, the cyanide removal rate of the commercial solid preparation for 24 hours is 91%, and the cyanide removal rate of the commercial solid preparation for 48 hours is 100%.

<110> Jingbo chemical research institute of yellow river delta Ltd

<120> cyanide degradation strain and method for degrading cyanide by using same

<160>1

<210>1

<211>1417

<212>DNA

<213> Bacillus cereus (Bacillus cereus)

<400>1

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agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca 300

ccggcagtca ccttaaagtg cccaacttaa tgatggcaac taagatcaag ggttgcgctc 360

gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt 420

cactctgctc ccgaaggaaa agccctatct ctagggttgt cagaagatgt caagacctgg 480

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aacttcagca ctaaagggcg gaaaccctct aacacttagc actcatcgtt tacggcgtgg 660

actaccaggg tatctaatcc tgtttgctcc ccacgctttc gcgcctcagt gtcagttaca 720

gaccagaaag tcgccttcgc cactggtgtt cctccatatc tctacgcatt tcaccgctac 780

acatggaatt ccactttcct cttctgcact caagtctccc agtttccaat gaccctccac 840

ggttgagccg tgggctttca catcagactt aagaaaccac ctgcgcgcgc tttacgccca 900

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gtggctttct ggttaggtac cgtcaaggtg ccagcttatt caactagcac ttgttcttcc 1020

ctaacaacag agttttacga cccgaaagcc ttcatcactc acgcggcgtt gctccgtcag 1080

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cgttacctca ccaactagct aatgcgacgc gggtccatcc ataagtgaca gccgaagccg 1260

cctttcaatt tcgaaccatg cggttcaaaa tgttatccgg tattagcccc ggtttcccgg 1320

agttatccca gtcttatggg caggttaccc acgtgttact cacccgtccg ccgctaactt 1380

cataagagca agctcttaat ccattcgctc gactgca 1417

Claims

1. A cyanide degrading strain with the biological preservation number of CGMCCNo.16151; designated YJY18-02, belongs to Bacillus cereus (Bacillus cereus) (ii) a The nucleotide sequence of the 16S rDNA is shown in SEQ ID NO. 1.

2. A method for degrading cyanide using the cyanide-degrading strain of claim 1, wherein: the method comprises the following specific steps:

the cyanide degradation strain of claim 1 is prepared into fermentation liquor or solid microbial inoculum, wastewater with cyanide concentration of less than 350mg/L can be treated, and the prepared microbial inoculum is only required to be added into a sewage treatment aerobic system for treatment according to an aerobic treatment process.

3. The method of cyanide degradation by a cyanide-degrading strain of claim 2, wherein: solid microbial inoculum is selected and added into a sewage treatment aerobic system.