Rhodococcus ruber and application thereof in degradation of tetrahydrofuran and indole

Abstract

The invention relates to Rhodococcus ruber, a microbial agent containing the Rhodococcus ruber and application of the Rhodococcus ruber, the Rhodococcus ruber is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31681. The Rhodococcus ruber can grow by using tetrahydrofuran as a unique carbon source and energy, and tetrahydrofuran is completely mineralized into CO2 and H2O. Under a pure culture condition, the bacterium can completely degrade 500uL / L of THF in an inorganic salt culture medium into CO2 and H2O within 144 hours, and can efficiently degrade the substrate while growing by taking tetrahydrofuran as a unique carbon source and energy source.

Description

Technical Field

[0001] The invention relates to a Rhodococcus erythrocyte and a microbial agent containing the same. The bacterium can efficiently degrade tetrahydrofuran and indole in water and belongs to the technical field of environmental microorganisms. Background Art

[0002] Tetrahydrofuran (THF) is a cyclic ether compound widely used in industry. Its excellent solubility makes it an ideal solvent for many compounds except polyethylene, polypropylene and fluororesin, so it enjoys the reputation of "universal solvent". In addition, THF also plays an important role in the field of drug synthesis. As a key raw material for anti-infective and hormone drugs, it can also be used to produce polytetrahydrofuran, and further manufacture spandex elastic fibers and polyurethane materials.

[0003] However, the potential toxicity of THF cannot be ignored. It is an inhibitor of cytochrome P450 and can enter the human body through the respiratory tract, digestive tract and skin, causing varying degrees of harm to the human body. At low concentrations, it may irritate the skin and mucous membranes; at high concentrations, it may produce an anesthetic effect, be toxic to the liver, and even be lethal. Long-term toxicity studies have also found that THF is carcinogenic to animals. In addition to its impact on human health, THF also has an inhibitory effect on microbial activity, can interfere with the dehydrogenase and aerobic respiration of microorganisms in activated sludge, and affect the activity of enzymes such as phosphatase, urease and catalase. This inhibitory effect further exacerbates the environmental pollution problem of THF.

[0004] At present, the treatment methods for THF pollution at home and abroad mainly include extraction-distillation method and pervaporation-vaporization permeation membrane method. However, although these methods are effective, the equipment investment and operating costs are high, and it is difficult to promote and apply them on a large scale. In addition, due to the particularity of THF structure, it was once considered to be a difficult biodegradable compound, making its pollution control more difficult.

[0005] In recent years, with the continuous development of biotechnology, researchers have begun to explore the possibility of using microorganisms to degrade THF. Since 1991, when German scholars Bernhardt and Diekmann first isolated the strain Rhodococcus ruber 219 that can grow with THF as the only carbon source, research on the biodegradation of THF has made significant progress. However, the THF-degrading bacteria reported so far still face many challenges in terms of degradation efficiency, substrate concentration and tolerance, which require further research and optimization.

[0006] Indole is a nitrogen heterocyclic aromatic compound with stable chemical properties and a strong malodor. The environmental pollution caused by indole compounds is becoming increasingly serious. Indole compounds are the main component of farm odor and are also one of the common pollutants in industrial and agricultural wastewater. Industrial production such as petroleum processing and drug production will produce a large amount of indole-containing wastewater, thereby polluting the soil, groundwater, surface water and even the indoor environment. Indole is also considered a mutagen and carcinogen, which brings long-term harm to human health and the ecological environment.

[0007] Traditional physical and chemical methods for degrading or eliminating indole have the problems of large reagent usage, high cost and chemical residues. Microorganisms have the characteristics of fast growth and reproduction, large biomass and high environmental adaptability. Therefore, screening and isolating microbial strains with strong indole degradation function has become a research hotspot at home and abroad in recent years.

[0008] If a microbial strain that can efficiently degrade both tetrahydrofuran and indole can be obtained, the pollution problems of both can be alleviated or solved, and the application scenarios and scope of the microbial agents will be broader. Summary of the invention

[0009] Aiming at the problems of low degradation activity, slow efficiency and low tolerance concentration of tetrahydrofuran and indole degrading bacteria in the prior art, the present invention provides a Rhodococcus erythrorhizium, a microbial agent containing the same and application thereof in degrading tetrahydrofuran and indole.

[0010] The present invention claims protection for a Rhodococcus ruber, whose 16S rDNA sequence is shown in SEQ ID No: 1, which is deposited in the General Microbiological Center of China Culture Collection Administration, the address of which is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 31681 and a deposit date of August 19, 2024.

[0011] Rhodococcus ruber has the biological characteristics of Gram-negative, orange-yellow colonies, round and opaque, rough and dry surface, spherical or short rod-shaped cells, no flagella, no movement, no spore production; aerobic, chemoheterotrophic. The bacteria can be produced using general fermentation equipment in the fermentation industry.

[0012] The mechanism of action of Rhodococcus erythrocytes degrading tetrahydrofuran of the present invention is as follows:

[0013] The carbon atom adjacent to the oxygen atom of THF is hydroxylated to generate 2-hydroxytetrahydrofuran, which is further oxidized to generate γ-butyrolactone, and then γ-butyrolactone is ring-opened to generate 4-hydroxybutyric acid. 4-Hydroxybutyric acid is further oxidized to generate succinic acid (succinic acid), and finally enters the tricarboxylic acid cycle. During the whole process, the metabolic changes of substances involve the continuous consumption and generation of energy. Finally, the energy generated by the completion of THF metabolism provides a guarantee for the growth of microbial cells.

[0014] The mechanism of indole degradation by Rhodococcus erythrocytes of the present invention is:

[0015] During the growth and metabolism of this strain, indole is first carbonylated to oxidized indole, followed by further carbonylation to produce indole, which is finally degraded to water and carbon dioxide.

[0016] The present invention also claims protection for a microbial agent comprising the above-mentioned Rhodococcus erythrorhizium.

[0017] The beneficial effects of the technical solution of the present invention are:

[0018] (1) Rhodococcus erythrocytes can use THF as the only carbon source and energy source for growth, completely mineralizing THF into CO 2 and H 2 Under pure culture conditions, the bacteria can completely degrade 500uL / L THF in inorganic salt medium into CO within 144h. 2 and H 2 O;

[0019] (2) The red coccus of the present invention can use indole as the only carbon source and energy source for growth, degrading indole into CO 2 and H 2 Under pure culture conditions, the bacteria can completely degrade 100uL / L of indole in inorganic salt medium into CO within 120h. 2 and H 2 O;

[0020] (3) The microbial agent of the present invention has a high number of viable bacteria and the addition amount can be as low as 200 ppm, which can reduce the cost of purchasing bacteria or microbial agents;

[0021] (4) The microbial agent of the present invention can be in solid or liquid form, and its cost is no higher than that of commercially available agents. It does not damage the original environment, does not cause secondary pollution, has good treatment effect, and is easy to operate. It has a wide range of applications and strong universality.

[0022] The fermentation method of Rhodococcus erythrocytes of the present invention comprises the following steps:

[0023] (1) Primary seed culture: under sterile conditions, inoculate Rhodococcus erythrorhizium into a nutrient medium, and culture at 25-35° C. and 180-300 rpm for 20-36 hours to obtain a primary seed culture solution;

[0024] (2) Secondary seed culture: under sterile conditions, the primary seed culture solution obtained in step (1) is inoculated into a nutrient medium at a ratio of 1-10 vol%, and cultured at 25-35° C. and 180-300 r / min for 20-36 hours to obtain a secondary seed culture solution;

[0025] (3) Fermentation: After the fermentation medium is disinfected, the secondary seed culture solution obtained in step (2) is inoculated into the fermentation medium at a ratio of 1-10 vol%, the temperature is controlled at 25-35° C., the rotation speed is 180-300 r / min, and the fermentation is carried out at a ventilation ratio of 1:(1-2). During the fermentation process, the pH is adjusted to 6.5-7.5. After 20-36 hours, the fermentation is stopped to obtain a fermentation liquid.

[0026] Furthermore, the composition of the nutrient medium is: 5-15 g / L tryptone, 3-8 g / L yeast extract powder, 5-15 g / L sodium chloride, the solvent is water, and the pH is 6.5-8.0.

[0027] Furthermore, the fermentation medium is composed of: 10-50 g / L carbon source, 5-15 g / L nitrogen source, K + 1.0-1.5g / L, Mg 2+ 0.02-0.5g / L, Ca 2+ 0.1-0.5g / L, Mn 2+ 0.01-0.5 g / L, the solvent is water, and pH=6.5-8.

[0028] Furthermore, the carbon source is selected from one or more of glucose, potassium sodium tartrate, sucrose, starch, succinic acid or sodium citrate.

[0029] Furthermore, the nitrogen source is selected from one or more of potassium nitrate, diammonium phosphate, urea, yeast powder, peptone or ammonium chloride.

[0030] The ions and salts involved in the culture medium can all be derived from inorganic compounds. + The source is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium sulfate, potassium chloride, and potassium nitrate, wherein the Mg 2+ The source is one or more of magnesium sulfate and magnesium chloride, wherein the Ca 2+ The source is one or more of calcium chloride and calcium nitrate, and the Mn 2+ The source is manganese sulfate.

[0031] The ventilation ratio described in the fermentation method refers to the ratio of the volume of air introduced into the fermenter per minute to the total volume of the fermentation liquid.

[0032] The present invention also claims protection for a method for purifying water using the above-mentioned activation solution of Rhodococcus or a microbial agent containing Rhodococcus, comprising the step of inoculating the activation solution of Rhodococcus or a microbial agent containing Rhodococcus into the water.

[0033] Furthermore, the inoculation amount of the Rhodococcus erythrorhizium activation solution and the microbial agent containing Rhodococcus erythrorhizium is 500 ppm or more, preferably 500-10000 ppm, further preferably 500-5000 ppm, and most preferably 500-1000 ppm.

[0034] Furthermore, the salinity of the water body is below 2%, preferably below 1%.

[0035] Furthermore, the temperature during the water purification process is controlled at 20-35°C, preferably 25-35°C, and most preferably 30°C.

[0036] The present invention also claims to protect the use of the above-mentioned Rhodococcus and microbial agent in the field of sewage and wastewater treatment. Preferably, the Rhodococcus and microbial agent are used to degrade tetrahydrofuran and indole in sewage and wastewater.

[0037] Furthermore, the concentration of tetrahydrofuran in the wastewater is below 5000uL / L, preferably below 1000uL / L.

[0038] In actual application, the form of the final Rhodococcus product can be determined according to actual use and storage needs. When a liquid product is needed, the fermentation broth can be diluted to the required concentration and used directly. When a solid product is needed, the fermentation broth can be centrifuged to obtain bacterial mud, and then freeze-drying technology is used to prepare solid bacterial powder. DETAILED DESCRIPTION

[0039] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0040] Example 1: Screening and separation of strains

[0041] Activated sludge from a sewage treatment pool of a chemical plant in Shandong was selected for strain enrichment and screening. 5 mL of activated sludge was added to 50 mL of enrichment solution containing 100 ppm tetrahydrofuran as the sole carbon source. The culture was shaken at 30°C and 200 r / min. After 4 days, 5 mL of the enrichment solution was transferred to the next round of enrichment solution containing 300 ppm tetrahydrofuran as the sole carbon source. After 5 days of culture, the enrichment solution was obtained.

[0042] The enriched solution was diluted to 10-3 , 10 -4 , 10 -5 The enrichment solution at the concentration was used for spreading and separation of strains. The plates were made of solid inorganic salt selection medium with tetrahydrofuran as the only carbon source (0.5 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.1 g / L sodium chloride, 1 g / L ammonium sulfate, 0.01 g / L ferrous sulfate heptahydrate, 0.01 g / L manganese sulfate monohydrate, 20 g / L agar, 500 ul / L tetrahydrofuran). After dilution and spreading, the plates were inverted in a 30°C incubator for static culture for 48 h. Single colonies were picked for purification on nutrient agar plates. The purified single bacteria were transferred to nutrient slant culture medium, cultured at 30°C for 24 h, and then transferred to a 4°C refrigerator for standby use.

[0043] The screened single bacteria were subjected to high-throughput sequencing and identification. After screening out pathogenic bacteria, three strains were finally obtained and named THF001, THF002 and THF003.

[0044] Preparation of activation solution: Under a sterile environment, three bacterial strains were selected and inoculated into a 250 mL Erlenmeyer flask containing 50 mL LB medium (10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, water as solvent, and pH = 7.0), and the flask was placed in a shaking incubator at 30 ° C and 200 r / min for 24 h to obtain the activation solution;

[0045] The evaluation culture medium (0.5 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.1 g / L sodium chloride, 1 g / L ammonium sulfate, 0.01 g / L ferrous sulfate heptahydrate, 0.01 g / L manganese sulfate monohydrate, 500 ul / L tetrahydrofuran) was dispensed into 250 mL conical flasks, with 50 mL of culture medium in each flask, and sterilized at 121°C for 20 min.

[0046] Evaluation method: 0.1 mL of activation solution of three strains was added to the evaluation culture medium respectively. Three parallel experiments were set up for each group of experiments. 0.1 mL of nutrient culture medium was added to the blank group. Samples were taken regularly and centrifuged to detect the content of tetrahydrofuran. Tetrahydrofuran was determined by COD test. The degradation effect of tetrahydrofuran is shown in Table 1.

[0047] Table 1 Tetrahydrofuran degradation effect of each strain

[0048]

[0049] As shown in Table 1, strains THF001, THF002 and THF003 all have tetrahydrofuran degradation effects. Compared with THF002 and THF003, strain THF001 has more prominent advantages and can achieve a tetrahydrofuran degradation efficiency of 90% within 144 hours, so THF001 was selected as the research object.

[0050] Example 2: Exploration of bacterial strain characteristics

[0051] Preparation of activation solution: Under a sterile environment, pick a slant of Rhodococcus and inoculate it into a 250mL Erlenmeyer flask containing 50mL of nutrient medium. Place the flask in a shaker at 30°C and 200r / min for 24h to obtain the activation solution of Rhodococcus for use.

[0052] 2.1 Temperature tolerance test

[0053] The experimental culture medium was prepared by weighing 10 g of tryptone, 5 g of yeast extract powder, and 10 g of sodium chloride, dissolving them in 1000 mL of distilled water, adjusting the pH to 6.5-7.0, and dispensing 50 mL of each into 250 mL Erlenmeyer flasks, sterilizing them at 121°C for 20 min and setting aside.

[0054] 1% of Rhodococcus erythrocyte activation solution was inoculated into each experimental culture medium under sterile conditions, and the reaction system was cultured at a speed of 200 r / min and different temperatures (10°C, 20°C, 30°C, and 40°C). Three parallel groups were set up for each experiment, and samples were taken after 48 hours to detect the OD 600 The values ​​were averaged to determine their growth.

[0055] Table 2 Growth of Rhodococcus at different temperatures

[0056]

[0057]

[0058] As shown in Table 2, the growth state of Rhodococcus is best when the temperature is 20-30℃. When the temperature exceeds this range, the growth of the bacteria is obviously inhibited. It can also be seen that the optimal growth temperature of the bacteria is 30℃.

[0059] 2.2 Salinity tolerance experiment

[0060] The experimental culture medium was prepared by weighing 10g of tryptone, 5g of yeast extract powder and different masses of sodium chloride and dissolving them in 1000mL of distilled water to prepare culture medium with different salinities (0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%). The pH was adjusted to 6.5-7.0, and 50mL of each was dispensed into 250mL conical flasks, sterilized at 121°C for 20min, and set aside. Under sterile conditions, 1% of the Rhodococcus erythrocyte activation solution was inoculated into the experimental culture medium, and the reaction system was cultured at 30°C and 200r / min. Three parallel groups were set up under each experimental condition, and samples were taken after 48h to detect their OD 600 The values ​​were averaged to determine their growth.

[0061] Table 3 Growth of Rhodococcus rubrum under different salinity conditions

[0062] salinity <![CDATA[OD 600 ]]> 0.5％ 2.89 1.0％ 2.13 2％ 1.49 3％ 0.88 4％ 0.35 5％ 0.21

[0063] As shown in Table 3, with the increase of salinity, the growth of Rhodococcus is inhibited, and the inhibition is also increasing with the increase of salinity. When the salinity exceeds 2.0%, its growth is almost completely inhibited. The salinity between 0.5-1.0% is more suitable for the growth of Rhodococcus.

[0064] 2.3 pH tolerance test

[0065] The experimental culture medium was prepared by weighing 10g of tryptone, 5g of yeast extract powder, and 10g of sodium chloride, dissolving them in 1000mL of distilled water, and taking 50mL of each into 250mL Erlenmeyer flasks. Sterilize at 121°C for 20min, and adjust the pH of the culture medium under aseptic conditions after the culture medium is cooled, setting the pH gradient to 5.5, 6.5, 7.5, and 8.5. Under aseptic conditions, 1% of the Rhodococcus erythrocyte activation solution was inoculated into each experimental culture medium, and the reaction system was cultured at 30°C and 200r / min. Three parallel groups were set under each experimental condition, and samples were taken after 48h to detect their OD 600 The values ​​were averaged to determine their growth.

[0066] Table 4 Growth of Rhodococcus erythrocytes under different pH conditions

[0067] pH <![CDATA[OD 600 ]]> 5.5 2.56 6.5 2.87 7.5 2.54 8.5 2.28

[0068] As shown in Table 4, the pH value between 5.5 and 8.5 had little effect on Rhodococcus erythrorhizium. 600 The value first increases and then decreases, and the optimum pH is 6.5.

[0069] Example 3: Laboratory simulated wastewater test

[0070] 3.1 Evaluation of tetrahydrofuran tolerance

[0071] (1) The evaluation culture medium (0.5 g / L potassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.1 g / L sodium chloride, 1 g / L ammonium sulfate, 0.01 g / L ferrous sulfate heptahydrate, 0.01 g / L manganese sulfate monohydrate) was dispensed into 250 mL conical flasks, 50 mL per flask, sterilized at 121°C for 20 min, and after sterilization and cooling, tetrahydrofuran solution was added thereto to make tetrahydrofuran into different concentration gradients (500 uL / L, 1000 uL / L, 2000 uL / L, 3000 uL / L, 5000 uL / L) for later use.

[0072] (2) Evaluation method: Rhodococcus erythrocyte powder was added to the evaluation culture medium at a dosage of 500 mg / L. Three parallel groups were set up for each experiment. The reaction system was placed at 30°C and 200 r / min. The residual amount of tetrahydrofuran was detected every day. The experimental results are shown in Table 5.

[0073] Experimental group 1: tetrahydrofuran 500uL / L;

[0074] Experimental group 2: tetrahydrofuran 1000uL / L;

[0075] Experimental group 3: tetrahydrofuran 2000uL / L;

[0076] Experimental group 4: tetrahydrofuran 3000uL / L;

[0077] Experimental group 5: tetrahydrofuran 5000uL / L;

[0078] Table 5 Degradation effect of Rhodococcus on different concentrations of tetrahydrofuran

[0079]

[0080] As shown in Table 5, the tolerance of Rhodococcus erythrocytes to tetrahydrofuran can reach 5000uL / L, and it has a good degradation effect on tetrahydrofuran within 5000uL / L. It can degrade 1000uL / L of tetrahydrofuran to 54uL / L in 144h, and the degradation efficiency is more than 90%. It can degrade 5000uL / L of tetrahydrofuran to 308uL / L in 192h, and the degradation rate is more than 90%.

[0081] 3.2 Evaluation of inoculum size

[0082] Different amounts of Rhodococcus erythrocyte powder (200 mg / L, 500 mg / L, 800 mg / L) were added to the evaluation culture medium with a tetrahydrofuran concentration of 500 uL / L. Three parallel experiments were set for each bacterial agent concentration. The reaction system was placed at 30°C and 200 r / min. The remaining amount of tetrahydrofuran was detected every day. The experimental results are shown in Table 6.

[0083] The specific experimental arrangements are as follows:

[0084] Control group: Rhodococcus rubrum 0 mg / L;

[0085] Experimental group 1: Rhodococcus erythrocytes powder 200 mg / L;

[0086] Experimental group 2: Rhodococcus erythrocytes powder 500 mg / L;

[0087] Experimental group 3: Rhodococcus erythrocytes powder 800 mg / L;

[0088] Table 6 Degradation effect of different inoculation amounts of Rhodococcus erythrocyte powder on tetrahydrofuran

[0089]

[0090] It can be seen from Table 6 that when the tetrahydrofuran concentration is 500uL / L, with the increase of the inoculation amount of Rhodococcus erythrorhizium, the degradation efficiency of tetrahydrofuran gradually increases. Considering the comprehensive economic efficiency, the dosage is set to 500mg / L, which is the best.

[0091] Example 4: Application experiment in actual wastewater

[0092] 4.1 Experimental Supplies

[0093] (1) Experimental water: effluent from the aerobic section of a chemical enterprise in Shandong

[0094] (2) Experimental bacterial agent: freeze-dried powder of Rhodococcus rubrum

[0095] 4.2 Evaluation Method

[0096] Take 50mL of experimental water and put it in a 250mL conical flask, add Rhodococcus erythrocyte powder into it, and the dosage is set to 500mg / L. Set up 3 parallel groups, and take another group of experimental water without adding bacterial agent as blank control. The reaction system is placed at 30℃ and 200r / min for evaluation, and the remaining amount of tetrahydrofuran is detected every day.

[0097] 4.3 Experimental Results

[0098] Table 7 Degradation effect of Rhodococcus on tetrahydrofuran in actual wastewater

[0099]

[0100]

[0101] It can be seen from Table 7 that after 144 hours of adding Rhodococcus powder, the concentration of tetrahydrofuran in the water sample dropped to 40uL / L, and the removal rate of tetrahydrofuran could reach more than 95%.

[0102] Example 5: Effect of Rhodococcus erythrocytes on indole degradation

[0103] (1) The evaluation culture medium (0.5 g / L potassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.1 g / L sodium chloride, 1 g / L ammonium sulfate, 0.01 g / L ferrous sulfate heptahydrate, 0.01 g / L manganese sulfate monohydrate) was dispensed into 250 mL conical flasks, 50 mL per flask, sterilized at 121°C for 20 min, and after sterilization and cooling, indole solution was added to the flask to make the indole concentration 100 uL / L for later use.

[0104] (2) Evaluation method: Rhodococcus erythrocyte powder was added to the evaluation culture medium at a dosage of 500 mg / L. Three parallel groups were set up for each experiment. The reaction system was placed at 30°C and 200 r / min, and the remaining amount of indole was detected every day. Indole was detected by spectrophotometry, and the results are shown in Table 8.

[0105] Table 8 Degradation effect of Rhodococcus erythrorhizium on indole

[0106]

[0107] As shown in Table 8, Rhodococcus erythrocytes has a good degradation effect on indole. In 120 hours, 100uL / L of indole can be degraded to 15uL / L, and the degradation efficiency is more than 90%.

[0108] Example 6. Production and post-processing process of Rhodococcus erythrorhizium

[0109] 1. Small-scale fermentation

[0110] 1) Primary shake flask activation

[0111] In a sterile environment, pick 1 ring of Rhodococcus species and inoculate it into a 250mL Erlenmeyer flask containing 100mL of nutrient medium (5g / L yeast extract, 10g / L trypsin, 10g / L sodium chloride, pH=7.0, sterilized at 121℃ for 20min), and culture it at 30℃ and 180r / min for 24h to obtain a first-level seed solution.

[0112] 2) Preparation of secondary seed solution

[0113] In a sterile environment, transfer 10 mL of the first-level seed liquid to four 1L Erlenmeyer flasks containing 500 mL of nutrient medium (5 g / L yeast extract, 10 g / L trypsin, 10 g / L sodium chloride, pH = 7.0, sterilized at 121°C for 20 min), and culture at 30°C and 180 r / min for 24 h to obtain the second-level seed liquid.

[0114] 3) 50L tank fermentation

[0115] The fermentation medium was sterilized at 121°C for 20 minutes. The final fermentation medium formula was: starch 10g / L, glucose 10g / L, yeast powder 10g / L, magnesium sulfate 0.2%, manganese sulfate 0.1%, calcium chloride 0.5%, dipotassium hydrogen phosphate 0.3%. The secondary seed culture solution was inoculated into the fermentation medium at an inoculation rate of 5-10vol%, the fermentation tank was filled with 70%, and the pH was initially adjusted to 7.0 with sodium hydroxide. The aeration ratio was 1:1.5 (m 3 ·min / m 3 ), 200rpm, and fermentation culture at 30°C. During the fermentation process, pH=7 was controlled by adding ammonia water. The fermentation cycle was about 30h. When the dissolved oxygen began to rise, the fermentation was stopped immediately. At this time, the fermentation was at the end of the logarithmic phase. The number of live bacteria counted in the fermentation liquid reached 13 billion CFU / mL, the bacterial activity was the strongest, the fermentation nutrients remained the least, and the number of live bacteria decayed less during storage.

[0116] 2. Post-treatment process of fermentation broth

[0117] In order to obtain bacterial powder, the fermentation liquid can be treated by freeze-drying post-processing technology, and the number of live bacteria counted in the freeze-dried bacterial powder can reach 45 billion CFU / g.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Rhodococcus ruber, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No.31681.

2. A microbial agent, characterized in that: The active ingredient comprises the Rhodococcus erythrocytes according to claim 1.

3. The fermentation method of Rhodococcus erythrocytes according to claim 1, characterized in that: The method comprises the following steps: (1) primary seed culture: inoculating Rhodococcus erythrorhizium into a nutrient medium under sterile conditions, and culturing the medium at 25-35° C. and 180-300 r / min for 20-36 hours to obtain a primary seed culture solution; (2) Secondary seed culture: under sterile conditions, the primary seed culture solution obtained in step (1) is inoculated into a nutrient medium at a ratio of 1-10 vol%, and cultured at 25-35° C. and 180-300 r / min for 20-36 hours to obtain a secondary seed culture solution; (3) Fermentation: After the fermentation medium is disinfected, the secondary seed culture solution obtained in step (2) is inoculated into the fermentation medium at a ratio of 1-10 vol%, the temperature is controlled at 25-35° C., the rotation speed is 180-300 r / min, and the fermentation is carried out at a ventilation ratio of 1:(1-2). During the fermentation process, the pH is adjusted to 6.5-7.

5. After 20-36 hours, the fermentation is stopped to obtain a fermentation liquid.

4. The fermentation method according to claim 3, characterized in that The composition of the nutrient medium is: 5-15 g / L tryptone, 3-8 g / L yeast extract powder, 5-15 g / L sodium chloride, the solvent is water, and the pH is 6.5-8.0; The fermentation medium is composed of: 10-50 g / L carbon source, 5-15 g / L nitrogen source, K + 1.0-1.5g / L, Mg 2+ 0.02-0.5g / L, Ca 2+ 0.1-0.5g / L, Mn 2+ 0.01-0.5 g / L, the solvent is water, and pH=6.5-8.

5. The fermentation method according to claim 4, characterized in that The carbon source is selected from one or more of glucose, sodium potassium tartrate, sucrose, starch, succinic acid or sodium citrate; The nitrogen source is selected from one or more of potassium nitrate, diammonium phosphate, urea, yeast powder, peptone or ammonium chloride.

6. A method for purifying water, comprising the steps of inoculating the activated liquid of the Rhodococcus strain of claim 1 into the water or applying the microbial agent of claim 2 into the water.

7. The method according to claim 6, characterized in that The inoculation amount of the activation solution or microbial agent of the Rhodococcus erythrorhizium strain is 500 ppm or more, preferably 500-10000 ppm, further preferably 500-5000 ppm, and most preferably 500-1000 ppm.

8. The method according to claim 6 or 7, characterized in that: The salinity of the water body is below 2%, preferably below 1%.

9. Use of the Rhodococcus according to claim 1 and the microbial agent according to claim 2 in the field of sewage and wastewater treatment. Preferably, the Rhodococcus according to claim 1 and the microbial agent according to claim 2 are used to degrade tetrahydrofuran and indole in sewage and wastewater.

10. The use according to claim 9, characterized in that: The concentration of tetrahydrofuran in the wastewater is below 5000uL / L, preferably below 1000uL / L.

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