Z13 strain of rhodococcus and applications thereof

Abstract

The present application relates to the field of microbial technology, and particularly to a Rhodococcus Z13 strain and application thereof.The screened Rhodococcus Z13 strain is identified as a new species of Rhodococcus genus, and is named as Rhodococcus ferula, with a preservation number of GDMCC No.62880; and the strain has the characteristics of high-temperature resistance, and can normally grow at a maximum temperature of 45 DEG C.Through experiments, it is proved that under the high-temperature condition of 45 DEG C, the Rhodococcus Z13 can completely degrade phenol with a concentration of 1300 mg / L within 30 h; it can be seen that the screened strain has high efficiency in the biological treatment of phenolic wastewater, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to Rhodococcus Z13 strain and application thereof.

BACKGROUND TECHNIQUE

[0002] Phenol is an important chemical raw material, which is widely used in the production of phenolic resin, oil refining, coke, dye, textile, insecticide, pesticide and medicine, and becomes the main pollutant in these industrial wastewater. The discharge of phenol-containing wastewater causes serious pollution to the environment, endangers human health and the growth and reproduction of organisms, so many countries have listed it in the blacklist of environmental priority control pollutants. At present, the main methods for treating phenol-containing wastewater include adsorption, extraction, oxidation-reduction and biodegradation. Among them, the biodegradation method uses microorganisms that can utilize phenol as carbon source to grow to degrade phenolic pollutants in wastewater, and the whole process basically occurs in biochemical reactions under the participation of microbial enzymes, which has the advantages of wide adaptability, low energy consumption, high treatment efficiency, less secondary pollution, low investment, etc., and is a relatively advanced and important method in the field of wastewater treatment.

[0003] In recent years, the phenol-degrading microorganisms isolated and identified at home and abroad mainly include bacteria and fungi. The bacteria mainly include Pseudomonas sp., Bacillus sp., Alcaligens sp. and Rhodococcus sp.; the fungi mainly include Saccharomyces sp., Candida sp. and Acinetobacter sp. The degradation activity is mostly 300-1000 mg / L, and with the increase of phenol concentration, the activity of microorganisms is greatly inhibited, and the degradation period is long, which limits the application of most microorganisms in wastewater treatment. Therefore, it is of great significance to isolate and screen microorganisms that can degrade high-concentration phenol and have high degradation efficiency.

[0004] In the prior art, some Rhodococcus strains can also degrade phenol, but the degradation rates are different. In addition, the temperature of the wastewater containing phenol discharged in actual production is too high, and the degradation capacity of the strain is easily destroyed.

SUMMARY OF THE INVENTION

[0005] Therefore, the present application aims to provide a new Rhodococcus Z13 strain and application thereof.

[0006] In order to achieve the above-mentioned purpose, the present application screens a Rhodococcus sp. Z13 strain, the preservation number of which is GDMCC No. 62880, the preservation date of which is October 13, 2022, and the preservation address of which is No. 59, Building 5, Guangzhou City, Guangdong Province, China, and the preservation unit of which is Guangdong Microbial Culture Collection Center (GDMCC).

[0007] The Rhodococcus Z13 strain (Rhodococcus ferula) of the present application is derived from a soil sample of a sugarcane field in Sanjiang County, Liuzhou City, Guangxi Zhuang Autonomous Region. Genomic DNA of the screened strain is extracted, PCR amplification is performed using universal primers 27F and 1492R, sequencing is performed, and the obtained 16S rRNA gene sequence is submitted to EzBiocloud for comparison. The results show that the Rhodococcus Z13 has the highest similarity (both 98.62%) with strains Rhodococcus zopfii (JCM 9919) and Rhodococcus phenolicus (DSM 44812). The evolutionary tree is constructed using the N-J method, and the results show that the Rhodococcus Z13 strain is clustered with strains Rhodococcus zopfii (JCM 9919) and Rhodococcus phenolicus (DSM 44812), and is clustered with Rhodococcus. According to the comparison results of the 16S rRNA gene sequence, the Rhodococcus Z13 strain is a strain of Rhodococcus. The nucleotide sequence of the 16S rDNA of the Rhodococcus Z13 strain is shown in the sequence table, and is 1521 bp long.

[0008] Further, the biological characteristics of the Rhodococcus Z13 are as follows: the morphology on the LB plate is pink, round with protrusions, moist, the edges are neat, and the diameter is about 2 mm; the cells are short rods, and the cell size is (0.75-1.05) x (1.2-1.5) μm.

[0009] Further, the physiological and biochemical characteristics of the Rhodococcus Z13 are as follows: the Tween 20 experiment result is negative, the oxidase, amylase, cellulose hydrolysis, nitrate reduction, milk coagulation and peptonization, tryptophan decomposition, hydrogen sulfide production, MR experiment, V-P experiment are negative, and the peroxidase, urease, Tween 40, Tween 80, gelatin liquefaction are positive.

[0010] The present application also provides a culture method of the Rhodococcus Z13 strain. After the Rhodococcus Z13 strain is activated, it is inoculated into a culture medium with a pH of 7.0-8.0, a temperature of 37-40℃, and a mass concentration of NaCl of 0-1%.

[0011] The application also provides application of the Rhodococcus Z13 strain in treating phenol wastewater.

[0012] The application also provides a microbial agent containing the Rhodococcus Z13 strain.

[0013] In summary, the application has the following advantages:

[0014] The Rhodococcus Z13 strain provided by the application is identified as a new species of Rhodococcus, and is named Rhodococcus ferula. The strain has the characteristics of high temperature resistance, and can normally grow at a maximum temperature of 45 DEG C. The optimal pH condition of the strain is 7.0-8.0, the optimal temperature is 37-40 DEG C, and the mass concentration of NaCl of Z13 is 0-1%. The experiment proves that the Rhodococcus Z13 can completely degrade phenol with a concentration of 1300 mg / L within 30 h under the condition of 45 DEG C high temperature. It can be seen that the strain screened by the application has high efficiency in the biological treatment of phenolic wastewater, and has wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Figure 1 is a cell morphology diagram of the Rhodococcus Z13 strain under an electron microscope.

[0016] Figure 2 Figure 3 is a growth condition diagram of the Rhodococcus Z13 strain under different T conditions.

[0017] Figure 3 Figure 5 is a growth condition diagram of the Rhodococcus Z13 strain under different pH conditions.

[0018] Figure 4 Figure 7 is a growth condition diagram of the Rhodococcus Z13 strain under different NaCl conditions.

[0019] Figure 5 Figure 9 is a polar lipid diagram of the Rhodococcus Z13 strain.

[0020] Figure 6 Figure 11 is a respiratory quinone HPLC peak diagram of the Rhodococcus Z13 strain, JCM 9919 and DSM 44812.

[0021] Figure 7 Figure 13 is a 16S rDNA sequence evolution tree diagram of the Rhodococcus Z13 strain and its closely related strains.

[0022] Figure 8 Figure 15 is a degradation comparison diagram of the strain Z13 on different initial concentrations of phenol under the condition of 45 DEG C.

[0023] Figure 9 Figure 5: Comparison of OD values of strain Z13 at different initial concentrations of phenol at 45°C.

DETAILED DESCRIPTION

[0024] All of the features disclosed in this specification, and / or all of the steps of any method or process specified in this specification, can be combined in any combination, unless specific excluded either in the claims or from the nature of the features or steps themselves.

[0025] Any of the features disclosed in this specification, unless explicitly stated otherwise, are examples of a generic series of equivalent or similar features.

[0026] Example 1: Screening and identification of strain Z13 of Rhodococcus

[0027] 1. Screening of the strain

[0028] The soil sample was collected from Sanjiang County, Liuzhou City, Guangxi. 1 g of the soil sample was added to a flask containing 100 mL of a medium with alkaline lignin as the sole carbon source, and enriched at 37°C, 200 rpm for 48 h. The enriched liquid was diluted with sterile water in a gradient of 10 times, and 100 μL of the 10-1-10-7 dilutions were spread on the medium with alkaline lignin as the sole carbon source and incubated at 30°C for 2 days. The growth conditions and morphological characteristics were observed, and the single colonies that grew were purified by streaking multiple times, and single colonies were picked and transferred to LB slant medium and stored at 4°C for preservation.

[0029] 2. Physiological and biochemical characteristics of the strain

[0030] (1) Morphological characteristics

[0031] The Z13 single colony was picked and streaked on an LB solid plate and incubated at 37°C for 24 h. The results showed that Z13 was pink, round with a protrusion, moist, with a neat edge, and about 2 mm in diameter on the LB plate.

[0032] The formula of the LB medium was as follows: tryptone 10 g, yeast extract 5 g, sodium chloride (NaCl) 10 g, and agar powder 20 g for solid medium, with double distilled water added to 1000 mL, and the pH value was 7.2.

[0033] Take 10-20 mL of bacterial solution, centrifuge at 10000 rpm, 4°C for 5 min to collect the bacterial body, wash with PBS buffer for 3 times, each time stand for 10 min, add 2.5% glutaraldehyde 1 mL to resuspend the bacterial body, stand at 4°C overnight. After fixation, centrifuge at 10000 rpm, 4°C for 5 min to collect the bacterial body, resuspend the bacterial body with ethanol solution with concentration gradient of 20%, 40%, 60%, 80% and 100% in turn, each concentration for 10 min, centrifuge at 10000 rpm for 5 min. Precool the bacterial body at-80°C, and then dry the bacterial body into powder using vacuum freeze dryer, spray gold after drying, and observe the cell morphology using scanning electron microscope Figure 1 The results show that the cell of the strain of Rhodococcus Z13 is short rod-shaped, and the cell size is (0.75-1.05) x (1.2-1.5) μm.

[0034] (2) Growth characteristics

[0035] The experiment of the effect of different temperatures on the growth of the strain of Rhodococcus Z13, LB liquid medium was configured, the bacterial solution was inoculated in a shaking flask, and the shaking culture was carried out at 20, 25, 28, 30, 35, 37, 42, 45 and 50°C respectively, and the shaking culture was carried out at 200 rpm for 24 h. The absorbance value was detected at 600 nm using ultraviolet spectrophotometer. As shown in Fig. 2, the temperature growth range of Z13 is 28-45°C, and the optimum temperature is 37-40°C. Figure 2 The results show that the temperature growth range of Z13 is 28-45°C, and the optimum temperature is 37-40°C.

[0036] The experiment of the effect of different pH values on the growth of the strain of Rhodococcus Z13, LB liquid medium was configured, and the pH was adjusted to 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 and 11 respectively, and the fresh bacterial solution was inoculated in a shaking flask, and the shaking culture was carried out at 200 rpm and 37°C for 24 h. The absorbance value was detected at 600 nm using ultraviolet spectrophotometer. As shown in Fig. 3, the pH growth range of Z13 is 5.5-9.0, and the optimum pH is 7.0-8.0. Figure 3 The results show that the pH growth range of Z13 is 5.5-9.0, and the optimum pH is 7.0-8.0.

[0037] The experiment of the effect of different NaCl concentrations on the growth of the strain of Rhodococcus Z13, LB liquid medium was configured, and the NaCl concentration was adjusted to 0, 0.5%, 1%, 2%, 3%, 4%, 5%, 8%, 10% and 15% respectively, and the fresh bacterial solution was inoculated in a shaking flask, and the shaking culture was carried out at 200 rpm and 37°C for 24 h. The absorbance value was detected at 600 nm using ultraviolet spectrophotometer. As shown in Fig. 4, the NaCl concentration range of Z13 is 0-8%, and the optimum concentration is 0-1%. Figure 4 The results show that the NaCl concentration range of Z13 is 0-8%, and the optimum concentration is 0-1%.

[0038] (3) Physiological and biochemical characteristics

[0039] The physiological and biochemical characteristics of the strain of Rhodococcus Z13 were determined according to the Common Bacteria System Identification Manual and Actinomycetes Systematics-Principles, Methods and Practice. As shown in Table 1, the Tween 20 test result of the strain of Rhodococcus Z13 was negative, while the Tween 20 test results of the reference strains JCM 9919 and DSM 44812 were positive, which was inconsistent. The results of the oxidase, amylase, cellulose hydrolysis, nitrate reduction, milk coagulation and peptonization, tryptophan decomposition, hydrogen sulfide production, MR test and V-P test of the strain of Rhodococcus Z13 were all negative, which were consistent with the results of the reference strains. The results of the peroxidase, urease, Tween 40, Tween 80 and gelatin liquefaction tests of the strain of Rhodococcus Z13 were all positive, which were consistent with the results of the reference strains.

[0040] Table 1 Physiological and biochemical characteristics of the strain of Rhodococcus Z13 and its reference type strains

[0041]

[0042] 3. Chemical component analysis

[0043] (1) Polar lipids

[0044] Polar lipid extraction: 1 g of wet bacterial cells was weighed into a 50 mL centrifuge tube with a screw cap, 15 mL of methanol was added, and the mixture was placed in a 100 ℃ water bath for 5 min and then naturally cooled. Then 10 mL of chloroform and 2% NaCl were added, and the mixture was shaken vigorously for 10 min and then centrifuged at 8000 rpm for 10 min. After centrifugation, the tube was vertically placed, and the lower layer of the liquid was taken after the layers were separated. The liquid was concentrated and dried using a nitrogen blowing instrument, and 200 μL of chloroform / methanol (2:1, v / v) was added twice, the mixture was shaken and mixed, and the liquid was transferred to a 1.5 mL centrifuge tube, which was centrifuged at 8000 rpm for 10 min to remove the precipitate. The resulting solution was stored at 4 ℃ for later use.

[0045] Two-dimensional thin layer chromatography and color development: 5-15 μL of the polar lipid extract was taken with a capillary tube and spotted on a 10×10 cm silica gel H plate (1.5×1.5 cm from the bottom edge); two-dimensional development (two perpendicular directions), first with chloroform / methanol / water solution (65:25:4, v / v, chromatographically pure) as the first phase development agent, and the plate was taken at 1.5 cm from the top edge after development, and naturally air-dried; then with chloroform / glacial acetic acid / methanol / water solution (80:18:12:5, v / v, chromatographically pure) as the second phase development agent, and the plate was taken at 1.5 cm from the top edge after development, and naturally air-dried. After the chromatography was completed, different color developing agents were sprayed to develop color and observe, and the results were compared with those of the reference type strains to determine the type of polar lipids of the test strain. As shown in the results, the strain of Rhodococcus Z13 mainly contains DPG, PE, PG, PI, PIM, GL, GPL and some unknown lipids. Figure 5 ​

[0046] (2) Respiratory quinones

[0047] Extraction and purification: 100 mg of freeze-dried bacterial cells were added to 40 mL of chloroform / methanol solution (2:1, v / v), wrapped with newspaper to avoid light, and shaken overnight for about 10 h. After filtration with filter paper, the filtrate was dried with a nitrogen blowing instrument, 1 mL of the same chloroform / methanol solution was added for redissolution, and long strip spotting was performed on a 10 x 20 cm GF254 silica gel plate (1.5 cm from the bottom). Toluene solution (analytical pure) was used as the developing agent, and the plate was taken out at 1.5 cm from the top after development. After natural air drying, the plate was observed under a 254 nm ultraviolet lamp. The dark brown band with a migration rate Rf value of about 0.8 was the position of methyl naphthoquinone (MK), and the dark brown band with a migration rate Rf value of about 0.5 was the position of ubiquinone (Q). The silica gel containing the target band was scraped off, redissolved in 1 mL of acetone, filtered with a membrane, and stored at low temperature in the dark.

[0048] High performance liquid chromatography analysis: Agilent 1260 Infinity II high performance liquid chromatograph (HPLC) and Agilent 5HC-C18 reversed-phase column (4.6 x 250 mm) were used for detection. The mobile phase was methanol / isopropanol solution (2:1, v / v; chromatographically pure), the flow rate was set to 1 mL / min, the column temperature was 40°C, and the ultraviolet detection wavelength was 250 nm and 270 nm. The respiratory quinone extract of the reference strain was used as the standard.

[0049] As Figure 6 Experimental results: The respiratory quinone of Rhodococcus Z13 strain was MK-8(H2), which was consistent with the reference strain.

[0050] 4. Genotype comparison

[0051] (1) 16S rRNA gene sequence comparison

[0052] Genomic DNA of Rhodococcus Z13 strain was extracted, and universal primers 27F and 1492R were used for PCR amplification and sequencing. The obtained 16S rRNA gene sequence (SEQ ID NO: 1) was submitted to EzBiocloud for comparison. The results showed that Rhodococcus Z13 strain had the highest similarity (both 98.62%) with strains Rhodococcus zopfii (JCM 9919) and Rhodococcus phenolicus (DSM 44812).

[0053] (2) Phylogenetic tree analysis

[0054] The phylogenetic tree was constructed using the N-J method, and the results showed that the Rhodococcus Z13 strain was clustered with Rhodococcus zopfii (JCM 9919) and Rhodococcus phenolicus (DSM 44812), and was clustered with the Rhodococcus genus, and the 16S rRNA gene sequence alignment was as follows Figure 7 It can be seen that the Rhodococcus Z13 strain is a strain of the Rhodococcus genus.

[0055] (3) ANI and dDDH analysis

[0056] ANI and dDDH analysis were performed on the Rhodococcus Z13 strain and the closest type strain Rhodococcus zopfii (JCM 9919) and Rhodococcus phenolicus (DSM 44812), and the results in Table 2 showed that the ANI and dDDH values of the Rhodococcus Z13 strain and Rhodococcus zopfii (JCM 9919) and Rhodococcus phenolicus (DSM 44812) were all below the inter-species boundary critical value (ANI 95-96%, dDDH 70%), thus it can be seen that the genomes of the Rhodococcus Z13 strain and the reference strains are significantly different.

[0057] Table 2. ANI and dDDH calculation results of the Rhodococcus Z13 strain and the reference strains, the upper row is dDDN and the lower row is ANI

[0058]

[0059] Based on the physiological and biochemical, chemical characteristics and genotypic analysis of the strains, the Rhodococcus Z13 strain and the type strain of the Rhodococcus genus have many similar characteristics, but also have many differences, thus it can be seen that the Rhodococcus Z13 strain is a new species of the Rhodococcus genus, and is named Rhodococcus ferula.

[0060] The identified Rhodococcus Z13 strain has been preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on October 13, 2022, with the preservation number GDMCC No. 62880, and the preservation address is No. 59 Building, 5th Floor, 100 Middle Xianlie Road, Guangzhou, China.

[0061] Example 2: Phenol degradation ability of Rhodococcus ferula Z13 strain

[0062] The bacterial strain Rhodococcus ferula Z13 was inoculated into an inorganic salt medium (NaCl 5 g, NH4NO3 1 g, MgSO4-7H2O 0.2 g, KH2PO4 0.5 g, K2HPO4 0.5 g, 1000 mL deionized water) and adjusted to pH 8.0. Different initial concentrations of phenol, 300, 500, 800, 1000, 1200 and 1300 mg / L, were added, respectively, and the culture was incubated at 45°C and 200 rpm. The phenol content was detected by sampling. The bacterial growth condition and residual phenol content were detected by sampling every 6 h. The bacterial growth condition was detected by measuring the absorbance of the bacterial suspension at 600 nm using a UV spectrophotometer. The residual phenol content was detected by centrifuging the sample at 12000 rpm for 5 min, filtering through a 0.22 μm nylon filter, and detecting the sample using HPLC. The experiment was repeated three times, and the results were averaged. Figure 8 , Figure 9 The results showed that Rhodococcus ferula Z13 could completely degrade phenol at a concentration of 1300 mg / L within 30 h under high temperature conditions.

[0063] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. Rhodococcus Rhodococcus sp.) Z13 strain in treating phenol wastewater, characterized in that: The Rhodococcus ( Rhodococcus sp.) Z13 strain, its deposit number is GDMCC No.62880, the deposit date is October 13, 2022, the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, and the deposit unit is: Guangdong Microbiological Culture Collection Center (GDMCC).

2. The Rhodococcus sp. strain Z13 of claim 1, wherein, The Rhodococcus ( Rhodococcus The biological characteristics of Z13 are as follows: the morphology on the LB plate is pink, round with convexities, moist, with neat edges, and a diameter of about 2 mm; the cells are short rod-shaped, with a cell size of (0.75-1.05) × (1.2-1.5) μm.

3. The Rhodococcus sp. strain Z13 of claim 1, wherein, The physiological and biochemical characteristics of the Rhodococcus sp. Rhodococcus The physiological and biochemical characteristics of the Rhodococcus sp. Z13 are as follows: the Tween 20 experiment result is negative, the oxidase, amylase, cellulose hydrolysis, nitrate reduction, milk coagulation and peptonization, tryptophan decomposition, hydrogen sulfide production, MR experiment, V-P experiment are negative, and the peroxidase, urease, Tween 40, Tween 80 and gelatin liquefaction are positive.

4. A method for culturing Rhodococcus strain Z13, characterized by, After the Rhodococcus Z13 strain of claim 1 or claim 2 is activated, it is inoculated into a culture medium with a pH of 7.0-8.0, a temperature of 37-40 ℃, and a mass concentration of NaCl of 0-1%.

5. A microbial agent comprising the Rhodococcus Z13 strain of claim 1.

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