Use of Erythrobacterium alternifolium in degrading pyrene and a method for degrading pyrene

Abstract

The present invention relates to the use of Erythrobacterium alternifolium in pyrene degradation and a method for degrading pyrene. The strain screened by the present invention can realize the continuous degradation of pyrene without adding exogenous nutrients, and can effectively degrade pyrene in petroleum-contaminated soil, coking wastewater, and oilfield wastewater. The degradation rate of pyrene in 30 days reaches 76% when petroleum hydrocarbons are used as the co-metabolism matrix, and the degradation rate of pyrene in 30 days reaches 65% when phenol and m-cresol are used as the co-metabolism matrix.

Description

Technical Field

[0001] The invention belongs to the field of environmental microbiology, and particularly relates to use of Erythrobacterium alternifolium in degrading pyrene and a method for degrading pyrene. Background Art

[0002] Pyrene is a stable organic compound composed of four fused benzene rings. As a representative of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs), pyrene is commonly found in oilfield wastewater, refinery wastewater, and coking wastewater. It is carcinogenic, teratogenic, mutagenic, and difficult to biodegrade. With the continuous deepening of oil and gas exploration and development in my country and the continued development of the coal chemical industry, residual PAHs in crude oil extraction, processing, transportation, storage, and coking wastewater treatment often cause water pollution.

[0003] At present, the main methods for treating wastewater include physical, chemical, and microbial methods. After treatment, the vast majority of low-molecular-weight organic pollutants in wastewater can be effectively removed, but high-molecular-weight polycyclic aromatic hydrocarbons usually remain in the water body due to their low solubility and difficulty in biodegradation. Among them, the residual amount of pyrene is particularly prominent, which poses a great threat to the ecological environment and human health. At present, the removal of pyrene mainly relies on the co-metabolism of microorganisms, that is, adding exogenous nutrients to the contaminated soil as co-metabolism substrates. Microorganisms use the added nutrients to improve their own metabolic performance and degrade pyrene at the same time. However, as the added nutrients are exhausted, the metabolic activity of microorganisms decreases, thereby limiting the degradation of pyrene. Summary of the Invention

[0004] In response to the problems existing in the prior art, obtaining pyrene-degrading bacteria that can use saturated hydrocarbons, aromatic hydrocarbons, and phenolic compounds as co-metabolizing substrates is the key to achieving efficient degradation of pyrene in petroleum-contaminated soil and coking wastewater. Therefore, the present invention provides a strain of Alterrythrobacter sp. for degrading pyrene, specifically Alterrythrobacter sp. N1, which is deposited in the General Microbiology Center of the China Culture Collection Administration under the accession number CGMCC NO. 23607 and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] The above-mentioned Erythrobacterium alternifolium N1 can be used to degrade pyrene in soil or water contaminated by crude oil, petroleum and / or phenolic compounds. Specifically, the phenolic compounds are phenol and / or m-cresol.

[0006] As common forms of pollution, the soil is petroleum-contaminated soil, and the water body is coking wastewater or oilfield wastewater.

[0007] The present invention also provides a method for degrading pyrene, specifically, inoculating the above-mentioned Erythrobacterium alternifolium into soil or water contaminated by crude oil, petroleum and / or phenolic compounds containing pyrene, wherein the inoculation amount of Erythrobacterium alternifolium N1 in the water is 4% (volume ratio), and the bacterial concentration in the bacterial solution is 1×10 6 -2×10 8 pcs / ml.

[0008] Beneficial effects of the present invention:

[0009] The strain screened by the present invention can achieve continuous degradation of pyrene without adding exogenous nutrients, and can effectively degrade pyrene in petroleum-contaminated soil, coking wastewater, and oilfield wastewater. When petroleum hydrocarbons are used as the cometabolism matrix, the 30-day pyrene degradation rate reaches 76%, and when phenol and m-cresol are used as the cometabolism matrix, the 30-day pyrene degradation rate reaches 65%. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a colony photo of Erythrobacterium alternifolium N1 of the present invention;

[0011] Figure 2 This is a transmission electron microscope photograph of Erythrobacterium alternifolium N1 of the present invention;

[0012] Figure 3 is a phylogenetic tree of the 16S rDNA sequence of Erythrobacterium alternatum N1 of the present invention;

[0013] Figure 4 The degradation effect of Erythrobacterium alternatum N1 of the present invention on pyrene in saturated hydrocarbons and aromatic crude oil as co-metabolism substrates;

[0014] Figure 5 The present invention shows the degradation effect of Erythrobacterium alternifolium N1 on pyrene when phenol and m-cresol are used as co-metabolism substrates. DETAILED DESCRIPTION

[0015] Unless otherwise specified, the terms used in the present invention generally have the meanings commonly understood by those skilled in the art.

[0016] Example 1

[0017] Screening of bacterial strains

[0018] (1) The pyrene-degrading bacteria were enriched by gradually increasing the pyrene content.

[0019] In September 2020, oil sludge from the soil surface was collected near an oil well in the Zhansan Block of Shengli Oilfield. 10 g of the oil sludge from the soil surface was added to an inorganic salt medium containing 100 mg / L pyrene. The culture was incubated in an air bath shaker at 35°C and 150 rpm for 7 days. After the incubation period, the supernatant was transferred to an inorganic salt medium containing 300 mg / L pyrene and continued to be incubated for 7 days. This procedure was repeated, with subsequent transfers to inorganic salt medium containing 500 mg / L and 700 mg / L pyrene.

[0020] (2) After the enrichment is completed, 0.1 ml of the culture medium is diluted in a gradient manner and then spread on an inorganic salt solid culture medium containing pyrene. After colonies grow, single colonies of different sizes and morphologies are picked and separated by streaking.

[0021] in,

[0022] LB medium: yeast powder 5.0 g / L, peptone 10.0 g / L, sodium chloride 10.0 g / L, pH = 7.0.

[0023] Mineral salt medium (MSM): KH2PO4 1.0g / L, CaCl2 0.01g / L, K2HPO4 1.0g / L, FeSO47H2O0.1g / L, MgSO4 0.5g / L, NH4NO3 1.0g / L, pH = 7.0. Add 2% agar powder to serve as a solid culture medium and sterilize at high pressure at 121°C for 20min.

[0024] Bacterial species identification and morphological analysis

[0025] The strain was cultured on LB solid medium by plate spreading method and the colony morphology (such as Figure 1 The colonies of the strain were light yellow in pyrene solid inorganic salt medium, and the colonies were round, smooth and shiny. The morphology of the bacteria was observed using a transmission electron microscope (as shown in Figure 2). Figure 2 The bacterial body is about 1.2 μm in size, rod-shaped, and has no flagella.

[0026] The bacterial genome was extracted using a bacterial genomic DNA extraction kit (the kit used in this example was: American Omega). The primer sequences used for 16SrDNA amplification were 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The reaction mixture (25 μL) was: 2 μL bacterial DNA, 1 μL 10 μ / L primer, 12.5 μL 2×Taq PCR master solution. The PCR amplification conditions were: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 53°C annealing for 30 s, 72°C extension for 90 s (35 cycles), and 72°C extension for 10 min. The amplified products were detected by agarose gel electrophoresis and sequenced. The sequence similarity analysis was performed using MEGA 7.0, and a phylogenetic tree was constructed based on the neighbor-joining method (such as Figure 3 ), the strain was inferred to be Altererythrobacter sp., named Altererythrobacter sp. N1, and the strain was submitted to the General Microbiology Center of China Culture Collection Administration on October 15, 2021, with the preservation number CGMCC NO.23607.

[0027] (3) Whole genome sequencing

[0028] The Erythrobacter alternatus N1 strain was inoculated into 50 ml of LB medium, cultured in a shaker at 35°C and 180 rpm for 7 days, and the cells were collected by centrifugation at 10,000 rpm for 5 minutes. According to the instructions, genomic DNA was extracted using the Qiagen DNA kit (Hilden, Germany). The quality of the extracted DNA was evaluated using Qubit Fluorometer and NanoDrop. After the high-quality DNA samples were sheared to an average fragment size of 500 bp or 6 kb, two whole-genome sequencing libraries of different fragment sizes were prepared, and the libraries were sequenced using Illumina MiSeq and PacBio sequencing systems. The offline data were subjected to genome estimation, sequence assembly, result evaluation and other analyses to obtain the genome sequence of the Erythrobacter alternatus N1 strain. Whole-genome analysis showed that the genome of Erythrobacter alternatus N1 contains 3,520 genes with a total length of 3,536,970 bp and a G+C content of 60.95%.

[0029] Example 2

[0030] (1) Degradation of pyrene using saturated hydrocarbons, aromatic hydrocarbons, and crude oil as co-metabolism substrates

[0031] The cometabolism performance of Erythrobacterium alternifolium N1 on pyrene was analyzed using saturated hydrocarbons, aromatic hydrocarbons and crude oil as cometabolism matrices.

[0032] Crude oil is separated into four components to obtain saturated hydrocarbons and aromatic hydrocarbons. The separation of components is based on the Petroleum and Natural Gas Industry Standard of the People's Republic of China (SY / T 5119-2006) "Analysis of Soluble Organic Matter in Rocks and Crude Oil Group Components".

[0033] 100 mg / L of saturated hydrocarbons, aromatic hydrocarbons, and crude oil were added to the inorganic salt medium containing 100 mg / L of pyrene as co-metabolism substrates, and the inorganic salt medium containing only 100 mg / L of pyrene was used as the control. After inoculation with Erythrobacter alternifolius N1 in the logarithmic growth phase, the inoculation amount was 4% (volume ratio) at 35°C, and the bacterial concentration in the bacterial solution was 2×10 6 The culture medium was incubated at 150 rpm and ethyl acetate was used to extract pyrene from the degradation solution. The pyrene content in the extracts after 7 days and 30 days was determined by gas chromatography with decafluorobiphenyl as the internal standard, and the pyrene degradation rate was calculated.

[0034] like Figure 4 As shown, the degradation rates of pyrene were highest in the crude oil and saturated hydrocarbon groups at 7 days, at 23.6% and 22.4%, respectively, while the degradation rate for aromatic hydrocarbons was only 16.7%, indicating that saturated hydrocarbons and crude oil were more effective than aromatic hydrocarbons in the degradation of co-metabolism. The overall degradation trend of the 30-day degradation rate was the same as that of the 7-day degradation rate, with a significant increase. The degradation rate of pyrene using crude oil as the co-metabolism substrate reached 76%, indicating that Erythrobacterium alternifolium N1 can utilize saturated hydrocarbons and aromatic hydrocarbons in crude oil as co-metabolism substrates to enhance the degradation of pyrene.

[0035] (2) Degradation of pyrene using phenol and m-cresol as co-metabolites

[0036] The cometabolism performance of Erythrobacterium alternifolium N1 on pyrene was analyzed using phenol, m-cresol, and phenol + m-cresol as cometabolism matrices.

[0037] Phenol and m-cresol are pollutants with high content in coking wastewater, among which phenol has the highest content, followed by m-cresol, and m-cresol is more toxic than phenol. Phenol and m-cresol were used to simulate phenolic pollutants in coking wastewater. 100 mg / L of phenol, 100 mg / L of m-cresol, and 50 mg / L of phenol + 50 mg / L of m-cresol were added to the inorganic salt medium containing 100 mg / L of pyrene as co-metabolism substrates, respectively. The inorganic salt medium containing only 100 mg / L of pyrene was used as the control. After inoculation with Erythrobacter alternifolius N1 in the logarithmic growth phase, the culture was inoculated at 35°C with an inoculation amount of 4% (volume ratio). The bacterial concentration in the bacterial solution was 2×10 6 The culture medium was incubated at 150 rpm and ethyl acetate was used to extract pyrene from the degradation solution. The pyrene content in the extracts after 7 days and 30 days was determined by gas chromatography with decafluorobiphenyl as the internal standard, and the pyrene degradation rate was calculated.

[0038] The results are as follows Figure 5As shown in the data, the degradation rates of pyrene in the phenol group and the phenol + m-cresol group were 26.6% and 29.4% after 7 days, respectively, which were higher than those in the m-cresol group, indicating that phenol and phenol + m-cresol were more effective than m-cresol in the degradation of pyrene using them as co-metabolites. The degradation rate increased significantly after 30 days, reaching 69% and 65% when using phenol and phenol + m-cresol as co-metabolites, respectively. This indicates that high pyrene degradation rates can be achieved even in the presence of multiple phenols, and that Erythrobacterium alternifolium N1 is able to utilize phenol and m-cresol in coking wastewater as co-metabolites to enhance pyrene degradation.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. Use of Erythrobacter alternatus in the degradation of pyrene, wherein the Erythrobacter alternatus is specifically Erythrobacter alternatus ( Alterrythrobacter sp. )N1, deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the accession number CGMCC NO.23607.

2. The method according to claim 1, wherein the Erythrobacterium alternifolium is used to degrade pyrene in soil or water contaminated by crude oil and / or phenolic compounds.

3. The method according to claim 2, wherein the phenolic compound is phenol and / or m-cresol.

4. The use according to claim 2 or 3, wherein the soil is petroleum-contaminated soil, and the water body is coking wastewater or oilfield wastewater.

5. A method for degrading pyrene, comprising inoculating soil or water contaminated by pyrene-containing crude oil and / or phenolic compounds with Erythrobacterium alternifolium N1, which is deposited in the General Microbiology Center of the China General Culture Collection Administration under the accession number CGMCC No. 23607.

6. The method according to claim 5, wherein the phenolic compound is phenol and / or meta-cresol.

7. The method according to claim 5 or 6, wherein the soil is petroleum-contaminated soil, and the water body is coking wastewater or oilfield wastewater.

8. The method according to claim 7, wherein the inoculation amount of Erythrobacterium alternifolium N1 in the water body is 4%, the 4% is a volume ratio, and the bacterial concentration in the bacterial solution is 1×10 6 -2×10 8 pcs / ml.

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