CN113512511B - Acinetobacter AVYS1 in sunstroke-prevention reef lagoon and application of acinetobacter AVYS1 in degradation of high-concentration diesel oil - Google Patents

Abstract

The invention discloses a heatstroke prevention reef cathartic Acinetobacter (Acinetobacter venenianus) AVYS1, wherein the preservation number of the strain is GDMCC NO: 61368, the preservation date is 12 months and 16 days in 2020, the preservation unit is Guangdong province microorganism strain preservation center, and the preservation address is Guangzhou, China. The strain AVYS1 is used for screening the environment of the microbial community of floating microalgae at the algal boundary of the lagoon of the south China sea sunstroke reef, has stronger degradation effect on diesel oil and has good environmental adaptability. The invention also discloses application of the acinetobacter Veneti AVYS1 in degrading high-concentration diesel oil.

Description

Acinetobacter AVYS1 in sunstroke-prevention reef lagoon and application of acinetobacter AVYS1 in degradation of high-concentration diesel oil

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a heatstroke reef lagoon Venice acinetobacter AVYS1 and application thereof in degrading high-concentration diesel.

Background

South China Sea (South China Sea) is used as the Sea area with the largest area, the most islands and the closest equator in China, not only contains abundant microbial resources, but also has important strategic position. The south sea has the characteristics of high water temperature, poor nutrition, dominant plankton and the like (Zhang Ji and the like, 2016), is rich in south sea microbial resources, and is an important factor for promoting substance circulation and energy flow of a south sea marine ecosystem. The south China sea island reef is used as the offshore land of the south China sea, and has important strategic significance for the relevant research of the south China sea island reef. At present, the mining of south China sea microbial resources focuses on evaluating the composition and functional gene diversity of microbial communities and mining secondary metabolites with specific biological activities from special habitats of south China sea water (Chang Bao Xuan, 2018), sediments (Yang Rui, 2019), deep sea and cold spring (Wang Kui et al, 2019; Liu Shi Qi, 2019) and coercion environments of coral and sponge (Fang Qian Yun, 2019; Chai Guang Jun et al, 2016). And relatively few researches on the excavation and utilization of culturable functional microbial resources in the south sea island and reef areas are carried out. Research shows that the metabolic activity of culturable floras of the water body in the island sea area of the south China sea to carbon sources is remarkably increased through oligotrophic culture (Zhuang kang et al, 2020).

Bioremediation, one of the most important methods for treating oil pollution in the ocean at present, is a bioremediation technique that degrades oil pollutants into carbon dioxide and water or other pollutants by using the degradation of organisms such as marine plants, marine animals and microorganisms (Zhengtianling et al, 2001). Among them, microorganisms play an important role in the degradation process of oil pollutants, and they degrade and utilize oil as a carbon source (Swannell et al, 1996), and play a decisive role in the degradation process. Marine oil-degrading bacteria have been reported to include: alcalivorax, Pseudomonas, Flavobacterium, Microbacterium, Actinomyces, Archrobacter, Nocardia, Alcaligenes, Acinetobacter, Rhodococcus, Bacillus, Vibrio, Halomonas, Mycobacterium, etc. (Whyte et al, 1997; Zhang Zhen et al, 2010).

Acinetobacter (Acinetobacter) is one of the important bacteria in marine environments for degrading oils (Hou et al, 2013, Sun et al, 2014). An Acinetobacter bacterium separated by the method in the Hemsleya et al (2010) can reduce the concentration of diesel oil from 3.75mg/L to 1.51mg/L within 72 h. The Acinetobacter bacterium separated by Zhouyi et al (2009) is cultured by taking diesel oil with the concentration of 100-500 mg/L as a unique carbon source, and the degradation rate on day 3 is 38.70% -57.20%. The diesel oil degradation rate after 10 days of the Acinetobacter bacteria evaluated by the King Seger et al (2011) in a diesel oil culture medium with an initial concentration of 1000mg/L is 58.6%. The petroleum hydrocarbon degrading bacteria Acinetobacter sp.B11 obtained by enriching the activated sludge by Ganzhi et al (ZL201510632607.6) has a degradation rate of 61.5% to 1000mg/L oily wastewater within 7 days.

The south China sea island is used as the offshore land of south China sea, ships frequently run, and the potential risk that the water body is polluted by oil is large. In order to excavate culturable microbial resources in the south sea island reef sea area, diesel oil is used as a unique carbon source, an indigenous strain capable of efficiently degrading high-concentration diesel oil is screened from the microbial community of the floating microalgae interstellar in the south sea Yongshu reef lagoon, the environmental adaptability and the diesel oil degradation effect are determined, indigenous microbial purification strain resources can be provided for ecological protection of the south sea island reef sea area environment, and the method has important environmental protection value and strategic significance.

Disclosure of Invention

The invention aims to provide an Acinetobacter lancinalis (Acinetobacter venenianus) AVYS1 strain which has a good degradation effect on high-concentration diesel oil (the initial oil concentration is more than 6500 mg/L) and has good environmental adaptability.

The invention also aims to provide the application of the acinetobacter Veneti AVYS1 in degrading high-concentration diesel.

The first object of the present invention can be achieved by the following technical solutions: the Acinetobacter winogrisensis (Acinetobacter venenianus) AVYS1 is a strain with the preservation number of GDMCC NO: 61368, the preservation date is 12 months and 16 days in 2020, the preservation unit is Guangdong province microorganism strain preservation center, and the preservation address is Guangzhou, China.

The Acinetobacter viniferus (Acinetobacter venenianus) AVYS1 was isolated from the planktonic microalgae algal community in the Yongshu reef lagoon.

Further, Acinetobacter venenatus (Acinetobacter venenianus) AVYS1 in the present invention is obtained by the following method: collecting seawater samples from the south China sea sunstroke reef lagoon, filtering floating microalgae and its intercellular microorganisms with polycarbonate membrane (pore diameter 0.22 μm), placing the filter membrane into a sterile centrifuge tube filled with marine bacteria seed retention liquid, and freezing at-20 deg.C for later use. And (3) putting the filter membrane into a rejuvenation culture solution, and carrying out shake culture for 5-7 d under the conditions that the illumination intensity is 2500-3000 lx, the temperature is 30-35 ℃, and the rotating speed is 150-200 rpm. Then 5mL of the culture solution is taken and transferred into a screening culture solution added with 1% diesel oil, and the shaking culture is carried out for 5-7 d under the conditions that the temperature is 30-35 ℃ and the rotating speed is 150-200 rpm. Then, the enrichment is carried out for 3 times according to the conditions, and the addition amounts of the diesel oil are 2 percent, 3 percent and 4 percent in sequence. And taking the enriched bacterial liquid, performing gradient dilution, coating the bacterial liquid on a screening solid culture medium, and culturing at the temperature of 30-35 ℃ for 5-7 days to obtain a single bacterial colony of the bacterial strain. And inoculating the strain obtained by enrichment and separation into a screening culture solution added with 1% of filtered and sterilized diesel oil, carrying out shake culture at the temperature of 30-35 ℃ and the rotating speed of 150-200 rpm for 7d, respectively measuring the diesel oil concentration at the 0d, 3d and 7d, and calculating the degradation rate. And screening the strain AVYS1 with better degradation effect on the diesel oil according to the degradation effect of the isolated strain on the diesel oil. The degradation effect of the strain AVYS1 on commercial 0# diesel oil for automobiles and ships is further evaluated.

The second object of the present invention can be achieved by the following technical solutions: the application of the Acinetobacter vinnetianus AVYS1 in degrading high-concentration diesel oil is provided.

Wherein the initial diesel oil concentration of the high-concentration diesel oil is over 6500 mg/L.

Preferably, the initial diesel concentration of the high-concentration diesel is 6500-8000 mg/L.

The culture conditions of the acinetobacter Veneti AVYS1 in the invention are as follows: the strain is cultured at salinity of 10-40, pH of 6-10, temperature of 10-40 ℃ and initial strain concentration of 10⁴～10⁷The cfu/mL can grow normally, and the optimal growth conditions are that the salinity is 10-40, the pH value is 6-10, the temperature is 20-30 ℃, and the initial bacteria concentration is 10⁴～10⁷cfu/mL. The bacterial quantity reaches 10 after 16h of culture in the screening culture solution⁸The number level of cfu/mL, the bacterial quantity of 24 h-168 h is stabilized at (2.45-5.30) multiplied by 10⁸cfu/mL. The strain has good growth condition.

When the initial pH is 8, the degradation effect of the strain AVYS1 is best, the concentration of the marine diesel oil is reduced from 7389.02mg/L to 494.63mg/L, compared with a control, the average degradation rate of 7d is 91.11-93.30%, and the bacterial load is (2.25-3.00) multiplied by 10⁸cfu/mL, significantly higher than the initial pH of 4, 6, 10 degradation effect and bacterial load (P)<0.05). When the initial pH values are 4, 6 and 10, the average degradation rates of the strain AVYS1 at the 7 th d on diesel oil are 10.68-12.19%, 46.85-55.36% and 57.73-59.58% respectively compared with the control.

When the salinity of the water body is 20-40, compared with the control, the degradation rate of the bacterial strain AVYS1 to diesel oil at 7d is 84.80-96.00%, and particularly, when the salinity is 30-40, the degradation rate exceeds 90%, and can reach 96% at most. The concentration of the diesel oil is reduced from 6736.75-6940.09 mg/L to 300.34-804.49 mg/L, the bacterial amount is (2.05-3.45) × 10⁸cfu/mL; when the salinity is 10, the degradation effect of the strain AVYS1 is poor, the diesel concentration is reduced from 6931.19mg/L to 2746.88mg/L, compared with the control, the average degradation rate of the 7 th strain is 52.71-66.30%, and the bacterial load is (1.85-2.25) multiplied by 10⁸cfu/mL。

When the water temperature is 20-30 ℃, compared with a control, the degradation rate of the bacterial strain AVYS1 to diesel oil at 7d is 88.86-95.47%. The concentration of the diesel oil is reduced from 7450.66-7638.71 mg/L to 332.26-654.47 mg/L, and the bacterial load is (2.15-3.60) × 10⁸cfu/mL; when the temperature is 10 ℃ and 40 ℃, the degradation effect of the strain AVYS1 is poor, the diesel concentration is reduced from 7348.20-7544.79 mg/L to 5052.72-6446.48 mg/L, compared with the control, the average degradation rate of the 7 th strain is 1.58-32.78%, and the bacterial load is (2.55-32.5) multiplied by 10⁷cfu/mL。

When the initial bacteria concentration is 10⁴～10⁷The bacterial AVYS1 has good degradation effect and growth effect on diesel oil when cfu/mL, the 7 th degradation rate is 94.73-97.11%, and no significant difference (P)>0.05). Compared with a control, the concentration of the diesel oil is reduced from 6585.51-7012.49 mg/L to 251.60-280.94 mg/L, and the bacterial load is (2.45-3.30) multiplied by 10⁸cfu/mL。

In summary, the acinetobacter Veneti AVYS1 has an initial pH of 8, a salinity of 30-40, a temperature of 20-30 ℃, and an initial bacteria concentration of 10⁴～10⁷Under the condition of cfu/mL, the strain AVYS1 has good degradation effect on high-concentration diesel oil (the initial diesel oil concentration is 6500-8000 mg/L). The diesel concentration can be reduced from 7638.71mg/L to 332.26mg/L, and compared with a control, the degradation rate of 7d can reach 97.11 percent at most.

Compared with the prior art, the invention has the following advantages:

(1) the acinetobacter Veneti AVYS1 has obvious degradation effect on high-concentration marine diesel oil, the concentration of the diesel oil can be reduced from 7638.71mg/L to 332.26mg/L, compared with a contrast, the degradation rate of 7d can reach 97.11 percent at most, and the strain is superior to other strains in the aspects of initial degradable diesel oil concentration, degradation amount and degradation rate;

(2) the acinetobacter Veneti AVYS1 is from the floating microalgae and interstellar microbial community in the sunstroke-prevention reef lagoon, has good environmental adaptability, and is suitable for the sea area of China, particularly the south sea; meanwhile, the mining of the south China sea culturable microbial resources has important strategic significance.

Drawings

FIG. 1 is a graph showing the growth of the strain AVYS1 in example 3 in the selection medium;

FIG. 2 shows the degradation rate of 3d and 7d strains AVYS1 on diesel oil under different initial pH conditions in example 3;

FIG. 3 is the diesel concentration change at 0d, 3d, 7d under different initial pH conditions in example 3;

FIG. 4 shows the change of the bacterial amounts of 0d, 3d and 7d strains AVYS1 under different initial pH conditions in example 3;

FIG. 5 shows the degradation rate of 3d and 7d strain AVYS1 on diesel oil under different salinity conditions in example 3;

FIG. 6 is the diesel concentration changes at 0d, 3d, 7d under different salinity conditions in example 3;

FIG. 7 shows the bacterial load change of 0d, 3d and 7d strain AVYS1 under different salinity conditions in example 3;

FIG. 8 shows the degradation rate of 3d and 7d strains AVYS1 on diesel oil under different temperature conditions in example 3;

FIG. 9 shows the concentrations of diesel oil at 0d, 3d and 7d under different temperature conditions in example 3;

FIG. 10 shows the change of the bacterial amounts of 0d, 3d and 7d strains AVYS1 under different temperature conditions in example 3;

FIG. 11 shows the degradation rate of 3d and 7d strains AVYS1 on diesel oil under different initial bacteria concentration conditions in example 3;

FIG. 12 shows the diesel concentrations at 0d, 3d and 7d under different initial bacteria concentrations in example 3;

FIG. 13 shows the change of the bacterial amounts of the 0 d-, 3 d-and 7 d-strains AVYS1 under different initial bacterial concentrations in example 3.

Detailed Description

The method of the present invention is further illustrated by the following examples. The following examples and drawings are illustrative only and are not to be construed as limiting the invention. Unless otherwise specified, the reagent raw materials used in the following examples are biochemical reagent raw materials which are conventionally commercially available or commercially available, and the laboratory instruments used are laboratory conventional instruments, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.

Example 1 enrichment screening of Marine Diesel-degrading bacteria

1. Material preparation

1.1 sample Collection

Samples of seawater were collected from the south sea sunstroke reef lagoon. Filtering floating microalgae and its interstellar microorganisms with polycarbonate membrane (pore diameter 0.22 μm), placing the membrane into sterile centrifuge tube containing marine bacteria seed-retaining liquid, and freezing at-20 deg.C for use.

1.2 culture Medium

(1) Rejuvenation culture solution: NaCl 24g, MgSO₄·7H₂O 7g，NH₄NO₃ 1g，KCl 0.7g，KH₂PO₄ 2g，Na₂HPO₄3g, 1000mL of distilled water, pH 7.6-7.8, 121 ℃,20 min. After sterilization, 5mL/L sterile microelement mixture is added.

And (3) mixing trace element liquid: CaCl₂ 2mg，FeCl₃·6H₂O 50mg，CuSO₄ 0.5mg，MnCl₂·4H₂O0.5mg，ZnSO₄·7H₂O10 mg, distilled water 1000 mL.

(2) Screening a culture solution: adding diesel oil which is filtered and sterilized by a 0.22 mu m filter membrane into the rejuvenation culture solution.

(3) Screening a solid culture medium: adding 1.5-2% of agar into a rejuvenation culture solution, wherein the pH value is 7.6-7.8; sterilizing at 121 deg.C for 20min, preparing solid plate culture medium, and uniformly coating 200 μ L diesel oil on the surface of the plate.

(4)2216E culture solution: 1L of old seawater, 5g of peptone, 1g of yeast extract and 0.01g of iron phosphate, wherein the pH value is 7.6-7.8, and the sterilization is carried out for 15min at 121 ℃.

2. Enrichment and screening of strains

2.1 rejuvenation culture of the microbial communities at the rhizosphere

Selecting a filter membrane sample of a microbial community of floating microalgae at an algal community of the microalgae collected from the south China sea Yongshu reef lagoon sea area, and putting the filter membrane sample into 100mL of rejuvenation culture solution, wherein the illumination intensity is set to be 2500-3000 lx, the temperature is 30-35 ℃, and the oscillation culture is carried out for 5-7 days under the condition of the rotation speed of 150-200 rpm.

2.2 enrichment of Diesel degrading bacteria

Transferring 5mL of culture solution into a screening culture solution added with 1% diesel oil, and performing shaking culture for 5-7 d at the temperature of 30-35 ℃ and the rotating speed of 150-200 rpm. Then, the enrichment is carried out for 3 times according to the conditions, and the addition amounts of the diesel oil are 2 percent, 3 percent and 4 percent in sequence.

2.3 isolation of Diesel degrading bacteria

And (3) taking the enriched bacterial liquid, carrying out gradient dilution, selecting a proper diluent, sucking 100 mu L of the diluent, coating the diluent on a screening solid culture medium, culturing for 5-7 d at the temperature of 30-35 ℃, and separating to obtain a single colony of the potential bacterial strain.

2.4 screening of Diesel degrading bacteria

And inoculating the strain obtained by enrichment and separation into 100mL of 2216E culture solution, and performing shake culture for 1-3 days at the temperature of 30-35 ℃ and the rotating speed of 150-200 rpm to prepare a seed solution. Taking 3mL of seed solution, transferring into 100mL of screening culture solution added with 1% of filtered and sterilized diesel oil, inoculating bacteria to a control group, carrying out shake culture for 7d under the conditions of 30-35 ℃ and 150-200 rpm of rotation speed, respectively measuring the diesel oil concentration at 0d, 3d and 7d, and calculating the degradation rate. And screening the strain AVYS1 with better degradation effect on the diesel oil according to the degradation effect of the isolated strain on the diesel oil.

2.5 degradation Effect on two kinds of Diesel

Commercially available 0# diesel oil for motor vehicles and marine diesel oil are respectively selected as unique carbon sources, and the degradation effect of the strain AVYS1 on the two diesel oils is tested. The result shows that by 7d, the degradation rates of the strain AVYS1 to the commercial 0# diesel oil for automobiles and the commercial diesel oil for ships are 89% and 92% respectively, and the effect is better than that of other strains. And further using marine diesel as a test material to evaluate the diesel degradation effect of the strain.

Example 2 identification of the Strain AVYS1

The invention carries out 16S rDNA molecular identification on the bacterial strain AVYS1 in example 1, and determines the species of the bacterial strain from molecular level and combining with the analysis of morphological characteristics and physiological and biochemical characteristics of bacteria. The 16S rDNA sequence analysis mainly comprises the following steps:

1. extraction of bacterial genomic DNA

(1) Inoculating the purified strain into 100mL of 2216E culture solution, placing the culture solution in a constant-temperature shaking box, and culturing for 1-2 d at 30 ℃ and 180 r/min;

(2) centrifuging 10mL of bacteria culture solution at 10000rpm (11500 Xg) for 1 minute, and sucking the supernatant as far as possible;

(3) adding 200 mu L of buffer solution GA into the thallus precipitate, and oscillating until the thallus is completely suspended;

(4) adding 20 mu L of proteinase K solution into the tube, and uniformly mixing;

(5) adding 220 μ L buffer solution GB, shaking for 15 s, standing at 70 deg.C for 10 min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover;

(6) adding 220 mu L of absolute ethyl alcohol, fully oscillating and uniformly mixing for 15 seconds, and centrifuging briefly to remove water drops on the inner wall of the tube cover;

(7) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12000rpm (13400 Xg) for 30 seconds, pouring off waste liquid, and placing an adsorption column CB3 into the collecting pipe;

(8) adding 500 μ L buffer GD into adsorption column CB3, centrifuging at 12000rpm (13400 × g) for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;

(9) adding 700 μ L of rinsing liquid PW into adsorption column CB3, centrifuging at 12000rpm (13400 × g) for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;

(10) adding 500 μ L of rinsing liquid PW into adsorption column CB3, centrifuging at 12000rpm (13400 × g) for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;

(11) the adsorption column CB3 was returned to the collection tube, and centrifuged at 12000rpm (13400 Xg) for 2 minutes to discard the waste liquid. Placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;

(12) transferring the adsorption column CB3 into a clean centrifugal tube, suspending and dripping 50-200 mu L of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 2-5 minutes, centrifuging at 12000rpm (13400 Xg) for 2 minutes, and collecting the solution into the centrifugal tube;

(13) the concentration and purity of the recovered DNA fragment were determined by agarose gel electrophoresis and UV spectrophotometer.

2. PCR amplification of 16S rDNA Gene

The bacterial universal primers used for the amplification of 16S rDNA were synthesized by Biotechnology engineering (Shanghai) GmbH, and the forward primers (8f) were: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; the reverse primer (1492r) is: 5'-GGTTACCTTGTTACGACTT-3' are provided.

The 50 μ L PCR reaction included: sterilized double distilled water (37. mu.L), primers (1. mu.L each), dNTPs (2.5mmol/L) 4. mu.L, Tapase (1. mu.L), 10 XPCR buffer (5. mu.L), DNA template (1. mu.L).

And (3) PCR reaction conditions: 3 minutes at 95 ℃; 30 cycles of 95 ℃ for 1 minute, 48 ℃ for 1 minute, and 72 ℃ for 2 minutes; 10 minutes at 72 ℃.

3. 16S rDNA sequencing

The amplified PCR product was detected by 1.0% agarose gel electrophoresis, and sent to Biotechnology engineering (Shanghai) Co., Ltd for sequencing, and the sequence was determined to be SEQ ID NO: 1, specifically as follows: AAGTCGGTCCTCCTTGCGGTTAGACTACCTACTTCTGGTGCAACAAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATTCTGATCCGCGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGATCGGCTTTTTGAGATTAGCATCCTATCGCTAGGTAGCAACCCTTTGTACCGACCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCGTCCCCGCCTTCCTCCAGTTTGTCACTGGCAGTATCCTTAAAGTTCCCATCCGAAATGCTGGCAAGTAAGGAAAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTATCTAGATTCCCGAAGGCACCAATCCATCTCTGGAAAGTTTCTAGTATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAGTCTTGCGACCGTACTCCCCAGGCGGTCTACTTATCGCGTTAGCTGCGCCACTAAAGCCTCAAAGGCCCCAACGGCTAGTAGACATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCATGCTTTCGTACCTCAGCGTCAGTATTAGGCCAGATGGCTGCCTTCGCCATCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCATCCTCTCCCATACTCTAGCCATCCAGTATCGAATGCAATTCCCAAGTTAAGCTCGGGGATTTCACATTTGACTTAAATGGCCGCCTACGCACGCTTTACGCCCAGTAAATCCGATTAACGCTCGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGGTGCTTATTCTGCGAGTAACGTCCACTATCCAGTAGTATTAATACTAGTAGCCTCCTCCTCGCTTAAAGTGCTTTACAACCATAAGGCCTTCTTCACACACGCGGCATGGCTGGATCAGGGTTCCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCGGATCATCCTCTCAGACCCGCTACAGATCGTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCCGACTTAGGCTCATCTATTAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCATTCCTTTCGGAATGTTGTCCCCCACTAATAGGCAGATTCCTAAGCATTACTCACCCGTCCGCCGCTAGGTCCAGTAGCAAGCTAC are provided.

4. Colony morphology and physiological characteristics of strain AVYS1

The colony morphology and physiological characteristics of the strain AVYS1 are shown in the following table 1.

TABLE 1 bacterial colony morphology, physiological characteristics of the strain AVYS1

5. Identification of Strain AVYS1

The 16S rDNA gene sequence of the strain is compared with the registered gene sequence in GenBank, and the result shows that the strain is Acinetobacter Venetiae AVYS1(Acinetobacter venetianus). The result of 16S rDNA gene sequence analysis, biochemical identification, morphological characteristics and the like is integrated, and the strain AVYS1 is determined to be Acinetobacter venenatus (Acinetobacter venetianus). According to the relevant data, no research report on the efficient degradation of high-concentration diesel oil (the initial diesel oil concentration is more than 6500 mg/L) by using Acinetobacter vineyi AVYS1(Acinetobacter venenianus) is available. The strain is preserved in Guangdong province microorganism strain collection center in 12 and 16 months in 2020, and the preservation number is as follows: GDMCC 61368, deposit address: china center for preservation of microbial strains in Guangdong province, No. 59 building, No. 5 building, Guangdong province, of Jie Zhou Dazhou No. 100, Jie, Guangzhou, China.

Example 3 degradation Effect of the Strain AVYS1 on high concentration Marine Diesel

1. Growth of the Strain

The strain AVYS1 obtained in example 1 is inoculated into a screening culture solution and cultured for 16h until the bacterial quantity reaches 10⁸Number of cfu/mLHorizontally, the bacterial quantity is stabilized at (2.45-5.30) multiplied by 10 after 24 hours⁸The growth curves of cfu/mL and the strain AVYS1 are shown in FIG. 1.

2. Degradation effect of strain on high-concentration diesel oil under different initial pH conditions

After sterilization, a screening culture solution (water salinity of 30 and pH of 8) of diesel oil which is filtered and sterilized by a 0.22 mu m filter membrane is added to serve as a test water control, and the Venetian acinetobacter AVYS1 is not added. Add bacterial group the A.veneris AVYS1 obtained in example 1 was used as 10⁷The cfu/mL concentration was inoculated into test bodies of water at different pH. Setting the pH values to be 4, 6, 8 and 10 respectively, carrying out shaking culture at 150-200 rpm and 30 ℃ for 7d, measuring the diesel oil concentration at 0d, 3d and 7d respectively, and calculating the degradation rate.

As shown in FIGS. 2 to 4, the results showed that when the initial pH was 8, the strain AVYS1 showed the best degradation effect, the diesel concentration was reduced from 7389.02mg/L to 494.63mg/L, and the 7 th day average degradation rate was 91.11% to 93.30% and the bacterial load was (2.25 to 3.00). times.10% as compared with the control⁸cfu/mL, significantly higher than the initial pH of 4, 6, 10 degradation effect and bacterial load (P)<0.05). When the initial pH values are 4, 6 and 10, the average degradation rates of the strain AVYS1 at the 7 th d on diesel oil are 10.68-12.19%, 46.85-55.36% and 57.73-59.58% respectively compared with the control.

3. Degradation effect of strain on high-concentration diesel oil under different initial salinity conditions

After sterilization, a screening culture solution (water salinity of 30 and pH of 8) of diesel oil which is filtered and sterilized by a 0.22 mu m filter membrane is added to serve as a test water control, and the Venetian acinetobacter AVYS1 is not added. Add bacterial group the A.veneris AVYS1 obtained in example 1 was used as 10⁷The cfu/mL concentration was inoculated into test waters of varying salinity. The salinity is respectively set to be 10, 20, 30 and 40, the shaking culture is carried out for 7d at the speed of 150-200 rpm and the temperature of 30 ℃, the diesel oil concentration is respectively measured at the 0d, the 3d and the 7d, and the degradation rate is calculated.

The results are shown in FIGS. 5-7, and show that when the salinity of the water body is 20-40, compared with the control, the degradation rate of the bacterial strain AVYS1 to the diesel oil at 7d is 84.80% -96.00%, and particularly, the degradation rate exceeds that of the bacterial strain AVYS1 to the salinity of 30-40Over 90%, up to 96%. The concentration of the diesel oil is reduced from 6736.75-6940.09 mg/L to 300.34-804.49 mg/L, and the bacterial load is (2.05-3.45) × 10⁸cfu/mL; when the salinity is 10, the degradation effect of the strain AVYS1 is poor, the diesel concentration is reduced from 6931.19mg/L to 2746.88mg/L, compared with the control, the average degradation rate of the 7 th strain is 52.71-66.30%, and the bacterial load is (1.85-2.25) multiplied by 10⁸cfu/mL。

4. Degradation effect of strain on high-concentration diesel oil under different initial temperature conditions

After sterilization, a screening culture solution (water salinity of 30 and pH of 8) of diesel oil which is filtered and sterilized by a 0.22 mu m filter membrane is added to serve as a test water control, and the Venetian acinetobacter AVYS1 is not added. Add bacterial group the A.veneris AVYS1 obtained in example 1 was used as 10⁷The concentration of cfu/mL was inoculated into the test water body. Setting different temperature gradients, specifically 10 ℃,20 ℃, 30 ℃ and 40 ℃, performing shaking culture at 150-200 rpm for 7d, respectively measuring the diesel oil concentration at 0d, 3d and 7d, and calculating the degradation rate.

The results are shown in FIGS. 8-10, and show that when the water temperature is 20-30 ℃, the degradation rate of the strain AVYS1 at 7d on diesel is 88.86% -95.47% compared with the control. The concentration of the diesel oil is reduced from 7450.66-7638.71 mg/L to 332.26-654.47 mg/L, and the bacterial load is (2.15-3.60) × 10⁸cfu/mL; when the temperature is 10 ℃ and 40 ℃, the degradation effect of the strain AVYS1 is poor, the diesel concentration is reduced from 7348.20-7544.79 mg/L to 5052.72-6446.48 mg/L, compared with the control, the average degradation rate of the 7 th strain is 1.58-32.78%, and the bacterial load is (2.55-32.5) multiplied by 10⁷cfu/mL。

5. Degradation effect of strain on high-concentration diesel oil under different initial bacterium concentration conditions

After sterilization, a screening culture solution (water salinity of 30 and pH of 8) of diesel oil which is filtered and sterilized by a 0.22 mu m filter membrane is added to serve as a test water control, and the Venetian acinetobacter AVYS1 is not added. Add groups the A.veneris AVYS1 obtained in example 1 was used as 10⁴cfu/mL、10⁵cfu/mL、10⁶cfu/mL、10⁷The concentration of cfu/mL was inoculated into the test water body. At 150-200 rpm and 30 DEG CShaking and culturing for 7d, measuring diesel oil concentration at 0d, 3d and 7d, respectively, and calculating degradation rate.

The results are shown in FIGS. 11-13, and show that the initial bacteria concentration is 10⁴～10⁷In cfu/mL, the bacterial strain AVYS1 has good degradation effect and growth effect on diesel oil, the 7 th degradation rate is 94.73-97.11%, and no significant difference (P)>0.05). Compared with a control, the concentration of the diesel oil is reduced from 6585.51-7012.49 mg/L to 251.60-280.94 mg/L, and the bacterial load is (2.45-3.30) multiplied by 10⁸cfu/mL。

In conclusion, the initial bacteria concentration is 10 at the initial pH of 8, the salinity of 30-40, the temperature of 30 DEG C⁴CFU/mL～10⁷Under the condition of CFU/mL, the strain AVYS1 has a good degradation effect on high-concentration diesel oil (the initial diesel oil concentration is 6500-8000 mg/L), and the degradation rate of 7d exceeds 90 percent, and can reach 97.11 percent at most.

The invention is not limited to the specific embodiments described above, which are intended to illustrate the use of the invention in detail, and functionally equivalent production methods and technical details are part of the disclosure. In fact, a person skilled in the art, on the basis of the preceding description, will be able to find different modifications according to his own needs, which modifications are intended to be within the scope of the claims appended hereto.

Sequence listing

<110> research institute for aquatic products in south China sea

GUANGZHOU XINHAI LISHENG BIOTECH Co.,Ltd.

<120> one strain of Acinetobacter Venezueli AVYS1 in sunstroke-forever reef lagoon and application thereof in degrading high-concentration diesel oil

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1386

<212> DNA

<213> Venetian Acinetobacter (Acinetobacter venenianus)

<400> 1

aagtcggtcc tccttgcggt tagactacct acttctggtg caacaaactc ccatggtgtg 60

acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcattctgat ccgcgattac 120

tagcgattcc gacttcatgg agtcgagttg cagactccaa tccggactac gatcggcttt 180

ttgagattag catcctatcg ctaggtagca accctttgta ccgaccattg tagcacgtgt 240

gtagccctgg ccgtaagggc catgatgact tgacgtcgtc cccgccttcc tccagtttgt 300

cactggcagt atccttaaag ttcccatccg aaatgctggc aagtaaggaa aagggttgcg 360

ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacagcc atgcagcacc 420

tgtatctaga ttcccgaagg caccaatcca tctctggaaa gtttctagta tgtcaaggcc 480

aggtaaggtt cttcgcgttg catcgaatta aaccacatgc tccaccgctt gtgcgggccc 540

ccgtcaattc atttgagttt tagtcttgcg accgtactcc ccaggcggtc tacttatcgc 600

gttagctgcg ccactaaagc ctcaaaggcc ccaacggcta gtagacatcg tttacggcat 660

ggactaccag ggtatctaat cctgtttgct ccccatgctt tcgtacctca gcgtcagtat 720

taggccagat ggctgccttc gccatcggta ttcctccaga tctctacgca tttcaccgct 780

acacctggaa ttctaccatc ctctcccata ctctagccat ccagtatcga atgcaattcc 840

caagttaagc tcggggattt cacatttgac ttaaatggcc gcctacgcac gctttacgcc 900

cagtaaatcc gattaacgct cgcaccctct gtattaccgc ggctgctggc acagagttag 960

ccggtgctta ttctgcgagt aacgtccact atccagtagt attaatacta gtagcctcct 1020

cctcgcttaa agtgctttac aaccataagg ccttcttcac acacgcggca tggctggatc 1080

agggttcccc ccattgtcca atattcccca ctgctgcctc ccgtaggagt ctgggccgtg 1140

tctcagtccc agtgtggcgg atcatcctct cagacccgct acagatcgtc gccttggtag 1200

gcctttaccc caccaactag ctaatccgac ttaggctcat ctattagcgc aaggcccgaa 1260

ggtcccctgc tttctcccgt aggacgtatg cggtattagc attcctttcg gaatgttgtc 1320

ccccactaat aggcagattc ctaagcatta ctcacccgtc cgccgctagg tccagtagca 1380

agctac 1386

Claims (2)

1. Acinetobacter (A. tumefaciens) for sunstroke prevention reef lagoonAcinetobacter venetianus) AVYS1, which is characterized in that: the preservation number of the strain is GDMCC NO: 61368, the preservation date is 12 months and 16 days in 2020, the preservation unit is Guangdong province microorganism strain preservation center, and the preservation address is Guangzhou, China.

2. The use of acinetobacter Veneti AVYS1 in claim 1 for degrading high-concentration diesel fuel, wherein the initial diesel fuel concentration of the high-concentration diesel fuel is 6500-8000 mg/L.

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