Medium- and long-chain alkane-degrading bacteria and their applications

Abstract

The present invention provides a medium- and long-chain alkane-degrading bacterium and its application. Among them, the medium- and long-chain alkane-degrading bacterium includes Gordonia sp. SD1, taxonomically named Gordonia sp., which was deposited at the China Center for Type Culture Collection (CCTCC) on June 17, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC M 2022916; and / or Sphingomonas sp. SD2, taxonomically named Sphingomonas sp., which was deposited at the China Center for Type Culture Collection (CCTCC) on June 17, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC M 2022917. It can solve the problem in the prior art of lacking strains that can adapt to contaminated soil and degrade medium- and long-chain alkanes, and is applicable to the field of microbial technology.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular, to a medium and long-chain alkane-degrading bacterium and its application. Background Art

[0002] Alkanes are an important component of petroleum pollutants. The content of straight-chain alkanes with carbon atoms within C 40 accounts for about 15%-20% of the total amount of petroleum. According to different relative molecular masses, alkanes in petroleum exist in three forms, namely gaseous, liquid, and solid. Under normal temperature and pressure, C1-C4 alkanes are gaseous; C5-C 16 alkanes are liquid; C 16 and above alkanes are solid. According to the number of carbon atoms, saturated alkanes can be divided into short-chain, medium-chain, and long-chain alkanes. Generally, short-chain alkanes are volatile, diffusible, and can be degraded by microorganisms; medium and long-chain alkanes are not volatile and are relatively difficult to be degraded by microorganisms. Therefore, the bioremediation of medium and long-chain alkanes plays an important role in the treatment of petroleum pollutants.

[0003] The methods for treating medium and long-chain alkane-contaminated soil mainly include physical methods, chemical methods, and biological methods. Physical methods require a large amount of work for high-concentration polluted sites, chemical methods are costly and prone to secondary pollution, while biological methods have the advantages of being economical, efficient, and environmentally friendly. Bioremediation is a method that uses the characteristics of microorganisms to decompose organic matter, so that organic macromolecules are converted into the substances of microorganisms themselves and other environmentally friendly products (CO2, H2O, CH4) through the microbial metabolism process. This method has the advantages of low cost, no secondary pollution, and simple operation, and has received extensive attention in the research of organic pollution remediation. The main bioremediation technologies include natural attenuation and bioaugmentation. The natural attenuation method is a method that uses the natural physical, chemical, and biological processes in polluted soil to reduce the concentration or total amount of pollutants. Bioaugmentation refers to a method of introducing microorganisms with specific functions into polluted soil to promote the degradation of recalcitrant organic compounds and thus improve the degradation efficiency.

[0004] In the bioaugmentation remediation of introducing exogenous strains for the enhanced degradation of pollutants, exogenous bacteria are often stressed by environmental adaptation pressure, competition from indigenous bacteria, and predation by protozoa, resulting in a decrease in their survival rate and loss of activity, which greatly limits the popularization and application of this technology. Therefore, developing strains that can adapt to polluted soil and degrade medium and long-chain alkanes is of great significance for the bioaugmentation treatment of medium and long-chain alkane-contaminated soil. Summary of the Invention

[0005] The main object of the present invention is to provide a medium and long-chain alkane-degrading bacterium and its application to solve the problem in the prior art that there is a lack of strains that can adapt to polluted soil and degrade medium and long-chain alkanes.

[0006] To achieve the above object, according to the first aspect of the present invention, a medium- and long-chain alkane-degrading bacterium is provided. The medium- and long-chain alkane-degrading bacterium includes Gordonia sp. SD1, taxonomically named Gordonia sp., deposited at the China Center for Type Culture Collection (CCTCC) on June 17, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC M 2022916; and / or Sphingomonas sp. SD2, taxonomically named Sphingomonas sp., deposited at the China Center for Type Culture Collection (CCTCC) on June 17, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC M 2022917.

[0007] To achieve the above object, according to the second aspect of the present invention, a microbial inoculant is provided. The microbial inoculant includes the above-mentioned medium- and long-chain alkane-degrading bacterium, and the microbial inoculant includes a solid inoculant or a liquid inoculant.

[0008] Further, the liquid in the liquid inoculant is LB liquid medium or PBS buffer.

[0009] Further, the concentration of the liquid inoculant is 1.1 - 1.2×10 8 cfu / g.

[0010] Further, the solid inoculant is the dried liquid inoculant.

[0011] Further, the drying method includes spray drying, vacuum drying or freeze drying.

[0012] Further, the water content of the solid inoculant is < 30%.

[0013] To achieve the above object, according to the third aspect of the present invention, an application of the above-mentioned medium- and long-chain alkane-degrading bacterium, or microbial inoculant, in degrading medium- and long-chain alkanes, or repairing medium- and long-chain alkane-polluted soil is provided. The medium- and long-chain alkanes include alkanes with C 16 and C 16 and above.

[0014] Further, the application includes applying the medium- and long-chain alkane-degrading bacterium, or microbial inoculant, to medium- and long-chain alkanes or soil.

[0015] Further, when applying to medium- and long-chain alkanes or soil, the concentration of the medium- and long-chain alkane-degrading bacterium, or microbial inoculant, is 1×10 6 -1×10 8 cfu / g.

[0016] Applying the technical solution of the present invention and using the above-mentioned medium- and long-chain alkane-degrading bacteria, the medium- and long-chain alkanes can be degraded, and the contaminated soil can be adapted, so as to achieve the remediation of the soil contaminated with medium- and long-chain alkanes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0018] Figure 1 Shows a schematic diagram of the strain phylogenetic tree according to Embodiment 1 of the present invention.

[0019] Figure 2 Shows the strain degradation of n-C according to Embodiment 2 of the present invention 16 Capability schematic diagram.

[0020] Figure 3 Shows the strain degradation of n-C according to Embodiment 2 of the present invention 21 Capability schematic diagram.

[0021] Figure 4 Shows the ability of the mixed strains to remediate n-C contaminated soil according to Embodiment 3 of the present invention 16 Capability schematic diagram.

[0022] Figure 5 Shows the ability of the mixed strains to remediate n-C contaminated soil according to Embodiment 3 of the present invention 21 Capability schematic diagram. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0024] Term Explanation:

[0025] Medium- and long-chain alkanes: Alkanes with a carbon chain length of 16 or more.

[0026] As mentioned in the background art, the treatment methods for medium- and long-chain alkane-contaminated soil include physical methods, chemical methods and biological methods. Compared with physical methods and chemical methods, biological methods have the advantages of economy, high efficiency, green environmental protection, etc. However, in the enhanced degradation of pollutants by introducing foreign aid bacteria, the foreign bacteria are often stressed by environmental adaptation pressure, competition with indigenous bacteria and predation by protozoa, resulting in a decrease in their survival rate and loss of activity.

[0027] Therefore, in this application, the inventors attempted to screen suitable indigenous functional strains (i.e., native microorganisms) from contaminated soil and use such native microorganisms to degrade medium- and long-chain alkanes, thereby overcoming the problem that exogenous strains are difficult to adapt to contaminated soil. Therefore, a series of protection schemes of this application were proposed.

[0028] In the first typical embodiment of this application, a medium- and long-chain alkane-degrading bacterium is provided. The medium- and long-chain alkane-degrading bacterium includes Gordonia sp. SD1, classified as Gordonia sp., deposited at the China Center for Type Culture Collection (CCTCC) on June 17, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC M 2022916; and / or Sphingomonas sp. SD2, classified as Sphingomonas sp., deposited at the China Center for Type Culture Collection (CCTCC) on June 17, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC M 2022917.

[0029] Both of the above-mentioned Gordonia sp. SD1 and Sphingomonas sp. SD2 have the activity of using medium- and long-chain alkanes as the sole carbon source. Gordonia sp. SD1 and Sphingomonas sp. SD2 can independently or jointly metabolize and consume medium- and long-chain alkanes and proliferate in a culture environment containing medium- and long-chain alkanes.

[0030] In the second typical embodiment of this application, a microbial inoculum is provided. The microbial inoculum includes the above-mentioned medium- and long-chain alkane-degrading bacterium, and the microbial inoculum includes a solid inoculum or a liquid inoculum.

[0031] In a preferred embodiment, the liquid in the liquid inoculum is LB liquid medium or PBS buffer.

[0032] In a preferred embodiment, the concentration of the liquid inoculum is 1.1 - 1.2×10 8 cfu / g.

[0033] In a preferred embodiment, the solid inoculum is the dried liquid inoculum.

[0034] In a preferred embodiment, the drying method includes spray drying, vacuum drying or freeze drying.

[0035] In a preferred embodiment, the water content of the solid inoculum is <30%.

[0036] In the above microbial inoculum, a large amount of Gordonia sp. SD1 and / or Sphingomonas sp. SD2 are contained, which can be directly added to the target environment during use, and the distribution amount of Gordonia sp. SD1 and / or Sphingomonas sp. SD2 in the target environment can be increased within a short time, so as to quickly achieve the consumption and removal of medium and long-chain alkanes in the target environment.

[0037] The solid inoculum is the dried liquid inoculum, which is beneficial to storage and transportation. During actual use, it can be diluted with water or culture medium before use, or directly dispersed in the target environment. By drying the liquid inoculum by the above drying method, the activity of the strains is prevented from being affected by high temperature.

[0038] In the third typical embodiment of the present application, an application of the above medium and long-chain alkane-degrading bacteria or microbial inoculum in degrading medium and long-chain alkanes or repairing soil polluted by medium and long-chain alkanes is provided. The medium and long-chain alkanes include C 16 and C 16 and above alkanes.

[0039] In a preferred embodiment, the application includes applying the medium and long-chain alkane-degrading bacteria or microbial inoculum to medium and long-chain alkanes or soil.

[0040] In a preferred embodiment, when applied to medium and long-chain alkanes or soil, the concentration of the medium and long-chain alkane-degrading bacteria or microbial inoculum is 1×10 6 -1×10 8 cfu / g.

[0041] Using Gordonia sp. SD1 and / or Sphingomonas sp. SD2, and the microbial inoculum composed of the above strains, can tolerate medium and long-chain alkanes and use medium and long-chain alkanes as a carbon source. And the above strains belong to indigenous microorganisms and can adapt to the polluted soil environment, so as to maintain activity and metabolize medium and long-chain alkanes in the soil.

[0042] The beneficial effects of the present application will be further explained in detail below with specific examples.

[0043] Example 1. Screening and identification of strains

[0044] Take petroleum-contaminated soil from around an oil well, put it into a sterile bag and transport it back to the laboratory for strain screening work.

[0045] (1) Design a liquid medium with crude oil, trace element mixture and inorganic salt medium (MSM). The composition of the trace element stock solution used is g·L -1: FeSO4·7H2O, 0.002; CuSO4·5H2O, 0.0004; MnSO4·H2O, 0.004; CoCl2·6H2O, 0.001; NiCl2·6H2O, 0.002; Vitamin B1, 0.0001. The composition of the MSM medium includes: K2HPO4 1 g, KH2PO4 1 g, NaCl 0.5 g, NH4Cl 1 g, CaCl2 0.015 g, MgSO4·7H2O 0.2 g, trace element stock solution 0.25 g, deionized water 1000 mL.

[0046] Before use, the pH of the medium was adjusted to about 7.0 and sterilized at 121 °C for 20 min before use. Crude oil-contaminated soil was added and cultured on a shaker at 140 rpm and 30 °C for 7 d. 5% of the culture solution was transferred to a new liquid medium identical to the above and cultured for 4 d. The above steps were continuously transferred 3 times in the same manner to enrich the degrading strains.

[0047] (2) Gradually dilute the enriched bacterial solution above, pipette the diluted solution and spread it on a solid medium with medium and long-chain alkanes as the sole carbon source, and place it in a biochemical incubator at 30 °C until single colonies are formed. Under sterile conditions, pick a single colony with good growth state and streak it on Luria-Bertani (LB) solid medium. The composition of the used LB medium includes: yeast powder 5.0 g, peptone 10.0 g, NaCl 10.0 g, 1.5% agar and deionized water 1000 mL. Before use, the pH of the medium was adjusted to about 7.0 and sterilized at 121 °C for 20 min, then streak plating was carried out and 2 single strains of medium and long-chain alkane degradation were repeatedly purified. The screened strains were determined by 16S rRNA molecular biology identification technology, and their genera were determined according to the sequencing results and a phylogenetic tree was constructed as Figure 1 shown. They were named Gordonia sp. SD1 (hereinafter referred to as SD1) and Sphingomonas sp. SD2 (hereinafter referred to as SD2) respectively.

[0048] Example 2. Test on the degradation ability of strains to medium and long-chain alkanes

[0049] Using representative medium and long-chain alkanes n-hexadecane (n-C 16 ) and n-heneicosane (n-C 21 ) as the sole carbon source, the degradation performance of 2 strains of medium and long-chain alkane-degrading bacteria screened with crude oil as the sole carbon source for n-C 16 and n-C 21 was tested. The experiment was carried out in a 150 mL conical flask, and the reaction system included 20 mL of MSM, 1×10 7 cfu·mL-1 Each bacterial suspension, 100 mg·L -1 of n-C 16 or n-C 21 , and in addition, a sterilization control CK (Control Check) group was set up, with 2 replicates in each group. All culture bottles were placed in a shaker at 25 °C and 160 rpm for light-shielding shaking culture. Samples were taken and extracted at 3 d and 6 d respectively, and then GC-MS concentration quantification was carried out.

[0050] Figure 2 and Figure 3 are the degradation residual concentration effect diagrams of each strain provided by the present invention for n-C 16 and n-C 21 . As can be seen from Figure 2 , after 3 d of culture, n-C 16 has been basically completely degraded by strain SD1 or SD2. Compared with the sterilization control CK group, the degradation rates of n-C 16 both reached more than 99%. It shows that both strains SD1 and SD2 have strong degradation ability for n-C 16 . As can be seen from Figure 3 , after 3 d of culture, in the treatments with strain SD1 or SD2 added, each strain showed a certain degree of degradation ability for n-C 21 ; after 6 d of culture, the degradation rates of strain SD1 and SD2 for n-C21 reached 96.81% and 94.00% respectively. It can be seen from this that both strains SD1 and SD2 have strong degradation ability for n-C 21 . In general, both strains SD1 and SD2 can degrade n-C 16 and n-C 21 , and can be used as enhanced degradation bacterial sources for bioremediation experiments of medium and long-chain alkanes.

[0051] Example 3. Remediation of crude oil-contaminated soil with mixed bacterial agents

[0052] Construct a bioremediation microcosm experiment for representative medium and long-chain alkanes n-C 16 and n-C 21 . The soil around the oil well in the oil field area collected was used as the test soil. The experimental groups were set as natural attenuation (NA) and bioaugmentation (BA) groups respectively, and in addition, a sterilization control (CK) group was set up.

[0053] In the NA group, 3 g of the above test soil, 10 mL of sterilized MSM and 1.5 mg of n-C 16 (or n-C 21 ) were added. On the basis of the NA group, the BA group was further added with 1×10 7 cfu·g -1 GS mixed bacteria (SD1 and SD2 were cultured separately and then mixed, and the concentrations were 0.5×10 7cfu·g -1 )。The CK group was sterilized with high-temperature steam at 120 °C for 30 min every day for 5 days on the basis of the NA group. Then, 1% (w / w) sodium azide (NaN3) was added, and the same amount of n-C as in the above experimental group was added. 16 (n-C 21 )。

[0054] Sodium azide is a commonly used microbial inhibitor. Since there is a possibility of partial microbial resurrection in the sterilized soil, sodium azide is used to inhibit microbial growth.

[0055] The preparation method of the GS mixed bacteria includes: preparing the bacterial suspensions of SD1 and SD2 respectively, measuring the OD of the diluted bacterial suspensions with an ultraviolet-visible spectrophotometer 600 , and adjusting it to OD 600 of about 1.0 (about 9.93×10 7 cfu / g) by the dilution factor method. The bacterial suspensions of SD1 and SD2 were mixed in equal volumes to obtain the GS mixed bacteria.

[0056] Each group of experiments was carried out in a 250 mL screw-capped bottle. Four replicates were set for each sample, and they were placed in a shaker at 25 °C and 160 rpm for dark shaking culture. The experimental samples with n-C 16 were cultured for 5 days, and the experimental samples with n-C 21 were cultured for 7 days. Ventilation was carried out regularly for 30 min every day. Samples were taken at regular intervals for GC-MS analysis. The concentration changes of n-C 16 and n-C 21 during the microbial remediation process were quantitatively detected by GC-MS, and the residual rate r / % was calculated. The formula is Ct / C0×100 (C0 and Ct are the concentrations of n-C 16 or n-C 21 measured at 0 day and t day of culture, respectively). The removal rate (R / %) calculation formula is (1 - Ct / C0)×100.

[0057] The change of the residual rate of n-C 16 is as shown in Figure 4 . On the 1st day, the residual rate of n-C 16 in BA was significantly lower than that in NA (P<0.05), indicating that the added GS mixed bacteria in BA played a certain promoting role in the degradation of n-C 16 . Both NA and BA achieved a removal of more than 95% of n-C 16 at 3 days, with R reaching 95.33% and 97.63% respectively, and the residual rates r being 4.67% and 2.37% respectively. The n-C 16There is also a certain degree of attenuation (the removal rate is about 40%), which may be caused by other abiotic processes (such as adsorption, volatilization processes, etc.), as well as the degradation of residual inactivated microorganisms.

[0058] n-C 21 The residual rate of Figure 5 is shown as follows. At 5 d, the R of n-C 21 reaches 97.22% in BA and 87.47% in NA, showing a significant difference (P < 0.05), indicating that the added GS mixed bacteria play a certain promoting role in the degradation of n-C 21 . After 7 d of cultivation, the R of n-C 21 in NA and BA reaches 98.00% and 99.68% respectively, and the residual rates r are 2% and 0.32% respectively. The n-C 21 in the CK group only shows a slight decrease (9.84%).

[0059] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: By screening out 2 strains of microorganisms, Gordonia sp. SD1 and Sphingomonas sp. SD2, which can degrade medium and long-chain alkanes, from long-term contaminated soil, using the above 2 strains or a microbial composition of these 2 strains respectively, they can proliferate with medium and long-chain alkanes such as n-C 16 , n-C 21 as the sole carbon source, realizing the tolerance to contaminated soil and the degradation of medium and long-chain alkanes. Adding the above strains, microbial composition or microbial inoculant to the contaminated site can play a role alone or jointly with the native strains in the contaminated site, increasing the degradation efficiency of medium and long-chain alkanes; and can effectively alleviate the stress faced by exogenous bacteria entering a new ecological environment, and can achieve the rapid degradation of target pollutants through the increase in functional microorganisms and the population effect, which is of great significance for the bioremediation of medium and long-chain alkane-contaminated soil and realizes the remediation of medium and long-chain alkane-contaminated soil.

[0060] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Medium- and long-chain alkane-degrading bacteria, characterized in that, The medium- and long-chain alkane-degrading bacteria include Gordonia sp. SD1, classified and named as the genus Gordonia Gordonia sp., was deposited at the China Center for Type Culture Collection (CCTCC) on June 17, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2022916; and / or Sphingomonas SD2, classified and named as Sphingomonas sp. , was deposited at the China Center for Type Culture Collection (CCTCC) on June 17, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2022917.

2. A microbial inoculant, characterized in that, The microbial agent includes the medium- and long-chain alkane-degrading bacteria described in claim 1, and the microbial agent includes a solid microbial agent or a liquid microbial agent.

3. The microbial inoculant according to claim 2, characterized in that, The liquid in the liquid microbial agent is an LB liquid medium or a PBS buffer solution.

4. The microbial inoculum according to claim 2, wherein The concentration of the liquid bacterial agent is 1.1 - 1.2×10 8 cfu / g.

5. The microbial inoculant according to claim 2, wherein The water content of the solid microbial agent is < 30%.

6. Use of the medium- and long-chain alkane-degrading bacteria described in claim 1 or the microbial agent described in any one of claims 2 to 5 in degrading medium- and long-chain alkanes or repairing soil contaminated with medium- and long-chain alkanes, wherein the medium- and long-chain alkanes are n-hexadecane or n-heneicosane.

7. The application according to claim 6, wherein The use includes applying the medium- and long-chain alkane-degrading bacteria or the microbial agent to the medium- and long-chain alkanes or the soil.

8. The application according to claim 7, characterized in that, When applied to the medium- and long-chain alkanes or the soil, the concentration of the medium- and long-chain alkane-degrading bacteria is 1×10 6 -1×10 8 cfu / g.

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