CN117448171A

CN117448171A - 3-strain filamentous fungus combination for synergistically degrading pyrene and application thereof

Info

Publication number: CN117448171A
Application number: CN202311180000.XA
Authority: CN
Inventors: 黄赛花; 吕亚辉; 高原雪; 李文改; 黄友良
Original assignee: Institute of Eco Environmental and Soil Sciences of Guangdong Academy of Sciens
Current assignee: Institute of Eco Environmental and Soil Sciences of Guangdong Academy of Sciens
Priority date: 2023-09-12
Filing date: 2023-09-12
Publication date: 2024-01-26

Abstract

The invention belongs to the technical field of organic pollution repair, and discloses a 3-strain filamentous fungus combination for synergistically degrading pyrene and application thereof. In particular discloses a multi-strain composition for degrading polycyclic aromatic hydrocarbon, which comprises Mucor niveus, mucor circinelloides and Aspergillus terreus. The multi-strain composition provided by the invention combines the Mucor niveus, mucor circinelloides and Aspergillus terreus, is used for polycyclic aromatic hydrocarbon (such as pyrene), has a degradation rate of 89.7% on high-concentration (6140 mg/L) polycyclic aromatic hydrocarbon in 5 days, is higher than that of a single strain, and has potential of being suitable for repairing environment polluted by high-concentration polycyclic aromatic hydrocarbon.

Description

3-strain filamentous fungus combination for synergistically degrading pyrene and application thereof

Technical Field

The invention belongs to the technical field of organic pollution repair, and particularly relates to a 3-strain filamentous fungus combination for synergistically degrading pyrene and application thereof.

Background

Polycyclic aromatic hydrocarbons (Polycyclic aromatic hydrocarbons, PAHs) are volatile hydrocarbons produced by incomplete combustion of organic substances such as coal, petroleum, wood, tobacco, organic high molecular compounds and the like, and are important persistent organic pollutants in the environment. PAHs can be divided into low molecular weight PAHs (2-3 rings) and high molecular weight PAHs (4-7 rings) according to the number of benzene rings, and compared with low-ring aromatic hydrocarbon, the 4-7-ring high-ring PAHs have the characteristics of stronger hydrophobicity, fat solubility, difficult degradability and the like, and stronger teratogenic, carcinogenic and mutagenic effects, the PAHs enter the environment through various ways such as natural sources, artificial sources and the like, are easy to enrich in organisms, have high ecological risks, and bring acute or chronic injury to human beings when being in the environment polluted by the PAHs for a long time. Therefore, how to effectively accelerate the elimination speed of PAHs in the environment, reduce the pollution of the PAHs to the environment, and have important significance for guaranteeing the ecological environment safety and the human health.

The bioremediation method has the characteristics of high efficiency, low cost, environmental friendliness, no secondary pollution and the like, becomes a research hotspot, and also becomes a pollution remediation technology with the most development potential in the current stage. To date, many microorganisms have been found to have the ability to degrade PAHs, and studies on microbial degradation of tetracyclic and higher macromolecular PAHs have progressed rapidly, and pyrene degrading bacteria which have been isolated include Acinetobacter Acinetobacter strain, bacillus licheniformis Bacillus licheniformis, achromobacter denitrificans Acinetobacter strain, white rot fungi Mycobacterium, pseudomonas, mycobacterium, bacillus Brevibacillus Brevis, and the like. Most of them are bacteria, rehmann et al (1998) isolated from PAHs contaminated soil to a strain of Mycobacterium sp.KR2 which has a degradation rate of 60% by 8 days for 500mg/L of pyrene. Habe et al (2004) separated a strain of Mycobacterium sp.MHP-1 from soil, degraded 1000mg/L pyrene in alkaline condition for 7 days, and the degradation rate was about 50%. PAHs polluted soil is collected by Zeng et al (2010), and 2 strains of pyrene degrading bacteria Mycobacterium NJS-1 and Mycobacterium NJS-P are screened after pyrene domestication, wherein the degradation rate of the two strains of bacteria to 100mg/L pyrene in 14 days is 87.9% and 92% respectively. The time for which Pseudomonas has the ability to degrade pyrene was found relatively late compared to Mycobacterium, but this genus has been studied more in recent decades. Zhang et al (2011) isolated a strain of Pseudomonas aeruginosa (Pseudomonas aeruginosa) DQ8 from petroleum contaminated soil with a 40mg/L pyrene degradation rate of 34.5% within 12 days. In 2013, ma et al (2013) screened a strain of Pseudomonas sp Jpyr-1 from activated sludge to degrade 200mg/L pyrene, the degradation rate was 64.7% for 48 hours, and 144 hours could be completely degraded. In addition, the pyrene degradation rates of Bacillus (Caulobacter sp.) T2A12002 at 37℃and 25℃for 18 days were 56% and 59% respectively for Burkholderia (Burkholderia fungorum) T3A13001 and for both temperatures, respectively, and 35% to 36% for Acetobacter (Caulobacter sp.) T2A12002 at both temperatures, respectively, were obtained by degrading 100mg/L pyrene at 37℃and 25 ℃. Adebusoye et al isolated a strain of Proteus vulgaris CPY1 from cow dung, and used the strain to carry out 18 days degradation test on 100mg/L pyrene, the maximum degradation rate is 87.72%. Animal bone carbon contaminated soil is taken as a sample, obayori et al screen a strain of Proteus mirabilis (Proteus mirabilis), and the degradation rate of 100mg/L pyrene can reach 87.92% within 30 days. Zhou et al isolated a strain of Propionibacterium (Thalassospira sp.) TSL5-1 from yellow sea beach soil, and utilized the strain to degrade 20mg/L pyrene for 25 days, with a degradation rate of 41.4% in a medium with pyrene as the sole carbon source.

According to the research results of screening and degrading capability of propyrene degrading bacteria, besides the strong degrading capability of few strains of mycobacterium and pseudomonas on pyrene, most of the known bacterial strains have weak degrading capability, mainly have the defects of low adaptation concentration, low degrading rate, long degrading period and the like, in fact, the degrading process of PAHs in the environment is mostly completed by one microorganism alone, but is the result of the synergistic effect of a plurality of microorganisms, the synergistic degrading efficiency of the plurality of microorganisms is higher than that of a single microorganism, and Shalma (2016) finds that the microorganism group consisting of Serratia marcescens L-11,Streptomyces rochei PAH-13 and white rot fungi Phanerochaete chrysosporium VV-18 repairs PAHs in situ, and the degrading rate is obviously improved to 56% -98% in 7 days. Aydin (2017) considers anaerobic and aerobic bacteria to be advantageous for fungal degradation or synergistic fungal degradation of PAHs. Zhang Hui et al (2020) found that 3 strains of degrading bacteria were inoculated in an inoculum size of 1:1:1 in a naphthalene degradation test, and the degradation rate was increased from 60.74% for single bacteria to 89.40%; barin et al (2014) found that the mixed bacteria Bacillus subtilis tb1 and Pseudomonas aeruginosa tb13 (surfactant producing strains) improved hydrocarbon removal rate in soil by 25% compared with the single strain of tb 1. Patel (2019) utilizes mixed bacteria (ASPF) composed of 22 genus bacteria groups to degrade PAHs, 400mg/L of phenanthrene and fluoranthene can be degraded in Bushnell-Haas culture medium, shalma (2016) reports that microorganism group composed of Serratia marcescens L-11,Streptomyces rochei PAH-13 and Phanerochaete chrysosporium VV-18 degrades about 60% -70% of PAHs in broth culture medium in stable culture condition, and the PAHs degradation rate is remarkably improved to 56% -98% in 7 days under in-situ repair condition, and 83.50% -100% in 30 days.

It is easy to see that fungi plays a very important role in combined degradation, and besides the advantages of general microbial degradation of PAHs, fungi also improve the exposure opportunity of the PAHs due to large biomass and strong penetrability of hyphae, and increase the contact area, so that the biological accessibility of the PAHs is changed; and secondly, the degradation efficiency is directly improved by high-activity enzymatic degradation, so that the PAHs are repaired by using fungi in a combined way, and the method is a low-consumption, high-efficiency and environment-friendly bioremediation technology.

The synergistic degradation mechanism among microorganisms is found to be mainly expressed in the aspects of mutual utilization of metabolites among strains, improvement of activity of high degrading enzyme, complementation of degrading enzyme, acceleration of electron transfer speed and the like, and finally, the biological accessibility is increased, the biological degradation rate is improved, so that the conversion of nondegradable PAHs in the environment is easier. Therefore, the selection of the high-efficiency combined degradation flora is the key for the success of the enhanced repair of the environmental organic pollution.

Disclosure of Invention

Aiming at the soil problem, the invention aims to provide a 3-strain filamentous fungus combination for synergistically degrading high-concentration pyrene, and the combination fungus can promote the degradation of pyrene more than single fungus.

The object of the first aspect of the present invention is to provide a multi-strain composition for degrading polycyclic aromatic hydrocarbons.

The object of the second aspect of the present invention is to provide a biological agent.

The object of the third aspect of the present invention is to provide a method for producing the biological agent of the second aspect of the present invention.

The object of the fourth aspect of the present invention is to provide the use of the multi-strain composition of the first aspect of the present invention and/or the biological agent of the second aspect of the present invention.

The fifth aspect of the invention aims to provide a soil remediation method polluted by polycyclic aromatic hydrocarbon.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided a multi-strain composition for degrading polycyclic aromatic hydrocarbons comprising Mucor niveus, mucor circinelloides and Aspergillus terreus.

In some embodiments of the invention, the multi-strain composition consists of myceliophthora nivea, mucor circinelloides and Aspergillus terreus.

In some embodiments of the invention, the polycyclic aromatic hydrocarbon is a 4 to 7 ring polycyclic aromatic hydrocarbon.

In some embodiments of the invention, the polycyclic aromatic hydrocarbon comprises at least one of pyrene, benzo (a) anthracene, benzo (b) fluoranthene, benzo (a) pyrene, indeno (1, 2, 3-cd) pyrene, and dibenzo (a, h) anthracene.

The mechanism of the synergistic degradation among the multiple bacterial strain compositions is the mutual utilization of metabolites and the acceleration of the electron transfer speed, so as to realize the efficient removal of the polycyclic aromatic hydrocarbon.

3-strain allied bacteria consisting of paecilomyces Paecilomyces niveus (also known as Byssochlamys nivea) (Pn for short), aspergillus (At) and Mucor circinelloides (Mc) have the capability of degrading high-concentration polycyclic aromatic hydrocarbon more than 6000mg/L, and currently, more reports of initial concentration of degraded pyrene within 1000mg/L, less reports of the initial concentration of degraded pyrene exceeding 2000mg/L, 6140mg/L of degraded pyrene exceeding 6000mg/L and 89.7% of degraded rate within 5 days are not reported yet.

In a second aspect of the invention, there is provided a biological agent comprising: the multi-strain composition or culture of the first aspect of the invention.

The active ingredients of the biological agent may be the culture (such as fermentation product) of the above-mentioned myceliophthora, mucor circinelloides and Aspergillus terreus, the above-mentioned mycelial myces, mucor circinelloides and Aspergillus terreus and/or the hyphae of the above-mentioned mycelial myces, mucor circinelloides and Aspergillus terreus, and the active ingredients of the above-mentioned microbial agent may also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the above-mentioned biological agent may be determined by those skilled in the art according to the effect of the agent.

The term "culture" refers to a generic term for liquid or solid products (all substances in the culture vessel, fermentation products) grown with a population of microorganisms after artificial inoculation and cultivation. I.e. the product obtained by growing and/or amplifying the microorganism, which may be a biologically pure culture of the microorganism, or may contain a certain amount of medium, metabolites or other components produced during the culture.

In the above biological agent, the biological agent contains a carrier in addition to the active ingredient. The carrier may be a carrier commonly used in the art (e.g., microorganisms, agriculture) and which is biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier can be mineral material, plant material or high molecular compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of corn flour, soy flour and starch; the polymer compound may be polyvinyl alcohol and/or polyglycol; the liquid carrier may be an organic solvent or water; the organic solvent may be decane and/or dodecane.

Among the above bacterial agents, the biological agent can be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

If necessary, binders, stabilizers (such as antioxidants), pH regulators, etc. may be added to the biological agent.

In a third aspect of the present invention, there is provided a method for preparing a biological agent according to the second aspect of the present invention, comprising the steps of: respectively inoculating the myceliophthora, the mucor circinelloides and the aspergillus terreus to a PDA (digital versatile array) culture medium for culture, and taking all bacterial mycelia to culture in a liquid culture medium to respectively obtain hypha culture solutions of the myceliophthora, the mucor circinelloides and the aspergillus terreus; mixing the mycelium culture solutions in proportion to obtain the biological preparation.

In some embodiments of the invention, the liquid medium comprises glucose, ammonium tartrate, KH ₂ PO ₄ 、MgSO ₄ .7H ₂ O、CaCl、Na ₂ HPO ₄ And trace elements.

In some embodiments of the invention, the liquid medium comprises 5-15 g/L glucose, 2-6 g/L, KH amine tartrate ₂ PO ₄ 2～4g/L、MgSO ₄ .7H ₂ O 0.1～0.5g/L、CaCl 0.1～0.5g/L、Na ₂ HPO ₄ 0.1-0.5 g/L and microelements, and the pH value is 5.0-6.0.

In some embodiments of the invention, the trace elements include MnSO ₄ 、NaCl、FeSO ₄ .7H ₂ O、ZnSO ₄ 、CuSO ₄ And Na (Na) ₂ MoO ₄ .2H ₂ O。

In some embodiments of the invention, the trace elements include MnSO ₄ 0.5～1g/L，NaCl 1～2g/L，FeSO ₄ .7H ₂ O 1～2g/L，ZnSO ₄ 0.1～0.5g/L，CuSO ₄ 0.1～0.5g/L，Na ₂ MoO ₄ .2H ₂ O 0.01～0.5g/L。

In some embodiments of the invention, the mixing ratio of the mycelium culture solutions is that the mycelium dry weight ratio of the myceliophthora nivea, the mucor circinelloides and the aspergillus terreus is 1 (1-2): 1.5-3.

In some embodiments of the invention, the culture conditions in the liquid medium are 150-180 r/min 25-30℃for 5-6 days.

In a fourth aspect, the present invention provides the use of a multi-strain composition according to the first aspect of the invention and/or a biological agent according to the second aspect of the invention in any one of (1) to (4)

(1) Degrading polycyclic aromatic hydrocarbon;

(2) Preparing a product for degrading polycyclic aromatic hydrocarbon;

(3) Preparing a surfactant;

(4) And (3) repairing the environment polluted by polycyclic aromatic hydrocarbon.

In some embodiments of the invention, the surfactant is a lipopeptidic surfactant, such as rhamnolipids.

In a fifth aspect of the present invention, there is provided a method for restoring soil contaminated with polycyclic aromatic hydrocarbons comprising the step of applying the multi-strain composition of claim 1 or 2 and/or the biological agent of claim 3 to the contaminated soil.

In some embodiments of the invention, the multi-strain composition or the biological agent is applied to the contaminated soil by means of application, wetting, drenching, atomizing, raining, spraying, soaking.

The beneficial effects of the invention are as follows:

the multi-strain composition provided by the invention combines the Mucor niveus, mucor circinelloides and Aspergillus terreus, is used for polycyclic aromatic hydrocarbon (such as pyrene), has a degradation rate of 89.7% on high-concentration (6140 mg/L) polycyclic aromatic hydrocarbon in 5 days, is higher than that of a single strain, and has potential of being suitable for repairing environment polluted by high-concentration polycyclic aromatic hydrocarbon.

According to the invention, 3 strains of combined fungi synergistic degradation mechanisms such as the myceliophthora, the Mucor circinelloides and the aspergillus terreus are researched by taking high-ring pyrene as target pollutants of PAHs, synergistic degradation is taken as a core, the influence of extracellular secretion on degradation is taken as a cut-in point, the biological effectiveness is improved, the electron transfer rate is accelerated and the like around the research thought, analysis technical means such as metabonomics, enzymatic dynamics and the like are adopted, the degradation of pyrene by single strains and 3 strains of allied bacteria in a high-concentration pyrene environment under PAHs stress is systematically researched, the influence of hypha dry bacteria, the reduction/oxidase activity, the oil ring size and the rhamnolipid on pyrene removal is deeply analyzed, and the chemical and microbial regulation mechanism of the high-concentration pyrene degradation is synergistically promoted by the 3 strains of allied bacteria, so that an important theoretical basis is provided for bioremediation application of PAHs.

Drawings

FIG. 1 is a bar graph of degradation rate of myceliophthora, mucor circinelloides, aspergillus terreus, or a combination thereof against pyrene on day 5.

FIG. 2 is a bar graph of hyphal dry weight of myceliophthora, mucor circinelloides, aspergillus terreus, or a combination thereof on day 5.

FIG. 3 is a bar graph of oil drain diameter for Chlamydomonas nivalis, mucor circinelloides, aspergillus terreus, or combinations thereof on day 5.

FIG. 4 is a bar graph of rhamnolipid content of myces niveus, mucor circinelloides, aspergillus terreus, or a combination thereof, on day 5.

FIG. 5 is a bar graph of quinone reductase activity of Chlamydomonas nivalis, mucor circinelloides, aspergillus terreus, or a combination thereof on day 5.

FIG. 6 is a bar graph of quinone oxidase activity of Chlamydomonas nivalis, mucor circinelloides, aspergillus terreus, or a combination thereof on day 5.

Detailed Description

The invention will now be described in detail with reference to specific examples, without limiting the scope of the invention.

The materials, reagents and the like used in this example are commercially available materials and reagents unless otherwise specified.

Example 1

1 materials and methods

1.1 strain: the oily sludge is taken, 1g/L of acetone pyrene solution is used for domestication for 7 days, two strains of filamentous fungi are separated and cultured by a PDA culture medium, the characteristics of a flat plate are observed by inoculating a culture medium of Chlamydomonas, wort and Chlamydia trachomatis, and the characteristics of the flat plate are respectively identified as paecilomyces (also known as Mucor niveum) Paecilomyces niveus (also known as Byssochlamys nivea) (Pn for short) and Aspergillus terreus Aspergillus terreus (At for short) by combining with ITS sequence alignment.

Mucor circinelloides H3 Mucor circinelloides (Mc for short, stored in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.11578 and China inventive patent number ZL 201511033977.4).

1.2 reagents

Standard pyrene solution: number GBW (E) 081022 is purchased from the national standard substance network, and the concentration is 261mg/L;

solid pyrene: purity of 98%, available from future utility company, inc. in Shanghai;

the rest are common inorganic and organic biochemical reagents, which are purchased from Guangzhou chemical reagent factories and are all analytically pure.

1.3 Medium:

PDA medium: 200 g of potato, 20 g of glucose, 15g of agar, 1000 ml of distilled water and natural pH.

Liquid medium: 10g/L glucose, 5g/L amine tartrate, KH ₂ PO ₄ 2g/L，MgSO ₄ .7H ₂ O 0.25g/L，CaCl 0.1g/L，Na ₂ HPO ₄ 0.25g/L, 10mL of trace elements, pH 5.0.

Trace elements: mnSO ₄ 0.5g/L，NaCl 1.0g/L，FeSO ₄ .7H ₂ O 1.0g/L，ZnSO ₄ 0.1g/L，CuSO ₄ 0.1g/L，Na ₂ MoO ₄ .2H ₂ O 0.01g/L。

1.4 Main instruments

Ultra-clean bench: SW-CJ-1C Suzhou purification plant Co., ltd; an electronic balance: GJJ200, double Jie group Co., ltd; portable pressure steam sterilizer: YX-280D Hefeihua medical devices Co., ltd; electrothermal constant temperature blast drying box: DL-101 Tianjin middle ring experiment electric furnace Co., ltd; constant temperature shaking incubator: HZC-250 Taicang city experimental facilities factory; ultraviolet visible spectrophotometer: TU-1810 Beijing general analysis general instruments Limited liability company; biochemical incubator: LRH-250A Guangdong province medical equipment works; gas chromatograph-mass spectrometer: GC/MS QP2010 Plus Shimadzu corporation.

1.5 measurement method:

1.5.1 hypha dry weight (g/L): and drying the beaker and the filter paper to constant weight at 80 ℃ for standby, filtering 50mL of bacterial liquid, then drying the filter residue and the filter paper, the beaker and the filter paper to constant weight at 80 ℃, and subtracting the beaker and the filter paper from the weighing result to obtain the mycelium dry weight.

1.5.2 quinone reductase activity: extracellular filtrate or intracellular cell disruption solution, which uses 1, 2-naphthoquinone as substrate and coenzyme NADPH as proton donor, can catalyze NADPH to add protons to naphthoquinone to obtain oxidized NADP ⁺ The latter has no light absorption at 340 nm. Detecting the change of the absorption value of the 340nm solution in real time in the reaction process, wherein the reaction temperature is 25 ℃, and the reaction system is as follows: 100mM Tris-HCl solution 1.5mL,0.2mM NADPH 0.5mL pH7.5, intracellular enzyme extract or filtrate 0.5mL,100mM 1, 2-naphthoquinone 0.5mL,1 enzyme activities were expressed as changes in absorbance at 1min340 nm.

1.5.3 quinone oxidase: 1, 2-naphthol is used as a substrate, and the principle and the process of the substrate react with quinone reductase reversely.

1.5.4 oil drain ring: taking a culture dish with the diameter of 9cm, filling water, dripping a drop of mineral oil on the water surface, sucking extracellular fluid by using a 1mL microscale syringe after a layer of oil film is formed on the surface, accurately dripping a drop of extracellular fluid in the middle of the culture dish by using a needle head, and measuring the diameter of an oil drain ring by using a ruler.

1.5.5 rhamnolipid assay: determination of the amount of rhamnose contained in rhamnolipids: adding 1mL of crude surfactant into a colorimetric tube, adding 4mL of anthrone color-developing agent, cooling, carrying out boiling water bath for 15min, cooling for 20min, colorimetric determination of a light absorption value at 620nm, comparing with a rhamnose standard curve, and calculating the content of rhamnose, namely the content of rhamnolipid.

Determination of 1.5.6 pyrene:

extraction of pyrene: the pyrene-containing contents of a 50mL Erlenmeyer flask were filtered together with filter paper in a 50mL high-type beaker, wherein the glass flask and filter paper were washed 2 times with 5mL of acetone, then with 2 times with 3mL of cyclohexane, and transferred into a 125mL separating funnel, each time with 7mL of cyclohexane and shake-extracted, under each shake 300, the cyclohexane layer was preserved, dehydrated with anhydrous sodium sulfate baked at 300℃for 2 hours, and washed with cyclohexane multiple times, all cyclohexane was collected into 25mL cuvettes with plugs, shaking at constant volume, then 1mL was pipetted into a 2mL sample bottle with a polytetrafluoroethylene gasket, 10. Mu.L of 100mg/L deuterated pyrene and 5. Mu.L of 100mg/L hexamethylbenzene were added, and pyrene was measured on the machine. The recovery rate of pyrene by the extraction method reaches 79.6-81.2%.

Measurement of pyrene content in the organic phase was measured by using a gas chromatograph-mass spectrometer (Shimadzu GC/MS QP2010 Plus, japan). Chromatographic conditions: the sample inlet has no split mode, and the temperature of the sample inlet is 270 ℃; the chromatographic column model is DB-5 elastic quartz capillary chromatographic column (30 m×0.25mm×0.25 μm); column flow 1.65mL/min; the furnace temperature is kept at 55 ℃ for 1min, and is raised to 200 ℃ at 25 ℃/min, is kept at 240 ℃ for 0.5min at 10 ℃/min, and is kept at 280 ℃ for 2min at 30 ℃/min.

1.6 experimental design:

pyrene solution: 6.2653g of pyrene with 98% content is dissolved with methanol to a volume of 1L, which is 6140mg/L, and this is a pyrene standby liquid.

Preparing PDA culture medium, inoculating separated degrading fungi respectively, culturing for 7 days, inoculating mycelium into a triangular flask with 50mL liquid culture medium, and culturing at 28deg.C for 6 days in shaking table 160r/min until mycelium pellet grows into solution; another 50mL triangular flask was taken, 8mL of mixed bacterial liquid containing mycelium pellets (1.60 mL of each bacterial was added for 3 bacterial species), 5mL of pyrene stock solution and 37mL of liquid culture medium were covered with a sterile double-layer filter membrane and sealed with rubber bands, 5 treatments were set for Mc, pn, at and Pn+At+Mc and the non-sterile control CK (Pn, at and Mc 8mL of mycelium dry weight were 0.0228g, 0.0488g and 0.0353 respectively), 2.67mL of each of Pn+At+Mc 3 bacterial species was added respectively, shaking was performed At a constant temperature of 160r/min for 5 days At 28 ℃, the mycelium pellet growth was observed, and the mycelium dry weight, oil drain ring, quinone oxidase, quinone reductase, rhamnolipid and pyrene concentration were measured respectively, 6 treatments were each in parallel, 3 treatments were ensured, and degradation products in the culture were qualitatively analyzed.

1.7 calculation and mapping

And taking the control without bacteria as a reference to calculate the degradation rate of pyrene.

Degradation (removal) rate (%) = (pyrene remaining in blank pyrene-solution)/blank pyrene;

all data were averaged and standard deviation calculated and plotted using the origin software.

2 results and analysis

2.1 The 3 strains of allium (pn+at+mc) produce biosurfactants comprising rhamnolipids, which promote the degradation of pyrene.

As can be seen from FIGS. 1, 3 and 4, the diameter of the oil drain circle of 3 strains of the strain (Pn+At+Mc) for 5 days is 0.45cm and 1.29 times of Pn, and the yield of rhamnolipid (1.88 mg/L) is lower than that of Pn (4.17 mg/L), but the degradation rate of 3 strains of the strain for 5 days on pyrene is 89.7% and higher than that of single strains of the strain (Pn, at and Mc are 81.8%, 86.6% and 86.6%, respectively).

The biosurfactant is a research hot spot for repairing the hydrophobic organic pollutants because of the characteristics of wide application range, various molecular structure types, excellent surface performance, extremely low biotoxicity, environmental friendliness and the like. Rhamnolipids are a common type of biosurfactant of glycolipids secreted by microorganisms in the metabolic process of specific conditions, the molecular structure of the biosurfactant generally takes fatty acyl chains as hydrophobic groups, the glycosyl groups as hydrophilic groups, the hydrophilic groups generally consist of 1-2 molecules of mouse Li Tanghuan, and the hydrophobic groups consist of 1-2 molecules of saturated or unsaturated fatty acids with different carbon chain lengths. Common glycolipid biosurfactants are rhamnolipids, sophorolipids, trehalose lipids and mannitol lipids. Among them, rhamnolipid is a glycolipid which is commercially successful at present, and is formed by connecting saturated or unsaturated fatty acids with 1-2 molecules of mouse Li Tanghuan, and the carbon chain length of the fatty acids is usually 8-14.

Rhamnolipids have the effect of solubilising and reducing surface tension, and in general, surfactant-enhanced biodegradation processes fall into the following categories: 1) The water-oil interfacial tension is reduced, which is beneficial to increasing the contact area of degrading microorganisms and pyrene and increasing accessibility; 2) The hydrophilic and hydrophobic properties of the cells on the surface of the microorganism are changed, the adsorption condition of pollutants is regulated, and the interaction is promoted; 3) The structure and the property of a cell membrane are changed, the mass transfer effect between microorganisms and pollutants is changed, the uptake of the pollutants is promoted, and the action mode is a main mechanism of the action of the biosurfactant on petroleum pollutants. 4) The concentration of the solubilizer is controlled to stimulate the physiological activities of microorganisms to different degrees, and the aim of removing pollutants is achieved by influencing the physiological activities of cells. 5) Solubilization is beneficial to increasing the water solubility of pyrene. The mechanism of the biological surfactant for promoting pyrene degradation is as follows: firstly, a biosurfactant links with pyrene through the hydrophobic effect of the biosurfactant to form a micelle containing pyrene, then contacts a cell membrane, increases the porosity of the cell membrane by utilizing the hydrophilicity of the biosurfactant, enables the pyrene to enter the cell, and is decomposed and utilized by mixed enzymes.

The biosurfactant may be spontaneously produced by microorganisms or may be induced in the presence of hydrophobic compounds or external pressure (e.g. pH, temperature, aeration, agitation speed or nitrogen source limitation, etc.), so that the composition of the medium, physicochemical conditions and its culture environment affect the properties and yield of the biosurfactant. The diameter of an oil drain ring of 3 strain allied bacteria for 5 days is 0.45cm and is larger than Pn; however, the yield of rhamnolipid is lower than Pn, the degradation rate of pyrene is 89.7% according to 3 strains of the allied bacteria for 5 days, and is higher than 81.8%, 86.6% and 86.6% of Pn, at and Mc, so that the surfactant generated by Pn is considered to be mainly rhamnolipid, the surfactant generated by 3 strains of the allied bacteria (Pn+at+Mc) contains rhamnolipid and other lipopeptides surfactant, and the culture condition of 3 strains of the allied bacteria can promote the degradation of pyrene.

2.2 The dry weight of 3 strain hyphae of the allied bacteria is not maximized, quinone oxidase is detected but quinone reductase is not detected, and the synergistic degradation of 3 strain allied bacteria on pyrene is larger than that of single strain enzyme degradation.

As can be seen from FIGS. 1, 5 and 6, the quinone reductase activity is only detected in At, 3 strains of the bacterium (Pn+at+Mc) and the other two single strains have no quinone reductase activity, but Pn+at+Mc and Mc have quinone oxidase activity, and in general, under the stress of specific pollutants, when hyphae grow to a certain extent, specific degrading enzymes, such as peroxidase systems produced by white rot fungi (Phanerochaete chrysosporium) under the condition of limited nitrogen and sulfur, are easy to produce under the condition that the mycelium is limited by certain nutrient elements in the environment, and the degrading enzymes have strong degrading capability on pollutants. Similarly, glucose is added into the experiment culture medium as a co-metabolism carbon source, in the early stage of pyrene stress, the microorganism preferentially utilizes glucose as the carbon source to increase the dry weight of hyphae, meanwhile, the environment of high-concentration pyrene plays a potential domestication and induction role in the growth and adaptation of the microorganism, and when the glucose consumption is complete, the dry weight of the hyphae is rapidly increased, various nutrient elements in the culture medium are limited, and the microorganism is stimulated to produce degrading enzymes.

Quinone reduction/oxidase (NADPH/NADP) is a kinase that drives one of the key enzymes of PAHs substance metabolism as a metabolite phenanthrenequinone substance formed in degradation. The metabolic pathways of pyrene are: the microorganism produces dioxygenase and monooxygenase, the dioxygenase oxidizes pyrene to cis-4, 5-pyrene dihydro-diol, then dehydrogenates to form 4, 5-dihydro-diol pyrene under the action of dihydro-diol dehydrogenase, or the monooxygenase and 4, 5-pyrene oxidase oxidize pyrene directly to 4, 5-dihydro-diol pyrene, then oxidize pyrene 4, 5-dione under the action of quinone reductase to form pyrene 4, 5-diacid, then further form 4-phenanthrene acid, continue oxidizing to phenanthrene-dihydro-diol-4-carboxylic acid under the action of dioxygenase, further generate 1-hydroxy-2-naphthoic acid, dehydrogenate to form 1-hydroxy-2-naphthoate under the action of acetaldehyde dehydrogenase, then enter naphthalene degradation path to form naphthalene 1, 2-diol, further form salicylaldehyde, oxidize to salicylic acid under the action of salicylaldehyde oxidase, then form gentisic acid under the action of salicylic acid-5-hydroxylase, and metabolize gentisic acid through the tyrosine path.

As can be seen from FIGS. 1 and 2, the result of the hypha dry fungus of 3 strains (Pn+at+Mc) for 5 days is 2.23g/L, which is higher than Pn and Mc but lower than 2.90g/L of At, but the removal of pyrene by 3 strains for 5 days is maximum, thereby deducing that the growth of the hypha dry weight of 3 strains limited by the total nutrition is mutually competitive and balanced, and metabolites are mutually utilized.

In summary, 3 strains of the allied bacteria (Pn+At+Mc) produced rhamnolipids, oil drain rings, quinone reductase and hyphae were not higher than single strains, only a small amount of quinone oxidase activity was detected, and the activity was lower than Mc, but the pyrene degradation rate was higher than that of 3 single strains. The general synergistic degradation is more efficient than single species degradation, such as Wang et al (2018) by introducing the rice soil component into red soil, creating a mixed soil microflora. The novel mixed microbial flora is found to be capable of effectively degrading high molecular weight PAHs-pyrene in the soil mixture. The initial removal rate of the red soil and the paddy soil to the pyrene is 19% and 98% respectively; the removal rate of pyrene by the mixed microbial community is obviously improved by increasing the dosage of the rice soil inoculant, and the removal rates of the pyrene in a rice soil/red soil mixed system with the mass ratio of 1/1, 3/7 and 1/9 are respectively 93%, 58% and 27%. The microorganisms often have the functions of mutual promotion and synergistic degradation, and the method is mainly characterized in that the activity of degrading enzymes is improved, the enzyme production environments of more than two key degrading enzymes are consistent, the degrading enzymes are complementary, metabolites are utilized, the electron transfer speed is accelerated and the like. Therefore, the mechanism of the 3-strain linkage strain inter-species synergistic degradation is the mutual utilization of metabolites and the acceleration of the electron transfer speed.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims

1. A multi-strain composition for degrading polycyclic aromatic hydrocarbons comprises Mucor niveus, mucor circinelloides and Aspergillus terreus.

2. The multi-strain composition of claim 1, wherein the polycyclic aromatic hydrocarbon is a 4-7 ring polycyclic aromatic hydrocarbon.

3. A biological agent comprising the multi-strain composition of claim 1 or 2 or a culture thereof.

4. A method of preparing the biologic of claim 3, comprising the steps of:

respectively inoculating the myceliophthora, the mucor circinelloides and the aspergillus terreus to a PDA (digital versatile array) culture medium for culture, and taking all bacterial mycelia to culture in a liquid culture medium to respectively obtain hypha culture solutions of the myceliophthora, the mucor circinelloides and the aspergillus terreus; mixing the mycelium culture solutions in proportion to obtain the biological preparation.

5. The method according to claim 4, wherein the liquid medium comprises glucose, amine tartrate, KH ₂ PO ₄ 、MgSO ₄ .7H ₂ O、CaCl、Na ₂ HPO ₄ And trace elements.

6. The method according to claim 5, wherein the trace elements include MnSO ₄ 、NaCl、FeSO ₄ .7H ₂ O、ZnSO ₄ 、CuSO ₄ And Na (Na) ₂ MoO ₄ .2H ₂ O。

7. The preparation method according to claim 6, wherein the mixture ratio of the mycelium culture solutions is 1 (1-2): 1.5-3 by weight of hypha of myceliophthora nivea, mucor circinelloides and aspergillus terreus.

8. Use of the multi-strain composition of claim 1 or 2 and/or the biological agent of claim 3 in any one of (1) to (4):

(1) Degrading polycyclic aromatic hydrocarbon;

(2) Preparing a product for degrading polycyclic aromatic hydrocarbon;

(3) Preparing a surfactant;

9. A method of restoring soil contaminated with polycyclic aromatic hydrocarbons comprising the step of applying the multi-strain composition of claim 1 or 2 and/or the biological agent of claim 3 to the contaminated soil.

10. The remediation method of claim 9, further characterized by applying, wetting, drenching, atomizing, sleeking, spraying, atomizing, soaking the multi-strain composition or the biological agent to act on contaminated soil.