CN110734882A

CN110734882A - phenanthrene efficient degradation bacterial strains and application thereof in environmental remediation

Info

Publication number: CN110734882A
Application number: CN201911164239.1A
Authority: CN
Inventors: 唐鸿志; 张莉鸽; 许平; 王伟伟; 柳宁; 陶飞
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2019-11-25
Filing date: 2019-11-25
Publication date: 2020-01-31
Anticipated expiration: 2039-11-25
Also published as: CN110734882B

Abstract

The invention discloses phenanthrene high-efficiency degradation bacterial strains and application thereof in environmental remediation, and relates to the technical field of environmental biology, wherein the bacterial strains can take phenanthrene as only carbon source and energy source, namely sphingolipid bacteria SZ1(Sphingobium sp.SZ1), the bacterial strains are stored in China center for type culture collection, the collection number is CCTCC M2019792, and the collection time is 2019, 10 months and 10 days.10 the sphingolipid bacteria SZ1 provided by the invention have high-efficiency degradation capability on phenanthrene, especially high-concentration (200 mg/L-800 mg/L) phenanthrene, and also have degradation capability on for other polycyclic aromatic hydrocarbons, such as naphthalene, fluorene, carbazole, dibenzofuran and dibenzothiophene, so that the biological environment remediation treatment effect of high efficiency, cleanness and no secondary pollution can be realized.

Description

phenanthrene efficient degradation bacterial strains and application thereof in environmental remediation

Technical Field

The invention relates to the technical field of environmental biology, in particular to phenanthrene efficient degradation strains and application thereof in environmental remediation.

Background

Polycyclic Aromatic Hydrocarbons (PAHs) are types of organic pollutants which are in the environment, the PAHs are types of Hydrocarbons consisting of two or more benzene rings, the PAHs are generally characterized by high melting point and high boiling point, are insoluble in water, are easily soluble in organic solvents such as benzene, ether, chloroform and the like, and along with the increase of the number of the benzene rings, the water solubility of the PAHs is reduced, and the bioavailability and the degradability of the PAHs are also reduced.

PAHs are mainly products produced by incomplete combustion or low-temperature treatment (100-300 ℃) of organic compounds under high-temperature conditions (500-800 ℃). With the rapid development of economy, the current situation of PAHs pollution in China is very severe. According to preliminary estimation, the annual emission of PAHs in China exceeds 25000 tons, and the average emission density of cities is 158kg/km²The local discharge density in rural areas is up to 479kg/km². Shanghai city PM_2.5The average content of 16 main PAHs in the sample is 34.021ng/m³(ii) a The average content of benzo (a) pyrene is 3.046ng/m³Is the maximum allowable value of 2.5ng/m of benzo (a) pyrene per 24h specified by national standard³1.22 times of. PAHs have the characteristics of cytotoxicity, immunotoxicity, carcinogenesis, teratogenicity and mutagenicity, and can affect human health in the modes of environmental accumulation, biological accumulation, biotransformation and the like after entering the environment, and even cause genetic defects. The toxicity of PAHs has attracted public attention from countries around the world. As early as 1979, 16 PAHs have been listed as the priority pollutants for monitoring by the united states national Environmental Protection Agency (EPA). Therefore, PAHs in various media in the environment are efficiently degraded, pollution of the PAHs is eliminated, and human health and environment are maintainedThe protection is of great significance.

At present, the main repair methods of PAHs include physical methods, chemical methods, incineration methods and microbial degradation methods. The physical method and the chemical method mainly comprise activated carbon adsorption, flocculation precipitation, photo-oxidation method, ultrasonic oxidation method and the like, and the incineration method is to decompose pollutants by using high temperature. Although the traditional treatment method is simple in operation, the traditional treatment method cannot deal with the problem of pollution which is nowadays increasingly severe. The methods have the advantages of insignificant treatment effect and easy generation of secondary pollution. The microbial degradation method which is clean, efficient and free of secondary pollution becomes the most effective method for treating environmental pollutants.

Numerous strains capable of growing on phenanthrene as the sole carbon and energy source have been discovered so far, most of which are isolated from contaminated soil, such as Pseudomonas, sphinganas, mycobacter, Rhodococcus and bacillus, but these strains are basically intolerant to high concentrations of phenanthrene (>200mg/L), and have long growth cycles, slow degradation rates and relatively poor practical environmental application effects.

Therefore, the technical personnel in the field are dedicated to developing phenanthrene high-efficiency degradation strains, can efficiently degrade high-concentration phenanthrene, has definite degradation capability on other polycyclic aromatic hydrocarbon compounds, and can realize clean, high-efficiency and secondary pollution-free environmental remediation effects.

Disclosure of Invention

In view of the above defects in the prior art, the technical problem to be solved by the invention is how to provide phenanthrene high-efficiency degradation strains, which can not only efficiently degrade high-concentration phenanthrene, but also have definite degradation capability on other polycyclic aromatic hydrocarbon compounds, thereby realizing clean, high-efficiency and secondary pollution-free environmental remediation.

In order to achieve the purpose, the invention provides phenanthrene high-efficiency degradation strains, the strains can use phenanthrene as only carbon source and energy source and are sphingolipid bacteria SZ1(Sphingobium sp. SZ1), the strains are preserved in China center for type culture collection, the preservation number is CCTCC M2019792, the preservation time is 2019, 10 months and 10 days, and the preservation address is Wuhan, China.

, the sphingolipid SZ1 is gram-negative bacteria, the thallus is in the shape of short rod, can move, has no spore, and the colony grows white, round and smooth edge on the basic salt (MSM) culture medium.

The invention also provides application of phenanthrene efficient degrading strains in environmental remediation.

, the application includes removing the pollutant phenanthrene in the environment, which includes the following steps:

step , culturing the sphingolipid bacteria SZ 1;

step two, inoculating the sphingolipid bacterium SZ1 cultured in the step into a contaminated sample containing phenanthrene, and culturing to remove the phenanthrene in the contaminated sample.

, the culturing step includes slant culture and seed culture, wherein the slant culture uses basic salt (MSM) solid culture medium and phenanthrene steam as carbon source, and the seed culture uses basic salt (MSM) liquid culture medium containing phenanthrene.

, the basic salt (MSM) solid culture medium is composed of the basic salt (MSM) liquid culture medium and agar powder, wherein the mass volume ratio of the agar powder is 1% -2%.

And , the contaminated sample in the second step includes a water body sample.

, the concentration range of the applicable substrate phenanthrene for degrading phenanthrene by the strain is 200 mg/L-800 mg/L.

Further , the phenanthrene degradation products of the strain include 3,4-dihydroxyphenanthrene (3, 4-dihydroxyphenylene), 1-hydroxy-2-naphthoic acid (1-hydroxy-2-naphthoic acid), 1-naphthol (1-naphthol), and salicylic acid (salicylic acid).

Further , the use further includes degrading environmental pollutants such as naphthalene, fluorene, carbazole, dibenzofuran, and dibenzothiophene.

Compared with the prior art, the invention at least has the following beneficial technical effects:

(1) the sphingolipid bacteria SZ1 provided by the invention can grow by taking phenanthrene as only carbon source and energy source, and has high-efficiency degradation effect on phenanthrene with the concentration as high as 200 mg/L-800 mg/L, so that the pollutant phenanthrene in the environment can be degraded efficiently, cleanly and without secondary pollution, and a good environment bioremediation treatment effect is achieved;

(2) the sphingolipid bacteria SZ1 provided by the invention can also show degree degradation capability to other polycyclic aromatic hydrocarbon pollutants including naphthalene, fluorene, carbazole, dibenzofuran and dibenzothiophene, so that a better biological remediation environmental effect is realized;

(3) the method utilizes the microbial sphingolipid bacteria SZ1 to carry out biocatalysis on phenanthrene, and has the advantages of low catalyst cost, simple and convenient operation, mild reaction conditions and energy conservation.

The concept, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.

Drawings

FIG. 1 is a scanning electron micrograph of the morphology of SZ1 of sphingolipid according to a preferred embodiment of the present invention ();

FIG. 2 is a dendrogram of preferred embodiments of the present invention, which is obtained by analyzing the 16S rRNA sequence of sphingolipid SZ 1;

FIG. 3 is a data graph showing the growth of preferred embodiments of sphingolipid strain SZ1 in MSM liquid medium containing phenanthrene at various concentrations;

FIG. 4 is a graph showing phenanthrene degradation data of preferred embodiments of sphingolipid bacteria SZ1 in MSM liquid medium containing different concentrations of phenanthrene according to the present invention;

FIG. 5 is a schematic diagram showing the degradation pathway of phenanthrene by sphingolipid strain SZ1 according to the present invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

The invention provides new strains capable of efficiently decomposing and metabolizing phenanthrene, namely sphingolipid bacteria SZ1(Sphingobiumsp. SZ1), which can grow in MSM liquid culture medium by taking 200-800 mg/L phenanthrene as only carbon source and energy source, degradation products of phenanthrene, such as 3,4-dihydroxyphenanthrene (3,4-dihydroxyphenanthrene), 1-hydroxy-2-naphthoic acid (1-hydroxy-2-naphthoic acid), 1-naphthol (1-naphthonol) and salicylic acid (salicylic acid), can be detected in the culture process, so that the way of metabolizing phenanthrene by sphingolipid bacteria SZ1 is that dioxygen is added at the 3-4-position of phenanthrene, then benzene rings are broken to generate 1-hydroxy-2-naphthoic acid, and the phenanthrene enters TCA circulation through 1-naphthol and salicylic acid sequentially, enters TCA circulation, so as to achieve the purpose that phenanthrene is completely mineralized, and the phenanthrene can be degraded in water with high concentration and can be degraded in % polluted water bodies after passing through 3680 days of environment.

Example screening and isolation of sphingolipid bacteria SZ1

1. Sampling a sample

Soil sample collection site: tianjin polycyclic aromatic hydrocarbons pollute petrochemical pollution sites.

2. Screening and isolation of strains

5g of soil sample is dissolved in 50mL of MSM liquid culture medium, and the soil solution is obtained after shaking for 5 minutes on a vortex oscillator. 5mL of soil solution is added into 50mL of MSM liquid culture medium, a small amount of phenanthrene solid is added, and the mixture is cultured for 5 days in a constant temperature shaking table at 30 ℃ and 200 rpm. And (3) inoculating 5mL of seed solution into a new MSM culture medium, culturing for 3 days under the same conditions, continuing to perform the inoculation, after passage for 3-4 times, diluting the culture solution, coating the culture solution on an MSM solid culture medium, performing steam culture on phenanthrene mother solution, and culturing for 5-10 days at 30 ℃. Selecting single colony to fresh MSM liquid culture medium containing 200mg/L phenanthrene, selecting the strain with the fastest growth, streaking and separating, and repeating for multiple times until obtaining purified single bacteria.

EXAMPLE two identification of sphingolipid bacteria SZ1

1. The thallus and colony morphology characteristics of the strain

As shown in figure 1, the thallus is in a short rod shape, the length of the thallus is about 0.6 mu m, the thallus can move and has no spores, a bacterial colony grows on a basic salt culture medium to be transparent white and round, the bacterial colony is small and smooth in edge and is easy to pick up, the sphingolipid SZ1 is a gram-negative bacterium which favors oxygen, and phenanthrene can be used as a carbon source and an energy source only for growth.

2. Physiological and biochemical characteristics of the strain

The strain is gram-negative bacteria, prefers oxygen and can grow by using phenanthrene as only carbon-nitrogen source.

The above bacteriological characteristics are consistent with the physiological and biochemical properties of sphingolipid bacteria documented in the literature (manual for identifying common bacteria, 2001). The strain was studied for phylogenetic use of 16S rRNA sequence, as shown in FIG. 2, and related to sphingolipid bacteria recently. Combined with the above physiological and biochemical characteristics and phylogenetic tree analysis, it was identified as sphingolipid strain SZ1(Sphingobium sp.

EXAMPLE III degradation Properties of Phosphatidylseb SZ1 on phenanthrene

1. Effect of phenanthrene concentration on Strain culture

2.5mL of the seed solution was inoculated into 50mL of MSM liquid medium containing phenanthrene (200mg/L, 350mg/L, 500mg/L, 650mg/L and 800mg/L) at different concentrations, and the growth of the strain and the degradation of phenanthrene were compared under the culture conditions of 30 ℃ and 200 rpm. It can be found that the strain can grow under the concentration of 200 mg/L-800 mg/L phenanthrene (as shown in figure 3) and can degrade phenanthrene relatively quickly (as shown in figure 4).

2. HPLC detection of phenanthrene content

The detection conditions of HPLC are as follows: an Agilent 1100 high performance liquid chromatograph is provided with an Eclipse XDB-C18 analytical column; the mobile phase is water: methanol 20: 80(v/v), the flow rate is 0.8mL/min, the ultraviolet detection wavelength is 254nm, and the column temperature is 30 ℃.

And (3) quantitatively detecting the concentration of the phenanthrene by using HPLC, preparing phenanthrene standard substances with different concentration gradients, measuring peak areas under corresponding concentrations, and drawing a phenanthrene standard curve. And (3) after the sample to be detected is diluted to a proper concentration, converting the concentration of phenanthrene from a standard curve according to the integral area of a peak.

Example four analysis of intermediates in degradation of phenanthrene by sphingolipid bacteria SZ1

1. Preparation of product sample of sphingolipid bacterium SZ1

1mL of the seed solution was inoculated into 20mL of MSM broth containing 200mg/L of phenanthrene, and cultured at 30 ℃ and 200 rpm. In the growth process of the strain, 20mL of bacterial liquid is periodically taken, evenly divided into two parts, the pH is respectively adjusted to 7 and 2, the two parts are respectively extracted and oscillated by 10mL of ethyl acetate for 1min, and then an upper organic phase is collected. The organic phase was dried after concentration after removal of water with excess anhydrous sodium sulfate and resuspended in 1mL chromatographic grade ethyl acetate for subsequent GC-MS analysis.

2. GC-MS detection conditions

The gas chromatography (Agilent 6850/5975C) inlet and detector temperatures were 280 deg.C, helium flow rate 1mL/min, and pre-column pressure 50 kPa. The temperature rise process is as follows: the initial sample introduction temperature is 75 ℃, and the sample is kept for 1 min; the temperature is raised to 300 ℃ at a rate of 14 ℃/min and kept for 8 min.

The GC-MS can detect intermediate metabolites such as 3,4-dihydroxyphenanthrene (3, 4-dihydroxyphenyl naphthalene), 1-hydroxy-2-naphthoic acid (1-hydroxy-2-naphthoic acid), 1-naphthol (1-naphthol), salicylic acid (salicylic acid) and the like.

Example five simulation application of sphingolipid bacteria SZ1 in environmental remediation

Because the actual polluted environment is generally severe, in order to simulate the application of sphingolipid SZ1 in removing phenanthrene from wastewater, the adaptive range (200 mg/L-800 mg/L) of the strain to the phenanthrene concentration has been tested. The degradation rates for various concentrations of phenanthrene over the 72 hour period are shown in the data in table 1.

The simulation application of the sphingolipid bacteria SZ1 in wastewater shows that the bacterial strain can realize more than 78% degradation of phenanthrene with different concentrations within 72 hours, and has higher tolerance concentration and degradation speed compared with other bacterial strains.

TABLE 1 degradation rate of phenanthrene of sphingolipid bacteria SZ1 on wastewater containing phenanthrene of different concentrations

Phenanthrene concentration	200mg/L	350mg/L	500mg/L	650mg/L	800mg/L
						Degradation rate of 72h phenanthrene	80.0％	94.1％	78.0％	93.3％	83.1％

EXAMPLE VI degradation Performance of sphingolipid bacteria SZ1 on other polycyclic aromatic hydrocarbons

The pollution of polycyclic aromatic hydrocarbons in the environment is often the combined pollution of a plurality of pollutants, so the influence of different polycyclic aromatic hydrocarbons on the culture of sphingolipid bacteria SZ1 is measured.

1. Use condition of sphingolipid bacteria SZ1 on different polycyclic aromatic hydrocarbons

Other polycyclic aromatic hydrocarbons are: naphthalene, fluorene, anthracene, fluoranthene, pyrene, benzo (a) anthracene, dibenzofuran, dibenzothiophene, carbazole, biphenyl, 11 in total.

2.5mL of the seed solution was inoculated into 50mL of MSM liquid medium containing 100mg/L of polycyclic aromatic hydrocarbon, and cultured at 30 ℃ and 200 rpm. After 72 hours, the growth of the strains under different polycyclic aromatic hydrocarbon culture conditions is compared.

2. Determination of growth

Fully extracting a liquid culture medium containing the thalli and ethyl acetate with the same volume at normal temperature, standing for a moment, taking 1mL of lower-layer water phase, detecting the light absorption value of the lower-layer water phase at the wavelength of 600nm, and taking the lower-layer water phase as the measurement standard of the growth state of the strain. "+" indicates that 0.2-0.4 optical densities are achieved after 72 hours, "+" indicates that 0.4-0.8 optical densities are achieved after 72 hours, and "-" indicates that the absorbance is substantially the same as before inoculation.

As shown in Table 2, sphingolipid strain SZ1 has different utilization degrees for naphthalene, fluorene, carbazole, dibenzofuran and dibenzothiophene, has no utilization ability for anthracene, fluoranthene, pyrene, benzo (a) anthracene and biphenyl, and has a substrate spectrum of .

TABLE 2 utilization of different polycyclic aromatic hydrocarbons by sphingolipid bacteria SZ1

Substrate	Whether or not to grow
		Naphthalene	+
Fluorene compounds	+
		Carbazole	++
Dibenzofurans	+
		Dibenzothiophenes	++
Anthracene	-
		Fluoranthene	-
Pyrene	-
		Benzo (a) pyrene	-
Benzo (a) anthracenes	-
		Biphenyl	-

EXAMPLE VII degradation Properties of sphingolipid SZ1 for fluorene, carbazole, dibenzofuran, and dibenzothiophene

1. Effect of fluorene, carbazole, dibenzofuran, dibenzothiophene on Strain culture

In the sixth example, it is known that sphingolipid strain SZ1 has good utilization ability for fluorene, carbazole, dibenzofuran, and dibenzothiophene, and then the specific degradation characteristics of sphingolipid strain SZ1 for these four polycyclic aromatic hydrocarbons were examined by steps.

Inoculating 2.5mL of seed liquid into 50mL of MSM liquid culture medium containing 100mg/L of polycyclic aromatic hydrocarbon, taking the liquid culture medium containing thalli after 72 hours, fully extracting the liquid culture medium and ethyl acetate with the same volume at normal temperature, standing for a moment, taking supernatant, and detecting the polycyclic aromatic hydrocarbon.

2. HPLC (high performance liquid chromatography) detection of content of polycyclic aromatic hydrocarbon

And (3) quantitatively detecting the concentration of the substrate by using HPLC (high performance liquid chromatography), preparing polycyclic aromatic hydrocarbon standard substances with different concentration gradients, measuring peak areas under corresponding concentrations, and drawing a standard curve. And (3) after the sample to be detected is diluted to a proper concentration, converting the concentration of the polycyclic aromatic hydrocarbon by a standard curve according to the integral area of the peak.

As shown in Table 3, sphingolipid SZ1 can be used for degrading fluorene, carbazole, dibenzofuran and dibenzothiophene, can degrade more than 45% of substrates in 72 hours under the condition that the initial concentration is 100mg/L, and has higher degradation activity compared with other similar strains.

TABLE 3 degradation rates of sphingolipid strain SZ1 for fluorene, carbazole, dibenzofuran, and dibenzothiophene

Substrate	Fluorene compounds	Carbazole	Dibenzofurans	Dibenzothiophenes
					Degradation rate of 72h	54.8％	51.8％	45％	53.3％

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

The phenanthrene high-efficiency degradation strain is characterized in that phenanthrene can be used as -only carbon source and energy source and is sphingolipid bacterium SZ1(Sphingobium sp.SZ1), the strain is preserved in China center for type culture collection with the preservation number of CCTCCM 2019792, and the preservation time is 2019, 10 months and 10 days.
2. The phenanthrene efficient degradation strain of claim 1, wherein the sphingolipid SZ1 is a gram-negative bacterium, the thallus of the bacterium is in a short rod shape, can move, has no spores, and the colony grows white, round and smooth edges on a basic salt (MSM) culture medium.
3, application of the phenanthrene efficient degradation strain of any of claims 1-2 in environmental remediation.
4. The application of the phenanthrene efficient degradation strain in environmental remediation, which is claimed in claim 3, comprises application of removing pollutants phenanthrene in the environment, and specifically comprises the following steps:

step , culturing the sphingolipid bacteria SZ 1;

step two, inoculating the sphingolipid bacterium SZ1 cultured in the step into a contaminated sample containing phenanthrene, and culturing to remove the phenanthrene in the contaminated sample.
5. The use of the phenanthrene highly efficient degradation strain in environmental remediation as claimed in claim 4, wherein the culturing in step includes slant culture and seed culture, wherein the slant culture uses a basic salt (MSM) solid medium and phenanthrene vapor as a carbon source, and the seed culture uses a basic salt (MSM) liquid medium containing phenanthrene.
6. The application of the phenanthrene efficient degradation strain in environmental remediation, which is claimed in claim 5, wherein the basic salt (MSM) solid culture medium consists of a basic salt (MSM) liquid culture medium and agar powder, and the mass volume ratio of the agar powder is 1% -2%.
7. The application of the phenanthrene efficient degrading strain in environmental remediation according to claim 4, wherein the polluted sample in the second step comprises a water body sample.
8. The application of the phenanthrene efficient degradation strain in environmental remediation as claimed in claim 4, wherein the concentration range of the phenanthrene applicable substrate for phenanthrene degradation of the strain is 200 mg/L-800 mg/L.
9. The use of the phenanthrene-degrading strain with high efficiency in environmental remediation, according to claim 4, wherein the phenanthrene-degrading product of the strain comprises 3,4-dihydroxyphenanthrene (3,4-dihydroxyphenanthrene), 1-hydroxy-2-naphthoic acid (1-hydroxy-2-naphthoic acid), 1-naphthol (1-naphthol), and salicylic acid (salicylic acid).
10. The application of the phenanthrene high-efficiency degradation strain in environmental remediation, which is characterized by further comprising degrading pollutants in the environment, namely naphthalene, fluorene, carbazole, dibenzofuran and dibenzothiophene.