CN114958670B - In-situ PAHs degradation functional bacterium LJB-135, and microbial inoculum and application thereof - Google Patents
In-situ PAHs degradation functional bacterium LJB-135, and microbial inoculum and application thereof Download PDFInfo
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- CN114958670B CN114958670B CN202210613942.1A CN202210613942A CN114958670B CN 114958670 B CN114958670 B CN 114958670B CN 202210613942 A CN202210613942 A CN 202210613942A CN 114958670 B CN114958670 B CN 114958670B
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- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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Abstract
The invention discloses an in-situ PAHs degradation functional bacterium LJB-135, a microbial inoculum and application thereof. The strain is Marinobacter sp.LJB-135, and is deposited in Guangdong province microorganism culture collection center with the deposition number of GDMCC No. 62388. Experiments prove that the strain LJB-135 can utilize phenanthrene as a unique carbon source and quickly degrade phenanthrene; after 3 days of culture in an inorganic salt culture medium with the initial concentration of phenanthrene of 100mg/L, the degradation rate is 56.1 percent; the degradation rate of the strain prepared into a microbial inoculum can reach 73.1 percent. Therefore, the strain has better application potential in bioremediation of polycyclic aromatic hydrocarbon polluted environment.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to an in-situ PAHs degradation functional bacterium LJB-135, a microbial inoculum and application thereof.
Background
Polycyclic aromatic hydrocarbons are a class of environmental pollutants, are ubiquitous in soil and water, and can form great harm to ecological environment and human health. Common PAHs include class 16, of which phenanthrene is often used as a PAH model compound for PAHs biodegradation studies. The main reasons are due to their unique chemical structure and their properties of persistence, carcinogenesis, teratogenicity, mutagenicity and bioaccumulation.
The microorganism can degrade PAHs in the environment so as to achieve the aim of restoration. To explore the homing of PAHs in the environment, several culture-based identification methods were able to identify and isolate microorganisms that were able to degrade them individually. To date, many functional microorganisms have been isolated which degrade PAHs, most of which are strains from the genera Paenibacillus, burkholderia, stenotrophoromonas, mycobacterium, sphingomonas, and the like. The pure culture method provides clues for determining the types, functional genes and metabolic characteristics of the degrading bacteria. However, in practical environments, only a few contaminant-functional degrading bacteria can be isolated; moreover, it is doubtful whether the isolated functional microorganism can function in a natural habitat. Since most microorganisms present in the natural environment are not culturable, moreover, this method greatly underestimates the prokaryotic microbial diversity, does not explain the complex interactions between individuals within the microbial community in the natural environment, and does not reflect the real situation of degradation of pollutants by functional microorganisms in a contaminated environment. Therefore, it is difficult to use laboratory culture hair to reveal the ecological functions of indigenous microorganisms in situ. In contrast, the use of non-culturable methods makes it possible to evaluate the metabolic response of functional microorganisms without the need for laboratory culture and to directly link their properties to functions in the natural habitat.
The Stable Isotope Probe (SIP) technology can combine the stable isotope labeling technology with molecular biological means such as high-throughput sequencing, TRFLP, etc., and can determine microorganisms with degradation function in an environmental sample by adding a stable isotope labeled target compound (13C or 15N) to the environmental sample and analyzing a stable isotope labeled biomarker (such as DNA, RNA, etc.) by using the molecular biological means. This technique can bypass the requirement of isolated culture and directly link microbial communities and functions. At present, various PAHs degradation functional bacteria have been successfully identified by applying SIP technology, but few researchers successfully isolate the PAHs degradation functional bacteria by a separation culture mode, so that the strains cannot be applied. Therefore, the research takes phenanthrene as a target compound, utilizes SIP to explore PAHs degrading microorganisms, sets a culture medium according to information such as the genetic relationship of identified functional bacteria, screens out the microorganisms with the function of degrading the PAHs in situ, and provides data support for the biological treatment of phenanthrene and other polycyclic aromatic hydrocarbons.
Disclosure of Invention
The first purpose of the invention is to provide a marine bacillus lipolyticus (Marinobacter lipolyticus) LJB-135 with a polycyclic aromatic hydrocarbon degradation function. The strain was deposited in the Guangdong province culture Collection (GDMCC) at 14/4/2022, address: building 5 of first furnance, large yard, 100, building 59, guangdong province, guangzhou, china, zip code: 510070, the preservation number is: GDMCC No:62388.
the reported strain is Marinobacter lipolyticus (Marinobacter lipolyticus) LJB-135, which is a novel strain of the Marinobacter separated and identified in an eastern petroleum-polluted field in 2021, but the strain has not been reported at home and abroad for the research on PAHs degradation. The research adopts SIP-pure culture technology to explore, domesticate and separate 1 high-efficiency strain taking phenanthrene as a carbon source from the oil field of Shandong Dongying City in Shandong province, identifies the strain, researches the growth characteristic and the degradation characteristic of the strain on phenanthrene, simultaneously prepares the strain into a microbial agent and explores the degradation characteristic of the strain on phenanthrene, and provides reference for bioremediation of PAHs polluted environment.
The second purpose of the invention is to provide the application of the Marinobacter lipolyticus LJB-135 in degrading polycyclic aromatic hydrocarbons.
Preferably, the polycyclic aromatic hydrocarbon is polycyclic aromatic hydrocarbon in degraded soil or sewage.
Preferably, the polycyclic aromatic hydrocarbon is a phenanthrene.
The third purpose of the invention is to provide the application of the Marinobacter lipolyticus LJB-135 in the preparation of the polycyclic aromatic hydrocarbon degrading microbial inoculum.
The fourth purpose of the invention is to provide a polycyclic aromatic hydrocarbon degrading microbial inoculum, which comprises the marine bacillus lipolyticus (Marinobacter lipolyticus) LJB-135 as an active ingredient.
Preferably, the preparation method of the polycyclic aromatic hydrocarbon degrading microbial inoculum comprises the following steps:
s1: preparing sea bacillus lipolyticus (Marinobacter lipolyticus) LJB-135 into OD 600 Bacterial suspension of = 1;
s2: mixing the corn straw biochar and the bacterial suspension according to the proportion of 1:20w/v, shaking and culturing for 8 hours at 28 ℃ and 180r/min, then centrifuging and cleaning twice with sterile water, centrifuging for 5min under the centrifugation condition of 4000r/min, taking precipitate to obtain adsorbed thalli, adding sterile water into the adsorbed thalli, and fixing the volume to 20mL to obtain an adsorbed thalli solution;
s3: respectively weighing 10g of polydienol and 0.5g of sodium alginate, adding 60mL of distilled water, heating and stirring at 70 ℃ to dissolve the polydienol and the sodium alginate, sterilizing at 121 ℃ for 15min, and cooling to obtain an embedding agent solution;
s4: weighing 5g Ca (NO) 3 ) 2 Adding the mixture into 100mL of deionized water, stirring to dissolve the mixture, and adjusting the pH value to 7.0 to obtain a cross-linking agent solution;
s5: adding 20mL of adsorbed thallus solution into the embedding agent solution, diluting to 100mL with sterile water, uniformly mixing, taking 50mL of mixed solution by using a 50mL syringe with the caliber of 2mm, dropwise adding the mixed solution into 100mL of cross-linking agent solution from a height of 30cm, and fixing for 1 hour to obtain the polycyclic aromatic hydrocarbon degrading microbial inoculum.
The fifth purpose of the invention is to provide the application of the Marinobacter lipolyticus LJB-135 and the polycyclic aromatic hydrocarbon degrading microbial inoculum in bioremediation of polycyclic aromatic hydrocarbon polluted environment.
Preferably, the polycyclic aromatic hydrocarbon polluted environment comprises polycyclic aromatic hydrocarbon polluted water and/or soil. The polycyclic aromatic hydrocarbon is phenanthrene.
The sixth purpose of the invention is to provide a method for degrading polycyclic aromatic hydrocarbon, which is to sprinkle the Marinobacter lipolyticus LJB-135 or the polycyclic aromatic hydrocarbon degrading microbial inoculum in an environment containing polycyclic aromatic hydrocarbon to degrade the polycyclic aromatic hydrocarbon.
The invention adopts a DNA Stable Isotope Probe (SIP) technology to explore a microorganism which belongs to the genus Helminthobium and is represented by OTU-5 and belongs to MarinobaThe similarity of 16S rRNA of cter lipolyticus is as high as 99.7%. Starting from the identified genetic relationship of functional bacteria, a specific culture medium is set, a degradation strain LJB-135 taking phenanthrene as a carbon source is successfully obtained by domestication and separation from contaminated soil of a Shengli oil field, and the strain is identified to be Marinobacter lipolyticus according to strain morphology, physiological characteristics, gram reaction, 16S rDNA gene sequencing analysis and phylogenetic analysis. According to the similarity of LJB-135 and OTU-5 (99.7%; greater than the 16S rRNA OTU standard clustering threshold (97%)), the strain is confirmed to be a typical strain represented by the in situ Fei Jiang functional microorganism. The optimal environmental conditions for the growth of LJB-135 are: the temperature is 35 ℃, the pH value is 7.5, and the NaCl accounts for 7 percent (mass fraction); the 16S rRNA gene sequencing analysis showed that the strain closest to LJB-135 was Marinobacter lipolyticus SYP-11 (99.9%). LJB-135 can utilize phenanthrene as a sole carbon source and degrade phenanthrene rapidly; the initial mass concentration of phenanthrene is 100 mg.L -1 After 3 days of culture, the degradation rate is 56.1%; the degradation rate of the strain prepared into a microbial inoculum can reach 73.1 percent. Therefore, the strain has better application potential in the aspect of bioremediation of petroleum-polluted soil.
Marinobacter lipolyticus (Marinobacter lipolyticus) LJB-135, deposited at 14/4/2022 at the guangdong collection of microorganisms and cell cultures (GDMCC), address: building 5 of first furios middle way 100 large yard 59, guangdong province, guangzhou, zip code: 510070, the preservation number is: GDMCC No:62388.
drawings
FIG. 1 shows the phylogenetic information of the strain LJB-135.
FIG. 2 is a growth morphology of strain LJB-135.
FIG. 3 shows phenanthrene degradation characteristics of strain LJB-135 and its microbial inoculum.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Example 1: isolation and characterization of Haemophilus lipolyticus (Marinobacter lipolyticus) LJB-135
1. Sample collection
The contaminated soil used in the experiment is from Shandong Yingli oil field, and is stored at 4 ℃ after sampling for later use.
2. SIP Microcosmic culture System construction
In the experiment, 5g of contaminated soil and 20mL of sterile water were added to a 150mL serum bottle, and then unlabelled phenanthrene (A) was added to the bottle by a syringe 12 C-PHE) or 13 C-phenanthrene (C) 13 C-PHE) (99%, cambridge Isotrope Laboratories, inc., tewksbury, MA, USA) to a final concentration of 10mg/L phenanthrene, sealed with a rubber stopper and aluminum cap. All treatment groups of the experiment included: control group without phenanthrene addition, sterile control group with filter-sterilized soil, and addition 12 C-PHE or 13 C-PHE soil samples. Three parallel groups were set up for all experiments. Samples were taken on day 3 after incubation for chemical analysis and DNA extraction.
3. DNA extraction and ultracentrifugation, sequencing
Strong soil DNA extraction kit Using MOBIO (A)
DNA Isolation Kit) total genomic DNA was extracted from the different treatment samples (Tillett and Neilan, 2000) in three parallel sets per sample. After DNA extraction, the DNA was ultracentrifuged after the concentration was determined by ND-1,000UV-Vis UV-visible spectrophotometer (NanoDrop Technologies, wilmington, DE, USA). The centrifugation condition is that the mixture is ultracentrifuged for 48 hours at 20 ℃ and the rotating speed is 178000g. After centrifugation, the DNA solution was fractionated into 14 layers using a Beckman fractionating apparatus. Each layer of DNA was subsequently assayed and purified and sequenced.
After sequencing data are obtained, mothur is used for sequence splicing to obtain a target sequence, and finally, the target sequence is subjected to flow analysis through QIIME, sequences with similarity larger than 97% are classified into the same OTU (Operational taxomic Unit), and species classification information is divided. Comparison of 12 C-phenanthrene and 13 after the C-phenanthrene sample is centrifuged, the relative abundance of each OTU in each layer is combined with the buoyancy of each separation layerThe density was analyzed. Culturing the sample for a period of time, adding 13 The DNA corresponding to certain functional microbial communities in the C-phenanthrene soil sample can be enriched in the heavy layer, so that the relative abundance ratio of the communities in the heavy layer is obvious 12 High in C-phenanthrene-treated group. The most abundant 100 OTUs were selected for subsequent analysis in this study. Finally, by comparative analysis 12 C-phenanthrene and 13 the abundance of each OTU of different abscission layers in the C-phenanthrene treatment group is changed, and 1 OTU (OTU _5;>1%) which belongs to the genus Marinobacter lipolyticus. Phylogenetic Information of the degrading functional microorganisms was determined based on BLAST (National Center for Biotechnology Information, bethesda, md., USA) and MEGA.
The functional bacteria identified by SIP are microorganisms represented by OTU-5, and the similarity of the functional bacteria and the 16S r RNA of Marinobacter lipolyticus is up to 99.7 percent according to the sequence comparison result.
4. Enrichment, separation and growth condition optimization of phenanthrene degradation microorganisms
According to the SIP result, in order to separate the high-efficiency strain for degrading phenanthrene in situ in the polluted soil, the research carries out directional enrichment, separation and screening on phenanthrene degrading bacteria in the collected soil sample, and the main steps are as follows:
4.1 enrichment of phenanthrene-degrading bacteria
According to the previous culture research on Marinobacter lipolyticus, the traditional inorganic salt culture medium is modified. Not only are various mineral elements and microbial elements added, but also a class of antibiotics are added to inhibit the growth of other microorganisms. The composition of the modified inorganic salt culture medium is shown in the following table 1, and the preparation method comprises the steps of adding the components into water, stirring and uniformly mixing, and sterilizing.
TABLE 1 inorganic salt media formulation
(a)
(b)
(c)
We added collected 5g of soil to the above inorganic salt medium, unlabeled phenanthrene (f) at a concentration of 100mg/L 12 C-PHE) as a substrate, and placing the substrate in an incubator at 35 ℃ in a dark place for shake culture. And (3) domesticating the phenanthrene degrading bacteria by using a modified inorganic salt culture medium taking phenanthrene as a carbon source, wherein 7d is a domesticating period. The inoculum size of 10% by volume fraction was transferred to fresh mineral salts medium with the same culture system and the enrichment process was repeated until three times.
4.2 isolation of phenanthrene-degrading bacteria
The fourth-generation enriched sample obtained above was subjected to coating separation by the dilution plate method, and the sample was separated with the above inorganic salt medium to which agar was added in a mass fraction of 1.5%. And (3) placing the coated flat plate at 35 ℃ for culturing for 4d, growing obvious single colonies on the surface of the culture medium, selecting various different single colonies according to the characteristics of the colonies, such as morphology size, transparency, color and the like, and carrying out streak purification on the inorganic salt culture medium flat plate. If single colonies of different types can still grow on the streaked and purified plate, the single colonies are picked out and streaked again until single colonies of different characteristics cannot be observed on the same plate. 10 pure strains taking phenanthrene as a carbon source are obtained in the experiment, a strain degradation capability experiment is carried out, and finally 1 novel strain LJB-135 which has good degradation performance on phenanthrene and is related to in-situ phenanthrene degradation is obtained by screening from 10 degradation strains. Strain LJB-135 is from Marinobacter lipolyticus, and has a gene similarity of 99.7% with the 16S rRNA of OTU-5, which is greater than the standard clustering threshold (97%) of 16S rRNA OTU. Accordingly, the strain is considered to be a functional microorganism with phenanthrene degradation in situ. And (3) selecting the purified single bacterial colony to be cultured in an inorganic salt culture medium until the logarithmic phase is reached, mixing the bacterial liquid and sterile glycerol, subpackaging the mixture into a sterile 2mL freezing tube (the concentration of the glycerol is 15 percent by mass), and placing the tube at the temperature of minus 80 ℃ for long-term storage.
4.3 identification of phenanthrene-degrading bacteria
(1) And preliminarily identifying the strain LJB-135 according to morphological characteristics of the strain LJB-135. Activating and separating the obtained strains, respectively inoculating the strains on inorganic salt culture medium plates by a streaking separation mode, culturing the strains for 48 hours under the condition of the optimal temperature (35 ℃), and observing the characteristics of the formed single colony such as morphological size, color and the like. Meanwhile, several staining methods such as flagella staining, gram staining, spore staining and the like are selected to perform staining experiments on the cultured bacteria, the stained cells are observed under an optical microscope, and the gram staining of the cells is positive or negative and whether the cells have flagella and spores or not is observed and distinguished.
After being activated, the LJB-135 is streaked on a flat plate made of an inorganic salt culture medium, and is cultured for 48 hours under the aerobic condition at 35 ℃ to form milky white colonies with the diameter of 1.0-2.5mm, smooth surfaces, slightly upward bulges, neat edges, opaqueness, no flagella, no spores, negative gram stain and rod-shaped individual strains (figure 2). The bacteria are obligate aerobic bacteria, and the cell size is about (0.3-0.6) × (2.0-3.4) μm.
(2) Before sequencing bacteria and constructing phylogenetic trees, the genomic DNA of the bacteria is extracted. The bacterial genome DNA rapid extraction kit is used for extracting DNA in experiments. In order to study the taxonomy of unknown bacteria, it is usually necessary to amplify a 16S rRNA gene fragment, which is a DNA fragment encoding prokaryotic rRNA, with high conservation and specificity, and to construct phylogenetic trees, which are usually used for detection and identification of bacteria.
Polymerase Chain Reaction (PCR) is mainly used to amplify different gene segments, and the primers used to amplify the 16S rRNA gene segment are 27F and 1492R (27F. PCR reactionThe system comprises 10 XBuffer 2.5 mu L of Mg 2+ 1.5. Mu.L (25 mmol/L), 0.25. Mu.L Taq enzyme, 0.3. Mu.L dNTP (25 mmol/L), 0.5. Mu.L front primer (10 mmol/L), 0.5. Mu.L rear primer (10 mmol/L), 0.1. Mu.L template DNA, 19.35. Mu.L deionized water. The PCR amplification conditions were: denaturation at 95 deg.C, annealing at 55 deg.C, extension at 72 deg.C, circulating for 30 times, extension at 72 deg.C for 10min, and storing at 4 deg.C after PCR reaction. Preparing a gel block by adding a proper amount of nucleic acid stain GelRed into 1% agarose, adding a proper amount of PCR products and DNA markers (DNA marker) containing fragments with various lengths into the gel block, placing the gel block into an electrophoresis apparatus containing TBE (Tris boric acid) buffer solution, taking out the gel block after working for 20min under a certain voltage, and placing the gel block into a gel imaging system for observation to check whether the PCR amplification reaction is successful or not. The PCR product was then sent to Huada Gene science and technology, inc. for sequencing using the same primers as those used for amplification. To obtain more accurate sequence results, the 16S rRNA gene fragment was introduced into a plasmid vector, followed by TA cloning experiments and sequencing again.
The sequences obtained were spliced and subsequently uploaded to EzTaxon-e (http:// EzTaxon-e. Ezbiocloud. Net /) (Kim et al, 2012), which site would align the submitted DNA fragments with the 16s rRNA gene sequences of typical strains of the species that have been recognized, giving information on the similarity and homology between the sequences. Analyzing the sequence comparison result, selecting a typical strain with higher similarity as a model bacterium of the strain obtained by the experiment separation, simultaneously obtaining a 16S rRNA gene sequence of the model bacterium, and further performing phylogenetic analysis to prove that the functional bacterium separated by the experiment is different from the model bacterium, thereby identifying the strain obtained by the separation. The MEGA 5.05 program is used for constructing a phylogenetic tree, and the phylogenetic tree is usually constructed by adopting an adjacency method, a maximum reduction method or a minimum evolution method, wherein the adjacency method is most commonly used, and the self-expansion value is usually set to be repeatedly calculated for 1000 times.
The 16S rRNA gene sequence of the strain LJB-135 (the sequence is shown in SEQ ID NO. 1) is obtained by PCR amplification and gene sequencing. A phylogenetic tree is made by using the 16S rRNA gene sequence and a part of the 16S rRNA gene sequence with higher similarity, so as to obtain a homology result between the 16S rRNA genes. The phylogenetic tree constructed by the orthotopic grafting method is shown in FIG. 1. The strain LJB-135 and Marinobacter lipolyticus SYP-11 can be intuitively observed on the same branch through a phylogenetic tree, and the strain and the Marinobacter lipolyticus strain have higher homology. Since the similarity of 16S rRNA sequences of LJB-135 and SYP-11 strains is 99.9%, the functional strain LJB-135 isolated in the experiment is considered to be Marinobacter lipolyticus, which is named as Haemophilus lipolyticus (Marinobacter lipolyticus) LJB-135, and is deposited in Guangdong province culture Collection of microorganisms (GDMCC) at 14.4.2022, address: building 5 of first furnance, large yard, 100, building 59, guangdong province, guangzhou, china, zip code: 510070, the preservation number is: GDMCC No:62388.
example 2: growth conditions of sea bacillus lipolyticus (Marinobacter lipolyticus) LJB-135
1. Measurement of growth temperature: preparing an inorganic salt culture medium (formula shown in table 1), and sterilizing in a sterilizing pot after preparation. The activated fresh LJB-135 bacterial liquid is inoculated into a culture medium (an experimental group), the culture medium without inoculated strains is used as a control group, the culture medium is placed into incubators at different temperatures for light-shielding shake culture for 7d, each sample has three repetitions, the growth condition of bacteria every day is observed, when a result which cannot be distinguished by naked eyes is met, a visible-ultraviolet spectrophotometer is used for measuring the light absorption value (OD value) of the culture liquid at the position of wavelength lambda =600nm, and finally the growing temperature range and the optimal growing temperature of the strains are obtained. The test temperatures included 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, and 50 deg.C.
2. Measurement of growth pH: preparing an inorganic salt culture medium (the formula is shown in table 1), and adjusting the pH of the culture medium by using the following buffer systems, wherein the pH is 4.0-5.0,0.1mol/L citric acid and 0.1mol/L sodium citrate; pH 6.0-8.0,0.1mol/L NaOH and 0.1mol/L KH 2 PO 4 ；pH 9.0–10.0，0.1mol/L Na 2 CO 3 And 0.1mol/L NaHCO 3 . Inoculating activated fresh LJB-135 bacteria liquid into culture solution, setting three pH values in parallel, and using culture medium without inoculated strain as control groupAnd (3) carrying out shake culture in the dark at proper temperature for 7d, wherein each sample has three repetitions, observing the growth condition of bacteria every day, and when a result which cannot be distinguished by naked eyes is met, measuring the light absorption value (OD value) of the culture solution at the position of wavelength lambda =600nm by using a visible-ultraviolet spectrophotometer to finally obtain the pH range and the optimal growth pH of the strains. The tested pH included 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0.
3. Salt concentration tolerance: preparing an inorganic salt culture medium (formula shown in table 1), adjusting the salt concentration of the culture medium and sterilizing. Inoculating activated fresh LJB-135 bacterial liquid into a culture medium, setting three parallel salts in each concentration, taking the culture medium without inoculated strains as a control group, carrying out shake culture for 7d in the absence of light under the condition of the optimal temperature, repeating the samples for three times, observing the growth condition of bacteria every day, and measuring the light absorption value (OD value) of the culture solution at the position of wavelength lambda =600nm by using a visible-ultraviolet spectrophotometer when a result which cannot be distinguished by naked eyes is met, thereby finally obtaining the salt concentration range which can be tolerated by the strains. The salinity tested included 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% (mass fraction).
The result shows that the temperature of the optimal growth of the strain LJB-135 is 35 ℃, the pH of the optimal growth is 7.5, and the salinity of the optimal growth is 7 mass percent NaCl.
Example 3: experiment on degradation of phenanthrene
1. Preparation of LJB-135 microbial inoculum
(1) Somatic cells of the strain LJB-135 were collected, resuspended in 5mL of 1 XPBS buffer, and then OD thereof was adjusted 600 And =1, obtaining bacterial suspension for standby.
(2) Mixing the corn straw biochar and the LJB-135 bacterial suspension according to the proportion of 1.
(3) Respectively weighing 10g of polydienol and 0.5g of sodium alginate, adding 60mL of distilled water, heating and stirring at 70 ℃ to dissolve the polydienol and the sodium alginate, sterilizing at 121 ℃ for 15min, and cooling to obtain an embedding agent solution.
(4) Weighing 5g Ca (NO) 3 ) 2 The resulting solution was added to 100mL of deionized water, stirred to dissolve the resulting solution, and the pH was adjusted to 7.0 to obtain a crosslinking agent solution.
(5) Adding 20mL of adsorbed thallus solution into the embedding agent solution, using sterile water to fix the volume to 100mL, and uniformly mixing, wherein the final concentration of the polydienol and the sodium alginate is 10% and 5%, and the embedded microorganism amount is 10%. And (3) taking 50mL of the mixed solution by using a 50mL syringe with the caliber of 2mm, dripping the mixed solution into 100mL of the cross-linking agent solution from a height of 30cm, and fixing for 1 hour to obtain the LJB-135 microbial inoculum.
2. Degradation characteristics of degrading bacteria and microbial inoculum when phenanthrene is used as carbon source
Comparing the DNA-SIP exploration result with the indoor culture separation experiment result, whether the separated strain can degrade phenanthrene in situ in the polluted soil can be confirmed. Under the optimal growth conditions (optimal temperature, pH and salinity tolerance), the phenanthrene degradation condition of the in-situ phenanthrene degradation bacteria is explored.
According to the experimental results, the optimal growth condition of the strain LJB-135 is determined to be 35 ℃, the pH value is 7.5, and NaCl with the mass fraction of 7% is added. The degradation experiments of the bacterial strains LJB-135 and LJB-135 in the phenanthrene concentration of 100mg/L are carried out under the condition. Inoculating strain LJB-135 or LJB-135 bacteria at logarithmic growth phase at volume fraction of 10% (obtaining original bacteria solution containing cells by conventional plate counting method with the number of 1 × 10 8 CFU·mL -1 ) Respectively inoculated into an inorganic salt medium (formulation shown in Table 1, added with 7% by mass of NaCl, pH 7.5) containing an initial phenanthrene concentration of 100mg/L, shake-cultured at 35 ℃ for 3d, and subjected to 3 times of parallel experiments. The control treatment is the treatment without adding pure Marinobacter sp.LJB-135 and LJB-135 bacteria.
Taking each processing sample for chemical analysis, and the specific steps are as follows: (1) sample pretreatment: adding dichloromethane into each culture sample for extraction, simultaneously adding 5 μ L recovery rate indicator (shown in Table 2, including naphthalene-d 8, acenaphthene-d 10, phenanthrene-d 10, and chrysene-d 12) with concentration of 200mg/L, shaking fully, transferring into a separating funnel, and standing. And collecting organic phases after layering, putting the lower layer liquid back to a shake flask, repeatedly extracting with dichloromethane of the same volume, combining the extracts, transferring the combined extracts to a flat-bottomed flask containing a proper amount of activated copper sheets for rotary evaporation, concentrating to about 2mL, adding a small amount of n-hexane (about 5 mL), carrying out rotary evaporation to 2mL, repeatedly washing for three times, and replacing the organic solvent with the n-hexane. The concentrate after the displacement was purified by means of a glass-packed column (diameter: about 9 mm). The column packing was from bottom to top 3cm 3% deactivated neutral alumina, 3cm 3% deactivated silica gel and 1cm anhydrous sodium sulfate. The column is activated by using a proper amount of n-hexane, the packed column is rinsed by using 15mL of mixed reagent of n-hexane/dichloromethane (volume ratio is 1). 5 mul internal standard hexamethylbenzene was added before the machine measurement, and the concentration was 200mg/L. (2) instrumental analysis: and determining the content of PAHs in each processed sample by adopting an Agilent 7890 gas chromatograph-5975 mass spectrometer. The column used was an Agilent DB 5-MS capillary column (column length 30m, inner diameter 0.25mm, film thickness 0.25 μm). An Agilent 7890 gas chromatograph-5975 mass spectrometer is used for measuring the content of PAHs. The separation analysis was carried out using an Agilent DB 5-MS (column length 30m, inner diameter 0.25mm, film thickness 0.25 μm) capillary chromatography column. The injection port temperature is 290 ℃, the injection is performed in a non-split mode by 1 mu L, and the solvent is delayed for 10min. High purity helium gas was used as a carrier gas, and the flow rate was set to 1.83mL/min. The temperature-raising program is set as follows: keeping at 60 deg.C for 5min, and heating at 4 deg.C/min to 290 deg.C/min for 30min. The mass spectrum uses an EI source as an ion source, and the temperature is set to 230 ℃. The detector performs a full scan in the range of 50 to 500 m/z. The obtained data were processed with an agilent chromatography workstation and the quantification of PAHs was performed with a 6-point calibration curve and an internal standard method.
TABLE 2 recovery indicators and internal standards for PAHs determination
The result shows that the strain LJB-135 can grow under the condition of high concentration phenanthrene and can degrade phenanthrene rapidly, and the degradation rate is 56.1% after the strain is cultured in an inorganic salt culture solution with the initial concentration of phenanthrene of 100mg/L for 3 days; the degradation rate of the strain prepared into a microbial inoculum can reach 73.1 percent (figure 3). The results show that the strain LJB-135 is a strain with strong in-situ phenanthrene degradation capability and has good application potential in the aspect of in-situ bioremediation of polycyclic aromatic hydrocarbon polluted sites.
The above are only preferred embodiments of the present invention, and it should be noted that the above preferred embodiments should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Guangzhou geochemistry institute of Chinese academy of sciences
<120> in-situ PAHs degradation functional bacterium LJB-135, and microbial inoculum and application thereof
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> sea bacillus LJB-135 (Marinobacter sp. LJB-135)
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aggcgacgat ccgtagctgg tctgagagga tgatcagcca catcgggact gagacacggc 240
ccgaactcct acgggaggca gcagtgggga atattggaca atgggggcaa ccctgatcca 300
gccatgccgc gtgtgtgaag aaggctttcg ggttgtaaag cactttcagt gaggaggaag 360
gctcaaaggc taataccctt tgggattgac gttactcaca gaagaagcac cggctaactc 420
cgtgccagca gccgcggtaa tacggagggt gcaagcgtta atcggaatta ctgggcgtaa 480
agcgcgcgta ggtggtttga taagcgagat gtgaaagccc cgggcttaac ctgggaacgg 540
catttcgaac tgtcaggcta gagtgtggta gagggtagtg gaatttcctg tgtagcggtg 600
aaatgcgtag atataggaag gaacaccagt ggcgaaggcg gctacctgga ccaacactga 660
cactgaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 720
aaacgatgtc aactagccgt tgggactctt gaagtcttag tggcgcagct aacgcactaa 780
gttgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga cgggggcccg 840
cacaagcggt ggagcatgtg gtttaattcg acgcaacgcg aagaacctta cctggccttg 900
acatgcagag aactttccag agatggattg gtgccttcgg gaactctga 949
Claims (8)
1. Bacillus lipolyticus (A. Lipolytica)Marinobacter lipolyticus) LJB-135 with the deposit number: GDMCC No. 62388.
2. The Haemophilus lipolytic bacterium of claim 1 (Haemophilus lipolytic.) (Marinobacter lipolyticus) The application of LJB-135 in degrading phenanthrene.
3. Use according to claim 2, wherein the degrading phenanthrene is a degrading phenanthrene in soil or sewage.
4. The method of claim 1 Bacillus lipolyticus of (A), (B), (C)Marinobacter lipolyticus) The application of LJB-135 in the preparation of phenanthrene degradation microbial inoculum.
5. A phenanthrene degrading bacterial agent, comprising the Bacillus stearolysis bacterium (I) of claim 1Marinobacter lipolyticus) LJB-135 asAn active ingredient.
6. The Haemophilus lipolytic bacterium of claim 1 (Haemophilus lipolytic.) (Marinobacter lipolyticus) Use of LJB-135 or the bacterial agent of claim 5 in bioremediation of phenanthrene-contaminated environments.
7. The use of claim 6, wherein the phenanthrene-contaminated environment is a phenanthrene-contaminated water body and/or soil.
8. A method for degrading phenanthrene, comprising subjecting the Haemophilus lipolyticus of claim 1 (A)Marinobacter lipolyticus) The LJB-135 or the bacterial agent of claim 5 sprayed in an environment containing phenanthrene to degrade the phenanthrene.
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