CN106995786B - Fusarium strain and application thereof - Google Patents
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- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 title claims abstract description 15
- 241000223218 Fusarium Species 0.000 title claims abstract description 15
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- 239000005708 Sodium hypochlorite Substances 0.000 description 1
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- NIFIFKQPDTWWGU-UHFFFAOYSA-N pyrite Chemical compound [Fe+2].[S-][S-] NIFIFKQPDTWWGU-UHFFFAOYSA-N 0.000 description 1
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract
The invention belongs to the technical field of biological engineering, and particularly discloses a fusarium strain and application thereof. The Fusarium sp is identified by the ITS gene sequence, and the preservation number is CCTCC M2016582. The invention utilizes the inactivated FP-JCCW thalli to research the adsorption behavior of the thalli to thallium to obtain the optimal adsorption condition: pH5.0, initial concentration of 100mg/L, contact time of 90min, rotation speed of 150r/min, biomass of 2.5 g/L. And the adsorption of the composite material is more in line with Langmuir isothermal model and second-order kinetic equation, is monomolecular layer adsorption and is mainly chemical adsorption. FT-IR, SEM and XRD characterization and analysis show that FP-JCCW has porous surface, thallium adsorption mainly depends on hydroxyl, carbonyl and the like on cell walls, and the crystal structure of thalli is changed before and after adsorption. It is concluded that thallium adsorption by the inactivated microbial cell FP-JCCW is completed through electrostatic adsorption and surface complexation. The inactivated microorganism FP-JCCW provided by the invention can be used as an economic, environment-friendly and sustainable adsorbent to treat heavy metal thallium pollution.
Description
Technical Field
The invention belongs to the technical field of biological engineering, relates to microorganisms and application thereof, and particularly relates to a Fusarium sp strain FP-JCCW and application of inactivated microorganisms thereof as an adsorbent in treatment of heavy metal thallium pollution.
Background
With the development of the industrialized process, the problem of heavy metal treatment after large-scale mining is increasingly highlighted. Heavy metal thallium has strong toxicity, is easy to enrich in sulfide, often migrates when mined in a mining area, can enter a human body along with a food chain, and has potential threat to human life.
For mining, particularly thallium ore and pyrite, effective treatment of heavy metals is a key to sustainable development of the mining industry. At present, the treatment of heavy metals is mainly carried out by physical and chemical methods, but compared with microbial treatment, the treatment method has the defects of high manufacturing cost, long treatment period, easy secondary pollution and the like, and is difficult to treat a large amount of low-concentration pollution. The microbial treatment is a very active research field in China at present, and the research on thallium adsorption by microorganisms such as pseudomonas, high-tolerance fungi, deep flocculation microorganisms and the like has a good effect, and the good application possibility of heavy metal pollution treatment is shown. However, most of the studies are conducted to adsorb thallium by using an active strain, and few studies have been conducted on adsorption of inactive microorganisms, which ignore the inhibitory effect of microorganisms themselves on rejection of heavy metals.
Disclosure of Invention
Aiming at the technical defects existing in the treatment of heavy metal thallium pollution by the existing method, the invention provides a Fusarium sp strain FP-JCCW and an inactivated microbial body of the Fusarium sp strain used as an economic, environment-friendly and sustainable adsorbent for treating heavy metal thallium pollution.
In order to achieve the purpose, the invention adopts the technical scheme that:
a Fusarium sp strain is FP-JCCW, which is an excellent anti-thallium fungus screened by the inventor from polluted plants in Shaoguan Dabaoshan. The preservation information of the strain (biological material) is:
name: fusarium sp.fp-JCCW;
the preservation number is: CCTCC NO: m2016582;
the preservation unit: china Center for Type Culture Collection (CCTCC);
and (4) storage address: wuhan university, Wuhan, China, zip code 430072;
preservation time: 2016, 10 months and 20 days.
The invention identifies the classification of FP-JCCW strains through ITS gene sequences. The ITS gene PCR amplification primers are as follows: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). BLAST comparison is carried out on the gene sequence of the strain and related strain sequences in an NCBI database, the FP-JCCW strain and Fusarium sp belong to the same branch, so the strain is named as Fusarium sp.
The FP-JCCW strain has very high homology with Fusarium sp according to the identification result of ITS gene sequence, so that the strain is named as Fusarium sp.
The FP-JCCW strain can be used for preparing an adsorbent.
Furthermore, an adsorbent can be prepared by using the inactivated FP-JCCW bacteria. The preparation method of the adsorbent comprises the following steps: carrying out liquid culture on the FP-JCCW strain, and filtering to obtain FP-JCCW mycelia; then placing the mycelium in an oven for drying; and grinding the dried mycelium into powder to obtain the adsorbent.
In some embodiments, the method of making the above adsorbent is further optimized as: carrying out liquid culture on the FP-JCCW strain for 72h, and filtering by using 8 layers of gauze to obtain FP-JCCW mycelia; then transferring the mycelium into an enamel tray, and placing the enamel tray in an oven at 80 ℃ for baking for 6 hours; and (3) placing the dried mycelia in a mortar and grinding into powder to obtain the adsorbent.
The adsorbent can be used as an economic, environment-friendly and sustainable biological adsorption material for treating heavy metal thallium pollution.
Compared with the prior art, the invention has at least the following beneficial effects or advantages:
(1) the invention separates and screens a strain with strong resistance to thallium from plants in a Shaoguan Dabaoshan polluted mining area, and the strain is inferred to be fusarium by BLAST comparison homology and identified as Fusarium.sp.FP-JCCW with the accession number of KX 349437. The FP-JCCW strain can be used as an economic, efficient and environment-friendly biological adsorption material for treating heavy metal thallium pollution. The invention provides the adsorption behavior and mechanism of the inactivated FP-JCCW strain to the heavy metal thallium, and provides more theoretical basis for the application and treatment of thallium in the future.
(2) The invention defines the optimal adsorption condition for the inactivated microorganism FP-JCCW to adsorb thallium: pH5.0, initial concentration of 100mg/L, contact time of 90min, rotation speed of 150r/min, biomass of 2.5 g/L.
(3) According to the invention, through establishing and comparing adsorbed Langmuir and Freundlich models, the adsorption process of the FP-JCCW strain is more consistent with a Langmuir isothermal model, which indicates that the adsorption process is monomolecular layer adsorption and probably mainly occurs on cell walls. Establishing an adsorption primary and secondary kinetic equation, finding that the secondary kinetics is more consistent with the adsorption process, and indicating that the adsorption process is dominant in chemical adsorption and surface complex reaction is likely to occur.
(4) After infrared spectrum analysis, in the adsorption process of FP-JCCW strain, -OH, C-H, C ═ O, -NH2C-O, etc. groups play a major contributing role. The inactivated microorganism FP-JCCW before and after adsorption is analyzed through SEM-EDS observation and X-ray diffraction, the cell surface before adsorption is not smooth and has pores, the cell appearance after adsorption is obviously changed, and crystals are generated. After characterization and analysis, the FP-JCCW adsorption is completed through electrostatic adsorption and surface complexation.
Drawings
FIG. 1 is a phylogenetic tree of the FP-JCCW strain of the present invention.
FIG. 2 is the pH vs. Tl for the adsorption test described in the examples+Influence curve of adsorption.
FIG. 3 shows Tl in the adsorption test described in the examples+Initial concentration versus adsorption.
FIG. 4 is a graph of the effect of contact time on adsorption in the adsorption test described in the examples.
FIG. 5 is a graph showing the effect of the rotational speed of the rocking platforms on adsorption in the adsorption test described in the examples.
FIG. 6 is a graph of the effect of biomass on adsorption in the adsorption experiments described in the examples.
FIG. 7 is a Langmuir adsorption isotherm model established for the adsorption experiments described in the examples.
FIG. 8 is a Freundlich adsorption isotherm model established for the adsorption experiments described in the examples.
FIG. 9 is a first order kinetic equation established for the adsorption experiments described in the examples.
FIG. 10 is a second order kinetic equation established for the adsorption experiments described in the examples.
FIG. 11 shows adsorption of Tl by FP-JCCW strain according to the present invention+Comparison of Fourier infrared spectrum change of the former A and the post-adsorption B.
FIG. 12 shows adsorption of Tl by FP-JCCW strain according to the present invention+Comparative scanning electron microscope change of the front A and the adsorption B.
FIG. 13 shows adsorption of Tl by FP-JCCW strain according to the present invention+Spectrometer analysis of front a and post-adsorption B.
FIG. 14 shows adsorption of Tl by FP-JCCW strain according to the present invention+X-ray diffraction patterns of pre-A and post-adsorption B.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following examples.
Example 1: isolation and characterization of FP-JCCW Strain
The FP-JCCW strain is a resistant strain screened from a Shaoguan Dabaoshan mine area, and specifically comprises the following steps:
(1) preparation of culture Medium
PDA culture medium: 200g of potato (peeled), 20g of glucose, 20g of agar and 1000mL of distilled water, and the pH value is natural.
The specific preparation process of the PDA culture medium comprises the following steps: peeling and bud eyes of 200g of potatoes, cutting the potatoes into small pieces, putting the small pieces into 1000mL of distilled water, boiling until the potatoes are soft and not rotten, filtering the potato pieces by using 8 layers of gauze, pouring the filtrate into a pot again, adding agar and glucose, uniformly stirring, replenishing water to 1000mL, and finally subpackaging the potato pieces into 250mL triangular flasks for later use.
(2) Acclimatization culture
Preparing an endophytic fungus strain which is freshly cultured to logarithmic phase into spore suspension, coating 1mL of spore suspension into a solid culture medium containing thallium at a certain concentration, placing the solid culture medium in a constant-temperature incubator at 25 ℃ for culturing for 72h, observing whether colonies grow out, if the colonies grow, sucking 1mL of spore suspension again, inoculating the spore suspension into a fresh solid culture medium with a higher thallium concentration, continuing culturing, observing the growth condition of the colonies, and determining the minimum inhibition concentration of thallium ions.
(3) Separating and purifying
Washing with tap water, collecting silt on the surface of root, stem and leaf of plant, sterilizing in 95% ethanol and 0.5% sodium hypochlorite for 1min, washing with sterilized normal saline for 3 times, drying with sterilized absorbent paper, cutting into 0.5 × 0.5cm pieces under aseptic condition, inoculating to fresh PDA culture medium, and standing in 25 deg.C incubator. And after 3-7d of culture, if hyphae grow on the edge of the fragment, selecting hyphae with good edge growth by using an inoculating needle, inoculating the hyphae on a new solid culture medium, after the newly inoculated hyphae grow into strains, selecting the hyphae on the edge of the newly inoculated hyphae, culturing, and repeatedly purifying in the way to obtain the purified strain.
And (II) carrying out classification and identification on the FP-JCCW strain by adopting an ITS gene sequencing method.
The ITS gene PCR amplification primers are as follows: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3').
The amplification procedure was: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 40s, extension at 72 ℃ for 60s, 34 cycles, and extension at 72 ℃ for 10 min.
After the gene amplification product is purified, through Shanghai's engineering sequencing, the obtained sequence is subjected to BLAST comparative analysis with the existing ITS sequence in GENBANK, MEGA5.2 is used for multiple sequence comparison, and an NJ (Neighbor-join) phylogenetic tree is built by adopting a Kimura-2 model.
The DNA of the FP-JCCW strain was PCR amplified and its gene sequence was BLAST-aligned with the relevant strain sequences in the NCBI database. The results show that this strain belongs to the same branch as Fusarium sp, thus it was named Fusarium sp, and MEGA5.2 software was used to construct a NJ (neighbor-joining) phylogenetic tree (FIG. 1), the sequence of which has been submitted to the GENBANK database of NCBI, with the strain gene sequence accession number KX 349437.
Example 2: performance and action mechanism of FP-JCCW strain for adsorbing heavy metal thallium
In order to further determine the adsorption characteristic and the preliminary mechanism of the FP-JCCW strain to the heavy metal thallium, in this example, thallium is used as a research object, the adsorption characteristic of the microbial strain to thallium is researched under different environmental conditions, and the preliminary mechanism of the strain to adsorb the heavy metal thallium is researched by using methods such as an electron microscope and infrared spectroscopy.
Preparation of FP-JCCW strain adsorbent
Carrying out liquid culture on the FP-JCCW strain for 72h, and filtering by using 8 layers of gauze to obtain FP-JCCW mycelia. Then transferring the thalli to an enamel tray, and placing the enamel tray in an oven at 80 ℃ for baking for 6 hours. And (3) placing the dried mycelium in a mortar and grinding the mycelium into powder to obtain the inactivated microorganism FP-JCCW (adsorbent).
The liquid medium for culturing the FP-JCCW strain is PDA liquid medium. The preparation of PDA liquid culture medium is: cutting 200g peeled potato into pieces, decocting in 1000ml distilled water for 30min, filtering with gauze, adding 20g glucose, stirring to dissolve, diluting to 1000ml, packaging into conical flask, and sterilizing at 121 deg.C for 30 min.
(II) adsorption test method and results
(1) pH to Sr2+Influence of adsorption
Tl at a concentration of 20mg/L in a volume of 20ml+To the solution, 0.02g of the inactivated microorganism FP-JCCW was added. Slowly dripping nitric acid or sodium hydroxide while oscillating, respectively adjusting pH to 3, 4, 5, 6, 7, 8, 9, and oscillating at constant temperature of 30 deg.C and 150r/min for 60 min. Centrifuging the adsorbed solution at 8000r/min for 5min, digesting the supernatant, and measuring its Tl+And (4) concentration.
pH is an important factor in microbial adsorption. As can be seen from FIG. 2, the adsorption process of the FP-JCCW strain varies with pH. At pH<5, the adsorption amount increases with the increase of pH, probably because the pH is so low that water and hydrogen ions (H) are generated3O+) Occupies the adsorption sites and blocks the inactive microorganisms from acting on Tl+Absorption of (2). The highest degree of adsorption was achieved at pH5, with an adsorption capacity of 120.9 mg/g. The amount of adsorption subsequently decreases with increasing pH, which may lead to the formation of hydroxide colloids in the solution, resulting in a decrease in the amount of adsorption.
(2) Effect of thallium initial concentration on adsorption
Respectively at Tl concentration of 20mg/L, 40mg/L, 60mg/L, 80mg/L, 100mg/L and volume of 20ml+Adding 0.02g of inactive microorganism FP-JCCW into the solution, and keeping the temperature at 30 ℃ at 150r/min oscillate for 60 min. Centrifuging the adsorbed solution at 8000r/min for 5min, digesting the supernatant, and measuring its Tl+And (4) concentration.
As can be seen from FIG. 3, the adsorption amount of FP-JCCW increases with the increase of the initial concentration. When the volume of the solution is constant, the concentration of the metal ions increases, and the adsorption amount per unit adsorbent also increases, possibly because the adsorption sites of the adsorbent are not fully occupied by the metal ions. When Tl is in solution+The maximum adsorption capacity was 229.8mg/g at a concentration of 100 mg/L.
(3) Effect of contact time on adsorption
Tl at a concentration of 20mg/L in a volume of 20ml+To the solution, 0.04g of the inactivated microorganism FP-JCCW was added. Oscillating at constant temperature of 30 deg.C and 150r/min for 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, and 120min, respectively. Centrifuging the adsorbed solution at 8000r/min for 5min, digesting the supernatant, and measuring the residual Tl+And (4) concentration.
As can be seen from FIG. 4, the adsorption amounts increased with the increase of the contact time, and reached almost equilibrium by 90 min. However, the longer the contact time is, the more likely thallium ions are precipitated. Therefore, 90min is the best condition, and the adsorption quantity is 116.8 mg/g.
(4) Influence of the rotational speed of the rocking bed on the adsorption
Tl at a concentration of 20mg/L in a volume of 20ml+To the solution, 0.04g of the inactivated microorganism FP-JCCW was added. Oscillating at 30 deg.C for 60min at 30r/min, 60r/min, 90r/min, 120r/min, 150r/min and 180r/min respectively. Centrifuging the adsorbed solution at 8000r/min for 5min, digesting the supernatant, and measuring the residual Tl+And (4) concentration.
As can be seen from FIG. 5, the rotation speed is less than 150r/min, and the adsorption amount increases as the rotation speed increases. The adsorption peak was reached at 150r/min, the adsorbed amount was 117.3mg/g, and then the adsorbed amount suddenly decreased at 180 r/min. The rotation speed is increased, so that the contact resistance of thallium ions with the FP-JCCW strain is reduced, the chance of contact with the adsorption sites is increased, and therefore the adsorption amount is increased. The rotational speed is too high, and the adsorption sites of the adsorbent may be damaged, resulting in a decrease in the amount of adsorption.
(5) Effect of Biomass on adsorption
Tl at a concentration of 20mg/L in a volume of 20ml+0.5g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L of inactive microorganism FP-JCCW are respectively added into the solution, and the solution is shaken for 60min at the constant temperature of 30 ℃ and 150 r/min. Centrifuging the adsorbed solution at 8000r/min for 5min, digesting the supernatant, and measuring the residual Tl+And (4) concentration.
As can be seen from FIG. 6, in the range of biomass of 0.5g/L to 4g/L, the adsorption amount increased as the biomass increased. This is probably because the biomass increases, the adsorption sites increase, and the adsorption amount also increases. After a biomass of more than 2.5g/L, the adsorption capacity decreases with increasing biomass. The adsorption resistance of thallium ions to the adsorption sites may increase due to insufficient ion concentration. The reason why the adsorption amount reaches 117.6mg/g at maximum when the biomass is 1g/L is that the adsorption amount per the adsorbent FP-JCCW is decreased because the biomass is increased due to the increase in biomass with a constant metal ion concentration.
(6) Isothermal adsorption study
According to the experimental data of the influence of the initial concentration on the adsorbent FP-JCCW, Langmuir and Freundlich models are established to analyze the adsorption, and the analysis results are shown in FIG. 7 and FIG. 8.
As can be seen from FIGS. 7 and 8, the adsorption process of the adsorbent FP-JCCW is closer to the Langmuir isothermal adsorption model, and the adsorption of thallium ions by the adsorbent is mainly based on monolayer adsorption. Correlation coefficient R of Langmuir isotherm20.9911, the theoretical adsorption saturation B is 129.87mg/g, the adsorption constant K is 0.0246, and the adsorption is substituted into the Langmuir adsorption empirical formula Co/W＝1/KB+CoB, to obtain Co/W＝3.2034+0.0077Co。
(7) Study of adsorption kinetics
According to the experimental data of the influence of the contact time on the adsorbent FP-JCCW, a first-order kinetic equation and a second-order kinetic equation are established to analyze the adsorption, and the analysis results are shown in FIG. 9 and FIG. 10.
As can be seen from FIGS. 9 and 10, the adsorption process of the adsorbent FP-JCCW is more consistent with the quasi-second order kinetic fit, and the adsorption process is mainly chemical adsorption. First order kinetics are fitted with data from 10-80min, R20.9672, can be better describedInitial phase of FP-JCCW adsorption. Secondary kinetics describes the state of the FP-JCCW adsorption overall process, R20.9879. Therefore, the adsorption process is combined with physical adsorption and chemical adsorption, the physical adsorption mainly acts in the initial stage of adsorption, and electrostatic adsorption may occur. While chemisorption dominates the overall process and surface complexation may occur.
(III) SEM and FT-IR analysis of thallium by FP-JCCW strain
(1) Infrared spectroscopy (FT-IR) analysis: 2mg of the dried adsorbent is added with 200mg of spectrally pure potassium bromide (mass ratio is 1:100) and ground in an agate mortar, the mixture is pressed into a uniform and transparent sheet, and a sample is analyzed by a Raman infrared spectrometer.
As can be seen from FIG. 11, there was a significant change in FP-JCCW before and after adsorption. 3500cm-1The absorption peak nearby is from 3385cm-1Moved to 3422cm-1It is shown that hydroxyl group (-OH) on cell wall participates in reaction in thallium ion environment. 2920cm-1Nearby 2926cm-1And 2854cm-1The intensity of the absorption peak is changed, which indicates that carbon-hydrogen bonds (C-H) on the aliphatic carbon chain participate in the reaction in the adsorption process. 1650cm-1Nearby absorption peak from 1624cm-1Moved to 1653cm-1The carbonyl group (C ═ O) located in the amide i band indicates that the carbonyl group (C ═ O) is active on the amide group and is involved in the adsorption reaction. 1450cm-11458cm nearby-1The absorption peak intensity has obvious change, which indicates that amino (-NH) exists in thallium ion environment2) Participates in the reaction. 1088cm-1The intensity of the nearby absorption peak also changes significantly, indicating that it is caused by the contraction vibration of the carbon-oxygen bond (C-O) on the carbohydrate or alcohol on the cell wall. FT-IR infrared analysis results show that-OH, C-H, C ═ O and-NH are mainly contained in the process of FW-JCCW thallium adsorption2Groups such as C-O and the like participate in the reaction.
(2) SEM-EDS analysis: the strain powder before and after adsorption is pretreated and then placed under an FEI Quanta400FEG type environment scanning electron microscope (Czech FEI company) for scanning observation at room temperature to obtain the shape structure of the sample.
As can be seen from FIG. 12, the scanning electron micrographs before and after adsorption of FW-JCCW were significantly changed, indicating that the inactivated microorganisms reacted with thallium ions. Before adsorption, the microorganism surface is not uniform and has a porous structure. After adsorption, the microorganisms are scattered and some crystals, possibly thallium-forming compounds, are distributed around them.
Putting the treated FP-JCCW before and after adsorption into an energy spectrum analyzer, and carrying out SEM and EDS analysis to study the existence of thallium deposited on the cell surface of the inactivated microorganism.
As can be seen from FIG. 13, C, O, P, S, Cl and K peaks were present on the cell surface before thallium ions were adsorbed by the inactivated microorganisms. After adsorption, the absorption peak of Tl appeared, while the peak of P, S, O element was clearly reduced and C increased, possibly indicating that the P, S, O-containing group formed a chelate. In addition, the K element peak disappears after adsorption, and then Na element peak appears, so that adsorption is possibly related to competitive inhibition of potassium ions.
(3) XRD analysis: the powder of the strain before and after the adsorption was tabletted and then subjected to X-ray diffraction analysis in D/max-2200/PC.
Putting the treated FP-JCCW before and after adsorption into an X-ray diffractometer, and carrying out XRD analysis. As can be seen from FIG. 14, characteristic peaks appear at 8.28 °, 19.69 °, 28.35 °, 40.68 ° and 47.22 ° for 2 θ before FP-JCCW adsorption. After adsorption, the peaks at 8.28 degrees and 19.69 degrees are shifted to the right by slightly 9.03 degrees and 19.22 degrees, and the peak values are greatly increased, which indicates that the crystal structure is changed, and FP-JCCW adsorption is probably realized through electrostatic adsorption and surface complexation.
The present invention has been further described with reference to the examples, but the present invention is not limited to the above-described embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
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