CN110734864B - Curvularia lunata and application thereof - Google Patents
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Abstract
The invention discloses Curvularia lunata and application thereof in treatment of chromium-containing wastewater, which is Curvularia lunata HT02, wherein the strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO. 17597. The invention acquires a water sample of a steel mill wastewater storage pool, obtains a strain with hexavalent chromium reduction capability through domestication, screening, separation and purification, and identifies the strain as curvularia lunata, self-coded number HT 02. The strain has certain reduction treatment capacity on chromium-containing wastewater, and particularly has a better and obvious effect on hexavalent chromium wastewater with low concentration. Has certain application prospect and value in the aspect of microbial remediation of water body polluted by low-concentration hexavalent chromium.
Description
Technical Field
The invention relates to the technical field of environmental microbial remediation, in particular to Curvularia lunata and application thereof.
Background
With the development of modern industrial economic technology, chromium salt series products are widely applied to the industries of electroplating, steel, electroplating, leather manufacturing and the like. However, due to the non-standard industrial industry and the unsupervised supervision of related departments, a large amount of chromium-containing wastewater and waste residues are discharged and discarded randomly, and serious pollution is caused to the environment. Chromium, one of the common heavy metal pollutants, exists in multiple valence states in the environment, and is mainly trivalent chromium and hexavalent chromium. Among them, hexavalent chromium is highly toxic, dissolves in water, has strong mobility, easily causes pollution to groundwater and surface water, generates mutation and carcinogenesis to organisms, harms the ecological environment, and affects the health of human beings.
At present, the traditional chromium pollution remediation methods mainly comprise physical and chemical modes such as chemical reduction, electrochemical treatment, ion exchange and the like. However, the traditional restoration method has the problems of high treatment cost, large energy consumption and secondary pollution. Therefore, the popularization and the application are slow at present. With the rise of the bioremediation technology, the technology has the advantages of low cost, simple operation, no secondary pollution and the like, and gradually becomes a research hotspot and an attention object, thus being a green and natural remediation technology. In recent years, many researchers have written patents on reducing hexavalent chromium by microorganisms of different species, such as Alcaligenes sp.GTM2(CN201610426026.1), Cellulosimicrobium sp.GTM1(CN201610399636.7), Streptomyces puniciuststrin.LYzhlm (CN201610077641.6), Ochrobactrum sp.CTS-325(CN200710009694.5), Bacillus sp.Y04(CN201810198416.7), Enterobacter holtzeri CL.2013(CN201510212773.0), Penicillium oxalicum SL2(CN201510017900.1), and the like. However, these microorganisms have a strong reducing and degrading ability to high concentrations of hexavalent chromium (above 500mg/L), but have a low reducing and degrading ability to low concentrations of hexavalent chromium (below 50 mg/L), and cannot completely reduce hexavalent chromium, and the residual concentration of hexavalent chromium is still high. Therefore, it is very important and necessary to screen strains with high reduction capacity for low-concentration hexavalent chromium treatment.
Disclosure of Invention
In view of the above, the present invention aims to provide Curvularia lunata and applications thereof, particularly applications to treatment of low-concentration hexavalent chromium wastewater.
In order to achieve the above object, the Curvularia lunata of the present invention, the Latin name of the strain is Curvularia HT02, the preservation unit: china general microbiological culture Collection center, preservation date: 2019, 4, 15, deposit number: CGMCC NO. 17597.
To achieve the above object, the microbial agent of the present invention comprises Curvularia lunata as set forth in claim 1.
Further, the microbial inoculum is applied to the preparation of hexavalent chromium-containing wastewater products.
Further, the microbial inoculum is applied to the treatment of hexavalent chromium-containing wastewater.
Further, the microbial inoculum is applied to the preparation of a wastewater product containing low-concentration hexavalent chromium.
Further, the microbial inoculum is applied to treatment of wastewater containing low-concentration hexavalent chromium.
In order to achieve the above object, the method for treating wastewater containing hexavalent chromium of the present invention treats wastewater containing hexavalent chromium with the above Curvularia lunata or its bacterial suspension.
In some embodiments of the invention, Cr is included 6+ Are all prepared by adding potassium dichromate (K) 2 Cr 2 O 7 ) Obtaining;
in the embodiment of the invention, the hexavalent chromium is measured by a dibenzoyl dihydrazide spectrophotometric method for measuring the hexavalent chromium in GB-T7467-87 water.
In the examples of the present invention, the removal rate of hexavalent chromium in a water sample is (initial concentration of hexavalent chromium-concentration of hexavalent chromium after treatment) ÷ initial concentration of hexavalent chromium × 100%.
Compared with the prior art, the invention has the following beneficial advantages:
the Curvularia lunata HT02 screened by the method has a certain reduction treatment effect on hexavalent chromium wastewater, and particularly has a better and remarkable effect on low-concentration hexavalent chromium wastewater. Has certain application prospect and value in the aspect of microbial remediation of water bodies polluted by low-concentration hexavalent chromium.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to specific embodiments.
Curvularia lunata is Curvularia lunata HT02, and the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO. 17597.
The invention separates and screens curvularia lunata, re-screens strains and identifies strains
(1) Separation and screening:
collecting a sample: collecting a water sample from a wastewater storage pool of a certain steel plant in Maanshan;
domestication and culture: taking 10mL of water sample, inoculating to sterilized liquid Cnahs medium (containing 10mg/L Cr) 6+ ) Performing shake culture in an incubator at 28 ℃ at the rotation speed of 180 rpm; after culturing for 2d, taking 10mL of culture solution for acclimatization culture, and keeping the culture medium and culture conditions unchanged;
coating and screening: continuously acclimatizing and culturing for 3 times, diluting the acclimatized bacterial suspension with a dilution gradient of 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Coating 100. mu.L of diluted bacterial suspension with solid culture medium (containing 10mg/L Cr) 6+ ) (ii) a Transferring the coated flat plate into a constant-temperature incubator at 28 ℃, and standing for culture.
Scribing and separating: when colonies grow on the plate, colonies with different appearances, forms and front and back colors are selected, and the edge hyphae are taken to be inoculated into a new solid Czochralski medium plate (containing 10mg/L Cr 6+ ) Transferring the mixture into a constant-temperature incubator at 28 ℃, and standing and culturing; the above steps are repeated until each new colony on the plate is a pure strain.
6 pure colonies are obtained through the separation screening experiment; respectively inoculating the obtained strains into a flat plate and an inclined plane of a Chao's medium, standing and culturing at 28 ℃ until hyphae grow fully, and then placing in a refrigerator at 4 ℃ for preservation;
(2) bacterial strain rescreening
The 6 strains obtained by screening and separating are screened and selected again by detecting the reduction effect of the strains on hexavalent chromium, and the method comprises the following specific steps:
inoculating the strains from the plate into sterilized liquid Czochralski culture medium (50mL/250mL conical flask), and performing shake culture in an incubator at 28 ℃ at the rotation speed of 180 rpm; after 3 days of culture, 1mL of bacterial suspension is taken according to the inoculation amount of 1% (v/v), and inoculated to the medium containing 10mg/L Cr 6+ In a 50mL/250mL Erlenmeyer flask, and cultured at a rotation speed of 180rpm in an incubator at 28 ℃ with shaking. After 3d, sampling, centrifuging and removing Cr in the water sample 6+ The concentration was measured and the removal rate was calculated. Finally, the bacterial strain HT02 is determined to have obvious effect on removing hexavalent chromium, and the removal rate reaches 43 percent.
(3) Strain identification
By adopting a molecular biology PCR technology and universal primers ITS1 and ITS4, an ITS1+5.8S + ITS2 sequence of rDNA of the rDNA is amplified, and the rDNA sequence is compared with an NCBI GenBank database, the homology is 99 percent, and the rDNA sequence is identified as Curvularia lunata HT 02.
(4) Strain preservation
The inventor names the screened strain with hexavalent chromium reduction effect as Curvularia pinnatifida with the self-coding number of HT02, and sends the strain to the China general microbiological culture Collection center for registration and preservation, wherein the preservation number is CGMCC NO. 17597.
Example 1 reduction experiment of different addition amounts of microbial inoculum on low-concentration hexavalent chromium
(1) Preparation of microbial inoculum containing Curvularia alutata
A part of an agar block with hyphae was picked from a plate or a slant of Curvularia lunata HT02, inoculated into a sterilized liquid medium for Kirschner flask (50mL/250mL Erlenmeyer flask), and cultured with shaking in an incubator at 28 ℃ at a rotation speed of 180 rpm. Culturing for 3d to obtain seed liquid;
then inoculating the inoculum solution to a sterilized liquid Cnahs medium (100mL/500mL conical flask) according to the inoculation amount of 10% (v/v), and culturing for 5d to prepare a microbial inoculum; after 5 days, the bacterial strain grows well, and mycelium spheres grow in the conical flask, namely the required Curvularia lunata-containing HT02 microbial inoculum.
(2) Preparation of hexavalent chromium wastewater
Firstly, preparing a Chao's culture medium solution,adding potassium dichromate mother liquor to make Cr in the culture medium 6+ The concentration of the obtained product is 25mg/L, namely the prepared low-concentration hexavalent chromium wastewater.
(3) Reduction treatment
Adding the prepared microbial inoculum into the prepared hexavalent chromium wastewater according to the addition amount of 0.2%, 1% and 2% (v/v), and carrying out shaking culture at 180rpm at 28 ℃. And sampling every 3d, centrifuging, measuring the concentration of hexavalent chromium in the supernatant, and calculating the removal rate of the hexavalent chromium. The results are given in the following table:
the experimental effect shows that the curvularia lunata microbial inoculum provided by the invention has strong reducing capability on low-concentration hexavalent chromium, and the reducing capability on hexavalent chromium is gradually increased along with the increase of the addition amount of the microbial inoculum. 3d, treating, wherein the removal rate of hexavalent chromium in the wastewater is about 50%; in the 6d, the concentration of the residual hexavalent chromium in the wastewater is lower than 1 mg/L; at the 9d, under the addition of 1 percent and 2 percent (v/v), the hexavalent chromium is completely reduced, the removal rate can reach 100 percent, and under the addition of 0.2 percent (v/v), the residual hexavalent chromium is only 0.05mg/L and is lower than the limit standard (0.1mg/L) of the hexavalent chromium in the surface V-type water.
Example 2 reduction experiment of different addition amounts of microbial inoculum on high-concentration hexavalent chromium
(1) Preparation of microbial inoculum containing Curvularia HT02
Selecting partial agar block with hypha from flat plate or inclined plane of Curvularia lunata HT02, inoculating into sterilized liquid Chaochou culture medium (50mL/250mL conical flask), culturing at 28 deg.C under shaking at 180rpm for 3d to obtain seed solution;
then inoculating the inoculum solution to a sterilized liquid Cnahs medium (100mL/500mL conical flask) according to the inoculation amount of 10% (v/v), and culturing for 5d to prepare a microbial inoculum; after 5 days, the bacterial strain grows well, and mycelium spheres grow in the conical flask, namely the required Curvularia lunata-containing HT02 microbial inoculum.
(2) Preparation of hexavalent chromium wastewater
Preparing a Chao's culture medium solution, and adding a potassium dichromate mother solution to obtain Cr in the culture medium 6+ The concentration of the hexavalent chromium is 500mg/L, namely the prepared high-concentration hexavalent chromium wastewater.
(3) Reduction treatment
Adding the prepared microbial inoculum into the prepared hexavalent chromium wastewater according to the addition amount of 0.2 percent, 1 percent and 2 percent (v/v), and carrying out shaking culture at 180rpm at 28 ℃. And sampling every 3d, centrifuging, measuring the concentration of hexavalent chromium in the supernatant, and calculating the removal rate of the hexavalent chromium. The results are given in the following table:
according to the experimental results, the curvularia lunata reduction treatment effect on high-concentration hexavalent chromium wastewater (500mg/L) is general, although the removal rate of hexavalent chromium is increased with the increase of the addition amount of the microbial inoculum. However, after 9 days of treatment, the removal rate of hexavalent chromium reaches about 30% when the addition amount is 1% and 2% (v/v), and compared with the removal rate of 99.9% of low-concentration hexavalent chromium wastewater, the removal rate has a certain treatment effect difference.
Therefore, the curvularia lunata newly screened by the invention has certain reduction treatment effect on hexavalent chromium in the wastewater through the reduction treatment on the low-concentration and high-concentration hexavalent chromium wastewater, and the effect is better and obvious particularly on the low-concentration hexavalent chromium wastewater. Has certain application prospect and value in the aspect of microbial remediation of water bodies polluted by low-concentration hexavalent chromium.
The above embodiments are further illustrative of the present invention, but it should not be construed that the scope of the above subject matter is limited to the above embodiments. All the techniques realized based on the above experiments are within the scope of the present invention.
Claims (5)
1. Curvularia lunata (Sing.) RaddeCurvularia lunata ) HT02, characterized by the fact that the depository: china (China)General microbiological culture collection center of the committee for preservation management of microorganisms, preservation date: year 2019, month 4 and day 15, accession number: CGMCC NO. 17597.
2. A microbial preparation comprising Curvularia lunata as claimed in claim 1.
3. Use of the bacterial agent of claim 2 in the preparation of a product for treating hexavalent chromium-containing wastewater.
4. Use of the microbial inoculum according to claim 2 for the treatment of wastewater containing hexavalent chromium.
5. A method for treating hexavalent chromium-containing wastewater, which comprises treating hexavalent chromium-containing wastewater with Curvularia lunata or a bacterial suspension thereof according to claim 1.
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WO2017122227A1 (en) * | 2016-01-13 | 2017-07-20 | Shri Amm Murugappa Chettiar Research Centre (Mcrc) | Mrsa and vrsa resistant textile materials |
CN106085900A (en) * | 2016-06-07 | 2016-11-09 | 中国科学院生态环境研究中心 | Fiber germ and the application that chromate waste water is processed thereof |
CN108555007A (en) * | 2018-03-28 | 2018-09-21 | 中冶华天工程技术有限公司 | The reduction of soil-repairing agent and hexavalent chromium polluted soil stabilizes restorative procedure |
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