CN113583918A - River sediment degrading strain and application thereof - Google Patents
River sediment degrading strain and application thereof Download PDFInfo
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- CN113583918A CN113583918A CN202111029371.9A CN202111029371A CN113583918A CN 113583918 A CN113583918 A CN 113583918A CN 202111029371 A CN202111029371 A CN 202111029371A CN 113583918 A CN113583918 A CN 113583918A
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/06—Sludge reduction, e.g. by lysis
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Abstract
The invention discloses a river sediment degrading strain and application thereof in river sediment degradationMicrococcus luteus ZR3, which is preserved in China center for type culture Collection with the strain preservation number of M2021839 and the preservation date of 2021, 7 months and 8 days. The micrococcus luteusMicrococcus luteusThe ZR3 can effectively improve the degradation rate of organic matters in the bottom mud of the river, can be used for the reduction and stabilization treatment of the bottom mud in the treatment process of black and odorous water in the river, and has higher application value.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, and particularly relates to a strain for degrading bottom mud of a river and application thereof.
Background
With the acceleration of urbanization, a large amount of domestic and industrial wastewater is discharged into the river without proper treatment, and acute or chronic negative effects are caused on the river and surrounding soil. The river sediment containing a lot of accumulated inorganic matters, persistent organic matters and the like has the characteristics of complex components, difficult degradation and the like, and when the pollutants reach a certain concentration, the pollutants such as nitrogen, phosphorus, heavy metals, organic matters and the like can be continuously released from the sediment to the overlying water, so that the pollution of the river is aggravated. Pollutants in the sediment constitute a great threat to human health and ecological environment, and the comprehensive influence is still barely known.
At present, in order to effectively control the pollution of the bottom mud of the river channel, two technologies of heterotopic repair and in-situ repair can be adopted. In ex-situ remediation, because dredging causes a great amount of pollutants to migrate and has a great negative effect on the ecological environment, in-situ remediation technology or a combination of multiple remediation technologies is often used. The in-situ restoration is widely applied due to the characteristics of small environmental influence, low investment, good restoration effect and the like, and has good development prospect. The in-situ remediation technology of the sediment can be divided into three major categories of physics, chemistry and biology, and the in-situ physical remediation comprises the technologies of sewage interception, water diversion, covering, aeration and the like; the in-situ chemical remediation is a measure of adding a chemical reagent into the polluted bottom sediment, changing the oxidation-reduction potential, the pH value and the like of a water body to slow down the release of pollutants and make the bottom sediment solidified or harmless; in-situ bioremediation is to reduce the pollution of bottom mud by means of microbial degradation or plant purification and the like. The in-situ bioremediation of the bottom sediment mainly comprises two types, one type is to promote the degradation of pollutants by organisms by restoring the bottom sediment environment, and the other type is also called a biological growth promotion technology; the other is to add microbial agents or introduce plants to reduce the pollution of bottom mud, also called a biological strengthening technology. The biological strengthening technology is mainly characterized in that efficient microbial strains capable of degrading pollutants are screened and domesticated and added into polluted bottom mud, and the pollutants are decomposed, converted and degraded by using the life metabolism of microorganisms, so that the concentration of the pollutants in the bottom mud is reduced, and the ecological environment of a river channel is improved. The microorganism can completely degrade organic pollutants into CO in aerobic environment2、N2、H2Inorganic of O or the likeA compound (I) is provided. Meanwhile, the heavy metal can be converted into an inert state and fixed in the bottom mud, so that the migration and release of the heavy metal are reduced. The microbial remediation technology has the characteristics of small environmental influence, good treatment effect, low investment and the like, and can be widely applied to river sediment treatment.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention provides a river sediment degrading strain and application thereof, which are used for solving the problems in the prior art.
In order to achieve the above objects and other related objects, the present invention provides in a first aspect a river sediment-degrading bacterial species identified as Micrococcus luteusMicrococcus luteusThe culture is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address of Wuhan university in eight roads of Wuchang district in Wuhan city, Hubei province. Name of bacterial speciesMicrococcus luteusZR3, preservation date is 2021, 7 months and 8 days, and preservation number is CCTCC NO: M2021839.
Micrococcus luteusMicrococcus luteusZR3, which is gram-positive bacterium, and the colony morphology is light yellow circle, neat edge, and smooth and moist surface.
Micrococcus luteusMicrococcus luteusZR3, containing the gene sequence shown in SEQ ID NO. 1.
In a second aspect, the invention provides a liquid microbial inoculum, which comprises micrococcus luteusMicrococcus luteusZR3 Micrococcus luteusMicrococcus luteusThe concentration of ZR3 is at least 1 × 108 cfu/mL。
The third aspect of the invention provides a preparation method of a microbial inoculum, which comprises the following steps: micrococcus luteusMicrococcus luteusThe pure strain of ZR3 is inoculated in LB liquid culture medium for culture, and liquid microbial inoculum is obtained after the culture is finished.
In a fourth aspect, the present invention provides Micrococcus luteusMicrococcus luteusThe ZR3 is applied to the degradation of bottom mud of a river channel.
Micrococcus luteusMicrococcus luteusThe degradation rate of ZR3 to VS in the bottom sediment of the river reaches 24.8 percent.
The fifth aspect of the invention provides a method for degrading bottom mud of a river channel, which comprises the following steps:
(1) micrococcus luteusMicrococcus luteusInoculating a liquid microbial inoculum of ZR3 to the bottom mud of the river channel;
(2) and carrying out aerobic culture under the conditions of proper temperature, pH value and DO (dissolved oxygen content) to degrade the bottom sludge of the river channel.
The inoculation amount of the inoculation is 0.5-5% of the volume of the sediment, and is preferably 1-3%;
the temperature is 5-40 ℃, and preferably 20-30 ℃;
the pH value is 5-9, and preferably 6-8;
the DO value is 2-5 mg/L, preferably 4-5 mg/L.
As mentioned above, the river sediment degrading strain and the application thereof have the following beneficial effects:
(1) micrococcus luteus is used in the inventionMicrococcus luteusThe degradation rate of ZR3 to VS in the bottom mud of the river reaches 24.8%, the organic matters in the bottom mud can be efficiently degraded, the degradation rate of VS in the bottom mud is effectively improved, the reduction and stabilization of the bottom mud of the river are realized, and the application value is high;
(2) the invention has high treatment efficiency, good economic benefit, convenient operation and no pollution.
Drawings
FIG. 1 shows a phylogenetic tree map of the river sediment degrading strain ZR3 of the present invention.
FIG. 2 shows the morphology of the growing colonies of the river sediment-degrading strain ZR3 of the present invention on LB plates.
FIG. 3 shows the effect of the river sediment degrading strain ZR3 of the invention on the degradation of VS in the river sediment.
Fig. 4 shows the change of the river sediment degrading strain ZR3 of the present invention in VS degradation rate of river sediment.
Detailed description of the preferred embodiments
Example 1: separation screening and performance determination of river sediment degrading strain ZR3
The media and components used were as follows:
LB liquid medium: 10 g/L sodium chloride, 10 g/L tryptone, 5 g/L yeast extract and a pH value of 7.0-7.5.
LB solid medium: 10 g/L sodium chloride, 10 g/L tryptone, 5 g/L yeast extract and 15 g/L agar powder, and the pH value is 7.0-7.5.
River sediment liquid culture medium: adding water into the river sediment (the total solid TS content is 11%, the volatile solid VS content is 7.8% of TS, and the mass fractions) to adjust the TS content of the sediment to be 5% and the pH value to be 7.0.
River sediment solid culture medium: adding water into the river sediment (the TS content is 11%, the VS content is 7.8% of the TS content, and the mass fractions respectively) to adjust the TS content of the sediment to be 5%, 15 g/L agar powder and the pH value to be 7.0.
The prepared culture medium is sterilized by high pressure steam at 121 deg.C for 20 min.
Separation and purification: collecting sediment samples from different river channels, taking 5 mL of sediment to 45 mL of sterilized LB liquid culture medium, uniformly mixing, and placing in a shaking table at 20 ℃ and 180 r/min for enrichment culture for 48 h; after the cultured enrichment solution is subjected to gradient dilution, uniformly coating the enrichment solution on an LB solid culture medium; after culturing for 48 hours in a constant temperature incubator at 20 ℃, selecting monoclonals with different colony morphologies, and marking, purifying and preserving the monoclonals. A total of 12 strains are obtained through separation and purification.
Primary screening: inoculating the pure strains obtained by separation and purification to a river sediment solid culture medium by adopting a point grafting method, carrying out primary screening, culturing the inoculated flat plate in a constant temperature incubator at 20 ℃ for 48-96 h, selecting strains which grow well on the culture medium, carrying out streak purification, numbering and preserving. 5 strains are obtained through primary screening.
And (3) determining the degradation performance of the bottom mud: the experimental group inoculates 5 strains obtained by primary screening into LB liquid culture medium, cultures for 24 h in a shaking table at 20 ℃ and 180 r/min, uses 2% (volume ratio) bacterial liquid to wash and resuspend with sterile normal saline after centrifugation, and inoculates into riverway sediment liquid culture medium. The control group was administered the same volume of sterile saline. Culturing in a shaking table at 20 ℃ and 100 r/min for 7 d, determining the degradation rate of VS in the bottom sediment after the reaction is finished, and evaluating the bottom sediment degradation performance of each strain compared with a control group without bacteria. As can be seen from FIG. 3, the degradation of the strain ZR3 to the sediment VS is the highest, and reaches 24.8%, and compared with the VS degradation rate of a control group, the VS degradation rate is improved by 34.1%. The strain ZR3 has obvious river sediment degradation effect.
Example 2: biological identification of river sediment degrading strain ZR3
The strain identification adopts a 16S rDNA sequence alignment method. Genomic DNA of strain ZR3 was extracted according to the prior art and used as template for amplification of strain 16S rDNA using a pair of universal primers (27F, 1492R). The forward primer was 27F (5'-AGAGTTTGATCCTGGCTCA-3') and the reverse primer was 1492R (5'-GGTTACCTTGTTACGACTT-3').
The PCR reaction (20. mu.L) was as follows: 0.5 μ L of template DNA, 10 μ L of PCR Taqmix, 0.6 μ L of each of the upstream and downstream primers, and ddH2O to the reaction system was 20. mu.L.
PCR procedure: pre-denaturation at 94 deg.C for 5 min, denaturation at 94 deg.C for 30 s, annealing at 55 deg.C for 30 s, and extension at 72 deg.C for 1 min and 30 s, circulating for 30 times, extension at 72 deg.C for 10 min, and storing at 4 deg.C.
The 16S rDNA sequence of the obtained strain is shown in SEQ ID NO.1 by the purification and sequencing of PCR products by Shanghai Jili biotechnology limited. The 16S rDNA sequence obtained by sequencing was submitted at NCBI, homology sequence alignment analysis was performed with GenBank by software, and MEGA 7 software was used to construct phylogenetic tree of the strain (FIG. 1).
The effective gene sequence length of the strain ZR3 is 1409 bp, which is shown in a gene sequence table SEQ ID NO. 1. The sequence was compared with NCBI databaseMicrococcus luteus(NCBI accession number: MN 540712.1) homology is up to 99%, other biological characteristics of the strain are integrated, the strain ZR3 is gram-positive bacteria, the colony morphology is light yellow, round, neat in edge, smooth and moist in surface, and the colony morphology is shown in figure 2. Identifying the strain as micrococcus luteusMicrococcus luteusFinally, it was named Micrococcus luteusMicrococcus luteus ZR3。
Example 3: preparation of liquid microbial inoculum of river sediment degrading strain ZR3
Inoculating ZR3 pure strain into 10 mL LB liquid culture medium, culturing for 24 h in constant temperature shaking table of 20 deg.C and 180 r/min, then inoculating into the liquid culture medium of the next stage of amplification culture with the inoculum size of 5% of the volume of the liquid culture medium, and performing multistage amplification culture under the same conditions to obtain the liquid microbial inoculum of the strain.
Example 4: degradation effect of river sediment degrading strain ZR3 on river sediment
The river water and bottom mud are taken from a river in Shanghai city, wherein the bottom mud is mainly grey black sludge which is formed by depositing pollutants from life and industry at the bottom of a river bed through physical, chemical and biological actions and is rich in organic matters and nutrient salts. The TS content of the bottom mud is 13%, the VS content is 9.3% of the TS, and the pH value is 6.8.
The experimental group inoculates the liquid microbial inoculum of the strain ZR3 in a substrate sludge mixed system (the volume ratio of the riverway water to the substrate sludge is 1: 1) according to the volume ratio of 2 percent, and the control group is inoculated into a liquid culture medium with the same volume under the conditions of DO value of a water body of 3 mg/L, temperature of 20 ℃ and pH value of 6.8. Sampling every 24 h in the reaction process, and measuring the VS degradation rate of the river sediment. Fig. 4 shows the degradation condition of the strain ZR3 on the bottom sediment of the river within 120 h, and it can be seen that the highest value of the VS degradation rate of the strain ZR3 on the bottom sediment of the river reaches 33.7%, which is 53.9% higher than the VS degradation rate of the control group without inoculation of bacteria, which is 21.9%. The strain ZR3 has obvious degradation effect on the bottom sediment of the river.
Sequence listing
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Claims (9)
1. Micrococcus luteus (A) and (B)Micrococcus luteus) ZR3 with the preservation number of CCTCC NO: M2021839.
2. Micrococcus luteus (A) according to claim 1Micrococcus luteus) ZR3, wherein Micrococcus luteus (A), (B) or (C)Micrococcus luteus) ZR3 has the gene sequence shown in SEQ ID NO. 1.
3. A microbial preparation comprising Micrococcus luteus (Micrococcus luteus) according to claim 1Micrococcus luteus）ZR3。
4. The microbial inoculum according to claim 3, which is a liquid microbial inoculum comprising Micrococcus luteus (Micrococcus luteus) ((Micrococcus luteus))Micrococcus luteus) The concentration of ZR3 is at least 1 × 108 cfu/mL。
5. The microbial preparation according to claim 4, wherein the microbial preparation is prepared by subjecting Micrococcus luteus (Micrococcus luteus)) (Micrococcus luteus) Inoculating the pure strain of ZR3 into liquid culture medium, and culturing to obtain liquid microbial inoculum.
6. A Micrococcus luteus(s) according to claim 1Micrococcus luteus) The ZR3 is applied to the degradation of bottom mud of a river channel.
7. The use of claim 6, wherein Micrococcus luteus (A), (B), (C) or (C)Micrococcus luteus) The degradation rate of ZR3 to VS in the bottom sediment of the river reaches 24.8 percent.
8. A method for degrading bottom mud of a river channel is characterized by comprising the following steps:
micrococcus luteus (A) to (B)Micrococcus luteus) The ZR3 CCTCC NO is M2021839 which is prepared by inoculating liquid microbial inoculum to the bottom mud of the river channel;
and carrying out aerobic culture under the conditions of proper temperature, pH value and DO value to degrade the bottom sludge of the river channel.
9. The method for degrading riverway sediment according to claim 8, further comprising the following conditions:
the inoculation amount of the inoculation is 0.1-5% of the volume of the bottom mud of the river channel;
the temperature is 5-40 ℃;
the pH value is 5-9;
the DO value is 2-5 mg/L.
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| CN114350575A (en) * | 2022-03-08 | 2022-04-15 | 佛山市玉凰生态环境科技有限公司 | Anaerobic riverway bottom mud degrading strain and application thereof |
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| CN110343630A (en) * | 2019-01-31 | 2019-10-18 | 贵州大学 | A kind of micrococcus luteus and its application |
| CN110468079A (en) * | 2019-08-28 | 2019-11-19 | 中国科学院上海高等研究院 | A kind of sludge degradation strain and its application |
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| CN102747023A (en) * | 2012-07-27 | 2012-10-24 | 长安大学 | Micrococcus luteus and application thereof |
| CN110343630A (en) * | 2019-01-31 | 2019-10-18 | 贵州大学 | A kind of micrococcus luteus and its application |
| CN110468079A (en) * | 2019-08-28 | 2019-11-19 | 中国科学院上海高等研究院 | A kind of sludge degradation strain and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114350575A (en) * | 2022-03-08 | 2022-04-15 | 佛山市玉凰生态环境科技有限公司 | Anaerobic riverway bottom mud degrading strain and application thereof |
| CN114350575B (en) * | 2022-03-08 | 2022-05-27 | 佛山市玉凰生态环境科技有限公司 | Anaerobic riverway bottom mud degrading strain and application thereof |
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