阅读说明：本技术 一种土著型微生物菌剂及其制备方法 (Indigenous microbial agent and preparation method thereof ) 是由 王蕾 于 2021-08-11 设计创作，主要内容包括：本发明公开了一种土著型微生物菌剂及其制备方法,属于河湖水治理技术领域,包括以下步骤：S1：准备；S2：取样；S3：总DNA提取；S4：PCR扩增；S5：DGGE分离；S6：测序和鉴定,确定微生物群落；S7：提取已确定微生物,经过纯化,形成不同微生物纯种菌群；S8：准备；S9：不同微生物纯种菌群分别等量倾倒在若干倾倒有等量水样的无菌观察箱内并标注；S10：观察并记录,筛选出多个土著型有益微生物纯种菌群；S11：复配；S12：准备；S13：不同复合型微生物菌群分别等量倾倒在若干倾倒有等量水样的无菌观察箱内并标注；S14：观察并记录,筛选出最优土著型微生物菌群；S15：二次扩配、复壮,应用于本土水环境治理；本发明能够快速且有效的改善本体水环境的质量。(The invention discloses an indigenous microbial agent and a preparation method thereof, belonging to the technical field of river and lake water treatment and comprising the following steps: s1: preparing; s2: sampling; s3: extracting total DNA; s4: PCR amplification; s5: DGGE separation; s6: sequencing and identifying, and determining microbial community; s7: extracting determined microorganisms, and purifying to form pure microbial floras of different microorganisms; s8: preparing; s9: respectively pouring different pure microbial floras into a plurality of aseptic observation boxes with equivalent water samples in an equivalent manner, and labeling; s10: observing and recording, and screening a plurality of indigenous beneficial microorganism pure flora; s11: compounding; s12: preparing; s13: respectively pouring different compound microbial floras into a plurality of aseptic observation boxes with the same amount of poured water samples in an equal amount and labeling; s14: observing and recording, and screening out the optimal indigenous microbial community; s15: secondary expanding preparation and rejuvenation, and is applied to the treatment of the native water environment; the invention can quickly and effectively improve the quality of the body water environment.)

1. An indigenous microbial agent and a preparation method thereof are characterized by comprising the following steps:

s1: taking a plurality of sterile water sample collecting bags with the same shape and size and numbering;

s2: respectively taking water samples to be detected from a plurality of positions in the local water environment, respectively injecting the water samples to be detected into sterile water sample collecting bags with different numbers, and respectively marking the water taking positions and the water taking time of the water samples to be detected on the sterile water sample collecting bags with different numbers;

s3: respectively extracting total DNA from the water samples to be detected in the plurality of sterile water sample collecting bags;

s4: amplifying specific sequences in the extracted total DNAs by polymerase chain reaction;

s5: separating a plurality of amplification products by using DGGE respectively;

s6: sequencing, identifying and analyzing the DGGE bands to determine microbial communities in the local water environment;

s7: extracting determined microorganisms from the native water environment, and forming different pure microbial floras after screening, purification, domestication, expansion and rejuvenation;

s8: taking a plurality of sterile observation boxes with the same shape and size and numbering;

s9: sampling from the same position in the local water environment, pouring a water sample into a plurality of aseptic observation boxes in equal quantity, respectively pouring different pure microbial floras into the plurality of aseptic observation boxes in equal quantity, and respectively labeling the names of the poured pure microbial floras on the aseptic observation boxes with different numbers;

s10: observing and recording the conditions of water samples in the plurality of aseptic observation boxes, obtaining the influence of different pure microbial floras on the water samples according to the recorded water sample conditions, and screening out a plurality of indigenous beneficial pure microbial floras;

s11: compounding two or more pure flora of indigenous beneficial microorganisms to form a plurality of compound microbial flora;

s12: taking a plurality of sterile observation boxes with the same shape and size and numbering;

s13: sampling from the same position in the local water environment, pouring a water sample into a plurality of aseptic observation boxes in equal amount, pouring different compound microbial floras into the plurality of aseptic observation boxes in equal amount respectively, and labeling the names of the poured compound microbial floras on the aseptic observation boxes with different numbers respectively;

s14: observing and recording the conditions of water samples in a plurality of aseptic observation boxes, comparing the directional digestion capability of different compound microbial floras with the directional digestion capability of the pure indigenous beneficial microorganism floras, and screening out the optimal indigenous beneficial microorganism floras;

s15: and performing secondary expansion and rejuvenation on the screened optimal indigenous microbial flora to form an indigenous microbial agent, and applying the indigenous microbial agent to treatment of the indigenous water environment.

2. The indigenous microbial agent of claim 1, wherein: in the step S3, the total DNA extraction method of the water sample to be detected may adopt one or any combination of a freezing-thawing method, an enzymatic method, a chemical method, an ultrasonic disruption method, and a liquid nitrogen under-grinding method.

3. The indigenous microbial agent of claim 1, wherein: in step S6, if the identification analysis results of the microbial communities on the DGGE strips are the same, determining the final microbial community in the local water environment, and if the identification analysis results of the microbial communities on the DGGE strips are different, taking the water samples to be tested from different positions in the local water environment again, performing identification analysis according to the same steps, and so on until the identification analysis results on the DGGE strips are not different from the existing identification analysis results, determining the final microbial community in the local water environment.

4. The indigenous microbial agent of claim 1, wherein: in the step S10 and the step S14, the water quality detector is used as an instrument for observing and recording the conditions of the water samples in the plurality of sterile observation boxes.

5. The indigenous microbial agent of claim 1, wherein: in step S11, before two or more kinds of indigenous type pure flora of beneficial microorganisms are combined, related data are checked to screen out the indigenous type pure flora of beneficial microorganisms with conflict, and when combined, the indigenous type pure flora of beneficial microorganisms with conflict can be eliminated.

Technical Field

The invention belongs to the technical field of river and lake water treatment, and particularly relates to an indigenous microbial agent and a preparation method thereof.

Background

Along with the continuous development of social economy, the discharge amount of industrial sewage and wastewater and the discharge amount of urban domestic water are continuously increased, the pollution of river and lake water is increasingly serious, the life health of people is greatly threatened, and only when the treatment is started to improve the water quality environment from the pollution of the river and lake water, the limited water resource can be continuously and healthily circulated, so that the generation of the world provides a source for the survival and development of human beings.

The existing method for improving water quality environment by river and lake water treatment is to configure various beneficial microorganism flora into microbial inoculum which is put in the river and lake water, and because the water quality of the river and lake water in different areas is different, the beneficial microorganism flora in the microbial inoculum has directional digestion capability to the river and lake water in different areas, so that the pertinence is poor, the time for improving water quality environment by the river and lake water treatment is longer, and the effect is poor.

Disclosure of Invention

To solve the problems set forth in the background art described above. The invention provides an indigenous microbial agent and a preparation method thereof, and the indigenous microbial agent has the characteristics of strong pertinence and capability of quickly and effectively improving the quality of a body water environment.

In order to achieve the purpose, the invention provides the following technical scheme: an indigenous microbial agent and a preparation method thereof, comprising the following steps:

s1: taking a plurality of sterile water sample collecting bags with the same shape and size and numbering;

s2: respectively taking water samples to be detected from a plurality of positions in the local water environment, respectively injecting the water samples to be detected into sterile water sample collecting bags with different numbers, and respectively marking the water taking positions and the water taking time of the water samples to be detected on the sterile water sample collecting bags with different numbers;

s3: respectively extracting total DNA from the water samples to be detected in the plurality of sterile water sample collecting bags;

s4: amplifying specific sequences in the extracted total DNAs by polymerase chain reaction;

s5: separating a plurality of amplification products by using DGGE respectively;

s6: sequencing, identifying and analyzing the DGGE bands to determine microbial communities in the local water environment;

s7: extracting determined microorganisms from the native water environment, and forming different pure microbial floras after screening, purification, domestication, expansion and rejuvenation;

s8: taking a plurality of sterile observation boxes with the same shape and size and numbering;

s9: sampling from the same position in the local water environment, pouring a water sample into a plurality of aseptic observation boxes in equal quantity, respectively pouring different pure microbial floras into the plurality of aseptic observation boxes in equal quantity, and respectively labeling the names of the poured pure microbial floras on the aseptic observation boxes with different numbers;

s10: observing and recording the conditions of water samples in the plurality of aseptic observation boxes, obtaining the influence of different pure microbial floras on the water samples according to the recorded water sample conditions, and screening out a plurality of indigenous beneficial pure microbial floras;

s11: compounding two or more pure flora of indigenous beneficial microorganisms to form a plurality of compound microbial flora;

s12: taking a plurality of sterile observation boxes with the same shape and size and numbering;

s13: sampling from the same position in the local water environment, pouring a water sample into a plurality of aseptic observation boxes in equal amount, pouring different compound microbial floras into the plurality of aseptic observation boxes in equal amount respectively, and labeling the names of the poured compound microbial floras on the aseptic observation boxes with different numbers respectively;

s14: observing and recording the conditions of water samples in a plurality of aseptic observation boxes, comparing the directional digestion capability of different compound microbial floras with the directional digestion capability of the pure indigenous beneficial microorganism floras, and screening out the optimal indigenous beneficial microorganism floras;

s15: and performing secondary expansion and rejuvenation on the screened optimal indigenous microbial flora to form an indigenous microbial agent, and applying the indigenous microbial agent to treatment of the indigenous water environment.

Further, in the present invention, in step S3, the total DNA extraction method of the water sample to be detected may adopt one or a combination of several of a freezing-thawing method, an enzymatic method, a chemical method, an ultrasonic disruption method, and a liquid nitrogen under-grinding method.

In the present invention, in step S6, if the results of the identification and analysis of the microbial communities on the DGGE strips are the same, the final microbial community in the local water environment is determined, and if the results of the identification and analysis of the microbial communities on the DGGE strips are different, the water samples to be tested need to be taken from different positions in the local water environment again, and identification and analysis are performed according to the same procedure, and so on, until the results of the identification and analysis on the DGGE strips are not different from the results of the existing identification and analysis, the final microbial community in the local water environment is determined.

Further, in the present invention, in the step S10 and the step S14, the water quality detector is used as an instrument for observing and recording the conditions of the water samples in the plurality of aseptic observation boxes.

Further, in the present invention, in step S11, before two or more kinds of the indigenous type pure beneficial microorganism colonies are combined, related data are required to be looked up, and the indigenous type pure beneficial microorganism colonies with conflicts are screened out.

Compared with the prior art, the invention has the beneficial effects that:

the method comprises the steps of firstly screening a microbial sequence in the native water environment by adopting a denaturing gradient gel electrophoresis technology, determining the microbial species in the native water environment, then extracting microbes for screening, purifying, domesticating, expanding and rejuvenating to obtain various pure microbial floras, screening out the native beneficial microbial pure flora by observing the directional digestion capability of the various pure microbial floras, then compounding the native beneficial microbial flora, screening out the optimal native microbial flora by observing the directional digestion capability of the composite microbial flora and the native microbial pure flora, carrying out expansion and rejuvenation on the optimal native microbial flora, applying the expanded and rejuvenated microbial flora to the local river and lake water treatment engineering, and having strong pertinence and being capable of quickly and effectively improving the quality of the native water environment.

Drawings

FIG. 1 is a flow chart of the indigenous type microbial agents of the present invention and a method for preparing the same.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, the present invention provides the following technical solutions: an indigenous microbial agent and a preparation method thereof, comprising the following steps:

s1: taking a plurality of sterile water sample collecting bags with the same shape and size and numbering;

s2: respectively taking water samples to be detected from a plurality of positions in the local water environment, respectively injecting the water samples to be detected into sterile water sample collecting bags with different numbers, and respectively marking the water taking positions and the water taking time of the water samples to be detected on the sterile water sample collecting bags with different numbers;

s3: respectively extracting total DNA from the water samples to be detected in the plurality of sterile water sample collecting bags;

s4: amplifying specific sequences in the extracted total DNAs by polymerase chain reaction;

s5: separating a plurality of amplification products by using DGGE respectively;

s6: sequencing, identifying and analyzing the DGGE bands to determine microbial communities in the local water environment;

s7: extracting determined microorganisms from the native water environment, and forming different pure microbial floras after screening, purification, domestication, expansion and rejuvenation;

s8: taking a plurality of sterile observation boxes with the same shape and size and numbering;

s9: sampling from the same position in the local water environment, pouring a water sample into a plurality of aseptic observation boxes in equal quantity, respectively pouring different pure microbial floras into the plurality of aseptic observation boxes in equal quantity, and respectively labeling the names of the poured pure microbial floras on the aseptic observation boxes with different numbers;

s10: observing and recording the conditions of water samples in the plurality of aseptic observation boxes, obtaining the influence of different pure microbial floras on the water samples according to the recorded water sample conditions, and screening out a plurality of indigenous beneficial pure microbial floras;

s11: compounding two or more pure flora of indigenous beneficial microorganisms to form a plurality of compound microbial flora;

s12: taking a plurality of sterile observation boxes with the same shape and size and numbering;

s13: sampling from the same position in the local water environment, pouring a water sample into a plurality of aseptic observation boxes in equal amount, pouring different compound microbial floras into the plurality of aseptic observation boxes in equal amount respectively, and labeling the names of the poured compound microbial floras on the aseptic observation boxes with different numbers respectively;

s14: observing and recording the conditions of water samples in a plurality of aseptic observation boxes, comparing the directional digestion capability of different compound microbial floras with the directional digestion capability of the pure indigenous beneficial microorganism floras, and screening out the optimal indigenous beneficial microorganism floras;

s15: and performing secondary expansion and rejuvenation on the screened optimal indigenous microbial flora to form an indigenous microbial agent, and applying the indigenous microbial agent to treatment of the indigenous water environment.

Specifically, in step S3, the total DNA extraction method of the water sample to be detected may adopt one or a combination of several of a freezing-thawing method, an enzymatic method, a chemical method, an ultrasonic disruption method, and a liquid nitrogen under-grinding method.

Specifically, in step S6, if the identification analysis results of the microbial communities on the DGGE bands are the same, the final microbial community in the local water environment is determined, and if the identification analysis results of the microbial communities on the DGGE bands are different, the water samples to be tested need to be taken from different positions in the local water environment again, and identification analysis is performed according to the same steps, and so on, until the identification analysis results on the DGGE bands are not different from the existing identification analysis results, the final microbial community in the local water environment is determined.

Specifically, in step S10 and step S14, the water quality detector is used as the instrument for observing and recording the conditions of the water samples in the plurality of aseptic observation boxes.

Specifically, in step S11, before two or more kinds of indigenous pure strains of beneficial microorganisms are combined, relevant data are checked to screen out the indigenous pure strains of beneficial microorganisms with conflicts, and during the combination, the combination among the indigenous pure strains of beneficial microorganisms with conflicts can be eliminated.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.