CN112094772A

CN112094772A - Biological agent and preparation method and application thereof

Info

Publication number: CN112094772A
Application number: CN202010941239.4A
Authority: CN
Inventors: 李德镇; 高崎; 王丹丹; 朱国琴; 王军; 李朋朋; 张亚光
Original assignee: Sph Xing Ling Sci & Tech Pharmaceutical Co ltd
Current assignee: Sph Xing Ling Sci & Tech Pharmaceutical Co ltd
Priority date: 2020-09-09
Filing date: 2020-09-09
Publication date: 2020-12-18
Anticipated expiration: 2040-09-09
Also published as: CN112094772B

Abstract

The invention relates to the technical field of environmental microbiology, in particular to a biological agent and a preparation method and application thereof, wherein the biological agent comprises a liquid agent and a solid agent, and the preparation method of the liquid agent comprises the following steps: taking aerobic activated sludge generated by treating the ginkgo leaf extract industrial wastewater, standing, and taking supernatant to perform primary culture in a selective culture medium; taking the bacterial suspension after the primary culture to be cultured again in a new selective culture medium, and culturing the bacteria living in the selective culture medium again to be the flora in the liquid microbial inoculum; transferring the flora to enrichment medium to culture with distilled water as referenceTime OD₆₀₀It is preferably 0.8 or more. The biological agent has high efficiency of treating the ginkgo leaf extract industrial wastewater, good economic benefit, convenient operation and no pollution.

Description

Biological agent and preparation method and application thereof

Technical Field

The invention relates to the technical field of environmental microorganisms, in particular to a biological agent and a preparation method and application thereof.

Background

The ginkgo leaf extract can increase the blood flow of the cerebral vessels, improve the blood circulation function of the cerebral vessels, protect brain cells, expand coronary arteries, prevent angina and myocardial infarction, prevent thrombosis and improve the immunity of the organism, and is very beneficial to patients with coronary heart disease, angina, cerebral arteriosclerosis, senile dementia and hypertension. When the ginkgo leaf extract is extracted, more industrial wastewater is generated, the Chemical Oxygen Demand (COD) and ammonia nitrogen content in the industrial wastewater are higher, the original treatment process cannot meet the discharge requirement of the wastewater, along with the increasing of public environmental protection consciousness, the attention degree to the environmental problem is higher and higher, the wastewater treatment cannot reach the standard enough to block the development and growth of enterprises, and the ginkgo leaf extract becomes the bottleneck of enterprise development.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a biological agent, a preparation method and a use thereof, which are used for solving the problems in the prior art.

In order to achieve the above and other related objects, the method for preparing a liquid microbial inoculum provided by the invention comprises the following steps:

1) taking aerobic activated sludge generated by treating the ginkgo leaf extract industrial wastewater, standing, and sucking supernatant fluid to perform primary culture in a selective culture medium; sucking the bacterial suspension after primary culture into a new selective culture medium for secondary culture, wherein the bacteria surviving in secondary culture are the flora in the liquid microbial inoculum;

2) the flora of the step 1)Transferring to enrichment medium and culturing to OD with distilled water as reference₆₀₀Is 0.8 or more.

The liquid microbial inoculum provided by the invention is prepared by the method.

The solid microbial inoculum provided by the invention comprises a carrier and floras adsorbed on the carrier material, wherein the floras are obtained from the liquid preparation.

The carrier is a solid phase carrier. In one embodiment, the carrier is selected from one or more of wheat bran, corn flour and soybean meal.

The preparation method of the solid microbial inoculum provided by the invention comprises the following steps of mixing a carrier and a liquid microbial inoculum according to the mass ratio of 1: 80-1: 120, and mixing.

The invention provides application of the liquid microbial inoculum or the solid microbial inoculum in treating ginkgo leaf extract industrial wastewater.

The method for treating the ginkgo leaf extract industrial wastewater provided by the invention comprises the following steps: and adding the liquid microbial inoculum or the solid microbial inoculum into an aerobic pool, ginkgo leaf extract industrial wastewater or aerobic activated sludge containing the ginkgo leaf extract industrial wastewater.

As mentioned above, the biological agent, the preparation method and the application thereof have the following beneficial effects:

1) the efficiency of the activated sludge for treating organic matters in the wastewater is improved, and the problem that the wastewater cannot be effectively treated in time after the biochemical treatment system collapses is solved.

2) Provides an effective case for the treatment of the Chinese medicine wastewater with complex components, is beneficial to the green production and sustainable development of Chinese medicine pharmaceutical enterprises, and improves the ecological environment

3) Good economic benefit, convenient operation and no pollution.

Drawings

FIG. 1 shows the growth profile of the dominant bacteria population of the present invention.

FIG. 2 shows the effect of two activated sludges of the present invention on COD removal.

FIG. 3 shows the ammonia nitrogen removal effect of two activated sludges of the present invention.

FIG. 4 shows the effect of solid and liquid bacteria on the removal of COD from wastewater.

FIG. 5 shows the effect of solid and liquid bacteria on the removal of ammonia nitrogen from wastewater.

FIG. 6 shows the results of gel electrophoresis of PCR amplification (M in the figure shows the DNA Marker).

FIG. 7 shows the structure of a colony of bacteria at the phylum taxonomic level in the biological agent of the present invention.

FIG. 8 shows the structure of the colony of bacteria at the class classification level in the biological agent of the present invention.

FIG. 9 shows the colony structure of bacteria at the genus classification level in the biological agent of the present invention.

FIG. 10 is a flow chart showing the treatment of industrial wastewater containing ginkgo biloba extract.

Detailed Description

The biological agent comprises a liquid agent and a solid agent. The invention firstly provides a preparation method of the liquid microbial inoculum, which comprises the following steps:

1) taking aerobic activated sludge generated by treating the ginkgo leaf extract industrial wastewater, standing, and sucking supernatant fluid to perform primary culture in a selective culture medium; sucking the bacterial suspension after primary culture into a new selective culture medium for secondary culture, wherein the flora surviving in secondary culture is the flora in the liquid microbial inoculum;

2) transferring the flora obtained in the step 1) to an enrichment medium for culturing until OD is reached when distilled water is used as a reference₆₀₀Is 0.8 or more.

The folium Ginkgo extract is dry leaf extract of Ginkgo biloba (Ginkgo biloba L.) of Ginko, is light yellow brown flowable powder, and has effects of promoting blood circulation, removing blood stasis, and dredging collaterals.

The folium ginkgo extract industrial wastewater is industrial wastewater generated in the production of folium ginkgo extracts.

The activated sludge is a generic term for microbial populations and the organic and inorganic materials to which they are attached.

The aerobic activated sludge is obtained from an aerobic tank in the process of treating the ginkgo biloba extract industrial wastewater by adopting an anaerobic-aerobic combined method. The aerobic activated sludge is acclimated in the process of treating the ginkgo biloba extract industrial wastewater, and contains dominant bacteria capable of adsorbing and removing ammonia nitrogen and chemical oxygen demand. Specifically, the flow of treating the ginkgo biloba extract industrial wastewater is shown in fig. 10: the large solid particles such as ginkgo leaf residues and the like in the industrial wastewater are removed through the rotary drum grating, the wastewater enters the regulating tank through the water collecting tank to regulate the water quality and the water quantity, and then enters the anaerobic tank to carry out hydrolytic acidification reaction, so that the organic matter with high molecular weight and difficult degradation is converted into the organic matter with low molecular weight and easy degradation. The effluent of the anaerobic tank is subjected to denitrification treatment by activated sludge adsorption and degradation in the aerobic tank to generate a large amount of sludge, the effluent of the aerobic tank enters a secondary sedimentation tank for solid-liquid separation, and the precipitated activated sludge flows back to the aerobic tank for aerobic reaction. And (4) performing advanced oxidation advanced treatment on the effluent of the secondary sedimentation tank, feeding the effluent into a tertiary sedimentation tank, and discharging the effluent after the solid-liquid separation in the air floatation tank reaches the standard. The pH and chemical oxygen demand of each process stage are shown in Table 1.

TABLE 1 pH and chemical oxygen demand for each process step

In one embodiment, the selective media comprises ginkgo biloba extract industrial wastewater. The content of the ginkgo biloba extract industrial wastewater meets the carbon source required by the growth of microorganisms. In one embodiment, the volume of the ginkgo biloba leaf extract industrial wastewater is 5 to 25 percent of the total volume of the selection medium. Preferably, the volume of the ginkgo biloba extract industrial wastewater is 5-10%, 10-15%, 15-20% or 20-25% of the total volume of the selection culture medium. The selective medium also comprises other nutrients: peptone, sodium chloride, K₂HPO₄Magnesium sulfate heptahydrate and water. Because the concentration of organic pollutants in the ginkgo leaf extract industrial wastewater is high and the ginkgo leaf extract industrial wastewater can be used as a carbon source of dominant bacteria for efficiently degrading the organic pollutants in the metabolic wastewater, the ginkgo leaf extract industrial wastewater is used as the carbon source to screen the dominant bacteria, and a culture medium is placed in an autoclave for sterilization for 30 min.

In a kind of fruitIn embodiments, the selection medium comprises the following composition: peptone 2.00g, ginkgo leaf extract industrial wastewater 100mL, sodium chloride 1.00g, K₂HPO₄1.00g, magnesium sulfate heptahydrate 0.60g, and 900mL of ultrapure water. Wherein the ginkgo leaf extract industrial wastewater is to-be-treated industrial wastewater.

In one embodiment, the aerobic activated sludge is stirred uniformly and then allowed to stand during the primary culture. After standing, taking the supernatant into a selective medium, wherein the volume ratio of the supernatant to the selective medium is 1: 10-1: 30, and preferably 1: 20.

In one embodiment, the volume ratio of the bacterial suspension after primary culture to the selection medium is 1:10 to 1:30, preferably 1: 20.

The enrichment medium comprises peptone, beef extract, sodium chloride and ultrapure water. In one embodiment, the enrichment medium comprises peptone 10.00g/L, beef extract 3.00g/L, sodium chloride 5.00g/L, and ultrapure water.

Specifically, the preparation method further comprises one or more of the following characteristics:

a) the culture temperature is 30-40 ℃; preferably 36-38 ℃;

b) the pH value is 6-9, preferably 7 in the culture process;

c) the rotating speed is 100-180 r/min in the culture process; preferably 155 r/min.

According to the conditions, the primary culture time and the secondary culture time are 4-6 days; the enrichment culture time is 8-18 h, namely OD can be obtained when the flora in the enrichment culture medium takes distilled water as reference₆₀₀Is 0.8 or more.

The flora surviving the re-cultivation in the preparation process may also be referred to as dominant flora.

The liquid microbial inoculum provided by the invention is obtained by the preparation method.

The flora of the liquid microbial inoculum comprises the following bacteria: beta-Proteobacteria, gamma-Proteobacteria, alpha-Proteobacteria, Flavobacterium, Bacteromycetes, -Proteobacteria, WCHB1-03, -Proteobacteria, Ignavibacteria, Acidobacterium, holophaga, spirochaeta.

The flora of the liquid microbial inoculum comprises the following genera: thermomonas, Desloromonas, Flavobacterium (Flavobacterium), unclassified Sphingomaadaceae, norak Pseudomonas adaceae, sphingolipid (Novosphingobium), norak Bacteroidales, Hydrophilia (Hydrogenophaga), Rubivivax. The Thermomonas of Proteobacteria was the most abundant genus of all the bacteria, 13.9% of all the bacteria, followed by the Desloromonas of Proteobacteria, 8.4% of all the bacteria.

The liquid microbial inoculum also comprises an enrichment medium. The enrichment medium comprises peptone, beef extract, sodium chloride and ultrapure water. In one embodiment, the enrichment medium comprises peptone 10.00g/L, beef extract 3.00g/L, sodium chloride 5.00g/L, and ultrapure water.

In one embodiment, the support is a solid support. In one embodiment, the carrier is selected from one or more of wheat bran, corn flour and soybean meal.

Specifically, the mass ratio of the carrier to the liquid microbial inoculum is selected from any one of the following ranges: 1: 80-1: 90, 1: 90-1: 100, 1: 100-1: 110, 1: 110-1: 120.

The four-day removal rate of the liquid microbial inoculum of the invention on the COD of the ginkgo leaf extract industrial wastewater reaches more than 96 percent, and the four-day removal rate of ammonia nitrogen reaches more than 93 percent.

The four-day removal rate of COD was measured by the following method:

1) 250mL of ginkgo biloba extract industrial wastewater with a COD value of 2100mg/L and ammonia nitrogen of 37.4mg/L and 25mL of aerobic activated sludge liquid are added into both groups;

2) adding 30mL of liquid microbial inoculum into one group, and adding no liquid microbial inoculum into the other group;

3) two groups of aeration culture at 25 ℃, sampling every 24 hours, determining the COD value of the wastewater by using a national standard GB 11914-1989 dichromate method, and determining the ammonia nitrogen value by using a GB 7479-; and calculating the removal rate of COD and ammonia nitrogen by using a formula (I):

removal rate (%) [ initial value-detection value ]/initial value × 100% ((r))

Wherein the initial value is the initial COD concentration or the initial ammonia nitrogen concentration of the ginkgo biloba extract industrial wastewater, namely 2100mg/L and 37.4mg/L respectively; the detection value is the COD concentration or ammonia nitrogen concentration in the system detected when the sample is taken on the x day, and the four-day removal rate is the COD concentration or ammonia nitrogen concentration in the sampling system when x is 4.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

The media components used in the examples are as follows:

selecting a culture medium: peptone 2.00g, ginkgo leaf extract industrial wastewater 100mL, sodium chloride 1.00g, K₂HPO₄1.00g, magnesium sulfate heptahydrate 0.60g, and 900mL of ultrapure water.

Enrichment culture medium: 10.00g of peptone, 3.00g of beef extract, 5.00g of sodium chloride and 1000mL of ultrapure water.

Example 1 screening for dominant bacterial flora

Putting 50mL of ginkgo biloba extract production tail end aerobic activated sludge suspension into a beaker, placing the beaker into a magnetic stirrer, stirring the mixture evenly, standing the mixture for a period of time at room temperature, sucking 5mL of supernatant into a conical flask containing 100mL of sterilized selective medium, and culturing the mixture for 5 days at the temperature of 35 ℃ in a constant-temperature incubator and at the speed of 160 r/min. 5mL of the primary culture suspension was aspirated into a 100mL Erlenmeyer flask containing the sterilized selection medium, and cultured again under the same conditions for 5 days. The bacteria which can not degrade and metabolize the organic matters in the wastewater die, and the bacteria which can utilize the organic matters in the wastewater are reserved, so that the purpose of screening dominant bacteria is achieved.

Example 2 optimization of optimal growth conditions for dominant bacteria

The experiment in this group utilizes a uniform design method to determine the optimal growth conditions of the dominant flora. The growth of the strain is greatly influenced by environmental factors, and the rotating speed of a shaker, the initial pH value and the culture temperature are selected as three factors of the experiment to carry out five-level experiment. 2mL of the bacterial suspension of the dominant bacterial population of example 1 was aseptically added to a conical flask containing 100mL of sterilized enriched medium, and after a certain period of incubation, the absorbance of the broth was measured at 600nm using a spectrophotometer. The specific design is shown in table 2.

Table 2 homogeneous experimental design for optimization of dominant flora growth conditions

The results of the experiment are shown in table 3. And carrying out quadratic polynomial stepwise regression analysis on the experimental result through DPS software to obtain an equation: y-1.0372 +0.00085X₁+0.02213X₃-0.00007125X₃*X₃And the significance level P is 0.0299, the equation has significance, F is 605.7819, the correlation coefficient R is 0.9997, the adjusted correlation coefficient Ra is 0.9989, and the optimal combination is obtained according to the regression equation, wherein the culture temperature is 38 ℃, the pH value is 7, and the rotating speed of the shaking table is 155R/min.

TABLE 3 homogeneous test results for optimization of dominant flora growth conditions

Example 3 determination of growth curves of dominant flora

Under aseptic conditions, 2mL of the bacterial suspension of the dominant bacterial flora in example 1 was added to a conical flask containing 100mL of sterilized enrichment medium, and the initial pH value for enrichment, the culture temperature and the rotational speed of the shaker were determined according to the experimental results of the uniform design in example 2, i.e., the growth conditions of the dominant bacterial flora were set to 38 ℃, pH 7 and rotational speed of the shaker 155 r/min. Culturing bacteria for 72h, and measuring OD of bacterial liquid by sampling every 4h when the bacteria are cultured for 0-12 h₆₀₀Sampling and measuring every 6h for 12-36 h, sampling and measuring every 12h for 36-12 h, and then measuring the OD₆₀₀Values and sampling times were used to plot the growth curves of the dominant flora.

The results are shown in Table 4, based on the OD measured₆₀₀Values and sampling times growth curves for the dominant flora are plotted as shown in fig. 1. As can be seen from figure 1, the dominant bacteria are cultured under the optimal growth condition, 0-8 h is the adaptation period of the strain, then the strain enters the logarithmic growth phase, the number of the dominant bacteria is rapidly increased within 8-18 h, after 24h, because nutrient substances of the culture medium are rapidly consumed, a large amount of harmful metabolites are accumulated, the number of bacteria reproduction and the number of death tend to be balanced, and the increase speed of the number of bacteria is obviously slowed down. The growth curve is drawn, and the method has certain guiding significance to the production of the biological agent.

TABLE 4 measurement results of growth curves of dominant bacteria

Example 4 determination of Performance of liquid microbial inoculum for treating folium Ginkgo extract Industrial wastewater

The performance of the liquid microbial inoculum is determined by comparing the effect of the activated sludge added with the liquid microbial inoculum and the effect of the common activated sludge without the liquid microbial inoculum on treating the ginkgo leaf extract industrial wastewater. 500mL of ginkgo biloba extract industrial wastewater with a COD value of 2100mg/L and ammonia nitrogen of 37.4mg/L is respectively filled in two beakers, 50mL of activated sludge is added into the two beakers, 30mL of liquid microbial inoculum (dominant bacterial flora grows for 12 hours in an enrichment medium) is taken in one beaker, aeration culture is carried out at 25 ℃, the COD value of the wastewater in the beaker is measured every 24 hours, a curve of the COD value changing along with time is drawn, and the degradation effect of the activated sludge added with the liquid microbial inoculum and the degradation effect of the common activated sludge without the liquid microbial inoculum are observed.

The results of measuring COD and ammonia nitrogen values are shown in Table 5 and Table 6, and FIG. 2 and FIG. 3. As can be seen from fig. 2 and 3, the removal effect of the activated sludge strengthened by the liquid microbial inoculum on the wastewater COD and the ammonia nitrogen is better than that of the common activated sludge, the removal rate of the activated sludge strengthened by the liquid microbial inoculum on the wastewater COD and the ammonia nitrogen respectively reaches about 96% and 93%, compared with the common activated sludge, the activated sludge strengthened by the liquid microbial inoculum has obvious advantages, and the result shows that the liquid microbial inoculum can enhance the treatment capacity of the activated sludge, mainly because the screened advantageous flora can effectively utilize the organic pollutants in the ginkgo leaf extract wastewater, and the added advantageous flora in the activated sludge can increase the number of bacteria capable of utilizing the organic pollutants in the wastewater, and can also play a synergistic role with other bacteria.

TABLE 5 Effect of liquid microbial inoculum for enhancing COD removal of activated sludge and ordinary activated sludge

TABLE 6 liquid microbial inoculum for strengthening removal effect of activated sludge and common activated sludge on ammonia nitrogen

Example 5 examination of solid microbial inoculum Carrier Material

Because the liquid microbial inoculum is inconvenient to store and transport, the method for preparing the solid microbial inoculum by adsorbing dominant bacteria on carrier materials such as wheat bran, bean pulp, corn flour and the like with large bacteria adsorption capacity is adopted, and the problems are solved.

In the experiment, a solid material selected from wheat bran, corn flour and soybean meal is used as a carrier of the solid microbial inoculum. The specific operation is as follows: adding 1g of solid adsorption carrier material and 100mL of enrichment medium into a 250mL conical flask, sterilizing at 121 ℃ for 20min in an autoclave, cooling to room temperature, inoculating 2mL of bacterial suspension of liquid microbial inoculum under aseptic condition, culturing according to the growth condition optimized in the embodiment 3, and sampling and measuring once every 1 day for 0-5 days. After natural sedimentation, the culture medium in the conical flask is carefully poured out, the adsorption carrier material in the conical flask is washed by deionized water and then transferred to a 50mL centrifuge tube, the centrifuge tube is centrifuged at the rotation speed of 500rpm/min, the supernatant is poured out, and the washing and centrifugation are repeated for 3 times to remove the residual culture medium. After washing, the water is separatedAdding 5mL distilled water into the core tube, vortex vibrating for 2min, carefully pouring out the liquid, repeating for 3 times, diluting the poured out liquid to a constant volume of 20mL, and measuring OD with distilled water as reference₆₀₀. To some extent, the above measured OD₆₀₀The value is in positive correlation with the bacterial absorption quantity of the solid adsorption carrier material, and can reflect the bacterial absorption capacity of the carrier material.

The results of the experiment are shown in Table 7. The results show that under the same culture conditions, the bacteria adsorbing capacity of each solid material is as follows: wheat bran is more than corn flour and soybean meal.

TABLE 7 results of cell adsorption onto each solid material

Example 6 determination of Performance of solid microbial inoculum for treating folium Ginkgo extract Industrial wastewater

The experiment of the group determines the effect of the solid microbial inoculum (the carrier material is wheat bran) and the liquid microbial inoculum on treating the ginkgo leaf extract industrial wastewater, and the effect takes a COD value and an ammonia nitrogen value as indexes. 50mL of ginkgo biloba extract industrial wastewater with a COD value of about 2100mg/L and an ammonia nitrogen value of 37.4mg/L is filled into two beakers, 1.0g of solid microbial inoculum and 1.0g of liquid microbial inoculum precipitate are respectively added into the two beakers, aeration culture is carried out at 25 ℃, the COD value and the ammonia nitrogen value of the wastewater in the beakers are measured every 12 hours, curves of the COD value and the ammonia nitrogen value along with time change are drawn, and the effect of the solid microbial inoculum and the liquid microbial inoculum on treating the ginkgo biloba extract industrial wastewater is observed.

The results of the solid microbial inoculum and the liquid microbial inoculum on the degradation of COD and ammonia nitrogen in wastewater are shown in tables 8 and 9, and figures 4 and 5. As can be seen from fig. 4 and 5, the removal effect of the solid microbial inoculum on COD and ammonia nitrogen in the folium ginkgo extract industrial wastewater is stronger than that of the liquid microbial inoculum, and the solid microbial inoculum has obvious advantages in the folium ginkgo extract industrial wastewater. In the degradation process of COD, along with the increase of time, organic pollutants in the wastewater are greatly consumed, toxic and harmful metabolites increase, the activity and metabolic activity of microorganisms are influenced, and the degradation of COD in the wastewater is slowed down. In the degradation process of ammonia nitrogen, the removal rate of ammonia nitrogen in wastewater enters a plateau phase, which is probably because carbon sources in wastewater are rapidly consumed along with the increase of time, so that denitrifying bacteria do not have sufficient carbon sources to react, the removal rate of ammonia nitrogen does not change greatly, but the removal rate of ammonia nitrogen by solid microbial inoculum is slowly increased.

TABLE 8 removal effects of solid and liquid microbial agents on COD in wastewater

TABLE 9 removal effects of solid and liquid inocula on Ammonia Nitrogen

Example 7 analysis of bacterial colony Structure in biological agent

1 method

1.1 extraction of DNA of microorganisms in the biological agent

The colony structure analysis experiment is based on a liquid microbial inoculum, so the colony structure analysis experiment result of the liquid microbial inoculum can be applied to the solid microbial inoculum. Weighing 200mg of liquid microbial inoculum under the aseptic condition, placing the liquid microbial inoculum in a 1.5mL EP tube, and extracting the total DNA of bacteria in the liquid microbial inoculum by referring to the operation steps of an Ezup column type soil genome DNA extraction kit, wherein the specific steps are as follows:

(1) adding 400 mu L of Buffer SCL into the EP tube with the weighed sample, shaking in a vortex mode for 1min, uniformly mixing, placing in a water bath at 65 ℃ for 5min, centrifuging at the rotating speed of 1200rpm after the water bath for 3min at room temperature, and transferring the supernatant into a clean EP tube with the volume of 1.5 mL.

(2) Adding Buffer SP with the same volume as the liquid in the EP tube, shaking up, placing in an ice bath at 4 ℃ for 10min, then centrifuging at 12000rpm for 3min at room temperature, and transferring the supernatant into a clean 1.5mL EP tube.

(3) Adding 200 μ L chloroform into an EP tube, shaking for 1min by vortex, mixing well, centrifuging at 12000rpm for 5min at room temperature, taking out the EP tube, and transferring the upper aqueous phase into a clean 1.5mL EP tube.

(4) Adding Buffer SB with volume 1.5 times of that of EP tube liquid, shaking in vortex for 1min, mixing, transferring to adsorption column, standing for 2min, centrifuging at 1200rpm for 1min at room temperature, and removing waste liquid from the collecting tube.

(5) Putting the adsorption column into the collection tube again, adding 700 μ L Wash Solution, centrifuging at 12000rpm for 1min at room temperature, pouring off the waste liquid in the collection tube, and eluting again. The column was returned to the collection tube and centrifuged at 12000rpm for 2min at room temperature.

(6) Taking out the adsorption column, placing the adsorption column into a 1.5mL EP tube, adding 50 μ L TE Buffer into the center of an adsorption film of the adsorption column, standing at room temperature for 5min, centrifuging at 12000rpm for 2min, detecting the concentration and purity of the obtained DNA solution by using a NanoDrop One ultramicro spectrophotometer, and placing the DNA sample meeting the requirements of subsequent experiments in a refrigerator at-20 ℃ for storage or for subsequent experiments.

1.2 PCR amplification

The experiment adopts a general primer pair for amplifying V3-V4 regions of the 16S rDNA gene of bacteria: 314F (5 '-CCTACGGGNGGCWGCAG-3') (SEQ ID No.1) and 805R (5 '-GACTACHVGGGTATCTAATCC-3') (SEQ ID No.2) amplify the extracted DNA with a gene degeneracy, wherein W ═ a or T, H ═ A, C or T, V ═ A, C or G, N ═ A, C, G or T.

The PCR amplification system and conditions were as follows:

PCR amplification conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 20s, annealing at 52 ℃ for 20s, extension at 72 ℃ for 30s, and circulation for 25 times; finally, extension is carried out for 5min at 72 ℃. After the amplification product is subjected to gel electrophoresis, as shown in FIG. 6, the DNA band is clear and bright, and the requirements of subsequent experiments can be met.

The PCR amplified DNA was purified using a SanPrep column DNA gel recovery kit and then subjected to Illumina Miseq2500 high throughput sequencing.

1.3 high throughput sequencing data analysis

In order to ensure the quality and accuracy of sequencing data, 16S rDNA amplicon sequencing data obtained by Illumina Miseq2500 sequencing needs to be subjected to quality control and pretreatment, and then downstream data analysis can be carried out. The original data obtained by Miseq2500 sequencing is a double-ended sequence, effective sequences are distinguished by tag sequences and primer sequences at two ends of the sequence, and the direction of the sequence is adjusted. The sequence quality control method comprises the following steps: firstly removing a sequencing primer adaptor sequence, splicing paired sequences into a sequence according to the relation of overlapping regions (overlap) between the paired sequences, setting a 10bp window, filtering bases with the tail quality value of less than 20 of the sequence, and finally removing the sequence containing unknown bases and short sequences. And renaming the quality-controlled effective sequence according to a sequence tag sequence (barcode) and then cutting out the barcode of the sequence and a primer sequence to obtain a pure sequence for subsequent analysis. Due to the fact that the number of sequences is excessive, the repetition degree is high, and the statistical significance and the functional significance of the sequences which appear 1 time or several times are not large, the sequence redundancy removal and the low-abundance sequence filtration are greatly convenient for downstream analysis. And carrying OUT OTU clustering on the sequence subjected to redundancy removal according to 97% similarity, removing chimeras to generate high-quality OUT representative sequences for various OUT analyses.

The analysis of the data in this chapter is completed by means of USEARCH software, a Linux operating system, QIIME software and RStudio software, and the database used by the OTU for inosculating and species annotation data is a Silva database.

2 results

2.1 structural characteristics of bacterial communities in biological agents

52791 effective sequences are obtained by the Illumina Miseq2500 sequencing data after quality control, the effective sequences are distributed among 410-440 sequences, the average length is 418bp, and the downstream analysis requirements are met. OTU clustering is carried OUT on all effective sequences according to 97% similarity, 117OUT representative sequences are obtained after chimeric and low-abundance sequences are removed, and all OUT are classified into 13 phyla, 26 classes, 39 orders, 58 families and 83 genera through Silva database annotation classification.

2.2 phyla of colony Structure analysis at the Classification level

Through sequencing data analysis, the community structure of bacteria in the liquid microbial inoculum is shown in fig. 7 under the phylum classification level, 13 bacterial phyla are found on the phylum classification level, and the sequencing according to the richness is respectively as follows: proteobacteria (Proteobacteria), Bacteroides (Bacteroides), Actinomycetes (Actinobacterium), Caldisceria, Chlorobacteria (Chlorobi), Spirochaetes (Spirochaetes), Verrucomicrobia (Verrucomicrobia), Firmicutes (Firmicutes), TM7, Phycomycetes (Plancotycetes), and the like. Wherein the proteobacteria is the phylum with the largest relative abundance, which accounts for about 72.7% of all sequences, and secondly, bacteroidetes (15.7%), actinomycetemcomica (4.0%), Caldiscerica (2.7%), Chlorobacteria (2.0%), spirochete (1.7%), etc., and the phylum with the relative abundance of more than 1% is the dominant phylum.

Community structure analysis at class 2.3 classification level

Through sequencing data analysis, the colony structure of bacteria in the liquid microbial inoculum at class classification level is shown in fig. 8, and a total of 26 bacteriaceae at class classification level are found, and the colony structure mainly comprises the following bacteriaceae: beta-Proteobacteria (Betaproteobacteria), gamma-Proteobacteria (Gamma), alpha-Proteobacteria (Alphaproteobacteria), Flavobacter (Flavobacter), Bacteroides (Bacteroides), -Proteobacteria (Epsilonproteobacteria), WCHB1-03, -Proteobacteria (Deltaproteobacteria), Ignavibacter, Acidobacterium (Acidobacterium), holophaga (Holophagae), Spirochaetes (Spirochaetes), and the like. Wherein the Proteobacteria have the largest number of beta-Proteobacteria, which account for 23.8% of the class Benzillidae, secondly the relative abundances of gamma-Proteobacteria and alpha-Proteobacteria, which are ranked second and third, respectively, in proportions of 21.8% and 18.9% of the class Bacteridae, and fourthly the relative abundances of 8.2% of Flavobacterium, Bacteroides (6.7%), and-Proteobacteria (5.8%), WC 1-03 (2.7%), -Proteobacteria (2.5%), Ignavibacter (2.0%), Acidobacterium (1.7%), holophaga (1.4%), spirochaeta (1.3%), and bacteria of which the relative abundances are greater than 1% are dominant.

2.4 colony Structure analysis at the Category Classification level

The colony structure of bacteria in the liquid microbial inoculum on the genus level is shown in figure 9, and only the genera with the relative abundance of more than 1 percent are shown in the figure, wherein unclassified indicates that no classification information can be found in a database on the genus classification level, and norank indicates that no clear classification information and classification name are available on the genus classification level. The bacteria in the liquid microbial inoculum mainly comprise the following genera: thermomonas, Desloromonas, Flavobacterium (Flavobacterium), unclassified Sphingomaadaceae, norak Pseudomonas adaceae, sphingolipid (Novosphingobium), norak Bacteroidales, Hydrophilia (Hydrogenophaga), Rubivivax, and the like. Of these, Themomonas of Proteobacteria is the most abundant genus, accounting for about 13.9% of all the genera, followed by Desloromonas of Proteobacteria, accounting for about 8.4% of all the genera.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

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Claims

1. The preparation method of the liquid microbial inoculum is characterized by comprising the following steps:

1) taking aerobic activated sludge generated by treating the ginkgo leaf extract industrial wastewater, standing, and taking supernatant to perform primary culture in a selective culture medium; taking the bacterial suspension after primary culture to culture again in a new selective culture medium, wherein the flora surviving in secondary culture is the flora in the liquid microbial inoculum;

2. The method of claim 1, wherein the selective medium comprises ginkgo biloba extract industrial wastewater.

3. The method of claim 1, further comprising one or more of the following features:

a) the culture temperature is 30-40 ℃;

b) the pH value is 6-9 in the culture process;

c) the rotating speed is 100-180 r/min in the culture process;

d) the volume of the ginkgo biloba extract industrial wastewater in the selective culture medium is 5 to 25 percent of the total volume of the selective culture medium;

e) the enrichment medium comprises peptone, beef extract, sodium chloride and water.

4. A liquid microbial inoculum obtained by the preparation method according to any one of claims 1 to 3.

5. The liquid microbial inoculum of claim 4, wherein the flora of the liquid microbial inoculum comprises the following genera: thermomonas, Desloromonas, Flavobacterium, unclassified Sphingomonadaceae, norankpseudomonas adaceae, sphingomonas, norank bacteriodes, Hydrophilia and Rubivivax.

6. A solid bacterial preparation comprising a carrier and a population of bacteria adsorbed on the carrier material, wherein the population is obtained from the liquid preparation of any one of claims 4 to 5.

7. The solid microbial inoculum according to claim 6, wherein the carrier is a solid phase carrier; the carrier is preferably one or more of wheat bran, corn flour and soybean meal.

8. A preparation method of a solid microbial inoculum is characterized by comprising the following steps of mixing a carrier and the liquid microbial inoculum according to claim 4 or 5 in a mass ratio of 1: 80-1: 120, and mixing.

9. Use of the liquid microbial agent according to any one of claims 4 to 5 or the solid microbial agent according to any one of claims 6 to 7 for treating ginkgo biloba extract industrial wastewater.

10. A method for treating ginkgo biloba extract industrial wastewater is characterized by comprising the following steps: adding the liquid microbial inoculum according to any one of claims 4 to 5 or the solid microbial inoculum according to any one of claims 6 to 7 into an aerobic tank, ginkgo biloba extract industrial wastewater or aerobic activated sludge of ginkgo biloba extract industrial wastewater.