CN117736938B - Microorganism composite flora and application thereof - Google Patents

Microorganism composite flora and application thereof Download PDF

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CN117736938B
CN117736938B CN202410173579.5A CN202410173579A CN117736938B CN 117736938 B CN117736938 B CN 117736938B CN 202410173579 A CN202410173579 A CN 202410173579A CN 117736938 B CN117736938 B CN 117736938B
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bacillus
bacterial
cereus
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bacillus cereus
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CN117736938A (en
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陈文文
李伟科
李娜娜
李昂
冯杰
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Nanjing Wanrui Environmental Technology Co ltd
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Abstract

The invention discloses a microorganism composite flora and application thereof, wherein the microorganism composite flora comprises Bacillus subtilis (Bacillus velezensis), bacillus cereus (Bacillus cereus), bacillus subtilis and Bacillus licheniformis; bacillus belicus (Bacillus velezensis) was deposited at the China general microbiological culture Collection center, accession number: CGMCC No.29354; bacillus cereus (Bacillus cereus) was deposited in the China general microbiological culture Collection center, accession number: CGMCC No.29355. The composite flora can effectively degrade avermectin medicines in aquaculture tail water, has a degradation rate of up to 96% on ivermectin, doramectin or arbutin, has a higher removal effect on phosphate, total nitrogen and COD, has lower requirements on environmental temperature and pH, and has higher salt tolerance.

Description

Microorganism composite flora and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a microorganism composite flora and application thereof.
Background
With the rapid development of fishery production in China, the aquaculture industry plays an important role in water resource utilization, economic development and the like. However, this field is also faced with an increasingly serious problem of water pollution, wherein the discharge of the tail water of cultivation is one of the important causes of deterioration of water quality. Due to accumulation of additives such as feed, medicines, vaccine culture solution and the like used in the cultivation process, metabolites and the like of the cultivated objects, the tail water pollutants of the aquaculture are various, high in concentration and difficult to degrade.
Residues of veterinary drugs in aquaculture tail water can bring potential risks and hazards to the environment and human health, and are specifically expressed as follows: (1) effect on aquatic organisms: the residue of veterinary drugs in aquaculture tail water can cause eutrophication of aquaculture water, thereby causing red tide, water bloom and other aquatic organism disasters. These biological disasters can affect the stability of the aquatic ecosystem and the maintenance of biodiversity, and destroy the living environment of aquatic organisms; (2) effect on human health: residues of veterinary drugs in the tail water of cultivation can finally influence the food safety of human beings through accumulation of food chains and biological amplification effects. For example, eating fish and shrimp contaminated with veterinary drugs may have a chronic impact on human health, such as liver damage, nervous system damage, immune system damage, etc. (3) long term impact on environment: residues of veterinary drugs in aquaculture tail water can also have a long-term negative impact on the aqueous environment. These residues may alter the chemical nature of the body of water, disrupt the physical structure of the body of water, and have a persistent impact on the structure and function of the aquatic community.
Avermectin drugs (AVMs) are the most widely used antiparasitic drugs at present, have molecular structures similar to those of bacterial protease inhibitors, and can interfere with bacterial protein synthesis to inhibit bacterial growth and reproduction. In recent years, abamectin medicines have the characteristics of high efficiency, broad spectrum and the like, and are widely applied to the fields of livestock, veterinary medicine, aquaculture and the like. In aquaculture, avermectin medicines are mainly used for preventing and treating bacterial and parasitic diseases of aquatic animals and plants such as fish, shrimp and crab, etc., such as gill rot, enteritis, gill mildew, etc. In addition, avermectin medicines can also be used for controlling harmful microorganisms in water bodies, such as protozoa, algae and the like, and preventing excessive propagation of the harmful microorganisms, thereby causing water eutrophication and water quality deterioration. However, if the avermectin is excessively applied to aquaculture, the water quality is affected, and the surrounding land environment and the production of crops are further compromised.
Therefore, the culture tail water needs to be treated and then discharged, wherein the biological method treatment has unique advantages, but the environmental adaptation of a single strain is poor and is insufficient for completely degrading water pollutants independently, so that functional strains for removing avermectin medicines in a targeted manner are urgently needed to be found out and combined into a microorganism composite flora according to a certain proportion, and the microorganism composite flora is added into a tail water treatment system, so that the treatment efficiency is fully exerted.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a microorganism composite flora and application thereof, which can effectively degrade avermectin medicines in aquaculture tail water and has higher removal effect on phosphate, total nitrogen and COD.
In order to achieve the above object, the present invention adopts the following technical scheme:
A microbial complex population comprising Bacillus subtilis (Bacillus velezensis), bacillus cereus (Bacillus cereus), bacillus subtilis, and Bacillus licheniformis; bacillus belicus (Bacillus velezensis) was deposited at the China general microbiological culture Collection center, accession number: CGMCC No.29354; bacillus cereus (Bacillus cereus) was deposited in the China general microbiological culture Collection center, accession number: CGMCC No.29355.
Preferably, the ratio of the viable count of Bacillus subtilis to Bacillus licheniformis to Bacillus belicus (Bacillus velezensis) is (1-3): (3-5): (7-9): (5-7).
Preferably, the ratio of the viable count of the aforementioned Bacillus belicus (Bacillus velezensis), bacillus cereus (Bacillus cereus), bacillus subtilis and Bacillus licheniformis is 3:9:7:3.
A microbial composite microbial agent comprises the composite flora.
The application of the complex bacterial colony or the complex bacterial agent in degrading abamectin medicines in aquaculture tail water is that the abamectin medicines are one or more of ivermectin, doramectin or arbutin.
The temperature of the aquaculture tail water is 15-30 ℃, the pH value is 6.0-9.0, and the NaCl concentration is 150-300 mg/L.
The preparation method of the microbial composite microbial agent comprises the following steps:
S1, respectively placing Bacillus belicus (Bacillus velezensis), bacillus cereus (Bacillus cereus), bacillus subtilis and Bacillus licheniformis in an LB culture medium, and performing shake culture to prepare seed liquid of each strain;
S2, inoculating seed liquid of each strain into a fermentation medium, and performing expansion culture to obtain expansion culture products of each strain;
S3, taking the expanded culture products of the strains to respectively culture in a fermentation medium to obtain bacillus belicus bacterial liquid, bacillus cereus bacterial liquid, bacillus subtilis bacterial liquid and bacillus licheniformis bacterial liquid;
S4, mixing bacillus belicus bacterial liquid, bacillus cereus, bacillus subtilis bacterial liquid and bacillus licheniformis bacterial liquid to obtain composite bacterial suspension, centrifugally collecting bacterial mud, mixing the bacterial mud with zeolite powder, and drying to obtain the solid microorganism composite bacterial agent.
Preferably, in the aforementioned step S1, the LB medium comprises the following components: 5g of yeast extract, 10g of peptone, 5g of NaCl and 1L of water.
Preferably, in the foregoing step S2 and step S3, the components of the fermentation medium are: 5.0g/L yeast powder, 10.0g/L peptone, 5.0g/L sodium chloride, 1.0g/L dipotassium hydrogen phosphate, 1.0g/L monopotassium phosphate, 0.2g/L magnesium sulfate, 0.8g/L ammonium sulfate and 0.02g/L calcium chloride.
The invention has the advantages that: in the composite flora, bacillus belicus, bacillus cereus, bacillus subtilis and bacillus licheniformis can play a good synergistic effect, can effectively degrade avermectin medicines in aquaculture tail water, has a degradation rate of up to 96% on ivermectin, doramectin or arbutin, has a high removal effect on phosphate, total nitrogen and COD, has low requirements on environmental temperature and pH, and has high salt tolerance.
Drawings
FIG. 1 is a graph showing the degradation effect of complex flora on avermectin drugs;
FIG. 2 is a graph showing the effect of complex flora on ivermectin degradation at different pH values;
FIG. 3 is a graph showing the effect of complex flora on ivermectin degradation at different temperatures;
FIG. 4 is a graph showing the effect of complex flora on ivermectin degradation at different NaCl concentrations;
FIG. 5 is a graph of the effect of complex flora on degradation at various initial ivermectin concentrations;
FIG. 6 is a graph showing the effect of the composite microbial inoculum on the treatment of actual aquaculture tail water.
Detailed Description
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1: a microbial complex population comprising Bacillus subtilis (Bacillus velezensis), bacillus cereus (Bacillus cereus), bacillus subtilis, and Bacillus licheniformis; bacillus belicus (Bacillus velezensis) was deposited at the China general microbiological culture Collection center, 12 months and 18 days of 2023, with the deposit unit address: the accession number of the Gao Yuan 1, gao 3, north Star, beijing city is: CGMCC No.29354; bacillus cereus (Bacillus cereus) was deposited in the general microbiological center of the chinese microbiological bacterial culture collection center at 12/18 of 2023 with the deposit unit address: the accession number of the Gao Yuan 1, gao 3, north Star, beijing city is: CGMCC No.29355. Bacillus subtilis and Bacillus licheniformis are known strains and are commercially available.
Example 2: proportioning determination of composite flora
The ivermectin in the culture tail water is measured by adopting a high performance liquid chromatography-tandem mass spectrometry combined technology, the indexes of phosphate, ammonia nitrogen, total nitrogen and total phosphorus are measured by using a water quality analyzer, the COD is measured by using an alkaline potassium permanganate method, the phosphate is measured by adopting a phosphomolybdenum blue spectrophotometry, the ammonia nitrogen content is measured by adopting a hypobromite oxidation method, and the total nitrogen and the total phosphorus are measured by adopting a potassium persulfate oxidation method.
Inoculating each strain into 100mL LB culture medium (LB culture medium comprises 5g of yeast extract, 10g of peptone, 5g of NaCl and 1L of water), shake culturing for 24h in a 180 rpm shaking table at 30 ℃ to obtain seed liquid, putting each seed liquid into a centrifuge, centrifuging at 8000rpm for 3min, washing with PBS buffer solution for three times, diluting the OD600 value to 0.6, setting the inoculum size to 1%, 2%, 3%, 4% and 5% of five gradients, setting three parallel water samples without bacterial liquid as blank control, sampling and measuring the index of ivermectin, phosphate, ammonia nitrogen, total phosphorus and COD in each sample, and comparing to obtain the optimal removal effect of the water index under different dosage. Three different inoculum levels were designed based on the optimal results for each single inoculum, as shown in table 1, and then four-factor three-level orthogonal tests were designed based on level table 1, as shown in table 2.
TABLE 1 orthogonal design factors, level Meter
TABLE 2 orthogonal test analysis table
Bacterial suspensions were prepared according to tables 1 and 2, and added into 200mL of culture tail water in proportion, and the degradation effects of ivermectin, phosphate, ammonia nitrogen, total phosphorus and COD were measured in different proportions by taking a water sample without bacterial liquid as a blank control, as shown in table 3.
TABLE 3 degradation effects of contaminants in aquaculture tail water
As can be seen from Table 3, the experiment group 9 has the best effect of removing ivermectin, total nitrogen and COD, and the other indexes have small differences, so the optimal proportion of the compound bacteria is the experiment group 9, namely, the proportion of Bacillus belicus, bacillus cereus, bacillus subtilis and Bacillus licheniformis is 3:3:9:7.
Example 3: the preparation method of the composite microbial inoculum comprises the following specific steps:
S1, activating and expanding culture of each strain: placing each strain in 100 mL LB culture medium (comprising 5g of yeast extract, 10g of peptone, 5g of NaCl and 1L of water) at 30deg.C, shake culturing in 180 rpm shaker for 24 hr to obtain seed solution; the seed solution was inoculated into a fermenter of a fermentation medium (the composition of the fermentation medium comprises 5.0g/L of yeast powder, 10.0g/L of peptone, 5.0g/L of sodium chloride, 1.0g/L of dipotassium hydrogen phosphate, 1.0g/L of potassium dihydrogen phosphate, 0.2g/L of magnesium sulfate, 0.8g/L of ammonium sulfate and 0.02g/L of calcium chloride) at a volume ratio of 1% to conduct an expansion culture.
S2, fermenting each strain: the amplified culture products of the strains are respectively cultured in a fermentation medium (the components of the fermentation medium are yeast powder 5.0g/L, peptone 10.0g/L, sodium chloride 5.0g/L, dipotassium hydrogen phosphate 1.0g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.2g/L, ammonium sulfate 0.8g/L and calcium chloride 0.02 g/L) at 30 ℃ and pH value of 6-8, and the viable count of the obtained strains is 6 multiplied by 10 12~7×1012 CFU/mL.
S7, preparing a composite microbial agent: mixing 30g of bacillus belicus bacterial liquid, 30g of bacillus cereus bacterial liquid, 90g of bacillus subtilis bacterial liquid and 70g of bacillus licheniformis bacterial liquid to obtain a composite bacterial suspension, and centrifugally collecting bacterial mud at 4000 rpm; mixing the bacterial mud with zeolite powder according to the proportion of 1:3 volumes of the materials are mixed and dried at a low temperature of 35 ℃ to obtain the solid microbial inoculum.
Example 4: degradation effect of composite flora on avermectin medicines
Inoculating the complex flora into a culture medium containing ivermectin, doramectin or arbutin according to an inoculum size of 1% (v/v), wherein the concentration of each pollutant in the culture medium is 200mg/L, and the culture medium comprises the following components: the yeast extract (5 g), peptone (10 g), naCl (5 g) and water (1L) were incubated at 25℃and 200rpm for 2 days, and the concentration of ivermectin, doramectin or arbutin in the treated medium was measured to calculate the degradation rate, and the results are shown in FIG. 1.
As can be seen from FIG. 1, the degradation rate of the strain complex flora on ivermectin, doramectin and arbutin can reach more than 90% in 2d, and the degradation effect is excellent.
Example 5: degradation effect of complex flora on ivermectin at different pH
The pH of the ivermectin culture medium is respectively adjusted to 5, 5.5, 6,7, 8, 9, 9.5 and 10, and the culture medium comprises the following components: yeast extract 5g, peptone 10g, naCl 5g and water 1L, ivermectin was inoculated into the culture medium in an inoculum size of 1% (v/v), the initial concentration of ivermectin was 200mg/L, after 2d of culture at 25℃and at 200rpm, the concentration of ivermectin in the treated culture medium was measured, and the degradation rate was calculated, and the results are shown in FIG. 2.
As shown in FIG. 2, the complex bacterial colony can adapt to a wider pH range, the optimal pH value of the treatment effect is between 6.0 and 9.0, and the degradation rate of ivermectin can reach more than 80 percent.
Example 6: degradation effect of complex flora on ivermectin at different temperatures
The temperature of the ivermectin culture medium is respectively adjusted to 5, 10, 15, 20, 25, 30, 35 and 40, and the culture medium comprises the following components: yeast extract 5g, peptone 10g, naCl 5g and water 1L, pH 7.0, ivermectin was inoculated into the medium in an inoculum size of 1% (v/v), the initial concentration of ivermectin was 200mg/L, after 2d cultivation at 200rpm, the concentration of ivermectin in the medium after the treatment was detected, and the degradation rate was calculated, and the results are shown in FIG. 3.
As shown in FIG. 3, the complex bacterial colony has good adaptability to temperature, and can effectively degrade ivermectin at 15-30 ℃, the degradation rate can reach more than 80%, and the degradation rate of ivermectin at 35-40 ℃ is 60-75%, which indicates that the complex bacterial colony can still degrade avermectin medicines at a higher temperature.
Example 7: degradation effect of complex flora on ivermectin under different NaCl concentrations
NaCl is added into the ivermectin culture medium respectively, so that the NaCl concentration in the culture medium is 0, 1%, 1.5%, 2%, 3%, 4%, 5%, 5.5% and 6%, and the complex flora is inoculated into the culture medium according to the inoculum size of 1% (v/v), wherein the culture medium comprises the following components: the initial concentration of ivermectin was 200mg/L, pH 7.0, and after 2d incubation at 25℃and 200rpm, the concentration of ivermectin in the treated medium was measured and the degradation rate was calculated, and the results are shown in FIG. 4.
As shown in FIG. 4, the complex bacterial group has better salt tolerance, and the degradation rate of ivermectin is 60-75% at the salinity of 2-5%, and compared with the complex bacterial group with the salinity of 1% at the low salinity, the complex bacterial group can still effectively degrade ivermectin at the salinity of 6%.
Example 8: degradation effect of complex flora on ivermectin at different initial concentrations
Under the conditions that the initial concentration of ivermectin is 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, 300 mg/L, 350 mg/L, 400 mg/L and 450 mg/L respectively, the compound flora is inoculated into culture mediums containing different initial concentrations of ivermectin according to the inoculation amount of 1% (v/v), and the culture medium comprises the following components: the degradation effect was measured after 2d incubation at 25℃and 200rpm with 5g of yeast extract, 10g of peptone, 5g of NaCl and 1L of water at pH7.0, as shown in FIG. 5.
As shown in FIG. 5, when the initial concentration of ivermectin is 150-300 mg/L, the degradation rate is more than 90%, and the degradation effect is excellent.
Example 9: treatment effect of composite microbial inoculum on actual aquaculture tail water
Adding cool boiled water, brown sugar and a compound microbial inoculum into a container, uniformly stirring, maintaining the temperature at 30 ℃ for culture, continuously stirring for two days, pouring the mixture into 10m of aquaculture tail water according to the inoculum size of 5%, controlling the hydraulic retention time to be 8-16 h, controlling the concentration of dissolved oxygen to be greater than 4 mg/L in the aeration process, standing the sediment, and taking supernatant water to analyze the water purification effect, wherein the result is shown in figure 6.
As shown in FIG. 6, the degradation rate of the compound microbial inoculum on ivermectin, doramectin and arbutin in the tail water of aquaculture is over 92 percent, and phosphate, total nitrogen and COD in the tail water can be effectively removed.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the invention in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the invention.

Claims (8)

1. A microbial composite bacterium is characterized by comprising Bacillus beliae ((Bacillus velezensis), bacillus cereus, bacillus subtilis and Bacillus licheniformis, wherein Bacillus beliae (Bacillus velezensis) is preserved in China general microbiological culture collection center of China general microbiological culture Collection center for 12 months of 2023, the preservation number is CGMCC No.29354, bacillus cereus is preserved in China general microbiological culture collection center of 2023 for 12 months, the preservation number is CGMCC No.29355, and the ratio of the viable count of Bacillus beliae (Bacillus velezensis), bacillus cereus (Bacillus cereus), bacillus subtilis and Bacillus licheniformis is (1-3): (3-5): (7-9): (5-7).
2. The microorganism complex of claim 1, wherein the ratio of viable count of Bacillus belicus (Bacillus velezensis), bacillus cereus (Bacillus cereus), bacillus subtilis and Bacillus licheniformis is 3:3:9:7.
3. A microbial composite bacterial agent, which is characterized by comprising the microbial composite bacterial agent as claimed in claim 1.
4. The use of the microbial complex bacteria of claim 1 or the microbial complex bacteria of claim 3 for degrading avermectin in aquaculture tail water, wherein the avermectin is one or more of ivermectin, doramectin or arbutin.
5. The use according to claim 4, wherein the temperature of the aquaculture tail water is 15-30 ℃, the pH is 6.0-9.0, and the NaCl concentration is 150-300 mg/L.
6. The method for preparing the microbial composite agent according to claim 3, which is characterized by comprising the following steps:
S1, respectively placing Bacillus belicus (Bacillus velezensis), bacillus cereus (Bacillus cereus), bacillus subtilis and Bacillus licheniformis in an LB culture medium, and performing shake culture to prepare seed liquid of each strain;
S2, inoculating seed liquid of each strain into a fermentation medium, and performing expansion culture to obtain expansion culture products of each strain;
S3, taking the expanded culture products of the strains to respectively culture in a fermentation medium to obtain bacillus belicus bacterial liquid, bacillus cereus bacterial liquid, bacillus subtilis bacterial liquid and bacillus licheniformis bacterial liquid;
S4, mixing bacillus belicus bacterial liquid, bacillus cereus, bacillus subtilis bacterial liquid and bacillus licheniformis bacterial liquid to obtain composite bacterial suspension, centrifugally collecting bacterial mud, mixing the bacterial mud with zeolite powder, and drying to obtain the solid microorganism composite bacterial agent.
7. The method for preparing a microbial composite agent according to claim 6, wherein in the step S1, the LB medium comprises the following components: 5g of yeast extract, 10g of peptone, 5g of NaCl and 1L of water.
8. The method for preparing a microbial composite agent according to claim 6, wherein in the step S2 and the step S3, the components of the fermentation medium are: 5.0g/L yeast powder, 10.0g/L peptone, 5.0g/L sodium chloride, 1.0g/L dipotassium hydrogen phosphate, 1.0g/L monopotassium phosphate, 0.2g/L magnesium sulfate, 0.8g/L ammonium sulfate and 0.02g/L calcium chloride.
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