Microbial stimulant for chlorinated compound contaminated remediation and use thereof

Abstract

The application discloses a biological stimulant for chlorinated compound pollution remediation and application thereof, and the biological stimulant comprises polymeric ferric sulfate, and / or fulvic acid and Lactobacillus plantarum fermentation supernatant. The polymeric ferric sulfate is first selected as an iron source for the bacterial body, the fulvic acid provides carbon and nitrogen sources, and the Lactobacillus plantarum supernatant is used in cooperation, so that the degradation of the chlorinated compound by the microorganism, especially Acinetobacter, is promoted, the degradation efficiency of the chlorinated compound by the microorganism is improved, the fermentation waste liquid is reasonably utilized to achieve the purpose of waste treatment by waste, and the results show that the degradation rate of chlorophenol with an initial concentration of 20 mg / L is increased by 94% within 24 hours; the degradation efficiency of chlorobenzene with an initial concentration of 20 mg / L is increased by 79% within 45 days, and the biological aid for the biological remediation strategy of the chlorinated organic matter in the water environment is enriched.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental remediation, and in particular relates to a microbial stimulant for remediation of chlorine-containing compound pollution and application thereof. Background Art

[0002] Chlorinated organic compounds are organic solvents and chemical raw materials commonly used in industrial synthesis, and are widely used in multiple fields such as decontamination and textiles. Chlorinated organic compounds are often accompanied by odor and foul smell, and are extremely harmful to human health, causing acute and chronic poisoning of different organs. With improper discharge, chlorine-containing compounds with stable chemical properties continue to accumulate in the environment. Currently, the main remediation methods for chlorine-containing compound pollution include high-temperature incineration, physical methods, and chemical redox methods. However, these technologies have problems such as the production of toxic by-products, incomplete degradation, and high costs. Their economic and environmental benefits do not conform to the advocacy of "green production."

[0003] Microbial remediation technology primarily utilizes the metabolic activity of microorganisms, using pollutants as an energy source to convert them into low- or non-toxic substances. Microorganisms degrade the target pollutants by utilizing them as a carbon and energy source. Compared to other remediation technologies, microbial remediation is simple to operate, low-cost, and environmentally friendly. However, the low availability of organisms and the low activity of both indigenous and introduced pollution-degrading bacteria have become bottlenecks limiting its application. Summary of the Invention

[0004] The first aspect of the present invention aims to provide a composition for remediation of chlorine-containing compound pollution.

[0005] The second aspect of the present invention aims to provide applications of the above composition.

[0006] A third aspect of the present invention aims to provide a product.

[0007] A fourth aspect of the present invention aims to provide a method for degrading chlorine-containing compounds.

[0008] The technical solution adopted by the present invention is:

[0009] A first aspect of the present invention provides a composition comprising agent A and agent B; agent A comprises polyferric sulfate and / or fulvic acid, and agent B comprises Lactobacillus plantarum fermentation supernatant.

[0010] In some embodiments of the present invention, the concentration of the polyferric sulfate is 0.160-0.635 g / L.

[0011] In some preferred embodiments of the present invention, the concentration of the polyferric sulfate is 0.32-0.635 g / L.

[0012] In some embodiments of the present invention, the concentration of fulvic acid is 0.125-0.5 g / L.

[0013] In some preferred embodiments of the present invention, the concentration of fulvic acid is 0.25-0.5 g / L

[0014] In some embodiments of the present invention, the ratio of agent A:agent B is 4:1-4 (V / V).

[0015] In some preferred embodiments of the present invention, the ratio of agent A:agent B is 4:2-4 (V / V).

[0016] In some embodiments of the present invention, the Lactobacillus plantarum can be commercially available strains or can be screened from the environment. The implementation of the present invention is not limited by the source of the strains.

[0017] In some embodiments of the present invention, the Lactobacillus plantarum supernatant is a supernatant obtained by solid-liquid separation of Lactobacillus plantarum anaerobic fermentation at 35-39° C. for 6-8 h.

[0018] In some embodiments of the present invention, the culture medium is cultured until the number of viable cells is 1×10 9 / mL~1×10 11 / mL when solid-liquid separation occurs.

[0019] In some embodiments of the present invention, the solid-liquid separation method includes centrifugation.

[0020] In some preferred embodiments of the present invention, the solid-liquid separation method comprises: centrifuging the bacterial suspension after fermentation at 0-6°C and 6000-10000 rpm for 7-13 min to separate the bacterial cells and the supernatant.

[0021] In some embodiments of the present invention, the method for preparing the Lactobacillus plantarum supernatant specifically comprises: L. plants Inoculate the first-level culture medium at a rate of 0.2-2%, and incubate at 35-39°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 15-25% into the second-level culture medium, and incubate anaerobically at 35-39°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 9 / mL~1×10 11 / mL, and take the fermentation supernatant waste liquid.

[0022] The implementation of the present invention is not restricted by the species of Lactobacillus plantarum.

[0023] The second aspect of the present invention provides the use of the composition described in the first aspect of the present invention in improving the ability of microorganisms to degrade chlorine-containing compounds and / or preparing products that improve the ability of microorganisms to degrade chlorine-containing compounds.

[0024] In some embodiments of the invention, the microorganism comprises Acinetobacter.

[0025] In some embodiments of the present invention, the Acinetobacter can be a commercially available strain or can be screened from the environment. The implementation of the present invention is not limited by the source of the strain.

[0026] In some embodiments of the present invention, the Acinetobacter is Acinetobacter seifertii .

[0027] In some embodiments of the present invention, the sequence number of the Acinetobacter is shown as OP364051.1.

[0028] In some preferred embodiments of the present invention, the Acinetobacter was deposited in Guangdong Provincial Microbial Culture Collection Center on November 30, 2022, and was classified and named Acinetobacter seifertii , the deposit number is GDMCC No: 62994, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0029] In some embodiments of the present invention, the chlorine-containing compound includes chlorobenzene and chlorophenol compounds.

[0030] In some preferred embodiments of the present invention, the chlorophenol compound comprises o-chlorophenol.

[0031] The third aspect of the present invention provides a product, characterized in that the product contains the composition described in the first aspect of the present invention.

[0032] In some embodiments of the present invention, the product further comprises the microbial liquid described in the second aspect of the present invention.

[0033] In some embodiments of the invention, the microorganism comprises Acinetobacter.

[0034] In some embodiments of the present invention, the method for preparing the microbial liquid comprises: inoculating microorganisms into seed liquid and culturing at 27-33° C. and 120-180 rpm on a shaking table for 22-24 hours to obtain the microbial liquid.

[0035] In some embodiments of the present invention, the OD600 of the microbial culture liquid is 1.5-2.5.

[0036] In some preferred embodiments of the present invention, the OD600 of the microbial culture liquid is 1.5-2.0.

[0037] In some more preferred embodiments of the present invention, the OD of the microbial culture is 600 It is 1.8-2.0.

[0038] In some embodiments of the present invention, the Acinetobacter can be a commercially available strain or can be screened from the environment. The implementation of the present invention is not limited by the source of the strain.

[0039] In some embodiments of the present invention, the sequence number of the Acinetobacter is shown as OP364051.

[0040] In some embodiments of the present invention, the preparation method of the Acinetobacter comprises: aerating and culturing sludge from an organochlorine wastewater treatment plant, taking the supernatant after 20 to 28 hours and enriching and culturing it, acclimating the enriched cultured bacterial liquid multiple times in a culture medium containing chlorine-containing compounds, separating and purifying the acclimated bacterial liquid to obtain a single bacterium, and preserving it on a slant.

[0041] In some embodiments of the present invention, the nutrient solution used in aeration culture contains 3-7 g / L glucose, 2-4 g / L beef extract, 2.5-4.5 g / L sodium dihydrogen phosphate, 1-3 g / L dipotassium hydrogen phosphate, 0.1-0.3 g / L magnesium sulfate, 0.01-0.03 g / L manganese sulfate, and 0.005-0.015 g / L ferric chloride; the pH of the culture solution is 6.8-7.8.

[0042] In some embodiments of the present invention, the aeration culture time is 40-56 h.

[0043] In some preferred embodiments of the present invention, the aeration culture time is 44-52 h.

[0044] In some more preferred embodiments of the present invention, the aeration culture time is 46-50 h.

[0045] In some embodiments of the present invention, the slant solid culture medium for bacterial cell preservation contains 8-12 g / L tryptone, 8-12 g / L NaCl, 4-6 g / L yeast extract, and 16-24 g / L agar powder.

[0046] In some preferred embodiments of the present invention, the culture medium used in the enrichment culture contains 10 g / L tryptone, 10 g / L NaCl, and 5 g / L yeast extract.

[0047] In some embodiments of the present invention, the enrichment culture time is 20-28 h.

[0048] In some preferred embodiments of the present invention, the enrichment culture time is 22-26 h.

[0049] In some preferred embodiments of the present invention, the enrichment culture time is 23-25 ​​h.

[0050] In some embodiments of the present invention, the product may further include an auxiliary agent, and the auxiliary agent includes at least one of a stabilizer, a wetting agent, an emulsifier, an adhesive, and an isotonic agent.

[0051] In some embodiments of the present invention, the product may be in the form of at least one of tablets, granules, powders, capsules, solutions, suspensions, and freeze-dried preparations.

[0052] The fourth aspect of the present invention provides a method for degrading chlorine-containing compounds, characterized in that the product and / or microbial liquid described in the third aspect of the present invention is inoculated into an environment containing chlorine-containing compounds for degradation.

[0053] In some embodiments of the present invention, when the product does not contain microbial liquid, it needs to be added separately.

[0054] In some embodiments of the present invention, the OD600 of the microbial culture liquid is 1.5-2.5.

[0055] In some preferred embodiments of the present invention, the OD600 of the microbial culture liquid is 1.5-2.0.

[0056] In some more preferred embodiments of the present invention, the OD of the microbial culture is 600 It is 1.8-2.0.

[0057] In some embodiments of the present invention, the inoculation amount of the microbial liquid is 1% to 20%.

[0058] In some preferred embodiments of the present invention, the inoculation amount of the microbial liquid is 2% to 10%.

[0059] In some more preferred embodiments of the present invention, the inoculation amount of the microbial liquid is 4% to 7%.

[0060] In some embodiments of the present invention, the volume ratio of agent A, agent B and microbial liquid is 4:1~4.

[0061] In some embodiments of the present invention, the volume ratio of the composition to the microbial liquid is 1.25-2:0.25-5.

[0062] In some preferred embodiments of the present invention, the volume ratio of the composition to the microbial liquid is 1.25-2:0.5-2.5.

[0063] In some preferred embodiments of the present invention, the volume ratio of the composition to the microbial liquid is 1.25-2:1.25-2.5.

[0064] In some preferred embodiments of the present invention, the volume ratio of the composition to the microbial solution is 1.25 to 2:2.5.

[0065] In some embodiments of the present invention, the concentration of the chlorinated compound is 1-100 mg / L.

[0066] In some preferred embodiments of the present invention, the concentration of the chlorinated compound is 5-50 mg / L.

[0067] In some more preferred embodiments of the present invention, the concentration of the chlorinated compound is 10-30 mg / L.

[0068] In some embodiments of the present invention, the environment contains inorganic salts required for the growth of microorganisms.

[0069] In some embodiments of the present invention, the inorganic salt includes at least one of potassium salt, sodium salt, ammonium salt, and magnesium salt.

[0070] In some preferred embodiments of the present invention, the inorganic salt comprises at least one of 0.01-0.013 mol / L potassium salt, 0.02-0.04 mol / L sodium salt, 0.005-0.008 mol / L ammonium salt, and 0.0003-0.0005 mol / L magnesium salt.

[0071] In some preferred embodiments of the present invention, the inorganic salt further comprises at least one of 1.50 g / L potassium dihydrogen phosphate, 4.19 g / L disodium hydrogen phosphate, 0.83 g / L ammonium sulfate, and 0.05 g / L magnesium sulfate.

[0072] The composite composition of the present invention and the inorganic salts in the environment have a promoting effect on the growth, metabolism and degradation function of microorganisms, and can effectively improve the removal of chlorine-containing compounds in the water environment.

[0073] In some embodiments of the present invention, the degradation temperature is 20-40°C.

[0074] In some preferred embodiments of the present invention, the degradation temperature is 25-35°C.

[0075] In some more preferred embodiments of the present invention, the degradation temperature is 30°C.

[0076] In some embodiments of the present invention, the degradation time is 8-48 h.

[0077] In some preferred embodiments of the present invention, the degradation time is 10-36 h.

[0078] In some more preferred embodiments of the present invention, the degradation time is 24-36 h.

[0079] In some embodiments of the present invention, the degradation rotation speed is 50 to 250 rpm.

[0080] In some embodiments of the present invention, the degradation rotation speed is 100-200 rpm.

[0081] In some embodiments of the present invention, the degradation rotation speed is 140-160 rpm.

[0082] In some embodiments of the present invention, the pH of the environment containing chlorine compounds is 6.5-8.

[0083] In some preferred embodiments of the present invention, the pH of the environment containing chlorine compounds is 7-8.

[0084] In some embodiments of the present invention, the environment comprises an aquatic environment and / or soil.

[0085] The beneficial effects of the present invention are:

[0086] The present invention is the first to use polyferric sulfate to provide an iron source for the bacteria and fulvic acid to provide a carbon and nitrogen source. When used in combination with the supernatant of Lactobacillus plantarum, it can promote the degradation of chlorine-containing compounds by microorganisms, especially Acinetobacter, and improve the degradation efficiency of chlorine-containing compounds by microorganisms. At the same time, the fermentation waste liquid is rationally utilized to achieve the purpose of treating waste with waste. The results showed that within 24 hours, the degradation rate of chlorophenol with an initial concentration of 20 mg / L was increased to 94% at most; the degradation efficiency of chlorobenzene with an initial concentration of 20 mg / L was increased to 79% after 45 days, enriching the biological additives used in the bioremediation strategy of chlorine-containing organic matter in water environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 for Acinetobacter Kinetic fitting curve of the degradation of o-chlorophenol by sp. strain ZY1. DETAILED DESCRIPTION

[0088] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0089] In some embodiments of the present application, the polymeric ferric sulfate is purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., CAS No. 35139-28-7, with a Fe content of 21%, the fulvic acid is purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., and the Lactobacillus plantarum supernatant is from Guangzhou Microbial Research Company.

[0090] The separation and preparation method of the bacterial solution for degrading o-chlorophenol in the following examples and comparative examples is as follows:

[0091] A mixed solution was obtained by adding a nutrient solution to sludge taken from a sewage treatment plant of Lunan Pharmaceutical in Shandong, China, and o-chlorophenol was added to the mixed solution to make its content 20 mg / L, aeration was carried out for 48 h, after standing and layering, the supernatant was taken and inoculated into a seed solution, and after enrichment culture for 24 h, an initial bacterial solution was obtained. The initial bacterial solution was diluted 10 6 times, 100 μL of the bacterial solution after dilution was used for plate coating, and culture was carried out at 30℃ for 24 h. Single colonies grown on the plate were picked and plate streaking was carried out, and culture was carried out at 30℃ for 24 h to complete the purification of the strain. After strain identification, the strain was named Acinetobacter , and named Acinetobacter sp. strain ZY1 (NCBI Accession No: OP364051.1); its sequence has 99% similarity with the sequence of Acinetobacter seifertii, and it is identified as Acinetobacter seifertii. The bacterial strain was preserved in Guangdong Microbial Culture Collection Center on November 30, 2022, and was named Acinetobacter seifertii , with a preservation number of GDMCC No: 62994 and a preservation address of No. 59 Building, 5th Floor, 100 Middle Xianlie Road, Guangzhou, Guangdong Province.

[0092] The formula of the nutrient solution is: 5 g / L glucose, 3 g / L beef extract, 3.5 g / L sodium dihydrogen phosphate, 1.8 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.02 g / L manganese sulfate, 0.01 g / L ferric chloride, pH=7.2; the formula of the seed solution is: 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract.

[0093] The formula of the solid culture medium is: 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, 20 g / L agar powder.

[0094] Example 1

[0095] (1) A agent: 0.16 g of polymeric ferric sulfate was weighed on a balance and dissolved in 1000 mL of water to obtain A agent;

[0096] (2) B agent: 0.2 g of Lactobacillus plantarum was weighed on a balance and dissolved in 1000 mL of water to obtain B agent. L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0097] (3) The dosage of compound biostimulant A and B is 4% and 1% (V / V) respectively;

[0098] (4) Prepare the seed solution with the following formula: 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, and sterilize at 121°C for 20 min;

[0099] (5) Prepare an inorganic salt culture medium with the following formula: 1.50 g / L potassium dihydrogen phosphate, 4.19 g / L sodium dihydrogen phosphate, 0.83 g / L ammonium sulfate, 0.05 g / L magnesium sulfate, pH = 7.2 ± 0.1, sterilize at 121 °C for 20 min, and add o-chlorophenol sterilized by filtration with a 0.22 μm filter membrane to make the o-chlorophenol concentration in the culture medium 20 mg / L;

[0100] (6) Store in a 4℃ refrigerator with a slant surface A . strain ZY1 was inoculated with the seed solution in (4) and cultured in a shaking incubator at 30°C and 150 rpm for 22-24 hours to obtain liquid strains. OD 600 1.8-2.0;

[0101] (7) The liquid culture in (6) was inoculated into the inorganic salt culture medium prepared in (5) at a 10% (V / V) inoculation rate, and the compound biostimulant was added according to the addition ratio in (3). The culture system was cultured at 30°C and 150 rpm for 24 h;

[0102] (8) Detection of o-chlorophenol removal efficiency by high performance liquid chromatography: 1 mL of the sample after degradation for 0 h and 24 h was taken, centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was diluted 20 times with acetonitrile. After filtering through a 0.22 μm organic filter membrane, 1 mL was taken and placed in a brown injection vial. A 4.5 mm × 250 mm C18 column was used, the column temperature was 30°C, the mobile phase was 60%:40% acetonitrile: ultrapure water, the flow rate was 1 mL / min, and the UV detector was used for detection at a wavelength of 280 nm. The o-chlorophenol concentration after degradation for 0 h (undegraded) was subtracted from the concentration after degradation for 24 h, and the difference was divided by the o-chlorophenol concentration after degradation for 0 h (undegraded) to obtain the o-chlorophenol removal rate.

[0103] Example 2

[0104] (1) Agent A: Weigh 0.16 g of polyferric sulfate on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0105] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0106] (3) The dosage of compound biostimulant A and B is 4% and 2% (V / V) respectively;

[0107] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0108] Example 3

[0109] (1) Agent A: Weigh 0.16 g of polyferric sulfate on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0110] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0111] (3) The dosage of compound biostimulant A and B is 4% and 4% (V / V) respectively;

[0112] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0113] Example 4

[0114] (1) Agent A: Weigh 0.32 g of polyferric sulfate on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0115] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0116] (3) The dosage of compound biostimulant A and B is 4% and 1% (V / V) respectively;

[0117] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0118] Example 5

[0119] (1) Agent A: Weigh 0.32 g of polyferric sulfate on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0120] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×1010 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0121] (3) The dosage of compound biostimulant A and B is 4% and 2% (V / V) respectively;

[0122] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0123] Example 6

[0124] (1) Agent A: Weigh 0.32 g of polyferric sulfate on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0125] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0126] (3) The dosage of compound biostimulant A and B is 4% and 4% (V / V) respectively;

[0127] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0128] Example 7

[0129] (1) Agent A: Weigh 0.635 g of polyferric sulfate on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0130] (2) Agent B: Glycerol-preserved L. plantarumInoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0131] (3) The dosage of compound biostimulant A and B is 4% and 1% (V / V) respectively;

[0132] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0133] Example 8

[0134] (1) Agent A: Weigh 0.635 g of polyferric sulfate on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0135] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0136] (3) The dosage of compound biostimulant A and B is 4% and 2% (V / V) respectively;

[0137] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0138] Example 9

[0139] (1) Agent A: Weigh 0.635 g of polyferric sulfate on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0140] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0141] (3) The dosage of compound biostimulant A and B is 4% and 4% (V / V) respectively;

[0142] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0143] Example 10

[0144] (1) Agent A: Weigh 0.125 g of fulvic acid on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0145] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0146] (3) The dosage of compound biostimulant A and B is 4% and 1% (V / V) respectively;

[0147] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0148] Example 11

[0149] (1) Agent A: Weigh 0.125 g of fulvic acid on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0150] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0151] (3) The dosage of compound biostimulant A and B is 4% and 2% (V / V) respectively;

[0152] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0153] Example 12

[0154] (1) Agent A: Weigh 0.125 g of fulvic acid on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0155] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0156] (3) The dosage of compound biostimulant A and B is 4% and 4% (V / V) respectively;

[0157] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0158] Example 13

[0159] (1) Agent A: Weigh 0.25 g of fulvic acid on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0160] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0161] (3) The dosage of compound biostimulant A and B is 4% and 1% (V / V) respectively;

[0162] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0163] Example 14

[0164] (1) Agent A: Weigh 0.25 g of fulvic acid on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0165] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0166] (3) The dosage of compound biostimulant A and B is 4% and 2% (V / V) respectively;

[0167] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0168] Example 15

[0169] (1) Agent A: Weigh 0.25 g of fulvic acid on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0170] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0171] (3) The dosage of compound biostimulant A and B is 4% and 4% (V / V) respectively;

[0172] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0173] Example 16

[0174] (1) Agent A: Weigh 0.5 g of fulvic acid on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0175] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0176] (3) The dosage of compound biostimulant A and B is 4% and 1% (V / V) respectively;

[0177] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0178] Example 17

[0179] (1) Agent A: Weigh 0.5 g of fulvic acid on a balance and dissolve it in 1000 mL of water to obtain Agent A;

[0180] (2) B agent: glycerol-preserved L. plantarum According to the inoculation amount of 1%, it is added into the first medium, and it is cultured at 37°C for 20-24 h. The prepared first seed liquid is added into the second medium according to the inoculation amount of 20%, and it is anaerobically cultured at 37°C for 6-8 h, until the viable cell number in the medium is 1×10 10 / mL. The fermentation supernatant waste liquid is taken as the B agent. The first medium and the second medium have the following formula: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate, pH=6-7, sterilized at 121°C for 20 min;

[0181] (3) The adding amount of the compounded biological stimulant A agent and B agent is 4% and 2% (V / V) respectively;

[0182] The subsequent steps are carried out according to (4)-(8) in Example 1.

[0183] Example 18

[0184] (1) A agent: 0.5 g of fulvic acid is weighed on a balance and dissolved in 1000 mL of water to obtain the A agent;

[0185] (2) B agent: glycerol-preserved L. plantarum According to the inoculation amount of 1%, it is added into the first medium, and it is cultured at 37°C for 20-24 h. The prepared first seed liquid is added into the second medium according to the inoculation amount of 20%, and it is anaerobically cultured at 37°C for 6-8 h, until the viable cell number in the medium is 1×10 10 / mL. The fermentation supernatant waste liquid is taken as the B agent. The first medium and the second medium have the following formula: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate, pH=6-7, sterilized at 121°C for 20 min;

[0186] (3) The adding amount of the compounded biological stimulant A agent and B agent is 4% and 4% (V / V) respectively;

[0187] The subsequent steps are carried out according to (4)-(8) in Example 1.

[0188] Example 19

[0189] (1) A agent: 0.16 g of polyferric sulfate and 0.125 g of fulvic acid are weighed on a balance and dissolved in 1000 mL of water to obtain the A agent;

[0190] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0191] (3) The dosage of compound biostimulant A and B is 4% and 1% (V / V) respectively;

[0192] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0193] Example 20

[0194] (1) Agent A: Weigh 0.16 g of polyferric sulfate and 0.25 g of fulvic acid on a balance and dissolve them in 1000 mL of water to obtain Agent A;

[0195] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0196] (3) The dosage of compound biostimulant A and B is 4% and 2% (V / V) respectively;

[0197] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0198] Example 21

[0199] (1) Agent A: Weigh 0.16 g of polyferric sulfate and 0.5 g of fulvic acid on a balance and dissolve them in 1000 mL of water to obtain Agent A;

[0200] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0201] (3) The dosage of compound biostimulant A and B is 4% and 4% (V / V) respectively;

[0202] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0203] Example 22

[0204] (1) Agent A: Weigh 0.326 g of polyferric sulfate and 0.125 g of fulvic acid on a balance and dissolve them in 1000 mL of water to obtain Agent A;

[0205] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0206] (3) Prepare seed solution with the following formula: 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, and sterilize at 121°C for 20 min;

[0207] (3) The dosage of compound biostimulant A and B is 4% and 4% (V / V) respectively;

[0208] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0209] Example 23

[0210] (1) Agent A: Weigh 0.32 g of polyferric sulfate and 0.25 g of fulvic acid on a balance and dissolve them in 1000 mL of water to obtain Agent A;

[0211] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0212] (3) The dosage of compound biostimulant A and B is 4% and 2% (V / V) respectively;

[0213] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0214] Example 24

[0215] (1) Agent A: Weigh 0.32 g of polyferric sulfate and 0.5 g of fulvic acid on a balance and dissolve them in 1000 mL of water to obtain Agent A;

[0216] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0217] (3) The dosage of compound biostimulant A and B is 4% and 1% (V / V) respectively;

[0218] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0219] Example 25

[0220] (1) Agent A: Weigh 0.635 g of polyferric sulfate and 0.125 g of fulvic acid on a balance and dissolve them in 1000 mL of water to obtain Agent A;

[0221] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0222] (3) The dosage of compound biostimulant A and B is 4% and 2% (V / V) respectively;

[0223] The subsequent steps were carried out according to (4) to (8) in Example 1.

[0224] Example 26

[0225] (1) Agent A: Weigh 0.635 g of polyferric sulfate and 0.25 g of fulvic acid on a balance and dissolve them in 1000 mL of water to obtain Agent A;

[0226] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10The fermentation supernatant waste liquid is taken as the B agent, wherein the first-stage and second-stage medium formulations are as follows: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121°C for 20 min;

[0227] (3) The dosing amounts of the compounded biological stimulant A agent and B agent are 4% and 1% (V / V) respectively;

[0228] The subsequent steps are performed according to (4)-(8) in Embodiment 1.

[0229] Embodiment 27

[0230] (1) A agent: 0.635 g of polymeric ferric sulfate and 0.5 g of fulvic acid are weighed on a balance and dissolved in 1000 mL of water to obtain the A agent;

[0231] (2) B agent: 0.5 g of yeast extract is dissolved in 1000 mL of glycerol to obtain the B agent; L. plantarum According to the inoculation amount of 1%, the first-stage medium is inoculated and cultured at 37°C for 20-24 h, and the prepared first-stage seed liquid is inoculated into the second-stage medium according to the inoculation amount of 20% and anaerobically cultured at 37°C for 6-8 h, until the viable cell count in the medium is 1×10 10 The fermentation supernatant waste liquid is taken as the B agent, wherein the first-stage and second-stage medium formulations are as follows: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121°C for 20 min;

[0232] (3) The dosing amounts of the compounded biological stimulant A agent and B agent are 4% and 4% (V / V) respectively;

[0233] The subsequent steps are performed according to (4)-(8) in Embodiment 1.

[0234] Embodiment 28

[0235] (1) A agent: 0.635 g of polymeric ferric sulfate and 0.5 g of fulvic acid are weighed on a balance and dissolved in 1000 mL of water to obtain the A agent;

[0236] (2) B agent: 0.5 g of yeast extract is dissolved in 1000 mL of glycerol to obtain the B agent; L. plantarumInoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0237] (3) Prepare a nutrient medium containing C and N sources: glucose 5 g / L, sodium dihydrogen phosphate 3.5 g / L, potassium dihydrogen phosphate 1.8 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.02 g / L, ferric chloride 0.01 g / L, peptone 2 g / L, beef extract 3 g / L, pH = 7.2, sterilize at 121°C for 20 min;

[0238] (4) Sludge from the anaerobic section of the Lunan Pharmaceutical Wastewater Treatment Plant in Shandong, China was mixed with the nutrient medium in (3) at a ratio of 3:7 (V / V), and chlorobenzene solution was added to make the chlorobenzene concentration in the sludge-water mixture 20 mg / L;

[0239] (5) Add the compound biostimulants A and B into the mud-water mixture at a dosage of 4% and 4% (V / V);

[0240] (6) After sealing, incubate in a 30°C incubator for 45 days;

[0241] (7) Gas chromatography to detect the removal efficiency of chlorobenzene: 5 mL of the sample at 0 day and 45 days was taken and extracted with equal volumes of n-hexane multiple times. After filtering the extract through an organic filter membrane, 1 mL was taken into a brown injection vial. The RB-5 column was used with an injection volume of 5 μL. The injection port temperature was set at 260°C, the split ratio was 10:1, the column pressure was 80 kPa, the total flow rate was 30.5 mL / min, the column flow rate was 1 mL / min, and the purge flow rate was 3 mL / min. The temperature program was as follows: the column temperature was maintained at 50°C for 1 minute, and then increased to 180°C at a rate of 10°C / min. The sample was detected by FID detector at a detection temperature of 280°C. The chlorobenzene concentration at 0 day of degradation was subtracted from the chlorobenzene concentration at 45 days of degradation, and the difference was divided by the chlorobenzene concentration at 0 hour to obtain the chlorobenzene removal efficiency. After deducting the sludge adsorption efficiency, the biodegradation efficiency of chlorobenzene in the sludge-water mixed system was obtained.

[0242] Comparative Example 1

[0243] (1) Prepare seed solution with the following formula: 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, and sterilize at 121°C for 20 min.

[0244] (2) Prepare an inorganic salt culture medium with the following formula: 1.50 g / L potassium dihydrogen phosphate, 4.19 g / L sodium dihydrogen phosphate, 0.83 g / L ammonium sulfate, 0.05 g / L magnesium sulfate, pH = 7.2 ± 0.1, sterilize at 121°C for 20 min, and add o-chlorophenol sterilized by filtration with a 0.22 μm filter membrane to make the o-chlorophenol concentration in the culture medium 20 mg / L;

[0245] (3) Store in a 4℃ refrigerator with a slant surface A . strain ZY1 was inoculated into the seed solution in (1) and cultured in a shaking incubator at 30°C and 150 rpm for 22-24 hours to obtain liquid culture. OD 600 1.8-2.0;

[0246] (4) The liquid culture in (3) was inoculated into the inorganic salt culture medium prepared in (2) at a 10% (V / V) inoculation rate and cultured at 30°C and 150 rpm for 48 h;

[0247] (5) Detection of o-chlorophenol removal efficiency by high performance liquid chromatography: 1 mL of sample was taken after degradation for 0 h, 8 h, 10 h, 24 h, 36 h, and 48 h, and centrifuged at 4 °C and 8000 rpm for 10 min. The supernatant was diluted 20 times with acetonitrile, filtered through a 0.22 μm organic filter membrane, and 1 mL was taken into a brown injection vial. A 4.5 mm × 250 mm C18 column was used, the column temperature was 30 °C, the mobile phase was 60%:40% acetonitrile: ultrapure water, the flow rate was 1 mL / min, and the UV detector was used for detection at a wavelength of 280 nm. The concentration C0 at 0 h and the corresponding concentration C at each time t during the degradation process were obtained. t , fitting ln(C t The relationship between the degradation kinetics of o-chlorophenol by the strain (see Figure 1 ), the o-chlorophenol concentration at degradation time 0 h (undegraded) was subtracted from the o-chlorophenol concentration at degradation time 24 h, and the difference was divided by the o-chlorophenol concentration at degradation time 0 h (undegraded) to obtain the o-chlorophenol removal rate for 24 h.

[0248] Comparative Example 2

[0249] (1) Agent A: Weigh 0.16 g of polyferric sulfate and 0.125 g of fulvic acid on a balance and dissolve them in 1000 mL of water to obtain Agent A;

[0250] (2) Agent B: Glycerol-preserved L. plantarumInoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0251] (3) Prepare an inorganic salt culture medium with the following formula: 1.50 g / L potassium dihydrogen phosphate, 4.19 g / L sodium dihydrogen phosphate, 0.83 g / L ammonium sulfate, 0.05 g / L magnesium sulfate, pH = 7.2 ± 0.1, sterilize at 121°C for 20 min, and add o-chlorophenol sterilized by filtration with a 0.22 μm filter membrane to make the o-chlorophenol concentration in the culture medium 20 mg / L;

[0252] (4) The dosage of compound biostimulant A and B is 4% and 1% (V / V) respectively;

[0253] (5) Detection of o-chlorophenol removal efficiency by high performance liquid chromatography: 1 mL of the sample after degradation for 0 h and 24 h was taken, centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was diluted 20 times with acetonitrile. After filtering through a 0.22 μm organic filter membrane, 1 mL was taken and placed in a brown injection vial. A 4.5 mm × 250 mm C18 column was used, the column temperature was 30°C, the mobile phase was 60%:40% acetonitrile: ultrapure water, the flow rate was 1 mL / min, and the UV detector was used for detection at a wavelength of 280 nm. The o-chlorophenol concentration at degradation for 0 h (undegraded) was subtracted from the o-chlorophenol concentration at degradation for 24 h, and the difference was divided by the o-chlorophenol concentration at degradation for 0 h (undegraded) to obtain the o-chlorophenol removal rate.

[0254] Comparative Example 3

[0255] (1) Agent A: Weigh 0.32 g of polyferric sulfate and 0.25 g of fulvic acid on a balance and dissolve them in 1000 mL of water to obtain Agent A;

[0256] (2) Agent B: Glycerol-preserved L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10The fermentation supernatant waste liquid is taken as the B agent, wherein the formula of the primary and secondary culture medium is as follows: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121°C for 20 min;

[0257] (3) The inorganic salt culture medium is configured, and the formula is as follows: 1.50 g / L potassium dihydrogen phosphate, 4.19 g / L sodium phosphate dibasic, 0.83 g / L ammonium sulfate, and 0.05 g / L magnesium sulfate, pH = 7.2±0.1, sterilized at 121°C for 20 min, and o-chlorophenol filtered and sterilized by a 0.22 μm filter is added to make the o-chlorophenol concentration in the culture medium 20 mg / L;

[0258] (4) The dosing amount of the compounded biological stimulant A agent and B agent is 4% and 2% (V / V) respectively;

[0259] (5) The removal efficiency of o-chlorophenol is detected by high performance liquid chromatography: 1 mL of the sample degraded for 0 h and 24 h is centrifuged at 4°C and 8000 rpm for 10 min, the supernatant is diluted by 20 times with acetonitrile, filtered by a 0.22 μm organic filter, 1 mL is taken in a brown sample bottle, a 4.5 mm×250 mm C18 chromatographic column is used, the column temperature is 30°C, the mobile phase is 60%:40% acetonitrile:ultra-pure water, the flow rate is 1 mL / min, an ultraviolet detector is used to detect at a wavelength of 280 nm, the removal rate of o-chlorophenol is obtained by subtracting the concentration of o-chlorophenol degraded for 24 h from the concentration of o-chlorophenol degraded for 0 h (not degraded) and then dividing the difference by the concentration of o-chlorophenol degraded for 0 h (not degraded).

[0260] Comparative Example 4

[0261] (1) A agent: 0.635 g of polyferric sulfate and 0.5 g of fulvic acid are weighed on a balance and dissolved in 1000 mL of water to obtain the A agent;

[0262] (2) B agent: 10 g of glycerol-stored L. plantarum According to the inoculation amount of 1%, the primary culture medium is inoculated, and the prepared primary seed liquid is inoculated into the secondary culture medium according to the inoculation amount of 20%, and the secondary culture medium is anaerobically cultured at 37°C for 6-8 h, until the viable cell count in the culture medium is 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0263] (3) Prepare an inorganic salt culture medium with the following formula: 1.50 g / L potassium dihydrogen phosphate, 4.19 g / L sodium dihydrogen phosphate, 0.83 g / L ammonium sulfate, 0.05 g / L magnesium sulfate, pH = 7.2 ± 0.1, sterilize at 121°C for 20 min, and add o-chlorophenol sterilized by filtration with a 0.22 μm filter membrane to make the o-chlorophenol concentration in the culture medium 20 mg / L;

[0264] (4) The dosage of compound biostimulant A and B is 4% and 4% (V / V) respectively;

[0265] (5) Detection of o-chlorophenol removal efficiency by high performance liquid chromatography: 1 mL of the sample after degradation for 0 h and 24 h was taken, centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was diluted 20 times with acetonitrile. After filtering through a 0.22 μm organic filter membrane, 1 mL was taken and placed in a brown injection vial. A 4.5 mm × 250 mm C18 column was used, the column temperature was 30°C, the mobile phase was 60%:40% acetonitrile: ultrapure water, the flow rate was 1 mL / min, and the UV detector was used for detection at a wavelength of 280 nm. The o-chlorophenol concentration at degradation for 0 h (undegraded) was subtracted from the o-chlorophenol concentration at degradation for 24 h, and the difference was divided by the o-chlorophenol concentration at degradation for 0 h (undegraded) to obtain the o-chlorophenol removal rate.

[0266] Comparative Example 5

[0267] (1) Prepare a nutrient medium containing C and N sources: glucose 5 g / L, sodium dihydrogen phosphate 3.5 g / L, potassium dihydrogen phosphate 1.8 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.02 g / L, ferric chloride 0.01 g / L, peptone 2 g / L, beef extract 3 g / L, pH = 7.2, sterilize at 121°C for 20 min;

[0268] (2) Sludge from the anaerobic section of the Lunan Pharmaceutical Wastewater Treatment Plant in Shandong, China was mixed with the nutrient medium in (1) at a ratio of 3:7 (V / V), and chlorobenzene solution was added to make the chlorobenzene concentration in the sludge-water mixture 20 mg / L;

[0269] (3) After sealing, incubate in a 30°C incubator for 45 days;

[0270] (4) Gas chromatography to detect the removal efficiency of chlorobenzene: 5 mL of the sample at 0 day and 45 days was taken and extracted with equal volumes of n-hexane multiple times. After filtering the extract through an organic filter membrane, 1 mL was taken into a brown injection vial. The RB-5 column was used with an injection volume of 5 μL. The injection port temperature was set at 260°C, the split ratio was 10:1, the column pressure was 80 kPa, the total flow rate was 30.5 mL / min, the column flow rate was 1 mL / min, and the purge flow rate was 3 mL / min. The temperature program was as follows: the column temperature was maintained at 50°C for 1 minute, and then increased to 180°C at a rate of 10°C / min. The sample was detected by FID detector at a detection temperature of 280°C. The chlorobenzene concentration at 0 day of degradation was subtracted from the chlorobenzene concentration at 45 days of degradation, and the difference was divided by the chlorobenzene concentration at 0 hour to obtain the chlorobenzene removal efficiency. After that, the sludge adsorption efficiency was subtracted to obtain the biodegradation efficiency of chlorobenzene in the sludge-water mixed system.

[0271] Comparative Example 6

[0272] (1) Agent B: Preserve the L. plantarum Inoculate the first-level culture medium at a rate of 1% and incubate at 37°C for 20-24 h. Inoculate the prepared first-level seed solution at a rate of 20% into the second-level culture medium and incubate anaerobically at 37°C for 6-8 h until the number of viable bacteria in the culture medium reaches 1×10 10 / mL, and the fermentation supernatant waste liquid was taken as agent B, where the primary and secondary culture medium formulas were: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.58 g / L magnesium sulfate heptahydrate, 0.25 g / L manganese sulfate, pH = 6-7, sterilized at 121℃ for 20 min;

[0273] (2) The dosage of agent B is 4% (V / V);

[0274] (3) Prepare seed solution with the following formula: 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, and sterilize at 121°C for 20 min;

[0275] (4) Prepare an inorganic salt culture medium with the following formula: 1.50 g / L potassium dihydrogen phosphate, 4.19 g / L sodium dihydrogen phosphate, 0.83 g / L ammonium sulfate, 0.05 g / L magnesium sulfate, pH = 7.2 ± 0.1, sterilize at 121°C for 20 min, and add o-chlorophenol sterilized by filtration with a 0.22 μm filter membrane to make the o-chlorophenol concentration in the culture medium 20 mg / L;

[0276] (5) Store in a 4℃ refrigerator with a slant surface A. strain ZY1 was inoculated with the seed solution in (4) and cultured in a shaking incubator at 30°C and 150 rpm for 22-24 hours to obtain liquid strains. OD 600 1.8-2.0;

[0277] (6) The liquid culture in (6) was inoculated into the inorganic salt medium prepared in (5) at a 10% (V / V) inoculation rate, and agent B was added according to the addition ratio in (3). The culture system was cultured at 30°C and 150 rpm for 24 h;

[0278] (7) Detection of o-chlorophenol removal efficiency by high performance liquid chromatography: 1 mL of the sample after degradation for 0 h and 24 h was taken, centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was diluted 20 times with acetonitrile. After filtering through a 0.22 μm organic filter membrane, 1 mL was taken and placed in a brown injection vial. A 4.5 mm × 250 mm C18 column was used, the column temperature was 30°C, the mobile phase was 60%:40% acetonitrile: ultrapure water, the flow rate was 1 mL / min, and the UV detector was used for detection at a wavelength of 280 nm. The o-chlorophenol concentration at degradation for 0 h (undegraded) was subtracted from the concentration at degradation for 24 h, and the difference was divided by the o-chlorophenol concentration at degradation for 0 h (undegraded) to obtain the o-chlorophenol removal rate.

[0279] In Comparative Example 1, the o-chlorophenol degradation efficiency was 66.0% ± 6.2%. In Examples 1-27 (see Table 1), the o-chlorophenol degradation efficiency ranged from 65% to 94%, with 18 examples achieving an o-chlorophenol degradation efficiency exceeding 90%. In Comparative Examples 2-4, the o-chlorophenol removal efficiency was all <10%, indicating that the composite biostimulant of the present invention cannot directly remove o-chlorophenol in a reaction system uninoculated with Acinetobacter, but rather stimulates the biological action of microorganisms to affect o-chlorophenol removal. In Comparative Example 6, a degradation experiment using Agent B at a 4% addition rate resulted in an o-chlorophenol removal efficiency of 79.4% ± 3.0%, demonstrating that the composite biostimulant of the present invention is more effective. In Example 28, a composite biostimulant was applied to the biodegradation of chlorobenzene in a sludge-water system. Compared to the degradation of chlorobenzene in a sludge-water system without the composite biostimulant (Comparative Example 5), the degradation efficiency increased from 23.2% to 41.5%. The composite biostimulant proposed by this invention can also be used to stimulate the biodegradation of chlorobenzene, expanding the scope of application of the invention. The composite multi-matrix biostimulant proposed by this invention can effectively enhance the degradation of chlorine-containing compounds by stimulating microorganisms, overcoming the problem of low biodegradation efficiency.

[0280] Table 1 Degradation of o-chlorophenol in Examples 1-28 and Comparative Examples 1-6

[0281]

[0282] The above specific embodiments provide a detailed description of the present invention. However, the present invention is not limited to the above embodiments. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with each other unless there is a conflict.

Claims

1. A microbial stimulant for remediation of chlorine-containing compound pollution, characterized in that: The microbial stimulants are agent A and agent B; agent A is polyferric sulfate and / or fulvic acid, and agent B is Lactobacillus plantarum fermentation supernatant; the volume ratio of agent A to agent B is 4:1-4; the concentration of polyferric sulfate is 0.32-0.635 g / L; the concentration of fulvic acid is 0.125-0.5 g / L; the Lactobacillus plantarum fermentation supernatant is the supernatant obtained by solid-liquid separation after anaerobic fermentation of Lactobacillus plantarum at 35-39°C for 6-8 hours; the microorganism is Acinetobacter; and the chlorine-containing compounds are chlorobenzene and o-chlorophenol.

2. Use of the microbial stimulant according to claim 1 in improving the degradation ability of microorganisms on chlorine-containing compounds.

3. Use of the microbial stimulant according to claim 1 in the preparation of a product for improving the ability of microorganisms to degrade chlorine-containing compounds.

4. A product, characterized in that The product contains the microbial stimulant according to claim 1.

5. The product according to claim 4, characterized in that The product also contains Acinetobacter bacterial liquid.

6. A method for degrading chlorine-containing compounds, characterized in that: The product according to claim 4 or claim 5 is added to an environment containing chlorine-containing compounds for degradation.

7. The method according to claim 6, characterized in that The OD600 of the Acinetobacter bacterial solution is 1.8-2.

8. The method according to claim 6, characterized in that The volume ratio of the microbial stimulant to the Acinetobacter bacterial liquid is 1.25-2:0.5-2.

5.

9. The method according to claim 6, characterized in that The inoculation amount of the Acinetobacter bacterial liquid is 1% to 20%.

10. The method according to claim 6, characterized in that The concentration of the chlorine-containing compound is 10-50 mg / L.

11. The method according to claim 6, characterized in that The pH of the environment containing chlorine compounds is 6.5-8.

12. The method according to claim 6, characterized in that The degradation temperature is 20-40°C.

Images