CN116903415A - A method for producing a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis Zhanjiang - Google Patents

Abstract

The invention belongs to the technical field of microalgae cultivation, and specifically relates to a production method of a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis zhanjiang. The steps are: the present invention uses sub-powder and feather meal as fermentation raw materials, and selects strains with significant growth promotion effect on microalgae, and combines Lactobacillus buchneri, Lactobacillus curvature, Lactobacillus plantarum and Bifidobacterium with high acetic acid production. , Candida utilis, and Bacillus subtilis are used as fermentation strains; at the same time, a highly active keratinase engineering bacterium with high activity under acidic conditions is cloned and expressed, and the reducing agent sodium sulfite is added for collaborative fermentation. The final fertilizer and water agent obtained contains acetic acid, The chelation rate of peptides, amino acids, and minerals is high; in addition, the amino acids and minerals in the fertilizer and water agent can also be used as nutritional supplements for Ichrysochrysis zhanjiang. This method is suitable for any open or closed culture system. It is safe, environmentally friendly, low-cost, can avoid the large-scale reproduction of harmful bacteria, improves the aquaculture environment, and has broad application prospects.

Description

A method for producing a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis Zhanjiang

Technical field

The invention belongs to the technical field of microalgae cultivation, and specifically relates to a production method of a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis zhanjiang.

Background technique

In recent years, microalgae have grown slowly due to the high cost of autotrophic cultivation and the lack of carbon sources under low-light conditions such as rainy days and winter. As a result, shellfish lack natural live feeds such as Isochrysis Zhanjiang, making it difficult to achieve high-density cultivation. Studies have found that when Isochrysis Zhanjiang uses some carboxylic acids as organic carbon sources and is cultured in mixed culture, it can greatly increase the biological yield and specific growth rate and reduce the growth of bacteria. For aquaculture, fertilizers and aquatic products are used, especially after "water diversion" or water quality deterioration, a large number of algae will die, seriously affecting photosynthesis, and the pond will be hypoxic, thereby causing the death of aquatic animals. This It is necessary to use fertilizer and aquatic products to cause algae to multiply, so fertilizer and aquatic products play a vital role in the field of aquaculture.

At present, commonly used fertilizer and water products are divided into organic fertilizers and inorganic fertilizers. Inorganic fertilizers mainly include urea, calcium phosphate, ammonium carbonate, etc. Fertilizer and water products are effective quickly, but the disadvantage is that they do not last long; organic fertilizers mainly include green manure, manure, etc. , but its nutrition is single; and the preparation of single pure organic acids and minerals is cumbersome and costly, and is not suitable for economical cultivation of microalgae. Therefore, the development of an efficient fertilizer and water agent has very important production and practical significance.

Contents of the invention

In view of the existing problem of low yield of Isochrysis Zhanjiang, the present invention provides a high-efficiency fertilizer and water agent using feather meal and sub-powder as raw materials according to the growth needs of Isochrysis Zhanjiang. During the production process of the fertilizer and water agent, According to the growth needs of Isochrysis Zhanjiang, fermentation strains and minerals with high acetate production were creatively added. At the same time, a highly active keratinase engineering bacteria with high activity under acidic conditions was cloned and expressed, and the reducing agent sodium sulfite was added for collaborative fermentation, and finally obtained The finished fertilizer and water agent has a high chelation rate of acetic acid, small peptides, amino acids, and minerals, and has a significant effect on the growth of isochrysis Zhanjiang.

In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

(1) Mix feather powder and secondary powder and stir evenly to obtain a solid culture medium;

(2) Select lactic acid bacteria, yeast, and spore bacteria to inoculate them into the corresponding culture medium for activation and culture and obtain the corresponding lactic acid bacteria, yeast, and spore fermentation seed liquid;

(3) The keratinase gene sequence derived from Bacillus tequilensis strain Q7 was optimized according to the codon preference of Bacillus subtilis to obtain a gene encoding keratinase. The nucleotide sequence is shown in SEQ ID NO.1; this gene is The template is amplified by PCR, connected to the shuttle plasmid vector pMA and transformed into E. coli competent cells. After extracting the recombinant shuttle plasmid, it is introduced into Bacillus subtilis DB403 competent cells using chemical transformation to obtain recombinant keratinase spores. The engineering bacterium, namely DB403 pMA0911-kerQ7; culture the recombinant keratinase spore engineering bacterium to collect bacterial liquid, the bacterial liquid contains keratinase secreted extracellularly by the recombinant yeast engineering bacteria, and the obtained bacterial liquid is separated by solid-liquid separation by centrifugation Collect the supernatant and set aside;

(4) Inoculate the fermented seed liquid of lactic acid bacteria, yeast and spore bacteria in step (2) and the cultured supernatant of DB403pMA0911-kerQ7 in step (3) into the solid medium of step (1), and then add Minerals, sodium sulfite, and water are stirred evenly, and a high-efficiency fertilizer and water agent is obtained after fermentation.

Preferably, the dosage in step (1) is 10-30% of feather powder and 70-90% of secondary powder.

Preferably, the lactic acid bacteria in step (2) are Lactobacillus bucheri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium, the yeast is Candida utilis, and the spore is Bacillus subtilis.

Preferably, the temperature for culturing the recombinant keratinase spore engineered bacteria in step (3) is 30-37°C, the rotation speed is 180-220 rpm, and the time is 24-48 hours; the centrifugation conditions are 5000-8000 rpm, 5-10 min.

Preferably, the total bacterial liquid inoculum amount of the DB403 pMA0911-kerQ7 supernatant and lactic acid bacteria fermented seed liquid, yeast fermented seed liquid, and saccharomyces fermented seed liquid described in step (4) is 10 to 20% (V/W, ml/g); the volume ratio of the supernatant of DB403pMA0911-kerQ7, lactic acid bacteria fermented seed liquid, yeast fermented seed liquid and saccharomyces fermented seed liquid is 1:3:2:1; the lactic acid bacteria are Lactobacillus buchneri, When one or more of Lactobacillus curvature, Lactobacillus plantarum and Bifidobacterium are mixed, the volume ratio is 1 when multiple species are mixed.

Preferably, in step (4), the water content of the culture medium is controlled to be 25%-45% (V/W), the fermentation time is 5-12 days, and the fermentation temperature is 20-37°C.

Preferably, the amount of sodium sulfite added in step (4) is 0.1 to 2% (W/W) based on the mass of the solid culture medium; the minerals are ferrous sulfate, copper sulfate, manganese sulfate, zinc sulfate and calcium carbonate. , the addition amount of ferrous sulfate is 1.59-14.18g/kg, the addition amount of copper sulfate is 14.7-85.2mg/kg, the addition amount of manganese sulfate is 270-1080mg/kg, and the addition amount of zinc sulfate is 66-264mg /kg, the amount of calcium carbonate added is 3-30g/kg.

More preferably, the addition amount of ferrous sulfate is 7.95g/kg, the addition amount of copper sulfate is 36.75mg/kg, the addition amount of manganese sulfate is 540mg/kg, the addition amount of zinc sulfate is 132mg/kg, and the addition amount of calcium carbonate is 60g/kg. kg.

The high-efficiency fertilizer and water agent prepared by the present invention is used to promote the growth of Isochrysis Zhanjiang. The application method is: directly throw the prepared high-efficiency fertilizer and water agent into the breeding pond, once every 1 to 10 days, and the amount of application each time is 0.3g/L-1.2g/L.

Note: When the placement time is from December to February or on cloudy and rainy days, it is necessary to increase the amount by 10-30% of the original application amount; when the placement time is from March to May, September to November, it is necessary to increase the amount by 10-30% of the original application amount. Reduction.

The specific screening process for technical solutions is as follows:

(A) Screening of fermentation strains

The initial organic acid selection is: lactic acid, acetic acid, formic acid, propionic acid, citric acid, succinic acid, tartaric acid, malic acid;

Organic acids: lactic acid at 0, 0.05, 0.1, 0.15, 0.2g/L, acetic acid at 0, 0.1, 0.3, 0.5, 0.7g/L, formic acid at 0, 0.5, 0.1, 0.2, 0.3g/L, propylene The acid is 0, 0.05, 0.075, 0.1, 0.2g/L, the citric acid is 0, 0.05, 0.075, 0.1, 0.15g/L, the succinic acid is 0, 0.05, 0.1, 0.2, 0.3g/L, and the tartaric acid is 0 , 0.1, 0.3, 0.5, 0.7g/L, and malic acid were added to the microalgae culture medium (F/2 artificial seawater culture medium) at 0, 0.05, 0.075, 0.1, and 0.15g/L respectively to obtain different organic acids. and concentration of F/2 artificial seawater culture medium; adjust its pH to 8.0, and then transfer Isochrysis zhanjiang to the F/2 artificial seawater culture medium containing organic acids at an inoculum volume of 10%. The initial algae density Both were 5 × 10 ⁵ /mL, and the statistical results were cultured for 10 days; using algae density as an indicator, it can be seen that different organic acids have an impact on the growth of microalgae, among which the promotion effect of acetic acid and citric acid is particularly obvious. Further, By comparison, it was found that acetic acid has the most significant impact on the growth of Isochrysis zhanjiang, and its concentration is also crucial. The optimal concentration of acetic acid to promote the growth of Isochrysis zhanjiang is 0.5g/L. This is At that time, the algae density of Isochrysis Zhanjiang exceeded 10 ⁷ /mL algae density. The specific results are shown in Figure 1.

The formula of F/2 artificial seawater medium is: 75mg/L NaNO ₃ , 5mg/L NaH ₂ PO ₄ ·H ₂ O, 9.8μg/L CuSO ₄ ·5H ₂ O, 22μg/L ZnSO ₄ ·7H ₂ O, 10μg /L CoCl·6H ₂ O, 180μg/L MnCl ₂ ·4H ₂ O, 6.3μg/L Na ₂ MoO ₄ ·2H ₂ O, 4.36mg/L Na ₂ EDTA, 3.15mg/L FeCl ₃ ·6H ₂ O, 100μg/L VB ₁ , 0.5μg/LV _H , 0.5μg/L VB ₁₂ , 26g/L sea salt.

Then, HPLC was used to measure organic acids in the fermentation broth of lactic acid bacteria, yeasts, and Bacillus. Specifically, 15 experimental strains were selected, including Lactobacillus fermentum, Lactobacillus rhamnoides, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus casei, Bifidobacterium, Lactobacillus acidophilus, Candida primogeniture, Pichia pastoris, Saccharomyces cerevisiae, Bacillus licheniformis, Bacillus coagulans, Bacillus subtilis, Lactobacillus buchneri and Lactobacillus curvature, the determination results are shown in Table 1;

It can be seen from the experimental results in Table 1 that the organic acid content produced by different strains is significantly different. Lactobacillus bucheri, Lactobacillus curvature, Lactobacillus plantarum, Bifidobacterium, Candida primogenogens, and Bacillus subtilis were screened. Acetic acid production is higher than other organic acids, and the organic acid content is as follows in Table 1. The peak patterns of Lactobacillus bucheri and organic acid standards correspond to (a) and (b) of Figure 2 in sequence.

Table 1 Organic acid content of different bacterial strains

Note: S ₁ to _{S 15} are: Lactobacillus fermentum, Lactobacillus rhamnoides, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Bifidobacterium, Lactobacillus acidophilus, Candida primogens, Pichia pastoris Yeast, Saccharomyces cerevisiae, Bacillus licheniformis, Bacillus coagulans, Bacillus subtilis, Lactobacillus bucheri, Lactobacillus curvature.

(B) Screening of mineral forms and concentrations

Combine different forms of iron sources (ferric sulfate, ferrous sulfate, ferrous glycinate, ferrous lactate), zinc sources (zinc sulfate, zinc lactate, zinc glycinate), copper sources (copper sulfate, copper citrate, copper glycinate), Manganese sources (manganese chloride, manganese acetate, manganese glycinate), calcium sources (calcium chloride, calcium lactate, calcium aspartate) are respectively based on Fe=19umol/L, Zn=0.08unol/L, Cu=0.04umol/ L, Mn=0.91umol/L, Ca=0.04umol/L, were added in sequence to the F/2 artificial seawater culture medium without iron source, zinc source, copper source, copper source and calcium source, and then incubated with 10 % of the inoculum amount was inoculated with Isochrysis zhanjiang, and the initial algae density was controlled to 5× ¹⁰⁵ /mL. The results were statistically analyzed after 8 days of cultivation. The algae density is shown in Figure 3. The results show that Isochrysis zhanjiang was in chelation respectively. The growth is best under the conditions of iron source, zinc source, copper source and calcium source; further using algae density as an indicator to optimize the mineral concentration, it can be seen that its impact on the growth of microalgae is extremely significant under different concentration conditions. Only under Good results can only be obtained at specific concentrations; through experiments, it was obtained that 0.53-4.725mg/L ferrous glycinate is the iron source, 4.9-28.4ug/L copper glycinate is the copper source, and 90-360ug/L manganese glycinate is the manganese source. 22 -88ug/L zinc glycinate is the zinc source and 1-10mg/L calcium aspartate is the calcium source. The results are shown in Figure 4;

Based on iron source, copper source, manganese source, zinc source and calcium source, the corresponding minerals are selected as ferrous sulfate, copper sulfate, manganese sulfate, zinc sulfate and calcium carbonate. The addition of ferrous sulfate is determined according to the corresponding dosage conversion. The amount is 1.59-14.18g/kg (W/W), the amount of copper sulfate is 14.7-85.2mg/kg (W/W), the amount of manganese sulfate is 270-1080mg/kg (W/W), the amount of sulfuric acid The amount of zinc added is 66-264mg/kg (W/W), and the amount of calcium carbonate is 3-30g/kg (W/W).

(C) Obtaining acid-resistant keratinase-producing Bacillus subtilis (DB403 pMA0911-kerQ7)

S1. Submit the keratinase gene sequence of the Bacillus tequilensis strain in NCBI (GenBank: IDKP694221) to Nanjing Genscript Biotechnology Co., Ltd. for codon optimization based on the codon preference in Bacillus subtilis; after obtaining the codon optimization The recombinant keratinase (denoted as kerQ7) gene, its nucleotide sequence is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2;

S2. Use primer P1 (P1-F: GGAATTCCATATGTGTGTTAAGAAGAAGAACGTG, P1-R: CCGGATCCTTAATTGCTCGCCG) to PCR amplify the kerQ7 gene sequence. The reaction conditions are 94℃ 3min; 98℃ 10s, 58℃ 10S, 72℃ 1min: 30 cycles; 72℃ 10min; maintained at 4°C, the reaction system is Premix Ex Taq (2×) 12.5uL, Template 1uL, P1-F (10uM) 1uL, P1-R (10uM) 1uL, ddH2O 9.5uL.

S3. Then, perform double enzyme digestion on the PCR product (target gene kerQ7) and vector pMA0911. The enzyme digestion system for the target gene kerQ7 is: Nde I 1.0 μL, BamH I 1.0 μL, kerQ7 15 μL, 10× QuickCut ^TM Buffer 5.0 μL. , dd H ₂ O 28.0 μL, vector pMA0911 enzyme digestion system is Nde I 1.0 μL, BamH I 1.0 μL, pMA0911 plasmid 20 μL, 10×QuickCut ^TM Buffer 5.0 μL, dd H ₂ O 23.0 μL, and then add endo Enzyme kerQ7 and vector pMA were digested at 37°C for 15 minutes, 10×DNA Loading Buffer was added, and then the target fragment was recovered through gel recovery after agarose gel electrophoresis, and the concentration of the recovered fragment was measured with Onedrop.

S4. Use T4 DNA ligase to ligate the gel recovery product (target fragment) to obtain the enzyme ligation product; the enzyme ligation system is 1uL of T4 DNA ligase, 2uL of T4 DNA ligase buffer, 4uL of double enzyme digestion vector, and kerQ7 target fragment. 13uL, reaction conditions are 16℃, 3h; then, the enzyme conjugation product is directly transformed into the E.coli DH5α competent strain, spread on 100ug/ml ampicillin-resistant plate, select the positive transformant, expand and extract the plasmid and perform Verify by sequencing, the correct plasmid is named pMA0911-kerQ7, see Figure 5A; transfer pMA0911-kerQ7 to LB liquid culture medium, and after culturing to the logarithmic phase, add 300uL 50% to every 700uL bacterial solution on the ultra-clean workbench Glycerin is stored at -80°C.

S5. Transform the pMA0911-kerQ7 obtained in step 4 into the competent state of Bacillus subtilis DB403; the transformation method is: melt pMA0911-kerQ7 in a water bath at 45°C to form a competent state, and then add 5uL pMA0911--kerQ7 to the 500uL competent state again, and then Activate and culture in a constant temperature shaker for 1 hour, the culture conditions are 120 rpm, 37°C; then centrifuge at 5000 rpm for 5 minutes, remove 400uL supernatant, resuspend the remaining solution, apply it to an LB plate with 100ug/ml ampicillin to select transformants, and culture overnight. Pick a single colony to verify the transformants.

Then, the common vector pMA0911 primer P2 of Bacillus subtilis (P2F: gcgaaaatgcctcacatttgtgcc, P2R: cgggatctcagatctggtacgtacc) was used to amplify the gene. The PCR program was: 94°C for 3 minutes, 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, 30 cycles, and 72°C for 10 minutes. , the reaction system is Taq Master Mix (2×) 10uL, Template 1uL, P2-F (10μM) 0.8uL, P2-R (10μM) 0.8uL, ddH2O 7.4uL. The product was then subjected to agarose gel electrophoresis, and a bright band was visible at about 1300 bp, as shown in Figure 5B, proving that the transformation was successful, and the recombinant engineered bacterium was named DB403 pMA0911-kerQ7.

S6. Inoculate DB403 pMA0911-kerQ7 into 100ug/mL ampicillin LB medium, and culture it overnight on a shaker. The culture conditions are 180rpm and 37°C. Collect the fermentation broth and centrifuge it at 8000rpm for 10 minutes to collect the supernatant of the fermentation broth; perform Western -Blot verification, the target band appeared at 28Kda, proving that the expression was successful, see Figure 5C. The activity of the fermentation supernatant was measured to be 84U/mL.

The advantages and technical effects of the present invention are:

(1) According to the growth needs of Isochrysis Zhanjiang, the present invention creatively adds fermentation strains and minerals with high acetic acid production to meet the demand of Isochrysis Zhanjiang for acetic acid; at the same time, it clones and expresses a high activity under acidic conditions. Keratinase efficient engineered bacteria are added to the reducing agent sodium sulfite for collaborative fermentation. The final product of the fertilizer and water agent has a high chelation rate of acetic acid, small peptides, amino acids, and minerals. In addition, a large number of probiotics in the fertilizer and water agent can cooperate with the further transformation of microalgae. Utilize organic matter to increase the biological production of microalgae; the amino acids and minerals in the fertilizer and water agent can also be used as nutritional supplements for Isochrysis zhanjiang, which has a significant effect on the growth of Isochrysis zhanjiang.

(2) The present invention provides a method for bacterial enzymes to synergistically ferment feather meal, combined with the fertilizer and water agent process of sodium sulfite to degrade disulfide bonds, which not only promotes the production of microalgae, but also realizes the reduction of agricultural waste such as feather meal. conversion utilization.

(3) The present invention constructs an engineering strain of Bacillus subtilis to express keratinase and uses it for fermented feather meal feed, which greatly reduces the input cost of keratinase; it is also suitable for market production and has broad application prospects.

Description of the drawings

Figure 1 shows the measurement results of different organic acids on the growth of Isochrysis zhanjiang; A is the measurement result of different lactic acid contents on the growth of Isochrysis zhanjiang, and B is the measurement results of different acetic acid contents on the growth of Isochrysis zhanjiang. C is the measurement result of different formic acid content on the growth of Isochrysis zhanjiang, D is the measurement result of different propionic acid content on the growth of Isochrysis zhanjiang, E is the measurement result of different citric acid content on the growth of Isochrysis zhanjiang, F is the measurement result of different malic acid contents on the growth of Isochrysis zhanjiang; G is the measurement result of different tartaric acid contents on the growth of Isochrysis zhanjiang; H is the measurement result of different succinic acid contents on the growth of Isochrysis zhanjiang.

In Figure 2, 2a is the liquid chromatogram of Lactobacillus buchneri, and 2b is the liquid chromatogram of the organic acid standard.

Figure 3 shows the measurement results of different minerals on the growth of Isochrysis zhanjiang; where A is the measurement results of different forms of iron sources on the growth of Isochrysis zhanjiang; where Fe0: no iron source, Fe1: trichloride Iron, Fe2: ferrous sulfate, Fe3: ferrous lactate, Fe4: ferrous glycinate; B is the measurement result of different forms of copper sources on the growth of Isochrysis zhanjiang, where Cu0: no copper source, Cu1: copper sulfate, Cu2: copper citrate, Cu3: copper glycinate, CA: citric acid, GC: glycine; C is the measurement result of different forms of calcium sources on the growth of Isochrysis zhanjiang, where Ca0: no calcium source, Ca1: calcium chloride , Ca2: calcium lactate, Ca3: calcium aspartate, LA: lactic acid, ASP: aspartic acid; D is the measurement result of different forms of zinc sources on the growth of Isochrysis zhanjiang, where Zn0: no zinc source, Zn1: zinc sulfate, Zn2: zinc lactate, Zn3: zinc glycinate, LA: lactic acid, GC: glycine; E is the measurement result of different forms of manganese sources on the growth of Isochrysis zhanjiang, where Mn1: manganese chloride, Mn2: Manganese acetate, Mn3: manganese glycinate, AA: acetic acid, GC: glycine.

Figure 4 shows the measurement results of different chelated mineral contents on the growth of Isochrysis zhanjiang; A is the measurement result of different contents of ferrous glycinate on the growth of Isochrysis zhanjiang, and B is the measurement results of different contents of copper glycinate on the growth of Isochrysis zhanjiang. Determination results of the growth of Isochrysis algae, C Determination results of different contents of calcium aspartate on the growth of Isochrysis zhanjiang, D are the test results of different contents of zinc glycinate on the growth of Isochrysis zhanjiang, E are different Determination results of the content of manganese glycinate on the growth of Isochrysis zhanjiang.

In Figure 5, A is the plasmid map of the pMA0911-kerQ7 vector, B is the PCR verification of the Bacillus subtilis transformant colony, and C is the Western-Blot verification of the expression of Bacillus subtilis DB403 pMA0911-kerQ7.

Detailed ways

The present invention is further defined in the following examples. Based on the above description and these examples, those skilled in the art can determine the basic characteristics of the present invention, and can make various modifications to the present invention without departing from the spirit and scope of the present invention. Various modifications and changes are made to adapt it to various uses and conditions. The experimental raw materials used in the following examples can all be obtained through commercial channels unless otherwise specified. Unless otherwise noted, the present invention adopts the existing technology in this field.

Source of algae species: Zhanjiang Isochrysis was purchased from Fenggeng Algae Co., Ltd.;

Source of strains: Lactobacillus rhamnosus CGMCC 1.2467, Lactobacillus fermentum CGMCC 1.15608, Saccharomyces cerevisiae CGMCC 2.1527, Bacillus subtilis CGMCC 1.1086, Bacillus licheniformis ( Bacillus licheniformis)CGMCC1.813, Lactobacillus buchneriCGMCC 1.15607, Bifidobacterium lactisCGMCC 1.5091, Lactobacillus plantarumCGMCC 1.16089, Lactobacillus acidophilusCGMCC 1.12735 , cheese Lactobacillus casei CGMCC 1.8727, Lactobacillus paracei CGMCC 1.12731, Candida utilis CGMCC 2.3047, Bacillus coaguLans CGMCC 1.10823, Pichia pastoris )CGMCC 2.404 was from the China General Microbial Culture Collection Center, and Lactobacillus curvatus was from Ningbo Mingzhou Biotechnology Co., Ltd., numbered BMZ068373.

Note: The bacterial strains are all strains that can be obtained through existing channels. The description of the present invention is only for illustration and is not limiting; for example, the number of Lactobacillus plantarum (Lactobacillus plantarum) limited to the present invention is CGMCC 1.16089, and other Lactobacillus plantarum can be equivalent. replace.

The detection method adopted in the embodiment of the present invention may refer to the following description:

1. Determination of the density of Isochrysis algae in Zhanjiang

Take 100uL of algae solution, use a hemocytometer to count the board, follow the principle of "note up but not down, note left but not right", the number of algae cells per milliliter = 80 total cells in small squares/80×400×10 ⁴ × dilution factor .

2. Determination of organic acids in fermentation bacteria liquid

Cultivate the bacterial liquid to OD600nm=0.6, take 2 mL of bacterial liquid and centrifuge at 8000 rpm for 5 minutes. Take the supernatant and pass through the membrane for measurement. Chromatographic column: Yuexu OAA column; mobile phase: potassium dihydrogen phosphate: methanol=97:3; potassium dihydrogen phosphate concentration is 0.01mol/L, and 20% (V/V) phosphoric acid is used to adjust pH=2.7, detection wavelength: 210nm; isocratic elution, flow rate: 0.6mL/min.

3. Determination of organic acids in fertilizer and water agents

Weigh 1g of fermented fertilizer aqueous solution, add 30ml of deionized water, soak for 1 hour, ultrasonic for 30 minutes, and then centrifuge at 8000 rpm for 20 minutes. Take the supernatant and pass through the membrane for measurement. Chromatographic column: Yuexu OAA column; mobile phase: potassium dihydrogen phosphate: methanol=97:3; potassium dihydrogen phosphate concentration is 0.01mol/L, and 20% (V/V) phosphoric acid is used to adjust pH=2.7, detection wavelength: 210nm; isocratic elution, flow rate: 0.6mL/min.

4. Determination of crude protein and small peptide content in fertilizer and water agents

Weigh 0.2g of the dried fermented fertilizer aqueous solution for digestion and nitrogen determination for crude protein determination. Weigh 2.0g of dried fermented fertilizer aqueous solution, add 50mL of 15% (V/V) trichloroacetic acid and soak for 1 hour. Take 10mL of the filtrate for digestion and nitrogen determination for the determination of small peptides. Calculated as follows.

V ₀ : standard hydrochloric acid volume used in blank sample (mL); V ₁ : standard hydrochloric acid volume used in sample (mL); C: standard hydrochloric acid concentration (mol/L); M ₁ : sample mass (g); M ₂ : crude protein content(%)

5. Determination of mineral ion chelation rate of fertilizer and water agents

Weigh 1.00g of fertilizer and water agent and ashes it to white ash in a muffle furnace at 500-550°C. If the ashing is incomplete and there are black carbon particles, add a little nitric acid to moisten it after cooling. After drying on the electric hot plate, move it to a muffle furnace to continue ashing into white ash. After cooling, take it out, add 10 mL of 5% nitric acid solution to dissolve, and adjust the volume to 25 mL with deionized water. Do a blank experiment at the same time. The total contents of Fe, Cu, Mn, Zn, and Ca in the fertilizer and water agent were measured using an inductively coupled plasma spectrometer (ICP-OES), which were Fe ₀ , Cu ₀ , Mn ₀ , Zn ₀ , and Ca ₀ respectively.

Weigh 1.00g of fertilizer and water agent, add 30 ml of absolute ethanol, shake and soak for 30 minutes, filter with qualitative filter paper, and centrifuge the filtrate at 10,000 rpm for 10 minutes. Take 10 ml of the supernatant after centrifugation and place it in a muffle furnace to ashes (the same steps as above). Use ICP-OES to measure the contents of uncomplexed Fe, Cu, Mn, and Zn, which are Fe ₁ , Cu ₁ , Mn ₁ , and Zn ₁ respectively. The calculation of the complex rates of Fe, Cu, Mn and Zn is shown in formulas (5.1), (5.2) (5.3) (5.4) respectively.

Weigh 1.00g of fertilizer and water agent, add 50ml of 1% acetic acid solution, shake at 120rpm, soak for 30min, filter with qualitative filter paper, and centrifuge the filtrate at 10000rpm for 10min. Take 10 ml of the supernatant after centrifugation and place it in a muffle furnace to ashes (the same steps as above). The Ca content was measured using ICP-OES and was found to be Ca ₁ .

Weigh 1.00g of fertilizer and water agent, add 50 ml of deionized water, shake and soak for 30 minutes, filter with qualitative filter paper, and centrifuge the filtrate at 10,000 rpm for 10 minutes. Take 10 ml of the supernatant after centrifugation and place it in a muffle furnace to ashes (the same steps as above). The Ca content was measured using ICP-OES and was found to be Ca ₂ . The calculation of Ca complex rate is shown in formula (5.5).

6. Determination of free amino acid content of fertilizer and water agents

(1) Establishment of amino acid standard curve

Prepare mixed standards of 15 amino acids at 50 mg/L, 100 mg/L, 250 mg/L, 400 mg/L, and 500 mg/L. Use the mass concentration and peak area as the abscissa and ordinate to establish a standard curve.

(2) Sample extraction and derivatization

Mix 6.00g fermented feed with 40mL 0.1mol/L HCl, sonicate for 20min, shake and soak for 1h, centrifuge at 4000rpm for 20min, take 100uL supernatant and add 200uL derivatization buffer, shake and mix, then add 200uL derivatization agent, shake and mix, 60℃ React in water bath for 1 hour, filter with 0.45 μm filter membrane, and wait for testing.

(3) Calculation

Substitute the peak area of the measured sample into the standard curve formula to calculate the content of each free amino acid. The total free amino acid content is the sum of the contents of each free amino acid.

(4) Chromatographic conditions

Mobile phase: 0.05mol/L sodium acetate solution and 50% acetonitrile; chromatographic column: C18 column (Welch, AQ-C18, 5.0μm); detection wavelength: 360nm; flow rate: 1.0mL/min, gradient elution conditions are shown in Table 2 .

Table 2 Amino acid determination gradient elution conditions

Time(min) 0.05mol/L sodium acetate solution (%) 50% acetonitrile (%) 0 84 16 0.3 84 16 4 69 31 9.5 64 36 17 45 55 28 40 60 34 0 100 38 0 100 39 84 16 46 84 16

7.Method for determination of keratinase activity

Take 1.0 mL and add 2.0 mL 0.05 mol/L Tris-HCl buffer (pH 7.0), then add 10 mg feather powder as a substrate, react in a 30°C constant temperature shaker, and add 2.0 mL 10% trichloroacetic acid (TCA) after 1 hour. ) to terminate the reaction, centrifuge at 10,000 rpm and 4°C for 10 min, and measure the absorbance of the supernatant at 280 nm. A TCA-treated reaction tube was added before the reaction as a control. Enzyme activity is defined as a 0.01 increase in absorbance at 280 nm under the above reaction conditions, which is defined as 1 enzyme activity unit (U).

In the following examples, the lactic acid bacteria are Lactobacillus buchneri, Lactobacillus curvature, Lactobacillus plantarum and Bifidobacterium; the yeast is Candida primogenogens, and the spore is Bacillus subtilis.

Example 1:

(1) Activation culture and preparation of fermentation seed liquid of Lactobacillus bucheri, Lactobacillus curvature, Lactobacillus plantarum, Bifidobacterium bifidum, Candida primogenogens, and Bacillus subtilis, inoculated into the corresponding culture medium for activation culture and obtained Corresponding fermented seed liquid.

A. Preparation of culture medium

Liquid culture medium for yeast and spores: Add 5g yeast extract powder, 10g NaCl, and 10g tryptone to each liter of distilled water, sonicate for 5 minutes. After complete dissolution, sterilize at 121°C for 30 minutes, cool and set aside.

Liquid culture medium for lactic acid bacteria: add 10g beef extract, 10g soy peptone, 20g glucose, 5g yeast extract powder, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g anhydrous sodium acetate, 1g Tween 80, per liter of distilled water. 0.58g magnesium sulfate, 0.25g manganese sulfate, ultrasonic for 10 minutes, until completely dissolved, sterilize at 121°C for 30 minutes, cool and set aside.

B. Preparation of lactic acid bacteria, yeast and spore fermentation seed liquid:

Yeast was inoculated into LB medium with an inoculation amount of 1%, and cultured at 30°C with a shaker at 150rpm until OD600nm=0.6; Bacillus was inoculated into LB medium with an inoculum amount of 1% and cultured at 37°C with a shaker at 150rpm. to OD600nm = 0.6; lactic acid bacteria were inoculated into MRS culture medium at an inoculation amount of 1%, and cultured anaerobically at 37°C until OD600nm = 0.6 to obtain fermentation seed liquid.

(2) Mix 30% feather meal and 70% feather meal and stir evenly to make a solid culture medium;

(3) Obtain the fermentation supernatant of the recombinant keratinase engineering strain DB403 pMA0911-kerQ7 (the operation is the same as the previous screening step C);

(4) Add the fermentation seed liquid of step (1) and the supernatant of step (3) to the solid culture medium of step (2). The inoculation amount of the bacterial liquid is 6%, in which the fermentation supernatant of DB403 pMA0911-kerQ7 , the volume ratio of lactic acid bacteria fermented seed liquid, yeast fermented seed liquid, and spore fermented seed liquid is 1:3:2:1, and the lactic acid bacteria are Lactobacillus bucheri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium, among which The volume ratio of the fermented seed liquid of Lactobacillus reuteri, Lactobacillus curvature, Lactobacillus plantarum and Bifidobacterium is 1:1:1:1; the water content is controlled to 45%;

According to the mass of the solid culture medium in step (2): add 0, 1.59, 7.95, 14.18, 28.16g/kg (W/W) ferrous sulfate and stir evenly. Use the fertilizer and water agent without adding ferrous sulfate as the control group. Carry out anaerobic fermentation at a fermentation temperature of 30°C and a fermentation time of 7 days. After the fermentation is completed, a high-efficiency fertilizer and water agent (fermentation fertilizer and water agent) is obtained;

Determine the acetic acid content, total organic acid content, crude protein content, small peptide content, amino acid content, and iron chelation rate of the fertilizer and water agent. For the measurement methods, see Test Methods 2, 3, 4, 5, and 6. The measurement results are shown in Table 3; The results show that compared with the fertilizer and water agent without adding ferrous sulfate, as the amount of ferrous sulfate added increases, the chelation rate of iron increases. When 7.94g/kg ferrous sulfate is added, the acetic acid content, total organic acid content, small peptide content, and amino acid content of the fermented fertilizer aqueous solution reach the optimal level. At 28.16g/kg of ferrous sulfate, although the chelation rate of iron is increased, too much iron leads to a decrease in fermentation products related to fertilizer and water agents, including a decrease in the content of organic acids, small peptides and free amino acids, so ferrous sulfate is chosen. The iron addition amount is 7.95g/kg to prepare a fermented fertilizer aqueous solution, which is used for the cultivation operation in the subsequent step (5).

Table 3. Measurement results of the fermented fertilizer aqueous agent itself in Example 1

(5) Transfer the Isochrysis Zhanjiang seed solution to 10L of F/2 artificial seawater medium at an inoculum volume of 10%, and control the initial algae density to 5×10 ⁵ /mL. Isochrysis Zhanjiang seeds The preparation of the liquid is as shown in S1 below; then add the fermented fertilizer aqueous solution containing 7.95g/kg ferrous sulfate in step (4) to the F/2 artificial seawater culture medium of Isochrysis Zhanjiang at 0.3g/L; Unfermented fertilizer aqueous agent (Comparative Example 1) and no fertilizer aqueous agent were used as the control group; the pH value was adjusted to 8.0, the culture temperature was 26±1°C, the light intensity was 8000lux, the light-dark ratio was 12h:12h, and after 8 days of cultivation, the algae was Cell counting, the counting method is shown in Determination Method 1, and the results are shown in Table 4; in the culture medium of Isochrysis zhanjiang added with 7.95g/kg ferrous sulfate fermented fertilizer aqueous agent, the algae density is F/2 artificial seawater culture medium ( 2 times that without fertilizer and water agent).

S1. Preparation of Isochrysis zhanjiang seed liquid:

After culturing Isochrysis zhanjiang to the logarithmic growth phase, transfer it to 1L F/2 artificial seawater culture medium with an inoculum volume of 10% (V/V) for cultivation, and culture it to the logarithmic growth phase to obtain Zhanjiang Isochrysis seed liquid; F/2 artificial seawater culture medium: 75mg/L NaNO ₃ , 5mg/L NaH ₂ PO ₄ ·H ₂ O, 9.8μg/L CuSO ₄ ·5H ₂ O, 22μg/L ZnSO ₄ · 7H ₂ O, 10μg/LCoCl·6H ₂ O, 180μg/L MnCl ₂ ·4H ₂ O, 6.3μg/L Na ₂ MoO ₄ ·2H ₂ O, 4.36mg/L Na ₂ EDTA, 3.15mg/LFeCl ₃ ·6H ₂ O, 100μg/L VB ₁ , 0.5μg/LV _H , 0.5μg/L VB ₁₂ , 26g/L sea salt.

Comparative Example 1: The culture medium is the same as the solid culture medium in step (2) of Example 1, and the water content is controlled to 45%; and based on the mass of the solid culture medium in step (2): add 7.95g/kg (W/W ) ferrous sulfate and stir evenly to obtain unfermented fertilizer aqueous solution.

Table 4. Effect of fertilizer and water agents on the growth of microalgae

Example 2:

(1) Preparation of fermented seed liquid of Lactobacillus bucheri, Lactobacillus curvature, Lactobacillus plantarum, Bifidobacterium bifidum, Candida primogenogens, and Bacillus subtilis, the steps are the same as in Example 1;

(2) The preparation of solid culture medium, the mass proportion of each component is the same as in Example 1;

(3) Perform the same operation as in Example 1 to obtain the fermentation supernatant of DB403 pMA0911-kerQ7;

(4) Add the fermentation seed liquid of step (1) and the supernatant of step (3) to the solid culture medium of step (2). The inoculation amount of the bacterial liquid is 6%, in which the fermentation supernatant of DB403 pMA0911-kerQ7 , the volume ratio of lactic acid bacteria fermented seed liquid, yeast fermented seed liquid, and spore fermented seed liquid is 1:3:2:1; the lactic acid bacteria are Lactobacillus buchneri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium, among which The volume ratio of the fermentation seed liquid of Lactobacillus reuteri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium is 1:1:1:1; the water content is controlled to be 45%; calculated according to the mass of the solid culture medium in step (2): Add 7.95g/kg (W/W) ferrous sulfate, and 0, 14.7, 36.75, 85.2, 170.4mg/kg (W/W) copper sulfate and stir evenly. Use the fertilizer and water agent without copper sulfate as the control group. Carry out anaerobic fermentation at a fermentation temperature of 30°C and a fermentation time of 7 days. After the fermentation is completed, a high-efficiency fertilizer and water agent (fermentation fertilizer and water agent) is obtained;

Determine the acetic acid content, total organic acid content, crude protein content, small peptide content, amino acid content, and iron chelation rate of the fertilizer and water agent. The measurement methods are shown in Test Methods 2, 3, 4, 5, and 6. The measurement results are shown in Table 2. Compared with the fertilizer and water agent without adding copper sulfate, as the amount of copper sulfate added increases, the chelation rate of copper increases. When 36.75 mg/kg copper sulfate is added, the acetic acid content, total organic acid content, small peptide content, and amino acid content of the fermented fertilizer aqueous solution reach the optimal level. At 170.4mg/kg copper sulfate, although the chelation rate of copper is increased, too much copper sulfate leads to a decrease in fermentation products related to fertilizer and water agents, including a decrease in the content of organic acids, small peptides and free amino acids, so fermented fertilizer and water are selected. The copper sulfate addition amount of the agent is 36.75 mg/kg, which is used for the culture of step (5).

Table 5. Measurement results of the fermented fertilizer aqueous agent itself in Example 2

(5) Transfer the Isochrysis Zhanjiang seed liquid (preparation steps are the same as Example 1) to 10L of F/2 artificial seawater culture medium at an inoculum volume of 10%, and control the initial algae density to 5×10 ⁵ / mL, the preparation of Isochrysis Zhanjiang seed liquid is as shown in S1, and then the fermented fertilizer aqueous agent with 36.75 mg/kg copper sulfate added in step (4) is added to the F/2 of Isochrysis Zhanjiang at 0.3g/L. In the artificial seawater culture medium; unfermented fertilizer and water agent (Comparative Example 2) and no fertilizer and water agent were used as the control group; the pH value was adjusted to 8.0, the culture temperature was 26±1°C, the light intensity was 8000lux, and the light-dark ratio was 12h:12h. After cultivating for 8 days, count the algal cells. The counting method is shown in Determination Method 1, and the results are shown in Table 6. In the culture medium of Isochrysis zhanjiang with the addition of 36.75 mg/kg copper sulfate fermentation fertilizer, the algae density is F/2 2.25 times under artificial seawater medium (without fertilizer and water agent).

Comparative Example 2: The culture medium is the same as the solid culture medium in step (2) of Example 2, and the water content is controlled to 45%; and according to the mass of the solid culture medium in step (2): add 7.95g/kg (W/W ) ferrous sulfate and 36.75mg/kg copper sulfate and stir evenly to obtain an unfermented fertilizer aqueous solution.

Table 6. Effect of fertilizer and water agents on the growth of microalgae

Example 3:

(1) Preparation of activated culture and fermentation seed liquid of Lactobacillus buchneri, Lactobacillus curvature, Lactobacillus plantarum, Bifidobacterium bifidum, Candida primogenogens, and Bacillus subtilis. The steps are the same as in Example 1.

(2) Preparation of solid culture medium, the mass proportion of each component is the same as in Example 1, and stirred evenly.

(3) As in Example 1, obtain the fermentation supernatant of DB403 pMA0911-kerQ7.

(4) Add the fermentation seed liquid of step (1) and the supernatant of step (3) to the solid culture medium of step (2). The inoculation amount of the bacterial liquid is 6%, in which the fermentation supernatant of DB403 pMA0911-kerQ7 , the volume ratio of lactic acid bacteria fermented seed liquid, yeast fermented seed liquid, and spore fermented seed liquid is 1:3:2:1, and the lactic acid bacteria are Lactobacillus bucheri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium, among which The volume ratio of the fermentation seed liquid of Lactobacillus reuteri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium is 1:1:1:1; the water content is controlled to be 45%; calculated according to the mass of the solid culture medium in step (2): Add 7.95g/kg (W/W) ferrous sulfate, 36.75mg/kg (W/W) copper sulfate and 0, 270, 540, 1080, 2160mg/kg (W/W) manganese sulfate and stir evenly. The fertilizer and water agent added with copper sulfate was used as the control group, and anaerobic fermentation was carried out. The fermentation temperature was 30°C and the fermentation time was 7 days. After the fermentation, a high-efficiency fertilizer and water agent (fermentation fertilizer and water agent) was obtained;

Determine the acetic acid content, total organic acid content, crude protein content, small peptide content, amino acid content, and iron chelation rate of the fertilizer and water agent. For the measurement methods, see Test Methods 2, 3, 4, 5, and 6. The measurement results are shown in Table 7; The results showed that compared with the fertilizer and water agent without manganese sulfate added, as the amount of manganese sulfate added increased, the chelation rate of manganese increased. When 540 mg/kg manganese sulfate is added, the acetic acid content, total organic acid content, small peptide content, and amino acid content of the fermented fertilizer aqueous solution reach the optimal level. At 2160mg/kg manganese sulfate, although the chelation rate of manganese is increased, too much manganese sulfate leads to a decrease in fermentation products related to fertilizer and water agents, including a decrease in the content of organic acids, small peptides and free amino acids, so fermented fertilizer and water agents are selected. The added amount of manganese sulfate is 540mg/kg, which is used for the culture of step (5).

Table 7. Measurement results of the fermented fertilizer liquid itself in Example 3

(5) Transfer the Isochrysis Zhanjiang seed liquid (preparation steps are the same as Example 1) to 10L of F/2 artificial seawater culture medium at an inoculum volume of 10%, and control the initial algae density to 5×10 ⁵ / mL, the preparation of Isochrysis Zhanjiang seed liquid is as shown in S1; then add the fermented fertilizer aqueous solution of 540 mg/kg manganese sulfate in step (4) to the F/2 artificial Isochrysis Zhanjiang at 0.3g/L. In seawater culture medium; use unfermented fertilizer aqueous agent (Comparative Example 3) and no fertilizer aqueous agent as the control group; adjust the pH value to 8.0, the culture temperature to 26±1°C, the light intensity to 8000lux, and the light-dark ratio to 12h:12h. After 8 days, count algae cells. The counting method is shown in Determination Method 1, and the results are shown in Table 8. In the culture medium of Isochrysis zhanjiang added with 540 mg/kg manganese sulfate fermentation fertilizer, the algae density is F/2 artificial seawater. 2.45 times under culture medium (without fertilizer and water agent).

Comparative Example 3: The culture medium is the same as the solid culture medium in step (2) of Example 3, and the water content is controlled to 45%; and based on the mass of the solid culture medium in step (2): add 7.95g/kg (W/W ) Ferrous sulfate, 36.75mg/kg copper sulfate, 540mg/kg manganese sulfate, stir evenly to obtain an unfermented fertilizer aqueous agent.

Table 8. Effects of fertilizers and water agents on the growth of microalgae

Example 4:

(1) Activation culture and preparation of fermentation seed liquid of Lactobacillus bucheri, Lactobacillus curvature, Lactobacillus plantarum, Bifidobacterium bifidum, Candida primogenogens, and Bacillus subtilis, the steps are the same as in Example 1.

(2) Preparation of solid culture medium, the mass proportion of each component is the same as in Example 1, and stirred evenly.

(3) As in Example 1, obtain the fermentation supernatant of DB403 pMA0911-kerQ7.

(4) Add the fermentation seed liquid of step (1) and the supernatant of step (3) to the solid culture medium of step (2). The inoculation amount of the bacterial liquid is 6%, in which the fermentation supernatant of DB403 pMA0911-kerQ7 , the volume ratio of lactic acid bacteria fermented seed liquid, yeast fermented seed liquid, and spore fermented seed liquid is 1:3:2:1, and the lactic acid bacteria are Lactobacillus bucheri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium, among which The volume ratio of the fermentation seed liquid of Lactobacillus reuteri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium is 1:1:1:1; the water content is controlled to be 45%; calculated according to the mass of the solid culture medium in step (2): Add 7.95g/kg (W/W) ferrous sulfate, 36.75mg/kg copper sulfate, 540mg/kg manganese sulfate and 0, 66, 132, 264, 528mg/kg (W/W) zinc sulfate and stir evenly. The fertilizer and water agent without zinc sulfate was used as the control group. Anaerobic fermentation was carried out at a fermentation temperature of 30°C and a fermentation time of 7 days. After the fermentation, a high-efficiency fertilizer and water agent (fermented fertilizer and water agent) was obtained;

Determine the acetic acid content, total organic acid content, crude protein content, small peptide content, amino acid content, and iron chelation rate of the fertilizer and water agent. For the measurement methods, see Test Methods 2, 3, 4, 5, and 6. The measurement results are shown in Table 9. The results showed that compared with the fertilizer and water agent without adding zinc sulfate, as the amount of zinc sulfate added increased, the chelation rate of zinc increased. When 132 mg/kg zinc sulfate is added, the acetic acid content, total organic acid content, small peptide content, and amino acid content of the fermented fertilizer aqueous solution reach the optimal level. At 528 mg/kg of zinc sulfate, although the chelation rate of zinc is increased, too much zinc leads to a decrease in fermentation products related to fertilizer and water agents, including a decrease in the content of organic acids, small peptides and free amino acids. Therefore, fermented fertilizer and water agents are selected. The addition amount of zinc sulfate is 132mg/kg, which is used in step (5).

Table 9. Measurement results of the fermented fertilizer liquid itself in Example 4

(5) After culturing the Isochrysis Zhanjiang seed liquid (the preparation steps are the same as in Example 1) to the logarithmic growth phase, transfer it to 10L of F/2 artificial seawater culture medium with an inoculum volume of 10% to control the initial algae The density is 5 × 10 ⁵ /mL. The preparation of Isochrysis Zhanjiang seed liquid is as shown in S1, and then the fermented fertilizer aqueous solution added with 132 mg/kg zinc sulfate in step (4) is added to Zhanjiang et al. at 0.3g/L. In the F/2 artificial seawater culture medium of Phytophthora chrysalis; use unfermented fertilizer aqueous agent (Comparative Example 4) and no fertilizer aqueous agent as the control group; adjust the pH value to 8.0, the culture temperature to 26±1°C, and the light intensity to 8000lux. The light-dark ratio is 12h: 12h. After 8 days of culture, algae cells are counted. The counting method is shown in Measurement Method 1. The results are shown in Table 10; Isochrysis zhanjiang is in the culture medium with 132mg/kg zinc sulfate fermented fertilizer aqueous agent added. The algae density is 2.6 times that of F/2 artificial seawater medium (without fertilizer and water agent).

Comparative Example 4: The culture medium is the same as the solid culture medium in step (2) of Example 4, and the water content is controlled to 45%; and based on the mass of the solid culture medium in step (2): add 7.95g/kg (W/W ) Ferrous sulfate, 36.75mg/kg copper sulfate, 540mg/kg manganese sulfate, 132mg/kg zinc sulfate, stir evenly to obtain an unfermented fertilizer solution.

Table 10. Effect of fertilizer and water agents on the growth of microalgae

Example 5:

(1) Activation culture and preparation of fermentation seed liquid of Lactobacillus bucheri, Lactobacillus curvature, Lactobacillus plantarum, Bifidobacterium bifidum, Candida primogenogens, and Bacillus subtilis, the steps are the same as in Example 1.

(2) Preparation of solid culture medium, the mass proportion of each component is the same as in Example 1, and stirred evenly.

(3) As in Example 1, obtain the fermentation supernatant of DB403 pMA0911-kerQ7.

(4) Add the fermentation seed liquid of step (1) and the supernatant of step (3) to the solid culture medium of step (2). The inoculation amount of the bacterial liquid is 6%, in which the fermentation supernatant of DB403 pMA0911-kerQ7 , the volume ratio of lactic acid bacteria fermented seed liquid, yeast fermented seed liquid, and spore fermented seed liquid is 1:3:2:1, and the lactic acid bacteria are Lactobacillus bucheri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium, among which The volume ratio of the fermentation seed liquid of Lactobacillus reuteri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium is 1:1:1:1; the water content is controlled to be 45%; calculated according to the mass of the solid culture medium in step (2): Add 7.95g/kg (W/W) ferrous sulfate, 36.75mg/kg copper sulfate, 540mg/kg manganese sulfate, 132mg/kg zinc sulfate and 0, 3, 15, 30, 60g/kg (W/W) Stir the calcium carbonate evenly, use the fertilizer and water agent without copper sulfate as the control group, and conduct anaerobic fermentation. The fermentation temperature is 30°C and the fermentation time is 7 days. After the fermentation is completed, a high-efficiency fertilizer and water agent (fermented fertilizer and water agent) is obtained;

Determine the acetic acid content, total organic acid content, crude protein content, small peptide content, amino acid content, and iron chelation rate of the fertilizer and water agent. For the measurement methods, see Test Methods 2, 3, 4, 5, and 6. The measurement results are shown in Table 9; The results showed that compared with the fertilizer and water agent without adding calcium carbonate, as the amount of calcium carbonate added increased, the chelation rate of calcium increased. When 15g/kg calcium carbonate is added, the acetic acid content, total organic acid content, small peptide content, and amino acid content of the fermented fertilizer aqueous solution reach the optimal level. At 60g/kg calcium carbonate, although the chelation rate of calcium is increased, too much calcium carbonate leads to a decrease in fermentation products related to fertilizer and water agents, including a decrease in the content of organic acids, small peptides and free amino acids, so fermented fertilizer and water agents are chosen. The addition amount of calcium carbonate is 60g/kg, which is used in step (5).

Table 11. Measurement results of the fermented fertilizer aqueous agent itself in Example 5

(5) Transfer the Isochrysis Zhanjiang seed liquid (preparation steps are the same as Example 1) to 10L of F/2 artificial seawater culture medium at an inoculum volume of 10%, and control the initial algae density to 5×10 ⁵ / mL, the preparation of the Isochrysis Zhanjiang seed liquid is as shown in S1, and then the fermented fertilizer aqueous solution with 60g/kg calcium carbonate added in step (4) is added to the F/2 artificial Isochrysis Zhanjiang at 0.3g/L. In seawater culture medium; use unfermented fertilizer and water agent (Comparative Example 5) and no fertilizer and water agent as the control group; adjust the pH value to 8.0, the culture temperature to 26±1°C, the light intensity to 8000lux, and the light-to-dark ratio to 12h:12h. After 8 days, count the algae cells. The counting method is shown in Determination Method 1, and the results are shown in Table 12. In the culture medium of Isochrysis zhanjiang with the addition of 60g/kg calcium carbonate fermentation fertilizer, the algae density is F/2 artificial seawater. 2.75 times under culture medium (without fertilizer and water agent).

Comparative Example 5: The culture medium is the same as the solid culture medium in step (2) of Example 5, and the water content is controlled to 45%; and according to the mass of the solid culture medium in step (2): add 7.95g/kg (W/W ) Ferrous sulfate, 36.75mg/kg copper sulfate, 540mg/kg manganese sulfate, 132mg/kg zinc sulfate and 60g/kg calcium carbonate, stir evenly to obtain an unfermented fertilizer aqueous solution.

Table 12. Effect of fertilizer and water agents on the growth of microalgae

The type and dosage of minerals have a very important impact on the growth of Isochrysis Zhanjiang. The present invention determines through screening that the minerals are ferrous sulfate, copper sulfate, manganese sulfate, zinc sulfate and calcium carbonate, and further The dosage was screened. In Example 1, by optimizing the addition amount of ferrous sulfate, 7.95g/kg (W/W) ferrous sulfate was determined to be the optimal dosage. Under these conditions, a fermented fertilizer aqueous formulation was obtained. The acetic acid content, total organic acid content, small peptide content, amino acid content, and iron chelation rate have all been significantly improved; and the algae density of Isochrysis zhanjiang in the culture medium added with fermented fertilizer water agent is 1.70 × 10 ⁷ /mL, which is twice that of F/2 artificial seawater culture medium (without fertilizer and water agent).

Then, based on the conditions for preparing the fertilizer and water agent in Example 1, the dosage of copper sulfate was further determined in Example 2, and copper sulfate at 36.75 mg/kg (W/W) was the optimal dosage; Isochrysis zhanjiang was added The algae density in the culture medium of the fermented fertilizer aqueous solution containing 7.95g/kg (W/W) ferrous sulfate and 36.75mg/kg (W/W) copper sulfate is 1.78×10 ⁷ /mL, which is F/ 2.25 times under the artificial seawater medium (without fertilizer and water agent), which is 1.05 times higher than that in Example 1.

In Example 3, 540 mg/kg (W/W) manganese sulfate was added. Isochrysis zhanjiang was added with 7.95g/kg (W/W) ferrous sulfate and 36.75mg/kg (W/W). The algae density in the culture medium of copper sulfate and 540 mg/kg (W/W) manganese sulfate fermented fertilizer and water agent is 1.95×10 ⁷ /mL, which is the density of F/2 artificial seawater culture medium (without fertilizer and water agent). 2.45 times, which is 1.10 times higher than that of Example 2.

Example 4 adds different doses of zinc sulfate, the optimal dosage is 132mg/kg; based on 7.95g/kg (W/W) ferrous sulfate, 36.75mg/kg (W/W) copper sulfate, 540mg/kg The algae density in the culture medium of fermented fertilizer aqueous solution containing (W/W) manganese sulfate and 132 mg/kg (W/W) zinc sulfate is 2.05 × 10 ⁷ /mL, which is an F/2 artificial seawater culture medium (without (fertilizer and water agent), which is 1.05 times higher than that of Example 3.

Finally, Example 5 added different doses of calcium carbonate, and the optimal dosage was 15 mg/kg (W/W); Isochrysis zhanjiang added 7.95g/kg (W/W) of ferrous sulfate, 36.75mg Fertilizer liquid containing /kg (W/W) copper sulfate, 540mg/kg (W/W) manganese sulfate, 132mg/kg (W/W) zinc sulfate, 15mg/kg (W/W) calcium carbonate The algae density in the culture medium is 2.17×10 ⁷ /mL, which is 2.75 times that under the F/2 artificial seawater culture medium (without fertilizer and water agent), 1.06 times higher than that of Example 4, and 1.28 times higher than that of Example 1 times.

In summary, the minerals defined in the present invention have achieved significant promotion effects and have good synergistic effects; at the same time, according to the growth needs of Isochrysis Zhanjiang, fermentation strains with high acetic acid production are creatively added to meet the needs of Zhanjiang and other The demand for acetic acid by Phinochrysis algae; and cloning and expression of a high-efficiency engineering bacterium for keratinase with high activity under acidic conditions, and adding the reducing agent sodium sulfite for collaborative fermentation. The final fertilizer and water agent obtained contains acetic acid, small peptides, amino acids, and mineral chelates. The synthesis rate is high; on the other hand, the minerals can also be used as nutritional supplements for Isochrysis zhanjiang, forming a highly efficient fertilizer and water agent as a whole, which has important production and practical significance.

Note: The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; therefore, although the present invention has been described in detail with reference to the above-mentioned embodiments, those of ordinary skill in the art should It is understood that the present invention can still be modified or equivalently substituted; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered by the claims of the present invention.

The sequences involved in the present invention are as follows:

1. SEQ ID NO.1

ATGTGTGTTAAGAAGAAGAACGTGATGACATCAGTGCTGCTTGCGGTGCCGCTTCTG

TTTTCAGCAGGCTTTGGAGGCAGCATGGCGAATGCAGAAAGAGTTAGCAAAACAGA

TTCAGAAAAAAGCTATATCGTCGGCTTTAAAGCAAGCGCAACAACAAATTCATCAAA

AAAGCAAGCAGTTATCCAGAATGGAGGAAAACTTGAAAAACAGTATAGACTGATTA

ACGCGGCACAAGTTACAATGAGCGAACAGGCAGCGAAAAAACTTGAACATGATCCG

AGCATTGCcTATGTTGAAGAAGATCATAAAGCGGAAGCGTATGCTCAGACAGTGCCG

TATGGACAGGAACAGATTAAaGATCCGGCAGTGCATGCGCAAGGATATAAAGGAGCA

AATGTGAAAGTGGCAGTTCTGGATACAGGCATTCATGCAGCACATCCGGATCTGAAT

GTTGCAGGAGGCGCGTCATTTGCGCCGTCAGAACCGAATGCGACACAAGATTTTCAA

TCACATGCACAACATGTTGCAGGAACAATTGCAGCACTTGATAATACAATTGGAGTT

CTGGGCGTGGCACCGAGCGCAAGCCTGTATGCGGTGAAAGTTCTTGATAGAAATGG

AGATGGCCAATATTCATGGATTATTTCAGGCATTGAATGGGCGGTGGCAAATAATATG

GATGTTATTAATATGAGCCTGGGCGGACCGAATGGCAGCACAGCGCTTAAAAAATGCG

GTGGATACAGCGAATAATAGAGGCGTTGTTGTTGTTGCAGCAGCGGGAAATAGCGGC

AGCTTTGGCAGCACATCAACAGTGGGATATCCGGCAAAATATGATTCAACAGGAGCG

GTTGCAAATGTGAAcTCAAATAATGTGAACAACAGCAGCAGCAGCGCGGGACCGGA

ACTGGATGTTAGCGCGCCGGGCACAAGCATTCTGAGCACAGTTCCGTCATCAGGATA

TACATCATATACAGCGACATCAATGGCATCACCGCATGTTGCGGGAGCAGCAGCGGTT

ATTCTTTCAAAATATCCGAATCTGACAAACAGCCAGGTTAGACAGCAACTGGAAAAT

ACAGCAACACCGCTTGGCGATAGCTTTTATTATGGCAAAGGACTTATTAACGTCCAG

GCGGCGAGCAATTAA

2. SEQ ID NO.2

MCVKKKNVMTSVLLAVPLLFSAGFGGSMANAERVSKTDSEKSYIVGFKASATTNSSKK

QAVIQNGGKLEKQYRLINAAQVTMSEQAAKKLEHDPSIAYVEEDHKAEAYAQTVPYGQ

EQIKDPAVHAQGYKGANVKVAVLDTGIHAAHPDLNVAGGASFAPSEPNATQDFQSHAQ

HVAGTIAALDNTIGVLGVAPSASLYAVKVLDRNGDGQYSWIISGIEWAVANNMDVINMS

LGGPNGSTALKNAVDTANNRGVVVVAAAGNSGSFGSTSTVGYPAKYDSTGAVANSN

NVNNSSSSAGPELDVSAPGTSILSTVPSSGYTSYTATSMASPHVAGAAAVILSKYPNLTNS

QVRQQLENTATPLGDSFYYGKGLINVQAASN

Claims (10)

1. A method for producing a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis zhanjiang, characterized in that the preparation steps are as follows: (1) Mix feather powder and secondary powder and stir evenly to obtain a solid culture medium; (2) Select lactic acid bacteria, yeast, and spore bacteria to inoculate them into the corresponding culture medium for activation and culture and obtain the corresponding fermentation seed liquid of lactic acid bacteria, yeast, and spore bacteria; (3) The keratinase gene sequence derived from Bacillus tequilensis strain Q7 was optimized according to the codon preference of Bacillus subtilis to obtain a gene encoding keratinase. The nucleotide sequence is shown in SEQ ID NO.1; this gene is The template is amplified by PCR, connected to the shuttle plasmid vector pMA and transformed into E. coli competent cells. After extracting the recombinant shuttle plasmid, it is introduced into Bacillus subtilis DB403 competent cells using chemical transformation to obtain recombinant keratinase spores. The engineering bacterium, namely DB403 pMA0911-kerQ7; cultivate the recombinant keratinase spore engineering bacterium to collect bacterial liquid, the bacterial liquid contains keratinase secreted from the extracellular secretion of the recombinant keratinase spore engineering bacterium, and the obtained bacterial liquid is centrifuged to solid-liquid After separation, collect the supernatant for later use; (4) Inoculate the fermented seed liquid of lactic acid bacteria, yeast and spore bacteria in step (2) and the cultured supernatant of DB403pMA0911-kerQ7 in step (3) into the solid medium of step (1), and then add Stir the minerals, sodium sulfite and water evenly, and obtain a high-efficiency fertilizer and water agent after fermentation; the minerals are ferrous sulfate, copper sulfate, manganese sulfate, zinc sulfate and calcium carbonate, of which the added amount of ferrous sulfate is 1.59-14.18g/kg , the addition amount of copper sulfate is 14.7-85.2mg/kg, the addition amount of manganese sulfate is 270-1080mg/kg, the addition amount of zinc sulfate is 66-264mg/kg, and the addition amount of calcium carbonate is 3-30g/kg. 2. The production method of a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis Zhanjiang according to claim 1, characterized in that the dosage described in step (1) is 10-30% of feather meal and 70-70% of secondary powder. 90%. 3. The production method of a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis zhanjiang according to claim 1, characterized in that the lactic acid bacteria described in step (2) are Lactobacillus bucheri, Lactobacillus curvature, and plant milk. Bacillus, Bifidobacterium, yeast is Candida primogen, and spore is Bacillus subtilis. 4. The production method of a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis Zhanjiang according to claim 1, characterized in that the temperature for culturing the recombinant keratinase spore engineering bacteria in step (3) is 30-37°C, The rotation speed is 180-220 rpm, and the time is 24-48 hours; the centrifugation conditions are 5000-8000 rpm, 5-10 minutes. 5. The production method of a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis Zhanjiang according to claim 1, characterized in that the supernatant of DB403 pMA0911-kerQ7 described in step (4) and the lactic acid bacteria fermentation seed liquid, The total inoculum amount of yeast fermented seed liquid and spore fermented seed liquid is 10-20%; among them, the supernatant of DB403 pMA0911-kerQ7, lactic acid bacteria fermented seed liquid, yeast fermented seed liquid and spore fermented seed liquid are The volume ratio is 1:3:2:1; the lactic acid bacteria are one or more of Lactobacillus buchneri, Lactobacillus curvature, Lactobacillus plantarum, and Bifidobacterium. When multiple species are mixed, the volume ratio is 1 . 6. The production method of a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis Zhanjiang according to claim 1, characterized in that in step (4), the water content of the culture medium is controlled to be 25%-45%, and the fermentation time is 5-12 days, fermentation temperature 20-37℃. 7. The production method of a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis Zhanjiang according to claim 1, characterized in that, based on the quality of the solid culture medium, the addition amount of sodium sulfite in step (4) is 0.1 ~2%. 8. The production method of a high-efficiency fertilizer and water agent that promotes the growth of Isochrysis Zhanjiang according to claim 1, characterized in that the ferrous sulfate addition amount is 7.95g/kg, and the copper sulfate addition amount is 36.75mg/kg. , the addition amount of manganese sulfate is 540mg/kg, the addition amount of zinc sulfate is 132mg/kg, and the addition amount of calcium carbonate is 60g/kg. 9. A high-efficiency fertilizer and water agent prepared according to the method of any one of claims 1 to 8. 10. The use of the high-efficiency fertilizer and water agent according to claim 9 for promoting the growth of Isochrysis Zhanjiang, characterized in that the application method is: directly throwing the prepared high-efficiency fertilizer and water agent into the breeding pond every 1 to 10 days. Once, the dosage each time is 0.3g/L-1.2g/L.