CN111690576A - Bacillus belgii MMB-51 for producing biosurfactant and application thereof - Google Patents

Abstract

The invention discloses a Bacillus belgii MMB-51 for producing a biosurfactant and application thereof, wherein the Bacillus belgii MMB-51 is named as Bacillus velezensis MMB-51 in a classification way, the preservation number is CGMCC No.19099, and the steps for preparing a foliar fertilizer synergist are as follows: inoculating the strain into a seed liquid culture medium, and performing shake culture at 180rpm and 37 ℃ for 12-16h to obtain a seed liquid; transferring the seed liquid into a fermentation medium according to the proportion of 1% (v/v), and carrying out shake culture at 180rpm and 37 ℃ for 36-48h to obtain a fermentation liquid; centrifuging and collecting supernatant; adding a proper amount of HCl into the supernatant to enable the pH value of the supernatant to be 2.0, uniformly mixing, standing overnight at 4 ℃, centrifuging at 4 ℃, collecting precipitates, removing the supernatant, adjusting the residual liquid of the precipitates to be neutral by using 1M NaOH solution, and freeze-drying to obtain a biosurfactant product; the biological surfactant product is dissolved in water to prepare a solution which is used as a foliar fertilizer synergist.

Description

Bacillus belgii MMB-51 for producing biosurfactant and application thereof

Technical Field

The invention relates to the technical field of agricultural microorganisms, in particular to a bacillus belgii MMB-51 for producing a biosurfactant and application thereof.

Background

The biosurfactant is a metabolite which is secreted by microorganisms in the fermentation growth process and has certain surface activity, is a biodegradable surface active component, and has the common properties of the surfactants, such as solubilization, emulsification, dispersion, surface tension reduction and the like. Compared with the chemically synthesized surfactant, the biosurfactant also has the advantages of good safety, no toxicity, no harm, high surface activity and the like, has good compatibility with human bodies and environment, and is widely applied to the fields of detergents, petroleum exploitation, cosmetics and the like.

The surfactant can be produced by chemical synthesis and microbial fermentation, wherein the chemical synthesis method has the disadvantages of more reaction steps, high toxicity, difficult biodegradation and the like; therefore, in recent years, the production of biosurfactants by microbial fermentation has received more and more attention in domestic and foreign research. Biosurfactants can be classified into lipopeptides and lipoproteins, glycolipids, phospholipids, neutral lipids, and polymers, depending on their structure. There are many microorganisms in nature that are capable of producing surface active substances by fermentation metabolism, and they mainly include Pseudomonas (Pseudomonas sp.), Bacillus (Bacillus sp.), Arthrobacter (Arthrobacter sp.), Mycobacterium (Mycobacterium sp.), and Clostridium butyricum (Clostridium butyricum). The pseudomonas and bacillus are reported more at present, wherein the pseudomonas and bacillus mainly produce rhamnolipid biosurfactants; the latter mainly produces lipopeptide biosurfactant, and both of them have good surface activity and application prospect. The biosurfactant yield of biosurfactant producing bacteria separated and screened from an environmental sample is generally low, and the generated parts of the biosurfactant are combined with thalli of the producing bacteria, so that the extraction difficulty is increased, and the extraction purity of the biosurfactant is influenced. The low yield and the complex extraction process result in a relatively high cost of the biosurfactant, which limits the application range of the biosurfactant. In order to reduce the production cost of the biosurfactant, the current research mainly focuses on breeding high-yield strains, constructing high-yield genetic engineering bacteria, optimizing fermentation conditions and process parameters, improving extraction process and the like to improve the yield and quality of the biosurfactant, but the current really industrialized application has fewer strains, and screening high-yield functional strains and expanding the application range of the biosurfactant are still one of the key research works in the field.

China is a big agricultural country, the application of chemical fertilizers and pesticides is an important means for ensuring high yield of grains, and the contribution rate of the pesticides and the chemical fertilizers to the grain yield of China reaches 40-60%, so the importance of the pesticides and the chemical fertilizers to agriculture is self-evident. The foliar fertilizer is a fertilizer which aims at the absorption of the leaf surface of the plant and directly applies nutrients required by crops to the leaf surface, has the advantages of fast absorption, strong action, high efficiency and the like, and can be used as an important supplement for the fertilization of the root of the plant. The foliar fertilizer is mainly used for spraying nutrient elements on the surfaces of crop leaves in a liquid mode, and the nutrient elements enter the leaves through air holes on the surfaces of the leaves, so that the leaves are promoted to absorb nutrients, and the functions of promoting growth and increasing yield are exerted. Increasing the contact area of the unit foliar fertilizer liquid amount and the foliar surface or improving the adhesion of the foliar fertilizer liquid on the foliar surface is an effective way to improve the foliar fertilizer application efficiency and reduce the foliar fertilizer dosage, however, the research on the foliar fertilizer synergist is very little at present.

Disclosure of Invention

One of the objects of the present invention is to provide a strain of Bacillus beiLeisi MMB-51 which produces a biosurfactant.

The invention also aims to provide the application of the Bacillus belgii MMB-51 producing the biosurfactant in preparing a foliar fertilizer synergist.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: the Bacillus belgii MMB-51 for producing the biosurfactant is classified and named as Bacillus belgii velezensis MMB-51, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the preservation address: beijing in China, the preservation date is 12 months and 5 days in 2019, and the preservation number is CGMCC No. 19099.

The strain Bacillus velezensis MMB-51 is separated from environmental sample activated sludge in the eastern city of Daqing city of Heilongjiang province, the 16S rRNA is shown as SEQ ID No.1, and the sequence length is 1441 bp.

The invention also provides an application of the Bacillus belgii MMB-51 producing the biosurfactant in preparing a foliar fertilizer synergist, which comprises the following specific steps:

step 1: inoculating a strain Bacillus velezensis MMB-51 into a seed liquid culture medium, and performing shaking culture at constant temperature of 180rpm and 37 ℃ for 12-16 hours to obtain a seed liquid;

step 2: transferring the seed solution obtained in the step 1 into a fermentation medium which takes cane sugar as a carbon source and yeast powder as a nitrogen source according to the proportion of 1% (v/v), and carrying out shake cultivation at the constant temperature of 37 ℃ at 180rpm for 36-48 hours to obtain a fermentation solution;

and step 3: centrifuging the fermentation liquor, and collecting supernatant;

and 4, step 4: adding a proper volume of 6M HCl into the supernatant obtained in the step 3 to ensure that the pH value of the supernatant is 2.0, uniformly mixing, standing overnight at 4 ℃, centrifuging at 4 ℃, collecting precipitates, removing the supernatant, regulating the residual liquid of the precipitates to be neutral by using a 1M NaOH solution, and freeze-drying to obtain a biosurfactant product;

and 5: and (4) dissolving the biosurfactant product prepared in the step (4) in water to prepare a solution with the concentration of 5mg/L to 16mg/L, wherein the solution is used as a foliar fertilizer synergist.

Preferably, the seed liquid culture medium in step 1 comprises the following components: 2.0g/L glucose, 2.0g/L starch, 2.0g/L acid hydrolyzed casein, 2.0g/L yeast powder, 2.0g/L tryptone, 0.2g/L magnesium sulfate heptahydrate, 1.3g/L dipotassium hydrogen phosphate and high-temperature high-pressure steam sterilization at 115 ℃ for 30 min.

Preferably, the fermentation medium in step 3 comprises the following components: 10.0g/L of sucrose, 10.0g/L of yeast powder, 0.05g/L of magnesium sulfate heptahydrate, 0.325g/L of dipotassium hydrogen phosphate, and sterilizing by high-temperature high-pressure steam (115 ℃ for 30 min).

Preferably, the centrifugation speed of step 3 is 6000-; and 4, centrifuging for 15min at the centrifugal rotation speed of 10000rpm in the step 4.

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

1. the strain Bacillus velezensis MMB-51 used in the invention has the advantages of high growth speed, short fermentation period, and high activity and yield of the biosurfactant;

2. the biosurfactant produced by the Bacillus belgii has high activity, and can be directly applied to the application of a foliar fertilizer synergist.

Drawings

FIG. 1 is a phylogenetic tree of strain MMB-51 constructed based on the 16S rRNA gene sequence;

FIG. 2 is a photograph of colony morphology of the strain MMB-51;

FIG. 3 is a process flow diagram for the production of biosurfactant and foliar feed synergist.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples.

The experimental procedures in the following examples are, unless otherwise specified, conventional molecular biological procedures.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 screening and identification of strains

1.1 screening of strains

Collecting environmental sample activated sludge in the east city area of Daqing city of Heilongjiang province, filling the environmental sample activated sludge into a sterile self-sealing bag, sealing the bag, and taking the bag back to a laboratory to perform strain separation in time.

The screening process is as follows:

(1) taking 0.5g of activated sludge, adding 1mL of physiological saline (0.9% NaCl), vortex, shaking and mixing uniformly, and performing gradient dilution on the mixed solution by 10 and 10²、10³、10⁴And 10⁵Doubling;

(2) in a clean bench, 100. mu.L of the above gradient dilutions were pipetted to perform plate coating operation, and the plate was incubated in a constant temperature incubator (37 ℃) for 1 day;

(3) selecting different single colonies in a super clean bench, respectively inoculating in a seed liquid culture medium, placing in a shaking table (37 ℃, 180rpm), and culturing for 1 day; wherein the seed liquid culture medium comprises the following components: 2.0g/L of glucose, 2.0g/L of starch, 2.0g/L of acid hydrolyzed casein, 2.0g/L of yeast powder, 2.0g/L of tryptone, 0.2g/L of magnesium sulfate heptahydrate and 1.3g/L of dipotassium hydrogen phosphate;

(4) 1mL of the culture medium in the shaker was taken out into a 1.5mL centrifuge tube, 50. mu.L of the supernatant after centrifugation (12000rpm, 1min) was taken and vertically suspended in a plate (3mL soybean oil +60mL ddH)₂O) slightly puncturing the liquid drop in the center of the oil ring, and observing the diameter of the oil ring;

(5) if the diameter of the oil expansion ring is more than 4cm, the strain is purified and then preserved in a 50% glycerol seed preservation pipe (-80 ℃ refrigerator).

1.2 identification of the strains

(1) And (3) strain morphological identification: inoculating the strain to LB solid plate, culturing at 37 deg.C for 24 hr, observing colony morphology, and observing colony to obtain round, wet, and light yellow colony (as shown in FIG. 2);

(2)16S rRNA gene sequencing and phylogenetic analysis: scraping thallus from LB solid plate, extracting and amplifying 16S rRNA gene of strain, carrying out gel electrophoresis analysis on PCR product, and if target band appears, sending PCR product to Producer (Shanghai) company Limited for sequencing. And comparing the sequencing result on NCBI to preliminarily identify a specific genus, then calling a typical species with the nearest similarity of the genus on an EzBioCloud website to construct an evolutionary tree, performing sequence comparison and analysis by using Mega 6.0 software, and finally constructing a phylogenetic tree by using an adjacency method and performing phylogenetic analysis.

(3) Sequencing of 16S rRNA gene of strain and phylogenetic analysis:

the length of the 16S rRNA base sequence of the MMB-51 strain is 1441bp through sequencing, and is shown as SEQ ID NO. 1. The 16SrRNA gene sequence alignment finds that the strain belongs to a member of Bacillus (Bacillus), a typical species with the closest similarity of the genus is called on an EzBio Cloud website, the alignment finds that the strain has the highest similarity with the effective publishing species Bacillus belief of the genus, and the strain is gathered on an independent evolutionary branch (figure 1). Therefore, through comprehensive morphological observation and 16S rDNA sequence analysis, the strain MMB-51 which belongs to the members of the Bacillus is preliminarily identified to be the Bacillus belgii and named as the Bacillus velezensis MMB-51, and is preserved in the China general microbiological culture Collection center with the preservation date of 2019, 12 months and 5 days and the preservation number of CGMCC No. 19099.

EXAMPLE 2 production of biosurfactants

As shown in fig. 3, the specific steps are as follows:

step 1, preparing seed liquid: inoculating a strain Bacillus velezensis MMB-51 into a seed liquid culture medium, and culturing at 37 ℃ overnight by a shaking table at 180 rpm; wherein the seed liquid culture medium comprises the following components: 2.0g/L of glucose, 2.0g/L of starch, 2.0g/L of acid hydrolyzed casein, 2.0g/L of yeast powder, 2.0g/L of tryptone, 0.2g/L of magnesium sulfate heptahydrate and 1.3g/L of dipotassium hydrogen phosphate.

Step 2, fermentation production of the biosurfactant: inoculating the seed solution obtained in the step 1 into a fermentation culture medium according to the proportion of 1% (v/v), and performing shake fermentation culture at 37 ℃ and 180rpm for 36-48 h; wherein the fermentation medium comprises the following components: 10.0g/L of sucrose, 10.0g/L of yeast powder, 0.05g/L of magnesium sulfate heptahydrate and 0.325g/L of dipotassium hydrogen phosphate.

Step 3, extracting a biosurfactant product: centrifuging the fermentation liquor obtained in the step 2 at 6000-10000rpm for 10min, collecting supernatant, and adding the thallus precipitate obtained after centrifugation into a new fermentation medium to realize subsequent continuous fermentation; and (3) adding a proper volume of 6M HCl into the supernatant obtained in the step (3) to ensure that the pH value of the supernatant is 2.0, gently mixing the mixture uniformly, placing the mixture in a refrigerator at 4 ℃ for 12-24h, then centrifuging the mixture at 4 ℃ and 10000rpm for 15min, collecting precipitates, discarding the supernatant, adjusting the residual liquid of the precipitates to be neutral by using a 1M NaOH solution, and freeze-drying the precipitate by using a vacuum freeze-drying machine to obtain a biosurfactant product.

The experimental procedure for the product performance is as follows:

(1) under optimal conditions (37 deg.C, shaking table fermentation at 180rpm for 36-48h), collecting 1mL of culture solution in 1.5mL centrifuge tube, collecting 50 μ L of supernatant after centrifugation (12000rpm, 1min), and vertically suspending in a plate (3mL soybean oil +60mL ddH)₂O) slightly puncturing the liquid drop in the center of the oil ring, and measuring the diameter (cm) of the oil ring; the diameter of the oil expansion ring of the biosurfactant can reach more than 7 cm.

(2) Pouring the supernatant into a new 50mL centrifuge tube, adding a proper volume of 6M HCl to ensure that the pH value of the supernatant is 2.0, gently mixing the supernatant and the supernatant evenly, placing the mixture in a refrigerator at 4 ℃ for 12 to 24 hours, then centrifuging the mixture for 15min at 4 ℃ and 10000rpm, collecting precipitates, removing the supernatant, regulating the residual liquid of the precipitates to be neutral by using a 1M NaOH solution, and freeze-drying the precipitates by using a vacuum freeze-drying machine to obtain the biosurfactant product. At a concentration of 11mg/L, the surface tension value (ST) can be reduced to 28.5 mN/m.

Example 3 application of biosurfactant in preparation of foliar fertilizer synergist

Preparing a mixed solution of a foliar fertilizer and a biosurfactant with the concentration of 5mg/L to 16mg/L, and measuring the spreading area of the mixed solution on the leaf surfaces of the plants (a blank control is purified water); gradually inclining the leaf surface to enable the leaf surface fertilizer to slowly flow down the leaf surface, and calculating the residual quantity of the mixed liquid on the leaf surface. The effect of the biosurfactant as the foliar fertilizer synergist is evaluated by comparing the contact area of the foliar fertilizer mixture and the foliar surface and the residual amount of the foliar fertilizer mixed liquid on the foliar surface.

Analysis by experimental results: the uniform application amount is 200 mu L, the leaf surface spreading diameter of the control group (water) is about 1cm, and the residual amount is about 9.9 percent; the leaf surface spreading diameter of the mixed liquid of the leaf surface fertilizers in the experimental group is about 2cm, and the residual quantity can reach more than 39%.

Sequence listing

<110> university of Jiangsu profession

<120> Bacillus belezii MMB-51 for producing biosurfactant and application thereof

<160>1

<170>SIPOSequenceListing 1.0

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<211>1441

<212>DNA

<213> Bacillus belgii (Bacillus velezensis)

<400>1

ggcgcgtgct ataatgcagt cgagcggaca gatgggagct tgctccctga tgttagcggc 60

ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc cgggaaaccg 120

gggctaatac cggatggttg tttgaaccgc atggttcaga cataaaaggt ggcttcggct 180

accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg ctcaccaagg 240

cgacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag acacggccca 300

gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc tgacggagca 360

acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg gaagaacaag 420

tgccgttcaa atagggcggc accttgacgg tacctaacca gaaagccacg gctaactacg 480

tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag 540

ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg gggagggtca 600

ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg tagcggtgaa 660

atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct gtaactgacg 720

ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa 780

acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta acgcattaag 840

cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac gggggcccgc 900

acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga 960

catcctctga caatcctaga gataggacgt ccccttcggg ggcagagtga caggtggtgc 1020

atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080

ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt gacaaaccgg 1140

aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct 1200

acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca caaatctgtt 1260

ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg 1320

gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 1380

agagtttgta acacccgaag tcggtgaggt aacctttagg agccagccgc cgaagtgaca 1440

a 1441

Claims (6)

1. The Bacillus belgii MMB-51 for producing the biosurfactant is characterized by being classified and named as Bacillus belgii velezensis MMB-51, being preserved in the China general microbiological culture Collection center with the preservation date of 2019, 12 and 5 days and the preservation number of CGMCC No. 19099.

2. The use of bacillus beleisi MMB-51 producing biosurfactant according to claim 1 in the preparation of foliar fertilizer synergists.

3. The application of the bacillus beleisi MMB-51 producing biosurfactant in preparing a foliar fertilizer synergist according to claim 2 is characterized by comprising the following specific steps:

step 1: inoculating a strain Bacillus velezensis MMB-51 into a seed liquid culture medium, and performing shaking culture at constant temperature of 180rpm and 37 ℃ for 12-16 hours to obtain a seed liquid;

step 2: transferring the seed solution obtained in the step 1 into a fermentation medium which takes cane sugar as a carbon source and yeast powder as a nitrogen source according to the proportion of 1 percent by volume, and carrying out shake cultivation at the constant temperature of 180rpm and 37 ℃ for 36-48 hours to obtain a fermentation liquid;

and step 3: centrifuging the fermentation liquor, and collecting supernatant;

and 4, step 4: adding a proper volume of 6M HCl into the supernatant obtained in the step 3 to ensure that the pH value of the supernatant is 2.0, uniformly mixing, standing overnight at 4 ℃, centrifuging at 4 ℃, collecting precipitates, removing the supernatant, regulating the residual liquid of the precipitates to be neutral by using a 1M NaOH solution, and freeze-drying to obtain a biosurfactant product;

and 5: and (4) dissolving the biosurfactant product prepared in the step (4) in water to prepare a solution with the concentration of 5mg/L to 16mg/L, wherein the solution is used as a foliar fertilizer synergist.

4. The use of bacillus belgii MMB-51 producing biosurfactant for the preparation of foliar fertilizer synergists as claimed in claim 3 wherein the seed liquid culture medium in step 1 comprises the following components: 2.0g/L of glucose, 2.0g/L of starch, 2.0g/L of acid hydrolyzed casein, 2.0g/L of yeast powder, 2.0g/L of tryptone, 0.2g/L of magnesium sulfate heptahydrate and 1.3g/L of dipotassium hydrogen phosphate.

5. The use of bacillus belgii MMB-51 producing biosurfactant for the preparation of foliar fertilizer synergists as claimed in claim 3 wherein the fermentation medium in step 3 comprises the following components: 10.0g/L of sucrose, 10.0g/L of yeast powder, 0.05g/L of magnesium sulfate heptahydrate and 0.325g/L of dipotassium hydrogen phosphate.

6. The application of the Bacillus beleisi MMB-51 producing biosurfactant in preparing a foliar fertilizer synergist as claimed in claim 3, wherein the centrifugation rotation speed of step 3 is 6000-; and 4, centrifuging for 15min at the centrifugal rotation speed of 10000rpm in the step 4.

Images