CN110846250A - Bacillus subtilis capable of producing gamma-PGA in high yield and application thereof - Google Patents

Abstract

The invention belongs to the field of microorganisms, and particularly relates to a bacillus subtilis capable of producing gamma-PGA in a high yield and application thereof. The main technical scheme is as follows: provides a new bacillus subtilis with the preservation number as follows: CGMCC NO. 17215; the 16Sr DNA sequence is shown in SEQ ID NO 1. And based on the bacillus subtilis, an application method for improving soil environment and promoting plant growth is provided. By using the bacillus subtilis and the application method provided by the invention, high-yield gamma-PGA can be obtained at a lower temperature, so that the production energy consumption is greatly saved; effectively improve the growth performance of the plants and reduce the absorption of the plants to the heavy metals in the soil.

Description

Bacillus subtilis capable of producing gamma-PGA in high yield and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a bacillus subtilis capable of producing gamma-PGA in a high yield and application thereof.

Background

Gamma-polyglutamic acid (the English name is poly-gamma-glutamic acid, which is abbreviated as gamma-PGA) is a naturally occurring water-soluble homopolyamide, which has a large number of free carboxyl groups on its side chain and can adsorb metal cations. The research shows that the metal ions adsorbed by the gamma-PGA are mainly Ni²⁺、Cd²⁺、 Pb²⁺、Cu²⁺、Mn²⁺、Cr²⁺And the like. Based on the method, the gamma-PGA not only can be used as a functional polymer substance for environmental treatment, but also can be used for concentrating radionuclides and recovering heavy metals from the solution.

Pb has high toxicity and non-degradability, can be accumulated in soil, and can cause harm to human bodies through food chains, so heavy metal pollution has attracted wide attention of broad scholars. In recent decades, Pb pollution in the natural environment has increased dramatically due to excessive fertilization, improper disposal of industrial waste, increased vehicle exhaust emissions, and the like; particularly, the absorption of Pb by crops and vegetables in contaminated soil poses serious hazards to food safety. Therefore, the remediation of Pb-contaminated soil is very necessary and urgent for the protection of human health.

Since γ -PGA has a multi-cathode property, it is excellent for Pb²⁺、Cu²⁺The heavy metal ions have excellent chelating effect, and can be used as a heavy metal absorbent to prevent crops from absorbing excessive toxic heavy metals in soil. In addition, the gamma-PGA belongs to a pure natural biological preparation, can be naturally decomposed after entering soil to generate amino acid, and is finally absorbed and utilized by crops, so that secondary pollution to the environment can not be caused. The microporous structure and the surface functional groups of the biochar have important influence on the balance and regulation of soil nutrient ions.

However, the existing gamma-PGA is generally obtained by microbial fermentation, has the defects of long fermentation period, high fermentation energy consumption, low efficiency and the like, and cannot be produced on a large scale.

Therefore, the strain capable of realizing low energy consumption and high yield of the gamma-PGA is provided, is used in the fields of crop growth promotion, soil treatment and the like, and has important practical significance for promoting the sustainable development of agriculture in China.

Disclosure of Invention

The invention aims to provide a bacillus subtilis with high yield of gamma-PGA and application thereof, and mainly aims to improve the soil environment and promote the growth of plants.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: the bacillus subtilis is preserved in China general microbiological culture Collection center with the preservation name: the Bacillus subtilis8-2 has a preservation number of: CGMCC NO. 17215.

Correspondingly, the 16Sr DNA sequence of the bacillus subtilis is shown as SEQ ID NO 1.

Correspondingly, the bacillus subtilis is applied to the production of gamma-PGA.

Preferably, the application temperature is 28 ℃.

Accordingly, an application of the bacillus subtilis in promoting plant growth comprises the following steps:

(1) obtaining pretreated soil by mixing soil of a plant to be planted with charcoal;

(2) plants were planted in the pretreated soil and then irrigated with the bacillus subtilis fermentation broth.

Preferably, the ratio of the soil to the biochar is 95:5 by mass.

Preferably, the fermentation temperature of the bacillus subtilis fermentation liquor is 28 ℃.

Preferably, the culture medium for culturing the bacillus subtilis to obtain the fermentation broth is an E-type fermentation culture medium, and the formula of the culture medium comprises: 16g/L of citric acid, 50g/L of glutamic acid, 12g/L of ammonium sulfate, 50g/L of glycerol and K₂HPO₄2g/L，MgSO₄·7H₂O0.5g/L，FeCl₃·6H₂O 0.02g/L，MnSO₄·H₂O 0.1g/L，CaCl₂0.2g/L, solvent water, pH 7.0.

Preferably, the concentration of gamma-PGA in the soil after irrigation using the fermentation broth of Bacillus subtilis is 0.02 g/kg.

The invention has the following beneficial effects:

1. the optimum fermentation temperature of the screened bacillus is 28 ℃, and the fermentation temperature is obviously lower than the common culture temperature of 37 ℃; and the yield of the gamma-PGA produced by fermentation at 28 ℃ can reach 55g/L, which is much higher than the common yield of about 25 g/L. Therefore, the bacillus provided by the invention can obtain high-yield gamma-PGA with low energy consumption, and is very suitable for industrial production.

2. In the present invention, glycerol is used as a carbon source for culturing Bacillus. Unlike the general use of conventional carbon sources such as glucose, glycerol also stimulates polyglutamyl synthase in bacillus, which helps to catalyze the polymerization of L-glutamic acid to produce γ -PGA, thereby increasing the yield of γ -PGA. Meanwhile, the glycerol can also improve the permeability of the cell membrane of the bacillus to the gamma-PGA, thereby promoting the bacillus to secrete the gamma-PGA and further improving the yield of the gamma-PGA.

3. The gamma-PGA can promote the absorption of the celery cabbage to cations in the nutrient solution, but does not influence the absorption of the celery cabbage to anions, and the gamma-PGA has large molecular weight and contains more α -carboxyl, so that a large amount of nutrient elements such as cations and the like can be chelated to promote the absorption.

The invention also provides a biological agent for improving Pb-polluted soil and a using method thereof on the basis of the bacillus. The biological agent can be naturally degraded in soil to generate amino acid, and finally, the amino acid is completely absorbed and utilized by crops, so that secondary pollution to the environment can not be caused.

Meanwhile, the biochar used in the biological agent is activated by gamma-PGA. The biochar is not only an organic matter, but also a high-quality adsorption material, and has rich pores and large specific surface area; the pH can be increased to improve the respiratory metabolism rate of a microbial community and improve the utilization pattern of the microbes on a matrix, so that the soil fertility is improved; meanwhile, the CEC (Cation Exchange Capacity) of the soil is improved, and the absorption effect of the plants on the nutrient elements is promoted; and can also be subjected to precipitation reaction with heavy metal ions to convert the heavy metals from an effective state to a residue state. The biochar contains rich aromatic ring structures, hydroxyl carboxyl groups and other groups, so that the ion exchange sites are obviously increased, and the surface exchange activity is improved. And the cation chelated by the gamma-PGA can further react with a group on the surface of the biochar, so that the effect of passivating the heavy metal in the soil by the biochar and the plant growth promoting effect are further improved.

Drawings

FIG. 1 is a schematic diagram showing the colony morphology in LB medium of Bacillus 8-2;

FIG. 2 is a schematic diagram of the cell morphology of Bacillus 8-2 under a scanning electron microscope;

FIG. 3 is a phylogenetic tree of Bacillus 8-2;

FIG. 4 is a diagram showing the yield of γ -PGA produced by Bacillus 8-2 under different temperature conditions;

FIG. 5 is a schematic diagram showing the effect of biochar and gamma-PGA on the fresh weight of pakchoi;

FIG. 6 is a schematic diagram showing the effect of biochar and gamma-PGA on the root length of pakchoi;

FIG. 7 is a schematic view showing the effect of biochar and gamma-PGA on the cross-sectional area of a root of pakchoi;

FIG. 8 shows that the charcoal and gamma-PGA absorb Pb in pakchoi²⁺Schematic diagram of the effect of (c).

Detailed Description

The culture medium and solution of the invention are as follows:

1. type E isolation medium: 50g/L of glycerin, 10g/L of citric acid, 10g/L of sodium glutamate, (NH)₄)₂SO₄6g/L，K₂HPO₄1g/L，MgSO₄·7H₂O 0.5g/L，FeCl₃·6H₂O 0.02g/L， CaCl₂0.2g/L，MnSO₄·H₂O0.05g/L, 20g/L of agar and 7.2-7.5 of pH.

2. Type E fermentation medium: 16g/L of citric acid, 50g/L of glutamic acid, 12g/L of ammonium sulfate, 50g/L of glycerol and K₂HPO₄2g/L，MgSO₄·7H₂O 0.5g/L，FeCl₃·6H₂O 0.02g/L， MnSO₄·H₂O 0.1g/L，CaCl₂0.2g/L, solvent water, pH 7.0.

3. Seed culture medium: 5g/L of yeast extract, 10g/L of peptone, 10g/L of NaCl and 7.0 of pH.

4. LB slant preservation culture medium: 5g/L of yeast extract, 10g/L of peptone, 10g/L of NaCl, 7.0 of pH and 18-20 g/L of agar.

5. Slant activation medium: 5g/L beef extract, 10g/L peptone, 5g/L NaCl, water as solvent and 7.0-7.4 of pH; the activation culture conditions are as follows: culturing for 24-48 h at 25-37 ℃.

The first embodiment is as follows: obtaining and identifying Bacillus subtilis

1. Obtaining of Strain

(1) Extracting landfill leachate from Chengdu certain garbage treatment plant, adding 10g of leachate into 90mL of sterile water to obtain 10^-1Dilution of dilution. Keeping the diluted solution in 80 deg.C water bath for 15min to kill most of mixed bacteria, and collecting 1mL 10^-1The diluted solution is put into 9mL of sterile water to obtain 10^-2Dilution of dilution. Diluting sequentially as above to obtain 10^-3、10^-4、10^-5、10^-6、10^-7、10^-8The subsequent dilution of the diluent with the dilution degree can be directly carried out without carrying out water bath heat preservation. And (3) respectively sucking 0.2mL of the diluent of each dilution, coating the diluent on a corresponding E-type separation culture medium plate, making corresponding labels (the name of the culture medium, the name of a sample, the dilution multiple and the culture time), and then performing inverted culture at 35 ℃ in an incubator (culture at 25-37 ℃).

(2) After the strains grow on the flat plate, selecting different strains with larger colonies, viscous surfaces and wire drawing capability, inoculating the strains into a new E-type separation culture medium flat plate for strain purification, and selecting a single colony inclined plane for storage after the purified strains are purified to obtain each purified single strain.

(3) And respectively inoculating each purified strain into a corresponding fermentation medium for shake flask fermentation culture, wherein each sample is subjected to three repetitions. The culture conditions were: and (3) filling the 500mL triangular flask with 100mL samples, culturing at 35 ℃ and 240r/min for 3d, and measuring the yield of the gamma-PGA in the fermentation liquor. The determination method comprises the following steps: taking fermentation liquor, filtering to remove thallus, adding absolute ethyl alcohol with the volume of 3 times that of the fermentation liquor, centrifugally purifying the gamma-PGA, hydrolyzing the gamma-PGA for 24 hours at 110 ℃ in vacuum, and measuring the gamma-PGA by high performance liquid chromatography after derivatization. Selecting the strain with the determined result that the gamma-PGA yield is more than 20g/L, namely the strain required by the invention.

2. Identification of strains

(1) Physiological and biochemical characterization

The morphological observation and physiological and biochemical identification are determined according to the manual of identification of common bacteria systems. The strain was observed for colony morphology in plate culture, as shown in FIG. 1. The strain can grow in a large amount in 1d after being cultured at 25-35 ℃ on a beef extract peptone agar medium plate and an E-type fermentation medium agar plate, the surface of a bacterial colony is convex, the surface of the bacterial colony is not smooth and has wrinkles, the bacterial colony is white and translucent, the edge of the bacterial colony is irregular and circular, the surface of the bacterial colony has transparent liquid drops, the viscosity of the bacterial colony is high, and the bacterial colony is in a filamentous shape after being picked up.

Observing the shape of the cells under a scanning electron microscope, as shown in FIG. 2, the cells were rod-shaped and had a size of (1.25-2) × (0.7-0.9) μm, spores were formed by culturing at 37 ℃ for 2-3 days, the size of the spores was (0.4-0.6) × (0.8-1.5) μm, and the spores were oval. Gram staining is carried out on the bacterial liquid by using Solarbio gram staining solution, and the bacterial strain is gram positive bacteria.

The physiological and biochemical properties of the strain are shown in table 1. In Table 1, "+" indicates positive reaction, and "-" indicates negative reaction.

TABLE 1 comparison table of physiological and biochemical properties of the strains

Detecting items	Results	Detecting items	Results
				Fermentation of glucose	+	H2O2Enzyme assay	+
Lactose fermentation	-	Citrate test	+
				Fermentation of mannitol	-	V.P test	+
Fermentation of maltose	+	M.R test	-
				Fermentation of xylose	-	Indole test	-
Arabinose fermentation	-	Starch hydrolysis	+
				2％NaCI	++	Liquefaction of gelatin	-
5％NaCI	+	4℃	-
				7％NaCI	+	37℃	+
10％NaCI	+	45℃	+
				Anaerobic growth	-	50℃	+

(2) Molecular identification

The total bacterial DNA is extracted by a DNA kit, and is used as a template to carry out PCR amplification by adopting universal primers 27F and 1492R. And performing gel electrophoresis separation on the product, finally handing the PCR product to Shanghai bio-chemical company for sequencing, comparing the sequencing result with BLAST in NCBI database, and identifying the strain as Bacillus, which is named as Bacillus subtilis 8-2. Submitting the gene sequence obtained by sequencing to a GenBank database, selecting a 16Sr DNA sequence with higher homology, performing comparison analysis by adopting MEGA 7.0 software, and drawing a strain phylogenetic tree as shown in figure 3.

The strain is preserved in China general microbiological culture Collection center (CGMCC), and the preservation name is as follows: bacillus subtilis 8-2. The preservation number is: CGMCC NO. 17215. Hereinafter referred to simply as strain 8-2. The 16Sr DNA sequence of the strain 8-2 is shown as SEQ ID NO 1.

Example two: optimum fermentation temperature display of Strain 8-2

1. Inoculating the strain 8-2 into a slant culture medium for activation, and culturing at 35 ℃ for 12 h. Inoculating the strain activated by the inclined plane into a seed culture medium, and culturing for 12h at 35 ℃ with the rotating speed of a shaking table of 180rpm to obtain activated seed liquid. Inoculating the activated seed solution into a shake flask fermentation culture medium, wherein the inoculation amount is 5% (v/v), the liquid loading amount of the shake flask is 60mL/500mL, the rotation speed of a shaking table is 220rpm, and the activated seed solution is cultured for 24 hours at different temperatures (25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃ and 40 ℃) respectively.

And after the fermentation is finished, adding 6mol/L hydrochloric acid into the fermentation liquor to adjust the pH value to be below 3, filtering out thalli, adding 6mol/L sodium hydroxide to adjust the pH value to be neutral, adding 3 times of volume of absolute ethyl alcohol, and centrifugally purifying the gamma-PGA. And hydrolyzing with acid at 110 deg.C for 24 hr, performing derivatization and color development, and measuring gamma-PGA by high performance liquid chromatography. As a result, as shown in FIG. 4, the strain 8-2 produced γ -PGA at 28 ℃ at the highest yield of 55 g/L.

Example three: demonstration of plant growth promoting effect after gamma-PGA activated charcoal

The soil to be tested is sampled from farmland soil in double-flow county in cities of Sichuan province, the soil sample is sampled from soil layer with the surface layer of 0-20 cm, and 100mg/kg Pb is added²⁺. The Pb²⁺Is carried out by a metal solution which is Pb (NO)₃)₂. And fully mixing the metal solution with the soil, and passivating for three months. And adding biochar into the soil polluted by the heavy metal, wherein the using amount of the biochar is 5% of the mass of the soil. 2kg of the mixed soil sample is respectively weighed and placed in a plastic basin with the upper diameter of 20.5cm and the height of 12cm, and the biochar used in the embodiment is rice hull biochar.

Firstly, seedlings are grown in seedling raising pots. When the Chinese cabbage is planted, 3-4 plantlets of the Chinese cabbage with insignificant appearance difference are selected, 8mL of gamma-PGA (0, 5g/L, 10g/L and 20g/L) with different concentrations are respectively taken, and the final concentrations of the gamma-PGA are respectively (0, 0.02g/kg, 0.04g/kg and 0.08 g/kg). It should be noted that γ -PGA herein does not mean pure γ -PGA, but means a fermentation broth of strain 8-2 containing γ -PGA at a specific concentration. The specific obtaining method comprises the following steps: the strain 8-2 is fermented by shaking a flask at 28 ℃ and 220 r/min. Sampling after obtaining the fermentation liquid, adding absolute ethyl alcohol with the volume being three times of that of the fermentation liquid to purify and precipitate, and measuring under 216nm ultraviolet light to determine the accurate content of the gamma-PGA in the fermentation liquid. After the Chinese cabbages are planted and survive, top irrigation is carried out by respectively using gamma-PGA with various concentrations, and simultaneously, blank groups are set: CK, i.e., soil without γ -PGA treatment.

After the cabbage of each group grows out true leaves, thinning is carried out, and each pot keeps 2 cabbages with the same growth vigor. After thinning, continuously culturing for one week, and observing the growth condition of each group of pakchoi. Then taking out each group of pakchoi completely, cleaning with tap water and distilled water, separating aerial parts from roots, air drying, weighing, and measuring fresh weight of aerial parts and underground parts respectively. The effect of various concentrations of gamma-PGA activated charcoal on fresh weight of the overground and underground parts of pakchoi is shown in FIG. 5. When 0.02g/kg of gamma-PGA is used, the influence on the yield of the pakchoi is the largest, which shows that trace gamma-PGA has better growth promotion effect on the growth of plants. The influence of biochar and gamma-PGA with different concentrations on the root length of the pakchoi is shown in figure 6, the growth of the roots can be promoted after the gamma-PGA is added, and the growth promoting effect of the roots is the best when the gamma-PGA with the concentration of 0.02g/kg is added. The influence of biochar and gamma-PGA on the root section area of the pakchoi is shown in figure 7, which shows that the gamma-PGA can not only promote the root length, but also increase the root section diameter, and has a significant effect on promoting the plant growth.

Then, each group of pakchoi is dried in the air, put into an oven at 105 ℃ for deactivation of enzymes for 30min, and then dried at 70 ℃ to constant weight (24h), and the dry weights of the overground part and the root part are respectively measured. Pulverizing with pulverizer, sieving with 50 mesh sieve, placing in plastic sealing bag, placing in drier for testing, and determining Pb with different forms by improved BCR extraction method²⁺The content of (a). The improved BCR extraction method is carried out by referring to a method in the research on the comparison of extracting Pb and Cd in the paddy soil by using the improved BCR method and the Tessier method. The absorption of heavy metals in the stem and root of pakchoi by the treatment of gamma-PGA with different concentrations is shown in FIG. 8, and it can be seen that the best effect of adding 0.02g/kg of gamma-PGA activated charcoal can be seen, and the Pb on the stem and root of pakchoi can be remarkably reduced²⁺Absorption of (2).

Sequence listing

<110> institute of biological research of Chengdu of Chinese academy of sciences

<120> bacillus subtilis capable of highly producing gamma-PGA and application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1452

<212>DNA

<213> Bacillus subtilis

<400>1

ccactgcgca tgcctataca tgcagtcgag cggacagatg ggagcttgct ccctgatgtt 60

agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120

aaaccggggc taataccgga tggttgtttg aaccgcatgg ttcagacata aaaggtggct 180

tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240

ccaaggcaac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300

ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360

ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gttagggaag 420

aacaagtgcc gttcaaatag ggcggcacct tgacggtacc taaccagaaa gccacggcta 480

actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540

gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga 600

gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc 660

ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720

ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780

ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840

attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900

gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960

tcttgacatc ctctgacaat cctagagata ggacgtcccc ttcgggggca gagtgacagg 1020

tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080

caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca 1140

aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200

cgtgctacaa tggacagaac aaagggcagc gaaaccgcga ggttaagcca atcccacaaa 1260

tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta 1320

atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380

accacgagag tttgtaacac ccgaagtcgg tgaggtaacc ttttaggagc cagccgccga 1440

agtgacagaa tg 1452

Claims (9)

1. The bacillus subtilis is preserved in China general microbiological culture Collection center with the preservation name: the Bacillus subtilis8-2 has the preservation number: CGMCC NO. 17215.

2. A bacillus subtilis strain is characterized in that: the 16Sr DNA sequence is shown in SEQ ID NO 1.

3. Use of the Bacillus subtilis of claim 1 or 2 for the production of γ -PGA.

4. Use according to claim 3, characterized in that: the application temperature was 28 ℃.

5. Use of the Bacillus subtilis of claims 1-4 for promoting plant growth, wherein: the application comprises the following steps:

(1) obtaining pretreated soil by mixing soil of a plant to be planted with charcoal;

(2) plants were planted in the pretreated soil and then irrigated with the bacillus subtilis fermentation broth.

6. Use according to claim 5, characterized in that: according to the mass ratio, the ratio of the soil to the biochar is 95: 5.

7. Use according to claim 5, characterized in that: the fermentation temperature of the bacillus subtilis fermentation liquid is 28 ℃.

8. Use according to claim 5, characterized in that: the culture medium for culturing the bacillus subtilis to obtain the fermentation broth is an E-type fermentation culture medium, and the formula comprises: 16g/L of citric acid, 50g/L of glutamic acid, 12g/L of ammonium sulfate, 50g/L of glycerol and K₂HPO₄2g/L，MgSO₄·7H₂O 0.5g/L，FeCl₃·6H₂O 0.02g/L，MnSO₄·H₂O 0.1g/L，CaCl₂0.2g/L, solvent water, pH 7.0.

9. Use according to claim 5, characterized in that: after the fermentation liquor of the bacillus subtilis is used for irrigation, the concentration of the gamma-PGA in the soil is 0.02 g/kg.

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