CN116286461B - Bacillus salicinus with soil improvement effect, screening method and application thereof - Google Patents
Bacillus salicinus with soil improvement effect, screening method and application thereof Download PDFInfo
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- 239000002689 soil Substances 0.000 title claims abstract description 114
- 230000006872 improvement Effects 0.000 title claims abstract description 27
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title abstract description 16
- 230000000694 effects Effects 0.000 title abstract description 15
- 238000012216 screening Methods 0.000 title abstract description 7
- 239000003208 petroleum Substances 0.000 claims abstract description 16
- 241000971627 Halobacillus dabanensis Species 0.000 claims abstract description 5
- 150000003839 salts Chemical class 0.000 abstract description 60
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 abstract description 32
- 239000003513 alkali Substances 0.000 abstract description 31
- 230000015556 catabolic process Effects 0.000 abstract description 20
- 238000006731 degradation reaction Methods 0.000 abstract description 20
- 239000011780 sodium chloride Substances 0.000 abstract description 19
- 230000012010 growth Effects 0.000 abstract description 16
- 239000002283 diesel fuel Substances 0.000 abstract description 14
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 abstract description 10
- 230000001105 regulatory effect Effects 0.000 abstract description 4
- 239000001384 succinic acid Substances 0.000 abstract description 4
- 150000007524 organic acids Chemical class 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 241000516792 Bacillus salis Species 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
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- 238000011282 treatment Methods 0.000 description 47
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 240000006394 Sorghum bicolor Species 0.000 description 2
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- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 2
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
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- 235000015097 nutrients Nutrition 0.000 description 2
- 239000003415 peat Substances 0.000 description 2
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- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
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- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
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- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- 241001673062 Achromobacter xylosoxidans Species 0.000 description 1
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- 241000192125 Firmicutes Species 0.000 description 1
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- 241001467552 Mycobacterium bovis BCG Species 0.000 description 1
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- 229910019142 PO4 Inorganic materials 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000006383 Salimicrobium halophilum Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
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- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- -1 salt ion Chemical class 0.000 description 1
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- 239000011975 tartaric acid Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- General Engineering & Computer Science (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Abstract
Bacillus salicifolius with soil improvement effect, screening method and application thereof, named NBL-BS214 (Halobacillus dabanensis strain), wherein the strain is preserved in China Center for Type Culture Collection (CCTCC) in the year 2022, and the preservation number is CCTCC NO:20221370 the bacillus salis NBL-BS214 has strong environmental adaptability, has certain tolerance to salt, PH and petroleum, has proper growth of sodium chloride concentration of 5-15%, and has 15% of optimal salt content; the growth PH range is 4-11, and the optimum PH is 7; the diesel oil can grow when the content is within 15%, wherein the diesel oil is most suitable for growth when the addition amount is 1%, the bacillus salicifolius NBL-BS214 is high in yield of organic acid, the succinic acid content is highest, the degradation rate of salt reaches 43.88%, the degradation rate of PH reaches 20.19%, the degradation rate of soil petroleum is more than 76%, and the bacillus salicifolius NBL-BS214 has the capabilities of reducing salt, alkali and petroleum and has the function of regulating the pH value of soil, so that the soil is improved.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus salicillus with soil improvement function, a screening method and application thereof.
Background
Saline and alkaline earth are collectively referred to as saline-alkaline land, and the soil contains too much salt (greater than 0.3%) to cause the aboveground crops to fail to grow or the yield to be significantly reduced. Soil salinization is one of the world problems, and severely restricts agricultural development. It is counted that saline-alkali soil is not less than 9.5 hundred million hectares worldwide, of which 1 hundred million hectares exist in our country, and are mainly distributed in northeast, northwest, north China and coastal areas. At present, soil salinization has become one of the research hot problems of global researchers, and people and researchers are very important to consider the improvement and sustainable utilization of saline soil.
Currently, the measures for treating saline-alkali soil include four aspects: (1) The water conservancy is improved, and the soil salt is continuously leached and removed by means of irrigation, drainage, desilting, rice planting and other measures, so that the defect of waste of water resources is overcome; (2) The physical measures are improved, mainly methods such as leveling land, deep ploughing and fine cropping, fertilization, rotation cropping and interplanting are adopted, the structure and permeability of the soil can be effectively improved, and the investment is large; (3) The chemical mode improvement is mainly to apply an improvement substance, such as gypsum, calcium sulfite and the like, which can improve the physicochemical property of soil and reduce or eliminate salt to a certain extent, but the performance is single, and the chemical mode improvement can achieve good effect only by being mixed with other methods; (4) Biological improvement mainly comprises the measures of salt-tolerant crop selection, effective microorganism utilization, bio-organic fertilizer application and the like, has the advantages of natural and natural property, less damage to the ecological environment and the like, protects ecological balance, restores and improves salty soil compared with other improvement measures, and is popular with researchers in recent years.
In the prior art, the Chinese patent application No. 201710644204.2 discloses a method for improving saline-alkali soil by utilizing beneficial microorganism bacteria, which comprises the steps of domesticating the beneficial microorganism bacteria, inoculating the mixture of biochar, peat moss, corncob and coconut husk, taking the mixture of the biochar, the peat moss, the corncob and the coconut husk as an attached substrate of the beneficial microorganism bacteria, effectively improving the activity of the microorganism bacteria, increasing the number of the beneficial microorganism bacteria, and then scattering the beneficial microorganism bacteria into the saline-alkali soil to be improved, thereby effectively improving the activity of the saline-alkali soil. The Chinese patent application No. 201010519297.4 discloses a microbial composite microbial agent for repairing petroleum-polluted saline-alkali soil, which is prepared by mixing 3 strains of bacillus megatherium P9 strain, pseudomonas P4 strain and Achromobacter xylosoxidans P2 strain, microbial thalli obtained by liquid culture with nutrients and compounding a surfactant, and has higher removal efficiency on petroleum pollution in the saline-alkali soil and simultaneously reduces the PH of the saline-alkali soil.
Most of the bacteria used in the prior saline-alkali soil improvement patents are mixed strains, mainly exert the aim of saline-alkali soil improvement by improving the microbial activity in soil, and lack core strains capable of simultaneously reducing salt, alkali and petroleum.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide bacillus salicillus with soil improvement function, a screening method and application thereof.
The technical scheme adopted for solving the technical problems is as follows: bacillus salicinus, named NBL-BS214 (Halobacillus dabanensis strain), which is preserved in China center for type culture Collection (CCTCC NO) at 20/09/2022: 20221370, the amino acid sequence of which is shown in SEQ ID NO. 1.
The screening method of the bacillus salicilis comprises the following steps:
(1) Sample collection:
saline-alkali soil samples are collected from areas of Clamma in the Xinjiang, petroleum pollution area, xinjiang Hetian area, tian Xian bus and Pacun, tian Xianmu Ganla town, tian Xiansi Krural, pishan county Koch Ke Fenghu Krural, pishan county Du Wazhen, tian Xianhan Ai Rike town, shandong Dongying, liaoning Jilin and the like respectively for later use;
(2) Preparing a culture medium:
the salt-containing medium contains: 10g/L of peptone, 3.0g/L of beef extract powder, 10g/L of sodium chloride, 5g/L of potassium chloride, 1g/L of sodium carbonate, 2g/L of magnesium sulfate and 0.1g/L of calcium chloride, and adjusting the PH to 9.0, wherein the content of agar in a solid culture medium is 2%;
(3) Isolation and purification of strains:
carrying out gradient dilution on the collected soil sample to obtain a diluted sample of 10 -1 ～10 -7 3 repeats are arranged on each gradient, the gradient is coated on a solid agar culture medium containing salt, the culture is carried out at 30 ℃ until single colonies grow, a inoculating loop is used for picking single colonies which grow fast and have regular colonies, scribing and separating are carried out on a corresponding solid agar culture medium plate, the culture is carried out at 30 ℃, the scribing experiment is repeated for 2-3 times, and after single colonies which have good growth vigor and stable passage appear on the plate, the single colonies are preserved on an inclined plane at 4 ℃.
The application of bacillus salicillus in soil improvement.
Further, the soil is salinized soil or petroleum polluted soil.
The beneficial effects of the invention are as follows: (1) The bacillus calmette-guerin NBL-BS214 is separated from a petroleum pollution area of the Kramam in Xinjiang, has strong environmental adaptability, has certain tolerance to salt, PH and petroleum, has proper growth of sodium chloride concentration of 5-15%, and has optimal salt content of 15%; the growth PH range is 4-11, and the optimum PH is 7; the growth can be carried out when the diesel oil content is within 15%, wherein the growth is most suitable when the addition amount is 1%.
(2) The bacillus campaigns NBL-BS214 of the invention produces organic acid with high yield, and succinic acid, citric acid, acetic acid, lactic acid and the like, wherein the content of succinic acid is the highest and is 21.92g/L, the degradation rate of salt reaches 43.88%, the degradation rate of PH reaches 20.19%, and the bacillus campaigns NBL-BS214 has the functions of reducing salt, reducing alkali and regulating the pH value of soil, thereby achieving the functions of improving salinization, salinization and alkalization of the soil.
(3) The bacillus salicillus NBL-BS214 can generate surface active substances, emulsify diesel oil, and has the degradation rate of more than 76 percent on petroleum hydrocarbon in soil, thereby achieving the effect of improving the soil polluted by petroleum.
Drawings
FIG. 1 is a colony morphology of Bacillus halophilus NBL-BS214 of the present invention.
FIG. 2 shows the microscopic examination of Bacillus salicini NBL-BS214 according to the present invention.
FIG. 3 is a tree analysis of B.campaigns NBL-BS214 of the present invention.
FIG. 4 shows the emulsification of diesel oil with different contents by Bacillus salicillus NBL-BS214 of the present invention.
FIG. 5 shows the emergence rate and plant height of sorghum according to the invention in three kinds of soil with different CK, T1 and T2.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Bacillus salicinus, designated NBL-BS214 (Halobacillus dabanensis strain), which has been deposited with China Center for Type Culture Collection (CCTCC) No. at 2022, 09 and 20: 20221370, the amino acid sequence of which is shown in SEQ ID NO. 1.
The screening method of the bacillus salicilis comprises the following steps:
(1) Sample collection:
saline-alkali soil samples are collected from the areas of the Clamma oil pollution area, the Xinjiang Hetian area, tian Xian Basilanpun, tian Xianmu Ganlawen, tian Xiansi Krural, pishan county Ke-Tiaoke county, pishan county Du Wazhen, tian Xianhan Ai Rike town, shandong Dongying, liaoning Jilin and the like respectively for later use.
(2) Preparing a culture medium:
the salt-containing medium contains: 10g/L peptone, 3.0g/L beef extract powder, 10g/L sodium chloride, 5g/L potassium chloride, 1g/L sodium carbonate, 2g/L magnesium sulfate, 0.1g/L calcium chloride, and adjusting the pH to 9.0, wherein the content of agar in the solid culture medium is 2%.
(3) Isolation and purification of strains:
carrying out gradient dilution on the collected soil sample to obtain a diluted sample of 10 -1 ～10 -7 3 repeats are arranged on each gradient, the gradient is coated on a solid agar culture medium containing salt, the culture is carried out at 30 ℃ until single colonies grow, a inoculating loop is used for picking single colonies which grow fast and have regular colonies, scribing and separating are carried out on a corresponding solid agar culture medium plate, the culture is carried out at 30 ℃, the scribing experiment is repeated for 2-3 times, and after single colonies which have good growth vigor and stable passage appear on the plate, the single colonies are preserved on an inclined plane at 4 ℃.
(4) Results: the obtained 152 salt-tolerant strains were initially screened.
To verify the performance of Bacillus salicini NBL-BS214, the inventors conducted a number of laboratory research experiments, each of which was as follows:
1. identification of species
(1) Morphological identification: the bacterial strain NBL-BS214 is cultured for 24 hours at the temperature of 30 ℃ on a saline culture medium, the bacterial colony is light yellow, round, moist and smooth in surface, opaque, clean in edge, and clear in cell morphology, the gram positive bacteria are rod-shaped, round at two ends and 0.9-1.0 mu m multiplied by 1.3-2.1 mu m, single or paired or short-chain appears, oval endophytic spores, mesogenesis and cyst expansion are carried out, and the size of the spores is 0.93-0.97 mu m multiplied by 1.39-1.66 mu m.
(2) Molecular genetics identification: the genome of the strain NBL-BS214 is used as a template, the 16SrRNA gene universal primers 27F and 1492R are used for amplification, and the amplified fragments are used for sequence determination. The 16S rRNA gene sequencing result of the strain NBL-BS214 is as follows, and the result is compared with sequences in GenBank, and shows that the similarity of the strain and Halobacillus sp. reaches 96.99% -99.66%, the obtained 16S rDNA sequence is subjected to BLAST search in NCBI, and the known strain 16SrDNA sequence closest to the obtained 16S rDNA sequence is downloaded and used for constructing a phylogenetic tree. Multiple sequence alignment was performed using clustalW, the alignment was performed using MEGA5.10 software and using Neighbor-Joining statistical methods, bootstrap values were set to 1000, and 16SrDNA sequence based phylogenetic trees were constructed using Construct/Test Neighbor-Joining Tree base substitution patterns. The genetic evolutionary distance was calculated using the method d of transitions+transitions in MEGA5.10, and the specific phylogenetic tree is shown in FIG. 3, and the result shows that NBL-BS214 has the closest evolutionary relationship with NR 042860.1Halobacillus dabanensis strain D-8, thus the NBL-BS214 strain was named Halobacillus dabanensis strain.
2. Determination of saline-alkali reducing ability of strain
(1) Fermentation of bacteria: inoculating the bacteria on the inclined plane into a liquid saline culture medium (taking the saline culture medium without inoculating bacteria as a reference), culturing at 30 ℃ and 150rpm for 48 hours, and measuring the salt content and the pH value in the fermentation liquor.
(2) And (3) measuring the salt content of the fermentation liquor: by adopting a silver nitrate precipitation titration method, 2.0mL of sample liquid is taken in a 100mL volumetric flask, distilled water is added to fix the volume to a scale (if the salinity is low, the sample liquid can be properly sucked up). 25mL of the sample solution was taken up in a conical flask, two drops of phenolphthalein were added, the solution was neutralized with dilute NaOH solution (pink), 1mL of potassium caseinate solution (5% (W/V)) was added, titrated with silver nitrate until a brick red precipitate appeared, the volume of consumed silver nitrate was recorded, and three parallel experiments were performed.
The salt content calculation formula is:
wherein: x represents the content of sodium chloride in each hundred milliliters of sample, and the unit is g (g/V);
c represents the actual concentration of the silver nitrate standard titration solution, and the unit is mol/L;
v represents the volume of the sucked sample in mL;
V 0 the volume of the silver nitrate standard solution consumed in the blank test is expressed in mL;
V 1 the volume of the silver nitrate standard solution consumed in the sample is expressed in mL;
25 represents the sample taken per titration in mL;
100 represents the total volume of the sample diluent in mL;
0.05844 it shows that each 1.0mL of silver nitrate titrant (0.1 mol/L) corresponds to 0.05844g of sodium chloride.
(3) And (3) measuring the pH value of the fermentation liquor: a Sidoris PB-10 acidometer was used.
(4) The method for measuring the salt content and the PH degradation rate comprises the following steps:
salt degradation% = (salt content of non-inoculated bacteria-salt content in fermentation broth of inoculated bacteria)/salt content of non-inoculated bacteria ×100
PH degradation Rate = (pH value of non-inoculated bacteria-pH value of fermentation broth of inoculated bacteria)/pH value of non-inoculated bacteria × 100
The measurement results are shown in Table 1: wherein 6 strains of bacteria have a salt degradation rate of more than 25%, a PH value degradation rate of more than 18%, the best effect is BS214, the salt degradation rate of 43.88%, and the PH degradation rate of 20.19%, and the strain is separated from a region polluted by petroleum in Kramam in Xinjiang.
TABLE 1 determination of the salinity and PH degradation Capacity of different strains
| Strain numbering | Salt degradation rate% | PH degradation rate% |
| BS165 | 26.40 | 18.93 |
| BS125 | 25.30 | 18.37 |
| BS162 | 27.50 | 19.15 |
| BS141 | 33.98 | 18.82 |
| BS137 | 37.39 | 20.27 |
| BS214 | 43.88 | 20.19 |
3. Acid energy production measurement of strain
(1) Reagent: oxalic acid standard, tartaric acid standard, malic acid standard, lactic acid standard, acetic acid standard, citric acid standard, succinic acid standard, monoammonium phosphate, and chromatographic methanol. The above standards were purchased from Shanghai Seiyaka Biotechnology Co.
(2) Chromatographic separation conditions:
chromatographic column: c18 column 4.6mm×200mm;
mobile phase: phosphate solution (20 mM monoammonium phosphate, pH 2.3): 100% methanol=98:2;
flow rate: 0.8mL/min;
detection wavelength: 276nm;
liquid inlet amount: 10 mu L.
(3) Acid energy production of strain is measured: the strain is inoculated into a saline liquid culture medium, fermented for 2d, the fermentation broth is centrifuged, the supernatant is filtered by a sterile filter membrane, 10 mu L of the supernatant is accurately absorbed and injected into a high performance liquid chromatograph for separation and measurement, the retention time of a standard peak is used for qualitative, and the peak area external standard method is used for quantitative determination.
(4) The measurement results are shown in Table 2:
TABLE 2 determination of acid production by strains
The results indicate that NBL-BS214 produces high levels of organic acids, producing succinic acid, citric acid, acetic acid, lactic acid, etc., with the highest succinic acid content being 21.92g/L.
4. Evaluation of tolerance to bacterial species
(1) Test method
1) Tolerance to salts: the culture medium is taken as a basic culture medium, the concentration of sodium chloride in the culture medium is adjusted to be 0%, 5%, 10%, 15%, 20%, 25% and 30% respectively, BL-BS214 strain is inoculated, and the OD value of fermentation liquid is measured after fermentation for 24 hours by taking non-inoculation as a control.
2) Tolerance to PH: the OD value of the fermentation broth was measured by inoculating BL-BS214 strain to a basic medium, which was a salt-containing medium, and adjusting pH to 4, 5, 6, 7, 8, 9, 10, 11, and by using the non-inoculated strain as a control.
3) Tolerance to petroleum: inorganic salt culture medium (disodium hydrogen phosphate 3.8g/L, potassium dihydrogen phosphate 1g/L, sodium chloride 5g/L, ammonium chloride 0.5g/L, magnesium sulfate 0.5g/L, pH7.0 is regulated), diesel oil content is 1%, 3%, 5%, 10% and 15% respectively, and non-inoculation is used as a control, and OD value of fermentation liquid for 24h is measured.
(2) The test results are shown in tables 3 to 5:
TABLE 3 OD of fermentation broths containing different concentrations of sodium chloride in the medium
TABLE 4 OD values of fermentation broths containing different pHs in the medium
| pH of the culture Medium | OD value of fermentation liquor |
| 4 | 0.43±0.03 |
| 5 | 0.51±0.01 |
| 6 | 0.73±0.02 |
| 7 | 1.39±0.05 |
| 8 | 1.14±0.02 |
| 9 | 1.03±0.01 |
| 10 | 0.85±0.02 |
| 11 | 0.69±0.01 |
TABLE 5 OD values of fermentation broths of different diesel oil contents in the medium
| Diesel content of culture medium | OD value of fermentation liquor |
| 1％ | 1.35±0.04 |
| 3％ | 1.11±0.03 |
| 5％ | 0.74±0.02 |
| 10％ | 0.65±0.01 |
| 15％ | 0.42±0.02 |
As can be seen from the above tables 3 to 5, the strain NBL-BS214 has certain tolerance to salt, PH and petroleum, the sodium chloride concentration is more suitable for growth at 5 to 15 percent, and the optimal salt content is 15 percent;
the growth PH range is 4-11, and the optimum PH is 7;
the growth can be carried out when the diesel oil content is within 15%, wherein the growth is most suitable when the addition amount is 1%.
5. Determination of Petroleum degrading ability of bacterial strain
(1) Inorganic salt culture medium (disodium hydrogen phosphate 3.8g/L, potassium dihydrogen phosphate 1g/L, sodium chloride 5g/L, ammonium chloride 0.5g/L, magnesium sulfate 0.5g/L, pH7.0 is regulated) is adopted, diesel oil content is respectively 3% and 10%, and non-inoculation is used as a reference, diesel oil emulsification phenomenon occurs in the fermentation process, and as shown in figure 4, the surface active substances are estimated to be generated.
(2) Determination of degradation capability of BS214 on diesel oil in petroleum soil
300g of soil is added into a 500mL triangular flask, 15mL of diesel oil is added, and the mixture is uniformly mixed, so that the diesel oil content in the soil is 5%. And 10% of BS214 fermentation liquor (the viable count is 1×10) is inoculated into the soil 6 cfu/mL) with non-inoculated soil as a control. Culturing at room temperature, and observing the degradation effect of the diesel oil in the soil. 2g of soil sample is taken each time. Three replicates were set for each treatment. The removed 2g soil sample was air dried in a fume hood while a 100mL Erlenmeyer flask was prepared and dried to constant weight in an oven and weighed as W1. The soil sample was air-dried and then mixed with an equal amount of anhydrous sodium sulfate, and the solvent was extracted with 30mL of methylene chloride. And then collecting the extract liquid in a triangular flask after drying and weighing, rotationally evaporating the solvent by using a rotary evaporator, weighing after drying again, wherein the weight is W2, and the weight difference between the two times is W2-W1, namely the weight of total petroleum hydrocarbon.
Calculating the content of residual total petroleum hydrocarbon in the sample according to a formula:
total petroleum hydrocarbon content (mg/kg) = (W2 (g) -W1 (g)) ×10 6 /2g
Total petroleum hydrocarbon degradation rate (%) = [ W0- (W2 (g) -W1 (g)) ×10 6 /2g]/W0
Wherein W0 is the total petroleum hydrocarbon content in the non-inoculation soil, mg/kg
Results: the degradation rate of the BS214 to the total petroleum hydrocarbon in the soil reaches more than 76 percent.
6. Alleviating crop salt stress
(1) Test reagent: 3% sodium hypochlorite, mixed salt (NaHCO) 3 8.41, na 2 CO 3 10.6g of Na 2 SO 4 14.2g of NaCl 5.85g
(2) Test treatment: soil treatment the soil of gathering the plough layer, through air-drying, grinding, sieving (20 mesh), set up 5 treatment groups, respectively: the CK 0-soil is not added with salt; adding mixed salt into CK1 and T1-soil to enable the salt concentration in the soil to reach 0.6%; the mixed salt is added into the CK2 and T2-soil, so that the salt concentration in the soil reaches 1.2%.
Seed disinfection treatment: 5 repetitions are arranged for each treatment, 20 seeds are sowed for each repetition, 3% sodium hypochlorite is adopted for disinfection for 3min for each treatment group of wheat seeds, alcohol disinfection is carried out for 30s, and clean water is used for washing; then, the seeds of the groups CK0, CK1 and CK2 are soaked in clear water for 2 hours and sown into soil; BS214 bacterial liquid for T1 and T2 groups (number of viable bacteria is 1×10) 6 cfu/mL) was soaked for 2 hours and sown into the soil.
And (3) sowing and post-treatment: the seeds are covered with soil after sowing, and the seeds are placed at room temperature for greenhouse culture with a light-dark period of 14hr/10hr, and tap water is properly poured in the period. Spraying BS214 bacterial liquid (the number of viable bacteria is 1 multiplied by 10) on the T1 and T2 groups 1d and 7d after sowing 6 cfu/mL), the CK0, CK1, CK2 group did not do this. And counting the germination rate of the wheat after 20 days, and measuring the corresponding index and the soil index of the wheat plants.
(3) And (3) measuring indexes:
A. germination percentage =germination number/seeding number ×100
B. Plant height: the whole wheat is dug out, the root system is cleaned and the water is sucked by filter paper. Wheat was placed flat on a bench and the length of the plant from the rhizosphere to the tip of the top leaf blade was measured using a ruler and recorded.
C. Root length: straightening the root system by forceps, and selecting the longest main root and measuring the length of the root system by a ruler.
D. Soil salinity detection: determination of soil soluble salt content by oven drying residue method
(4) Test results
1) Influence of different treatments on wheat germination rate
TABLE 6 influence of different treatments on wheat germination rate
| Treatment of | Germination percentage (%) |
| CK0 | 89.4±5.8 |
| CK1 | 46.3±4.3 |
| T1 | 75.8±6.3 |
| CK2 | 27.1±3.5 |
| T2 | 42.5±1.4 |
As can be seen from table 6, the addition of 0.6% and 1.2% salt stress resulted in a 48.21% and 69.69% decrease in wheat germination rate, respectively, compared to normal soil. Under the salt stress of 0.6% and 1.2%, the germination rate can be respectively improved by 63.71% and 56.83% by soaking the seed-combined bacterial liquid with the BS214 bacterial liquid to treat the soil.
2) Influence of different treatments on wheat growth index
TABLE 7 influence of different treatments on wheat growth index
| Treatment of | Height of plant (cm) | Root length (cm) |
| CK0 | 15.3±2.8 | 4.8±0.7 |
| CK1 | 6.4±4.3 | 3.5±0.9 |
| T1 | 10.8±1.3 | 5.8±0.4 |
| CK2 | 4.1±3.5 | 2.2±0.6 |
| T2 | 7.5±1.4 | 3.5±0.8 |
As can be seen from Table 7, under the salt stress of 0.6% and 1.2%, the soil is treated by soaking the seed binding bacterial liquid with the BS214 bacterial liquid, so that the plant height of wheat can be increased by 68.75% and 82.93%, and the root length can be increased by 65.71% and 59.09%, respectively.
3) Influence of different treatments on soil salinity
TABLE 8 influence of different treatments on the salt content in the soil
| Treatment of | Salt (%) |
| CK0 | 0.09±0.02 |
| CK1 | 0.54±0.13 |
| T1 | 0.38±0.04 |
| CK2 | 1.10±0.25 |
| T2 | 0.87±0.07 |
As shown in Table 8, the salt reduction rates of the soil treated with the BS214 bacterial liquid under the salt stress conditions of 0.6% and 1.2% were 29.63% and 20.91%, respectively.
7. Saline-alkali soil improvement potting test evaluation
(1) Test sample: collecting east nutrient saline-alkali soil, wherein the soil salinity is 0.82%, and the PH value is 8.3-8.5.
(2) Test bacterial agent: activating BS214 with NB liquid culture medium, fermenting at 30deg.C, culturing at 180r/min for 48h, centrifuging at 4000r/min in 4 deg.C refrigerated centrifuge for 15min, drying the centrifuged thallus precipitate to obtain bacterial powder, and making the bacterial powder into 1×10 with purified water 6 The cfu/mL bacterial liquid is reserved.
(3) And (3) test design: the test set up 3 groups of: the CK group is pure saline-alkali soil, the T1 group is treated by bacillus salicifolius NBL-BS214, and the T2 group is a salt washing group soaked in large water. Sowing sorghum seeds, wherein the CK group is normally subjected to water management during the period, the T1 group is subjected to water management equal to the CK group, bacterial liquid is sprayed every 7d, and the T2 group is subjected to soaking and salt washing treatment by adopting water with the quantity which is 5 times that of the CK group every 7 d.
(4) Detection index
1) Soil salinity determination: the salt content in the soil of different groups before and after treatment is measured by adopting a silver nitrate precipitation titration method.
2) Soil pH value measurement: section 2 was tested using NYT1121.2-2006 soil: the method in the determination of soil pH detects the pH value of the soil of different groups before and after treatment.
3) Determination of emergence Rate and average plant height
(5) The test results are shown in Table 9,
TABLE 9 emergence rates and average plant heights at different pH and salinity
The results show that after the saline-alkali soil is treated by bacillus salicilis NBL-BS214, the salt content is reduced, the emergence rate and the average plant height are obviously improved compared with those of a control group, the effect is better than that of a large water soaking salt-washing group, and the average plant height is improved by 43 percent, as shown in figure 5.
8. Salt-tolerant alkali-reducing bacterium improved saline-alkali soil field test
1. Test protocol
Simulating salinization: the method simulates saline-alkali soil by adding exogenous saline-alkali into test soil, and is used for an exemplary test of improving the saline-alkali soil by the salt-tolerant alkali-reducing bacteria. According to the salt content and various salt ion contents of the salt alkaline soil of Huang-Huai-Hai plain (mg/kg: na) + 0.2656-6.8978、K +
0.005-0.117、Mg 2+ 0.5、Ca 2+ 0.099-0.527、HCO 3 2- 0.2、SO 4 2- 0.276-1.334、Cl - 0.175-14.744), sodium chloride, calcium chloride, magnesium sulfate, sodium bicarbonate and sodium hydroxide are added into the soil in proportion to salinize the soil.
The treatment method comprises the following steps: the test area was divided into 5 cells, which were treated for CK1, CK2, T1, T2, T3, respectively. Wherein, CK1 is normal soil which is not subjected to salinization treatment, and CK2, T1, T2 and T3 are subjected to corresponding improvement treatment after salinization. Wheat is planted after the treatment is finished, and the variety is Shengmai 711.
Table 10 salt-tolerant alkali-degrading bacteria improved saline-alkali soil field test
| Treatment mode (the following are the addition amount per mu) | |
| CK1 | Deep ploughing, normal agronomic management |
| CK2 | Deep turning and watering for 20 tons |
| T1 | After deep ploughing, 25L of salt-tolerant alkali-reducing bacteria liquid is irrigated with 20 tons of water |
| T2 | 50L of salt-tolerant alkali-reducing bacteria liquid after deep ploughing is irrigated with 20 tons of water |
| T3 | After deep ploughing, 75L of salt-tolerant alkali-reducing bacteria liquid is irrigated with 20 tons of water |
Salt-tolerant alkali-reducing bacteria liquid: BS214 adopts NB liquid culture medium to perform activation and fermentation culture, and culture at 30deg.C and 180r/min for 48h to make viable count reach 10 8 cfu/mL。)
And (3) measuring indexes: soil index: and respectively measuring the pH value, the EC and the water-soluble total salt content of the original soil, the soil after salinization and the alkali-resistant salt-reducing bacteria of the test plot after 7d and 21 d.
Crop indexes: the emergence rate of wheat, the plant height of 60d and the post-harvest yield index (spike number, thousand grain weight, yield). Theoretical yield (kg/mu) =ear count (ten thousand/mu) ×ear count (grain) ×thousand grain weight (g)/100×85%.
2. Test results:
(1) Influence of different treatments on wheat soil index
TABLE 11 effects of different treatments on soil pH changes
| Treatment of | Raw soil | After salinization | 7d after improvement | 21d after improvement |
| CK1 | 7.73 | 7.63 | 7.53 | 7.65 |
| CK2 | 7.78 | 8.27 | 8.09 | 8.31 |
| T1 | 7.64 | 8.33 | 7.97 | 7.87 |
| T2 | 7.61 | 8.38 | 7.94 | 7.85 |
| T3 | 7.65 | 8.26 | 7.77 | 7.74 |
TABLE 12 effects of different treatments on soil conductivity
| Treatment of | Raw soil | After salinization | 7d after improvement | 21d after improvement |
| CK1 | 352us/cm | 162.8us/cm | 289us/cm | 209us/cm |
| CK2 | 289us/cm | 5.91ms/cm | 3.85ms/cm | 4.02ms/cm |
| T1 | 305us/cm | 5.74ms/cm | 2.96ms/cm | 2.77ms/cm |
| T2 | 453us/cm | 6.85ms/cm | 1.89ms/cm | 1.57ms/cm |
| T3 | 352us/cm | 6.18ms/cm | 1.73ms/cm | 1.46ms/cm |
TABLE 13 Effect of different treatments on soil Water soluble Total salt content
Results: the pH, EC value and water-soluble total salt content of the soil are obviously reduced after each treatment improvement scheme is implemented. The pH values of T1, T2 and T3 are respectively reduced by 0.36, 0.44 and 0.49 units before the salt-tolerant alkali-reducing bacterial agent is applied for 7 days, and the reduction degree of the alkalinity is positively correlated with the application amount of the salt-tolerant alkali-reducing bacterial agent. The CK2 is only reduced by 0.14 units compared with the CK before improvement, which shows that the salt-tolerant alkali-reducing microbial inoculum has better effect on reducing the alkalinity of saline-alkali soil than that of large water-washed salt. The EC value and the water-soluble total salt content of the three treatments T1, T2 and T3 are also obviously reduced, the reduction degree is increased along with the increase of the salt-tolerant alkali-degrading bacterial liquid, and the medium salinization degree of the three treatments before being improved is reduced to light salinization. The EC value and the water-soluble total salt content are obviously reduced after CK2 treatment and large water washing salt treatment, but the reduction degree is lower than that of T1, T2 and T3 treatment, and the water-soluble total salt content after improvement is 0.571 percent and still is the moderate salinization degree. The alkalinity and the salt content of the soil still tend to be reduced after the salt-tolerant alkali-reducing bacteria are applied for 21d, and the improvement of the salt-tolerant alkali-reducing bacteria on the saline-alkali soil can be seen to be long-lasting.
(2) Influence of different treatments on wheat index
TABLE 14 Effect of different treatments on wheat emergence rates
| Treatment of | CK1 | CK2 | T1 | T2 | T3 |
| Emergence rate of seedlings | 92.2％ | 75.8％ | 83.7％ | 84.2％ | 87.6％ |
TABLE 15 influence of different treatments on the 60d plant height of wheat growth
| Treatment of | CK1 | CK2 | T1 | T2 | T3 |
| 60d | 17.59 | 15.95 | 16.77 | 17.18 | 17.31 |
TABLE 16 influence of different treatments on wheat yield
Results: after improvement of salt-tolerant alkali-reducing bacteria, the emergence rate of the wheat seeds treated by the three treatments of T1, T2 and T3 reaches more than 80%, wherein the emergence rate of the T3 reaches more than 87.6%, the emergence rate is close to the emergence level of normal soil CK1, and the emergence rate of CK2 is only 75.8%. The plant height of wheat growing for 60d is equal to that T1, T2 and T3 which are subjected to salt-tolerant alkali-degrading bacteria treatment are superior to CK2 treatment. After the salt-tolerant alkali-degrading bacteria are applied to the saline-alkali soil, the saline-alkali degree of the soil is reduced, and the tolerance of the wheat plants to the saline-alkali soil is improved. As can be seen from the wheat yield index, the yields of T1, T2 and T3 are respectively improved by 23.9%, 36.0% and 42.4% compared with CK2, the yield is positively correlated with the addition amount of salt-tolerant alkali-reducing bacteria, and the treatment of T3 is only 4.8% lower than that of normal soil CK 1. The indexes such as the spike number, the spike grain weight, the thousand grain weight and the like also basically have the same trend.
The foregoing description is only of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention.
Claims (3)
1. Bacillus sakaguchi, designated NBL-BS214 (Halobacillus dabanensis strain), was deposited at China center for type culture Collection (CCTCC NO) at year 09 and 20 of 2022: 20221370.
2. use of bacillus sakaguchi according to claim 1 for soil improvement.
3. The use of bacillus sakaguchi according to claim 2 for soil improvement, wherein the soil is salinized soil or alkalinized soil or petroleum contaminated soil.
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| WO2022227239A1 (en) * | 2021-04-30 | 2022-11-03 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Halophilic petroleum-hydrocarbon-degrading bacterium and use thereof |
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