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Strong bacillus G25-1-2 and applications thereof

A technology of bacillus and bacillus, applied in the field of microorganisms, can solve the problems of nitrogen fixation function that has not yet been discovered, achieve the effects of nitrogen cycle and maintenance of higher productivity levels, good nitrogen fixation effect, and convenient long-term storage and transportation

Inactive Publication Date: 2010-06-16
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The bacterium of Paenibacillus validus found in existing research has PAHs degradation function (Daane LL, 2002, PAH-degradation by Paenibacillus spp. and description of Paenibacillus naphthalenovorans sp nov., a naphthalene-degrading bacterium from the rhizosphere of Salt marsh plants, International journal of systematic and evolutionary microbiology, 2002, 52(1), 131-139) and growth-promoting function (Ulrich Hildebrandt, 2006, The bacterium Paenibacillus validus stimulates growth of the arbuscular mycorrhizal fungus Glomus to the intraradices formation of fertile spores, FEMS Microbiology Letters, 254(2), 258-267), etc., but no nitrogen fixation function has been found

Method used

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  • Strong bacillus G25-1-2 and applications thereof
  • Strong bacillus G25-1-2 and applications thereof
  • Strong bacillus G25-1-2 and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Separation and purification of strains

[0029] The sampling location of this example is the seaweed (18°15′08.6”N; 109°30′51.1”E) grown after coral degradation in the Sanya Bay Coral Reef Reserve, Sanya City, Hainan Province, China. The main seaweed species is Thalassia hemprichii). After the samples are collected, each sample is classified and numbered separately, put into a sterile sealed polyethylene bag, placed in an ice box and taken back to the laboratory, and stored at -20°C.

[0030] Take the seaweed samples collected above, take the rhizosphere (ie leaf sheath and rhizome), cut into small pieces of about 2cm with sterilized scissors, and put them into selective nitrogen fixation liquid medium (the medium contains 25g NaCl, 0.56 KCl g, MgSO 4 -7H 2 O 4.8g, MgCl 2 -6H 2 O 4.0g, K 2 HPO 4 0.01g, FeSO 4 -7H 2 00.001g, Tris 0.48g, peptone 4.0g, yeast extract 2.0g, glycerol 2.0ml and distilled water 1L, final pH is 8.2) for enrichment culture (30℃, 180rpm, cons...

Embodiment 2

[0033] Example 2 Determination of nitrogenase activity of strains

[0034] The nitrogenase activity of the bacteria in this example was measured by gas chromatograph for the Acetylene-reducing Activity (ARA) of nitrogen-fixing bacteria (refer to Capone 1993, Capone DG (1993) Determination of nitrogenase activity in aquatic samples using the acetylene reduction procedure. Aquatic Microbial Ecology, 621-631).

[0035] Take 40 μL of the reacted gas and measure the peak value of ethylene on a GC-17A gas chromatograph (Shimadzu). The concentration of ethylene in the standard gas is 138 ppm. The working conditions of the gas chromatograph are set as follows: the detection room temperature is 100℃, the column temperature is 60℃, the injection port is 60, the column length is 1m, and the head carrier gas pressure is 0.95kg·cm. -2 , Hydrogen is 0.8kg·cm -2 , The air is 0.6kg·cm -2 . The calculation method of acetylene reduction activity is:

[0036] ARA(nmol C H·H -1 ·Culture -1 )=Vst×Cst×A...

Embodiment 3

[0044] Example 3 Strain identification

[0045] Template preparation: select the pure strain obtained in Example 1 for DNA extraction and purification. For the extraction and purification of the DNA, refer to Dongxiuzhu's "Manual for Identification of Common Bacterial Systems" P 409 The DNA extracted and purified is the template used for subsequent PCR amplification in this example.

[0046] 1. Amplification of nifH gene

[0047] The universal primer PolF / PolR (refer to Poly et al., 2001) of the nifH gene of nitrogen-fixing bacteria is used for PCR amplification:

[0048] PCR reaction system: the total system is 25uL, 1×PCR Buffer, 200umol L -1 dNTPs, 2.0mmol L -1 Mg 2+ , 1umol L -1 The primers, 4ug of BSA and 2.0U of Taq DNA polymerase, template 1ul.

[0049] PCR amplification conditions: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing temperature at 55°C for 30s, extension at 72°C for 30s, and extension at 72°C for 10 minutes after 30 cycles.

[0050] Ta...

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Abstract

The invention discloses a strong bacillus G25-1-2 and applications thereof. The bacillus has been preserved by China Center for Type Culture Collection in Wuhan University (short for CCTCC) on 4th, June, 2009, and the preservation number is CCTCC NO:M209122. The bacillus is a pure bacillus with high-efficiency nitrogen-fixing activity, and is separated and sieved from a seaweed ecological system. When the strong bacillus G25-1-2 is used for processing seaweed, compared with a test control group, the processed seaweed has higher plant height, stem diameter average value, carotene content, carotenoid content and soluble saccharide content, thereby the result shows that the bacillus has good growth promotion effect on the seaweed. The strong bacillus G25-1-2 can be used for seaweed nursing and restoration of the whole seaweed ecological system, and has good promotion functions on the nitrogen circulation of the coral reef-seaweed bed composite ecological system and the maintenance of high production level.

Description

Technical field [0001] The present invention relates to the technical field of microorganisms, in particular to microbial nitrogen fixation, in particular to a Bacillus robustus G25-1-2 and applications thereof. Background technique [0002] The wetland ecosystem is a special kind of ecosystem, which produces an average of 9g / m of protein per year 2 , It is 3.5 times that of the terrestrial ecosystem, and it also has the functions of regulating water sources, stabilizing coastlines, regulating climate, purifying water quality and fixing nutrients, preserving species, providing wildlife habitats and other basic ecological benefits, providing industry and agriculture , Energy, medical industry, etc. to provide mass production raw materials. [0003] Coral reefs and seagrass beds are important components of tropical marine wetland ecosystems. High productivity and important ecological significance are their basic characteristics, but they also have the fragility and variability of wet...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01N63/02A01P21/00C05F11/08C12R1/01
Inventor 董俊德凌娟张燕英李丽璇王友绍张偲
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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