CN106399156B - Bacillus amyloliquefaciens and application thereof in gulfweed biodegradation - Google Patents
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Abstract
The invention discloses a bacillus HB12274 and application thereof in gulfweed biodegradation, belonging to the technical field of microorganisms. The Bacillus is Bacillus amyloliquefaciens subsp. The strain is preserved in the China general microbiological culture collection center for culture collection management in 2016, 5 months and 16 days, and the preservation number is CGMCC No. 12455. The bacillus HB12274 has the activity of degrading algin, cellulose and starch, and can effectively degrade gulfweed. The bacillus HB12274 and the fermentation liquor thereof can be used as a new microbial resource for degrading the gulfweed, and have good application and development prospects in the aspect of processing and utilization of the gulfweed.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus amyloliquefaciens HB12274 and application thereof in gulfweed biodegradation.
Background
Sargassum is a common large brown algae, belongs to Phaeophyta (Phaephhyta), Sargassaceae (Sargasacea) and Sargassum (Sargassum), is mostly warm water type, and is widely distributed in warm water and warm water sea areas. The gulfweed has high economic value, has great development potential in agriculture, industry and other aspects, and is an important fertilizer, feed and industrial raw material. The gulfweed cell wall is mainly composed of cellulose and algin, has stable structure and is difficult to damage. Degrading cellulose and algin in the cell wall of the gulfweed is the key for efficiently degrading and utilizing the gulfweed. The gulfweed is usually degraded by mechanical crushing and chemical degradation method in industry, but the method has the defects of high energy consumption, environmental pollution caused by waste and the like. The biodegradation method is to degrade cellulose and algin in the alga body by using cellulase and algin lyase produced by microorganisms so as to achieve the purpose of degrading the alga body, and is concerned about high efficiency and environmental protection.
Bacillus is an aerobic or facultative anaerobic bacterium, can produce stress-resistant endospores, and is one of the common microecological preparations. The bacillus can produce various extracellular enzymes, such as alginate lyase, cellulase, protease, amylase and the like, and can decompose various organic matters such as alginate, cellulose, protein, starch and the like, thereby being widely applied. The bacillus amyloliquefaciens HB12274 adopted by the invention is separated from the bottom mud of Hongkong mangrove forest of east village in Hainan province, has the activity of efficiently degrading algin, starch and cellulose, can effectively degrade sargassum fronds, and has good application and development prospects in the aspect of sargassum processing and utilization.
Disclosure of Invention
The invention aims to provide a bacillus amyloliquefaciens HB12274 capable of degrading algin, cellulose and starch, which can effectively degrade the algin, the cellulose and the starch and can be used as a novel microbial resource for degrading sargassum.
In order to achieve the purpose, the invention adopts the following technical scheme:
the bacillus amyloliquefaciens HB12274 with the activity of degrading algin, starch and cellulose, which is provided by the invention, has been subjected to strain preservation in China general microbiological culture Collection center (CGMCC) at 2016, 5, 16 and 16 days, and is proved to be alive, and the preservation registration number is CGMCC No. 12455. The saved address is: the institute of microbiology, institute of academy of sciences, China, West Lu No.1, Beijing, Chaoyang, North Chen. The Bacillus amyloliquefaciens HB12274 belongs to the bacterial domain, the phylum firmicutes, the class Bacillaceae, the order Bacillales, the family Bacillaceae, the genus Bacillus.
The process for obtaining the bacillus amyloliquefaciens HB12274 comprises the following steps: collecting substrate sludge samples from Hongshu in Dongzhai province, Hainan province, performing gradient dilution on the samples, performing water bath treatment at 80 ℃ for 20min to kill non-spore bacteria, and distributing the samples on a bacteria isolation culture medium plate in the future. And then carrying out activity determination on the algin-producing lyase, the amylase and the cellulase of the bacillus obtained by separation, and finally screening the bacillus amyloliquefaciens HB12274 with three enzyme activities.
The bacillus amyloliquefaciens HB12274 has the following biological characteristics: after the strain is subjected to streak culture on a wort nutrient agar culture medium for 3d, the bacterial colony is circular, irregular in edge, dull, uneven, milky white and opaque in surface; the bacteria are long and rod-shaped and produce spores. Gram staining, VP measurement and catalase test are positive, MR test is negative, nitrate can be reduced, starch, algin and cellulose can be hydrolyzed, and gelatin can be liquefied.
The 16S rDNA sequence of the bacillus amyloliquefaciens HB12274 has an accession number of KU529689 in GenBank. The strain is identified as Bacillus amyloliquefaciens by heterogeneous classification. The strain is easy to grow, has the capacity of degrading algin, starch and cellulose, can effectively degrade sargassum frond, and can be developed into a novel microbial agent applied to sargassum biodegradation.
The application of the bacillus amyloliquefaciens HB12274 in the biological degradation of the gulfweed comprises the following steps:
(1) strain activation: inoculating the bacillus amyloliquefaciens HB12274 into a nutrient agar culture medium, and culturing at 37 ℃ for 24-48 h. Culture medium: 8-10 g/L of peptone, 2-3 g/L of beef extract powder, 18-20 g/L of agar powder, 5-35 g/L of sodium chloride and pH 6.5-7.5.
(2) Liquid culture: inoculating the activated strain into a liquid seed culture medium, and performing shaking culture at 30-37 ℃ and 120-200 r/min for 12-24 h to prepare a seed solution. Culture medium: 1-3 g/L beef extract, 5-10 g/L peptone, 5-35 g/L sodium chloride and pH 6.5-7.5.
(3) Fermentation culture: inoculating the seed solution into a liquid fermentation culture medium according to the volume ratio of 2-10%, and carrying out shaking culture at 30-37 ℃ for 48-96 h at 150-200 r/min to obtain the liquid microbial inoculum of the bacillus amyloliquefaciens HB 12274. Culture medium: 1-3 g/L beef extract, 5-10 g/L peptone, 5-35 g/L sodium chloride and pH 6.5-7.5.
(4) Inoculating the liquid microbial inoculum with the inoculation amount of 5-10% into a conical flask filled with a sargassum powder fermentation culture medium, and performing shake cultivation for 10-15 days at the temperature of 30-37 ℃ under the condition of 150-200 r/min. Culture medium: 25-50 g/L sargassum powder, 5-10 g/L peptone, 5-35 g/L sodium chloride and pH 6.5-7.5.
Experiments show that the addition of the bacillus amyloliquefaciens HB12274 CGMCC No.12455 can effectively degrade gulfweed fronds, the weight loss rate of a solid is 50.02 percent after the sargassum fronds are cultured for 10 days, and the increment of reducing sugar in liquid is 65.6 mu g/mL. Therefore, the bacillus amyloliquefaciens HB12274 CGMCC No.12455 can be applied to the biological degradation of gulfweed fronds.
The invention is further described with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a photograph, magnification 1000, of the morphological characteristics of the strain HB12274 according to the invention.
FIG. 2 is a 16S rDNA-based phylogenetic tree of strain HB12274 according to the invention.
Detailed Description
The invention will be further illustrated with reference to specific examples:
example 1: separation of bacillus amyloliquefaciens HB12274 CGMCC No.12455
A5-point sampling method is adopted to collect sediment samples from Hongshan Hongkong nationality of east village in Hainan province. Dissolving 10g sample in 90ml sterile water, placing in constant temperature shaking table at 37 deg.C and 200r/min, oscillating for 30min, standing for 5min, and making into 10-2～10-4The series of soil suspensions. Treating with 80 deg.C water bath for 20min to kill non-spore bacteria, sucking 0.1ml of serial suspension, spreading on bacteria isolation culture medium, and placing in 37 deg.C incubator for inverted culture. When the bacterial colony grows out, selecting a single bacterial colony and streaking and purifying for 2-3 times according to the phenotypic characteristics of the shape, the color, the edge state, the transparency, the surface dry and wet state and the like of the bacterial colony until pure culture is obtained. After preliminary weight removal, numbering and storing.
Example 2 species identification of Bacillus amyloliquefaciens HB12274 CGMCC No.12455
(1) Morphological and cultural characteristics
The strain is subjected to streak culture on a wort nutrient agar culture medium, the strain grows rapidly, the diameter of a bacterial colony is 3-5 mm, the bacterial colony is circular, the edge is irregular, the surface is not glossy, uneven, milky white and opaque; the bacteria are rod-shaped, the gram staining result is positive, spores are produced, and one end of the bacteria is enlarged (figure 1).
(2) Physiological and biochemical characteristics
Strain HB12274 can utilize glucose, sorbitol, maltose, sucrose, cellobiose, galactose, mannitol, fructose, lactose and arabinose. Gram staining, VP determination, catalase test are positive, MR test is negative, nitrate can be reduced, and gelatin can be liquefied.
(3)16S rDNA sequence determination and phylogenetic analysis
The 16S rDNA sequence 1450bp of the strain HB12274 is obtained by PCR amplification and sequencing, and the accession number of the nucleotide sequence in Genbank is KU 529689. Comparing the sequence with the sequence in an EzBioCloud database, finding that the similarity of the strain HB12274 and the sequence of Bacillus amyloliquefaciens subsp. plantarum is the highest and reaches 99.5%, and the similarity of the strain HB12274 and the sequence of Bacillus amyloliquefaciens and Bacillus methylotrophicus is 99.4% and 99.3% respectively. Selection of related strains with high homology phylogenetic Tree was constructed in MEGA5.0 by Neighbor-join method (FIG. 2), and it can be seen that HB12274 and Bacillus amyloliquefaciens subspTOn the same branch, the two are closer in relationship. Comparing the morphological characteristics and physiological and biochemical characteristics of the two, finding out that the morphological characteristics are similar and the physiological and biochemical characteristics are consistent.
In summary, the strain HB12274(CGMCC No.12455) related to the invention is identified as Bacillus amyloliquefaciens, which has been subjected to strain preservation in China general microbiological culture Collection center (CGMCC) at 5-16 months in 2016 and is proved to survive, and the preservation registration number is CGMCC No. 12455. The preservation address is microbial research institute of Chinese academy of sciences, West Lu No.1 of Beijing, Chaoyang, Beijing.
Example 3: preparation of strain HB12274 CGMCC No.12455 liquid bacterial agent
(1) Strain activation: the bacillus amyloliquefaciens HB12274 is inoculated in a nutrient agar solid culture medium and cultured for 24 hours at 37 ℃. Culture medium: 10.0g of peptone, 3.0g of beef extract powder, 18.0g of agar powder, 16.0g of sodium chloride, 1000mL of agar powder, pH7.5 and sterilization at 121 ℃ for 20 min.
(2) Liquid culture: inoculating the activated strain into nutrient agar liquid culture medium, and shake culturing at 37 deg.C and 200r/min for 24 hr to obtain seed liquid.
(3) Fermentation culture: inoculating the seed solution into a nutrient agar liquid culture medium according to the volume ratio of 3%, and performing shaking culture at 37 ℃ and 200r/min for 72h to obtain the liquid microbial inoculum of the bacillus amyloliquefaciens HB 12274.
The liquid microbial inoculum of the bacillus amyloliquefaciens HB12274 is prepared according to the steps.
Example 4: activity measurement of strain HB12274 CGMCC No.12455 for degrading algin, cellulose and starch
The degradation activity of the strain HB12274 on algin, cellulose and starch was measured by the clear circle method, and the results are shown in Table 1.
And (3) measuring the produced algin lyase: taking a single colony, and spot-inoculating to sodium alginate culture medium (medium composition: sodium alginate 5.0g, (NH)4)2SO4 5.0g，K2HPO4 2.0g，NaCl 16.0g，MgSO4·7H2O 1.0g，FeSO4·7H20.01g of O, 18.0g of agar, 1000mL of distilled water and pH7.5), adding a proper amount of 95% alcohol after carrying out inverted culture at 37 ℃ for 3d, standing, wherein if the bacterial strain produces the alginate lyase, a hydrolysis ring is formed around a bacterial colony, and the ratio (Hc value) of the diameter of the hydrolysis ring to the diameter of the bacterial colony reflects the relative size of the alginate lyase. The result shows that the strain HB12274 has stronger activity of degrading sodium alginate, and the Hc value is 6.25.
And (3) amylase production determination: taking a single colony, inoculating the single colony to a starch hydrolysis culture medium (the culture medium comprises 10.0g of protein, 2.0g of soluble starch, 5.0g of beef extract, 16.0g of sodium chloride, 20.0g of agar, 1000ml of water and pH7.0), carrying out inverted culture at 37 ℃ for 3 days, and adding Lugol iodine solution into the plate. As starch turns blue when encountering iodine, if the strain produces amylase, a colorless transparent ring is displayed around a colony, and the ratio of the diameter of the transparent ring to the diameter of the colony (Hc value) reflects the relative size of the amylase activity. The result shows that the strain HB12274 has stronger starch degrading activity and the Hc value is 3.20.
And (3) determination of cellulase production: taking a single colony, and spot-inoculating to sodium carboxymethylcellulose culture medium (sodium carboxymethylcellulose 10.0g, NH)4NO3 1.0g，(NH4)2SO4 1.0g，MgSO4·7H20.5g of O, 16.0g of NaCl, 18.0g of agar, 1000mL of distilled water and natural pH), carrying out inverted culture at 37 ℃ for 3d, dyeing with 1% Congo red for 30min, and decoloring with 1mol/L sodium chloride for 20min (2 times), wherein if the bacterial strain produces cellulase, a colorless transparent ring is arranged around a bacterial colony, and the ratio (Hc value) of the diameter of the hydrolysis ring to the diameter of the bacterial colony reflects the relative size of the activity of the cellulase. The result shows that the strain HB12274 has stronger cellulose degrading activity and the Hc value is 4.80.
TABLE 1 enzyme-producing Activity and relative enzyme Activity of Strain HB12274
Example 5: degradation effect of sargassum alga by strain HB12274 CGMCC No.12455
Bacillus amyloliquefaciens HB12274 CGMCC No.12455 was prepared as in example 3. The liquid microbial inoculum is inoculated with 5 percent of inoculation amount into a 250mL conical flask filled with 100mL of sargassum powder fermentation medium (the medium comprises 50g/L of sargassum powder, 5g/L of peptone, 16g/L of sodium chloride and about pH7.5), and is subjected to shake cultivation for 10d at 37 ℃ and 200 r/min.
Experiments show that the addition of the bacillus amyloliquefaciens HB12274 CGMCC No.12455 can effectively degrade gulfweed fronds, the weight loss rate of a solid is 50.02 percent after the sargassum fronds are cultured for 10 days, and the increment of reducing sugar in liquid is 65.6 mu g/mL.
Claims (5)
1. A Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HB12274, characterized in that the strain is preserved in the China general microbiological culture Collection center (CGMCC) at 2016, 5, 16, with the following addresses: the collection number of the strain is CGMCC NO.12455, No. 3 of Xilu No.1 of Beijing, Chaoyang, Beijing, and institute of microbiology of Chinese academy of sciences.
2. The bacillus amyloliquefaciens of claim 1, wherein the 16S rDNA sequence is:
3. the fermentation liquid of bacillus amyloliquefaciens of claim 1, which is prepared by strain activation, seed culture and fermentation culture of the bacillus amyloliquefaciens, and specifically comprises the following steps:
(1) strain activation: inoculating the bacillus amyloliquefaciens HB12274 into a nutrient agar culture medium, and culturing at 37 ℃ for 24-48 h, wherein the culture medium comprises: 8-10 g/L of peptone, 2-3 g/L of beef extract powder, 18-20 g/L of agar powder, 5-35 g/L of sodium chloride and pH 6.5-7.5,
(2) liquid culture: inoculating the activated strain into a liquid seed culture medium, and carrying out shaking culture at 30-37 ℃ and 120-200 r/min for 12-24 h to prepare a seed solution, wherein the culture medium is as follows: 1-3 g/L beef extract, 5-10 g/L peptone, 5-35 g/L sodium chloride, pH 6.5-7.5,
(3) fermentation culture: inoculating the seed solution into a liquid fermentation culture medium according to the volume ratio of 2-10%, and carrying out shaking culture at 30-37 ℃ for 48-96 h at 150-200 r/min to obtain a liquid microbial inoculum of the bacillus amyloliquefaciens HB12274, wherein the culture medium comprises: 1-3 g/L beef extract, 5-10 g/L peptone, 5-35 g/L sodium chloride and pH 6.5-7.5.
4. The use of the bacillus amyloliquefaciens strain according to claim 1, wherein the strain is used for degrading algin, cellulose and starch.
5. The use of bacillus amyloliquefaciens according to claim 1, wherein the strain and a fermentation broth thereof are used for degrading sargassum sp.
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| CN101748078A (en) * | 2008-12-05 | 2010-06-23 | 农业药物毒物试验所 | Novel Bacillus amyloliquefaciens strain and application thereof |
| CN103881952A (en) * | 2014-04-03 | 2014-06-25 | 大连理工大学 | Bacillus amyloliquefaciens and an application thereof as well as method for preparing kelp feed |
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| CN101748078A (en) * | 2008-12-05 | 2010-06-23 | 农业药物毒物试验所 | Novel Bacillus amyloliquefaciens strain and application thereof |
| CN103881952A (en) * | 2014-04-03 | 2014-06-25 | 大连理工大学 | Bacillus amyloliquefaciens and an application thereof as well as method for preparing kelp feed |
| CN105316266A (en) * | 2015-12-08 | 2016-02-10 | 领先生物农业股份有限公司 | Bacillus amyloliquefaciens LX-J1 and application thereof |
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