CN106399156A - Bacillus amyloliquefaciens subsp.plantarum and application thereof to scagassum biodegradation - Google Patents
Bacillus amyloliquefaciens subsp.plantarum and application thereof to scagassum biodegradation Download PDFInfo
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Abstract
The invention discloses a bacillus HB12274 and an application thereof to the scagassum biodegradation, and belongs to the technical field of microorganisms. The bacillus is bacillus amyloliquefaciens subsp.plantarum HB12274 and is separated from a bottom sediment sample of Mangrove at Dongzhai Harbor in Hainan. The strain is collected in China General Microbiological Culture Collection Center (CGMCC) in May 16, 2016 and has a collection number of CGMCC No.12455. The bacillus HB12274 has the activity of degrading algin, cellulose and starch, and can effectively degrade scagassum. The bacillus HB12274 and a fermentation liquor thereof can serve as new microbial resources used for the scagassum biodegradation, and have good application and development prospects in the aspects of scagassum processing and utilization.
Description
Technical field
The invention belongs to microbial technology field is and in particular to a kind of bacillus amyloliquefaciens plant subspecies HB12274
And its application in sargassum is biodegradable.
Background technology
Sargassum is the common kelp of a class, belongs to Phaeophyta (Phaephyta), Sargassaceae
(Sargassaceae), Sargassum (Sargassum), great majority are water warm species, are distributed widely in warm water and warm water sea
Domain.Sargassum has very high economic worth, in aspect all great exploitation potential such as agriculture, industrial, is important fertilizer, feed
And the raw material of industry.Sargassum cell membrane is mainly made up of cellulose and algin, and Stability Analysis of Structures is it is more difficult to be destroyed.Degraded horse hair
Cellulose in frustule wall and algin are efficient degradation the key using sargassum.Industrial frequently with mechanical crushing and
Chemical degradation method is degraded to sargassum, but there is the shortcomings of power consumption height and waste pollution environment.Biological degradation method is
Cellulose and the algin degraded using cellulase produced by microorganism and algin catenase in frond, thus reach
The purpose of degraded frond, receives much concern because of its high-efficiency environment friendly.
Bacillus is that a class is aerobic or facultative anaerobic bacteria, can produce resistance endospore, is conventional Tiny ecosystem system
One of agent.Bacillus can produce multiple ectoenzymes, such as algin catenase, cellulase, protease, amylase etc., can divide
The gas chromatographies such as solution algin, cellulose, protein and starch, thus be used widely.The solution starch that the present invention adopts
Bacillus plant subspecies HB12274, are isolatable from Hainan Province Dong Zhaigang mangrove bed mud, have efficient degradation algin, starch
And cellulolytic activity, sargassum frond of effectively degrading, there is in terms of sargassum processing and utilization good application and development prospect.
Content of the invention
It is an object of the invention to provide a kind of bacillus amyloliquefaciens with degraded algin, cellulose and starch is planted
Thing subspecies, this bacterial strain can effectively be degraded algin, cellulose and starch, can as a kind of new for the biological fall of sargassum
The microbial resources of solution.
For achieving the above object, the present invention employs the following technical solutions：
The bacillus amyloliquefaciens plant subspecies with degraded algin, starch and cellulolytic activity provided by the present invention
HB12274, on May 16th, 2016 at China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)
Carry out culture presevation, and proved to survive, its preservation registration number has been CGMCC No.12455.Preserving address is：Beijing is exposed to the sun
Area North Star West Road 1 institute, Institute of Microorganism, Academia Sinica.Described bacillus HB12274 belongs to bacterium domain, heavy wall bacterium
Door, bacillus guiding principle, bacillus mesh, Bacillaceae, Bacillus.
Bacillus amyloliquefaciens plant subspecies HB12274 of the present invention obtain process：From Hainan Province Dong Zhaigang mangrove
Collection bed mud sample, sample adopts 80 DEG C of water bath processing 20min to kill non-brood cell bacterium after gradient dilution, is distributed on the way thereafter
Bacteria distribution culture medium flat plate.Again the bacillus separating acquisition is carried out producing algin catenase, amylase and cellulase
Vitality test, finally filters out bacillus plant subspecies HB12274 being provided simultaneously with three kinds of enzymatic activitys.
Described bacillus HB12274 has following biological characteristics：Bacterial strain is on brewer's wort nutrient agar
After line culture 3d, bacterium colony is rounded, edge is irregular, mattness, out-of-flatness, milky, opaque；Thalline stock
Shape, produces brood cell.Gram's staining, VP measure, catalase experiment is positive, and MR experiment is negative, reducible nitrate, and hydrolyzable forms sediment
Powder, algin and cellulose, can make gelatin liquefaction.
The 16S rDNA sequence of bacillus HB12274 is KU529689 in the accession number of GenBank.Through polyphase sort mirror
It is set to Bacillus amyloliquefaciens subsp.plantarum (bacillus amyloliquefaciens plant subspecies).This bacterial strain
It is easy to grow, there is the ability of degraded algin, starch and cellulose, sargassum frond of effectively degrading, can be developed into one kind
New application is in the biodegradable microbial bacterial agent of sargassum.
Application in sargassum is biodegradable for the bacillus HB12274, comprises the steps：
(1) bacterial strain activation：Described bacillus amyloliquefaciens plant subspecies HB12274 are inoculated in nutrient agar culture
Base, cultivates 24～48h in 37 DEG C.Culture medium：Peptone 8～10g/L, beef extract powder 2～3g/L, agar powder 18～20g/L, chlorine
Change sodium 5～35g/L, pH6.5～7.5.
(2) Liquid Culture：The bacterial strain of activation is accessed in liquid seed culture medium, in 30～37 DEG C, 120～200r/min
Shaken cultivation 12～24h, makes seed liquor.Culture medium：Beef extract 1～3g/L, peptone 5～10g/L, sodium chloride 5～35g/
L, pH6.5～7.5.
(3) fermented and cultured：Seed liquor 2～10% is inoculated in liquid fermentation medium by volume, 30～37 DEG C, 150
～200r/min shaken cultivation 48～96h, that is, be obtained the liquid bacterial agent of bacillus amyloliquefaciens plant subspecies HB12274.Culture
Base：Beef extract 1～3g/L, peptone 5～10g/L, sodium chloride 5～35g/L, pH6.5～7.5.
(4) liquid bacterial agent is inoculated in the conical flask equipped with sargassum powder fermentation medium with 5%～10% inoculum concentration
In, shaking table culture 10～15d under the conditions of 30～37 DEG C, 150～200r/min.Culture medium：Sargassum powder 25～50g/L, egg
White peptone 5～10g/L, sodium chloride 5～35g/L, pH6.5～7.5.
Experiment shows, adds bacillus HB12274 CGMCC No.12455 and can effectively degrade sargassum frond, culture
After 10d, solid content weight-loss ratio is 50.02%, and in liquid, reduced sugar increment is 65.6 μ g/mL.As can be seen here, the brood cell of the present invention
It is biodegradable that bacillus HB12274 CGMCC No.12455 can be applicable to sargassum frond.
With reference to specification drawings and specific embodiments, the invention will be further described.
Brief description
Fig. 1 is the morphological feature photo of bacterial strain HB12274 of the present invention, × 1000.
Fig. 2 is the phylogenetic tree based on 16S rDNA of bacterial strain HB12274 of the present invention.
Specific embodiment
With reference to specific embodiment, the invention will be further described：
Embodiment 1：The separation of bacillus HB12274 CGMCC No.12455
Using 5 point samplings, gather bed mud sample from Hainan Province Dong Zhaigang mangrove.10g sample is taken to be dissolved in 90ml aseptic
In water, it is placed in 37 DEG C, vibrates 30min in the constant-temperature table of 200r/min, after standing 5min, be configured to 10-2～10-4Series
Soil supension.The method adopting 80 DEG C of water bath processing 20min, to kill non-brood cell bacterium, is drawn serial suspension 0.1ml, is coated
Bacteria distribution culture medium, is placed in 37 DEG C of culture carton upside down cultures.When bacterium colony grows, according to colony shape, color, edge shape
State, transparency, surface dry and wet state isophenous feature, picking single bacterium colony line purifying 2～3 times, until obtain pure culture.Through just
After step re-scheduling, numbering, preserve.
The Species estimation of embodiment 2 bacillus HB12274 CGMCC No.12455
(1) form and cultural character
Bacterial strain is rule on brewer's wort nutrient agar culture, strain growth is rapid, colony diameter 3～5mm, circle
Shape, edge is irregular, mattness, out-of-flatness, and milky is opaque；Thalline elongated rod shape, Gram's staining result is sun
Property, produce brood cell, (Fig. 1) is expanded in one end.
(2) physiological and biochemical property
Bacterial strain HB12274 can be using glucose, sorbierite, maltose, sucrose, cellobiose, galactolipin, mannitol, really
Sugar, lactose and arabinose.Gram's staining, VP measure, catalase experiment is positive, and MR experiment is negative, reducible nitrate, energy
Make gelatin liquefaction.
(3) 16S rDNA sequencing and Phylogenetic Analysis
Expanded by PCR, being sequenced obtains 16S rDNA sequence 1450bp of HB12274, nucleotides sequence is listed in Genbank's
Accession number is KU529689.This sequence is compared with the sequence in EzBioCloud database, find bacterial strain HB12274 with
With Bacillus amyloliquefaciens subsp.plantarum sequence similarity highest, reach 99.5%, with
Bacillus siamensis and Bacillus methylotrophicus sequence similarity are respectively 99.4% and 99.3%.
The high related strain of homology is selected to utilize Neighbor-joining method phylogenetic tree construction (Fig. 2) in MEGA5.0, can
Find out, HB12274 and Bacillus amyloliquefaciens subsp.plantarum FZB42TIt is in same branch
On, the two affiliation is nearer.Relatively the two morphological feature, physiological and biochemical property, finds that morphological feature is similar, Physiology and biochemistry is special
Levy consistent.
Based on the above results, bacterial strain HB12274 (CGMCC No.12455) according to the present invention, is accredited as Bacillus
Amyloliquefaciens subsp.plantarum (bacillus amyloliquefaciens plant subspecies), exists on May 16th, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) has carried out culture presevation, and proves to survive, its
Preservation registration number is CGMCC No.12455.Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the micro- life of the Chinese Academy of Sciences
Thing research institute.
Embodiment 3：The preparation of bacterial strain HB12274 CGMCC No.12455 liquid bacterial agent
(1) bacterial strain activation：Described bacillus amyloliquefaciens plant subspecies HB12274 are inoculated in the training of nutrient agar solid
Foster base, cultivates 24h in 37 DEG C.Culture medium：Peptone 10.0g, beef extract powder 3.0g, agar powder 18.0g, sodium chloride 16.0g,
1000mL, pH7.5,121 DEG C of sterilizing 20min.
(2) Liquid Culture：The bacterial strain of activation is accessed in nutrient agar fluid nutrient medium, in 37 DEG C, 200r/min vibration
Culture 24h, makes seed liquor.
(3) fermented and cultured：Seed liquor 3% is inoculated in nutrient agar fluid nutrient medium, 37 DEG C, 200r/min by volume
Shaken cultivation 72h, that is, be obtained the liquid bacterial agent of bacillus amyloliquefaciens plant subspecies HB12274.
The liquid bacterial agent of bacillus amyloliquefaciens plant subspecies HB12274 is obtained by above step.
Embodiment 4：Bacterial strain HB12274 CGMCC No.12455 degraded algin, cellulose, amylase activity measure
Using transparent circle method measure bacterial strain HB12274 to algin, cellulose, starch degrading activity, the results are shown in Table 1.
Produce algin catenase to measure：Take single bacterium colony, point is connected to sodium alginate culture medium, and (culture medium forms：Sodium alginate
5.0g, (NH4)2SO45.0g, K2HPO42.0g, NaCl 16.0g, MgSO4·7H2O 1.0g, FeSO4·7H2O 0.01g, agar
18.0g, distilled water 1000mL, pH7.5), after being inverted culture 3d in 37 DEG C, plus appropriate 95% alcohol, standing, if bacterial strain generation is brown
Phycocolloid lyases, then periphery of bacterial colonies have hydrolysis circle, the ratio (Hc value) of hydrolytic circle/colony diameter reacted algin and split
The relative size of solution enzyme.Result shows, bacterial strain HB12274 has stronger degraded sodium alginate activity, and Hc value is 6.25.
Produce determination of amylase：Take single bacterium colony, point is connected to Starch Hydrolysis culture medium, and (culture medium forms：Peptone 10.0g, can
Soluble starch 2.0g, beef extract 5.0g, sodium chloride 16.0g, agar 20.0g, water 1000ml, pH7.0), it is inverted culture 3d for 37 DEG C
Afterwards, in flat board with Lugol's iodine solution.Iodine is met due to starch and becomes blue, if bacterial strain produces amylase, periphery of bacterial colonies can show colourless
Transparent circle, the ratio (Hc value) of transparent loop diameter/colony diameter has reacted the relative size of amylase activity.Result shows, bacterium
Strain HB12274 has stronger degradable starch activity, and Hc value is 3.20.
Cellulase-producing measures：Take single bacterium colony, point is connected to sodium carboxymethylcellulose culture medium (sodium carboxymethylcellulose
10.0g, NH4NO31.0g, (NH4)2SO41.0g, MgSO4·7H2O 0.5g, NaCl 16.0g, agar 18.0g, distilled water
1000mL, natural pH), it is inverted culture 3d for 37 DEG C, with 1% congo red staining 30min, with 1mol/L sodium chloride decolouring 20min (2
Secondary), if bacterial strain cellulase-producing, periphery of bacterial colonies has water white transparency circle, the ratio (Hc of hydrolytic circle/colony diameter
Value) react the relative size of cellulase activity.Result shows, bacterial strain HB12274 has stronger degraded cellulose activity,
Hc value is 4.80.
The inulinase-producing activity of table 1 bacterial strain HB12274 and enzyme activity
Embodiment 5：Bacterial strain HB12274 CGMCC No.12455 degraded sargassum frond effect
Bacillus HB12274 CGMCC No.12455 is prepared as described in Example 3.By liquid bacterial agent with 5% connect
The amount of kind is inoculated in equipped with 100ml sargassum powder fermentation medium (culture medium composition：Sargassum powder 50g/L, peptone 5g/L, chlorine
Change sodium 16g/L, pH 7.5 about) 250mL conical flask in, shaking table culture 10d under the conditions of 37 DEG C, 200r/min.
Experiment shows, adds bacillus HB12274CGMCC No.12455 and can effectively degrade sargassum frond, cultivates 10
After it, solid content weight-loss ratio is 50.02%, and in liquid, reduced sugar increment is 65.6 μ g/mL.
Sequence table
Sequence table (1450bp).
Claims (6)
1. a kind of bacillus is it is characterised in that described bacillus is bacillus amyloliquefaciens plant subspecies Bacillus
Amyloliquefaciens subsp.plantarum, bacterial strain code name is HB12274, and this bacterial strain was in May 16 preservation in 2016
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica, culture presevation number is CGMCC NO.12455.
2. bacillus described in claim 1 is characterised by, the sequence of its 16S rDNA is：
AAGACGGGGTGCTATAATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGA
GTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAAC
CGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGG
TAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAG
ACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGA
AGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCT
AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG
GCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACT
GGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCA
GTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCT
GGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAG
CACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATG
TGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCC
TTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA
GCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGT
GGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCG
AAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTG
GAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAC
GAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGACAGAGCA.
3. a kind of zymotic fluid of the bacillus described in claim 1 is it is characterised in that with described bacillus amyloliquefaciens plant
Subspecies HB12274 are made through bacterial strain activation, seed culture, fermented and cultured.Specifically include following steps：
(1) bacterial strain activation：Described bacillus amyloliquefaciens plant subspecies HB12274 are inoculated in nutrient agar, in
37 DEG C of culture 24～48h.Culture medium：Peptone 8～10g/L, beef extract powder 2～3g/L, agar powder 18～20g/L, sodium chloride 5
～35g/L, pH6.5～7.5.
(2) Liquid Culture：The bacterial strain of activation is accessed in liquid seed culture medium, in 30～37 DEG C, 120～200r/min vibration
Culture 12～24h, makes seed liquor.Culture medium：Beef extract 1～3g/L, peptone 5～10g/L, sodium chloride 5～35g/L,
PH6.5～7.5.
(3) fermented and cultured：Seed liquor 2～10% is inoculated in liquid fermentation medium by volume, 30～37 DEG C, 150～
200r/min shaken cultivation 48～96h, that is, be obtained the liquid bacterial agent of bacillus amyloliquefaciens plant subspecies HB12274.Culture medium：
Beef extract 1～3g/L, peptone 5～10g/L, sodium chloride 5～35g/L, pH6.5～7.5.
4. the application of bacillus described in claim 1 is it is characterised in that this bacterium has degraded algin, cellulose and starch
Activity.
5. the application of bacillus described in claim 1, is further characterized in that this bacterium can effectively be degraded sargassum.
6. the application of the bacillus described in claim 1 is it is characterised in that described bacterial strain and its zymotic fluid are biological in sargassum
Application in degraded.
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| CN111206010A (en) * | 2020-03-03 | 2020-05-29 | 杭州丰海生物科技有限公司 | Recombinant bacillus amyloliquefaciens secreting extracellular alginate lyase and cellulase and application thereof |
| CN111996140A (en) * | 2020-08-17 | 2020-11-27 | 华中农业大学 | Screening and application of buffalo rumen source bacillus amyloliquefaciens |
| CN113652379A (en) * | 2021-09-27 | 2021-11-16 | 中国科学院广州能源研究所 | Bacillus cell wall depolymerized GIEC and application thereof |
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| CN101748078B (en) * | 2008-12-05 | 2013-02-27 | 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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| CN111206010B (en) * | 2020-03-03 | 2022-11-04 | 杭州丰海生物科技有限公司 | Recombinant bacillus amyloliquefaciens secreting extracellular alginate lyase and cellulase and application thereof |
| CN111996140A (en) * | 2020-08-17 | 2020-11-27 | 华中农业大学 | Screening and application of buffalo rumen source bacillus amyloliquefaciens |
| CN111996140B (en) * | 2020-08-17 | 2023-04-07 | 华中农业大学 | Screening and application of buffalo rumen source bacillus amyloliquefaciens |
| CN113652379A (en) * | 2021-09-27 | 2021-11-16 | 中国科学院广州能源研究所 | Bacillus cell wall depolymerized GIEC and application thereof |
| CN113652379B (en) * | 2021-09-27 | 2023-11-03 | 中国科学院广州能源研究所 | Bacillus cell wall depolymerization GIEC and application thereof |
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