CN108753643A - A kind of vibrios H11 and its application - Google Patents
A kind of vibrios H11 and its application Download PDFInfo
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- CN108753643A CN108753643A CN201810563945.2A CN201810563945A CN108753643A CN 108753643 A CN108753643 A CN 108753643A CN 201810563945 A CN201810563945 A CN 201810563945A CN 108753643 A CN108753643 A CN 108753643A
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Abstract
The present invention relates to a kind of vibrios H11 and its applications, are preserved in China typical culture collection center, and deposit number is CCTCC NO：M 2018172.The present invention screens the bacterial strain of one plant of effectively hydrolyzing sea grass polysaccharide, and the reduced sugar that its hydrolysis obtains can utilize generation biological energy source substance by energy bacterial strain, compared with the single bacterial strain of existing hydrolysis sea grass polysaccharide, the hydrolysis ability of the vibrios H11 in the present invention is most strong.The present invention is combined by the hydrolysis of vibrios with the anaerobic fermentation of clostridium, to realize the higher value application of Enteromorpha.
Description
Technical field
Microbial biotechnology and biological resources technical field of the present invention more particularly to a kind of vibrios H11 and its application.
Background technology
Enteromorpha (Enteromorpha) is a kind of large-scale green alga, belongs to Chlorophyta (Chlorophyta) Ulvales
(Ulvales) plant of Ulvaceae (Ulvaceae).In recent years due to global warming, seawater eutrophication, cause Enteromorpha rapid
Growth, strong influence is brought to the marine eco-environment.Landfill and burning of the early stage to Enteromorpha, to the water body and soil of surrounding
Serious pollution is caused, and causes the waste of biomass resource.How Enteromorpha this explosive resource is efficiently used, it has also become
Improve the research hotspot of the marine eco-environment and seaweed comprehensive utilization.
Enteromorpha is a kind of potential reproducible bio-fuel source as a kind of large-scale green alga, can be used for preparing bio-oil,
Biogas or ethyl alcohol etc..However since sea grass polysaccharide is complex in composition and unclear, cause researchers for the high-valued of Enteromorpha
The research utilized is also fewer, to Enteromorpha and sea grass polysaccharide in terms of activated oligosaccharide or bioenergy application or it is seldom.Mesh
Before, mainly there are hydro-thermal method, fast pyrogenation method and fermentation method to the processing method of Enteromorpha.But what hydro-thermal method and fast pyrogenation method obtained
Fuel oil is difficult to be directly utilized；So microbial enzyme method is favored by numerous researchers.Alcohol is produced being currently used in microorganism
Class, the primary carbon source of acids are too high for monosaccharides, costs such as glucose, xylose, fructose.But this explosive resource of Enteromorpha can
Be hydrolyzed into can fermentation reducing sugar, as a kind of novel generation energy in raw material.
Invention content
The purpose of the present invention is to provide a kind of vibrios H11 and its application, with solve current sea grass polysaccharide it is complex in composition and
It is unclear that causing researchers also fewer for the research of the higher value application of Enteromorpha, so the side that research Enteromorpha utilizes
Case is relatively simple, presently mainly physicochemical method, and method has the problems such as significant limitation.
To solve the above problems, the present invention provides a kind of vibrios H11 (Vibrio sp.H11), March 31 in 2018
Day is preserved in " China typical culture collection center ", and deposit number is：CCTCC NO：M 2018172, depositary institution address
For：No. 299 Wuhan University's collections of Wuhan City, Hubei Province Wuchang District Bayi Road.The invention bacterial strain can be unique with sea grass polysaccharide
It is grown in the culture medium of carbon source, and it is Gram-negative bacterium；Physiological and biochemical property has VP positive, and MR is positive, gelatin
Liquefaction is negative.It can be grown using " green tide " Enteromorpha biomass as sole carbon source and the energy and be hydrolyzed into the fermentable reduction of clostridium
Sugar generates biological hydrogen and biological organic solvent etc. for anaerobic fermentation.
The cultural method of above-mentioned vibrios H11 is：The vibrios H11 bacterium are inoculated into sea grass polysaccharide to grow for sole carbon source,
Culture medium group becomes (1L) (NH4)2SO4, 1.0g；Na2HPO4, 0.8g；KH2PO4, 0.2g；MgSO4·7H2O, 0.2g；
CaCl2·2H2O, 0.1g；FeCl3·6H2O, 5mg； (NH4)6Mo7O24·4H2O, 1mg；ddH2O 1L；Sea grass polysaccharide, 20g；Pancreas
Peptone is 30 DEG C in temperature in 5g/L, and pH is 8.71 culture 12h.The first 20min that sterilizes at 121 DEG C of culture medium.
The application of vibrios H11 can be used for hydrolyzing " green tide " biomass.Being mainly used for Enteromorpha being hydrolyzed into clostridium can ferment
Reduced sugar.The anaerobic fermentation that sea grass polysaccharide in Enteromorpha is hydrolyzed to clostridium after reduced sugar by vibrios H11 is converted into bioenergy
Substance.Application process includes mainly：It is added containing described in the anaerobic fermentation culture medium containing Enteromorpha powder or sea grass polysaccharide
The crude enzyme liquid of the concentration of vibrios H11, and 40 DEG C, hydrolyze 48h in 200rpm shaking tables.Inoculate clostridium anaerobic fermentation.Clostridium can be with
For Clostridium acetobutylicum WA bacterial strains.
Sea grass polysaccharide is a kind of complicated heteroglycan, only knows its main composition, structure there is presently no
Clearly report.Hydrolysis for sea grass polysaccharide, mixed bacterial hydrolysis ability is preferable, but flora composition is easily varied, and does not allow
It is easy to maintain.For Vibrio sp.H11 bacterial strains in the present invention as single bacterial strain, the ability of hydrolysis sea grass polysaccharide is strongest.
Comprehensive treatment for " green tide " Enteromorpha this biomass and higher value application, current physico-chemical process is to cost requirement
Height, and the waste liquid generated is easy pollution environment.It converts Enteromorpha to biological energy source eventually by the method for microorganism in the present invention
Source substance, at low cost and environmental nonpollution.Currently, this is to convert Enteromorpha to biological energy source using the method for microbial fermentation for the first time
Source substance.
Compared with prior art, the present invention screens the bacterial strain of one plant of effectively hydrolyzing sea grass polysaccharide, and it hydrolyzes acquisition
Reduced sugar can be by energy bacterial strain using generation biological energy source substance, compared with the single bacterial strain of existing hydrolysis sea grass polysaccharide
Compared with the hydrolysis ability of the vibrios H11 in the present invention is most strong.The present invention is mutually tied by the hydrolysis and the anaerobic fermentation of clostridium of vibrios
It closes, to realize the higher value application of Enteromorpha.
Description of the drawings
Fig. 1 is the phylogenetic tree of Vibrio sp.H11；
Fig. 2 is the electron microscope of Vibrio sp.H11；
Fig. 3 is the Lugol's iodine solution colored graph of Vibrio sp.H11；
Fig. 4 be Vibrio sp.H11 growth curve and fermentation supernatant in content of reducing sugar curve；
Fig. 5 is Vibrio sp.H11 crude enzyme liquid enzyme activity curve graphs；
Fig. 6 be C.acetobutylicum WA bacterial strains using it is different can the biology that generates of fermentation reducing sugar anaerobic fermentation
Amounts of hydrogen；
Fig. 7 be C.acetobutylicum WA bacterial strains using the sea grass polysaccharide of different content can fermentation reducing sugar generate
Biological solutions amount；
Fig. 8 be C.acetobutylicum WA bacterial strains using the Enteromorpha powder of different content can fermentation reducing sugar generate
Biological solutions amount.
Specific implementation mode
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing
Step ground detailed description.
Embodiment 1
(1) screening and culturing of Vibrio sp.H11
Early period obtains green alga sample in Hainan and uses sterile mortar by sample after sterile saline rinses
It is ground into homogenate, is added in sea grass polysaccharide minimal medium with liquid-transfering gun gentle aspiration supernatant 1mL after standing, 25 DEG C,
After 150rpm is cultivated one day；Zymotic fluid to the new sea grass polysaccharide minimal medium of 1mL of transferring again continues to cultivate, repetitive operation
3 times.The zymotic fluid of acquisition is pressed 10-3、10-4、 10-5、10-6、10-7Gradient dilution is carried out, sea grass polysaccharide tablet is then coated on
Upper 25 DEG C are inverted culture one day.Form is observed, picking single bacterium colony and repeatedly scribing line obtain pure culture.Then by sea grass polysaccharide without
In machine salt culture medium zymotic fluid content of reducing sugar (DNS methods) come primarily determine the bacterium whether have sea grass polysaccharide hydrolysis ability.Most
One plant of vibrios H11, enzyme activity highest are obtained eventually.
Main group of sea grass polysaccharide minimal medium therein becomes:1L(NH4)2SO4, 1.0g； Na2HPO4, 0.8g；
KH2PO4, 0.2g；MgSO4·7H2O, 0.2g；CaCl2·2H2O, 0.1g； FeCl3·6H2O, 5mg；(NH4)6Mo7O24·4H2O,
1L sea grass polysaccharide leaching liquors.
(2) identification of Vibrio sp.H11
Vibrio sp.H11 are Gram-negative bacterial strain；Physio-biochemical characteristics have VP positive, and MR is positive, gelatin
Liquefaction is negative；Using following substance as carbon source：D-Fructose, D-Glucose, D- xyloses, D- galactolipins, sucrose, mannitol,
Citric acid, starch；The results are shown in Table 1.
The physio-biochemical characteristics of table 1Vibrio sp.H11
Using colony polymerase chain reaction (PCR) method, single bacterium colony is directly taken to carry out PCR as template.Utilize the primer of PCR amplification 16S rDNA
For a pair of of universal primer, 27F：5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492R：5'-GGTTACCTTGTTACGACT-
3'.PCR reaction conditions are：94℃3min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, 30 cycles；72℃10min.PCR is expanded
Increase production object after purification, sequencing result is sent to carry out Blast analyses, sequence length 1385bp, the present invention with the sequence on Genbank
There is prodigious homology with molten coral vibrios (Vibrio coralliilyticus)；Its 16S rDNA sequence such as SEQ ID:
Shown in NO.1.The sequence is compared with sequence in Genbank, chooses similar bacterial strain, with MEGA7 software building systems
Chadogram, constructed dendrogram are as shown in Figure 1.Meanwhile by the Electronic Speculum graph discovery of vibrios H11, be form be it is rod-shaped,
And it is as shown in Figure 2 with amphitrichous.
After vibrios H11 is cultivated 12 hours on the minimal medium tablet containing 2% sea grass polysaccharide, Lugol's iodine solution is used
Dyeing has apparent transparent circle, as shown in Figure 3.
Vibrios H11 is grown using sea grass polysaccharide as sole carbon source, generates relevant glycoside hydrolase, is broken sea grass polysaccharide
In different type glycosidic bond, the oligosaccharides to generate low molecular weight provides carbon source for own growth.It is broken what glycosidic bond generated
Reduction end can be detected by DNS methods.The invention bacterial strain in using sea grass polysaccharide as the culture medium of sole carbon source growth curve and
Content of reducing sugar curve graph in fermentation supernatant is as shown in Figure 4.It was found that vibrios H11 grows bacteria concentration highest in 14h, with culture
Changes of Reducing Sugar Content is consistent in base.But in zymotic fluid remaining content of reducing sugar cannot directly react crude enzyme liquid enzyme activity it is big
It is small.So then using the fermentation supernatant of the invention bacterial strain as crude enzyme liquid and 1% sea grass polysaccharide solution reaction, when surveying its different fermentations
Between crude enzyme liquid enzyme activity (U/mL), the results are shown in Figure 5.Before 12h, with gradually increasing for fermentation time, zymotic fluid
The enzyme activity of middle crude enzyme liquid gradually increases, and in 12h, enzyme activity highest, is declined slightly thereafter.
(3) optimization culture of Vibrio sp.H11
In order to improve the enzyme activity of crude enzyme liquid in zymotic fluid, culture medium prescription and condition of culture to vibrios H11 carry out
Condition optimizing.Sea grass polysaccharide grows for sole carbon source, and the group of the culture medium after optimization becomes (1L)： (NH4)2SO4, 1.0g；
Na2HPO4, 0.8g；KH2PO4, 0.2g；MgSO4·7H2O, 0.2g； CaCl2·2H2O, 0.1g；FeCl3·6H2O, 5mg；
(NH4)6Mo7O24·4H2O, 1mg；ddH2O 1L, pH 8.71；Cultivation temperature is 30 DEG C.
After condition optimizing, the bacterial strain is cultivated under optimal conditions and measures enzyme activity, enzyme activity is reachable
8.43U/mL, hence it is evident that higher than the enzyme activity before optimization.Table 2 be it has been reported that sea grass polysaccharide hydrolyze the hydrolysis energy of relevant bacterial strain
The vibrios H11 of power and the present invention compare the hydrolysis ability of sea grass polysaccharide.Wherein the crude enzyme liquid of Vibrio sp. H11 is than single
The enzyme activity of the corresponding enzymes of strains A lteromonas sp.A321, Alteromonas macleodii B7 is high；Compare Mixed Microbes simultaneously
The enzyme activity of group Micro-community D2-1 is high.Although lower than the enzyme activity of Micro-community H1,
Vibrio sp.H11 are single bacterial strains, more more stable than it, it is easier to be preserved；Micro-community H1 are in incubation
Bacterial community is easy to happen variation.
2 different strains of table (group) hydrolyze the comparison of sea grass polysaccharide ability
(4) application of Vibrio sp.H11
It is obtained in crude enzyme liquid after optimization culture based formulas and condition of culture, acts on 20g/L (2%), 50 g/L
(5%) the anaerobic fermentation culture medium (being free of reducing agent) of Enteromorpha powder, sea grass polysaccharide.The hair is cultivated under optimum culture condition
Bright bacterial strain H11 takes out sample when fermenting 26h.In order to obtain more crude enzyme liquid enzyme amount, sea grass polysaccharide or Enteromorpha powder is made to fill
It is hydrolyzed with dividing, crude enzyme liquid is concentrated by ultrafiltration at 4 DEG C collects crude enzyme liquid herein, and measure the enzyme activity of crude enzyme liquid.Containing
Crude enzyme liquid (the Enteromorpha powder or sea grass polysaccharide of 50ml of concentration is added in the anaerobic fermentation culture medium of Enteromorpha powder or sea grass polysaccharide
Anaerobic fermentation culture medium (be free of reducing agent) solution in the crude enzyme liquid of 2ml is added), and 40 DEG C, hydrolyze in 200rpm shaking tables
48h.Inoculate Clostridium acetobutylicum WA bacterial strain anaerobic fermentations.
When Enteromorpha content of powder is 2%, the biological amounts of hydrogen of generation is 1553.4mL/L, and biological solutions are a concentration of
3.46g/L.When Enteromorpha content of powder is 5%, the biogas scale of construction of generation is 1682mL/L, and biological solutions amount is 4.53g/L.It says
Bright the concentration of Enteromorpha powder is higher in a certain range, and the yield of hydrogen and biological solutions is higher.When the content of sea grass polysaccharide
When being 2%, it is 1473.4mL/L to generate in biological amounts of hydrogen, and biological solutions amount is 4.77g/L；The content of sea grass polysaccharide is 5%
When, the biological amounts of hydrogen of generation is 3217 mL/L, and biological solutions amount is 6.74g/L.Illustrate in a certain range, sea grass polysaccharide
Concentration it is higher, the yield of hydrogen and biological solutions is higher.Meanwhile Enteromorpha powder and sea grass polysaccharide are compared, it finds same
Under concentration, sea grass polysaccharide generates more hydrogen and biological solutions.As a result such as Fig. 6, Fig. 7, shown in Fig. 8.Comprehensive Comparison, waterside
Tongue polysaccharide has more the value of generation fuel.
Hydrolysis for sea grass polysaccharide, mixed bacterial hydrolysis ability is preferable, but flora composition is easily varied, and is not easy to protect
It deposits.For Vibrio sp.H11 bacterial strains in the present invention as single bacterial strain, the ability of hydrolysis sea grass polysaccharide is strongest.
SEQUENCE LISTING
<110>University Of Shantou
<120>A kind of vibrios H11 and its application
<130> 2018
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1385
<212> DNA
<213>Artificial sequence
<400> 1
tcttttgcag cccactccca tggtgtgacg ggcggtgtgt acaaggcccg ggaacgtatt 60
caccgtggca ttctgatcca cgattactag cgattccgac ttcatggagt cgagttgcag 120
actccaatcc ggactacgac gcactttttg ggattcgctc actttcgcaa gttggccgcc 180
ctctgtatgc gccattgtag cacgtgtgta gccctactcg taagggccat gatgacttga 240
cgtcgtcccc accttcctcc ggtttatcac cggcagtctc cctggagttc ccgactttac 300
tcgctggcaa acaaggataa gggttgcgct cgttgcggga cttaacccaa catttcacaa 360
cacgagctga cgacagccat gcagcacctg tctctcagtt cccgaaggca caagactgtc 420
tccagtctct tctgaggatg tcaagagtag gtaaggttct tcgcgttgca tcgaattaaa 480
ccacatgctc caccgcttgt gcgggccccc gtcaattcat ttgagtttta atcttgcgac 540
cgtactcccc aggcggtcta cttaacgcgt tagctccgaa agccacggct caaggccaca 600
acctccaagt agacatcgtt gacggcgtgg actaccaggg tatctaatcc tgtttgctcc 660
ccacgctttc gcatctgagt gtcagtatct gtccaggggg ccgccttcgc caccggtatt 720
ccttcagatc tctacgcatt tcaccgctac acctgaaatt ctacccccct ctacagtact 780
ctagcctgcc agtttcaaat gcaattccga ggttgagccc cgggctttca catctgactt 840
aacaaaccac ctgcatgcgc tttacgccca gtaattccga ttaacgctcg caccctccgt 900
attaccgcgg ctgctggcac ggagttagcc ggtgcttctt ctgcagctaa cgtcaaacga 960
atgcgctatt aacacatccg ccttcctcac tgctgaaagt actttacaac ccgaaggcct 1020
tcttcataca cgcggcatgg ctgcatcagg cttgcgccca ttgtgcaata ttccccactg 1080
ctgcctcccg taggagtctg gaccgtgtct cagttccagt gtggctgatc atcctctcag 1140
accagctagg gatcgtcgcc ttggtgagcc cttacctcac caactagcta atcccacctg 1200
ggcatatcct gacgcgagag gcccgaaggt ccccctcttt ggcccgaagg catcatgcgg 1260
tattagccat cgtttccaat ggttatcccc cacatcaggg caatttccca ggcattactc 1320
acccgtccgc cgctcgccgc ccttaacgtt ccccgaaggt tcagttaagt cgctgccgct 1380
cgact 1385
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
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agagtttgat cctggctcag 20
<210> 3
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<212> DNA
<213>Artificial sequence
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ggttaccttg ttacgact 18
Claims (6)
1. a kind of vibrios H11, which is characterized in that be preserved in China typical culture collection center, deposit number CCTCC
NO：M 2018172.
2. the cultural method of vibrios H11 according to claim 1, which is characterized in that the cultural method is by the vibrios
H11 bacterium are inoculated into sea grass polysaccharide and are grown for sole carbon source, and culture medium group becomes (1L) (NH4)2SO4, 1.0g；Na2HPO4, 0.8g；
KH2PO4, 0.2g；MgSO4·7H2O, 0.2g；CaCl2·2H2O, 0.1g；FeCl3·6H2O, 5mg；(NH4)6Mo7O24·4H2O,
1mg；ddH2O 1L；Sea grass polysaccharide, 20g；Tryptone, in 5g/L；It it is 30 DEG C in temperature, pH is 8.71 culture 12h.
3. the application of vibrios H11 according to claim 1, which is characterized in that for hydrolyzing " green tide " biomass.
4. applying according to claim 3, which is characterized in that for Enteromorpha to be hydrolyzed into the fermentable reduced sugar of clostridium.
5. applying according to claim 4, which is characterized in that after Enteromorpha is hydrolyzed to reduced sugar by the vibrios H11, in conjunction with shuttle
The anaerobic fermentation of bacterium is converted into biological energy source substance.
6. applying according to claim 5, which is characterized in that application process includes mainly：Containing Enteromorpha powder or Enteromorpha
The crude enzyme liquid of the concentration containing the vibrios H11, and 40 DEG C, water in 200rpm shaking tables are added in the anaerobic fermentation culture medium of polysaccharide
48h is solved, clostridium anaerobic fermentation is inoculated.
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| CN114480201A (en) * | 2022-02-15 | 2022-05-13 | 青岛中尚奇生物科技有限公司 | Vibrio natriegens capable of strongly degrading enteromorpha protein and application of vibrio natriegens in preparation of organic fertilizer |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111500484A (en) * | 2020-02-14 | 2020-08-07 | 上海交通大学 | Three newly separated vibrio capable of growing quickly and its application |
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| CN114410542A (en) * | 2022-02-15 | 2022-04-29 | 青岛中尚奇生物科技有限公司 | Vibrio sky blue and application thereof in enteromorpha degradation |
| CN114480201A (en) * | 2022-02-15 | 2022-05-13 | 青岛中尚奇生物科技有限公司 | Vibrio natriegens capable of strongly degrading enteromorpha protein and application of vibrio natriegens in preparation of organic fertilizer |
| CN114480201B (en) * | 2022-02-15 | 2023-08-11 | 青岛中尚奇生物科技有限公司 | Vibrio natriurus capable of strongly degrading enteromorpha protein and application of vibrio natriurus in preparation of organic fertilizer |
| CN114410542B (en) * | 2022-02-15 | 2023-08-11 | 青岛中尚奇生物科技有限公司 | Vibrio paradise and application thereof in degradation of enteromorpha |
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