CN107129949A - One plant promotes the sugared bacillus of Eichhornia crassipes matrix enzymolysis production and its application - Google Patents
One plant promotes the sugared bacillus of Eichhornia crassipes matrix enzymolysis production and its application Download PDFInfo
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Abstract
The invention provides bacillus (Bacillus axarquiensis) CTS BQ1 that one plant promotes Eichhornia crassipes enzymolysis production sugar, and further provide the method for promoting Eichhornia crassipes matrix enzymolysis production sugar and the microbial bacterial agent for promoting Eichhornia crassipes matrix enzymolysis production sugar.Bacterial strain provided by the present invention enriches wild degumming bacterium genetic resources, expands the standby storehouse of degumming bacterium full-length genome breeding.The bacterium can more improve Eichhornia crassipes enzymolysis output of sugar than the report preferable whiterot fungi of effect, and efficiency high than business pectase economy and effect significantly, has broad application prospects.
Description
Technical field
Field is utilized the invention belongs to solid waste resource recovery, and in particular to a bacillus (Bacillus
Axarquiensis) Effect of Pretreatment of CTS-BQ1 and its promotion Eichhornia crassipes matrix enzymolysis production sugar, can be applied to Eichhornia crassipes high level
Change the pre-treatment utilized.
Background technology
Eichhornia crassipes grows quick and strong adaptability as a kind of invasive water plant, on the one hand its, putrid and deteriorated rear easy
Polluted-water；On the other hand it is grown in water, and yield is big, is not take up land area, and its cellulose, hemicellulose level are high,
It is the raw materials for production of more potential biological energy source substance.But because its moisture content is high, lignin parcel cellulose makes it more difficult release
Put, be difficult to be utilized, it is pre-processed, and cost is high, and the sugared producing and ethanol efficiency of production is low.Biological Pretreatment method has that action condition is gentle, energy
The low, environmental pollution of consumption is small, and the low advantage of processing cost is the preprocess method of great potential.But current Biological Pretreatment is ground
Lignin degradation microbe species are still had in studying carefully few, degradation condition harshness, the enzyme activity of lignin decomposition enzyme's class is low, effect
Cycle is long, the problems such as portion of cellulose and hemicellulose can be consumed by bacterium in processing procedure；Biology enzyme financial cost is high,
Enzyme pretreatment substantially increases production cost so that Biological Pretreatment is difficult to industrial applications.How above mentioned problem is overcome,
Finding suitable pretreated fermentation bacterial strain turns into the focus of Biological Pretreatment research.
Degumming bacterium is used for the research to ramie, by remove the gum components in crudefiber crop material and meanwhile retain its fiber into
Point, strengthen bast-fibre performance, this Degumming method rarely has for other types plant to be related to.Forefathers are used for reference to ramie degumming bacterium
Screening technique, the strain excellent of screening degraded Eichhornia crassipes non-cellulose from Eichhornia crassipes different growing environment, and to its producing enzyme
Characteristic is probed into.The degumming strain reported at present has actinomyces, cellulomonas cartae, Shigella and bacillus subtilis
Deng, these are all the degumming strains to crudefiber crop, and to Bacillus axarquiensis report it is less.
The content of the invention
To improve the deficiencies in the prior art, the invention provides a kind of bacillus and its application, the bacillus is located in advance
Reason Eichhornia crassipes enzymolysis production sugar, which has, remarkably promotes effect.
First aspect present invention provide a kind of bacillus (Bacillus axarquiensis) CTS-BQ1, in
It is preserved in China typical culture collection center on May 18th, 2017, preserving number is CCTCC NO：M2017273, address：In
State, Wuhan, Wuhan University.
Second aspect of the present invention answering in Eichhornia crassipes matrix enzymolysis production sugar is promoted there is provided above-mentioned bacillus CTS-BQ1
With.
Third aspect present invention provides a kind of method for promoting the enzymolysis production of Eichhornia crassipes matrix sugared, and step includes：Adopt gemma
Mother bacterial liquid is obtained after bacillus CTS-BQ1, amplification cultivation, is then seeded to mother bacterial liquid in the fermentation flask for filling the fresh sample of Eichhornia crassipes.
Fourth aspect present invention provides a kind of microbial bacterial agent for promoting the enzymolysis production of Eichhornia crassipes matrix sugared, the microorganism
The active component of microbial inoculum includes above-mentioned bacillus CTS-BQ1.
The beneficial effects of the invention are as follows：Bacterial strain provided by the present invention enriches wild degumming bacterium genetic resources, expands
The standby storehouse of degumming bacterium full-length genome breeding.The bacterium can more improve Eichhornia crassipes enzymolysis production sugar than the report preferable whiterot fungi of effect
Amount, efficiency high than business pectase economy and effect significantly, has broad application prospects.Using detection matrix enzymolysis production sugar
The mode of efficiency is detected after bacillus CTS-BQ1 pretreatment Eichhornia crassipes in the experiment of the sugared effect of matrix enzymolysis production, in enzymolysis liquid
Maximum candy output is 142mg/g raw material butts, and the sugared effect of production improves notable.
Brief description of the drawings
Fig. 1：72h enzymolysis output of sugar compares after isolated strains pretreatment Eichhornia crassipes.
Fig. 2：The pretreatment fluid sugared content figure of isolated strains.
Fig. 3：The sugared ratio chart of production consumption of isolated strains.
Fig. 4：Different pretreatments condition pre-processes the shadow of the enzymolysis candy output of the fresh sample matrix of Eichhornia crassipes to bacterial strain CTS-BQ1
Ring.
Fig. 5：The enzymolysis output of sugar of different biological pre- place's Eichhornia crassipes matrix compares.
Embodiment
First aspect present invention provide a kind of bacillus (Bacillus axarquiensis) CTS-BQ1, in
It is preserved in China typical culture collection center on May 18th, 2017, preserving number is CCTCC NO：M2017273, address：In
State, Wuhan, Wuhan University.
Above-mentioned bacillus CTS-BQ1 is to utilize isolation medium using pectin as sole carbon source from the life near pig farm
One plant of obtained bacterium is screened in the pond water of long Eichhornia crassipes.The colony morphology characteristic of the bacillus CTS-BQ1：Bacterium colony is
Dirty white, the smooth moistening in surface, edge are irregular, gram-positive bacteria.
Bacillus CTS-BQ1 16S rDNA sequences are as shown in sequence table SEQ ID No.1, with Genbank databases
In sequence be compared, it is final to determine that bacterial strain CTS-BQ1 is bacillus.
Second aspect of the present invention answering in Eichhornia crassipes matrix enzymolysis production sugar is promoted there is provided above-mentioned bacillus CTS-BQ1
With.
In one embodiment of the invention, bacillus is detected by way of detecting the sugared efficiency of matrix enzymolysis production
Matrix enzymolysis produces the effect of sugar after CTS-BQ1 pretreatment Eichhornia crassipes, and specific method is：Using above-mentioned bacillus CTS-BQ1, expand
Mother bacterial liquid is obtained after increasing culture, mother bacterial liquid is inoculated into culture medium containing degumming by 2% inoculum concentration, shaken in 35 DEG C, 145rpm
Swing culture 3 days, separation of solid and liquid, by solid in being dried at 65 DEG C, with the cellulase load enzymolysis drying solid of 30FPU/g substrates
Candy output is reduced in 72h, DNS method detection enzymolysis liquid.Maximum candy output is 142mg/g raw material butts, the sugared effect of production in enzymolysis liquid
Improve notable.It can be seen that bacillus CTS-BQ1 is promoting Eichhornia crassipes matrix enzymolysis production sugar to have a good application prospect.
Third aspect present invention provides a kind of method for promoting the enzymolysis production of Eichhornia crassipes matrix sugared, and step includes：Adopt gemma
Mother bacterial liquid is obtained after bacillus CTS-BQ1, amplification cultivation, is then seeded to mother bacterial liquid in the fermentation flask for filling the fresh sample of Eichhornia crassipes.
It is preferred that, the mother bacterial liquid is seeded to by 2% inoculum concentration in the fermentation flask for filling the fresh sample of Eichhornia crassipes, 35 DEG C,
Cultivated 3 days under conditions of initial pH=9.
Fourth aspect present invention provides a kind of microbial bacterial agent for promoting the enzymolysis production of Eichhornia crassipes matrix sugared, the microorganism
The active component of microbial inoculum includes above-mentioned bacillus CTS-BQ1.
Promote the gemma bar that Eichhornia crassipes matrix enzymolysis produces sugar below in conjunction with one plant that specific embodiment is provided the present invention
Bacterium and its application are further described.The embodiments described below is exemplary, is only used for explaining the present invention, and can not be managed
Solve as limitation of the present invention.
Experimental method in following embodiments, is conventional method unless otherwise specified.Reality used in following embodiments
Test material unless otherwise specified, be that market is commercially available.
Involved culture medium prescription is as follows in embodiment：
Isolation medium：(Ⅰ)K2SO40.01%0.1g, CaCl2 0.2g, NaCl 0.2g, MgSO47H2O 0.3g,
KNO30.5g, (NH4)2SO40.5g, K2HPO41g, agar 20g, distilled water 1L；(II) pectin 1g, agar 5g, pH7.0, steam
Distilled water 1L.In culture dish fall double-layer plate, top layer be 5mL culture mediums II, basic unit be 10mL culture mediums I.
Hydrolysis circle determines culture medium：(Ⅰ)FeSO40.01g, CaCl20.2g, MgSO4·7H2O 0.5g, NaCl 0.5g,
KH2PO41g, NH4NO33g, pectin 5g, agar 20g, pH7.0, distilled water 1L；(II) 20g agar, distilled water 1L.In culture
In ware fall double-layer plate, top layer be 10mL culture mediums I, basic unit be 10mL culture mediums II.
Degumming culture medium：The fresh sample 5g of Eichhornia crassipes, salting liquid 150mL, pH are about 7.0；Salting liquid： MgSO4·7H2O
0.5g, K2HPO40.5g, (NH4)2SO45g, sterilized water 1L.
Slant medium：Beef extract-peptone solid medium.
Whiterot fungi pre-processes basic culture solution：MgSO4.7H2O 0.05g/L, KH2PO40.2g/L, CaCl20.01 g/L,
Vitamin B1 100mg/L, Tween-80 0.05%, 1mL inorganic salt solutions.Inorganic salt solution：0.5 g/L MnSO4·H2O,
0.1g/L FeSO4·7H2O, 0.1g/L CoCl2·6H2O, 0.1g/LZnSO4·7H2O, 0.01g/L CuSO4·5H2O,
0.01g/L AlK(SO4)2·12H2O, 0.01g/L H3BO3, 0.01g/LNa2MoO4·2H2O, 3.0g/LMgSO4·7H2O。
Embodiment 1
The present embodiment has carried out the preliminary screening of degumming microorganism, and specific screening process is as follows:
Gather pond water near Hua Zhong Agriculture University pig farm in the pond of growth Eichhornia crassipes, sediment of pond, South Lake water,
South Lake bed mud, wild root of Dahurian angelica lake water, wild root of Dahurian angelica lakebed mud, fresh pig dung and the vigorous Eichhornia crassipes bacterial strain of growth.Eichhornia crassipes bacterial strain is cleaned, sheared
Into 1~2cm segment, surface moisture is air-dried.10g bed muds (aqueous) are weighed in the sterile triangular flask equipped with 90mL aqua sterilisas,
In using two layers of filtered through gauze after the hunting speed vibration 1h of 125r/min under normal temperature, bed mud suspension is obtained；
The fresh sample 5g of Eichhornia crassipes after air-drying is weighed in sterile triangular flask, 30mL water samples, bed mud suspension is separately added into, then
270mL sterilized waters are added, 35 DEG C of cultures are scattered to Eichhornia crassipes fiber in constant incubator, and about 5d obtains one-step growth culture
Liquid.The fresh sample of 5g Eichhornia crassipes separately is weighed in sterile triangular flask, is separately added into one-step growth nutrient solution, the pond bottom of 30mL pond waters
The one-step growth nutrient solution of mud, adds 270mL sterilized waters, and 35 DEG C of cultures are scattered to Eichhornia crassipes fiber in constant incubator,
About 4d, obtains secondary Multiplying culture liquid.Bis- Multiplying culture liquid of 1mL are drawn in the sterile centrifugation tube containing 9mL sterilized waters, rifle
Head rinse 3 times repeatedly, shake up, produce 10-1The dilution of concentration.The dilution of 1mL 10-1 concentration is drawn in sterile containing 9mL
In the sterile centrifugation tube of water, pipette tips rinse 3 times repeatedly shake up, produce 10-2The dilution of concentration.Repeat to prepare 10 with this-1
~10-7The dilution bacteria suspension of concentration.Each sample draws 0.1mL 10 respectively-5、10-6、10-7The dilution bacterium solution of concentration, Yu Yiguo
Glue is placed in 35 DEG C of inversion cultures in constant incubator to be coated with the isolation medium of sole carbon source after surface moisture is blotted
3d.The preferable 4 plants of bacterial strains of growing way in culture medium are taken after 3d, by 4 plants of bacterial strains of acquisition on beef extract-peptone slant medium
Line, purifying 4~10 times.Above-mentioned isolated bacterial strain point is connected to hydrolysis circle and determines culture medium, 35 DEG C of cultures by bacterium colony primary dcreening operation
2d.The isolated bacterial strain point of enrichment culture is connected into hydrolysis circle to determine on culture medium, 3d is cultivated at 35 DEG C, is growing bacterium colony
On culture medium, covering quality concentration is 1mg/mL Congo red solution, after 10~15min, removes Congo red solution, adds material
Amount concentration be lmol/L NaCl solution, outwell NaCl solution after 15min, now, the periphery of bacterial colonies for producing pectase will
There is transparent circle.The colony diameter and hydrolytic circle of bacterium are measured, the bacterial strain for producing substantially hydrolysis circle is named as successively
XZF-HQ、CTDN-RQB、 CTS-BQ1、NHS-S1、CTS-S1、CTDN-HQ、YZHS-BQ1、YZHS-BQ2。
Embodiment 2
The present embodiment has carried out secondary screening to the degumming microorganism that embodiment 1 is obtained, and detailed process is as follows：
The bacterial strain that primary dcreening operation is obtained is inoculated into degumming culture medium respectively, 35 DEG C, 145rpm, is cultivated 3 days, while to be not added with
The liquid phosphorus decomposing culture medium of any bacterium is as a control group.Separation of solid and liquid after the completion of culture, liquid surveys total reducing sugars amount with DNS methods,
Solid digests reduced sugar in 72h, DNS methods detection enzymolysis liquid in being dried at 65 DEG C, with the cellulase load of 30FPU/g substrates and produced
Amount.As a result as shown in figure 1, having rush to the enzymolysis production sugar of matrix after above-mentioned 8 plants of bacterium pretreatment Eichhornia crassipes that the present invention is screened
Enter effect, while having a certain degree of consumption to reduced sugar in pretreatment fluid, as a result as shown in Figure 2；To be reduced in pretreatment
The consumption of sugar handled for bacterial strain in consumption sugar amount, using the sugared increment in enzymolysis liquid as pretreated output of sugar, make to produce sugar and
The ratio of consumption sugar is shown in Fig. 3, wherein maximum with CTS-BQ1 ratios, consuming minimum sugar has the output of maximum.
Embodiment 3
The present embodiment makes identification to bacterial strain CTS-BQ1
(1) bacterial strain CTS-BQ1 colonial morphology and property
Bacterium colony is dirty white, the smooth moistening in surface, and edge is irregular, gram-positive bacteria.
(2) bacterial strain CTS-BQ1 molecular biology identification
The CTS-BQ1 bacterial strains that above-mentioned screening is obtained are identified using molecular biology method, the DNA of the bacterial strain is made
Use universal primer 27F, 1492R to be expanded by PCR, amplified production carries out sequencing through sequencing company, will be sequenced row and
Sequence in GenBank databases carries out BLAST comparisons, as a result shows, the bacterial strain and bacillus (Bacillus
Axarquiensis similarity) is more than 99%.Combining form feature, cultural characteristic and 16S rRNA sequence analyses, the bacterium
Strain is defined as bacillus (Bacillus axarquiensis).
Embodiment 4
The influence that different condition of culture promote bacterial strain CTS-BQ1 Eichhornia crassipes matrix enzymolysis to produce sugar is present embodiments provided,
Detailed process is as follows：
Design the horizontal quadrature of 4 factor 4 experiment probe into temperature (25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C), initial pH value (3,5,7,
9), inoculum concentration (2%, 4%, 6%, 8%) and processing time (1d, 3d, 5d, 7d) pre-process phoenix eyes to bacillus CTS-BQ1
The influence that lotus digests production sugar to matrix is tested.Eichhornia crassipes matrix reduction candy output pre-processing the varying level of single factor test
Variance analysis result as shown in figure 4, enzymolysis reduction candy output with temperature rise have between increased trend, different temperatures level
There was no significant difference.Enzymolysis production sugar is not made significant difference when initial pH is 3,5,7, and when pH is 9, enzymolysis output of sugar is more other
Level is dramatically increased.The optimum condition that bacillus CTS-BQ1 pretreatments are obtained after analysis is temperature 35, initial pH 9, inoculation
Amount 2%, processing time 3d, enzymolysis output of sugar reaches 142mg/g under the conditions of managing in this place.
Embodiment 5
Present embodiments provide different biologic pretreatment methods and the influence for producing sugar is digested to Eichhornia crassipes, detailed process is as follows：
The fresh sample 15g of the Eichhornia crassipes after grinding (moisture content is 92.6%) is weighed in 250mL conical flasks, by solid-to-liquid ratio 1:
2.5 add basic culture solution, and natural pH is sealed after sterilizing at 121 DEG C.It is cooled to the yellow spore that a diameter of 1cm is inoculated with after room temperature
The flat lead fungi strain block of raw wool 5, in culture 10 days at 28 DEG C.After the completion of culture take out inoculation block, by sample clean, 65 DEG C drying
Weigh, determine 72h enzymolysis reduction candy outputs.Not to be inoculated with Phanerochaete chrysosporium sample as control, each processing sets 3 weights
It is multiple.
The fresh sample 15g of the Eichhornia crassipes after grinding (moisture content is 92.6%) is weighed in 250mL triangular flasks, by solid-to-liquid ratio 1:30
PH3.5 citric acid-sodium citrate buffer 30mL is added, sealing is after sterilizing at 121 DEG C.Room temperature is cooled to after 50 DEG C
Preheated in water-bath, pectase enzyme liquid is added according to the enzyme load of 20U/g substrates, in being reacted under 125r/min hunting speeds, in advance
Handle 72h.After the completion of processing, separation of solid and liquid, solid matter milli-Q water to neutrality is dried at 65 DEG C and weighed.To be not added with
Enzyme liquid is control treatment, and each processing sets 3 repetitions.
The fresh sample 15g of the Eichhornia crassipes after grinding (moisture content is 92.6%) is weighed in 250mL conical flasks, salting liquid is added
150mL, sterilize 30min at 121 DEG C, is cooled to room temperature.It is 9 to adjust pH, and CTS-BQ1 mother bacterial liquids are accessed by 2% inoculum concentration
(control adds 2%LB nutrient solutions), 35 DEG C on shaking bath, 3d is handled under 145r/min hunting speeds.After the completion of processing,
Separation of solid and liquid is carried out with 0.45 μm of filter membrane, solid is washed to neutrality, weighed in drying at 65 DEG C, each 3 repetitions of processing.
The sugared effect of Eichhornia crassipes matrix enzymolysis production after three kinds of Biological Pretreatments is as shown in Figure 5.In 3 kinds of Biological Pretreatments, fruit
Glue enzyme pre-processes output of sugar highest, and CTS-BQ1 takes second place, and Phanerochaete chrysosporium is minimum.Different disposal and its production of contrast ratio raw material
Sugar amount is significantly improved, and Phanerochaete chrysosporium is not significantly different between being compareed with it, and CTS-BQ1 compares the sugared difference of production with it
Significantly, that the sugared difference of production is compareed with it is not notable for pectase.It is the most notable that effect is improved after CTS-BQ1 pretreatments.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
<110>Hua Zhong Agriculture University
<120>One plant promotes the sugared bacillus of Eichhornia crassipes enzymolysis production and its application
<160> 1
<170>PatentIn version 3.3
<210> 1
<211>1447
<212>RNA
<213>Bacillus（Bacillus axarquiensis）CTS-BQ1
<400> 1
attctgtcac cttcggcggc tggctcctaa aaggttacct caccgacttc gggtgttaca 60
aactctcgtg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg 120
ctgatccgcg attactagcg attccagctt cacgcagtcg agttgcagac tgcgatccga 180
actgagaaca gatttgtggg attggcttaa cctcgcggtt tcgctgccct ttgttctgtc 240
cattgtagca cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac 300
cttcctccgg tttgtcaccg gcagtcacct tagagtgccc aactgaatgc tggcaactaa 360
gatcaagggt tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac 420
aaccatgcac cacctgtcac tctgcccccg aaggggacgt cctatctcta ggattgtcag 480
aggatgtcaa gacctggtaa ggttcttcgc gttgcttcga attaaaccac atgctccacc 540
gcttgtgcgg gcccccgtca attcctttga gtttcagtct tgcgaccgta ctccccaggc 600
ggagtgctta atgcgttagc tgcagcacta aggggcggaa accccctaac acttagcact 660
catcgtttac ggcgtggact accagggtat ctaatcctgt tcgctcccca cgctttcgct 720
cctcagcgtc agttacagac cagagagtcg ccttcgccac tggtgttcct ccacatctct 780
acgcatttca ccgctacacg tggaattcca ctctcctctt ctgcactcaa gttccccagt 840
ttccaatgac cctccccggt tgagccgggg gctttcacat cagacttaag aaaccgcctg 900
cgagcccttt acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg 960
ctggcacgta gttagccgtg gctttctggt taggtaccgt caaggtgccg ccctatttga 1020
acggcacttg ttcttcccta acaacagagc tttacgatcc gaaaaccttc atcactcacg 1080
cggcgttgct ccgtcagact ttcgtccatt gcggaagatt ccctactgct gcctcccgta 1140
ggagtctggg ccgtgtctca gtcccagtgt ggccgatcac cctctcaggt cggctacgca 1200
tcgtcgcctt ggtgagccgt tacctcacca actagctaat gcgccgcggg tccatctgta 1260
agtggtagcc gaagccacct tttatgtctg aaccatgcgg ttcaaacaac catccggtat 1320
tagccccggt ttcccggagt tatcccagtc ttacaggcag gttacccacg tgttactcac 1380
ccgtccgccg ctaacatcag ggagcaagct cccatctgtc cgctcgactg cattatagac 1440
ccgcccc 1447
Claims (5)
1. a kind of bacillus (Bacillus axarquiensis) CTS-BQ1, is deposited in China typical culture collection
The heart, preserving number is CCTCC NO：M2017273.
2. applications of the bacillus CTS-BQ1 described in claim 1 in Eichhornia crassipes matrix enzymolysis production sugar is promoted.
3. a kind of method for promoting the enzymolysis production of Eichhornia crassipes matrix sugared, it is characterised in that：Step includes：Using described in claim 1
Bacillus CTS-BQ1, obtain mother bacterial liquid after amplification cultivation, then mother bacterial liquid be seeded to the fermentation flask for filling Eichhornia crassipes
In.
4. promote the method for Eichhornia crassipes matrix enzymolysis production sugar as claimed in claim 3, it is characterised in that：The mother bacterial liquid is pressed
2% inoculum concentration is seeded in the fermentation flask for filling Eichhornia crassipes, is cultivated 3 days under conditions of 35 DEG C, initial pH=9.
5. a kind of microbial bacterial agent for promoting the enzymolysis production of Eichhornia crassipes matrix sugared, it is characterised in that：The activity of the microbial bacterial agent
Composition includes bacillus CTS-BQ1 described in claim 1.
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