CN105274037B - One plant of lignocellulosic substance efficient degrading bacteria K24 and its application - Google Patents
One plant of lignocellulosic substance efficient degrading bacteria K24 and its application Download PDFInfo
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Abstract
One plant of lignocellulosic substance efficient degrading bacteria K24 and its application, it is related to one plant of lignocellulosic substance efficient degrading bacteria K24 and its application.It is Huo Shi enterobacterias (Enterobacter hormaechei) K24, on December 12nd, 2014 in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, preserving number CGMCC No.10169.It is used for lignocellulose degradation substance.Bacterial strain of the present invention has increment rapidly, adaptable, is widely used；The weight-loss ratio of Bag Material agaric waste material after Huo Shi enterobacterias (Enterobacter hormaechei) K24 fermentation process is 39% or more, and the weight-loss ratio of the maize straw after Huo Shi enterobacterias (Enterobacter hormaechei) K24 fermentation process is 29% or more.
Description
Technical field
The present invention relates to one plant of lignocellulosic substance efficient degrading bacteria K24 and its applications.
Background technology
China is a large agricultural country, and agricultural wastes yield is extremely huge, due to universal by economic benefit and technology
Limitation, the mostly extensive inefficient utilization of agricultural wastes and idle situation is serious results in waste of resources and environmental pollution, waste
Have become the pollution sources of Largest In China.And in agricultural wastes, there is a large amount of lignocellulosics, it would be highly desirable to which people go to develop
It utilizes.
Lignocellulosic is second abundant organic renewable resource and the microorganism that cellulose is only second in nature
It is most difficult to one of the ingredient of degradation.In recent years, the fungi of certain lignin degradings has started to be applied in practice, but still needs
Further exploitation.The application of lignin microbial degradation mainly has：Paper industry；Feed industry；Fermentation and food industry；Biology
Object fertilizer environmental protection；Bio-bleaching technology of ligninase, etc..
Agricultural organic solid is discarded at present is usually cultivated by recovery energy, organic fertilizer, poultry and livestock feed, edible bacterium
Base-material and the raw material of industry are utilized.In the production practices using agricultural wastes, physics, chemistry and bioremediation warp
Often it is used in combination, and wherein bioremediation represents development trend from now on especially with microbiological treatment.Biology side
Method refers to just the lignin gone using lignin-degrading enzymes in degradation lignocellulosic material, to make lignin-hemicellulose-fibre
The plain structure of dimension is disintegrated, and cellulose is able to be exposed for subsequent step processing compared with traditional machinery, physical chemistry class method, raw
The advantages of object facture is that low energy consumption, and required environmental condition is mild, avoids the energy consumptions such as traditional chemical processing, mechanical treatment technology
More, the shortcomings of there are environmental pollutions, consider from cost and equipment angle, biological delignification's method occupies unique advantage.But
It is that current biological treatment limits its application there are one prodigious weakness, and here it is the angled key in biological treatment
The activity of color-lignin-degrading enzymes is not universal high, relatively low so as to cause treatment effeciency, if can be using genetic engineering and traditional
Biotechnology is transformed strain and enzyme, improves enzyme activity, reduces enzyme cost, and bioanalysis delignification rule is expected to be applied to
Large-scale industrial production.
And most widely used at present is microbial-bacterial fertilizer, microbial-bacterial fertilizer as a kind of new agrotechnical measure,
Effect in development an agriculture featuring high yields, fine quality and high efficiency is gradually recognized by people.Traditional microbial manure is own through being pushed away in large area
In wide application, novel microbial-bacterial fertilizer kind is continually developed out.Microorganism fertilizer is by the microorganism warp with special efficiency
Everfermentation and specified microorganisms product that is manufactured, having specific fertilizer efficiency containing a large amount of beneficial microbes, to crop.Microbial manure
Using the vital movement of microorganism, it converts the substance that crop cannot be absorbed and utilized to the nutrients that can be absorbed by crops and utilize,
Improve the nutritional condition of crop, some effects for having stimulation plant growth concurrently or enhancing disease resistance improve to improve crop yield
Quality of agricultural product.It can increase agricultural product go out to earn foreign exchange day and industrial or agricultural debirs efficiently use, prevent environment dirty
Dye improves soil texture, the increase soil fertility great social profit to circulate benignly with protecting ecology and ecological benefits.
Most of domestic research is fungus degrading lignocellulosic.But it is universal using fungus degrading lignocellulosic
There is a problem of that enzyme activity is relatively low.Since bacterial reproduction is very fast, fermentation period is short, can be applied to industrial production, and bacterium generates
Cellulase general action condition be neutral or meta-alkalescence, this pollutes the useless of industry in slurrying, papermaking and detergent industry etc.
There is potential application prospect on water harnessing, therefore filter out effective strain from bacterium applied to ligocellulose degradation to have
Certain practical significance and development prospect.
Invention content
The purpose of the invention is to provide one plant of lignin efficient degrading bacteria K24 and its application.
One plant of lignin efficient degrading bacteria of the present invention is Huo Shi enterobacterias (Enterobacter hormaechei) K24,
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is Chaoyang District, Beijing City North Star west
The institute 3 of road 1, the deposit date is on December 12nd, 2014, preserving number CGMCC No.10169.
One plant of lignin efficient degrading bacteria K24 of the present invention is used for lignin degrading.
Beneficial effects of the present invention are as follows：
Huo Shi enterobacterias (Enterobacter hormaechei) K24 increments of the present invention are rapid, adaptable, application
Extensively；Continuous passage culture 10 times, strain growth situation, producing enzyme situation and enzyme activity are stablized, no degradation phenomena；To Bag Material wood
Ear waste material and maize straw have apparent degradation effect, are sent out through Huo Shi enterobacterias (Enterobacter hormaechei) K24
The weight-loss ratio of ferment treated Bag Material agaric waste material is 39% or more, through Huo Shi enterobacterias (Enterobacter
Hormaechei) weight-loss ratio of the maize straw after K24 fermentation process is 29% or more；With significant lignin degradation ability
With hemicellulose degradation ability, it is particularly possible to high-efficiency lignin degrading, the maize straw through complex micro organism fungicide fermentation process
Middle content of lignin is wooden in the maize straw through Huo Shi enterobacterias (Enterobacter hormaechei) K24 fermentation process
2 times of lignin content, as single bacterial strain Huo Shi enterobacterias (Enterobacter hormaechei) K24 to the drop of lignin
Solution ability is better than complex micro organism fungicide；When degrading sample, the essential element content in sample is not influenced, will not lead to sample
In full nitrogen, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content variation, preserve sample fertilizer efficiency.
It to be unique with lignin that the present invention is separated from Bag Material agaric waste material, woodland rotten wood and forest fieid soil
The original strain of carbon source for growth determines degradation capability and race relation to lignin, is complex micro organism fungicide structure in future
It builds and lays the foundation.Strong help is provided by being utilized for the validation of China's agricultural resource.
Description of the drawings
Fig. 1 is that Huo Shi enterobacterias of the present invention (Enterobacter hormaechei) K24 cultivates the scanning electron after 12h
Microscope figure (× 20,000)；
Fig. 2 is that the agarose gel electrophoresis detection of 16S rDNA sequence PCR amplifications is schemed wherein, and 1 swimming lane is Maker
DL2000,2 swimming lanes are bacterial strain K24；
Fig. 3 is that the PCR product agarose gel electrophoresis detection after recycling is schemed wherein, and 1 swimming lane is Maker DL2000,2 swimming
Road is bacterial strain K24；
Fig. 4 is that the agarose gel electrophoresis detection of positive clone molecule screening is schemed wherein, and 1 swimming lane is Maker DL2000,2 swimming
Road is bacterial strain K24；
Fig. 5 is the systematic evolution tree of Huo Shi enterobacterias of the present invention (Enterobacter hormaechei) K24；
Fig. 6 is weightlessness of Huo Shi enterobacterias of the present invention (Enterobacter hormaechei) K24 to Bag Material agaric waste material
Rate figure；
Fig. 7 is weight-loss ratios of Huo Shi enterobacterias of the present invention (Enterobacter hormaechei) K24 to maize straw
Figure；
Fig. 8 is in maize straw after Huo Shi enterobacterias of the present invention (Enterobacter hormaechei) K24 strains for degrading
Each component content histogram；Wherein, 1 is content of lignin block diagram；2 be content of cellulose block diagram；3 be hemicellulose level
Block diagram；
Fig. 9 is corn stalk after the processing of Huo Shi enterobacterias of the present invention (Enterobacter hormaechei) K24 strain fermentations
Total nitrogen and total phosphor phosphorus and full potassium content block diagram in stalk；Wherein, 1 is total nitrogen content block diagram；2 be content of tatal phosphorus block diagram；3 be full potassium
Content histogram；
Figure 10 is corn after the processing of Huo Shi enterobacterias of the present invention (Enterobacter hormaechei) K24 strain fermentations
Available phosphorus contents block diagram in stalk；
Figure 11 is corn after the processing of Huo Shi enterobacterias of the present invention (Enterobacter hormaechei) K24 strain fermentations
Stalk effective K content histogram.
Specific implementation mode
Specific implementation mode one：One plant of lignocellulosic substance efficient degrading bacteria K24 of present embodiment, it is Huo Shi
Enterobacteria (Enterobacter hormaechei) K24, is deposited in China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and the deposit date is on December 12nd, 2014, preserving numbers
CGMCC No.10169。
Huo Shi enterobacterias (Enterobacter hormaechei) K24 of present embodiment is Gram-negative bacteria, the bacterium
Strain thalli morphology is rod-shaped, and thalline size is 0.52~0.56 × 1.3~1.5 μm, does not form gemma, atrichia and pod membrane；
Form that round, opaque, milky, protrusion be smooth, bacterium colony (as shown in Figure 1) of neat in edge on LB culture mediums.
The biochemical reactions result of the bacterial strain totally 18,28 indexs；Combining form and biochemical reactions as a result, than
It is right《Primary Jie Shi Bacteria Identifications handbook》Determine the kind of the bacterial strain, the results are shown in Table 1.
According to《Common bacteria system identification handbook》With《The outstanding bacterium handbook of uncle》To the enterobacteria isolated
(Enterobacter sp.) K24 carries out carry out Gram's staining, oxidizing ferment, catalase, fluorchrome, methyl red, aesculin
Hydrolysis, gelatin liquefaction, litmus milk peptonize and produce acid, lipase, Starch Hydrolysis, V.P. measurement, citrate utilization, cellulose drop
Solution, 3- ketone groups lactose utilization, phenylalanine deaminase, tryptophan deaminase, heat-resisting hot, salt tolerance bio-chemical characteristics inspection
It surveys and identifies.The result shows that enterobacteria (Enterobacter sp.) K24 is Gram-negative bacteria, salt tolerance is salinity 8%,
Heat resistance be 50 DEG C, catalase can be generated, but cannot generate oxidizing ferment, lipase, phenylalanine deaminase, tryptophan deaminase and
Fluorchrome, methyl red, citrate utilize, aesculin dissolving solution and litmus milk production acid show as the positive, and V.P. experiments are formed sediment
Powder hydrolysis, gelatin liquefaction, litmus milk peptonizes, cellulose degradation and 3- ketone group lactose utilizations show as feminine gender.
The morphological feature of 1 bacterial strain K24 of table and Physiology and biochemistry qualification result
The lignin efficient degrading bacteria of present embodiment is Huo Shi enterobacterias (Enterobacter hormaechei) K24
Screening technique it is as follows：
1, screening technique
It takes 10g samples to be aseptically fully ground, is added in the triangular flask equipped with 90mL sterile waters (with bead),
Vibrate 20min.It takes in triangular flask of the 5mL sample suspensions access equipped with 100mL LB liquid mediums, 37 DEG C, 180r/min oscillations
Cultivate 8h；The sample liquid that dilution is 10-1,10-2,10-3,10-4,10-5,10-6 is respectively prepared in bacterium solution, 200 μ L is respectively taken to apply
In lignin screening and culturing medium tablet, 37 DEG C of constant temperature incubation 48h adjust dilution gradient according to bacterium colony growing state.Keep culture
Condition is constant, the scribing line purifying repeatedly on lignin screening and culturing medium tablet of the single bacterium colony after 48h on each tablet of picking.Picking is pure
Single bacterium drop point after change is connected to lignin aniline blue culture medium flat plate, and 37 DEG C are protected from light constant temperature incubation 48h, measure the transparent of each bacterial strain
Loop diameter H and colony diameter C, filters out the larger bacterial strain of H/C values.Squamous subculture, continuous passage 10 are carried out to the bacterial strain of acquisition
It is secondary, it observes the upgrowth situation of bacterial strain and measures H/C values.According to bacterial strain respectively for colonial morphology, colony diameter, transparent loop diameter and
H/C value sizes determine strain growth situation and are protected to -80 DEG C of Freezing Glycerine methods of stability use of lignin degradation effect
It deposits, isolated strains preserve 3 pipes, write label (strain number, separately point, Habitat Types and holding time) exactly.
2, bacterial strain H/C values measure
Bacterial strain point is connected to lignin aniline blue culture medium flat plate, 37 DEG C are protected from light constant temperature incubation 48h, measure the saturating of each bacterial strain
Bright loop diameter H and colony diameter C.
3, interpretation of result：
3.1, bacterial strain screening
By above-mentioned separation screening process, 134 plants of bacterial strains that can be grown on lignin screening and culturing medium are obtained altogether.It will
This 134 plants of bacterial strains are inoculated on lignin aniline blue culture medium and are screened after purification, according to transparent circle generation time, clarity
Lignin-degrading bacteria is filtered out with H/C value sizes.Bacterial strain K24 transparent circles generate rapid and clear, the larger bacterial strain of H/C values,
It is determined as efficient lignin-degrading bacteria, according to the sample source of bacterium, numbers respectively.Above-mentioned bacterial strains are subjected to 10 passages
After culture, the H/C values of bacterial strain bacterium colony growing state, mode of appearance and bacterial strain have no significant change, show strain growth situation,
Producing enzyme situation and enzyme activity are stablized, no degradation phenomena.
3.2, bacterial strain H/C values measure
The generation of bacterial strain K24 transparent circles is clear rapid, and H/C values are larger；After 10 secondary cultures, the growth of the bacterial strain
Situation, producing enzyme situation and enzyme activity are stablized, and no degradation phenomena surveys the transparent loop diameter H and colony diameter C of this plant of bacterium
Fixed and data analysis.A diameter of 0.61 ± the 0.03cm of average colony of bacterial strain K24, maximum colony diameter reach 0.64cm；
Averagely degradation loop diameter is 1.25 ± 0.05cm, and most degradation loop diameter can reach 1.30cm.The average H/C values of bacterial strain K24 are
2.05 ± 0.08, maximum H/C values can reach 2.24.
Table 2K24 strains for degrading effects
4, the extraction of genomic DNA
The bacterial strain K24 genomic DNAs that above-mentioned screening is obtained are extracted using hot broken wall method.1mL is taken to be inoculated in the training of LB liquid
The bacteria suspension of 37 DEG C of 180r/min shaken cultivations of base for 24 hours is supported, is squeezed into 1.5mL centrifuge tubes, 5000r/min centrifuges 5min, abandons
Clearly, 1mL ddH are added2O, suction are beaten uniformly, and thalline is made to suspend, and 5000r/min centrifuges 5min, abandon supernatant, and 200 μ L ddH are added2O,
Suction is beaten uniformly, and thalline is made to suspend；8~10min in boiling water bath, 10000r/min centrifuge 10min.Aspirate supernatant is transferred to
In another 1.5mL centrifuge tube, 5 μ L point samples are taken, are Marker with λ EcoT14, the detection of 1% agarose gel electrophoresis, remaining -20
DEG C preserve.
5, the PCR amplification of 16S rDNA
Using 16S rDNA universal primers, using the strain gene group DNA extracted as template, according to following reaction system and
Amplification condition is expanded.Primer sequence reaction system and amplification condition are respectively as shown in table 3, table 4.PCR product with 1% fine jade
Lipolysaccharide electrophoresis detection.
The primer sequence of 3 16S rDNAPCR amplifications of table
The reaction system and response procedures of the PCR amplification of 4 16S rDNA of table
The recovery purifying of 5.1PCR products
By the PCR product whole point sample (80 holes μ L/, total holes) containing target stripe, 1.5% Ago-Gel electricity
Swimming, electrophoretic band are recycled with Tiangeng Ago-Gel DNA QIAquick Gel Extraction Kits, are as follows：
(1) column equilibration step：To adsorption column CA2In (being put into collecting pipe) be added 500 μ L equilibrium liquids BL, 12000r/
Min centrifuges 1min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.
(2) single target DNA band is cut from Ago-Gel and (cuts off redundance as possible) be put into it is clean
In centrifuge tube, weight is weighed.Formula：(weight of centrifuge tube before weight-dress glue after dress glue) × 1000=" 1000 times of volumes " μ
L。
(3) " 1000 times of volumes " sol solutions PN is added into blob of viscose, 10min is placed in 50 DEG C of water-baths, therebetween constantly leniently
Centrifuge tube is spun upside down, it is cooling to ensure that blob of viscose fully dissolves, sol solution temperature is down to room temperature upper prop again, 2 are stopped after upper prop
~5min.
(4) an adsorption column CA is added in previous step acquired solution2In, adsorption column is put into collecting pipe, 13000r/
Min centrifuges 1min, outwells the waste liquid in collecting pipe, adsorption column is reentered into collecting pipe.
(5) 600 μ L rinsing liquids PW (please first checked whether before use and absolute ethyl alcohol has been added) are added into adsorption column, stop
2min, 13000r/min centrifuge 1min, outwell waste liquid, adsorption column is reentered into collecting pipe.
(6) 500 μ L rinsing liquids PW, 13000r/min centrifugation 30 seconds is added into adsorption column, outwells waste liquid.Centrifugation is adsorbed
Column CA2It is put into collecting pipe, 13000r/min centrifuges 2min, removes rinsing liquid as possible.Adsorption column is placed in 50 DEG C of baking ovens and is dried
It several minutes, thoroughly dries, to prevent remaining rinsing liquid from influencing the experiment (influencing organic efficiency and DNA mass) of next step.
(7) adsorption column is put into a clean centrifuge tube and (cuts cap), 30 μ are vacantly added dropwise to adsorbed film centre position
L elution buffer EB, are placed at room temperature for 2min, and 13000r/min centrifuges 1min and collects DNA solution.
(8), can be by the obtained solution of centrifugation again add-back centrifugal adsorbing column in order to improve the yield of DNA, 13000r/
Min centrifuges 1min and collects DNA solution, is repeated 3 times elution.
(9) DNA solution is positioned in the centrifuge tube of lid (when last time elutes), -20 DEG C is stored in, to prevent DNA
Degradation.The DNA solution after a small amount of recycling is taken to verify its purity and content with 1% agarose electrophoresis.
The connection of 5.2 target fragments and cloning vector
The DNA segment that previous step PCR recovery purifyings obtain is uniformly mixed with pMD18-T carriers, 4 DEG C of reactions are overnight.Even
Junctor system is as follows：
Carrier after connection is transferred in E.coli DH5 α competent cells, after shaking training, coated plate, picking white colony connects
Kind 37 DEG C in the LB culture mediums containing Amp, 180r/min shaking table cultures 10~12 hours.
5.3 positive clone molecules detect
PCR amplification, primer sequence (referring to pMD18-T Vector specifications), reactant are carried out by template of gained bacterium solution
As shown in table 5 and table 6, product is detected with 1% agarose electrophoresis for system and amplification condition difference.
Primer sequence used in 5 recon PCR detections of table
The reaction system and response procedures of 6 recon PCR detections of table
5.4 16SrDNA sequence analyses
Obtained positive colony is sent to Shanghai life work (Sangon Biotech) bioengineering limited liability company to carry out
Sequencing analyzes sequencing result with 7.09 softwares of BioEdit, amputates primer sequence, the sequence results of acquisition are submitted to
GenBank databases obtain accession number, pass through BLASTn programs (http://www.ncbi.nlm.nih.gov/) it carries out online
The sequence of type strain of the similitude more than 90% is downloaded in analysis, is used in combination Clustal X softwares to carry out Multiple sequence alignments, so
The Neighbor-Joining phylogenetic tree constructions in software MEGA 5.03 are used afterwards, determine the race relation of bacterial strain.
6, bacterial strain 16S rDNA Sequence Identification results
6.1,16S rDNA sequences PCR amplification
16S rDNA sequences pcr amplification products are with 1% agarose gel electrophoresis inspection, and the results are shown in Figure 2.Bacterial strain K24
16S rDNA genetic fragment length be about 1500bp.
6.2, PCR product recycles
Electrophoresis carried out with 1.5% Ago-Gel to the 16S rDNA sequences pcr amplification products in 6.1, electrophoretic band with
DNA plastic recovery kits recycle.PCR product recycles electrophoresis pattern as shown in figure 3, according to band brightness it is found that this experiment has become
Work(is recovered to enough purified pcr products, can be used for follow-up test progress.
6.3, positive clone molecule is screened
16S rDNA pcr amplification products after purification are connect with carrier T, conversion to competent escherichia coli cell, with
Gained thalline is that template carries out PCR amplification, and the results are shown in Figure 4, has obtained the positive clone molecule with recombinant plasmid.
6.4,16S rDNA nucleotide sequencings
The 16S rDNA nucleotide sequencing results of each bacterial strain are shown in sequence table 1, phylogenetic tree such as Fig. 5 institutes of each bacterial strain
Show.Combining form and Physiology and biochemistry qualification result, determine the kind of each bacterial strain, the results are shown in Table 7.
The kind of 7 bacterial strain of table
By Morphological Identification, Physiology and biochemistry identification and 16S rDNA Molecular Identifications, determine that the bacterial strain of above-mentioned screening is intestines
Bacillus (Enterobacter sp.) bacterium K24.
Specific implementation mode two：The application of one plant of lignocellulosic substance efficient degrading bacteria of present embodiment, it is used
In lignocellulose degradation substance.
Following functions detection is carried out to the bacterial strain of the present invention：
By Huo Shi enterobacterias (Enterobacter hormaechei) K24 of specific implementation mode, it is useless to carry out Bag Material agaric
Material is measured with maize straw weight-loss ratio and lignin, cellulose and hemicellulose degradation, to verify its distinctive function.Specifically such as
Under：
1, Bag Material agaric waste material and maize straw weight-loss ratio measure
1.1 corn stalk powder
Maize straw is derived from the examination of this laboratory of Harbin, Heilongjiang Province Heilongjiang University Hulan school district in October, 2012
Field is tested, is dried, 40 mesh sieve is crushed, it is spare.
1.2 Bag Material agaric waste materials
Bag Material agaric waste material is provided by Heilongjiang Academy of Agricultural Sciences Mudanjiang branch.
1.3 control microbial inoculums
" the organic matter decomposing inoculant that Zhongnong Lvkang (Beijing) Biotechnology Co., Ltd. produced on October 27th, 2011
(straw type) ".
1.4 culture medium
Liquid fermentation medium：Glucose 5g, peptone 2g, NH4NO31.0g, CaCl20.2g, K2HPO40.5g,
FeCl30.02, MgSO4·7H2O 0.5g, NaCl 1.0g, distilled water 1000mL, pH 7.0.
Shake flask fermentation basal medium：Peptone 2g, NH4NO31.0g, CaCl20.2g, K2HPO40.5g, FeCl3
0.02, MgSO4·7H2O 0.5g, NaCl 1.0g, distilled water 1000mL, pH 7.0.
1.5 test method
1.5.1 Bag Material agaric waste material weight-loss ratio measures
Specific implementation mode one is screened to obtained Huo Shi enterobacterias (Enterobacter hormaechei) K24 inoculations
Into 5mL liquid fermentation mediums, 37 DEG C of 180r/min shaken cultivation 12h, centrifugation abandons supernatant and obtains thalline.Take 500 μ L shaking flasks
Fermentation basal medium makes thalline suspend, and bacteria suspension is accessed the shake flask fermentation basal medium containing 5% Bag Material agaric waste material
In, wherein Bag Material agaric waste material is sterilized using tyndallization, 121 DEG C of moist heat sterilization 30min, 37 DEG C of 180r/min shaken cultivations
After 30d, precipitation is washed with deionized in centrifugation, and after washing repeatedly three times, drying is weighed, and it is useless to calculate Bag Material agaric with Subtraction method
Expect weight-loss ratio, the data obtained is analyzed by SPSS19.0 softwares, Multiple range test is carried out using Duncan methods, as a result with marker word
Mother law indicates that the significance difference of each bacterial strain is anisotropic.Five groups of blank control groups and five groups of positive controls, i.e. blank control group are set not
Strain is connect, positive controls access 5% control microbial inoculum, other operations are identical as aforesaid operations.
Weight-loss ratio calculation formula is as follows：
1.5.2 maize straw weight-loss ratio measures
Specific implementation mode one is screened to obtained Huo Shi enterobacterias (Enterobacter hormaechei) K24 inoculations
Into 5mL liquid fermentation mediums, 37 DEG C of 180r/min shaken cultivation 12h, centrifugation abandons supernatant and obtains thalline.Take 500 μ L shaking flasks
Fermentation basal medium makes thalline suspend, and bacteria suspension is accessed in the shake flask fermentation basal medium containing 5% corn stalk powder,
Wherein corn stalk powder is sterilized using tyndallization, 121 DEG C of moist heat sterilizations 30min, 37 DEG C of 180r/min shaken cultivations 30d
Afterwards, it centrifuges, precipitation is washed with deionized, after washing repeatedly three times, drying is weighed, and it is weightless to calculate maize straw with Subtraction method
Rate analyzes the data obtained by SPSS19.0 softwares, Multiple range test is carried out using Duncan methods, as a result with marker word mother law mark
The significance difference of bright each bacterial strain is anisotropic.Five groups of blank control groups and five groups of positive controls are set, i.e. blank control group does not connect strain,
Positive controls access 5% complex micro organism fungicide, other operations are identical as aforesaid operations.
Weight-loss ratio calculation formula is as follows：
1.6 results and analysis
1.6.1 Bag Material agaric waste material weight-loss ratio measures
After bacterial strain K24 degradation Bag Material agaric waste materials, weight-loss ratio measurement result is as shown in table 8 and Fig. 6.
The Bag Material agaric waste material weight-loss ratio of 8 bacterial strain K24 of table
Note：The weight-loss ratio of Bag Material agaric waste material and maize straw after 30d liquid fermentations.Blank control group does not access bacterium
Kind, positive controls access the complex micro organism fungicide of 5% Zhongnong Lvkang (Beijing) Biotechnology Co., Ltd. production.Using
Duncan methods carry out Multiple range test.Significance p=0.05 is with lowercase letter, n=3.
By table 8 and Fig. 6 it is found that after culture 30d, ferment through Huo Shi enterobacterias (Enterobacter hormaechei) K24
Afterwards, the weight-loss ratio of Bag Material agaric waste material is 22.60 ± 0.24%；Blank control group Bag Material agaric waste material weight-loss ratio be 21.60 ±
0.82%；Positive controls Bag Material agaric waste material weight-loss ratio is 38.53 ± 0.87%.Through Huo Shi enterobacterias (Enterobacter
Hormaechei) weight-loss ratio of Bag Material agaric waste material is more than blank control group and positive controls after K24 degradations.According to notable water
Flat p=0.05 analyses, through Huo Shi enterobacterias (Enterobacter hormaechei) K24 fermentation process Bag Material agaric waste materials
Weight-loss ratio significant difference compared with the Bag Material agaric waste material weight-loss ratio of blank control group, the Bag Material agaric waste material with positive controls
Weight-loss ratio is not notable compared to difference.Huo Shi enterobacterias (Enterobacter hormaechei) K24 to Bag Material agaric waste material slightly
It is better than the degradation capability of complex micro organism fungicide in positive controls to Bag Material agaric waste material, the two is substantially at same level.
Huo Shi enterobacterias (Enterobacter hormaechei) K24 has very strong degradation capability to Bag Material agaric waste material.Through Huo Shi
The weight-loss ratio of Bag Material agaric waste material after enterobacteria (Enterobacter hormaechei) K24 fermentation process is 23% or more.
1.6.2 maize straw weight-loss ratio measures
After bacterial strain K24 degrading maize straws, weight-loss ratio measurement result is as shown in table 8 and Fig. 7.
By table 8 and Fig. 7 it is found that after culture 30d, ferment through Huo Shi enterobacterias (Enterobacter hormaechei) K24
Afterwards, the weight-loss ratio of maize straw is 48.52 ± 0.19%；Blank control group maize straw weight-loss ratio is 25.80 ± 0.63%；Sun
Property control group maize straw weight-loss ratio be 44.81% ± 1.02%.Through Huo Shi enterobacterias (Enterobacter hormaechei)
The weight-loss ratio of maize straw is more than blank control group after K24 degradations, is less than positive controls.According to level of signifiance p=0.05 points
Analysis, the maize straw weight-loss ratio through Huo Shi enterobacterias (Enterobacter hormaechei) K24 fermentation process and blank control
The maize straw weight-loss ratio of group compares significant difference, the significant difference compared with the maize straw weight-loss ratio of positive controls.Huo Shi
Enterobacteria (Enterobacter hormaechei) K24 has stronger degradation capability to maize straw.Through Huo Shi intestines bars
The weight-loss ratio of maize straw after bacterium (Enterobacter hormaechei) K24 fermentation process is 49% or more.
1.7 conclusion
Huo Shi enterobacterias (Enterobacter hormaechei) K24 filtered out through specific implementation mode one is to Bag Material
Agaric waste material and maize straw have apparent degradation effect.Through Huo Shi enterobacterias (Enterobacter hormaechei) K24
The weight-loss ratio of Bag Material agaric waste material after fermentation process is 23% or more；Through Huo Shi enterobacterias (Enterobacter
Hormaechei) weight-loss ratio of the maize straw after K24 fermentation process is 49% or more.Huo Shi enterobacterias (Enterobacter
Hormaechei) K24 is better than in positive controls complex micro organism fungicide to the drop of Bag Material agaric waste material to Bag Material agaric waste material
Solution ability.Bag Material agaric waste material and maize straw after Huo Shi enterobacterias (Enterobacter hormaechei) K24 degradations
Weight-loss ratio is all higher than the Bag Material agaric waste material of blank control group and the weight-loss ratio of maize straw；Wherein, Huo Shi enterobacterias
The weight-loss ratio of the Bag Material agaric waste material of (Enterobacter hormaechei) K24 fermentation process is more than positive controls Bag Material
Agaric waste material weight-loss ratio.
Huo Shi enterobacterias (Enterobacter hormaechei) K24 to the degradation effect of lignocellulosic substance with
Strain growth situation and sample composition have much relations, Bag Material agaric waste material and maize straw to be washed using Fan Shi (Van Soest)
It washs after fibre analysis method measures, each component content in maize straw and Bag Material agaric waste material is shown in Table 9, Bag Material agaric waste material and jade
The content of lignin, cellulose and hemicellulose in rice stalk is significantly different.Same bacterial strain is to Bag Material agaric waste material and jade
Rice stalk has different degradation effects, the reason is that containing due to lignin in Bag Material agaric waste material and maize straw and cellulose
The growth and breeding for measuring Different Effects bacterial strain cause strain growth situation different, and the secretion capacity of enzyme has differences, and then causes
Bacterial strain is different to the Utilization ability of catabolite, influences its degradation capability.
9 Bag Material agaric waste material of table and maize straw each component content
2, lignin, cellulose and hemicellulose degradation situation measure
2.1 culture mediums are prepared：
Liquid fermentation medium：Glucose 5g, peptone 2g, NH4NO31.0g, CaCl20.2g, K2HPO40.5g,
FeCl30.02, MgSO4·7H2O 0.5g, NaCl 1.0g, distilled water 1000mL, pH 7.0.
Shake flask fermentation basal medium：Peptone 2g, NH4NO31.0g, CaCl20.2g, K2HPO40.5g, FeCl3
0.02, MgSO4·7H2O 0.5g, NaCl 1.0g, distilled water 1000mL, pH 7.0.
Maize straw is derived from the examination of this laboratory of Harbin, Heilongjiang Province Heilongjiang University Hulan school district in October, 2012
Field is tested, is dried, 40 mesh sieve is crushed, it is spare.
2.2 liquid fermentation
Huo Shi enterobacterias (Enterobacter hormaechei) K24 of specific implementation mode one is inoculated into liquid hair
The bacteria suspension of OD600=0.5 is made in ferment culture medium, it is then with 5% (mL/mL) inoculum concentration that bacterium solution access is beautiful containing 7.5%
In the shake flask fermentation basal medium of rice straw powder, corn stalk powder is sterilized using tyndallization, 121 DEG C of moist heat sterilizations
30min, if repeating three times, 37 DEG C of 180r/min shaken cultivation 30d are dried using lyophilization.It is arranged one group
Blank control group and one group of positive controls, i.e. blank control group do not connect strain, and positive controls access 5% (g/mL) microbial inoculum,
Other operations are identical as aforesaid operations.
2.3 lignin, cellulose and hemicellulose degradation situation measure
Lignocellulosic each component content is measured using Fan Shi (Van Soest) washings fibre analysis method.Detailed mistake
Journey is as follows：
2.3.1, neutral detergent fiber (NDF) measures
By FiberCap specimen cups in 105 DEG C of oven drying 30min, taking-up is transferred in drier, after being cooled to 5min,
It weighs (being W1).It accurately weighs the sample after 2.000g (being W2) liquid fermentation to be placed in FiberCap specimen cups, by sample
Product cup is put into extraction beaker, and 400mL neutral detergents and 1mL decahydronaphthalenes and 2g anhydrous sodium sulfites is added.It will be on beaker sleeve
Condensation dress is placed in heating plate, boils 10min, continues slightly boiling 60min.After boiling, washed repeatedly with fresh hot water
Three times.Specimen cup is put into baking oven after 130 DEG C of drying 2h, is cooled to room temperature in drier, is weighed (being W3).
2.3.2, acid detergent fiber (ADF) measures
The FiberCap specimen cups containing neutral detergent fiber are placed in extraction beaker after above-mentioned drying is weighed, and are added
100mL acid detergents and 1mL decahydronaphthalenes and 2g anhydrous sodium sulfites.Condensation dress on beaker sleeve is placed in heating plate,
10min is boiled, slightly boiling 60min is continued.After boiling, three times with fresh hot water repeated washing.Specimen cup is put into baking oven
In after 130 DEG C of drying 2h, be cooled to room temperature, weigh (be W4) in drier.
2.3.3, acidic cleaning lignin (ADL) measures
The FiberCap specimen cups containing neutral detergent fiber are placed in extraction beaker after above-mentioned drying is weighed, and are added
72% sulfuric acid filters after 20 DEG C of digestion 3h, three times with fresh hot water repeated washing.Specimen cup is put into 130 DEG C of bakings in baking oven
It after dry 2h, is cooled to room temperature, weighs (being W5) in drier.
2.3.4, acid insoluble ash (AIA) measures
By specimen cup be placed in predrying and weigh (W6) ashing crucible (45 × 60mm) in, 600 DEG C of ashes in Muffle furnace
Change 4h.When crucible slowly cools to about 200 DEG C, taking-up is put in drier；It weighs after being cooled to room temperature (W6).
The data obtained is analyzed by SPSS19.0 softwares, Multiple range test is carried out using Duncan methods, as a result with marker word
Mother law indicates that the significance difference of each bacterial strain is anisotropic.Each calculation formula is as follows：
Neutral detergent fiber (NDF) content：
NDF (%)=(W3-W1)/W2 × 100%；
Acid detergent fiber (ADF) content：
ADF (%)=(W4-W3)/W2 × 100%；
Acidic cleaning lignin (ADL) content：
ADL (%)=W5/W2 × 100%；
Hemicellulose (Hemicellulose) content：
Hemicellulose (%)=NDF (%)-ADF (%)；
Cellulose (Cellulose) content：
Cellulose (%)=ADF (%)-W5/W2 × 100%；
Lignin (Lignin) content：
Lignin (%)=W5/W2 × 100%-W6/W2 × 100%；
Lignin proportionality coefficient：
2.4 results and analysis
2.4.1 lignin, cellulose and hemicellulose level measure
Maize straw is after strain fermentation handles 30d, cellulose, hemicellulose and content of lignin measurement result such as table 10
Shown in Fig. 8.
Each component average content in maize straw after 10 strains for degrading of table
Note：Blank control group does not access strain, and positive controls access the limited public affairs of the green health of 5% middle peasant (Beijing) biotechnology
Take charge of the complex micro organism fungicide of production.Multiple range test is carried out using Duncan methods.P=0.01 and p=0.05 points of significance
It is not indicated with upper and lower case letter, n=3.
By table 10 and Fig. 8 it is found that maize straw ferments through Huo Shi enterobacterias (Enterobacter hormaechei) K24
After handling 30d, content of lignin is 7.62 ± 0.72%, maize straw far smaller than in blank control group and positive controls
Content of lignin；It is analyzed with level of signifiance p=0.05, at Huo Shi enterobacterias (Enterobacter hormaechei) K24 fermentations
Content of lignin and content of lignin comparing difference in blank control group maize straw are notable in the maize straw of reason, right with the positive
It is notable according to content of lignin comparing difference in group maize straw；It is analyzed with level of signifiance p=0.01, Huo Shi enterobacterias
Content of lignin and blank control group corn stalk in the maize straw of (Enterobacter hormaechei) K24 fermentation process
Content of lignin comparing difference is notable in stalk, notable with content of lignin comparing difference in positive control maize straw group；Explanation
Huo Shi enterobacterias (Enterobacter hormaechei) K24 can be with high-efficiency lignin degrading.
Maize straw is analyzed, Huo Shi enterobacterias (Enterobacter after liquid fermentation 30d with level of signifiance p=0.05
Hormaechei) content of cellulose and content of cellulose in blank control group maize straw in the maize straw of K24 fermentation process
Comparing difference is not notable, not notable with corn stalk fiber cellulose content comparing difference in positive controls；With level of signifiance p=
0.01 analyzes, content of cellulose in the maize straw of Huo Shi enterobacterias (Enterobacter hormaechei) K24 fermentation process
Not notable, the fiber with maize straw in positive controls with the content of cellulose comparing difference of maize straw in blank control group
Cellulose content comparing difference is not notable；Illustrate that Huo Shi enterobacterias (Enterobacter hormaechei) K24 is unable to degradation of fibers
Element.
Maize straw is analyzed, Huo Shi enterobacterias (Enterobacter after liquid fermentation 30d with level of signifiance p=0.05
Hormaechei) hemicellulose level and hemicellulose in blank control group maize straw in the maize straw of K24 fermentation process
Comparision contents difference is not notable, notable with hemicellulose level comparing difference in positive controls maize straw；With level of signifiance p
=0.01 analyzes, the hemicellulose in Huo Shi enterobacterias (Enterobacter hormaechei) K24 fermentation process maize straws
Content and hemicellulose level comparing difference in blank control group maize straw be not notable, in positive controls maize straw half
Content of cellulose comparing difference is notable；Illustrating Huo Shi enterobacterias (Enterobacter hormaechei) K24 cannot degrade half fiber
Dimension element.
Huo Shi enterobacterias (Enterobacter hormaechei) K24 has significant lignin degradation ability, Ke Yigao
Imitate lignin degrading.The degradation capability of lignin is better than as single bacterial strain enterobacteria (Enterobacter sp.) K24 multiple
Microbial bacterial agent is closed, content of lignin is through Huo Shi enterobacterias in the maize straw through complex micro organism fungicide fermentation process
1.6 times of content of lignin in the maize straw of (Enterobacter hormaechei) K24 fermentation process.
2.5 conclusion
Huo Shi enterobacterias (Enterobacter hormaechei) K24 filtered out through specific implementation mode one has aobvious
The lignin degradation ability and hemicellulose degradation ability of work, it is particularly possible to high-efficiency lignin degrading.Through complex micro organism fungicide
Content of lignin is fermented through Huo Shi enterobacterias (Enterobacter hormaechei) K24 in the maize straw of fermentation process
1.6 times of content of lignin in the maize straw of processing, as single bacterial strain Huo Shi enterobacterias (Enterobacter
Hormaechei) K24 is better than complex micro organism fungicide to the degradation capability of lignin.
3, full nitrogen, full phosphorus, full potassium, rapid available phosphorus and available potassium measure
3.1, material and reagent
3.1.1, culture medium
Liquid fermentation medium：Glucose 5g, peptone 2g, NH4NO31.0g, CaCl20.2g, K2HPO40.5g,
FeCl30.02, MgSO4·7H2O 0.5g, NaCl 1.0g, distilled water 1000mL, pH 7.0.
Shake flask fermentation basal medium：Peptone 2g, NH4NO31.0g, CaCl20.2g, K2HPO40.5g, FeCl3
0.02, MgSO4·7H2O 0.5g, NaCl 1.0g, distilled water 1000mL, pH 7.0.
3.1.2, corn stalk powder
Maize straw is derived from the examination of this laboratory of Harbin, Heilongjiang Province Heilongjiang University Hulan school district in October, 2012
Field is tested, is dried, 40 mesh sieve is crushed, it is spare
3.2, test method
3.2.1, liquid fermentation
Huo Shi enterobacterias (Enterobacter hormaechei) K24 that specific implementation mode one filters out is inoculated into
OD is made in liquid fermentation medium600Then=0.5 bacteria suspension is contained bacterium solution access with 5% (mL/mL) inoculum concentration
In the shake flask fermentation basal medium of 7.5% corn stalk powder, corn stalk powder is sterilized using tyndallization, and 121 DEG C damp and hot
Sterilize 30min, and each bacterial strain is set to be repeated three times, and 37 DEG C of 180r/min shaken cultivation 30d are done using lyophilization
It is dry.One group of blank control group and one group of positive controls are set, i.e. blank control group does not connect strain, positive controls access 5%
(g/mL) microbial inoculum, other operations are identical as aforesaid operations.
3.2.2, sample solution prepare
Sample solution is carried out with reference to People's Republic of China (PRC) agricultural industry criteria NY525-2012, but is improved to some extent.
The accurate sample 0.5g (being accurate to 0.001g) weighed after being freeze-dried in 3.2.1, is placed in kjeldahl flask bottom, uses
A small amount of water flushing attaches the sample in bottle wall, adds 5mL sulfuric acid and 1.5mL hydrogen peroxide, carefully shakes up, bottleneck is put small with curved neck
Funnel is stood overnight, and being to slowly warm up to sulfuric acid on adjustable electric furnace smolders, and removes, and few cold plus 15 drop hydrogen peroxide gently shake
Kjeldahl flask heats 10min, removes, and the drop hydrogen peroxide of 5 drops of being in after slightly cold~10 and disappearing by several times boils, until solution is in colourless
Or after faint yellow clear liquid, continues to heat 10min, eliminate remaining hydrogen peroxide.It removes slightly cold, carefully adds water to 30mL, heat
To boiling.Cooling is removed, the curved small funnel of neck is rinsed with a small amount of water, washing lotion is put into former kjeldahl flask, and the boil liquid that will disappear moves into 100mL
In volumetric flask, add water constant volume, is filled into without phosphorus filter paper in dry blue lid reagent bottle, it is spare.Three groups of blank control groups are set,
In addition to being not added with sample, other operations are identical as aforesaid operations.
3.2.3, full nitrogen determination
Full nitrogen determination method with reference to People's Republic of China (PRC) agricultural industry criteria NY525-2012 and NY/T297-1995 into
Row, but improve to some extent.
Disappearing for being prepared in absorption 3.2.2 boils clear liquid 10mL in 50mL volumetric flasks, and 2mL boric acid is added and 200 μ L mixing refer to
Show agent mixed liquor, water is added to be settled to 50mL.It is distilled using kjeldahl apparatus, distillate is titrated with sulfuric acid standard solution, by
It is terminal, record consumption sulfuric acid titer volume (mL) that blue, which fades to aubergine,.The consumed sulfuric acid titer volume of blank determination
0.1mL is must not exceed, is otherwise redeterminated.Nitrogen (N) content is indicated with g/kg entirely, according to the following formula：
In formula：
V --- the volume of test solution titration consumption sulfuric acid standard solution, mL；
V0--- blank titration consumes the volume of sulfuric acid standard solution, mL；
C --- the concentration of sulfuric acid standard solution, mol/L；
0.014 --- with 1.00mL sulfuric acid (1/2H2SO4) the comparable nitrogen in grams of standard solution quality；
D --- point take multiple, constant volume/point take volume, 100/10；
M --- weigh sample mass, g；
1000 --- it is converted into the content of every kilogram of sample.
3.2.4, full phosphorus measures
Full phosphorus determination method with reference to People's Republic of China (PRC) agricultural industry criteria NY525-2012 and NY/T298-1995 into
Row, but improve to some extent.
Absorption phosphorus standard solution 0,1.00,2.00,3.00,4.00,5.00,6.00mL are respectively placed in 7 50mL volumetric flasks
In, the blank solution isometric with sample solution is drawn is added, adds water to 30mL, adds 400 μ L 2,6- dinitrophenol dinitrophenolate indicator molten
Liquid, it is in yellowish to adjust solution just with sodium hydroxide solution and sulfuric acid solution, adds 10.0mL vanadium ammonium molybdate reagents, shakes up, use water
It is settled to 50mL.This solution is the standard liquid series of 0,1.00,2.00,3.00,4.00,5.00,6.00 μ g of 1mL phosphorous (P).
After placing 20min under 15 DEG C of conditions above of room temperature, 2cm optical path cuvettes are used at spectrophotometer wavelength 440nm, with blank
Solution conditioning instrumentation zero carries out colorimetric, reads absorbance, draws standard curve according to phosphorus concentration and absorbance, finds out straight line
Regression equation.Disappearing for being prepared in absorption 3.2.2 boils clear liquid 10mL in 50mL volumetric flasks, 30mL is added water to, with standard solution system
Row read absorbance with condition colour developing, colorimetric.Content of tatal phosphorus indicates with g/kg, according to the following formula：
In formula：
C --- developing solution phosphorus concentration, μ g/mL are acquired by regression equation；
V --- color volume, 50mL；
D --- point take multiple, constant volume/point take volume, 100/10；
M --- weigh sample mass, g；
10-3--- μ g/g are converted into the factor of g/kg.
3.2.5, full potassium measures
Full potassium assay method with reference to People's Republic of China (PRC) agricultural industry criteria NY525-2012 and NY/T299-1995 into
Row, but improve to some extent.
Draw potassium standard solution 0,2.50,5.00,7.50,10.00mL be respectively placed in 5 50mL volumetric flasks, be added with
Draw the isometric placebo solution of sample solution, with water constant volume, this solution be 1mL containing potassium (K) 0,5.00,10.00,
15.00, the standard liquid series of 20.00 μ g.On flame photometer, with blank solution conditioning instrumentation zero, with standard solution
The standard solution of maximum concentration adjusts full value and indexes out to 80 in series.Again successively by low concentration to high measurement of concetration other standards
Solution, register instrument indicating value.Calibration curve is drawn according to potassium concn and instrument indicating value or finds out linear regression equation.It draws
3.2.2 disappearing for being prepared in boils clear liquid 5.00mL in 50mL volumetric flasks, with water constant volume.With standard liquid series with condition in flame
It is fixed on the upside of photometer, register instrument indicating value.It needs to be rectified an instrument with potassium standard solution after often measuring 5 samples.Full potassium content is with g/
Kg expressions, according to the following formula：
In formula：
C --- it is acquired by regression equation and measures liquid concentration, μ g/mL；
V --- volume is measured, this operation is 50mL；
D --- point take multiple, constant volume/point take volume, 100/5；
M --- weigh sample mass, g；
10-3--- the factor of g/kg is scaled by μ g/g.
3.2.6, rapid available phosphorus measure
Full potassium assay method is carried out with reference to People's Republic of China (PRC) agricultural industry criteria NY/T300-1995, but is changed
Into.
The accurate sample 1.00g weighed after being freeze-dried in 3.2.1 is placed in 50mL triangular flasks, is added 25 DEG C of 20mL's
Citric acid solution is jumped a queue, and vibrates 30min at 25 DEG C, is filtered into dry blue lid reagent bottle with without phosphorus filter paper, spare.If
Three groups of blank control groups are set, in addition to being not added with sample, other operations are identical as aforesaid operations.Assay method is identical as 3.2.4.It is quick-acting
Phosphorus content indicates with mg/kg, according to the following formula：
In formula：
C --- developing solution phosphorus concentration, μ g/mL are acquired by regression equation；
V --- color volume, 50mL；
D --- point take multiple, sample extracting liquid volume/point take volume, 20/5；
M --- weigh sample mass, g.
3.2.7, available potassium measure
The accurate sample 1.00g weighed after being freeze-dried in 3.2.1 is placed in 50mL triangular flasks, and it is molten that 10mL nitric acid is added
Liquid plugs small funnel, and the slightly boiling 10min on electric furnace is filtered while hot in 50mL volumetric flasks, is washed 5 times with hot water, fixed after cooling
Hold.Three groups of blank control groups are set, and in addition to being not added with sample, other operations are identical as aforesaid operations.Assay method and phase in 3.2.5
Together.Quick-acting potassium content indicates with mg/kg, according to the following formula：
In formula：
C --- it is acquired by regression equation and measures liquid potassium concn, μ g/mL；
V --- measure volume, 50mL；
M --- weigh sample mass, g.
3.3, result and analysis
3.3.1, full nitrogen, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content measure
Maize straw carries out full nitrogen, full phosphorus, full potassium, rapid available phosphorus and available potassium and surveys after strain liquid fermentation process 30d
It is fixed, as a result as shown in table 11 and Fig. 9~Figure 11.Maize straw is after liquid fermentation, according to the horizontal p=0.05 of the significance of difference
It is analyzed with p=0.01, it is the full nitrogen of Huo Shi enterobacterias (Enterobacter hormaechei) K24 fermentation process groups, full phosphorus, complete
Potassium, rapid available phosphorus and the quick-acting potassium content equal difference compared with blank control group is not notable.Therefore, bacterial strain degrades to maize straw
When do not interfere with essential element content therein, sample still preserves original fertilizer efficiency.
Essential element content in maize straw after 11 fermentation process of table
Note：Blank control group does not access strain.Multiple range test is carried out using Duncan methods.Significance p=0.01 and p
=0.05 is indicated with upper and lower case letter respectively, n=3.
3.4 conclusion
After Huo Shi enterobacterias (Enterobacter hormaechei) K24 handles 30d to maize straw liquid fermentation, examination
Full nitrogen, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content in sample have no significant change.Huo Shi enterobacterias (Enterobacter
Hormaechei) K24 will not cause the essential element content in sample when degrading sample, will not cause full nitrogen in sample,
Full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content variation, sample still preserve original fertilizer efficiency.
Claims (2)
1. one plant of lignocellulosic substance efficient degrading bacteria K24, it is characterised in that it for Huo Shi enterobacterias (Enterobacter hormaechei) K24, it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is north
The institute 3 of the Chaoyang Districts Jing Shi North Star West Road 1, the deposit date is on December 12nd, 2014, preserving number CGMCC No.10169.
2. the application of one plant of lignocellulosic substance efficient degrading bacteria K24 as described in claim 1, it is characterised in that it is used
The lignocellulosic substance in degradation Bag Material agaric waste material and maize straw.
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