CN105385632B - One plant of lignocellulosic substance efficient degrading bacteria S1 and its application - Google Patents

One plant of lignocellulosic substance efficient degrading bacteria S1 and its application Download PDF

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CN105385632B
CN105385632B CN201510863793.4A CN201510863793A CN105385632B CN 105385632 B CN105385632 B CN 105385632B CN 201510863793 A CN201510863793 A CN 201510863793A CN 105385632 B CN105385632 B CN 105385632B
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lignin
corn stover
enterobacter hormaechei
huo shi
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CN105385632A (en
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杨峰山
刘春光
孙永帅
吴奇
丁仕波
杨微
王鲲鹏
王庆华
魏才强
曲金玲
张慧
于文全
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Beijing middle peasant Guotai Technology Co., Ltd.
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Beijing Deruifeng Agriculture Technology Co ltd
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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02W30/00Technologies for solid waste management
    • Y02W30/40Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Abstract

One plant of lignocellulosic substance efficient degrading bacteria S1 and its application, it is related to one plant of lignocellulosic substance efficient degrading bacteria S1 and its application.It is Huo Shi enterobacteria (Enterobacter hormaechei) S1, on December 12nd, 2014 in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number CGMCC No.10165.It is used for lignocellulose degradation substance.Bacterial strain of the present invention has proliferation rapidly, adaptable, is widely used;The weight-loss ratio of Bag Material agaric waste material after Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process is 31% or more, and the weight-loss ratio of the corn stover after Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process is 33% or more.

Description

One plant of lignocellulosic substance efficient degrading bacteria S1 and its application
Technical field
The present invention relates to one plant of lignocellulosic substance efficient degrading bacteria S1 and its applications.
Background technique
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 there is a large amount of lignocellulosics for agricultural wastes, it would be highly desirable to which people go exploitation benefit With.
Lignin is that second organic renewable resource abundant that cellulose is only second in nature and microorganism are most difficult to One of ingredient of degradation.In recent years, the fungi of certain lignin degradings has started to be applied in practice, but still needs into one Step exploitation.The application of lignin microbial degradation mainly has:Paper industry;Feed industry;Fermentation and food industry;Biological object fertilizer Expect environmental protection;Bio-bleaching technology of ligninase, etc..
Agricultural organic solid is discarded at present usually passes through recovery energy, organic fertilizer, poultry and livestock feed, the culture of 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 especially with microbiological treatment represents development trend from now on.At biology Reason method just refers to the lignin gone in degradation lignocellulosic material using lignin-degrading enzymes, to make lignin-hemicellulose Element-cellulosic structure disintegrates, and cellulose is able to be exposed for subsequent step processing and traditional machinery, physical chemistry class method phase It is that low energy consumption than the advantages of, biological treatment, required environmental condition is mild, avoids traditional chemical processing, mechanical treatment technology Deng the disadvantages of more, there are environmental pollutions of consuming energy, consider from cost and equipment angle, biological delignification's method is occupied unique excellent Gesture.But current biological treatment has a very big weakness to limit its application, here it is the passes in biological treatment Bond angle color-lignin-degrading enzymes activity is not universal high, lower so as to cause treatment effeciency, if genetic engineering and biography can be utilized The biotechnology of system is transformed strain and enzyme, improves enzyme activity, reduces enzyme cost, and bioanalysis delignification rule is expected to answer For large-scale industrial production.
And most widely used at present is microbial-bacterial fertilizer, the microbial-bacterial fertilizer agrotechnical measure new as one kind, The effect developed in an agriculture featuring high yields, fine quality and high efficiency is gradually recognized by people.Traditional microbial manure is own to be 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 it is manufactured, containing a large amount of beneficial microbes, have the specified microorganisms product of specific fertilizer efficiency to crop.Microbial manure Using the vital movement of microorganism, the nutrients utilized can be absorbed by crops by converting the substance that crop cannot be absorbed and utilized to, 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 not only can increase earning foreign exchange day out for agricultural product, but also has the effective use of industrial or agricultural debirs, prevents environment dirty Dye, the great social profit and ecological benefits for improving soil texture, increasing soil fertility with protecting ecology benign cycle.
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 lower.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 is useless the pollution industry such as slurrying, papermaking and detergent industry There is potential application prospect on water harnessing, therefore filter out effective strain from bacterium to have applied to ligocellulose degradation Certain practical significance and development prospect.
Summary of the invention
The purpose of the invention is to provide one plant of lignin efficient degrading bacteria S1 and its application.
One plant of lignin efficient degrading bacteria of the invention is Huo Shi enterobacteria (Enterobacter hormaechei) S1, 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, deposit number CGMCC No.10165.
One plant of lignin efficient degrading bacteria S1 of the invention is used for lignin degrading.
Beneficial effects of the present invention are as follows:
Huo Shi enterobacteria (Enterobacter hormaechei) S1 proliferation of the invention is rapidly, adaptable, using wide General, salt tolerance is NaCl concentration 8%, and heat resistance is 50 DEG C;Continuous passage culture 10 times, strain growth situation, producing enzyme situation with And enzyme activity is stablized, no degradation phenomena;There is apparent degradation effect to Bag Material agaric waste material and corn stover, through Huo Shi intestines bar The weight-loss ratio of Bag Material agaric waste material after bacterium (Enterobacter hormaechei) S1 fermentation process is 31% or more, through suddenly The weight-loss ratio of corn stover after family name enterobacteria (Enterobacter hormaechei) S1 fermentation process is 33% or more;Tool There are apparent lignin degradation ability and hemicellulose degradation ability, it is particularly possible to high-efficiency lignin degrading.Through complex microorganism Content of lignin is sent out through Huo Shi enterobacteria (Enterobacter hormaechei) S1 in the corn stover of bacteria fermentation processing 2 times of content of lignin in the corn stover of ferment processing, as single bacterial strain Huo Shi enterobacteria (Enterobacter Hormaechei) S1 is better than complex micro organism fungicide to the degradation capability of lignin;When degrading sample, do not influence in sample Essential element content not will lead to full nitrogen, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content variation in sample, save sample fertilizer Effect.
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 in future structure It builds and lays the foundation.It is provided strong help being utilized for the validation of China's agricultural resource.
Detailed description of the invention
Fig. 1 is that the scanning electron after Huo Shi enterobacteria of the present invention (Enterobacter hormaechei) S1 culture 12h is aobvious Micro mirror figure (× 20,000);
Fig. 2 is that the agarose gel electrophoresis detection of 16S rDNA sequence PCR amplification is schemed wherein, and 1 swimming lane is Maker DL2000,2 swimming lanes are bacterial strain S1;
Fig. 3 is that PCR product agarose gel electrophoresis after the recovery detection is schemed wherein, and 1 swimming lane is Maker DL2000,2 swimming Road is bacterial strain S1;
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 S1;
Fig. 5 is the systematic evolution tree of Huo Shi enterobacteria of the present invention (Enterobacter hormaechei) S1;
Fig. 6 is weightlessness of Huo Shi enterobacteria of the present invention (Enterobacter hormaechei) S1 to Bag Material agaric waste material Rate figure;
Fig. 7 is weight-loss ratio figure of Huo Shi enterobacteria of the present invention (Enterobacter hormaechei) S1 to corn stover;
Fig. 8 is in corn stover after Huo Shi enterobacteria of the present invention (Enterobacter hormaechei) S1 strains for degrading Each component content histogram;Wherein, 1 is content of lignin histogram;2 be content of cellulose histogram;3 be hemicellulose level Histogram;
Fig. 9 is corn stalk after the processing of Huo Shi enterobacteria of the present invention (Enterobacter hormaechei) S1 strain fermentation Total nitrogen and total phosphor phosphorus and full potassium content histogram in stalk;Wherein, 1 is total nitrogen content histogram;2 be content of tatal phosphorus histogram;3 be full potassium Content histogram;
Figure 10 is corn stalk after the processing of Huo Shi enterobacteria of the present invention (Enterobacter hormaechei) S1 strain fermentation Available phosphorus contents histogram in stalk;
Figure 11 is corn stalk after the processing of Huo Shi enterobacteria of the present invention (Enterobacter hormaechei) S1 strain fermentation Stalk effective K content histogram.
Specific embodiment
Specific embodiment one:One plant of lignocellulosic substance efficient degrading bacteria S1 of present embodiment, it is Huo Shi Enterobacteria (Enterobacter hormaechei) S1, 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, deposit numbers CGMCC No.10165。
Huo Shi enterobacteria (Enterobacter hormaechei) S1 of present embodiment is Gram-negative bacteria, the bacterium Strain thalli morphology be it is rod-shaped, thallus size be 0.55~0.58 × 1.4~1.5 μm, do 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 medium.The bacterial strain Biochemical reactions result totally 18,28 indexs;Combining form is with biochemical reactions as a result, comparing《Primary Jie Shi bacterium mirror Determine 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 Huo Shi enterobacteria isolated (Enterobacter hormaechei) S1 carry 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, fibre Tie up plain degradation, 3- ketone group lactose utilization, phenylalanine deaminase, tryptophan deaminase, heat-resisting hot, salt tolerance Physiology and biochemistry reality The detection and identification tested.The result shows that Huo Shi enterobacteria (Enterobacter hormaechei) S1 is Gram-negative bacteria, it is resistance to Salt is NaCl concentration 8%, and heat resistance is 50 DEG C, can produce catalase and urase, but cannot generate oxidizing ferment, lipase, phenylpropyl alcohol Propylhomoserin deaminase, tryptophan deaminase and fluorchrome, methyl red, V.P. test, citrate utilizes, aesculin dissolves, sugar Alcohol fermentation (mannose), litmus milk, which are peptonized, shows as the positive, Starch Hydrolysis, gelatin liquefaction, cellulose with litmus milk production acid Degradation and 3- ketone group lactose utilization show as feminine gender.
The morphological feature and Physiology and biochemistry qualification result of 1 bacterial strain S1 of table
The lignin efficient degrading bacteria of present embodiment is Huo Shi enterobacteria (Enterobacter hormaechei) S1's Screening technique is as follows:
1, screening technique
It takes 10g sample to be aseptically fully ground, is added in the triangular flask equipped with 90mL sterile water (with bead), Vibrate 20min.Take the access of 5mL sample suspension equipped in the triangular flask of 100mL LB liquid medium, 37 DEG C, 180r/min vibrates 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 plate, 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 plate of the single colonie after 48h on each plate 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, measures the transparent of each bacterial strain Loop diameter H and colony diameter C, filters out the biggish bacterial strain of H/C value.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 value.According to bacterial strain respectively for colonial morphology, colony diameter, transparent loop diameter and H/C value size determines strain growth situation and protects to -80 DEG C of Freezing Glycerine methods of stability use of lignin degradation effect It deposits, isolated strains save 3 pipes, write label (strain number, separately point, Habitat Types and holding time) exactly.
2, bacterial strain H/C value measures
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 after purification and are screened, according to transparent circle generation time, clarity Lignin-degrading bacteria is filtered out with H/C value size.It is rapidly and clear that bacterial strain S1 transparent circle generates, the biggish bacterial strain of H/C value, really It is set to efficient lignin-degrading bacteria, according to the sample source of bacterium, numbers respectively.Above-mentioned bacterial strains are subjected to 10 passages training After supporting, the H/C value of bacterial strain bacterium colony growing state, mode of appearance and bacterial strain has no significant change, and shows strain growth situation, produces Enzyme situation and enzyme activity are stablized, no degradation phenomena.
3.2, bacterial strain H/C value measures
The generation of bacterial strain S1 transparent circle is clear rapid, and H/C value is 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 The analysis of fixed and data.The average colony diameter of bacterial strain S1 is 0.40 ± 0.10cm, and maximum colony diameter reaches 0.50cm;It is flat Degradation loop diameter is 1.21 ± 0.03cm, and most degradation loop diameter can reach 1.24cm.The average H/C value of bacterial strain S1 is 3.00 ± 0.02, maximum H/C value can reach 3.02.
2 S1 strains for degrading effect of table
4, the extraction of genomic DNA
The bacterial strain S1 genomic DNA that above-mentioned screening is obtained is extracted using hot broken wall method.1mL is taken to be inoculated in LB Liquid Culture The bacteria suspension of 37 DEG C of 180r/min shaken cultivations of base for 24 hours, is squeezed into 1.5mL centrifuge tube, and 5000r/min is centrifuged 5min, abandons supernatant, 1mL ddH is added2O, suction are beaten uniformly, and thallus is made to suspend, and 5000r/min is centrifuged 5min, abandon supernatant, and 200 μ L ddH are added2O inhales It beats uniformly, thallus is made to suspend;8~10min in boiling water bath, 10000r/min are centrifuged 10min.Aspirate supernatant is transferred to another In one 1.5mL centrifuge tube, 5 μ L point samples are taken, are Marker with λ EcoT14,1% agarose gel electrophoresis detects, and remaining -20 DEG C It saves.
5, the PCR amplification of 16S rDNA
Using 16S rDNA universal primer, using extracted strain gene group DNA 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 of table amplification
The reaction system and response procedures of the PCR amplification of 4 16S rDNA of table
The recovery purifying of 5.1PCR product
By the PCR product whole point sample (80 holes μ L/, totally two holes) containing target stripe, 1.5% Ago-Gel electricity Swimming, electrophoretic band are recycled with Tiangeng Ago-Gel DNA QIAquick Gel Extraction Kit, and specific step is as follows:
(1) column equilibration step:To adsorption column CA2In (being put into collecting pipe) be added 500 μ L equilibrium liquid BL, 12000r/ Min is centrifuged 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 (excision redundance as far 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 sufficiently dissolves, sol solution temperature is down to room temperature upper prop again, stops 2 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 is centrifuged 1min, outwells the waste liquid in collecting pipe, adsorption column is reentered into collecting pipe.
(5) 600 μ L rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column, stop 2min, 13000r/min are centrifuged 1min, outwell waste liquid, adsorption column is reentered into collecting pipe.
(6) 500 μ L rinsing liquid PW, 13000r/min are added into adsorption column centrifugation 30 seconds, outwell waste liquid.Centrifugation is adsorbed Column CA2It is put into collecting pipe, 13000r/min is centrifuged 2min, as far as possible removing rinsing liquid.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 recovery 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 middle position L elution buffer EB, is placed at room temperature for 2min, and 13000r/min is centrifuged 1min and collects DNA solution.
(8) in order to improve the yield of DNA, the solution that can obtain centrifugation is again in add-back centrifugal adsorbing column, 13000r/ Min is centrifuged 1min and collects DNA solution, is repeated 3 times elution.
(9) DNA solution is placed in the centrifuge tube of lid (when last time elutes), -20 DEG C is stored in, to prevent DNA Degradation.A small amount of DNA solution after the recovery is taken to verify its purity and content with 1% agarose electrophoresis.
5.2 the connection of target fragment and cloning vector
The DNA segment that previous step PCR recovery purifying obtains is uniformly mixed with pMD18-T carrier, 4 DEG C of reactions are overnight.Even Junctor system is as follows:
Carrier after connection is transferred in E.coli DH5 α competent cell, after shaking training, coated plate, picking white colony is connect Kind is 37 DEG C in the LB culture medium containing Amp, 180r/min shaking table culture 10~12 hours.
The detection of 5.3 positive clone molecules
PCR amplification, primer sequence (referring to pMD18-T Vector specification), 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.
5 recon PCR of table detects used primer sequence
The reaction system and response procedures of 6 recon PCR of table detection
5.416SrDNA sequence is analyzed
Obtained positive colony is sent to raw work (Sangon Biotech) the bioengineering limited liability company in Shanghai to carry out Sequencing analyzes sequencing result with 7.09 software of BioEdit, amputates primer sequence, the sequence results of acquisition are submitted to GenBank database obtains accession number, passes through BLASTn program (http://www.ncbi.nlm.nih.gov/) it carries out online Analysis, the sequence of type strain of the downloading similitude greater than 90%, and Multiple sequence alignments are carried out with Clustal X software, so Afterwards using the Neighbor-Joining phylogenetic tree construction in software MEGA 5.03, the race relation of bacterial strain is determined.
6, bacterial strain 16S rDNA Sequence Identification result
6.1,16S rDNA sequence PCR amplification
16S rDNA sequence pcr amplification product is with 1% agarose gel electrophoresis inspection, as a result as shown in Figure 2.Bacterial strain S1's 16S rDNA genetic fragment length is about 1500bp.
6.2, PCR product recycles
Electrophoresis carried out with 1.5% Ago-Gel to the 16S rDNA sequence pcr amplification product in 6.1, electrophoretic band with The recycling of DNA plastic recovery kit.PCR product recycles electrophorogram as shown in figure 3, according to band brightness it is found that this experiment has become Function 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 product after purification is connect with carrier T, conversion to competent escherichia coli cell, with Gained thallus is that template carries out PCR amplification, as a result as shown in figure 4, having obtained the positive clone molecule with recombinant plasmid.
6.4,16S rDNA nucleotide sequencing
The 16S rDNA nucleotide sequencing result of each bacterial strain is shown in sequence table 1, the phylogenetic tree of each bacterial strain such as Fig. 5 institute 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
7, the optimal culture condition of bacterial strain
With five pH, temperature, nitrogen source, incubation time, revolving speed experimental factors, by orthogonal arrage L18(35) design orthogonal test, Optimal culture condition to determine lignin-degrading bacteria is as shown in table 8.
8 orthogonal test experimental factor of table and level
Bacterial strain S1 orthogonal experiments, are shown in Table 9.
9 bacterial strain S1 orthogonal experiments of table
By Morphological Identification, Physiology and biochemistry identification and 16S rDNA Molecular Identification, determine that the bacterial strain of above-mentioned screening is suddenly Family name enterobacteria (Enterobacter hormaechei) S1.
The optimal culture condition that lignin degradation bacterial strain M1 growth is obtained by orthogonal design table acquired results is pH= 6.5,30 DEG C of temperature, nitrogen source is ammonium tartrate, time 6d, revolving speed 180r/min.
Specific embodiment two:The application of one plant of lignocellulosic substance efficient degrading bacteria S1 of present embodiment, it For lignocellulose degradation substance.
Following functions detection is carried out to bacterial strain of the invention:
By Huo Shi enterobacteria (Enterobacter hormaechei) S1 of specific embodiment, it is useless to carry out Bag Material agaric Material is measured with corn stover 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 corn stover weight-loss ratio measure
1.1 corn stalk powder
Corn stover is derived from the examination of this laboratory of Harbin, Heilongjiang Province Heilongjiang University Hulan school district in October, 2012 Field is tested, dries, crushed 40 meshes, 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, FeCl30.02, 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 embodiment one is screened into obtained enterobacteria (Enterobacter sp.) S1 and is inoculated into 5mL liquid fermentation In culture medium, 37 DEG C of 180r/min shaken cultivation 12h, centrifugation abandons supernatant and obtains thallus.The 500 L shake flask fermentation bases μ are taken to cultivate Base makes thallus suspend, and bacteria suspension is accessed in the shake flask fermentation basal medium containing 5% Bag Material agaric waste material, wherein Bag Material wood Ear waste material is sterilized using tyndallization, after 121 DEG C of moist heat sterilizations 30min, 37 DEG C of 180r/min shaken cultivation 30d, is centrifuged, is used Deionized water washing precipitating, after washing repeatedly three times, drying weighing calculates Bag Material agaric waste material weight-loss ratio with Subtraction method, by institute It obtains data to analyze by SPSS19.0 software, carries out Multiple range test using Duncan method, each bacterium is as a result indicated with marker word mother law The significance difference of 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 right According to a group control microbial inoculum for access 5%, other operations are identical as aforesaid operations.
Weight-loss ratio calculation formula is as follows:
1.5.2 corn stover weight-loss ratio measures
Specific embodiment one is screened obtained Huo Shi enterobacteria (Enterobacter hormaechei) S1 to be inoculated into In 5mL liquid fermentation medium, 37 DEG C of 180r/min shaken cultivation 12h, centrifugation abandons supernatant and obtains thallus.500 μ L shaking flasks are taken to send out Ferment basal medium makes thallus suspend, and bacteria suspension is accessed in the shake flask fermentation basal medium containing 5% corn stalk powder, Middle corn stalk powder is sterilized using tyndallization, after 121 DEG C of moist heat sterilizations 30min, 37 DEG C of 180r/min shaken cultivation 30d, Precipitating is washed with deionized in centrifugation, and after washing repeatedly three times, drying weighing calculates corn stover weight-loss ratio with Subtraction method, will The data obtained is analyzed by SPSS19.0 software, is carried out Multiple range test using Duncan method, is as a result indicated respectively with marker word mother law The significance difference of 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 Control group accesses 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
Bacterial strain S1 degrades after Bag Material agaric waste material, and 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 S1 of table
Note:The weight-loss ratio of Bag Material agaric waste material and corn stover after 30d liquid fermentation.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 method carries out Multiple range test.Significance p=0.05 indicates with lowercase, n=3.
By table 8 and Fig. 6 it is found that fermenting after culture 30d through Huo Shi enterobacteria (Enterobacter hormaechei) S1 Afterwards, the weight-loss ratio of Bag Material agaric waste material is 30.56 ± 0.79%;Blank control group Bag Material agaric waste material weight-loss ratio be 21.6 ± 0.82%;Positive controls Bag Material agaric waste material weight-loss ratio is 38.53 ± 0.87%.Through Huo Shi enterobacteria (Enterobacter Hormaechei) weight-loss ratio of Bag Material agaric waste material is greater than blank control group after S1 degradation, is less than positive controls.According to significant Horizontal p=0.05 analysis, through Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process Bag Material agaric waste material 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 compares significant difference.Huo Shi enterobacteria (Enterobacter hormaechei) S1 to Bag Material agaric waste material have compared with Strong degradation capability.Bag Material agaric waste material after Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process Weight-loss ratio 31% or more.
1.6.2 corn stover weight-loss ratio measures
After bacterial strain S1 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 fermenting after culture 30d through Huo Shi enterobacteria (Enterobacter hormaechei) S1 Afterwards, the weight-loss ratio of corn stover is 33.47 ± 0.25%;Blank control group corn stover weight-loss ratio is 25.80% ± 0.63%; Positive controls corn stover weight-loss ratio is 44.81% ± 1.02%.Through Huo Shi enterobacteria (Enterobacter Hormaechei) weight-loss ratio of corn stover is greater than blank control group after S1 degradation, is less than positive controls.According to the level of signifiance P=0.05 analysis, corn stover weight-loss ratio through Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process with The corn stover weight-loss ratio of blank control group compares significant difference, and difference is aobvious compared with the corn stover weight-loss ratio of positive controls It writes.Huo Shi enterobacteria (Enterobacter hormaechei) S1 has stronger degradation capability to corn stover.Through Huo Shi The weight-loss ratio of corn stover after enterobacteria (Enterobacter hormaechei) S1 fermentation process is 33% or more.
1.7 conclusion
Huo Shi enterobacteria (Enterobacter hormaechei) S1 filtered out through specific embodiment one is to Bag Material wood Ear waste material and corn stover have apparent degradation effect.It is dropped through Huo Shi enterobacteria (Enterobacter hormaechei) S1 Bag Material agaric waste material and the weight-loss ratio of corn stover are all larger than the Bag Material agaric waste material and corn stover of blank control group after solution Weight-loss ratio.The weightlessness of Bag Material agaric waste material after Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process Rate is 31% or more;The mistake of corn stover after Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process Rate is 33% or more again.
Degradation effect and bacterium of Huo Shi enterobacteria (Enterobacter hormaechei) S1 to lignocellulosic substance Strain growing state and sample composition have much relations, Bag Material agaric waste material and corn stover to wash using Fan Shi (Van Soest) After the measurement of fibre analysis method, each component content in corn stover and Bag Material agaric waste material is shown in Table 9, Bag Material agaric waste material and corn The content of lignin, cellulose and hemicellulose in stalk is significantly different.Same bacterial strain is to Bag Material agaric waste material and corn Stalk has different degradation effects, the reason is that due to the content of lignin and cellulose in Bag Material agaric waste material and corn stover The growth and breeding of Different Effects bacterial strain cause strain growth situation different, and the secretion capacity of enzyme has differences, and then leads to bacterium Strain is different to the Utilization ability of catabolite, influences its degradation capability.
9 Bag Material agaric waste material of table and corn stover each component content
2, lignin, cellulose and the measurement of hemicellulose degradation situation
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, FeCl30.02, MgSO4·7H2O 0.5g, NaCl 1.0g, distilled water 1000mL, pH 7.0.
Corn stover is derived from the examination of this laboratory of Harbin, Heilongjiang Province Heilongjiang University Hulan school district in October, 2012 Field is tested, dries, crushed 40 meshes, it is spare.
2.2 liquid fermentation
Huo Shi enterobacteria (Enterobacter hormaechei) S1 of specific embodiment one is inoculated into liquid fermentation The bacteria suspension of OD600=0.5 is made in culture medium, bacterium solution access is then contained by 7.5% corn with 5% (mL/mL) inoculum concentration In the shake flask fermentation basal medium of straw powder, corn stalk powder is sterilized using tyndallization, 121 DEG C of moist heat sterilization 30min, If repeating three times, 37 DEG C of 180r/min shaken cultivation 30d are dried using lyophilization.One group of blank pair is set According to group and one group of positive controls, i.e. blank control group does not connect strain, and positive controls access 5% (g/mL) microbial inoculum, other behaviour Make identical as aforesaid operations.
2.3 lignin, cellulose and the measurement of hemicellulose degradation situation
Lignocellulosic each component content is measured using Fan Shi (Van Soest) washing fibre analysis method.Detailed mistake Journey is as follows:
2.3.1, neutral detergent fiber (NDF) measures
By FiberCap specimen cup in 105 DEG C of oven drying 30min, taking-up is transferred in drier, after being cooled to 5min, It weighs (as W1).Sample after accurately weighing 2.000g (as W2) liquid fermentation is placed in FiberCap specimen cup, by sample Product cup is put into extraction beaker, and 400mL neutral detergent and 1mL decahydronaphthalenes and 2g anhydrous sodium sulfite is added.It will be on beaker sleeve Condensation dress is placed in heating plate, boils 10min, continues slightly boiled 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 (as W3).
2.3.2, acid detergent fiber (ADF) measures
FiberCap specimen cup containing neutral detergent fiber after above-mentioned drying weighing is placed in extraction beaker, is added 100mL acid detergent and 1mL decahydronaphthalenes and 2g anhydrous sodium sulfite.Condensation dress on beaker sleeve is placed in heating plate, 10min is boiled, slightly boiled 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 (as W4) in drier.
2.3.3, acidic cleaning lignin (ADL) measures
FiberCap specimen cup containing neutral detergent fiber after above-mentioned drying weighing is placed in extraction beaker, is 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 (as 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;(W6) is weighed after being cooled to room temperature.
The data obtained is analyzed by SPSS19.0 software, Multiple range test is carried out using Duncan method, 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%;
2.4 results and analysis
2.4.1 lignin, cellulose and hemicellulose level measurement
Corn stover is after strain fermentation handles 30d, cellulose, hemicellulose and content of lignin measurement result such as table 10 With shown in Fig. 8.
Each component average content in corn stover 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 method.Significance p=0.01 and p=0.05 points It is not indicated with upper and lower case letter, n=3.
By table 10 and Fig. 8 it is found that corn stover is through Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation After managing 30d, content of lignin is 6.03 ± 0.91%, respectively less than Spruce lignin in blank control group and positive controls Content;With level of signifiance p=0.05 analysis, the jade of Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process Content of lignin and content of lignin comparing difference in blank control group corn stover be not significant in rice stalk, with positive controls Content of lignin comparing difference is significant in corn stover;With level of signifiance p=0.01 analysis, Huo Shi enterobacteria Content of lignin and blank control group corn stover in the corn stover of (Enterobacter hormaechei) S1 fermentation process Middle content of lignin comparing difference is not significant, significant with content of lignin comparing difference in positive control corn stover group;Explanation Huo Shi enterobacteria (Enterobacter hormaechei) S1 can be with lignin degrading.
Corn stover after liquid fermentation 30d, content of cellulose be 35.63 ± 5.20%, be all larger than blank control group and Corn stalk fiber cellulose content in positive controls;With level of signifiance p=0.05 analysis, Huo Shi enterobacteria (Enterobacter Hormaechei) content of cellulose and content of cellulose ratio in blank control group corn stover in the corn stover of S1 fermentation process It is not significant compared with difference, it is not significant with corn stalk fiber cellulose content comparing difference in positive controls;With level of signifiance p=0.01 It analyzes, content of cellulose and sky in the corn stover of Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process The content of cellulose comparing difference of corn stover is not significant in white control group, contains with the cellulose of corn stover in positive controls It is not significant to measure comparing difference;Illustrate that Huo Shi enterobacteria (Enterobacter hormaechei) S1 is unable to degraded cellulose.
For corn stover after liquid fermentation 30d, hemicellulose level is 18.04 ± 0.65%, is less than in blank control group Technique of Hemicellulose from Cornstalk content is greater than Technique of Hemicellulose from Cornstalk content in positive controls;With level of signifiance p=0.05 points It analyses, hemicellulose level and sky in the corn stover of Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process Hemicellulose level comparing difference is not significant in white control group corn stover, contains with hemicellulose in positive controls corn stover It is not significant to measure comparing difference;With level of signifiance p=0.01 analysis, Huo Shi enterobacteria (Enterobacter hormaechei) S1 Hemicellulose level comparing difference is not in hemicellulose level in fermentation process corn stover and blank control group corn stover Significantly, not significant with hemicellulose level comparing difference in positive controls corn stover;Illustrate Huo Shi enterobacteria (Enterobacter hormaechei) S1 can be with degradation of hemicellulose.
Huo Shi enterobacteria (Enterobacter hormaechei) S1 has lignin degradation ability and hemicellulose degradation Ability, it is particularly possible to high-efficiency lignin degrading.As single bacterial strain Huo Shi enterobacteria (Enterobacter hormaechei) S1 Complex micro organism fungicide is better than to the degradation capability of lignin, it is wooden in the corn stover through complex micro organism fungicide fermentation process Cellulose content is content of lignin in the corn stover through Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process 2 times.
2.5 conclusion
Huo Shi enterobacteria (Enterobacter hormaechei) S1 filtered out through specific embodiment one has obvious Lignin degradation ability and hemicellulose degradation ability, it is particularly possible to high-efficiency lignin degrading.It is sent out through complex micro organism fungicide Content of lignin is through Huo Shi enterobacteria (Enterobacter hormaechei) S1 fermentation process in the corn stover of ferment processing Corn stover in 2 times of content of lignin, as single bacterial strain Huo Shi enterobacteria (Enterobacter hormaechei) S1 Complex micro organism fungicide is better than to the degradation capability of lignin.
3, full nitrogen, full phosphorus, full potassium, rapid available phosphorus and available potassium measurement
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, FeCl30.02, MgSO4·7H2O 0.5g, NaCl 1.0g, distilled water 1000mL, pH 7.0.
3.1.2, corn stalk powder
Corn stover is derived from the examination of this laboratory of Harbin, Heilongjiang Province Heilongjiang University Hulan school district in October, 2012 Field is tested, dries, crushed 40 meshes, it is spare
3.2, test method
3.2.1, liquid fermentation
Huo Shi enterobacteria (Enterobacter hormaechei) S1 that specific embodiment one filters out is inoculated into liquid OD is made in body fermentation medium600Then bacterium solution access is contained 7.5% with 5% (mL/mL) inoculum concentration by=0.5 bacteria suspension In the shake flask fermentation basal medium of corn stalk powder, corn stalk powder is sterilized using tyndallization, 121 DEG C of moist heat sterilizations 30min, each bacterial strain is set to be repeated three times, and 37 DEG C of 180r/min shaken cultivation 30d are dried using lyophilization. One group of blank control group and one group of positive controls are set, i.e. blank control group does not connect strain, and positive controls access 5% (g/ ML) microbial inoculum, other operations are identical as aforesaid operations.
3.2.2, sample solution preparation
Sample solution is carried out referring to People's Republic of China's agricultural industry criteria NY525-2012, but is improved to some extent.
The sample 0.5g (being accurate to 0.001g) after being freeze-dried in 3.2.1 is accurately weighed, kjeldahl flask bottom is placed in, is used 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 is to slowly warm up to sulfuric acid on adjustable electric furnace and is smoldered, removes, and few cold plus 15 drop hydrogen peroxide gently shake Kjeldahl flask heats 10min, removes, and 5 drops of being in after slightly cold~10 drop hydrogen peroxide and disappearing by several times boil, 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, rinses the curved small funnel of neck 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 sample is not added, other operations are identical as aforesaid operations.
3.2.3, full nitrogen determination
Full nitrogen determination method referring to People's Republic of China's agricultural industry criteria NY525-2012 and NY/T297-1995 into Row, but improve to some extent.
Disappearing for preparing in absorption 3.2.2 boils clear liquid 10mL in 50mL volumetric flask, and 2mL boric acid is added and 200 μ L mixing refers to Show agent mixed liquor, water is added to be settled to 50mL.It is distilled using kjeldahl apparatus, titrates distillate 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 It must not exceed 0.1mL, otherwise redeterminate.Nitrogen (N) content is indicated entirely with g/kg, 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, phosphorus measures entirely
Full phosphorus determination method referring to People's Republic of China's agricultural industry criteria NY525-2012 and NY/T298-1995 into Row, but improve to some extent.
Draw phosphorus standard solution 0,1.00,2.00,3.00,4.00,5.00,6.00mL be 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 is molten Liquid, with sodium hydroxide solution and sulfuric acid solution adjust solution be just in it is yellowish, add 10.0mL vanadium ammonium molybdate reagent, shake up, use water It is settled to 50mL.This solution is the standard liquid series of 1mL phosphorous (P) 0,1.00,2.00,3.00,4.00,5.00,6.00 μ g. After placing 20min under 15 DEG C of conditions above of room temperature, 2cm optical path cuvette is used, at spectrophotometer wavelength 440nm with blank Solution conditioning instrumentation zero point carries out colorimetric, reads absorbance, draws standard curve according to phosphorus concentration and absorbance, finds out straight line Regression equation.Disappearing for preparing in absorption 3.2.2 boils clear liquid 10mL in 50mL volumetric flask, 30mL is added water to, with standard solution system Column 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 is converted into the factor of g/kg.
3.2.5, potassium measures entirely
Full potassium measuring method referring to People's Republic of China's 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 point, 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 preparing in boils clear liquid 5.00mL in 50mL volumetric flask, 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 5 samples of every measurement.Full potassium content is with g/ Kg expression, according to the following formula:
In formula:
C --- measurement liquid concentration, μ g/mL are acquired by regression equation;
V --- measurement volume, this operation are 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 measurement
Full potassium measuring method is carried out referring to People's Republic of China's agricultural industry criteria NY/T300-1995, but is changed Into.
It accurately weighs the sample 1.00g after being freeze-dried in 3.2.1 to be placed in 50mL triangular flask, is added 25 DEG C of 20mL Citric acid solution is jumped a queue, and vibrates 30min at 25 DEG C, is filtered with without phosphorus filter paper into dry blue lid reagent bottle, spare.If Three groups of blank control groups are set, in addition to sample is not added, other operations are identical as aforesaid operations.Measuring 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 measurement
It accurately weighs the sample 1.00g after being freeze-dried in 3.2.1 to be placed in 50mL triangular flask, it is molten that 10mL nitric acid is added Liquid plugs small funnel, and the slightly boiled 10min on electric furnace is filtered while hot in 50mL volumetric flask, is washed 5 times with hot water, fixed after cooling Hold.Three groups of blank control groups are set, and in addition to sample is not added, other operations are identical as aforesaid operations.Measuring method and phase in 3.2.5 Together.Quick-acting potassium content indicates with mg/kg, according to the following formula:
In formula:
C --- measurement liquid potassium concn, μ g/mL are acquired by regression equation;
V --- measurement volume, 50mL;
M --- weigh sample mass, g.
3.3, result and analysis
3.3.1, nitrogen, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content measurement entirely
Corn stover 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.Corn stover 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 enterobacteria (Enterobacter hormaechei) S1 fermentation process group, full phosphorus, complete Potassium, rapid available phosphorus and the quick-acting potassium content equal difference compared with blank control group is not significant.Therefore, bacterial strain degrades to corn stover When will not influence essential element content therein, sample still saves original fertilizer efficiency.
Essential element content in corn stover after 11 fermentation process of table
Note:Blank control group does not access strain.Multiple range test is carried out using Duncan method.Significance p=0.01 and p =0.05 is indicated respectively with upper and lower case letter, n=3.
3.4 conclusion
After Huo Shi enterobacteria (Enterobacter hormaechei) S1 handles 30d to corn stover liquid fermentation, sample In full nitrogen, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content have no significant change.Huo Shi enterobacteria (Enterobacter Hormaechei) S1 will not cause the essential element content in sample when degrading sample, not will lead to full nitrogen in sample, Full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content variation, sample still save original fertilizer efficiency.

Claims (2)

1. one plant of lignocellulosic substance efficient degrading bacteria S1, it is characterised in that it is Huo Shi enterobacteria (Enterobacter Hormaechei) S1, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation address is Beijing The institute 3 of city Chaoyang District North Star West Road 1, the deposit date is on December 12nd, 2014, deposit number CGMCC No.10165.
2. the application of one plant of lignocellulosic substance efficient degrading bacteria S1 as described in claim 1, it is characterised in that it is used The lignocellulosic substance in degradation Bag Material agaric waste material and corn stover.
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