CN109234208A - The purposes of Friedlander's bacillus M1 degradation agricultural wastes - Google Patents
The purposes of Friedlander's bacillus M1 degradation agricultural wastes Download PDFInfo
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Abstract
The present invention relates to the purposes of a Klebsiella pneumoniae M1 and its agricultural wastes of degrading.Friedlander's bacillus (Klebsiella pneumoniae) M1, on December 12nd, 2014 in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number CGMCC No.10162.Bacterial strain proliferation of the present invention is rapidly, adaptable, is widely used；The weight-loss ratio of Bag Material agaric waste material after Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process is 31% or more, and the weight-loss ratio of the corn stover after Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process is 30% or more.
Description
The application is the divisional application of following patent application, the application number of original application: 201510862142.3, the applying date:
2015-12-01, denomination of invention: one plant of lignocellulosic substance efficient degrading bacteria M1 and its application.
Technical field
The present invention relates to the purposes of a Klebsiella pneumoniae M1 and its agricultural wastes of degrading.
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 object of the present invention is to provide the purposes of one plant of lignin efficient degrading bacteria M1 and its agricultural wastes of degrading.
One plant of lignin efficient degrading bacteria of the invention is Friedlander's bacillus (Klebsiella pneumoniae)
M1, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation address is Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, the deposit date is on December 12nd, 2014, deposit number CGMCC No.10162.
Friedlander's bacillus M1 of the invention is for agricultural wastes of degrading, in particular for Bag Material agaric waste material of degrading
Or corn stover.
Beneficial effects of the present invention are as follows:
Friedlander's bacillus (Klebsiella pneumoniae) M1 increment of the invention is rapidly, adaptable, applies
Extensively, salt tolerance is NaCl concentration 8%, and heat resistance is 70 DEG C；Continuous passage culture 10 times, strain growth situation, producing enzyme situation
And enzyme activity is stablized, no degradation phenomena；There is apparent degradation effect to Bag Material agaric waste material and corn stover, through pneumonia gram
The weight-loss ratio of Bag Material agaric waste material after the primary Salmonella of thunder (Klebsiella pneumoniae) M1 fermentation process 31% or more,
The weight-loss ratio of corn stover after Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process 30% with
On；It, can be with high-efficiency lignin degrading with significant lignin degradation ability.Corn through complex micro organism fungicide fermentation process
Content of lignin is the corn stover through Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process in stalk
2 times of middle content of lignin, as single bacterial strain Friedlander's bacillus (Klebsiella pneumoniae) M1 to wooden
The degradation capability of element is better than complex micro organism fungicide；When degradation sample, the essential element content in sample is not influenced, do not will lead to
Full nitrogen, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content variation in sample, save 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 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 Friedlander's bacillus of the present invention (Klebsiella pneumoniae) M1 cultivates the scanning electron after 12h
Microscope figure (× 20,000)；
Fig. 2 is that the agarose gel electrophoresis of 16S rDNA sequence PCR amplification detects figure；Wherein, 1 swimming lane is Maker
DL2000,2 swimming lanes are bacterial strain M1；
Fig. 3 is PCR product agarose gel electrophoresis after the recovery detection figure；Wherein, 1 swimming lane be Maker DL2000,2
Swimming lane is bacterial strain M1；
Fig. 4 is that the agarose gel electrophoresis of positive clone molecule screening detects figure；Wherein, 1 swimming lane be Maker DL2000,2
Swimming lane is bacterial strain M1；
Fig. 5 is the systematic evolution tree of Friedlander's bacillus of the present invention (Klebsiella pneumoniae) M1；
Fig. 6 is mistake of Friedlander's bacillus of the present invention (Klebsiella pneumoniae) M1 to Bag Material agaric waste material
Rate figure again；
Fig. 7 is weight-loss ratio of Friedlander's bacillus of the present invention (Klebsiella pneumoniae) M1 to corn stover
Figure；
Fig. 8 is corn stover after Friedlander's bacillus of the present invention (Klebsiella pneumoniae) M1 strains for degrading
Middle each component content histogram；Wherein, 1 is content of lignin histogram；2 be content of cellulose histogram；3 contain for hemicellulose
Measure histogram；
Fig. 9 is corn after the processing of Friedlander's bacillus of the present invention (Klebsiella pneumoniae) M1 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 complete
Potassium content histogram；
Figure 10 is corn after the processing of Friedlander's bacillus of the present invention (Klebsiella pneumoniae) M1 strain fermentation
Available phosphorus contents histogram in stalk；
Figure 11 is corn after the processing of Friedlander's bacillus of the present invention (Klebsiella pneumoniae) M1 strain fermentation
Stalk effective K content histogram.
Specific embodiment
Specific embodiment 1: one plant of lignocellulosic substance efficient degrading bacteria M1 of present embodiment, it is pneumonia
Klebsiella (Klebsiella pneumoniae) M1, it is commonly micro- to be deposited in China Committee for Culture Collection of Microorganisms
Bio-Centers, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, and the deposit date is on December 12nd, 2014, preservations
Number CGMCC No.10162.
Friedlander's bacillus (Klebsiella pneumoniae) M1 of present embodiment is Gram-negative bacteria, should
Bacterial strain thalli morphology be it is rod-shaped, thallus size be 0.62~0.71 × 1.5~1.6 μm, do not form gemma, atrichia has pod membrane；
Form that round, opaque, yellow, protrusion be smooth, bacterium colony (as shown in Figure 1) of neat in edge on LB culture medium.
The biochemical reactions result 20 items of the bacterial strain, 30 indexs；Combining form and biochemical reactions as a result, than
The kind of the bacterial strain is determined to " primary Jie Shi Bacteria Identification handbook ", the results are shown in Table 1.
According to " common Klebsiella system identification handbook " and " the outstanding Klebsiella handbook of uncle " to the Cray isolated
Primary Salmonella (Klebsiella sp.) M1 carries out carry out Gram's staining, oxidizing ferment, catalase, fluorchrome, methyl red, seven leaves
Glycosides dissolution, gelatin liquefaction, litmus milk peptonize and produce acid, lipase, Starch Hydrolysis, V.P. measurement, citrate utilization, cellulose
It is degradation, 3- ketone group lactose utilization, phenylalanine deaminase, tryptophan deaminase, heat-resisting hot, salt tolerance bio-chemical characteristics
Detection and identification.The result shows that Klebsiella (Klebsiella sp.) M1 is Gram-negative bacteria, salt tolerance is that NaCl is dense
Degree 8%, heat resistance are 70 DEG C, can produce catalase, urase and tryptophan deaminase, but cannot generate oxidizing ferment, lipase, phenylpropyl alcohol
Propylhomoserin deaminase and fluorchrome, methyl red, citrate utilization, Starch Hydrolysis, gelatin liquefaction, sugar alcohol fermentation (mannose),
Aesculin dissolution and litmus milk produce acid and show as the positive, and V.P. test, litmus milk peptonize, cellulose degradation and 3- ketone group are newborn
Sugar utilizes and shows as feminine gender.
The morphological feature and Physiology and biochemistry qualification result of 1 bacterial strain M1 of table
The lignin efficient degrading bacteria of present embodiment is Friedlander's bacillus (Klebsiella pneumoniae) M1
Screening technique it 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；It is 10 that dilution, which is respectively prepared, in bacterium solution-1、10-2、10-3、10-4、10-5、10-6Sample liquid, respectively take 200 μ L to be applied to wood
Quality screening and culturing medium plate, 37 DEG C of constant temperature incubation 48h adjust dilution gradient according to bacterium colony growing state.Keep condition of culture
It is constant, single colonie scribing line purifying repeatedly on lignin screening and culturing medium plate after 48h on each plate of picking.Picking is after purification
Single bacterium drop point be connected to lignin aniline blue culture medium flat plate, 37 DEG C are protected from light constant temperature incubation 48h, and the transparent circle for measuring each bacterial strain is straight
Diameter H and colony diameter C, filters out the biggish bacterial strain of H/C value.Squamous subculture is carried out to the bacterial strain of acquisition, continuous passage 10 times, is seen
It examines 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 saves to -80 DEG C of Freezing Glycerine methods of stability use of lignin degradation effect, divides
3 pipes are saved from bacterial strain, 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 M1 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 M1 transparent circle is clear rapid, and H/C value is larger；After 10 secondary cultures, the growth feelings of the bacterial strain
Condition, producing enzyme situation and enzyme activity are stablized, and no degradation phenomena is measured the transparent loop diameter H and colony diameter C of this plant of bacterium
And data analysis.The average colony diameter of bacterial strain M1 is 0.81 ± 0.02cm, and maximum colony diameter reaches 0.84cm；It is average
Degradation loop diameter is 1.26 ± 0.01cm, and most degradation loop diameter can reach 1.27cm.The average H/C value of bacterial strain M1 be 1.55 ±
0.05, maximum H/C value can reach 1.61.
2 M1 strains for degrading effect of table
4, the extraction of genomic DNA
The bacterial strain M1 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 ddH2O is added, suction is beaten uniformly, and thallus is made to suspend, and 5000r/min is centrifuged 5min, abandons supernatant, 200 μ L ddH2O are added,
Suction is beaten uniformly, and thallus is made to suspend；8~10min in boiling water bath, 10000r/min are centrifuged 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 save.
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 5.1 PCR products
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, the specific steps are 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: the 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.
The connection of 5.2 target fragments 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
The analysis of 5.4 16SrDNA sequences
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, is carried out by BLASTn program (http://www.ncbi.nlm.nih.gov/) 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 M1
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 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 M1 orthogonal experiments, are shown in Table 9.
9 bacterial strain M1 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 lung
Scorching Klebsiella (Klebsiella pneumoniae) Klebsiella M1.
Specific embodiment 2: the application of one plant of lignocellulosic substance efficient degrading bacteria M1 of present embodiment, it
For agricultural wastes of degrading, in particular for the lignocellulosic substance in degradation Bag Material agaric fertilizer and corn stover.
Following functions detection is carried out to bacterial strain of the invention:
By Friedlander's bacillus (Klebsiella pneumoniae) M1 of specific embodiment, Bag Material agaric is carried out
Waste material and corn stover weight-loss ratio and lignin, cellulose and hemicellulose degradation measure, to verify its distinctive function.Specifically
It is as follows:
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
(stalk 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
0.02g, 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 to obtained Friedlander's bacillus (Klebsiella pneumoniae) M1 inoculation
Into 5mL liquid fermentation medium, 37 DEG C of 180r/min shaken cultivation 12h, centrifugation abandons supernatant and obtains thallus.Take 500 μ L shaking flasks
Fermentation basal medium makes thallus 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, precipitating is washed with deionized in centrifugation, and after washing repeatedly three times, it is useless to calculate Bag Material agaric with Subtraction method for drying weighing
Expect weight-loss ratio, 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.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 corn stover weight-loss ratio measures
Specific embodiment one is screened to obtained Friedlander's bacillus (Klebsiella pneumoniae) M1 inoculation
Into 5mL liquid fermentation medium, 37 DEG C of 180r/min shaken cultivation 12h, centrifugation abandons supernatant and obtains thallus.Take 500 μ L shaking flasks
Fermentation basal medium makes thallus 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 cultivation 30d
Afterwards, it is centrifuged, precipitating is washed with deionized, after washing repeatedly three times, it is weightless to calculate corn stover with Subtraction method for drying weighing
Rate analyzes the data obtained by SPSS19.0 software, Multiple range test is carried out using Duncan method, 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
Bacterial strain M1 degrades after Bag Material agaric waste material, and weight-loss ratio measurement result is as shown in table 10 and Fig. 6.
The Bag Material agaric waste material weight-loss ratio of 10 bacterial strain M1 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 10 and Fig. 6 it is found that being sent out after culture 30d through Friedlander's bacillus (Klebsiella pneumoniae) M1
After ferment, the weight-loss ratio of Bag Material agaric waste material is 31.21 ± 0.36%；Blank control group Bag Material agaric waste material weight-loss ratio is
21.60% ± 0.82%；Positive controls Bag Material agaric waste material weight-loss ratio is 38.53 ± 0.87%.Through Friedlander's bacillus
The weight-loss ratio of Bag Material agaric waste material is greater than blank control group after (Klebsiella pneumoniae) M1 degradation, and it is positive right to be less than
According to group.It is analyzed according to level of signifiance p=0.05, at Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation
The weight-loss ratio of reason Bag Material agaric waste material significant difference compared with the Bag Material agaric waste material weight-loss ratio of blank control group, with positive control
The Bag Material agaric waste material weight-loss ratio of group compares significant difference.M1 pairs of Friedlander's bacillus (Klebsiella pneumoniae)
Bag Material agaric waste material has very strong degradation capability.It ferments through Friedlander's bacillus (Klebsiella pneumoniae) M1
The weight-loss ratio of treated Bag Material agaric waste material is 31% or more.
1.6.2 corn stover weight-loss ratio measures
After bacterial strain M1 degrading maize straws, weight-loss ratio measurement result is as shown in table 10 and Fig. 7.
By table 10 and Fig. 7 it is found that being sent out after culture 30d through Friedlander's bacillus (Klebsiella pneumoniae) M1
After ferment, the weight-loss ratio of corn stover is 30.10 ± 0.17%；Blank control group corn stover weight-loss ratio be 25.80% ±
0.63%；Positive controls corn stover weight-loss ratio is 44.81% ± 1.02%.Through Friedlander's bacillus (Klebsiella
Pneumoniae) weight-loss ratio of corn stover is greater than blank control group after M1 degradation, is less than positive controls.According to the level of signifiance
P=0.05 analysis, the corn stover weight-loss ratio through Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process
Difference is not significant compared with the corn stover weight-loss ratio of blank control group, poor compared with the corn stover weight-loss ratio of positive controls
It is different significant.Friedlander's bacillus (Klebsiella pneumoniae) M1 has stronger degradation energy to corn stover
Power.The weight-loss ratio of corn stover after Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process exists
30% or more.
1.7 conclusion
Friedlander's bacillus (Klebsiella pneumoniae) M1 filtered out through specific embodiment one is to Bag Material
Agaric waste material and corn stover have apparent degradation effect.Through Friedlander's bacillus (Klebsiella pneumoniae)
The weight-loss ratio of Bag Material agaric waste material after M1 fermentation process is 31% or more；Through Friedlander's bacillus (Klebsiella
Pneumoniae) weight-loss ratio of the corn stover after M1 fermentation process is 30% or more.Friedlander's bacillus (Klebsiella
Pneumoniae) M1 is better than degradation of the complex micro organism fungicide to Bag Material agaric waste material in positive controls to Bag Material agaric waste material
Ability.Bag Material agaric waste material and corn stover after Friedlander's bacillus (Klebsiella pneumoniae) M1 degradation
Weight-loss ratio is all larger than the Bag Material agaric waste material of blank control group and the weight-loss ratio of corn stover；Wherein, Friedlander's bacillus
The weight-loss ratio of the Bag Material agaric waste material of (Klebsiella pneumoniae) M1 fermentation process is greater than positive controls Bag Material agaric
Waste material weight-loss ratio
Friedlander's bacillus (Klebsiella pneumoniae) M1 to the degradation effect of lignocellulosic substance with
Strain growth situation and sample composition have much relations, Bag Material agaric waste material and corn stover to wash using Fan Shi (Van Soest)
After washing the measurement of fibre analysis method, each component content in corn stover and Bag Material agaric waste material is shown in Table 11, Bag Material agaric waste material and
The content of lignin, cellulose and hemicellulose in corn stover is significantly different.Same bacterial strain to Bag Material agaric waste material and
Corn stover has different degradation effects, the reason is that due to lignin in Bag Material agaric waste material and corn stover and cellulose
The growth and breeding of content Different Effects bacterial strain cause strain growth situation different, and the secretion capacity of enzyme has differences, and then leads
It causes bacterial strain different to the Utilization ability of catabolite, influences its degradation capability.
11 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,
FeCl30.02g, 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.
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
Friedlander's bacillus (Klebsiella pneumoniae) M1 of specific embodiment one is inoculated into liquid hair
OD is made in ferment culture medium600Then bacterium solution access is contained 7.5% corn with 5% (mL/mL) inoculum concentration by=0.5 bacteria suspension
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 12
With shown in Fig. 8.
Each component average content in corn stover after 12 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 12 and Fig. 8 it is found that corn stover is fermented through Friedlander's bacillus (Klebsiella pneumoniae) M1
After handling 30d, content of lignin is 5.73 ± 0.10%, corn stover far smaller than in blank control group and positive controls
Content of lignin；With level of signifiance p=0.05 analysis, Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation
Content of lignin and content of lignin comparing difference in blank control group corn stover are significant in the corn stover of processing, with the positive
Content of lignin comparing difference is significant in control group corn stover；With level of signifiance p=0.01 analysis, Friedlander's bacillus
In the corn stover of (Klebsiella pneumoniae) M1 fermentation process in content of lignin and blank control group corn stover
Content of lignin comparing difference is significant, significant with content of lignin comparing difference in positive control corn stover group；Illustrate pneumonia
Klebsiella (Klebsiella pneumoniae) M1 can be with high-efficiency lignin degrading.
Corn stover after liquid fermentation 30d, content of cellulose be 35.11 ± 3.64%, be all larger than blank control group and
Corn stalk fiber cellulose content in positive controls；With level of signifiance p=0.05 analysis, Friedlander's bacillus
In the corn stover of (Klebsiella pneumoniae) M1 fermentation process in content of cellulose and blank control group corn stover
Content of cellulose comparing difference is not significant, not significant with corn stalk fiber cellulose content comparing difference in positive controls；With aobvious
It writes horizontal p=0.01 to analyze, in the corn stover of Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process
Content of cellulose and the content of cellulose comparing difference of corn stover in blank control group be not significant, with corn in positive controls
The content of cellulose comparing difference of stalk is not significant；Illustrate that M1 is or not Friedlander's bacillus (Klebsiella pneumoniae)
It can degraded cellulose.
For corn stover after liquid fermentation 30d, hemicellulose level is 22.68 ± 2.14%, 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
Analysis, in the corn stover of Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process hemicellulose level with
Hemicellulose level comparing difference is not significant in blank control group corn stover, with hemicellulose in positive controls corn stover
Comparision contents significant difference；With level of signifiance p=0.01 analysis, Friedlander's bacillus (Enterobacter
Hormaechei) hemicellulose level in M1 fermentation process corn stover contains with hemicellulose in blank control group corn stover
It is not significant to measure comparing difference, it is not significant with hemicellulose level comparing difference in positive controls corn stover；Illustrate pneumonia gram
The primary Salmonella of thunder (Klebsiella pneumoniae) M1 being capable of degradation of hemicellulose.
Friedlander's bacillus (Klebsiella pneumoniae) M1 has significant lignin degradation ability, can be with
High-efficiency lignin degrading.As single bacterial strain Friedlander's bacillus (Klebsiella pneumoniae) M1 to lignin
Degradation capability is better than complex micro organism fungicide, in the corn stover through complex micro organism fungicide fermentation process content of lignin be through
2 times of content of lignin in the corn stover of Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process.
2.5 conclusion
Friedlander's bacillus (Klebsiella pneumoniae) M1 filtered out through specific embodiment one has aobvious
The lignin degradation ability of work, can be with high-efficiency lignin degrading.It is wooden in corn stover through complex micro organism fungicide fermentation process
Lignin content is lignin in the corn stover through Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process
2 times of content, as single bacterial strain Friedlander's bacillus (Klebsiella pneumoniae) M1 to the degradation energy of lignin
Power is better than complex micro organism fungicide.
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,
FeCl30.02g, MgSO47H2O 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
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
Friedlander's bacillus (Klebsiella pneumoniae) M1 that specific embodiment 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 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 --- the quality with the comparable nitrogen in grams of 1.00mL sulfuric acid (1/2H2SO4) standard solution；
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 13 and Fig. 9~Figure 11.Corn stover is after liquid fermentation, according to the horizontal p=of the significance of difference
0.05 and p=0.01 analysis, it is the full nitrogen of Friedlander's bacillus (Klebsiella pneumoniae) M1 fermentation process group, complete
Phosphorus, full potassium, rapid available phosphorus and the quick-acting potassium content equal difference compared with blank control group is not significant.Therefore, bacterial strain to corn stover into
It will not influence essential element content therein when row degradation, sample still saves original fertilizer efficiency.
Essential element content in corn stover after 13 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 indicates respectively with upper and lower case letter, n=3.
3.4 conclusion
After Friedlander's bacillus (Klebsiella pneumoniae) M1 handles 30d to corn stover liquid fermentation, examination
Full nitrogen, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content in sample have no significant change.Friedlander's bacillus (Klebsiella
Pneumoniae) M1 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.
Sequence table
<110>Beijing Deruifeng Agriculture Technology Co., Ltd.
<120>purposes of Friedlander's bacillus M1 degradation agricultural wastes
<141> 2018-11-16
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1498
<212> DNA
<213>Friedlander's bacillus (Klebsiella pneumoniae)
<400> 1
agagtttgat cctggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgagc 60
ggtagcacag agagcttgct ctcgggtgac gagcggcgga cgggtgagta atgtctggga 120
aactgcctga tggaggggga taactactgg aaacggtagc taataccgca taacgtcgca 180
agaccaaagt gggggacctt cgggcctcat gccatcagat gtgcccagat gggattagct 240
agtaggtggg gtaacggctc acctaggcga cgatccctag ctggtctgag aggatgacca 300
gccacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg gggaatattg 360
cacaatgggc gcaagcctga tgcagccatg ccgcgtgtgt gaagaaggcc ttcgggttgt 420
aaagcacttt cagcggggag gaaggcggtg aggttaataa cctcatcgat tgacgttacc 480
cgcagaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga gggtgcaagc 540
gttaatcgga attactgggc gtaaagcgca cgcaggcggt ctgtcaagtc ggatgtgaaa 600
tccccgggct caacctggga actgcattcg aaactggcag gctagagtct tgtagagggg 660
ggtagaattc caggtgtagc ggtgaaatgc gtagagatct ggaggaatac cggtggcgaa 720
ggcggccccc tggacaaaga ctgacgctca ggtgcgaaag cgtggggagc aaacaggatt 780
agataccctg gtagtccacg ctgtaaacga tgtcgatttg gaggttgtgc ccttgaggcg 840
tggcttccgg agctaacgcg ttaaatcgac cgcctgggga gtacggccgc aaggttaaaa 900
ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa 960
cgcgaagaac cttacctggt cttgacatcc acagaacttt ccagagatgg attggtgcct 1020
tcgggaactg tgagacaggt gctgcatggc tgtcgtcagc tcgtgttgtg aaatgttggg 1080
ttaagtcccg caacgagcgc aacccttatc ctttgttgcc agcggttagg ccgggaactc 1140
aaaggagact gccagtgata aactggagga aggtggggat gacgtcaagt catcatggcc 1200
cttacgacca gggctacaca cgtgctacaa tggcatatac aaagagaagc gacctcgcga 1260
gagcaagcgg acctcataaa gtatgtcgta gtccggattg gagtctgcaa ctcgactcca 1320
tgaagtcgga atcgctagta atcgtagatc agaatgctac ggtgaatacg ttcccgggcc 1380
ttgtacacac cgcccgtcac accatgggag tgggttgcaa aagaagtagg tagcttaacc 1440
ttcgggaggg cgcttaccac tttgtgattc atgactgggg tgaagtcgta acaaggta 1498
<210> 2
<211> 24
<212> DNA
<213>artificial sequence ()
<220>
Claims (3)
1. the purposes of Friedlander's bacillus M1 degradation agricultural wastes, which is characterized in that the agricultural wastes are Bag Material wood
Friedlander's bacillus M1 is inoculated into liquid fermentation medium and OD is made by ear waste material or corn stover600=0.5 bacterium is outstanding
Then bacteria suspension is accessed the fermentation containing 5-7.5%m/V Bag Material agaric waste material or corn stalk powder with 5%V/V inoculum concentration by liquid
In basal medium, 37 DEG C of 180r/min shaken cultivation 30d；
Wherein the deposit number of the Friedlander's bacillus M1 is CGMCC No.10162；
Wherein the Bag Material agaric waste material or corn stalk powder are sterilized using tyndallization, 121 DEG C of moist heat sterilization 30min.
2. purposes as described in claim 1, which is characterized in that the liquid fermentation medium g/L: glucose 5, peptone 2,
NH4NO31.0, CaCl20.2, K2HPO40.5, FeCl30.02, MgSO4·7H2O 0.5, NaCl 1.0, distilled water are prepared,
pH 7.0。
3. purposes as described in claim 1, which is characterized in that the fermentation basal medium g/L: peptone 2, NH4NO3
1.0, CaCl20.2, K2HPO40.5, FeCl30.02, MgSO4·7H2O 0.5, NaCl 1.0, distilled water are prepared, pH 7.0.
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| CN111471617A (en) * | 2020-04-08 | 2020-07-31 | 陕西省微生物研究所 | Cellulose degradation composite flora and screening thereof and application of saprophytic fungi residue for decomposing |
| CN112898095A (en) * | 2021-03-22 | 2021-06-04 | 广西农垦西江乳业有限公司 | Preparation method of cow dung organic fertilizer |
| CN114561323A (en) * | 2022-02-23 | 2022-05-31 | 浙江台州秀川科技有限公司 | Salt-tolerant Klebsiella pneumoniae capable of efficiently degrading gallic acid and application thereof |
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| CN109234208B (en) * | 2015-12-01 | 2021-12-31 | 海林市中农国泰生物科技有限公司 | Application of Klebsiella pneumoniae M1 in degrading agricultural wastes |
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| CN105385631B (en) | 2018-11-30 |
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