CN105462875A - Strain L252 capable of efficiently degrading lignocellulose substances and application of strain - Google Patents
Strain L252 capable of efficiently degrading lignocellulose substances and application of strain Download PDFInfo
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Abstract
The invention relates to a strain L252 capable of efficiently degrading lignocellulose substances and application of the strain. The stain is Klebsiella pneumoniae L252 which is preserved in China General Microbiological Culture Collection Center on Dec. 12th, 2014, and the preservation number is CGMCC No.10161. The strain is used for degrading the lignocellulose substances. The strain can be proliferated rapidly, the adaptability is high, and application is wide. The weight-loss ratio of bagged agaric waste subjected to fermentation treatment of the Klebsiella pneumoniae L252 is 30% or above, and the weight-loss ratio of maize straw subjected to fermentation treatment of the Klebsiella pneumoniae L252 is 26% or above.
Description
Technical field
The present invention relates to strain lignocellulose material efficient degrading bacteria L252 and an application thereof.
Background technology
China is a large agricultural country, agricultural wastes generation is extremely huge, and due to the restriction popularized by economic benefit and technology, the mostly extensive poor efficiency of agricultural wastes utilizes and idle situation is serious, cause the wasting of resources and environmental pollution, waste has become the source of pollution of Largest In China.And there is a large amount of lignocelluloses at agricultural wastes, urgently people go to develop.
Lignocellulose is that occurring in nature is only second to cellulosic second abundant organic renewable resources, is also one of composition of the most difficult degradation of microorganism.In recent years, the fungi of some lignin degrading has started to be applied in practice, but still needs to be developed further.The application of xylogen microbiological deterioration mainly contains: paper industry; Fodder industry; Fermentation and foodstuffs industry; The environment protection of biological thing fertilizer; The bio-bleaching technology of ligninase, etc.
Current agriculture organic solid is discarded to be utilized by recovery energy, organic fertilizer, poultry and livestock feed, edible bacterium culture medium material and industrial raw material usually.In the production practice utilizing agricultural wastes, physics, chemistry and bioremediation are often combined, and wherein bioremediation especially utilizes microbiological treatment to represent development trend from now on.Biological method just refers to and utilizes lignin-degrading enzymes to remove the xylogen of degrading in lignocellulosic material, thus xylogen-hemicellulose-cellulose structure is disintegrated, Mierocrystalline cellulose is come out for subsequent step process compared with traditional machinery, physical chemistry class method, the advantage of biological treatment is that energy consumption is low, required envrionment conditions is gentle, avoid traditional chemical process, mechanical treatment technology etc. power consumption more, there is the shortcomings such as environmental pollution, consider from cost and equipment angle, biological delignification method occupies unique advantage.But, current biological treatment has a very large weakness to limit its application, the activity of the pivotal player-lignin-degrading enzymes in Here it is biological treatment is general not high, thus cause processing efficiency lower, if genetically engineered and traditional biotechnology can be utilized to transform bacterial classification and enzyme, improve enzyme activity, reduce enzyme cost, biological process delignification rule is expected to be applied to large-scale commercial production.
And most widely used be at present microbial-bacterial fertilizer, microbial-bacterial fertilizer as a kind of new agrotechnical measure, development an agriculture featuring high yields, fine quality and high efficiency in effect gradually be familiar with by people.Traditional microbial fertilizer is own through in large scale application, and novel microbial-bacterial fertilizer kind is continually developed out.Microorganism fertilizer is made up by fermentation of the microorganism with special usefulness, containing a large amount of beneficial microorganism, specified microorganisms goods crop being had to specific fertilizer efficiency.Microbial fertilizer utilizes the vital movement of microorganism, the Substance Transformation that can not be absorbed by crop is can the nutrition of utilization absorbed by crops, and improve the nutritional condition of crop, some has the effect stimulating plant growth or enhance disease resistance concurrently, to improve crop yield, improve quality of agricultural product.The day that goes out that it can increase agricultural-food earns foreign exchange, and has again industrial or agricultural organic waste and effectively utilizes, prevents the pollution of the environment, improves Soil structure, increases soil fertility and the eucyclic great social profit of protecting ecology and ecological benefits.
Domestic research great majority are fungus degrading lignocellulose.But, utilize the problem that fungus degrading lignocellulose ubiquity enzyme activity is lower.Because bacterial reproduction is very fast, fermentation period is short, can be applicable to industrial production, and bacteriogenic cellulase general action condition is neutral or meta-alkalescence, this pollutes on the waste water treatment of industry at slurrying, papermaking and detergent industry etc. potential application prospect, from bacterium, therefore filters out effective strain be applied to ligocellulose degradation and have certain practical significance and DEVELOPMENT PROSPECT.
Summary of the invention
The object of the invention is to provide strain xylogen efficient degrading bacteria and an application thereof.
A strain xylogen efficient degrading bacteria of the present invention is Klebsiella pneumonia (Klebsiellapneumoniae) L252, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on December 12nd, 2014, preserving number CGMCCNo.10161.
A strain xylogen efficient degrading bacteria of the present invention is for lignin degrading.
Beneficial effect of the present invention is as follows:
Klebsiella pneumonia of the present invention (Klebsiellapneumoniae) L252 rises in value rapidly, and strong adaptability, is widely used, and salt tolerance is salt concn 8%, and thermotolerance is 50 DEG C; Continuous passage cultivates 10 times, and strain growth situation, product enzyme situation and enzyme activity are stablized, without degradation phenomena; To Bag Material auricularia auriculajudae waste material and maize straw, there is obvious degradation effect, the rate of weight loss of the Bag Material auricularia auriculajudae waste material after Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing is more than 30%, and the rate of weight loss of the maize straw after Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing is more than 26%; There is significant lignin degradation ability, can high-efficiency lignin degrading, in the maize straw of complex micro organism fungicide fermentative processing, content of lignin is 1.3 times of content of lignin in the maize straw of Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing, is better than complex micro organism fungicide as the degradation capability of single bacterial strain Klebsiella pneumonia (Klebsiellapneumoniae) L252 to xylogen; When degrading sample, not affecting the principal element content in sample, the full nitrogen in sample, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content change can not be caused, preserve sample fertilizer efficiency.
It can be the original strain that sole carbon source grows with xylogen that the present invention separates from Bag Material auricularia auriculajudae waste material, woodland rotten wood and forest fieid soil, determines the degradation capability to xylogen and race relation, lays the foundation for complex micro organism fungicide in future builds.Effectuation for China's agricultural resource is utilized strong help is provided.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope diagram (× 20,000) after Klebsiella pneumonia of the present invention (Klebsiellapneumoniae) L252 cultivates 12h;
Fig. 2 be the agarose gel electrophoresis detection figure of 16SrDNA sequence pcr amplification wherein, 1 swimming lane is MakerDL2000, and 2 swimming lanes are bacterial strain L252;
Fig. 3 be reclaim after PCR primer agarose gel electrophoresis detection figure wherein, 1 swimming lane is MakerDL2000, and 2 swimming lanes are bacterial strain L252;
Fig. 4 be positive colony screening agarose gel electrophoresis detection figure wherein, 1 swimming lane is MakerDL2000, and 2 swimming lanes are bacterial strain L252;
Fig. 5 is the systematic evolution tree of Klebsiella pneumonia of the present invention (Klebsiellapneumoniae) L252;
Fig. 6 is the rate of weight loss figure of Klebsiella pneumonia of the present invention (Klebsiellapneumoniae) L252 to Bag Material auricularia auriculajudae waste material;
Fig. 7 is the rate of weight loss figure of Klebsiella pneumonia of the present invention (Klebsiellapneumoniae) L252 to maize straw;
Fig. 8 is each component concentration histogram in maize straw after Klebsiella pneumonia of the present invention (Klebsiellapneumoniae) L252 strains for degrading; Wherein, 1 is content of lignin histogram; 2 is content of cellulose histogram; 3 is hemicellulose level histogram;
Fig. 9 is total nitrogen and total phosphor phosphorus and full potassium content histogram in maize straw after the process of Klebsiella pneumonia of the present invention (Klebsiellapneumoniae) L252 strain fermentation; Wherein, 1 is total nitrogen content histogram; 2 is content of tatal phosphorus histogram; 3 is full potassium content histogram;
Figure 10 is available phosphorus contents histogram in maize straw after the process of Klebsiella pneumonia of the present invention (Klebsiellapneumoniae) L252 strain fermentation;
Figure 11 is maize straw effective K content histogram after the process of Klebsiella pneumonia of the present invention (Klebsiellapneumoniae) L252 strain fermentation.
Embodiment
Embodiment one: a strain lignocellulose material efficient degrading bacteria of present embodiment, it is Klebsiella pneumonia (Klebsiellapneumoniae) L252, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on December 12nd, 2014, preserving number CGMCCNO.10161.
Klebsiella pneumonia (Klebsiellapneumoniae) L252 of present embodiment is Gram-negative bacteria, and this bacterial strain thalli morphology is shaft-like, and thalline size is 0.57 ~ 0.62 × 1.4 ~ 1.5 μm, does not form gemma, and atrichia has pod membrane; On LB substratum, formation is circular, opaque, oyster white, projection are smooth, the bacterium colony of neat in edge (as shown in Figure 1).
Biochemical reactions result totally 18,28 indexs of this bacterial strain; Combining form and biochemical reactions result, the kind of this bacterial strain is determined in comparison " uncle Jie Shi Bacteria Identification handbook ", and result is as shown in table 1.
According to " common bacteria system identification handbook " and " the outstanding bacterium handbook of uncle ", gramstaining is carried out to isolated Klebsiella pneumonia (Klebsiellapneumoniae) L252, oxydase, catalase, fluorochrome, methyl red, Vitamin C2 dissolves, gelatine liquefication, litmus milk peptonizes and produces acid, lipase, Starch Hydrolysis, V.P. measure, Citrate trianion utilizes, cellulose degradation, 3-ketone group lactose utilization, phenylalanine deaminase, tryptophane desaminase, heat-resisting hot, the detection of salt tolerance bio-chemical characteristics and qualification.Result shows that Klebsiella pneumonia (Klebsiellapneumoniae) L252 is Gram-negative bacteria, salt tolerance is salt concn 8%, thermotolerance is 50 DEG C, catalase can be produced, but oxydase, lipase, phenylalanine deaminase, tryptophane desaminase and fluorochrome can not be produced, methyl red, Citrate trianion utilize, litmus milk peptonizes, Vitamin C2 dissolves and the acid of litmus milk product shows as the positive, and V.P. test, Starch Hydrolysis, gelatine liquefication, cellulose degradation and 3-ketone group lactose utilization show as feminine gender.
The morphological feature of table 1 bacterial strain L252 and Physiology and biochemistry qualification result
The xylogen efficient degrading bacteria of present embodiment is that the screening method of Klebsiella pneumonia (Klebsiellapneumoniae) L252 is as follows:
1, screening method
Get 10g sample aseptically fully to grind, add in the triangular flask that 90mL sterilized water (band granulated glass sphere) is housed, vibration 20min.Getting the access of 5mL sample suspension is equipped with in the triangular flask of 100mL LB liquid medium, 37 DEG C, 180r/min shaking culture 8h; Bacterium liquid is made the sample liquid that extent of dilution is 10-1,10-2,10-3,10-4,10-5,10-6 respectively, respectively gets 200 μ L and be applied to xylogen screening culture medium flat board, 37 DEG C of constant temperature culture 48h, according to colony growth situation adjustment dilution gradient.Keep culture condition constant, the single bacterium colony after 48h on each flat board of picking is repeatedly rule purifying on xylogen screening culture medium flat board.Single bacterium colony point after picking purifying is connected to xylogen aniline blue culture medium flat plate, 37 DEG C of lucifuge constant temperature culture 48h, measures transparent circle diameter H and the colony diameter C of each bacterial strain, filters out the bacterial strain that H/C value is larger.Succeeding transfer culture is carried out, continuous passage 10 times to the bacterial strain obtained, observes the upgrowth situation of bacterial strain and measure H/C value.According to bacterial strain respectively for colonial morphology, colony diameter, transparent circle diameter and H/C value size, determine that strain growth situation and the stability to lignin degradation effect adopt-80 DEG C of Freezing Glycerine methods to preserve, isolated strains preserves 3 pipes, writes label (strain number, separation place, Habitat Types and shelf time) exactly.
2, bacterial strain H/C pH-value determination pH
Bacterial strain point is connected to xylogen aniline blue culture medium flat plate, 37 DEG C of lucifuge constant temperature culture 48h, measures transparent circle diameter H and the colony diameter C of each bacterial strain.
3, interpretation of result:
3.1, bacterial strain screening
By above-mentioned separation screening process, obtain the bacterial strain that 134 strains can grow in xylogen screening culture medium altogether.Be inoculated in the enterprising row filter of xylogen aniline blue substratum by after this 134 strain bacterial strain purifying, filter out lignin-degrading bacteria according to transparent circle generation time, sharpness and H/C value size.The generation of bacterial strain L252 transparent circle is rapid and clear, and the bacterial strain that H/C value is larger, is defined as efficient lignin-degrading bacteria, according to the sample source of bacterium, numbers respectively.After above-mentioned bacterial strains being carried out 10 Secondary Culture, the H/C value of bacterial strain colony growth situation, mode of appearance and bacterial strain has no significant change, and shows that strain growth situation, product enzyme situation and enzyme activity are stablized, without degradation phenomena.
3.2, bacterial strain H/C pH-value determination pH
It is rapidly clear that bacterial strain L252 transparent circle produces, and H/C value is larger; After 10 Secondary Culture, the growing state of this bacterial strain, produce enzyme situation and enzyme activity is stablized, without degradation phenomena, the transparent circle diameter H of this strain bacterium and colony diameter C is measured and data analysis.The average colony diameter of bacterial strain L252 is 0.17 ± 0.02cm, and maximum colony diameter can arrive 0.19cm; Average degraded loop diameter is 0.86 ± 0.01cm, and most degradation loop diameter can reach 0.87cm.The average H/C value of bacterial strain L252 is 5.21 ± 0.59, and maximum H/C value can reach 6.21.
Table 2L252 strains for degrading effect
4, the extraction of genomic dna
The bacterial strain L252 genomic dna above-mentioned screening obtained adopts hot broken wall method to extract.Get the bacteria suspension that 1mL is inoculated in LB liquid nutrient medium 37 DEG C of 180r/min shaking culture 24h, squeeze in 1.5mL centrifuge tube, the centrifugal 5min of 5000r/min, abandons supernatant, adds 1mLddH
2o, inhale and beat evenly, thalline is suspended, and the centrifugal 5min of 5000r/min, abandons supernatant, adds 200 μ LddH
2o, inhales and beats evenly, thalline is suspended; The centrifugal 10min of 8 ~ 10min, 10000r/min in boiling water bath.Aspirate supernatant, is transferred in another 1.5mL centrifuge tube, gets 5 μ L point samples, and with λ EcoT14 for Marker, 1% agarose gel electrophoresis detects, all the other-20 DEG C preservations.
5, the pcr amplification of 16SrDNA
Adopt 16SrDNA universal primer, with extracted strain gene group DNA for template, increase according to following reaction system and amplification condition.Primer sequence reaction system and amplification condition are respectively as shown in table 3, table 4.The PCR primer agarose electrophoresis of 1% detects.
The primer sequence of table 316SrDNAPCR amplification
The reaction system of the pcr amplification of table 416SrDNA and response procedures
The recovery purifying of 5.1PCR product
By whole for the PCR primer containing target stripe point sample (80 μ L/ holes, altogether holes), the agarose gel electrophoresis of 1.5%, electrophoretic band reclaims test kit with sky root sepharose DNA and reclaims, and concrete steps are as follows:
(1) column equilibration step: to adsorption column CA
2in (putting into collection tube) add 500 μ L balance liquid BL, 12000r/min, centrifugal 1min, outwell the waste liquid in collection tube, placed back in collection tube by adsorption column.
(2) single target DNA band is cut (excising redundance) from sepharose as far as possible put into clean centrifuge tube, take weight.Formula: (before the weight-dress glue after dress glue the weight of centrifuge tube) × 1000=" 1000 times of volumes " μ L.
(3) in blob of viscose, add " 1000 times of volumes " sol solutions PN, 10min is placed in 50 DEG C of water-baths, constantly leniently spins upside down centrifuge tube therebetween, to guarantee that blob of viscose fully dissolves, cooling, is down to room temperature upper prop again by sol solution temperature, stop 2 ~ 5min after upper prop.
(4) previous step gained solution is added an adsorption column CA
2in, adsorption column is put into collection tube, and the centrifugal 1min of 13000r/min, outwells the waste liquid in collection tube, is reentered in collection tube by adsorption column.
(5) in adsorption column, add 600 μ L rinsing liquid PW (please first check whether before use and added dehydrated alcohol), stop the centrifugal 1min of 2min, 13000r/min, outwell waste liquid, adsorption column is reentered in collection tube.
(6) in adsorption column, add 500 μ L rinsing liquid PW, centrifugal 30 seconds of 13000r/min, outwells waste liquid.By centrifugal adsorbing column CA
2put into collection tube, the centrifugal 2min of 13000r/min, removes rinsing liquid as far as possible.Adsorption column is placed in 50 DEG C of baking ovens and dries several minutes, dry up hill and dale, with the experiment (affecting organic efficiency and DNA quality) preventing the rinsing liquid remained from affecting next step.
(7) be put into by adsorption column in a clean centrifuge tube (cutting cap), to the unsettled dropping 30 in adsorption film mid-way μ L elution buffer EB, room temperature is placed the centrifugal 1min of 2min, 13000r/min and is collected DNA solution.
(8) in order to improve the yield of DNA, can by the centrifugal solution obtained again add-back centrifugal adsorbing column, the centrifugal 1min of 13000r/min collects DNA solution, repeats 3 wash-outs.
(9) DNA solution is positioned over (during last wash-out) in the centrifuge tube of lid, is stored in-20 DEG C, in case DNA degradation.The DNA solution taken a morsel after the reclaiming agarose electrophoresis of 1% verifies its purity and content.
The connection of 5.2 object fragments and cloning vector
Mixed with pMD18-T carrier by the DNA segment that previous step PCR recovery purifying obtains, 4 DEG C of reactions are spent the night.Linked system is as follows:
Proceed in E.coliDH5 α competent cell by the carrier after connecting, after shaking training, coated plate, picking white colony to be inoculated in the LB substratum containing Amp 37 DEG C, 180r/min shaking table cultivates 10 ~ 12 hours.
5.3 positive colony detect
With gained bacterium liquid for template carries out pcr amplification, respectively as shown in table 5 and table 6, the product agarose electrophoresis of 1% detects for primer sequence (see pMD18-TVector specification sheets), reaction system and amplification condition.
Table 5 recon PCR detects the primer sequence used
The reaction system that table 6 recon PCR detects and response procedures
5.416SrDNA sequential analysis
The positive colony obtained is sent to raw work (SangonBiotech) the biotechnology limited-liability company in Shanghai to check order, with BioEdit7.09 software analysis sequencing result, amputation primer sequence, the sequence results of acquisition is submitted to GenBank database and obtains accession number, on-line analysis is carried out by BLASTn program (http://www.ncbi.nlm.nih.gov/), download the sequence that similarity is greater than the type strain of 90%, and carry out Multiple sequence alignments with ClustalX software, then the Neighbor-Joining phylogenetic tree construction in software MEGA5.03 is adopted, determine the race relation of bacterial strain.
6, bacterial strain 16SrDNA Sequence Identification result
6.1,16SrDNA sequence pcr amplification
16SrDNA sequence pcr amplification product is with 1% agarose gel electrophoresis inspection, and result as shown in Figure 2.The 16SrDNA gene fragment length of bacterial strain L252 is about 1500bp.
6.2, PCR primer reclaims
Carry out electrophoresis to the 16SrDNA sequence pcr amplification product in 6.1 with 1.5% sepharose, electrophoretic band reclaims test kit with DNA glue and reclaims.PCR primer reclaims electrophoretogram as shown in Figure 3, and according to band brightness, this experiment is successfully recovered to enough purified pcr products, can be used for follow-up test and carries out.
6.3, positive colony screening
Be connected with carrier T by 16SrDNAPCR amplified production after purifying, be converted into large bacterium competent cell, with gained thalline for template carries out pcr amplification, result as shown in Figure 4, has obtained positive colony with recombinant plasmid.
6.4,16SrDNA nucleotide sequencing
The 16SrDNA nucleotide sequencing of each bacterial strain the results are shown in sequence table 1, and the phylogenetic tree of each bacterial strain as shown in Figure 5.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 table 7 bacterial strain
By Morphological Identification, Physiology and biochemistry qualification and 16SrDNA Molecular Identification, determine that the bacterial strain of above-mentioned screening is Klebsiella pneumonia (Klebsiellapneumoniae) bacterium L252.
Embodiment two: the application of a strain lignocellulose material efficient degrading bacteria of present embodiment, it is for lignocellulose degradation class material.
Following functions detection is carried out to bacterial strain of the present invention:
By Klebsiella pneumonia (Klebsiellapneumoniae) L252 of embodiment, carry out Bag Material auricularia auriculajudae waste material and maize straw rate of weight loss and xylogen, Mierocrystalline cellulose and hemicellulose degradation and measure, to verify its distinctive function.Specific as follows:
1, Bag Material auricularia auriculajudae waste material and maize straw rate of weight loss measure
1.1 corn stalk powder
Maize straw takes from this laboratory test field, school district, Harbin, Heilongjiang Province Heilongjiang University Hulan in October, 2012, dries, pulverizes 40 mesh sieves, for subsequent use.
1.2 Bag Material auricularia auriculajudae waste materials
Bag Material auricularia auriculajudae waste material is provided by Mudanjiang, Heilongjiang Academy of Agricultural Sciences branch.
1.3 contrast microbial inoculums
" organic matter decomposing inoculant (straw type) " that Zhongnong Lvkang (Beijing) Biotechnology Co., Ltd. produced on October 27th, 2011.
1.4 substratum
Liquid fermentation medium: glucose 5g, peptone 2g, NH
4nO
31.0g, CaCl
20.2g, K
2hPO
40.5g, FeCl
30.02, MgSO
47H
2o0.5g, NaCl1.0g, distilled water 1000mL, pH7.0.
Shake flask fermentation basic medium: peptone 2g, NH
4nO
31.0g, CaCl
20.2g, K
2hPO
40.5g, FeCl
30.02, MgSO
47H
2o0.5g, NaCl1.0g, distilled water 1000mL, pH7.0.
1.5 test method
1.5.1 Bag Material auricularia auriculajudae waste material rate of weight loss measures
Embodiment one being screened Klebsiella pneumonia (Klebsiellapneumoniae) L252 obtained is inoculated in 5mL liquid fermentation medium, and 37 DEG C of 180r/min shaking culture 12h are centrifugal, abandons supernatant and obtains thalline.Getting 500 μ L shake flask fermentation basic mediums makes thalline suspend, by bacteria suspension access containing in the shake flask fermentation basic medium of 5% Bag Material auricularia auriculajudae waste material, wherein Bag Material auricularia auriculajudae waste material uses tyndallization sterilizing, 121 DEG C of moist heat sterilization 30min, after 37 DEG C of 180r/min shaking culture 30d, centrifugal, precipitate with deionized water wash, after repeated washing three times, oven dry is weighed, Bag Material auricularia auriculajudae waste material rate of weight loss is calculated with Subtraction method, by the data obtained by SPSS19.0 software analysis, Duncan method is utilized to carry out multiple comparisons, result indicates the significance difference opposite sex of each bacterial strain with marker word mother law.Arrange five groups of blank groups and five groups of positive controls, namely blank group does not connect bacterial classification, and the contrast microbial inoculum of positive controls access 5%, other operations are all identical with aforesaid operations.
Rate of weight loss calculation formula is as follows:
1.5.2 maize straw rate of weight loss measures
Embodiment one being screened Klebsiella pneumonia (Klebsiellapneumoniae) L252 obtained is inoculated in 5mL liquid fermentation medium, and 37 DEG C of 180r/min shaking culture 12h are centrifugal, abandons supernatant and obtains thalline.Getting 500 μ L shake flask fermentation basic mediums makes thalline suspend, by bacteria suspension access containing in the shake flask fermentation basic medium of 5% corn stalk powder, wherein corn stalk powder uses tyndallization sterilizing, 121 DEG C of moist heat sterilization 30min, after 37 DEG C of 180r/min shaking culture 30d, centrifugal, precipitate with deionized water wash, after repeated washing three times, oven dry is weighed, maize straw rate of weight loss is calculated with Subtraction method, by the data obtained by SPSS19.0 software analysis, Duncan method is utilized to carry out multiple comparisons, result indicates the significance difference opposite sex of each bacterial strain with marker word mother law.Arrange five groups of blank groups and five groups of positive controls, namely blank group does not connect bacterial classification, and positive controls accesses 5% complex micro organism fungicide, and other operations are all identical with aforesaid operations.
Rate of weight loss calculation formula is as follows:
1.6 results and analysis
1.6.1 Bag Material auricularia auriculajudae waste material rate of weight loss measures
Bacterial strain L252 degrades after Bag Material auricularia auriculajudae waste material, and rate of weight loss measurement result is as shown in table 8 and Fig. 6.
The Bag Material auricularia auriculajudae waste material rate of weight loss of table 8 bacterial strain L252
Note: Bag Material auricularia auriculajudae waste material and the rate of weight loss of maize straw after 30d liquid fermenting.Blank group does not access bacterial classification, and positive controls accesses the complex micro organism fungicide that 5% Zhongnong Lvkang (Beijing) Biotechnology Co., Ltd. produces.Duncan method is adopted to carry out multiple comparisons.Significance level p=0.05
Represent with lowercase, n=3.
From table 8 and Fig. 6, after cultivating 30d, after Klebsiella pneumonia (Klebsiellapneumoniae) L252 ferments, the rate of weight loss of Bag Material auricularia auriculajudae waste material is 30.48 ± 0.49%; Blank group Bag Material auricularia auriculajudae waste material rate of weight loss is 21.60% ± 0.82%; Positive controls Bag Material auricularia auriculajudae waste material rate of weight loss is 38.53% ± 0.87%.After Klebsiella pneumonia (Klebsiellapneumoniae) L252 degrades, the rate of weight loss of Bag Material auricularia auriculajudae waste material is greater than blank group, is less than positive controls.Analyze according to conspicuous level p=0.05, through rate of weight loss significant difference compared with the Bag Material auricularia auriculajudae waste material rate of weight loss of blank group of Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing Bag Material auricularia auriculajudae waste material, significant difference compared with the Bag Material auricularia auriculajudae waste material rate of weight loss of positive controls.Klebsiella pneumonia (Klebsiellapneumoniae) L252 has stronger degradation capability to Bag Material auricularia auriculajudae waste material.The rate of weight loss of the Bag Material auricularia auriculajudae waste material after Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing is more than 30%.
1.6.2 maize straw rate of weight loss measures
After bacterial strain L252 degrading maize straws, rate of weight loss measurement result is as shown in table 8 and Fig. 7.
From table 8 and Fig. 7, after cultivating 30d, after Klebsiella pneumonia (Klebsiellapneumoniae) L252 ferments, the rate of weight loss of maize straw is 26.50 ± 0.54%; Blank group maize straw rate of weight loss is 25.80% ± 0.63%; Positive controls maize straw rate of weight loss is 44.81% ± 1.02%.After Klebsiella pneumonia (Klebsiellapneumoniae) L252 degrades, the rate of weight loss of maize straw is greater than blank group, is less than positive controls.Analyze according to conspicuous level p=0.05, maize straw rate of weight loss difference compared with the maize straw rate of weight loss of blank group through Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing is not remarkable, significant difference compared with the maize straw rate of weight loss of positive controls.Klebsiella pneumonia (Klebsiellapneumoniae) L252 has stronger degradation capability to maize straw.The rate of weight loss of the maize straw after Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing is more than 26%.
1.7 conclusion
Klebsiella pneumonia (Klebsiellapneumoniae) L252 filtered out through embodiment one has obvious degradation effect to Bag Material auricularia auriculajudae waste material and maize straw.After Klebsiella pneumonia (Klebsiellapneumoniae) L252 degrades, the rate of weight loss of Bag Material auricularia auriculajudae waste material and maize straw is all greater than the Bag Material auricularia auriculajudae waste material of blank group and the rate of weight loss of maize straw.The rate of weight loss of the Bag Material auricularia auriculajudae waste material after Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing is more than 30%; The rate of weight loss of the maize straw after Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing is more than 26%.
Klebsiella pneumonia (Klebsiellapneumoniae) L252 has much relations to the degradation effect of lignocellulose material and strain growth situation and sample composition, after Bag Material auricularia auriculajudae waste material and maize straw adopt Fan Shi (VanSoest) washing the fibre assay, each component concentration in maize straw and Bag Material auricularia auriculajudae waste material is in table 9, and the content of xylogen, Mierocrystalline cellulose and hemicellulose in Bag Material auricularia auriculajudae waste material and maize straw is all obviously different.Same bacterial strain has different degradation effects to Bag Material auricularia auriculajudae waste material and maize straw, reason is the Growth and reproduction due to xylogen in Bag Material auricularia auriculajudae waste material and maize straw and cellulosic content Different Effects bacterial strain, cause strain growth situation different, the secretion capacity of enzyme there are differences, and then cause bacterial strain different to the Utilization ability of degraded product, affect its degradation capability.
Table 9 Bag Material auricularia auriculajudae waste material and each component concentration of maize straw
2, xylogen, Mierocrystalline cellulose and hemicellulose degradation situation measure
2.1 substratum preparations:
Liquid fermentation medium: glucose 5g, peptone 2g, NH
4nO
31.0g, CaCl
20.2g, K
2hPO
40.5g, FeCl
30.02, MgSO
47H
2o0.5g, NaCl1.0g, distilled water 1000mL, pH7.0.
Shake flask fermentation basic medium: peptone 2g, NH
4nO
31.0g, CaCl
20.2g, K
2hPO
40.5g, FeCl
30.02, MgSO
47H
2o0.5g, NaCl1.0g, distilled water 1000mL, pH7.0.
Maize straw takes from this laboratory test field, school district, Harbin, Heilongjiang Province Heilongjiang University Hulan in October, 2012, dries, pulverizes 40 mesh sieves, for subsequent use.
2.2 liquid fermenting
Klebsiella pneumonia (Klebsiellapneumoniae) L252 of embodiment one is inoculated in liquid fermentation medium the bacteria suspension making OD600=0.5, then with 5% (mL/mL) inoculum size, the access of bacterium liquid is contained in the shake flask fermentation basic medium of 7.5% corn stalk powder, corn stalk powder uses tyndallization sterilizing, 121 DEG C of moist heat sterilization 30min, if three times are repeated, 37 DEG C of 180r/min shaking culture 30d, adopt lyophilization to carry out drying.Arrange one group of blank group and one group of positive controls, namely blank group does not connect bacterial classification, and positive controls access 5% (g/mL) microbial inoculum, other operations are all identical with aforesaid operations.
2.3 xylogen, Mierocrystalline cellulose and hemicellulose degradation situation measure
The each component concentration of lignocellulose adopts Fan Shi (VanSoest) washing the fibre analytical method to measure.Detailed process is as follows:
2.3.1, neutral detergent fiber (NDF) measures
By FiberCap sample cup in 105 DEG C of oven drying 30min, take out and transfer in moisture eliminator, after being cooled to 5min, weigh (being W1).The sample accurately taken after 2.000g (being W2) liquid fermenting is placed in FiberCap sample cup, sample cup is put into lixiviate beaker, adds 400mL neutral detergent and 1mL naphthane and 2g sodium sulphite anhydrous 99.3.Condensation dress on beaker sleeve is placed on hot-plate, boils 10min, continue micro-60min that boils.After boiling, with fresh hot water repeated washing three times.After sample cup being put into 130 DEG C, baking oven oven dry 2h, in moisture eliminator, be cooled to room temperature, weigh (being W3).
2.3.2, acid detergent fiber (ADF) measures
Be placed in lixiviate beaker containing the FiberCap sample cup of neutral detergent fiber after above-mentioned oven dry being weighed, add 100mL acid detergent and 1mL naphthane and 2g sodium sulphite anhydrous 99.3.Condensation dress on beaker sleeve is placed on hot-plate, boils 10min, continue micro-60min that boils.After boiling, with fresh hot water repeated washing three times.After sample cup being put into 130 DEG C, baking oven oven dry 2h, in moisture eliminator, be cooled to room temperature, weigh (being W4).
2.3.3, acidic cleaning xylogen (ADL) measures
Be placed in lixiviate beaker containing the FiberCap sample cup of neutral detergent fiber after above-mentioned oven dry being weighed, add 72% sulfuric acid, filter after 20 DEG C of digestion 3h, with fresh hot water repeated washing three times.After sample cup being put into 130 DEG C, baking oven oven dry 2h, in moisture eliminator, be cooled to room temperature, weigh (being W5).
2.3.4, acid insoluble ash (AIA) measures
Sample cup is placed in predrying and the ashing crucible (45 × 60mm) of weigh (W6), 600 DEG C of ashing 4h in retort furnace.When crucible slowly cools to about 200 DEG C, take out and be put in moisture eliminator; Weigh after being cooled to room temperature (W6).
By the data obtained by SPSS19.0 software analysis, utilize Duncan method to carry out multiple comparisons, result indicates the significance difference opposite sex of each bacterial strain with marker word mother law.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 xylogen (ADL) content:
ADL(％)＝W5/W2×100％；
Hemicellulose (Hemicellulose) content:
Hemicellulose(％)＝NDF(％)－ADF(％)；
Mierocrystalline cellulose (Cellulose) content:
Cellulose(％)＝ADF(％)－W5/W2×100％；
Xylogen (Lignin) content:
Lignin(％)＝W5/W2×100％－W6/W2×100％；
2.4 results and analysis
2.4.1 xylogen, Mierocrystalline cellulose and hemicellulose level measure
Maize straw is after strain fermentation process 30d, and Mierocrystalline cellulose, hemicellulose and content of lignin measurement result are as shown in table 10 and Fig. 8.
Each component average content in maize straw after table 10 strains for degrading
Note: blank group does not access bacterial classification, positive controls accesses the complex micro organism fungicide that 5% Zhongnong Lvkang (Beijing) Biotechnology Co., Ltd. produces.Duncan method is adopted to carry out multiple comparisons.Significance level p=0.01 and p=0.05 represents with upper and lower case letter respectively, n=3.
From table 10 and Fig. 8, maize straw is after Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing 30d, content of lignin is 9.02 ± 4.43%, is all less than Spruce lignin content in blank group and positive controls; Analyze with conspicuous level p=0.05, in the maize straw of Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing, in content of lignin and blank group maize straw, content of lignin comparing difference is remarkable, not remarkable with content of lignin comparing difference in positive controls maize straw; Analyze with conspicuous level p=0.01, in the maize straw of Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing, in content of lignin and blank group maize straw, content of lignin comparing difference is remarkable, not remarkable with content of lignin comparing difference in positive control maize straw group; Illustrate that Klebsiella pneumonia (Klebsiellapneumoniae) L252 can lignin degrading.
Maize straw is after liquid fermenting 30d, and content of cellulose is 33.65 ± 2.59%, is all greater than corn stalk fiber cellulose content in blank group and positive controls; Analyze with conspicuous level p=0.05, in the maize straw of Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing, in content of cellulose and blank group maize straw, content of cellulose comparing difference is remarkable, not remarkable with corn stalk fiber cellulose content comparing difference in positive controls; Analyze with conspicuous level p=0.01, in the maize straw of Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing, in content of cellulose and blank group, the content of cellulose comparing difference of maize straw is remarkable, not remarkable with the content of cellulose comparing difference of maize straw in positive controls; Illustrate that Klebsiella pneumonia (Klebsiellapneumoniae) L252 can not degraded cellulose.
Maize straw is after liquid fermenting 30d, and hemicellulose level is 24.33 ± 2.61%, is greater than Technique of Hemicellulose from Cornstalk content in blank group, is less than Technique of Hemicellulose from Cornstalk content in positive controls; Analyze with conspicuous level p=0.05, in the maize straw of Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing, in hemicellulose level and blank group maize straw, hemicellulose level comparing difference is remarkable, remarkable with hemicellulose level comparing difference in positive controls maize straw; Analyze with conspicuous level p=0.01, in hemicellulose level in Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing maize straw and blank group maize straw, hemicellulose level comparing difference is remarkable, not remarkable with hemicellulose level comparing difference in positive controls maize straw; Illustrate that Klebsiella pneumonia (Klebsiellapneumoniae) L252 can not degradation of hemicellulose.
Klebsiella pneumonia (Klebsiellapneumoniae) L252 has lignin degradation ability, can high-efficiency lignin degrading.Be better than complex micro organism fungicide as the degradation capability of single bacterial strain Klebsiella pneumonia (Klebsiellapneumoniae) L252 to xylogen, in the maize straw of complex micro organism fungicide fermentative processing, content of lignin is 1.3 times of content of lignin in the maize straw of Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing.
2.5 conclusion
Klebsiella pneumonia (Klebsiellapneumoniae) L252 filtered out through embodiment one has lignin degradation ability, can high-efficiency lignin degrading.In the maize straw of complex micro organism fungicide fermentative processing, content of lignin is 1.3 times of content of lignin in the maize straw of Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing, is better than complex micro organism fungicide as the degradation capability of single bacterial strain Klebsiella pneumonia (Klebsiellapneumoniae) L252 to xylogen.
3, full nitrogen, full phosphorus, full potassium, rapid available phosphorus and available potassium measure
3.1, material and reagent
3.1.1, substratum
Liquid fermentation medium: glucose 5g, peptone 2g, NH
4nO
31.0g, CaCl
20.2g, K
2hPO
40.5g, FeCl
30.02, MgSO
47H
2o0.5g, NaCl1.0g, distilled water 1000mL, pH7.0.
Shake flask fermentation basic medium: peptone 2g, NH
4nO
31.0g, CaCl
20.2g, K
2hPO
40.5g, FeCl
30.02, MgSO
47H
2o0.5g, NaCl1.0g, distilled water 1000mL, pH7.0.
3.1.2, corn stalk powder
Maize straw takes from this laboratory test field, school district, Harbin, Heilongjiang Province Heilongjiang University Hulan in October, 2012, dries, pulverizes 40 mesh sieves, for subsequent use
3.2, test method
3.2.1, liquid fermenting
Klebsiella pneumonia (Klebsiellapneumoniae) L252 embodiment one filtered out is inoculated in liquid fermentation medium and makes OD
600the bacteria suspension of=0.5, then with 5% (mL/mL) inoculum size, the access of bacterium liquid is contained in the shake flask fermentation basic medium of 7.5% corn stalk powder, corn stalk powder uses tyndallization sterilizing, 121 DEG C of moist heat sterilization 30min, each bacterial strain establishes three repetitions, 37 DEG C of 180r/min shaking culture 30d, adopt lyophilization to carry out drying.Arrange one group of blank group and one group of positive controls, namely blank group does not connect bacterial classification, and positive controls access 5% (g/mL) microbial inoculum, other operations are all identical with aforesaid operations.
3.2.2, sample solution preparation
Sample solution carries out with reference to People's Republic of China (PRC) agricultural industry criteria NY525-2012, but improves to some extent.
Accurately take the sample 0.5g (being accurate to 0.001g) in 3.2.1 after lyophilize, be placed in bottom kjeldahl flask, the sample attached on bottle wall is rinsed with a small amount of water, add 5mL sulfuric acid and 1.5mL hydrogen peroxide, carefully shake up, bottleneck is put with the little funnel of curved neck, placement is spent the night, adjustable electric furnace is slowly warming up to sulfuric acid smolder, take off, few coldly add 15 hydrogen peroxide, shake kjeldahl flask gently, heating 10min, take off, be in after slightly cold 5 ~ 10 hydrogen peroxide gradation disappears and boils, until solution is after colourless or faint yellow clear liquid, continue heating 10min, eliminate remaining hydrogen peroxide.Take off slightly cold, carefully add water to 30mL, be heated to boiling.Take off cooling, rinse the little funnel of curved neck with a small amount of water, washing lotion puts into former kjeldahl flask, and will disappear and boil in liquid immigration 100mL volumetric flask, add water constant volume, with without phosphorus filter paper filtering in the blue lid reagent bottle of drying, for subsequent use.Arrange three groups of blank groups, except not adding except sample, other operations are identical with aforesaid operations.
3.2.3, full nitrogen determination
Full nitrogen determination method carries out with reference to People's Republic of China (PRC) agricultural industry criteria NY525-2012 and NY/T297-1995, but improves to some extent.
Draw disappearing of preparing in 3.2.2 and boil clear liquid 10mL in 50mL volumetric flask, add 2mL boric acid and 200 μ L mixture indicator mixed solutions, add water and be settled to 50mL.Utilize kjeldahl apparatus to distill, with sulfuric acid standardized solution titration distillate, fading to red-purple by blueness is terminal, and record consumes sulfuric acid reference liquid volume (mL).Blank determination consumes sulfuric acid reference liquid volume and more than 0.1mL, otherwise must not redeterminate.Full nitrogen (N) content represents with g/kg, by following formulae discovery:
In formula:
V---test solution titration consumes the volume of sulfuric acid standardized solution, mL;
V
0---blank titration consumes the volume of sulfuric acid standardized solution, mL;
C---the concentration of sulfuric acid standardized solution, mol/L;
0.014---with 1.00mL sulfuric acid (1/2H
2sO
4) standardized solution suitable with a gram quality for the nitrogen represented;
D---point get multiple, constant volume/point get volume, 100/10;
M---take sample mass, g;
1000---be converted into the content of every kilogram of sample.
3.2.4, full phosphorus measures
Full phosphorus determination method carries out with reference to People's Republic of China (PRC) agricultural industry criteria NY525-2012 and NY/T298-1995, but improves to some extent.
Draw phosphorus standardized solution 0,1.00,2.00,3.00,4.00,5.00,6.00mL is placed in 7 50mL volumetric flasks respectively, add and the isopyknic blank solution of absorption sample solution, add water to 30mL, add 400 μ L2,6-dinitrophenol indicator solution, has been just micro-yellow with sodium hydroxide solution and sulphuric acid soln regulator solution, has added 10.0mL vanadium ammonium molybdate reagent, shake up, be settled to 50mL with water.This solution is the standardized solution series of 1mL phosphorous (P) 0,1.00,2.00,3.00,4.00,5.00,6.00 μ g.Place 20min under room temperature more than 15 DEG C conditions after, at spectrophotometer wavelength 440nm place with 2cm optical path cuvette, with blank solution conditioning instrumentation zero point, carry out colorimetric, read absorbancy, according to phosphorus concentration and absorbancy drawing standard curve, obtain linear regression equation.Draw in 3.2.2 disappearing of preparing and boil clear liquid 10mL in 50mL volumetric flask, add water to 30mL, develop the color with condition with standardized solution series, colorimetric, reading absorbancy.Content of tatal phosphorus represents with g/kg, by following formulae discovery:
In formula:
C---try to achieve nitrite ion phosphorus concentration by regression equation, μ g/mL;
V---color volume, 50mL;
D---point get multiple, constant volume/point get volume, 100/10;
M---take sample mass, g;
10
-3---μ g/g is converted into the factor of g/kg.
3.2.5, full potassium measures
Full potassium measuring method carries out with reference to People's Republic of China (PRC) agricultural industry criteria NY525-2012 and NY/T299-1995, but improves to some extent.
Draw potassium standardized solution 0,2.50,5.00,7.50,10.00mL is placed in 5 50mL volumetric flasks respectively, add and the isopyknic placebo solution of absorption sample solution, use water constant volume, this solution is the standardized solution series that 1mL contains potassium (K) 0,5.00,10.00,15.00,20.00 μ g.On flame photometer, with blank solution conditioning instrumentation zero point, full value to 80 calibration is regulated to go out with the standardized solution of maximum concentration in standardized solution series.Again successively by lower concentration paramount measurement of concetration other standards solution, recording instrumnet indicating value.Draw working curve according to potassium concn and instrument indicating value or obtain linear regression equation.Draw disappearing of preparing in 3.2.2 and boil clear liquid 5.00mL in 50mL volumetric flask, use water constant volume.Fixed on the upside of flame photometer with condition with standardized solution series, recording instrumnet indicating value.Need to rectify an instrument with potassium standardized solution after often measuring 5 samples.Full potassium content represents with g/kg, by following formulae discovery:
In formula:
C---tried to achieve by regression equation and measure liquid concentration, μ g/mL;
V---measure volume, be originally operating as 50mL;
D---point get multiple, constant volume/point get volume, 100/5;
M---take sample mass, g;
10
-3---the factor of g/kg is scaled by μ g/g.
3.2.6, rapid available phosphorus measure
Full potassium measuring method carries out with reference to People's Republic of China (PRC) agricultural industry criteria NY/T300-1995, but improves to some extent.
The sample 1.00g accurately taken in 3.2.1 after lyophilize is placed in 50mL triangular flask, adds the citric acid solution of 20mL25 DEG C, jumps a queue, and vibrate 30min at 25 DEG C, with without phosphorus filter paper filtering in dry blue lid reagent bottle, for subsequent use.Arrange three groups of blank groups, except not adding except sample, other operations are identical with aforesaid operations.Measuring method is identical with 3.2.4.Available phosphorus contents represents with mg/kg, by following formulae discovery:
In formula:
C---try to achieve nitrite ion phosphorus concentration by regression equation, μ g/mL;
V---color volume, 50mL;
D---point get multiple, sample extracting liquid volume/point get volume, 20/5;
M---take sample mass, g.
3.2.7, available potassium measure
The sample 1.00g accurately taken in 3.2.1 after lyophilize is placed in 50mL triangular flask, adds 10mL salpeter solution, plugs little funnel, micro-10min that boils on electric furnace, and filtered while hot, in 50mL volumetric flask, with hot wash 5 times, cools rear constant volume.Arrange three groups of blank groups, except not adding except sample, other operations are identical with aforesaid operations.Measuring method is identical with 3.2.5.Quick-acting potassium content represents with mg/kg, by following formulae discovery:
In formula:
C---tried to achieve by regression equation and measure liquid potassium concn, μ g/mL;
V---measure volume, 50mL;
M---take sample mass, g.
3.3, results and analysis
3.3.1, full nitrogen, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content measure
Maize straw is after strain liquid fermentative processing 30d, and carry out full nitrogen, full phosphorus, full potassium, rapid available phosphorus and available potassium mensuration, result is as shown in table 11 and Fig. 9 ~ Figure 11.Maize straw is after liquid fermenting, analyze according to horizontal p=0.05 and p=0.01 of the significance of difference, the full nitrogen of Klebsiella pneumonia (Klebsiellapneumoniae) L252 fermentative processing group, full phosphorus, full potassium, rapid available phosphorus are not remarkable with quick-acting potassium content equal difference compared with blank group.Therefore, can not affect principal element content wherein when bacterial strain is degraded on maize straw, original fertilizer efficiency still preserved by sample.
Principal element content in maize straw after table 11 fermentative processing
Note: blank group does not access bacterial classification.Duncan method is adopted to carry out multiple comparisons.Significance level p=0.01 and p=0.05 represents with upper and lower case letter respectively, n=3.
3.4 conclusion
Klebsiella pneumonia (Klebsiellapneumoniae) L252 is to after maize straw liquid fermenting process 30d, and the full nitrogen in sample, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content have no significant change.Klebsiella pneumonia (Klebsiellapneumoniae) L252 is when degrading sample, the principal element content in sample can not be caused, can not cause the full nitrogen in sample, full phosphorus, full potassium, rapid available phosphorus and quick-acting potassium content change, original fertilizer efficiency still preserved by sample.
Claims (2)
1. a strain lignocellulose material efficient degrading bacteria L252, it is characterized in that it is Klebsiella pneumonia (Klebsiellapneumoniae) L252, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on December 12nd, 2014, preserving number CGMCCNo.10161.
2. the application of a strain lignocellulose material efficient degrading bacteria L252 as claimed in claim 1, is characterized in that it is for lignocellulose degradation class material.
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