CN103602592A - Cellulose-degradation fungus and preparation of inoculum and application thereof - Google Patents

Cellulose-degradation fungus and preparation of inoculum and application thereof Download PDF

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CN103602592A
CN103602592A CN201310491950.4A CN201310491950A CN103602592A CN 103602592 A CN103602592 A CN 103602592A CN 201310491950 A CN201310491950 A CN 201310491950A CN 103602592 A CN103602592 A CN 103602592A
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penicillium oxalicum
cellulose
preparation
compost
conidium
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CN103602592B (en
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刘训理
刘鹏
姜红霞
周保强
鲍正宗
隋君康
赵旭
王晓辉
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Shandong Agricultural University
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Abstract

The invention provides a cellulose-degradation fungus, preparation of an inoculum and an application thereof. According to the invention, a strain is obtained from Mount Taishan woodland humus soil by artificial enrichment and screening; the strain is identified as penicillium oxalicum, which is named as penicillium oxalicum C2; the strain has good cellulose degradation capability, and high cellulose-production activity; after shake flask fermentation for 3 days, the filter paper cellulose activity is up to 132.26 U/mL; When used in silkworm excrement compost, the conidium inoculums prepared from the penicillium oxalicum C2 can increase the compost temperature, prolong the high temperature stage, facilitate water content reduction, promote organic matter decomposition and the increase of total nitrogen relative content, decrease the C/N ratio, raise the seed germination index, accelerate the composting process, improve decomposition efficiency and fertilizer quality; the preparation method of the inoculums is simple in process, low in cost, and suitable for industrial production.

Description

Preparation and the application of one strain cellulose degradation fungi and microbial inoculum thereof
Technical field
The present invention relates to preparation and the application of a strain cellulose degradation fungi and microbial inoculum thereof, belong to microbial technology field.
Background technology
Mierocrystalline cellulose is the abundantest class renewable energy source material of occurring in nature content, is extensively present in agricultural crop straw, feces of livestock and poultry, domestic waste and paper mill effluent.It is reported, the Mierocrystalline cellulose that the whole world produces by photosynthesis is every year approximately up to 4.0 * 10 10ton, wherein agricultural crop straw output is about 22 Yi Duodun， China as large agricultural country, and agricultural straw resource is very abundant, can reach every year 20%～30% of 6.5 hundred million tons of above ，Zhan world stalk ultimate productions.But, complex structure due to cellulosic molecule, natural degradation process is extremely slow, the lignocellulose utilising efficiency of China is very low at present, except small part is for the industries such as papermaking, building, weaving, all the other most of quilts are directly burned as fuel, or are arbitrarily piled up, abandon, not only waste resource, more caused environmental pollution and ecological damage.
As valuable renewable resources, cellulosic degraded not only plays an important role in natural carbon cycle, and all has important potential using value aspect a lot.Research shows, cellulose substances can be degraded to glucose, and is further converted to the materials such as biofuel, tropina, high-quality feed, food.Therefore, if these agricultural wastes can be transformed on a large scale and make full use of, this can not only alleviate energy shortage problem, more can solve a difficult problem for environmental pollution.
One of Main By product that silkworm excrement is produced as China's silkworm and mulberry, output is huge and contain abundant nutritive substance, is a kind of resource with good development prospect.In the numerous areas of the silkworm excrement utilization of resources, the compost treatment of silkworm excrement is current most widely used general and cost-effective method.But owing to containing the Mierocrystalline cellulose of a large amount of hard degradations in silkworm excrement, rely on merely microbiological deterioration in silkworm excrement and be difficult to produce a desired effect.Therefore, find and exploitation highly effective cellulose degrading microorganism, become the key that can cellulose resource efficiently be utilized.Cellulose-degrading bacteria in environment can and can not have the irreplaceable advantage of other processing modes to environment by the eccrine fiber element effective degraded cellulose of enzyme, has a extensive future.At present, from environment, separated and seed selection cellulase high-yield, excavates cellulase producing bacteria resource, utilizes the enzymolysis of microorganism, and solving environmental pollution or even being converted into the energy has become one of study hotspot both domestic and external.
Summary of the invention
In order to address the above problem, the invention provides preparation and the application of a strain cellulose degradation fungi and microbial inoculum thereof, can be used for the compost maturity of silkworm excrement.
One strain cellulose degradation fungi, derives from forest land, Mount Taishan vegetable mould, through artificial enrichment, screening, obtains.This identification of strains is penicillium oxalicum (Penicillium oxalicum), called after penicillium oxalicum C2, and bacterium colony is smooth, form shell, mycelia just gradually becomes sap green for after white, and conidiophore top forms the branch of broom shape, conidium is oval, smooth.
One strain penicillium oxalicum (Penicillium oxalicum), has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number: CGMCC NO.8335 on October 15th, 2013.Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica postcode 100101.
The microbiobacterial agent that utilizes above-mentioned penicillium oxalicum C2 to produce, its activeconstituents is the cellulase of penicillium oxalicum C2 conidium and generation thereof.Described microbiobacterial agent is solid powder.
The preparation method of penicillium oxalicum C2 conidium microbial inoculum, specifically comprises the steps:
(1) preparation of seed liquor: get the circular bacterium piece of the dull and stereotyped penicillium oxalicum C2 bacterial strain of cultivating of PDA, in inoculation liquid PDA substratum, 30, ℃ 200r/min shaking culture 18h～20h is as cellulose-degrading bacteria C2 seed liquor.
(2) fermentation culture: seed liquor is inoculated into and is produced on spore solid medium according to the inoculum size of relative solid medium massfraction 5%, under relative humidity 60%～80% condition, cultivate 4～6d for 28 ℃, to after culture natural air drying, pulverize and sieve, obtain penicillium oxalicum C2 conidium microbial inoculum.
Wherein said culture medium prescription is as follows:
PDA substratum: potato 200.0g, (NH 4) 2sO 41.0g, glucose 20.0g, extractum carnis 5.0g, MgSO 41.0g, KH 2pO 40.6g, CaCO 33.0g, agar 15g(liquid PDA substratum does not add agar), distilled water 1000mL;
Produce spore solid medium: wheat bran 40g, Semen Maydis powder 20g, sucrose 5g, KNO 31g, KH 2pO 40.5g, MgSO 40.1g, distilled water 300mL.
Conidium number containing penicillium oxalicum C2 in described penicillium oxalicum C2 conidium microbial inoculum is (4.8～5.6) * 10 8individual/g.
The present invention also provides the application of this penicillium oxalicum in silkworm excrement compost, can accelerate compost process, improves become thoroughly decomposed efficiency and fertilizer quality.
The present invention has the following advantages:
1. penicillium oxalicum C2 has good cellulose degradation ability, filter paper bar is carried out to after the degradation treatment of 5d the filter paper bar agglomerating pasty state that festers.
2. this penicillium oxalicum has good product Filter paper Cellulase ability, and after liquid fermenting 3d, Filter paper Cellulase vigor reaches 132.26U/mL.
3. this penicillium oxalicum bacteria growing is rapid, fermentation proterties is good.
4. this kind of preparation method's zymotechnique is simple, cost is low, is beneficial to suitability for industrialized production.
5. this penicillium oxalicum is applied in silkworm excrement compost, can accelerate compost process, improves become thoroughly decomposed efficiency and fertilizer quality.
Accompanying drawing explanation
Fig. 1 is bacterium colony, conidium and the conidiophore morphologic observation figure of this bacterial strain, and in figure, A is colonial morphology figure, and B is conidium aspect graph, and C is conidiophore aspect graph;
Fig. 2 is this bacterial strain 18S rDNA fragment 1% agarose gel electrophoresis result, and as can be seen from the figure, C2 bacterial strain 18SrDNA fragment length is about 600bp, meets conventional 18S rDNA sequence length;
Fig. 3 is the phylogenetic tree that utilizes this bacterial strain of 18S rDNA homology sequence and adjacent method structure, and as can be seen from the figure the genetic evolution of C2 bacterial strain and Penicillium is nearest;
Fig. 4 is the variation that conidium microbial inoculum is processed silkworm excrement compost temperature, as can be seen from the figure, the 3rd day temperature of experimental group heap body reaches 54 ℃ and enters the pliotherm period, top temperature reaches 65.5, ℃ pliotherm period continues 13d, and in compost, test group generally exceeds 5～9 than control group, after ℃ explanation inoculation conidium microbial inoculum, can accelerate to pile body and heat up, extend the pliotherm period.
Fig. 5 is that the water ratio that conidium microbial inoculum is processed in silkworm excrement compost changes, and as can be seen from the figure, experimental group water ratio declines by a big margin, and water ratio has reduced by 23.56%, is greater than the water ratio reducing amount 19.24% of control group;
Fig. 6 is the variation that conidium microbial inoculum is processed total content of organic carbon in silkworm excrement compost, and as can be seen from the figure, the fall of test group total content of organic carbon is 13.03%, is greater than the amplitude 10.53% that control group declines;
Fig. 7 is the variation that conidium microbial inoculum is processed total nitrogen content in silkworm excrement compost, and as can be seen from the figure, in composting process, the nitrogen content of test group is all the time higher than control group, and this this microbial inoculum of explanation inoculation has certain effect for compost keeping nitrogen tool;
Fig. 8 is the variation that conidium microbial inoculum is processed the C/N ratio in silkworm excrement compost, and as can be seen from the figure, the carbon-nitrogen ratio of test group and control group exists significant difference, and this explanation test group adds C2 microbial inoculum can accelerate compost maturity process;
Fig. 9 is that conidium microbial inoculum is processed the impact of silkworm excrement compost on seed germination index, as can be seen from the figure, on compost the 17th day, the seed germination index of test group reaches 53.14%, compost becomes thoroughly decomposed substantially, control group is on compost the 21st day, and seed germination index just reaches 51.05%, than test group late 4d; Test group was on compost the 29th day, and seed germination index reaches 81.24%, illustrates that product becomes thoroughly decomposed completely, and control group than experimental group late 12d just reach completely and become thoroughly decomposed.
Embodiment
Substratum involved in the present invention is as follows:
1. enrichment medium: peptone 10.0g, CMC-Na10.0g, K 2hPO 41.0g, Na 2cO 35.0g, MgSO 47H 2o0.1g, FeSO 47H 2o0.015g, MnSO 40.05g, yeast extract paste 10g, distilled water 1000mL, pH value 6.0;
2. carboxymethyl cellulose substratum (CMC substratum): CMC-Na15.0g, NH 4nO 31.0g, yeast extract paste 1.0g, MgSO 47H 2o0.5g, KH 2pO 41.0g, distilled water 1000mL, agar 15g;
3. the Congo red substratum of Mierocrystalline cellulose: K 2hPO 40.5g, Microcrystalline Cellulose 1.88g, MgSO 40.25g, gelatin 2.0g, Congo red 0.2g, agar 14.0g, distilled water 1000mL, agar 15g;
4. He Qixun substratum: KH 2pO 41.0g, CaCl 20.1g, MgSO 47H 2o0.3g, NaCl0.1g, FeCl 30.01g, NaNO 32.5g, pH7.2～7.3, distilled water 1000mL;
5. enzymatic production substratum: CMC-Na0.5g, peptone 1.0g, wheat bran 3.0g, NaCl0.5g, KH 2pO 40.1g, MgSO 47H 2o0.02g, (NH 4) 2sO 40.3g, distilled water 1000mL;
6. produce spore solid medium: wheat bran 40g, Semen Maydis powder 20g, sucrose 5g, KNO 31g, KH 2pO 40.5g, MgSO 40.1g, distilled water 300mL.
Penicillium oxalicum C2 bacterial strain seed liquor preparation method involved in the present invention is as follows:
With punch tool, beat and get circular bacterium piece (the diameter 5mm) 2 of penicillium oxalicum C2 bacterial strain that PDA culture medium flat plate is cultivated, be inoculated in 50mL liquid PDA substratum, 30, ℃ 200r/min shaking culture 18～20h is as seed liquor.
Embodiment mono-
The enrichment of cellulose-degrading bacteria is with separated
Get forest land, the Mount Taishan vegetable mould soil sample that 5g gathers and join in 45mL enrichment medium, 30, under ℃ 180r/min after shaking culture 3d, pipette 5mL nutrient solution and continue to cultivate 3d in another fills the triangular flask of fresh enrichment medium.
Get the nutrient solution after enrichment, with sterilized water, be diluted to 10 -4, 10 -5, 10 -6, 10 -74 concentration gradients.Each the 100 μ L of soil diluent that draw four kinds of concentration with pipettor are coated on CMC culture medium flat plate, and each concentration is carried out 3 times and repeated coated test, 28 ℃ of constant temperature culture 2d, and from substratum, picking list bacterium colony is in the separation and purification of ruling of CMC culture medium flat plate.
The bacterial strain that on CMC culture medium flat plate, purifying obtains is transferred on the Congo red culture medium flat plate of Mierocrystalline cellulose, 3 repetitions of each bacterial strain, cultivate 2d for 28 ℃, can there will be transparent circle clearly around by cellulolytic bacterial strain, according to the size of the Congo red dull and stereotyped transparent circle diameter of Mierocrystalline cellulose (D) and colony diameter (d), tentatively judge the size of strains for degrading Mierocrystalline cellulose ability.Through revision test repeatedly, obtain the maximum and bacterial strain rapidly of growing of a strain transparent circle diameter, called after penicillium oxalicum C2.
Embodiment bis-
1. filter paper bar Degrading experiment
Get the Erlenmeyer flask of six 250mL, be numbered respectively 1～No. 6; Wherein in 1～No. 3 Erlenmeyer flask, put into respectively the filter paper bar of 1cm * 6cm, respectively add 50mL He Qixun substratum, and the seed liquor of inoculating respectively 1mL cellulose-degrading bacteria C2 is as test group; In 4～No. 6 Erlenmeyer flasks, put into respectively the filter paper bar of 1cm * 6cm, respectively add 50mL He Qixun nutrient solution and the seed liquor of not inoculating cellulose-degrading bacteria C2 as a control group; Six Erlenmeyer flasks are all placed in to 30 ℃ of constant-temperature tables, and shaking culture 5d under 200r/min, estimates in 1～No. 3 Erlenmeyer flask the filter paper bar agglomerating pasty state that festers.
2. the mensuration of Filter paper Cellulase vigor
The present invention selects 3,5-dinitrosalicylic acid (DNS), under alkaline condition, generates colored compound with reducing sugar reaction, by spectrophotometer, carries out colorimetric estimation, determines that the method for the amount of low molecular sugar measures the vigor of cellulase.The mensuration basis thing that the yellow reagent of DNS is colorimetry with reducing sugar red-brown aminocompound 3-amino-5-NITROSALICYLIC ACID that thermal response generates altogether under alkaline condition.
The preparation of crude enzyme liquid: get penicillium oxalicum C2 bacterial strain seed liquor 1mL and join in enzymatic production substratum, 28, ℃ 180r/min shaking culture 3d, gets 5mL nutrient solution in Erlenmeyer flask, adds 45mL sodium citrate buffer solution (citric acid 10.5g, sodium hydroxide 5.0g, distilled water 1000mL), after 200r/min concussion 30min, the centrifugal 10min of 5000r/min, collect supernatant liquor and be crude enzyme liquid, save backup at 4 ℃.
Filter paper Cellulase vitality test: add filter paper bar in 25mL tool plug test tube, the citrate buffer that adds again 1.0mL infiltrates filter paper bar, be placed in 50 water ℃ bath water-bath balance 10min, then add crude enzyme liquid that 0.5mL suitably dilutes and the water of 5mL, electromagnetic oscillation 3s～5s, in 50 ℃ of water-baths, be incubated 60min, the DNS reagent that adds 2mL, then in boiling water bath, boil 5min, be cooled to room temperature, water is settled to 25mL, the glucose standardized solution of 0 μ g/mL of take is blank zeroising, under 540nm wavelength, measure absorbance A 1, measure the absorbance A 2 of the white sample of enzyme liquid air simultaneously, reference standard curve calculation enzyme activity.50, under ℃ pH5.5 condition, the 1mL crude enzyme liquid filter paper of degrading is produced to 1 μ g glucose as 1 enzyme activity unit in 1min, with (U/mL), represent.
The active calculation formula of Filter paper Cellulase:
X = [ ( A 1 - A 2 ) × K + b ] × D t
In formula:
X---sample fiber element enzyme activity (U/mL);
The absorbancy of A1---enzyme reaction solution;
The absorbancy of the blank sample of A2---enzyme;
The slope of K---typical curve;
The intercept of b---typical curve;
Total extension rate of D---sample;
T---the reaction times (min).
After measured, the cellulase activity of cellulose-degrading bacteria C2 bacterial strain crude enzyme liquid reaches 132.26U/mL, higher than many fungal bacterial strains of having reported, and far surpass < < microbial fertilizer that the national Ministry of Agriculture promulgates and produce bacterial strain quality evalution current techique and require in > > the regulation more than 70U/mL for cellulose-degrading bacteria cellulase-producing vigor, illustrate that this bacterium has good cellulase-producing ability.
Embodiment tri-
The evaluation of cellulose-degrading bacteria C2
1. colony characteristics and morphological features
(1) colony characteristics
This bacterial strain is grown rapidly on PDA flat board, and mycelia is dense, and bacterium colony is smooth, forms shell, is white when mycelia is initial, and along with the increase of incubation time, mycelia gradually becomes sap green, and the substratum back side is faint yellow; On CMC flat board, grow slower, mycelia is sparse, and mycelium is also shorter, and mycelia is smooth, is white when mycelia is initial, and along with the increase of incubation time, mycelia gradually becomes blue-greenish colour (see figure 1).
(2) morphological features
Vegetative hyphae has barrier film, conidiophore vertically bears from mycelia, top life be arranged in broom shape between branch, 7～9 of stigmas, (11.5～14.8) μ m * (3.0～3.4) μ m, conidium is oval, smooth, size (4.8～6.0) μ m * (3.2～3.9) μ m (seeing Fig. 1).
2.18S rDNA sequence and homology analysis
Extract cellulose-degrading bacteria C2 strain gene group DNA, adopt fungi rDNA transcribed spacer (ITS) universal primer ITS1 and ITS4 to carry out 18S rDNA fragment amplification; Primer sequence is: ITS1(5 '-TCCGTAGGTGAACCTGCGG-3 '), and ITS4(5 '-TCCTCCGCTTATTGATATGC-3 '); PCR reaction system (50 μ L): 10 * PCR Buffer5.0 μ L, MgCl 2(25m mol/L) 5.0 μ L, dNTP(10m mol/L) 2 μ L, Taq enzyme (5U/ μ L) 0.4 μ L, each 1 μ L of primer I TS1, ITS4, template DNA 1 μ L, ddH 2o34.6 μ L; Reaction conditions is: 95 ℃ of denaturation 5min, and 95 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 35 circulations, then 72 ℃ are extended 10min, 4 ℃ of termination reactions; The C2 bacterial strain 18S rDNA sequence length that utilizes primer I TS1/ITS4 amplification to obtain is about 600bp(Fig. 2), meet conventional 18S rDNA sequence length, reclaim this fragment and check order, sequence length is 595bp, and its 18S rDNA sequence is as shown in SEQ.ID.NO.1.
The 18S rDNA sequencing result of C2 bacterial strain is carried out to Blast comparison in GenBank database, from result, choose the 10 strains bacterial strain high with measuring bacterial strain sequence similarity and carry out Phylogenetic Analysis, with Mega4.0 software, take Neighbor-joining method constructing system evolutionary tree (see figure 3).As seen from the figure, the genetic evolution of C2 bacterial strain and Penicillium is nearest, reaches 99% with the 18S rDNA homology of known bacterial strain penicillium oxalicum (Penicillium oxalicum GU183174).In conjunction with its thalli morphology and colony characteristics observations, be accredited as Penicillium (Penicillium sp.).
Embodiment tetra-
The preparation of conidium microbial inoculum and the application in silkworm excrement compost
1. the preparation of conidium microbial inoculum
With punch tool, beat and get circular bacterium piece (the diameter 5mm) 2 of penicillium oxalicum C2 bacterial strain that PDA culture medium flat plate is cultivated, be inoculated in 50mL liquid PDA substratum, 30, ℃ 200r/min shaking culture 18～20h is as seed liquor.Seed liquor is inoculated into and is produced on spore solid medium according to the inoculum size of relative product spore solid medium mass ratio 5%, under relative humidity 60%～80% condition, cultivate 4～6d for 28 ℃, to after culture natural air drying, pulverize and sieve, obtain the conidium microbial inoculum of penicillium oxalicum C2 bacterial strain, the conidium content that adopts dilution spread flat band method to record the conidium microbial inoculum mesoxalic acid Penicillium notatum C2 of preparation is (4.8～5.6) * 10 8individual/g.
2. the application of conidium microbial inoculum in silkworm excrement compost
Four parts of silkworm excrements are set and carry out composting test, every part of silkworm excrement 100kg, and its water content is adjusted to 60%; Using the penicillium oxalicum C2 bacterial strain conidium microbial inoculum of inoculating respectively relative silkworm excrement massfraction 0.3% in two parts of silkworm excrements as (processing T for test group 1, process T 2); By adding respectively silkworm excrement massfraction in another two parts of silkworm excrements, be that 0.3% deactivation C2 bacterial strain conidium microbial inoculum (is CK as a control group 1, CK 2); The equal 4d turning of every group of heap body once.
During temperature measuring, selecting heap body center is point for measuring temperature, and the measuring point degree of depth is 30cm, and every day, 9:00 and 16:00 carried out temperature measuring, got respectively the mean value of test group and control group temperature as compost temperature, measured envrionment temperature (see figure 4) simultaneously.Result shows, after inoculation C2 bacterial strain conidium microbial inoculum, can accelerate to pile body heats up, the 3rd day temperature reaches 54 ℃ and enters the pliotherm period, top temperature reaches 65.5, ℃ pliotherm period continues 13d, at temperature raising period and high temperature interim trial group, than control group, generally exceeds 5～9, and the temperature of ℃ test group and control group compost exists significant difference (p<0.05); Measurement of water-content coefficient adopts atmosphere pressure desiccation, and result shows (see figure 5), and the heap body water ratio of test group and control group is all on a declining curve, declines the fastest at pliotherm period water ratio, and this is because the pliotherm period is conducive to moisture evaporation; Experimental group water ratio declines by a big margin, and water ratio has reduced by 23.56%, and the water ratio of control group has reduced by 19.24%.Statistical study shows, two groups of water ratio of processing compost exist utmost point significant difference (p<0.01), and this can accelerate to pile body with the C2 microbial inoculum adding in test group and heat up relevant with prolongation high-temperature time.
Every four days, two groups of compost (four heap bodies) are distinguished to once sampling, during sampling, getting 4 of heap body surroundings and top center place is sampling point, sampling depth 25～35cm, each point sampling quantity 100g, mixes laggard line parameter and measures (mean value that numerical value all adopts each group).Organic carbon determination adopts potassium bichromate titrimetric method, and full nitrogen determination adopts Micro-kjoldahl method.Result shows, the total content of organic carbon of test group and control group all reduces (see figure 6) along with the carrying out of compost, the fall of test group total content of organic carbon is 13.03%, control group fall is 10.53%, statistical analysis shows, test group and control group organic carbon content difference is (p<0.01) extremely significantly, and this explanation C2 microbial inoculum has promoted organic fast decoupled in silkworm excrement in composting process.The basically identical (see figure 7) of total nitrogen content variation tendency of test group and control group, statistical study shows, the nitrogen content difference of experimental group and control group compost is remarkable (p>0.05) not, but the nitrogen content of test group is all the time higher than control group, and this explanation inoculation C2 microbial inoculum has certain effect for compost keeping nitrogen tool.Along with compost carries out, middle organism constantly decomposes and full nitrogen relative content constantly rises, and C/N is than constantly declining, and the C/N ratio of final trial group and control group is respectively 13.5 and 14.7, all substantially becomes thoroughly decomposed.But test group C/N is faster than declining, and drops to below 20 at the 25th day, and control group drops to below 20 the 29th talent, than test group late 4d(see Fig. 8).There is significant difference (p<0.05) in the carbon-nitrogen ratio of test group and control group, this explanation test group adds C2 microbial inoculum can accelerate compost maturity process.
When carrying out seed germination assessment of indices, get the fresh sample of 20g compost and join in 200mL distilled water, fully after vibration, lixiviate is filtered.Get 6mL filtrate, join in the culture dish that is covered with filter paper.20 full Chinese cabbage seeds of program request in each culture dish, after dark culturing 48h, calculate percentage of germination, measure root long.Each is processed and repeats 3 times, contrasts as distilled water.The method of calculation of seed germination index (GI) are there is utmost point significant difference in seed germination index, on compost the 17th day, the seed germination index of test group reached 53.14%, and compost becomes thoroughly decomposed substantially, and control group is on compost the 21st day, and seed germination index just reaches 51.05%, than test group late 4d; Test group was on compost the 29th day, and seed germination index reaches 81.24%, illustrates that product becomes thoroughly decomposed completely, and control group than experimental group late 12d just reach the (see figure 9) of becoming thoroughly decomposed completely.
From piling external sight, observe, test group and control group heap body volume volume before and after compost all decline to some extent, and the heap body volume of test group reduces approximately 30%, and control group heap body volume reduction is less than test group, approximately 20%.From color, the external sight of the heap of test group is dry and comfortable, and quality is loose, even, and because the raised growth of fungi presents canescence; Control group is relatively moist, is grey black.At the composting process initial stage, test group and control group compost all frowziness produce, and along with the stink that carries out test group of compost alleviates gradually and disappears, there be fragrance and the earth breath of mulberry leaf in the later stage; Control group is frowziness generation always in composting process, and in composting process, produces a large amount of fly maggots.Above result shows, after interpolation C2 bacterial strain, can obviously accelerate compost process, improves become thoroughly decomposed efficiency and fertilizer quality.
Figure IDA0000398692580000011
Figure IDA0000398692580000021

Claims (3)

1. a strain preserving number is the penicillium oxalicum C2(Penicillium oxalicum of CGMCC NO.8335), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center; Its 18S rDNA sequence is as shown in SEQ.ID.NO.1.
2. the preparation method who utilizes penicillium oxalicum C2 conidium microbial inoculum as claimed in claim 1, is characterized in that comprising the steps:
(1) preparation of seed liquor: beat and get 2 of the circular bacterium pieces of the dull and stereotyped C2 bacterial strain of cultivating of PDA with punch tool, be inoculated in 50mL liquid PDA substratum, 28～30, ℃ 180～220r/min shaking culture, 18～20h is as seed liquor;
(2) fermentation culture: seed liquor is inoculated into and is produced on spore solid medium according to the inoculum size of relative product spore solid medium massfraction 5%, under relative humidity 60%～80% condition, cultivate 4～6d for 28 ℃; To after cultured material natural air drying, pulverize and sieve, obtain the solid spore microbial inoculum of penicillium oxalicum C2 bacterial strain;
Described product spore solid medium component is: wheat bran 40g, Semen Maydis powder 20g, sucrose 5g, KNO 31g, KH 2pO 40.5g, MgSO 40.1g, distilled water 300mL.
3. the application in silkworm excrement compost maturity according to the penicillium oxalicum C2 conidium microbial inoculum of claim 2 preparation.
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CN104593477A (en) * 2015-02-13 2015-05-06 江西省科学院微生物研究所 Method for quickly separating and screening cellulose decomposing fungi
CN107502553A (en) * 2017-07-11 2017-12-22 南京中医药大学 A kind of cellulase producing bacteria for being resistant to liquorice dregs and the method applied to liquorice dregs cellulase-producing
CN109207377A (en) * 2018-10-17 2019-01-15 湖南省林业科学院 One penicillium bacterial strain and its application in degradation cake of camellia oleifera seeds in cellulose
CN113151004A (en) * 2021-03-16 2021-07-23 黑龙江大学 Novel strain of Asira zeylanica and application thereof
CN113481103A (en) * 2020-10-16 2021-10-08 吉林省农业科学院 Preparation method of high-efficiency degraded cellulose penicillium griseum
CN114380629A (en) * 2022-02-17 2022-04-22 重庆市农业科学院 Composite microbial straw decomposing inoculant and preparation method thereof
CN114561327A (en) * 2022-03-31 2022-05-31 山东农业大学 Cellulose degradation composite microbial inoculum and preparation method and application thereof

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CN102559506A (en) * 2010-12-07 2012-07-11 中国农业科学院作物科学研究所 Penicillium oxalicum and application thereof

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Cited By (9)

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Publication number Priority date Publication date Assignee Title
CN104593477A (en) * 2015-02-13 2015-05-06 江西省科学院微生物研究所 Method for quickly separating and screening cellulose decomposing fungi
CN104593477B (en) * 2015-02-13 2016-08-03 江西省科学院微生物研究所 A kind of sharp separation screen fibre element decomposes the method for fungus
CN107502553A (en) * 2017-07-11 2017-12-22 南京中医药大学 A kind of cellulase producing bacteria for being resistant to liquorice dregs and the method applied to liquorice dregs cellulase-producing
CN109207377A (en) * 2018-10-17 2019-01-15 湖南省林业科学院 One penicillium bacterial strain and its application in degradation cake of camellia oleifera seeds in cellulose
CN113481103A (en) * 2020-10-16 2021-10-08 吉林省农业科学院 Preparation method of high-efficiency degraded cellulose penicillium griseum
CN113151004A (en) * 2021-03-16 2021-07-23 黑龙江大学 Novel strain of Asira zeylanica and application thereof
CN114380629A (en) * 2022-02-17 2022-04-22 重庆市农业科学院 Composite microbial straw decomposing inoculant and preparation method thereof
CN114561327A (en) * 2022-03-31 2022-05-31 山东农业大学 Cellulose degradation composite microbial inoculum and preparation method and application thereof
CN114561327B (en) * 2022-03-31 2023-09-12 山东农业大学 Cellulose degradation composite microbial inoculant, and preparation method and application thereof

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