CN102010844B - Perishable organic garbage degradation and elimination microbial agent, preparation method and used bacteria thereof - Google Patents
Perishable organic garbage degradation and elimination microbial agent, preparation method and used bacteria thereof Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a Bacillus subtilis with a preservation name of No.2 and a preservation unit of China General Microbiological Culture Collection Center as well as a preservation address of Institute of Microbiology Chinese Academy of Sciences NO.3, yard 1, West Beichen Road, Chaoyang District, Beijing 100101, China and a preservation data of June Ist 2010 and a preservation number of CGMCC NO.3882. The invention also discloses a perishable organic garbage degradation and elimination microbial agent I which is a composite liquid microbial agent of 3.0*10<8>-2.8*10<11>cfu/ml of Acetinmucor elegans with the preservation number of CGMCC No.3883 and 3.0*10<8>-2.7*10<11>cfu/ml of Bacillus subtilis with the preservation number of CGMCC No.3882. The invention also provides a preparation method of the microbial agent I.
Description
Technical field
The invention belongs to the domestic rubbish disposal field, relate to a kind of microbiological deterioration technology for eliminating that is used for perishable organic waste, the preparation method of microbial inoculum falls in the mikrobe that is specifically related to a kind of perishable organic waste.
Background technology
Along with the Chinese society rapid economy development, town dweller's domestic refuse not only quantity increases day by day, and comprises in the domestic refuse that the proportion of perishable organic wastes such as melon skin shell, leftovers leftovers is also increasing.Since putrescibility domestic refuse water ratio high (90%), incinerating combustion value low (2100～3100kJ/kg), after other municipal wastes compositions mix, adopt landfill to dispose not only to take valuable land resources but also can produce a large amount of percolates and polluted underground water is; Adopt burning electricity generation to dispose because of the thermal value that can not satisfy rubbish requires (being more than the 5000kJ/kg) and can cause the insufficient Dioxins that produces of incinerator burning, serious harm periphery resident is healthy.Because this type rubbish source disperses, therefore perishable smelly and contaminate environment in collecting transport process is difficult to collect from single household and gathers the back and realize recycling through feed processing, compost or biogas fermentation disposal technology.So the putrescibility domestic refuse has become the important source of pollution that influence the town environment quality.For this reason, research and develop a kind of innoxious, minimizing disposal new technology of height that this type rubbish produce in situ is degraded on the spot and eliminated, have important role for the Sustainable development of economic society and the safety of preserving the ecological environment.
Based on above-mentioned theory; At present China has developed some food refuse original position technology for eliminating, like the disclosure of the Invention of application number 03151167.8 a kind of garbage microbiological degradation handler, this machine is pulverized food refuses such as leftovers leftovers, melon skin shell earlier; Utilize set microorganism species that the food refuse particle is resolved into water, carbonic acid gas and mineral ion again; After the filtration of filtering granular layer, be liquid at last and enter water drain then, can not cause sewer line gambling plug.This technology has but increased treatment pressure of sewage when reducing the quantity discharged of solid refuse, therefore, fail fundamentally to solve the problem of environmental pollution of meal kitchen food refuse.Patent of invention is also arranged, and (application number: 02150972.7) disclose the domestic organic garbage handler of a kind of YB of application microbial function bacterium, this YB microbial function bacterium is made up of subtilis and Paracoccus denitrificans.
Summary of the invention
The technical problem that the present invention will solve provides a kind of perishable organic waste degraded elimination type microbiobacterial agent I and preparation method thereof and bacterial strain uses therefor.
In order to solve the problems of the technologies described above; The present invention provides a kind of subtilis (Bacillus subtilis), and this subtilis (Bacillus subtilis) preservation name is called No.2, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, preservation date: on June 1st, 2010, preserving number: CGMCC NO.3882.
The present invention also provides a kind of perishable organic waste degraded elimination type microbiobacterial agent I simultaneously, and this microbial inoculum I is 3.0 * 10
8～2.8 * 10
11Grace radiation Mucor (Actinomucor elegans) the CGMCC No.3881 and 3.0 * 10 of cfu/ml
8～2.7 * 10
11The composite fluid microbial inoculum of the subtilis of cfu/ml (Bacillus subtilis) CGMCC No.3882.
The present invention also provides the preparation method of above-mentioned perishable organic waste degraded elimination type microbiobacterial agent I simultaneously, may further comprise the steps:
1), slant culture:
Grace is radiated Mucor (Actinomucor elegans) CGMCC No.3881 be inoculated on the PDA inclined-plane, CGMCC No.3882 is inoculated in the beef extract-peptone inclined-plane with subtilis (Bacillus subtilis); Respectively at 15～43 ℃ of cultivation 24～72h, carry out the activation of bacterial strain;
2), primary seed solution is cultivated:
With the grace after the activation of step 1) gained radiation Mucor be inoculated in after PDA liquid nutrient medium, the gained activation subtilis be inoculated in the beef extract-peptone liquid nutrient medium, respectively at 15～43 ℃, 100～250r/min shaking table shaking culture, 48～120h; Respectively primary seed solution and the primary seed solution of subtilis of graceful radiation Mucor;
3), bacterial liquid fermentation culture:
In fermentor tank, place nutrient solution I, with step 2) primary seed solution of the subtilis of gained is inoculated among the nutrient solution I according to the inoculum size that accounts for nutrient solution I5～25% volume ratio, in 15 ℃～43 ℃, 30～100r/min fermentation culture, 48～72h; Obtain bacterium bacterium liquid;
4), fungi liquid fermentation culture:
In fermentor tank (another fermentor tank), place nutrient solution II; With step 2) primary seed solution of the grace of gained radiation Mucor is inoculated among the nutrient solution II according to the inoculum size that accounts for nutrient solution II 5～25% volume ratios, in 15 ℃～43 ℃, 30～100r/min fermentation culture, 48～72h; Obtain fungi bacterium liquid;
5), the bacterium bacterium liquid of step 3) gained is mixed with the fungi bacterium liquid of step 4) gained, obtain perishable organic waste degraded elimination type microbiobacterial agent I.
Improvement as the preparation method of perishable organic waste degraded elimination type microbiobacterial agent I of the present invention: the nutrient solution I in the step 3) is: with 10～200 times of monosodium glutamate waste liquid dilutions, and add adjusting PH with base to 7.1～7.3; Nutrient solution II in the step 4) is: with 10～200 times of monosodium glutamate waste liquid dilutions, and add adjusting PH with base to 5.4～5.6.
In the present invention:
Graceful radiation Mucor (Actinomucor elegans) No.1; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, preservation date: on June 1st, 2010, preserving number: CGMCC NO.3881.
Subtilis (Bacillus subtilis) No.2; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, preservation date: on June 1st, 2010, preserving number: CGMCC NO.3882.
The prescription and the preparation method of PDA solid medium are following: yam 200g peeling stripping and slicing adds 800g water boil 0.5h, filtered through gauze, and with sucrose 20g, agar 18g, adding distil water is to 1000mL; At pressure 1.05kg/cm
2, 121 ℃ of 20min that sterilize down of temperature.
The prescription and the preparation method of PDA liquid nutrient medium are following: yam 200g peeling stripping and slicing adds 800g water boil 0.5h, filtered through gauze, and with sucrose 20g, adding distil water is to 1000mL; At pressure 1.05kg/cm
2, 121 ℃ of 20min that sterilize down of temperature.
The prescription and the preparation method of beef extract-peptone solid medium are following: Carnis Bovis seu Bubali cream 3g, peptone 10g, NaCl5g, agar 18g, adding distil water transfer pH to 7.0～7.2 to 1000mL; At pressure 1.05kg/cm
2, 121.3 ℃ of 20min that sterilize down of temperature.
The prescription and the preparation method of beef extract-peptone liquid nutrient medium are following: Carnis Bovis seu Bubali cream 3g, peptone 10g, NaCl 5g, adding distil water transfer pH to 7.0～7.2 to 1000mL; At pressure 1.05kg/cm
2, 121 ℃ of 20min that sterilize down of temperature.
Monosodium glutamate waste liquid is to hand over waste liquid leaving of producing in the glutamate production; Nutritious, amino total content reaches about 10%, and heavy metal free pollutes simultaneously; Each item content is far below the agricultural control criterion of townm refuse; Therefore, have quality safety, nutritious characteristics, can reach the purpose of the treatment of wastes with processes of wastes against one another, utilization of waste as resource as the substratum utilization of microbiobacterial agent.For example, the monosodium glutamate waste liquid that can select for use the West Lake, Hangzhou monosodium glutamate Group Co.,Ltd in the glutamate production process, to be produced.
The preferred plan of perishable organic waste degraded elimination type microbiobacterial agent I of the present invention is: this microbial inoculum I is 4.3 * 10
9～2.8 * 10
11Grace radiation Mucor (Actinomucor elegans) the CGMCC No.3881 and 1.5 * 10 of cfu/ml
9～2.7 * 10
11The composite fluid microbial inoculum of the subtilis of cfu/ml (Bacillus subtilis) CGMCC No.3882.
Grace radiation Mucor (Actinomucor elegans) No.1 among the present invention separates, screens acquisition with subtilis (Bacillus subtilis) No.2 from nature, and they have or have concurrently the function of protein, starch, Mierocrystalline cellulose and fatty efficient degradation.
During the actual use of perishable organic waste degraded elimination type microbiobacterial agent I of the present invention, microbial inoculum with have good water-absorbing-retaining ability, open-textured solid support material mixes, thereby form perishable organic waste degradable solid matrix; Concrete degradation principles is following:
Perishable organic waste degradable solid matrix is placed in the garbage disposer,, build the optimal temperature and the aerobic environment of perishable organic waste degeneration system, drop into a certain amount of perishable rubbish every day through the operation of garbage disposer; In garbage degradation system operational process; The quick growth and breeding of microorganism species; Discharge a large amount of lytic enzyme albumen; Comprising lypase, glycase, cellulase, proteolytic enzyme etc., the fat in the perishable rubbish, starch, robust fibre, protein etc. are decomposed into short chain low molecule organic matters such as lipid acid, sugar and amino acid, flora goes out H as Nutrition and Metabolism
2O, CO
2And biological heat energy, breed rapidly with geometricprogression simultaneously.In the flora reproductive process, constantly eliminate organic waste, go round and begin again, realize that the innoxious and height minus of rubbish quantizes.
Generally speaking; In carrier, add perishable organic waste degraded elimination type microbiobacterial agent I of the present invention and constitute perishable organic waste degradable solid matrix according to 15%～30% weight ratio; Place then in the garbage degradation handler, the domestic organic garbage that adds is carried out degradation treatment through the operation of garbage disposer.
The nutrition source that the present invention prepares microbial inoculum is foodstuffs industry organic waste water a--monosodium glutamate waste liquid, and not only production technique is simplified, and cost is low, and has realized utilization of waste as resource.
Description of drawings
Do further explain below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is the bacteriolyze circle of the growth 3d of bacterial strain No.2 (subtilis Bacillus subtilis) on 3 kinds of substratum, the corresponding successively protein substratum of a～c, starch culture-medium, Mierocrystalline cellulose substratum;
Fig. 2 is the growth curve of bacterial strain No.2 (subtilis Bacillus subtilis).
Embodiment
Collection, separation, screening and the evaluation of embodiment 1, subtilis (Bacillus subtilis) No.2:
1), collection, separation, purifying
The fertilizer 10g that gets the fermentation of animal dung During High-Temperature Composting and get places in the 250mL triangular flask that 100mL beef extract-peptone liquid nutrient medium is housed 100r/min vibration 24h enrichment culture; Transfering loop dips in the suspension liquid of getting gained and on bacterium selective medium flat board, rules; Place 30 ℃ to cultivate 48h; The different single bacterium colony of the some forms of picking from the flat board, each bacterium colony is rule on bacterium selective medium flat board respectively, repeatedly for several times; Until each bacterium colony purifying, obtain some bacterial isolates.
The dull and stereotyped preparation of bacterium selective medium: Carnis Bovis seu Bubali cream 3g, peptone 10g, NaCl 5g, agar 18g, adding distil water transfer pH to 7.0～7.2 to 1000mL; At pressure 1.05kg/cm2,121.3 ℃ of 20min that sterilize down of temperature.Add nystatin 30 units/L, penicillium mould 2000 units/L after the cooling, fall dull and stereotyped.
2), bacteriolyze circle method screening
Seeing that the staple of food refuse is protein, fat, starch, Mierocrystalline cellulose, therefore should have or have concurrently the efficient degradation ability of these four big types of materials as the perishable garbage degradation function yeast of life.The some bacterial isolates that adopt bacteriolyze circle method to measure respectively to obtain in above-mentioned collection, separation, the purge process therefrom filter out efficient bacterial strain to the degradation capability of four types of materials:
The inclined-plane bacterium colony of the different isolates that separation and purification obtains in the inoculating needle picking bacterium selective medium flat board is connected to protein, starch, fat, Mierocrystalline cellulose culture medium flat plate respectively, observes the variation of bacteriolyze loop diameter with incubation time.Therefrom filter out the bigger bacterial isolates of bacteriolyze loop diameter, numbering NO.2.The bacteriolyze circle of on protein, starch, Mierocrystalline cellulose culture medium flat plate, cultivating 3d of isolate NO.2 is as shown in Figure 1.
Protein substratum: skim-milk 50g/L, Zulkovsky starch 10g/L, yeast extract paste 5g/L, KH
2PO
41g/L, MgSO
47H
2O 0.2g/L, agar 20g/L, all the other are zero(ppm) water, pH 7.0～7.2,121 ℃ of sterilization 20min, the cooling back is fallen dull and stereotyped.
Starch culture-medium: peptone 10g/L, Zulkovsky starch 2g/L, Carnis Bovis seu Bubali cream 5g/L, NaCl 5g/L, agar 20g/L, all the other are zero(ppm) water, pH 7.0～7.2,121 ℃ of sterilization 20min, the cooling back is fallen dull and stereotyped.
Fat substratum: (NH
4)
2SO
42g/L, K
2HPO
41g/L, KCl 0.5g/L, MgSO
47H
2O 0.5g/L, FeSO
40.01g/L, agar 20g/L, (sweet oil: 20g/L Z 150PH (PVA) is 1: 3 a volume ratio to sweet oil emulsion 12mL, rotating speed 1000r/min; 5min), purpurum bromocresolis 0.04g/L, all the other are zero(ppm) water; The pH nature, 121 ℃ of sterilization 20min, the cooling back is fallen dull and stereotyped.
Mierocrystalline cellulose substratum: K
2HPO
40.50g/L, MgSO
40.25g/L, CMC-Na 1.88g/L, Congo red 0.20g/L, agar 16.00g/L, gelatin 2.00g/L, all the other are zero(ppm) water, pH 7.0～7.2,121 ℃ of sterilization 20min, the cooling back is fallen dull and stereotyped.
3), the vitality test of proteolytic enzyme, lypase, glycase, cellulase
Growth curve is measured: the activation bacterium liquid 1mL of inoculation isolate No.2 is in 30mL beef extract-peptone liquid nutrient medium; 30 ℃, 120r/min shaking culture; Every at a distance from 2h get bacterium liquid in 560nm place the mensuration light absorption value (ultraviolet-visible pectrophotometer, Beckmann, DU640); Draw growth curve, as shown in Figure 2.
Be the degradation capability of further clear and definite isolate No.2 bacterial strain to 4 types of materials, measure 4 kinds of extracellular enzyme activity of this bacterial strain, the result lists in table 1.
I. protease activity is measured
Albumen seed culture fluid (Anshu Gupta, S K Khare, 2006): K
2HPO
47.0g/L, KH
2PO
42.0g/L, MgSO
47H
2O 0.2g/L, casein food grade 4.0g/L, yeast extract paste 6.0g/L, NaCl 0.5g/L, glycerine 7.0g/L, CaCl
2, 0.067g/L, all the other are zero(ppm) water, 7.0,121 ℃ of sterilizations of pH 20min.
Inoculation isolate No.2 logarithmic phase bacterium liquid 0.5mL is in 25mL albumen seed culture fluid; 30 ℃, 120r/min shaking culture; Every at a distance from the specified time sampling, press Folin-phenol reagent method (Cuiping Li et al, 2005) and measure the neutral protein enzymic activity: get spinning (10000g; 5min, 4 ℃) after supernatant be that crude enzyme liquid 0.5mL adds 0.5mL ddH
2O gets 1mL and contains the caseic 0.02M sodium phosphate buffer of 1% (W/V), places 5min at 30 ℃ of constant-temperature shaking casees respectively.Mix crude enzyme liquid and damping fluid, leave standstill 10min at 30 ℃.Slightly vibrate after adding 3mL 10%TCA (trichoroacetic acid(TCA)) then, again at the centrifugal 10min of 13000g (4 ℃).Get the 1mL supernatant, other adds 3mLNa
2CO
3(0.55M), add 1mLFolin-phenol reagent again, after the vibration, leave standstill 20min in 30 ℃.At last, survey its absorbancy in the 640nm place.With standard tyrosine concentration gradient as typical curve.Wherein Folin-phenol reagent disposes according to people (1951) methods such as Oliver H Lowry.The proteinase activity unit definition: every min catalysis casein hydrolysis obtains 1 μ gmL
-1The enzyme amount that tyrosine is required.
Ii. diastatic activity is measured
Starch seed nutrient solution: starch 10gL
-1, peptone 5gL
-1, K
2HPO
42gL
-1, NaCl 1gL
-1, CaCl
20.1gL
-1, MgSO
47H
2O 0.1gL
-1, all the other are zero(ppm) water, 121 ℃ of sterilization 20min.
Inoculation isolate No.2 logarithmic phase bacterium liquid 0.5mL is in 25mL starch seed nutrient solution, and 30 ℃, 120r/min shaking culture are whenever at a distance from the specified time sampling; Press DNS method (Gashaw Mamo, Amare Gessesse, 1997) and measure amylase activity: get the centrifugal (10000g of bacterium liquid; 5min; 4 ℃) supernatant is crude enzyme liquid 0.5mL, adds starch-phosphate buffered saline buffer mixed solution (50mM, pH 7.0 phosphoric acid buffers add 1% starch) 1.5mL; In 70 ℃ of water-baths, react 30min, add 3mL DNS reagent then and boil 5min.At last, measure light absorption value at the 540nm place.The powder enzyme activity unit is defined as: the hydrolysis of every min catalysis starch forms 1 μ gmL
-1Required enzyme amount during reducing sugar.
DNS reagent collocation method: accurately take by weighing anhydrous 3,5 dinitrosalicylic acid 6.5g and dissolve for use.No water sodium hydroxide 40g dissolving back moves into the 500mL volumetric flask, cooling back constant volume.Salicylic acid solution is moved into the 1000mL volumetric flask and adds the sodium hydroxide solution of 325mL, add the dissolving of 15mL USP Kosher again and be settled to 1000mL, be stored in the brown reagent bottle.
Iii. lipase activity is measured
Fatty seed nutrient solution: NaNO
32g/L, MgSO
47H
2O 0.3g/L, NaCl 10g/L, sweet oil 20g/L, CaCl
20.3g/L, NH
4Cl 0.3g/L, all the other are zero(ppm) water, 121 ℃ of sterilization 20min.
Inoculation isolate No.2 logarithmic phase bacterium liquid 1mL is in 50mL fatty seed nutrient solution; 30 ℃, 120r/min shaking culture; Sampling is at regular intervals measured lipase activity by volumetry (Pignede G et al, 2000): get the centrifugal (10000g of bacterium liquid then; 5min, 4 ℃) supernatant is that crude enzyme liquid 1mL adds 5mL emulsion and 4mL phosphate buffered saline buffer (Na
2HPO
4-KH
2PO
4, 100mM, pH 8.0) mixed solution in.37 ℃ of reactions of water-bath 10min.Use the ethanol stopped reaction of 15mL95% (V/V) then.Lipid acid with the generation of 50mM NaOH drop reaction.The lipase activity unit definition is: every min catalysis fat splitting produces 1 μ molmL
-1The enzyme amount that lipid acid is required.
Iv. cellulase activity (CMCase) is measured
Mierocrystalline cellulose seed culture fluid: Carnis Bovis seu Bubali cream 1.5g/L, peptone 1.0g/L, CaCO
32.0g/L all the other are zero(ppm) water; Every test tube is distributed into high 7cm deep layer, and puts into 1 of 1 * 6cm filter paper bar, 121 ℃ of sterilization 20min.
Inoculation isolate No.2 logarithmic phase bacterium liquid 1mL is in 50mL Mierocrystalline cellulose seed culture fluid; 30 ℃, 120r/min shaking culture; Sampling at regular intervals adopts 3, fixed sugared method (DNS) (the Miller GL of 5-dinitrosalicylic acid colorimetric; 1959) measure reducing sugar content in the enzymolysis solution, make typical curve with glucose solution.Concrete grammar is: get 1mL bacterium liquid and put into centrifuge tube, the centrifugal 5min of 10000g/min pipettes supernatant 0.5mL in test tube; Add citrate buffer solution (0.05mol/L, the pH 4.4) 1.5mL that contains 0.5%CMC-Na, then after 50 ℃ of water-baths accurately act on 30min; In vitro add 1.5mL DNS reagent immediately whenever, in boiling water, behind the immersion 5min, cool off with flowing water immediately again; Be settled to 25mL, measure OD value at the 520nm place.The contrast standard curve is tried to achieve its sugared content again.The cellulase activity unit definition: every min catalyzing cellulose hydrolysis forms 1 μ gmL
-1Required enzyme amount during glucose.
The result is as shown in table 1:
4 types of extracellular enzyme activity of table 1, isolate No.2
4), security is identified
The bacterium liquid of isolate No.2 (the sample title: No. 2 bacterium liquid) and graceful radiation Mucor (Actinomucor elegans) CGMCCNo.3881 send Zhejiang Academy of Medical Sciences hygiology institute; Carry out rat acute per os toxicity test, rat acute percutaneous toxicity test, the acute eye irritation/corrosion test of rabbit and rabbit skin irritation/corrosion test, the result is summarized as follows table 2:
Table 2
5), taxonomy is identified and preservation
The slant strains of isolate No.2 (strain number: No.2) send Institute of Microorganism, Academia Sinica to carry out taxonomy and identify; Must detect expert's conclusion: under this laboratory condition; According to microscopic morphology, Physiology and biochemistry data and 16S rDNA gene order data, (strain number: qualification result No.2) is Zhejiang University's censorship bacterial classification: bacterial strain No.2: be Bacillus subtilis subtilis.
No.2 has carried out preservation with this bacterial strain; Subtilis (Bacillus subtilis), strain number No.2 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, preservation date: on June 1st, 2010, preserving number: CGMCC NO.3882.
Embodiment 2: perishable organic waste degraded elimination type microbiobacterial agent I constitutes
Total count: 2.5 * 10
10Cfu/mL
Graceful radiation Mucor (Actinomucor elegans) No.1:1.4 * 10
10Cfu/mL
Subtilis (Bacillus subtilis)) No.2:1.1 * 10
10Cfu/mL
Embodiment 3: perishable organic waste degraded elimination type microbiobacterial agent I constitutes
Total count: 3.8 * 10
11Cfu/mL
Graceful radiation Mucor (Actinomucor elegans) No.1:2.8 * 10
11Cfu/mL
Subtilis (Bacillus subtilis) No.2:1.0 * 10
11Cfu/mL
Embodiment 4: perishable organic waste degraded elimination type microbiobacterial agent I constitutes
Total count: 5.6 * 10
10Cfu/mL
Graceful radiation Mucor (Actinomucor elegans) No.1:4.3 * 10
9Cfu/mL
Subtilis (Bacillus subtilis)) No.2:5.2 * 10
10Cfu/mL
Embodiment 5: perishable organic waste degraded elimination type microbiobacterial agent I constitutes
Total count: 3.4 * 10
10Cfu/mL
Graceful radiation Mucor (Actinomucor elegans) No.1:3.3 * 10
10Cfu/mL
Subtilis (Bacillus subtilis) No.2:1.5 * 10
9Cfu/mL
Embodiment 6: perishable organic waste degraded elimination type microbiobacterial agent I constitutes
Total count: 3.13 * 10
11Cfu/mL
Graceful radiation Mucor (Actinomucor elegans) No.1:4.3 * 10
10Cfu/mL
Subtilis (Bacillus subtilis) No.2:2.7 * 10
11Cfu/mL
Embodiment 7: the preparation of perishable organic waste degraded elimination type microbiobacterial agent I
(1) slant strains activation: the graceful radiation of picking one ring Mucor (Actinomucor elegans) No.1 is inoculated in the PDA solid medium; Picking one ring subtilis (Bacillus subtilis) No.2 is inoculated on the beef extract-peptone solid medium, under 30 ℃ of conditions, cultivates 2 days (48h) respectively, obtains the activation thalline.
(2) seed culture: grace radiation Mucor one ring after picking step (1) activation is inoculated in the PDA liquid nutrient medium of 100mL respectively; Subtilis one ring after the activation is inoculated in the beef extract-peptone liquid nutrient medium of 100mL; Respectively at 30 ℃ of condition 120r/min shaking table shaking culture 78h; Make each bacterial strain kind daughter bacteria liquid, that is, respectively primary seed solution and the primary seed solution of subtilis of graceful radiation Mucor.
(3) bacterial liquid fermentation culture: 85 times (promptly the zero(ppm) water of 1 parts by volume monosodium glutamate waste liquid+84 parts by volume dilutes) of monosodium glutamate waste liquid dilution, and, get nutrient solution I with 1mol NaOH or 1molHCl adjusting pH to 7.2; Nutrient solution I is placed through conventional disinfectant fermentor tank; After sterilization (jar temperature rise to the 121 ℃ maintenance 30min) cooling; Press the inoculum size of 10% volume ratio of nutrient solution I, the primary seed solution of subtilis is inoculated in fermentor tank, in 30 ℃, 60r/min; Venting pressure 0.6mPa condition fermentation culture 48h, obtaining total count is 2.2 * 10
10The bacterium bacterium liquid of cfu/mL.
(4) fungi liquid fermentation culture: 80 times of monosodium glutamate waste liquid dilutions, and, get nutrient solution II with 1mol NaOH or 1molHCl adjusting pH to 5.5; Place another through conventional disinfectant fermentor tank nutrient solution II, after sterilization (jar temperature rise to the 121 ℃ maintenance 30min) cooling, press the inoculum size of nutrient solution II11% volume ratio; The primary seed solution of grace being radiated Mucor is inoculated in fermentor tank; In 28 ℃, 80r/min, venting pressure 0.6mPa condition is carried out fermentation culture 60h, and obtaining total count is 2.8 * 10
10The fungi bacterium liquid of cfu/mL.
(5) the bacterium bacterium liquid with above-mentioned steps (3) gained mixes by 1: 1 volume ratio with the fungi bacterium liquid of step (4) gained, obtains perishable organic waste degraded elimination type microbiobacterial agent I, like embodiment 2.
Embodiment 8: the preparation of perishable organic waste degraded elimination type microbiobacterial agent I
(1) slant strains activation: the graceful radiation of picking one ring Mucor (Actinomucor elegans) No.1 is inoculated in the PDA solid medium; Picking one ring subtilis (Bacillus subtilis) No.2 is inoculated on the beef extract-peptone solid medium, under 28 ℃ of conditions, cultivates 2 days (48h) respectively, obtains the activation thalline.
(2) seed culture: grace radiation Mucor one ring after picking step (1) activation is inoculated in the PDA liquid nutrient medium of 100mL respectively; Subtilis one ring after the activation is inoculated in the beef extract-peptone liquid nutrient medium of 100mL; Respectively at 28 ℃ of condition 150r/min shaking table shaking culture 78h; Make each bacterial strain kind daughter bacteria liquid, that is, respectively primary seed solution and the primary seed solution of subtilis of graceful radiation Mucor.
(3) bacterial liquid fermentation culture: 75 times of monosodium glutamate waste liquid dilutions, and, get nutrient solution I with 1mol NaOH or 1molHCl adjusting pH to 7.2; Nutrient solution I is placed through conventional disinfectant fermentor tank; After sterilization (jar temperature rise to the 121 ℃ maintenance 30min) cooling; Press the inoculum size of 15% volume ratio of nutrient solution I, the primary seed solution of subtilis is inoculated in fermentor tank, in 32 ℃, 72r/min; Venting pressure 0.5mPa condition fermentation culture 60h, obtaining total count is 3.0 * 10
11The bacterium bacterium liquid of cfu/mL.
(4) fungi liquid fermentation culture: 70 times of monosodium glutamate waste liquid dilutions, and, get nutrient solution II with 1mol NaOH or 1molHCl adjusting pH to 5.5; Place another through conventional disinfectant fermentor tank nutrient solution II, after sterilization (jar temperature rise to the 121 ℃ maintenance 30min) cooling, press the inoculum size of nutrient solution II14% volume ratio; The primary seed solution of grace being radiated Mucor is inoculated in fermentor tank; In 30 ℃, 100r/min, venting pressure 0.55mPa condition fermentation culture 72h, obtaining total count is 4.2 * 10
11The fungi bacterium liquid of cfu/mL.
(5) the bacterium bacterium liquid with above-mentioned steps (3) gained mixes by 1: 2 volume ratio with the fungi bacterium liquid of step (4) gained, obtains perishable organic waste degraded elimination type microbiobacterial agent I, like embodiment 3.
Embodiment 9: the application of perishable organic waste degraded elimination type microbiobacterial agent I
Crumb, husk, the peat composed of rotten mosses are mixed with 5: 4: 1 volume ratio, constitute the solid support material of solid substrate in the garbage degradation system.In solid support material, add the perishable organic waste degraded elimination type microbiobacterial agent I (2 amount to heavy 4000g) that the foregoing description 7 account for solid support material gross weight 20% makes; Place in the garbage degradation handler of Ningbo City general-purpose plastics machine works manufacturing; Open and handle operation 2 days; Add the perishable organic waste of 1kg (compositions such as melon skin shell, leftovers leftovers) afterwards every day, move 3 months continuously, rubbish volume elimination factor reaches 97.6%.
Embodiment 10: the application of perishable organic waste degraded microbial liquid microbial inoculum I
Sawdust, cotton seed hull, the peat composed of rotten mosses are mixed with 3: 4: 1 volume ratio, constitute the solid support material of solid substrate in the garbage degradation system.In solid support material, add the perishable organic waste degraded elimination type microbiobacterial agent I (2 amount to heavy 3500g) that the foregoing description 8 account for solid support material gross weight 25% makes; Place in the garbage degradation handler of Ningbo City general-purpose plastics machine works manufacturing; Open and handle operation 2 days; Add the perishable organic waste of 0.8kg (compositions such as melon skin shell, leftovers leftovers) afterwards every day, move 3 months continuously, rubbish volume elimination factor reaches 98.1%.
The comparative example:
With the YB microbial inoculum that the liquid bacterial agent among the embodiment 10 makes into to inform in 02150972.7, rubbish volume elimination factor is merely 85.1%.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (3)
1. a subtilis (Bacillus subtilis); It is characterized in that: this subtilis (Bacillus subtilis) preservation name is called No.2; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on June 1st, 2010, preserving number: CGMCC NO.3882.
2. perishable organic waste degraded elimination type microbiobacterial agent I, it is characterized in that: this microbial inoculum I is 3.0 * 10
8～2.8 * 10
11Grace radiation Mucor (Actinomucor elegans) the CGMCC No.3881 and 3.0 * 10 of cfu/ml
8～2.7 * 10
11The composite fluid microbial inoculum of the subtilis of cfu/ml (Bacillus subtilis) CGMCC No.3882.
3. the preparation method of a perishable organic waste degraded elimination type microbiobacterial agent I as claimed in claim 2 is characterized in that may further comprise the steps:
1), slant culture:
Grace is radiated Mucor (Actinomucor elegans) CGMCC No.3881 be inoculated in the PDA inclined-plane, CGMCC No.3882 is inoculated in the beef extract-peptone inclined-plane with subtilis (Bacillus subtilis); Respectively at 28～32 ℃ of cultivation 24～72h, carry out the activation of bacterial strain;
2), primary seed solution is cultivated:
The subtilis that grace after the activation of step 1) gained radiation Mucor is inoculated in after the activation of PDA liquid nutrient medium, gained is inoculated in the beef extract-peptone liquid nutrient medium, respectively at 28～32 ℃, 100～250r/min shaking table shaking culture, 48～120h; Respectively primary seed solution and the primary seed solution of subtilis of graceful radiation Mucor;
3), bacterial liquid fermentation culture:
In fermentor tank, place nutrient solution I, with step 2) primary seed solution of the subtilis of gained is inoculated among the nutrient solution I according to the inoculum size that accounts for nutrient solution I5～25% volume ratio, in 28～32 ℃, 30～100r/min fermentation culture, 48～72h; Obtain bacterium bacterium liquid;
Said nutrient solution I is: with 10～200 times of monosodium glutamate waste liquid dilutions, and add adjusting PH with base to 7.1～7.3;
4), fungi liquid fermentation culture:
In fermentor tank, place nutrient solution II, with step 2) primary seed solution of the grace radiation Mucor of gained is inoculated among the nutrient solution II according to the inoculum size that accounts for nutrient solution II5～25% volume ratio, in 28～32 ℃, 30～100r/min fermentation culture, 48～72h; Obtain fungi bacterium liquid;
Said nutrient solution II is: with 10～200 times of monosodium glutamate waste liquid dilutions, and add adjusting PH with base to 5.4～5.6;
5), the bacterium bacterium liquid of step 3) gained is mixed with the fungi bacterium liquid of step 4) gained, obtain perishable organic waste degraded elimination type microbiobacterial agent I.
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