CN102010843B - Food waste degradation and elimination type microbial bacterial agent, preparation method thereof and bacteria used thereby - Google Patents
Food waste degradation and elimination type microbial bacterial agent, preparation method thereof and bacteria used thereby Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses Bacilus cereus of which the collection name is No.6, the collection unit is General Microbiology Center for China Committee for Culture Collection of Microorganisms, the collection address is Institute of Microbiology of Chinese Academy of Sciences, No.3, N0.1 yard, Beichen west road, Chaoyang district, Beijing city, the collection date is June 1st, 2010, and the collection number is CGMCCNO.3884. The invention also discloses a food waste degradation and elimination type microbial bacterial agent III, which is a liquid composite bacterial agent of actinomucor elegans with the microbial content of 3.0*10<8> to 1.4*10<11>cfu/ml and the Bacilus cereus with the microbial content of 3.0*10<8> to 1.8*10<11>cfu/ml. The invention also discloses a preparation method of the food waste degradation and elimination type microbial bacterial agent III. The microbial bacterial agent III is used for degrading food waste.
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 food refuse, the preparation method of microbial inoculum falls in the microorganism that is specifically related to a kind of food refuse.
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 food refuses such as melon skin shell, leftovers leftovers is also increasing.Since food refuse water ratio height (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 class 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 by feed processing, compost or biogas fermentation disposal technology.So food 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 class 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, China has developed some food refuse original position technology for eliminating at present, as 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 filtration after the filtration of 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 to be solved in the present invention provides a kind of food refuse degraded elimination type microbiobacterial agent III and preparation method thereof and used bacterium.
In order to solve the problems of the technologies described above, the invention provides a kind of bacillus cereus, this bacillus cereus (Bacilus cereus) preservation name is called No.6, 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.3884.
The present invention also discloses a kind of food refuse degraded elimination type microbiobacterial agent III simultaneously, and this microbial inoculum III is 3.0 * 10
8～1.4 * 10
11Grace radiation Mucor (Actinomucor elegans) the CGMCC No.3881 and 3.0 * 10 of cfu/ml
8～1.8 * 10
11The composite fluid microbial inoculum of the bacillus cereus of cfu/ml (Bacilus cereus) CGMCC No.3884.
The present invention also discloses the preparation method of above-mentioned food refuse degraded elimination type microbiobacterial agent III simultaneously, may further comprise the steps:
1), slant culture:
Grace is radiated Mucor (Actinomucor elegans) CGMCC No.3881 to be inoculated on the PDA inclined-plane, CGMCC No.3884 is inoculated in the beef extract-peptone inclined-plane with bacillus cereus (Bacilus cereus), respectively at 15～43 ℃ of cultivation 24～72h, carry out the activation of bacterial strain;
2), primary seed solution is cultivated:
The bacillus cereus that the grace radiation Mucor of step 1) activation back gained is inoculated in PDA liquid nutrient medium, activation back gained is inoculated in the beef extract-peptone liquid nutrient medium, in 15～43 ℃, 100～250r/min shaking table shaking culture, 48～120h; Respectively the primary seed solution of graceful radiation Mucor and the primary seed solution of bacillus cereus;
3), bacterial liquid fermentation culture:
In fermentor tank, place nutrient solution I, with step 2) primary seed solution of the bacillus cereus of gained is inoculated among the nutrient solution I according to the inoculum size that accounts for nutrient solution I 5～25% volume ratios, in 15 ℃～43 ℃, 30～100r/min fermentation culture, 48～72h; Obtain bacterium bacterium liquid;
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 II 5～25% volume ratios, in 15 ℃～43 ℃, 30～100r/min fermentation culture, 48～72h; Obtain fungi bacterium liquid;
5), the bacterium bacterium liquid with the step 3) gained mixes acquisition food refuse degraded elimination type microbiobacterial agent III with the fungi bacterium liquid of step 4) gained.
Improvement as the preparation method of food refuse of the present invention degraded elimination type microbiobacterial agent III:
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.
Bacillus cereus (Bacilus cereus) No.6, 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, protect good number: CGMCC NO.3884.
The prescription and the preparation method of PDA solid medium are as follows: potato 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 as follows: potato 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 as follows: extractum carnis 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 ℃ of 20min that sterilize down of temperature.
The prescription and the preparation method of beef extract-peptone liquid nutrient medium are as follows: extractum carnis 3g, peptone 10g, NaCl5g, 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 be produce in the glutamate production from handing over waste liquid, nutritious, amino total content reaches about 10%, heavy metal free pollutes simultaneously, every 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 version of food refuse degraded elimination type microbiobacterial agent III of the present invention is: this microbial inoculum III is 4.3 * 10
9～1.4 * 10
11Grace radiation Mucor (Actinomucor elegans) the CGMCC No.3881 and 1.1 * 10 of cfu/ml
9～1.8 * 10
11The composite fluid microbial inoculum of the bacillus cereus of cfu/ml (Bacilus cereus) CGMCC No.3884.
Grace radiation Mucor (Actinomucor elegans) No.1 among the present invention separates, screens acquisition with bacillus cereus (Bacilus cereus) No.6 from nature, and they have or have concurrently the function of protein, starch, Mierocrystalline cellulose and fatty efficient degradation.
During the actual use of food refuse of the present invention degraded elimination type microbiobacterial agent III, microbial inoculum III with have good water-absorbing-retaining ability, open-textured solid support material mixes, thereby composition food refuse degradable solid matrix; Concrete degradation principles is as follows:
Food refuse degradable solid matrix is placed in the garbage disposer,, build the optimal temperature and the aerobic environment of food refuse degeneration system, drop into a certain amount of food refuse every day by 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 lipase, amylase, cellulase, proteolytic enzyme etc., fat in the food refuse, 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 food refuse, go round and begin again, realize that the innoxious and height minus of food refuse quantizes.
Generally speaking, in carrier, add food refuse degraded elimination type microbiobacterial agent III of the present invention and constitute food refuse degradable solid matrix according to 15%～30% weight ratio, place then in the garbage degradation handler, the food refuse that adds is carried out degradation treatment by the operation of garbage disposer.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is grow on the protein substratum bacteriolyze circle of 3d of bacterial strain No.6 (bacillus cereus Bacilus cereus);
Fig. 2 is the growth curve chart of bacterial strain No.6 (bacillus cereus Bacilus cereus).
Embodiment
Collection, separation, screening and the evaluation of embodiment 1, bacillus cereus (Bacilus cereus)
1), collection, separation, purifying
Get the fertilizer 10g of animal dung During High-Temperature Composting fermentation gained, place 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 rules on bacterium selective medium flat board, places 30 ℃ to cultivate down 48h, the different single bacterium colony of the some forms of picking from the flat board, on bacterium selective medium flat board, rule respectively, for several times,, obtain some bacterial isolates repeatedly until each bacterium colony purifying.
The dull and stereotyped preparation of bacterium selective medium: extractum carnis 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 ℃ of 20min that sterilize down of temperature.Add nystatin 30 units/L, penicillin 2000 units/L after the cooling, fall dull and stereotyped.
2), bacteriolyze circle method screening
In view of the main component of food refuse is protein, fat, starch, Mierocrystalline cellulose etc., should have or have concurrently the efficient degradation ability of these four big class materials as food refuse degradation function bacterium.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 class 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, be connected to protein, starch, fat, Mierocrystalline cellulose culture medium flat plate respectively, observe of the variation of bacteriolyze loop diameter with incubation time, therefrom filter out the bigger bacterial isolates of bacteriolyze loop diameter, numbering NO.6.The bacteriolyze circle of cultivating on the protein culture medium flat plate of isolate NO.6 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 distilled water; PH 7.0～7.2,121 ℃ of sterilization 20min, and the cooling back is fallen dull and stereotyped.
Starch culture-medium: peptone 10g/L, Zulkovsky starch 2g/L, extractum carnis 5g/L, NaCl 5g/L, agar 20g/L, all the other are distilled water; PH 7.0～7.2,121 ℃ of sterilization 20min, and 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 polyvinyl alcohol (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 distilled 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 distilled 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, lipase, amylase, cellulase
Growth curve is measured: inoculation isolate No.6 bacterium liquid 1mL is in about 30mL beef extract-peptone liquid nutrient medium, 30 ℃, 120r/min shaking culture, get bacterium liquid every 2h and measure light absorption value (ultraviolet-visible pectrophotometer in the 560nm place, Beckmann, DU640), draw growth curve, as shown in Figure 2.
Be the degradation capability of further clear and definite isolate No.6 bacterial strain, measure 4 kinds of extracellular enzyme activity of this bacterial strain, the results are shown in table 14 class materials.
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 distilled water, 7.0,121 ℃ of sterilizations of pH 20min.
Inoculation isolate No.6 logarithmic phase bacterium liquid 0.5mL is in 25mL albumen seed culture fluid, 30 ℃, 120r/min shaking culture, take a sample every specified time, press Folin-phenol reagent method (Cuiping Li et al, 2005) measure the neutral protein enzymic activity: get centrifugation (10000g, 5min, 4 ℃) after supernatant liquor be that crude enzyme liquid 0.5mL adds 0.5mLddH
2O gets 1mL and contains the caseic 0.02M sodium phosphate buffer of 1% (W/V), places 5min at 30 ℃ of constant temperature vibration 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 liquor, 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 such as OliverHLowry (1951) method.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 distilled water, 121 ℃ of sterilization 20min.
Inoculation isolate No.6 logarithmic phase bacterium liquid 0.5mL is in 25mL starch seed nutrient solution, 30 ℃, 120r/min shaking culture, take a sample every specified time, press DNS method (Gashaw Mamo, Amare Gessesse, 1997) measure amylase activity: get the centrifugal (10000g of bacterium liquid, 5min, 4 ℃) supernatant liquor is crude enzyme liquid 0.5mL, add 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 stand-by.No water sodium hydroxide 40g dissolving back moves into the 500mL volumetric flask, cooling back constant volume.Salicylic acid solution moved into the 1000mL volumetric flask and add the sodium hydroxide solution of 325mL, add the dissolving of 15mL glycerol 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 distilled water, 121 ℃ of sterilization 20min.
Inoculation isolate No.6 logarithmic phase bacterium liquid 1mL is in 50mL fatty seed nutrient solution, 30 ℃, 120r/min shaking culture, sampling at regular intervals, then by volumetry (Pignede G et al, 2000) measure lipase activity: get the centrifugal (10000g of bacterium liquid, 5min, 4 ℃) supernatant liquor 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 15mL 95% (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: extractum carnis 1.5g/L, peptone 1.0g/L, CaCO
32.0g/L; Every test tube is distributed into high 7cm deep layer, and puts into 1 of 1 * 6cm filter paper bar, and all the other are distilled water, 121 ℃ of sterilization 20min.
Inoculation isolate No.6 logarithmic phase bacterium liquid 1mL is in 50mL Mierocrystalline cellulose seed culture fluid, 30 ℃, 120r/min shaking culture, sampling at regular intervals, adopt 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, pipette supernatant liquor 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 optical density 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 4 class extracellular enzyme activity of table 1, isolate No.6
4), security is identified
The bacterium liquid of isolate No.6 (the sample title: No. 6 bacterium liquid) and graceful radiation Mucor (Actinomucor elegans) CGMCC No.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.6 (strain number: No.6) 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, Zhejiang University's censorship bacterial classification (strain number: qualification result No.6) is: bacterial strain No.6:Baciluscereus bacillus cereus ".
This bacterial strain has been carried out preservation, bacillus cereus (Bacilus cereus), strain number No.6, be 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.3884;
Embodiment 2: food refuse degraded elimination type microbiobacterial agent III constitutes
Total count: 2.8 * 10
10Cfu/mL
Graceful radiation Mucor (Actinomucor elegans) No.1:1.3 * 10
10Cfu/mL
Bacillus cereus (Bacilus cereus) No.6:1.5 * 10
10Cfu/mL
Embodiment 3: food refuse degraded elimination type microbiobacterial agent III constitutes
Total count: 2.5 * 10
11Cfu/mL
Graceful radiation Mucor (Actinomucor elegans) No.1:1.4 * 10
11Cfu/mL
Bacillus cereus (Bacilus cereus) No.6:1.1 * 10
11Cfu/mL
Embodiment 4: food refuse degraded elimination type microbiobacterial agent III constitutes
Total count: 3.9 * 10
10Cfu/mL
Graceful radiation Mucor (Actinomucor elegans) No.1:4.3 * 10
9Cfu/mL
Bacillus cereus (Bacilus cereus) No.6:3.5 * 10
10Cfu/mL
Embodiment 5: food refuse degraded elimination type microbiobacterial agent III constitutes
Total count: 5.4 * 10
10Cfu/mL
Graceful radiation Mucor (Actinomucor elegans) No.1:5.3 * 10
10Cfu/mL
Bacillus cereus (Bacilus cereus) No.6:1.1 * 10
9Cfu/mL
Embodiment 6: food refuse degraded elimination type microbiobacterial agent III constitutes
Total count: 2.2 * 10
11Cfu/mL
Graceful radiation Mucor (Actinomucor elegans) No.1:4.3 * 10
10Cfu/mL
Bacillus cereus (Bacilus cereus) No.6:1.8 * 10
11Cfu/mL
Embodiment 7: food refuse degraded elimination type microbiobacterial agent III preparation
(1) slant strains activation: the graceful radiation of picking one ring Mucor (Actinomucor elegans) CGMCC No.3881 is inoculated in the PDA solid medium; Picking one ring bacillus cereus (Bacilus cereus) CGMCC No.3884 is inoculated on the beef extract-peptone solid medium, cultivates 2d (48h) under 30 ℃ of conditions, obtains activating thalline.
(2) seed culture: grace radiation Mucor one ring of picking step (1) activation gained is inoculated in the PDA liquid nutrient medium of 100mL, bacillus cereus one ring of activation gained is inoculated in the beef extract-peptone liquid nutrient medium of 100mL, at 30 ℃ of condition 120r/min shaking table shaking culture 78h, make each bacterial strain kind daughter bacteria liquid respectively; That is, respectively the primary seed solution of graceful radiation Mucor and the primary seed solution of bacillus cereus.
(3) fermentation using bacteria is cultivated: 65 times of monosodium glutamate waste liquid dilutions (distilled water that adds 64 parts by volume in the monosodium glutamate waste liquid of 1 parts by volume), and, get nutrient solution I with 1mol KOH 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 nutrient solution I volume 15%, the primary seed solution of bacillus cereus is inoculated in fermentor tank, in 32 ℃, 60r/min, venting pressure 0.6mPa condition fermentation culture 60h, obtaining total count is 2.2 * 10
11The bacterium bacterium liquid of cfu/mL.
(4) mycothallus fermentation culture: 85 times of monosodium glutamate waste liquid dilutions are also used 1mol NaOH or 1mol HCl adjusting pH to 5.5, get nutrient solution II; 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 II volume 13%, the primary seed solution of grace being radiated Mucor is inoculated in fermentor tank, in 30 ℃, 80r/min, venting pressure 0.6mPa condition is carried out fermentation culture 66h, and obtaining total count is 2.8 * 10
11The fungi bacterium liquid of cfu/mL.
(5) the bacterium bacterium liquid of step (3) gained and the fungi bacterium liquid of step (4) gained are mixed by 1: 1 volume ratio, obtain food refuse degraded elimination type microbiobacterial agent III, shown in embodiment 3.
Embodiment 8: food refuse degraded elimination type microbiobacterial agent III preparation
(1) slant strains activation: the graceful radiation of picking one ring Mucor (Actinomucor elegans) CGMCC No.3881 is inoculated in the PDA solid medium; Picking one ring bacillus cereus (Bacilus cereus) CGMCC No.3884 is inoculated on the beef extract-peptone solid medium, cultivates 2d (48h) under 30 ℃ of conditions, obtains activating thalline.
(2) seed culture: the graceful radiation of ring Mucor one ring of picking step (1) activation gained is inoculated in the PDA liquid nutrient medium of 100mL, bacillus cereus one ring of activation gained is inoculated in the beef extract-peptone liquid nutrient medium of 100mL, at 28 ℃ of condition 150r/min shaking table shaking culture 78h, make the kind daughter bacteria liquid of each bacterial strain respectively.That is, respectively the primary seed solution of graceful radiation Mucor and the primary seed solution of bacillus cereus.
(3) fermentation using bacteria is cultivated: 95 times of monosodium glutamate waste liquid dilutions, and regulate pH to 7.2, get nutrient solution I; 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 nutrient solution I volume 10%, the primary seed solution of bacillus cereus is inoculated in fermentor tank, in 30 ℃, 60r/min, venting pressure 0.6mPa condition fermentation culture 48h, obtaining total count is 5.2 * 10
10The bacterium bacterium liquid of cfu/mL.
(4) mycothallus fermentation culture: the monosodium glutamate waste liquid dilution is also regulated pH to 5.5 for 80 times, gets nutrient solution II; 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 II volume 9%, the primary seed solution of grace being radiated Mucor is inoculated in fermentor tank, in 25 ℃, 80r/min, venting pressure 0.6mPa condition is carried out fermentation culture 60h, and obtaining total count is 1.3 * 10
10The fungi bacterium liquid of cfu/mL.
(5) the bacterium bacterium liquid of step (3) gained and the fungi bacterium liquid of step (4) gained are mixed by 2: 1 volume ratio, obtain food refuse degraded elimination type microbiobacterial agent III, shown in embodiment 4.
Embodiment 9: the application of food refuse degraded elimination type microbiobacterial agent III
With wood chip, husk, the peat composed of rotten mosses mixes 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 food refuse degraded elimination type microbiobacterial agent III (2 amount to heavy 4200g) that the foregoing description 7 account for solid support material gross weight 20% makes, place in the garbage degradation handler (the garbage degradation handler that general-purpose plastics machine works in Ningbo City's makes), open and handle operation 2 days, add the 1kg food refuse afterwards every day (by the melon skin shell, compositions such as leftovers leftovers), operation is 3 months continuously, and rubbish volume elimination factor reaches 97.4%.
Embodiment 10: the application of food refuse degraded elimination type microbiobacterial agent III
Wood chip, 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 food refuse degraded elimination type microbiobacterial agent III (2 amount to heavy 3500g) that the foregoing description 8 account for solid support material gross weight 15% makes, place in the garbage degradation handler, open and handle operation 2 days, add the 0.8kg food refuse afterwards every day, move 3 months continuously, rubbish volume elimination factor reaches 96.5%.
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 only is 80.2%.
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 (2)
1. bacillus cereus, it is characterized in that: this bacillus cereus (Bacillus cereus) preservation name is called No.6, 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.3884.
2. food refuse degraded elimination type microbiobacterial agent III, it is characterized in that: this microbial inoculum III is 3.0 * 10
8～1.4 * 10
11Grace radiation Mucor (Actinomucor elegans) the CGMCC No.3881 and 3.0 * 10 of cfu/ml
8～1.8 * 10
11The composite fluid microbial inoculum of the bacillus cereus of cfu/ml (Bacillus cereus) CGMCC No.3884.
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| CN104745504B (en) * | 2015-03-06 | 2018-04-13 | 华南理工大学 | The Bacillus cercus in one plant of deep-sea source and its application in neutral proteinase is produced |
| CN105567604A (en) * | 2016-02-16 | 2016-05-11 | 江苏中通生物科技有限公司 | Compound microorganism bacterium agent and preparation method thereof |
| CN106986694A (en) * | 2017-04-18 | 2017-07-28 | 奥为（天津）环保科技有限公司 | A kind of preparation method containing high concentration potassium solubilizing bacteria organic fertilizer |
| CN107032836A (en) * | 2017-04-18 | 2017-08-11 | 奥为（天津）环保科技有限公司 | A kind of preparation method containing high concentration phosphate solubilizing bacteria organic fertilizer |
| CN110484597A (en) * | 2019-09-02 | 2019-11-22 | 河南大学 | A kind of method of intuitive measurement SOD enzyme activity |
| CN112226388B (en) * | 2020-10-23 | 2021-10-08 | 微米环创生物科技(北京)有限公司 | Novel enzyme-producing species of propionibacteriaceae and application thereof |
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