CN110452831A - A kind of kitchen garbage degradation bacteria and application - Google Patents
A kind of kitchen garbage degradation bacteria and application Download PDFInfo
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- CN110452831A CN110452831A CN201910597467.1A CN201910597467A CN110452831A CN 110452831 A CN110452831 A CN 110452831A CN 201910597467 A CN201910597467 A CN 201910597467A CN 110452831 A CN110452831 A CN 110452831A
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Abstract
The present invention relates to a kind of kitchen garbage degradation bacterias, entitled D1, specific name is bacillus Bacillllus sp., deposit number are as follows: CGMCC No.17861, preservation date: on May 28th, 2019, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.This D1 bacterial strain is to be applied in kitchen garbage for the first time, and the minimizing of single bacterium can reach 41.27%, under the synergistic effect of other three plants of bacterium, can be reduced 80%.
Description
Technical field
The invention belongs to technical field of waste treatment, especially a kind of kitchen garbage degradation bacteria and application.
Background technique
Ocean is the maximum habitat of organism other than land, hundreds of millions of biology of living in marine environment
Body.With the progress and development of the mankind, people start at sea more to be produced and development activities, the mankind at sea obtain
Huge destruction also is caused to marine environment while resource.In recent years, marine kitchen garbage progresses into the visual field of people,
Its processing mode also causes more and more to pay close attention to.It not only to be asked at which in view of processing cost problem and timeliness during reason
Topic, it is contemplated that occupied area problem, therefore a kind of method for searching out suitable disposition ocean platform kitchen garbage becomes to get over
Come more important.
Kitchen garbage is the very extensive concept of an extension, and Priceet etc. thinks food production, transport, distributes and disappear
The discarded part generated in expense belongs to kitchen garbage.Common kitchen garbage refers to residential households, catering industry and enterprise's thing
The food waste and remnants that industry unit dining room generates during food processing and dining, are the main compositions of domestic waste
Ingredient has moisture content high (up to 60%~80%), organic matter accounting big (account for dry weight 95%~98%), saliferous more
The features such as.Its state is generally rendered as solid-liquid mixing, and chemical composition is more complicated, mainly include water, inorganic salts and starch,
The organic compounds such as protein, cellulose, lipid, while also containing macronutrient, such as nitrogen, phosphorus, potassium, calcium.
Because the ingredient and land kitchen garbage ingredient of marine kitchen garbage are substantially similar, in line with nearest principle, so
It is expected that filtering out efficient microbial flora from the kitchen garbage of land to be applied to marine kitchen garbage, reach maximum
Minimizing.
By retrieval, following several patent publication us relevant to present patent application are found:
1, a kind of composite bacteria agent of efficient degradation kitchen garbage and Preparation method and use (CN102533718A), compound bacteria
Agent is made of starch degrading bacterium, grease degrading strain, protein degradation bacterium, cellulose-degrading bacteria and carrier；Preparation: each single bacterium
Strain enrichment culture, screening prepare carrier, then high-quality composite bacteria agent is mixed with carrier；The present invention makes full use of nature a variety of
The conspiracy relation of microorganism establishes the regulation method based on microorganism, reaches and utilize composite bacteria agent coupled fermentation kitchen rubbish
Rubbish carries out accelerating biogas fermentation process, improves traditional zymotic technique, improves biogas yield.
2, a kind of kitchen garbage fast degradation treatment in situ technique (CN109382394A) comprising following steps: sorting
The house refuse and percolate in kitchen garbage are isolated, and is 10~30mm's at diameter by the Domestic garbage crushing isolated
Particulate matter；Particulate matter is stirred, and extrusion dehydration is carried out to the particulate matter after stirring, makes moisture content between 40%~60%；It is logical
It crosses microbial degradation bacterium and generates a variety of enzymes, to carry out biodegrade to the kitchen castoff in the particulate matter after extrusion dehydration and produce
The raw organic fertilizer for recycling；The liquid that the percolate isolated, the broken dilution water generated, extrusion dehydration are generated
It is mixed into mixed liquor, to the processing of mixed liquor electrolysis, air bearing, hydrolysis acidification and biochemical process with water-oil separating mixed liquor, makes oil
The discharged wastewater met the national standard that water separation generates, and the grease that recycling and reusing water-oil separating generates.The present invention can make full use of meal
The surplus value of kitchen rubbish saves transportation cost, while having carried out quick degradation to kitchen garbage in time.
3, a kind of microbial bacterial agent for Chinese kitchen garbage of degrading and preparation method thereof (CN102586112A), including
The component of following mass ratio: protein degradation bacterium: starch degrading bacterium: fat acid decomposition bacterium: cellulose-degrading bacteria=1~2: 1~2: 1
~2: 2~3.Meanwhile the invention also discloses the preparation methods of mentioned microorganism microbial inoculum.The present invention is directed to domestic kitchen garbage
Component is gradually degraded using corresponding microbial bacteria, finally reaches the purpose dissolved completely, avoids traditional land-fill method
Be not thorough, time-consuming, the secondary pollution for burning mode is big, energy consumption is high, is suitble to current low-carbon, environmental protection, energy-efficient requirement.
4, a kind of complex microbial inoculum and its processing method and application (CN103756941A), the complex microbial inoculum
Composite thallus and solid fermentation auxiliary material including decomposing kitchen garbage, in which: the composite thallus for decomposing kitchen garbage includes fiber
10~15 parts of plain degradation bacteria, 15~30 parts of starch degrading bacterium, 15~20 parts of protein degradation bacterium, 15~20 parts of grease degrading strain,
10~15 parts of bacillus subtilis, 5~15 parts of pseudomonas aeruginosa；Solid fermentation ratio of adjuvant is 50~60kg of wheat bran, dregs of beans
15~20kg, 0.4~0.6kg of magnesium sulfate, 0.4~0.6kg of potassium dihydrogen phosphate, 0.1~0.2kg of disodium hydrogen phosphate, glucose 8~
10kg；Inoculum concentration is 0.8%~1% when the composite thallus of the decomposition kitchen garbage is inoculated with solid fermentation auxiliary material, through solid fermentation
Obtain the complex microbial inoculum.The complex micro organism fungicide not only has good point to grease, albumen, fiber substance
Effect is solved, and certain density salinity can be resistant to.When optimal, degradation rate can reach 90% or more.
By comparison, first in the preparation process of mix bacterium agent, the ratio of the ratio of each degradation bacteria and above-mentioned patent is not
Together；Secondly, bacterial strain D1 is to be applied in kitchen garbage for the first time, do not applied before, and the single bacterium minimizing of D1 can reach
41.27%, after other strains are added, synergistic effect is generated with other three plants of bacterium, the minimizing of kitchen garbage is made to reach 80%.
Therefore, there is the difference of essence in present patent application and above-mentioned patent publication us.
Summary of the invention
Place that the purpose of the present invention is to overcome the deficiency in the prior art, provides a kind of kitchen garbage degradation bacteria and application, should
D1 bacterial strain is to be applied in kitchen garbage for the first time, and the minimizing of single bacterium can reach 41.27%, is made in the collaboration of other three plants of bacterium
Under, 80% can be reduced.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of kitchen garbage degradation bacteria, entitled D1, specific name are bacillus Bacillllus sp., deposit number
Are as follows: CGMCC No.17861, preservation date: on May 28th, 2019, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation list
Position: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Moreover, the kitchen garbage bacterial isolation is from kitchen garbage.
Moreover, the basic physical and chemical of the kitchen garbage are as follows:
Moreover, the optimum growth temperature of the kitchen garbage degradation bacteria D1, pH, salinity, best inoculation time are as follows: 30 DEG C,
6,1%, 9~12, enzyme activity are as follows: 2832.4576IU/mL.
Kitchen garbage degradation bacteria as described above in terms of the kitchen garbage degradation in application.
Kitchen garbage degradation bacteria as described above in terms of prepare the kitchen garbage microbial bacterial agent with degradation capability in
Application.
The advantages of present invention obtains and good effect are as follows:
1, D1 bacterial strain of the present invention is to be applied in kitchen garbage for the first time, and the minimizing of single bacterium can reach 41.27%, at it
Under the synergistic effect of his three plants of bacterium, 80% can be reduced.
2, D1 bacterial strain of the present invention is applied when being degraded in kitchen garbage, compared with conventional compostation, in composting process
There is no the generation of foul smell.
3, D1 bacterial strain of the present invention is applied when being degraded in kitchen garbage, it will be apparent that the time of conventional compostation is shortened,
When 18 days or so, good reduction can be reached.
Detailed description of the invention
Fig. 1 is kitchen garbage degradation front and back comparison diagram in the present invention；Wherein, a is the kitchen garbage after degradation, wherein right side
For preceding kitchen garbage of degrading；B is the top view of kitchen garbage；
Fig. 2 is maltose solution canonical plotting in the present invention；
Fig. 3 is diastatic activity measurement result figure in the present invention；
Fig. 4 is tyrosine solution canonical plotting in the present invention；
Fig. 5 is protease activity measurement result figure in the present invention；
Fig. 6 is glucose solution canonical plotting in the present invention；
Fig. 7 is cellulase of the present invention measurement result figure living；
Fig. 8 is lipase activity measurement result figure in the present invention；
Fig. 9 is P-NP standard solution curve graph in the present invention；
Figure 10 is the optimum growth temperature figure of degradation bacteria strains in the present invention；Wherein, (a) is D1, is (b) DB26, (c) is
X2 (d) is Z7；
Figure 11 is the most suitable growth pH figure of degradation bacteria strains in the present invention；Wherein, (a) is D1, (b) is DB26, (c) is X2,
It (d) is Z7；
Figure 12 is the most suitable growth salinity figure of degradation bacteria strains in the present invention；Wherein, (a) is D1, is (b) DB26, (c) is
X2 (d) is Z7；
Figure 13 is the growth curve chart of degradation bacteria strains in the present invention；Wherein, (a) is D1, (b) is DB26, (c) is X2, (d)
For Z7；
Figure 14 is the kitchen garbage degradation effect figure of Mixed Microbes and single bacterium in the present invention.
Specific embodiment
The embodiment of the present invention is described in detail below, it should be noted that the present embodiment is narrative, is not limited
, this does not limit the scope of protection of the present invention.
Raw material used in the present invention is unless otherwise specified conventional commercial product；Used in the present invention
Method is unless otherwise specified the conventional method of this field.
A kind of kitchen garbage degradation bacteria, entitled D1, specific name are bacillus Bacillllus sp., deposit number
Are as follows: CGMCC No.17861, preservation date: on May 28th, 2019, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation list
Position: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
More preferably, the kitchen garbage bacterial isolation is from kitchen garbage.
More preferably, the basic physical and chemical of the kitchen garbage are as follows:
More preferably, the optimum growth temperature of the kitchen garbage degradation bacteria D1, pH, salinity, best inoculation time are as follows: 30
DEG C, 6,1%, 9~12, enzyme activity are as follows: 2832.4576IU/mL.
Kitchen garbage degradation bacteria as described above in terms of the kitchen garbage degradation in application.
Kitchen garbage degradation bacteria as described above in terms of prepare the kitchen garbage microbial bacterial agent with degradation capability in
Application.
Specific operation can be such that
1 materials and methods
1.1 kitchen garbage
Kitchen garbage is derived from the second students' dining hall of University Of Science and Technology Of Tianjin.(kitchen garbage used in sieve bacterium is from food to kitchen garbage
Hall fetch laboratory stack a couple of days kitchen garbage) basic physical and chemical it is as shown in table 1:
1 kitchen garbage physicochemical property of table
1.2 culture mediums and main agents
1.2.1 concentration and separation culture medium
Starch, protein-enriched isolation medium: tryptone 10g, yeast powder 5g, NaCl 10g, agar 20g, distillation
Water 1000mL.
Cellulose-degrading bacteria enriched medium: sodium carboxymethylcellulose 20.0g, Na2HPO42.5g, KH2PO40.5g, pancreas
Peptone 2.5g, yeast extract 0.5g, agar 20g, distilled water 1000mL.
Fat acid decomposition bacterium enriched medium: NaCl 5.0g, peptone 10g, beef extract 5.0g, soya-bean oil 10ml, agar 20g,
Distilled water 1000ml, pH7.2-7.4.
1.2.2 inclined-plane solid medium
Inclined-plane solid medium is LB culture medium: tryptone 10.0g, yeast powder 5.0g, NaCl 10g, agar 20g, is steamed
Distilled water 1000mL.
1.2.3 separation screening culture medium
(1) starch degrading bacterium
Primary dcreening operation culture medium: soluble starch 2.0g, NaCl 5g, tryptone 10.0g, beef extract 3.0g, agar 20.0g,
Distilled water 1000mL.
Secondary screening culture medium: NaCl 10g, tryptone 10g, rice 500g, potato decortication are cut into a 500g and (contain after sterilizing
Water is 65.41%), culture medium 30g+70g to be taken to make shaking table culture liquid.
(2) protein degradation bacterium
Primary dcreening operation culture medium: casein 5.0g, glucose 1.0g, MgSO4·7H2O 0.2g, K2HPO41.0g, KH2PO4
0.5g, agar 20g, distilled water 1000mL.
Secondary screening culture medium: remove the agar in primary dcreening operation culture medium.
(3) fat acid decomposition bacterium
Primary dcreening operation culture medium: NaCl 5.0g, KH2PO40.3g, MgSO4·7H2O 0.1g, K2HPO41.5g, (NH4)2SO4
1.0g, soya-bean oil 5mL, agar 20g, distilled water 1000mL
Secondary screening culture medium: tryptone 1.0g, NH4NO30.2g, K2HPO40.5g, KH2PO40.5g, MgSO4·7H2O
0.1g, soya-bean oil 3mL, distilled water 1000mL
(4) cellulose culture medium
Primary dcreening operation culture medium: KH2PO40.5g, MgSO4·7H2O 0.25g, sodium carboxymethylcellulose 2.0g, congo red
0.2g, agar 20g, distilled water 1000mL.
Secondary screening culture medium: KH2PO41.0g, MgSO4·7H2O 0.3g, NaCl 0.1g, FeCl30.01g, NaNO3
2.5g, sodium carboxymethylcellulose 30.0g, distilled water 1000mL.
1.2.4 amylase, protease, cellulase, lipase seed culture fluid
Amylase seed culture fluid: soluble starch 10g, peptone 5g, K2HPO42g, NaCl 1g, CaCl20.1g,
MgSO4·7H2O 0.1g, distilled water 1000mL
Protease seed culture fluid: K2HPO47g, KH2PO42g, MgSO4·7H2O 0.2g, casein 4g, yeast extract
6g, NaCl 0.5g, glycerol 7g, CaCl 0.067g, distilled water 1000mL
Lipase seed culture fluid: NaNO32g, MgSO4·7H2O 0.3g, NaCl 10g, olive oil 20g, CaCl
0.3g, NH4Cl 0.3g, distilled water 1000mL.
Cellulase seed culture fluid: CMC-Na 5g, peptone 5g, MgSO4·7H2O 0.5g, NaCl 1g, KH2PO4
1g, distilled water 1000mL
Separation, screening and the morphologic observation of 1.3 degradation bacterias
(1) it weighs 1.0g kitchen garbage to be placed in 250mL conical flask, 99mL sterile water is added under aseptic condition, after sealing
Conical flask is placed on 30 DEG C, shake culture 48h in the isothermal vibration incubator of 150r/min.To get to 10 after culture-1
Dilution, take 1mL supernatant to be added in the colorimetric cylinder of 50mL in aseptic superclean bench, add 9mL sterile water, make
Obtain 10-2Dilution, and so on, prepare 10-3、10-4、10-5、10-6、10-7、10-8Etc. a series of dilution of concentration gradients.System
The dilution obtained, which is placed in 4 DEG C of refrigerators, to be saved backup.Illustrate the screening of bacterial strain by taking protein degradation bacterium as an example below and isolates and purifies
Step, cellulose-degrading bacteria and starch degrading bacterium are the same as protein degradation bacterium.
(2) it takes above-mentioned 100 μ L of dilution to cultivate for 24 hours in solids enrichment culture medium and at 37 DEG C, prepares several 50mL
Enrichment culture liquid, then the independent bacterium colony of picking different shape is inoculated in liquid medium respectively from solids enrichment culture medium
In, conical flask is placed on 37 DEG C after sealing, is cultivated in the constant incubator of 150r/min for 24 hours, is then inoculated in primary dcreening operation culture medium
In.
(3) independent bacterium colony is constantly isolated and purified in picking primary dcreening operation culture medium in superclean bench, until obtaining shape
Obtained single thallus, is inoculated in the 150mL conical flask of the secondary screening culture solution equipped with 50mL by the consistent single thallus of state respectively
In, conical flask is placed on 37 DEG C after sealing, is cultivated in the constant incubator of 150r/min and is inoculated in inclined-plane solid medium afterwards for 24 hours
In, it is placed in 4 DEG C of refrigerators and saves.
(4) morphologic observation of bacterial strain is carried out using optical microscopy.
The screening of 1.4 dominant bacterias
1.4.1 transparent circle method
(1) screening technique of starch degrading bacterium
Starch degrading bacterium energy secreting amylase and by the Starch Hydrolysis in plating medium be maltose and glucose, hydrolysis
Starch afterwards, which meets iodine, no longer becomes blue, and occurs colourless transparent circle in periphery of bacterial colonies.Transparent loop diameter (D) and colony diameter
(d) ratio (D/d) can be used for judging that the size of starch degradation bacterial strain enzyme activity, size directly reflect Acarbose concentrations
Opposite height.
(2) screening technique of protein degradation bacterium
Protein degradation bacterium energy extracellular proteinase is to promote the decomposition of protein to make periphery of bacterial colonies transparent circle occur.It is transparent
The ratio (D/d) of loop diameter (D) and colony diameter (d) can be used for judging the size of protein degradation bacterial strain enzyme activity, size
Directly reflect the opposite height of protease concentration.
(3) screening technique of fat acid decomposition bacterium
Fat acid decomposition circle is observed using rhodamine flat band method, and rhodamine flat band method judges principle: rhodamine B energy and rouge
The various possible substrates (mono-, di- olein, oleic acid, enuatrol) of fat enzyme hydrolysis are combined into condensate, the condensate by
Ultraviolet photoactivation generates orange fluorescence.According to whether there is or not crocus, shade and hydrolysis circle sizes to determine whether producing fat
Enzyme and institute's yielding lipase vigor size is tentatively judged.
(4) screening technique of cellulose-degrading bacteria
Cellulose-degrading bacteria energy eccrine fiber element enzyme and by the cellulose degradation in plating medium at the oligomeric of small molecule
Sugar and cellobiose, glucose etc..Carbohydrate and Congo red dye particle shape after hydrolysis is at red precipitate.Therefore Congo red in cellulose
On culture medium, cellulose-degrading bacteria periphery of bacterial colonies can form red hydrolysis circle, hydrolytic circle (D) and colony diameter (d)
Ratio (D/d) can be used for judging the size of cellulose degradation strain enzyme activity, it is dense that size directly reflects cellulase
The opposite height of degree.1.4.2 enzyme activity determination
(1) the enzyme activity determination method of starch degrading bacterium
Amylase activity is measured using DNS method.It takes supernatant of bacteria solution liquid of the 0.5mL after being centrifuged to be added in test tube, sets first
5min is kept the temperature in 40 DEG C of water-baths, it is 1% starch solution (preparatory 40 DEG C of water-baths 5 minutes) that 0.5mL mass concentration, which is then added, is mixed
It is reacted in 40 DEG C of water-baths after even 10 minutes, 2mL DNS reagent is added in each test tube after reaction, then boiling water bath
5min is cooled to room temperature to test tube after the reaction was completed, is settled to 25mL.2mL DNS examination is first added in blank control in test tube
Agent, adding 1mL mass concentration is 1% starch solution, then boiling water bath 5min, the same sample cell of subsequent operation, is pair with blank
According to the measurement light absorption value at 520nm.One amylase activity unit definition is that 1mL enzyme solution is generated per minute needed for 1 μ g maltose
Enzyme amount.Make standard curve with maltose solution.
(2) the enzyme activity determination method of protein degradation bacterium
Neutral protease vigor, step are as follows: sample is after centrifuge is centrifugated are measured by Folin-phenol reagent method
Supernatant, that is, crude enzyme liquid, take 0.1mL enzyme solution be added test tube in, in 40 DEG C of water-baths preheat 3~5min, then to Guan Zhongjia
Entering 0.9mL mass concentration is 2% casein solution, and it is whole that 2mL 0.4M solution of trichloroacetic acid is added after 40 DEG C of heat preservation 20min immediately
It only reacts, is filtered after 15min with filter paper.1mL filtrate is drawn, 5mL 0.4M sodium carbonate and 1mL forint-phenol reagent are sequentially added,
It sufficiently shakes up, reacts 20min in 40 DEG C of water-baths.The method that measurement takes mean value three times is taken in experiment, is first added in blank control
2mL0.4M solution of trichloroacetic acid, adding 1mL mass concentration is 2% casein solution, is filtered after 15min with filter paper, subsequent behaviour
Make same sample cell.It is control with blank, its light absorption value is surveyed at 680nm.One enzyme-activity unit is defined as 1ml enzyme solution water per minute
Solve enzyme amount needed for casein generates 1 μ g tyrosine.Make standard curve with 100 μ g/mL tyrosine concentration gradient of standard.
(3) the enzyme activity determination method of fat acid decomposition bacterium
4mLp-NPP is drawn as substrate solution, then 40 DEG C of preheating 5min of water-bath are added 1mL enzyme solution and react 10min, stand
5mL0.5molL is added-1Trichloroacetic acid, after mixing place 5min terminate reaction, add 5.15mL 0.5molL- 1NaOH adjust pH value to react before unanimously (blank changes enzyme solution into distilled water), at 405nm measurement light absorption value.One enzyme
Unit definition living is the enzyme amount that lml enzyme solution releases 1 μm of ol p-nitrophenol per minute.Take a series of p-NP standard solution in suitable
When concentration gradient, draw one concentration relationship curve of absorbance.
(4) the enzyme activity determination method of cellulose-degrading bacteria
It is measured using the fixed sugared method (DNS) of 3,5 1 dinitrosalicylic acid colorimetrics.Method particularly includes: take bacterium solution to be put into centrifuge tube,
Supernatant 0.5mL is pipetted after centrifugation in test tube, and the citrate buffer solution 0.5mL for being 0.5%CMC-Na containing mass concentration is added,
Then after 50 DEG C of water-bath 30min, add 1.5mL DNS reagent in every test tube immediately, after then boiling 5min, use flowing water immediately
It is cooling, it is settled to 25mL.In blank control, 1.5mL DNS reagent is first added in test tube, 1mL is then added and contains mass concentration
For the citrate buffer solution of 0.5%CMC-Na, boiling water bath 5min, the same sample cell of subsequent operation.It is control with blank, in 540nm
Place's measurement light absorption value.Contrast standard curve again acquires its sugared content.It is per minute that one Cellulase Unit is defined as 1mL enzyme solution
Enzyme amount needed for hydrocellulose generates lmg glucose.Make standard curve with glucose solution.
The identification of 1.5 strains
Bacterium identifies category using the method for 16 sRNA genetic analysis, and fungi is identified using the method for ITS genetic analysis
Belong to.It is extracted respectively with DNA of the DNA extraction kit of fungi and bacterium to the strain filtered out.DNA with extraction is total
Template carries out PCR amplification, the PCR process of bacterium are as follows: the primer used is 27F primer
AGAGTTTGATCMTGGCTCAG, 1492R TACGGYTACCTTGTTACGAC, pcr amplification reaction system such as following table
It is shown:
Reaction process is as follows:
It is then delivered to Beijing Mei Jisangge biological medicine Science and Technology Ltd. and carries out purifying sequencing.The PCR process of fungi are as follows:
Primer: ITS 4 TCCTCCGCTTATTGATATGC of 1 TCCGTAGGTGAACCTGCGG, ITS, pcr amplification reaction system are as follows
Shown in table:
Reaction process is as shown in the table:
It is then delivered to Beijing Mei Jisangge biological medicine Science and Technology Ltd. and carries out purifying sequencing.
The growth conditions of 1.6 dominant bacterias measures
Strain after activation is inoculated into respectively in the 150mL conical flask equipped with 50mL LB culture solution, using single factor test reality
Proved recipe method, if other conditions are the same, respectively change temperature, pH, salinity carry out shaking table culture, cultivate 2d, using than
Turbid method measures light absorption value under the conditions of 600nm with ultraviolet specrophotometer, determines the optimum growing condition of every kind of bacterium.
Temperature, pH, salinity are respectively set as following gradient: (1) temperature: 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C；
(2) pH:4,5,6,7,8,9,10,11,12；(3) salinity: 0.5%, 1%, 2%, 3%, 4%, 5%.
The growth curve of 1.7 dominant bacterias measures
The empirical curve of micropopulation growth rhythm, referred to as growth curve in quantitative description fluid nutrient medium.When few
It measures after purebred unicellular microorganism is inoculated into the fluid nutrient medium of constant volume product, under the conditions ofs suitable temperature, ventilation etc., the bacterium
Group will be ascending, and generation regularly increases.Using turbidimetry for Determination, using incubation time as abscissa, absorbance value is
Ordinate, can draw a smoothed curve, which is known as the growth curve of bacterial strain.In this experiment, take 100 μ L bacterium solutions in being equipped with
In the conical flask of 200mL LB culture solution, 48h is continuously cultivated in 30 DEG C, the constant temperature oscillation box of 150r/min, is existed every 2h
Absorbance is measured under the conditions of 600nm, obtains volume of data, these data connections are arrived at a smooth curve should
The growth curve of bacterial strain.
The preparation of 1.8 complex micro organism fungicides
By four plants of bacterial strains, respective increased logarithmic phase is arrived in culture under the conditions of suitable temperature, salinity and pH, then by meal
Ratio, that is, starch of each nutritional ingredient in the rubbish of kitchen: protein: cellulose: fatty mass ratio is 2.65:1.78:1:1.14,
Draw the different bacterium solutions kitchen garbage microbial bacterial agent admixed together for being configured to that there is efficient degradation ability.
Weight-loss ratio of 1.9 degradation bacterias to kitchen garbage
The experiment for first carrying out single bacterium degradation kitchen garbage, takes every kind of bacterium solution 10mL to put into 50g under conditions of it is most suitable
Kitchen garbage in, hereafter daily be added 50g kitchen garbage, according to mass change measure kitchen garbage weight-loss ratio.Then
It takes composite bacteria liquid 10mL obtained above to be added in 50g kitchen garbage, 50g kitchen garbage is equally hereafter added daily, continuously
Measurement 25 days measures kitchen garbage weight-loss ratio by index of mass change.Measurement in every 2 or 3 days is primary, third day kitchen garbage and
The quality of food waste treater is denoted as m3, the quality of the kitchen garbage of addition is denoted as m, and the quality of food waste treater is denoted as
m0, then the weight-loss ratio of 0~3d can be calculated as follows:
m0The weight of kitchen rubbish kitchen garbage disposer
The kitchen weight that the kitchen rubbish that m- is added adds
mxThe weight of-the n-th day kitchen garbage and food waste treater
N- number of days.
2 results and discussion
The separation and morphologic observation of 2.1 kitchen garbage degradation bacterias
More than ten plants of starch, protein, cellulose, fat acid decomposition bacterium are isolated from kitchen garbage, pass through transparent circle and enzyme
Measurement living has selected 4 plants of best bacterial strains of degradation effect, is 1 plant of starch degrading bacterium respectively, and 1 plant of protein degradation bacterium, cellulose
1 plant of degradation bacteria, 1 plant of fat acid decomposition bacterium, number is D1, DB26, X2, Z7 respectively.By optical microscopy to single bacterium and bacterium colony
Observation, the results are shown in Table 2.
The morphologic observation of each bacterial strain of table 2
2.3 degradation bacteria transparent circle results
2.3.1 starch degrading bacterium
By the way that iodine solution is added dropwise in the culture medium with starch degrading bacterium, it can be observed that being formed in periphery of bacterial colonies transparent
Hydrosphere, starch degradation capacity of water is qualitatively judged according to the size of hydrosphere, and measurement result is as shown in table 3.
3 starch degrading bacterium transparent circle result of table
According to the ratio (D/d) of transparent loop diameter and colony diameter, to be greater than 1.5 as the mark for screening starch degradation bacterial strain
It is quasi-.The dominant bacteria with starch degradation ability filtered out has 5 plants, they are D1, D2, D3, D9, D12 respectively, then further according to
The strongest bacterial strain of enzyme activity further screening degradation capability measured.
2.3.2 protein degradation bacterium
Apparent transparent hydrosphere can be generated around protein degradation bacterium, can be laid eggs according to the size of hydrosphere to qualitatively judge
The size of white enzyme ability, measurement result are as shown in table 4.
4 protein degradation bacterium transparent circle result of table
According to the ratio (D/d) of transparent loop diameter and colony diameter, to be greater than 4.00 as screening protein degradation bacterial strain
Standard.The dominant bacteria with protein degradation ability filtered out has 5 plants, they are DB1, DB16, DB20, DB21, DB26 respectively,
Then further according to the strongest bacterial strain of enzyme activity further screening degradation capability measured.
2.3.3 fat acid decomposition bacterium
Fuzzy haloing can be generated around fat acid decomposition bacterium, can judge that yielding lipase is living according to the size of haloing
The size of power.
5 fat acid decomposition bacterium transparent circle of table
According to the ratio (D/d) of transparent loop diameter and colony diameter, to be greater than 2.00 as screening fat acid decomposition bacterial strain
Standard.The dominant bacteria with fat acid decomposition ability filtered out has 5 plants, they are Z6, Z7, Z16, Z21, Z22 respectively, then again
According to the strongest bacterial strain of enzyme activity further screening degradation capability measured.
2.3.4 cellulose-degrading bacteria
Because a circle can be generated around cellulose-degrading bacteria added with congo red in cellulose culture medium
Red hydrosphere can determine that the size of degradation capability, measurement result are as shown in table 5 with this.
5 cellulose-degrading bacteria transparent circle result of table
According to the ratio (D/d) of transparent loop diameter and colony diameter, to be greater than 1.60 as screen fibre element degradation bacteria strains
Standard.The dominant bacteria with cellulose degradation ability filtered out has 5 plants, they are X2, X3, X7, X12, X13 respectively, so
Afterwards further according to the strongest bacterial strain of enzyme activity further screening degradation capability measured.
The enzyme activity determination result of 2.4 degradation bacteria strains
2.4.1 starch degrading bacterium
The enzyme activity determination result of starch degrading bacterium is as shown in Figures 2 and 3, as seen from Figure 3 with transparent loop diameter with
The ratio of colony diameter is enzyme activity determination to be further carried out, the results show that the producing enzyme energy of D1 in 5 plants of bacterium that standard screen is selected
Power is most strong, has reached 2832.4576IU/mL, so D1 is selected to carry out in next step in next experiment as dominant strain
Research.
2.4.2 protein degradation bacterium
The enzyme activity determination result of protein degradation bacterium is as shown in Figure 4 and Figure 5, as seen from Figure 5, with transparent circle be according into
Row enzyme activity determination shows that the enzymatic productivity of DB26 is most strong in measurement result, has reached 185.2468IU/mL, so next
DB26 is selected to be studied as dominant bacteria in experiment.
2.4.3 cellulose-degrading bacteria
The enzyme activity determination result of cellulose-degrading bacteria is as shown in Figure 6 and Figure 7, from 5 plants of bacterium available in Fig. 7, X2's
Enzyme activity is best, has reached 4.091IU/mL, so X2 is selected to carry out in-depth study as dominant bacteria.
2.4.4 fat acid decomposition bacterium
The enzyme activity determination result of fat acid decomposition bacterium is as shown in Figure 8 and Figure 9, from 5 plants of bacterium available in Fig. 9, the enzyme of Z7
Work is best, has reached 26.98IU/mL, so Z7 is selected to carry out in-depth study as dominant bacteria.
The identification of 2.5 strains
Microbial molecular sequencing is realized by DNA sequencing, determines the category of 4 plants of bacterium, as shown in table 6.
The Molecular Identification result of 6 dominant bacteria of table
By qualification result it is found that D1 is A Yeboduoshi bacillus, X2 is trichoderma reesei, and DB26 is Ah's Mei Shi aspergillus,
Z7 is bacillus subtilis.Qualification result shows that D1, Z7 are bacteriums, and DB26 and X2 are fungies.In the three plants of bacterial strains screened
In, two plants are to apply in kitchen garbage for the first time, i.e. D1 and DB26, and this two plants enzymatic productivity is all very strong, so very
There are research significance and value.
The research of the growth conditions of 2.6 tetra- plants of degradation bacterias of D1, DB26, X2, Z7
2.6.1 the growth temperature of four plants of degradation bacterias
The optimum growth temperature of four plants of degradation bacterias is as shown in Figure 10, can be seen that D1 starch degrading bacterium from Figure 10 (a)
Optimum growth temperature range is 25 DEG C~30 DEG C, from the optimum growth temperature range of DB26 protein degradation bacterium available in (b)
Be 30 DEG C~50 DEG C, from (c) it can be concluded that the optimum growth temperature range of X2 cellulose-degrading bacteria be 20 DEG C~40 DEG C, from
(d) it can be concluded that the optimum growth temperature of Z7 fat acid decomposition bacterium is 20 DEG C in.
2.6.2 the growth pH of four plants of degradation bacterias
The most suitable growth pH of four plants of degradation bacterias is as shown in figure 11, is the research knot of the optimal pH of four plants of bacterial strains shown in Figure 11
Fruit, available D1 starch degrading bacterium, DB26 protein degradation bacterium, X2 cellulose-degrading bacteria, Z7 rouge from (a), (b), (c), (d)
The optimal pH of fat degradation bacteria is respectively 5~9,6~8,5~8,8~10.
2.6.3 the growth salinity of four plants of degradation bacterias
The most suitable growth salinity of four plants of degradation bacterias is as shown in figure 12, and the most suitable growth salt of four plants of bacterial strains has been obtained from Figure 12
Degree condition, the optimal salinity of available D1, DB26, X2, Z7 are all 0.5%~1%.
The growth curve of 2.7 4 plants of degradation bacterias
The growth curve of four plants of degradation bacterias is as shown in figure 13, from the result of the growth curve measured it is found that the life of four plants of bacterium
Long curve is substantially all divided into 3 processes: lag phase, increased logarithmic phase, stationary phase, because of the reason of measuring the time, there is no see
Measure the decline phase of bacterial strain.The increased logarithmic phase of D1 appears in 9~18h as we can see from the figure, and the increased logarithmic phase of DB26 goes out
The increased logarithmic phase of present 5~15h, X2 appear in 9~15h, and the increased logarithmic phase of Z7 appears in 5~14h.Increased logarithmic phase is
Thalli growth is metabolized stage the most vigorous, and the best period by seed culture medium large-scale inoculation to fermentation medium.
Therefore further progress research, obtains the best inoculation time of four plants of bacterium, as shown in the table:
7 four plants of best inoculation times of bacterial strain of table
Degradation effect of 2.8 degradation bacteria strains to kitchen garbage
As a result as shown in Fig. 1 and Figure 14, it will be seen from figure 14 that Mixed Microbes are obvious to the degradation effect of kitchen garbage
Higher than the degradation effect of other three plants of single bacteriums, reach 80% in the 25th day period.In 4 plants of single bacteriums, the drop of starch
It is best to solve effect, 41.27%, followed by protein degradation bacterium were reached in the 25th day period, is 29.33%, followed by rouge
Fat degradation bacteria 15.34%, worst is cellulose-degrading bacteria, only 10.59%.
3 conclusions
4 plants of bacterial strains with stronger degradation effect are filtered out from kitchen garbage, and the mirror of molecular level has been carried out to it
It is fixed, it is D1 A Yeboduoshi bacillus, DB26 Ah Mei Shi Aspergillus, X2 trichoderma reesei, Z7 bacillus subtilis respectively.It obtains
The optimum growth temperature of D1, pH, salinity, best inoculation time are to be respectively: 30 DEG C, 6,1%, 9~12；The most suitable growth of DB26
Temperature, pH, salinity, best inoculation time are respectively: 40 DEG C, 7,1%, 5~8；It is the optimum growth temperature of X2, pH, salinity, best
Inoculation time is: 30 DEG C, 8,1%, the optimum growth temperature of 10~13, Z7, pH, salinity, best inoculation time difference
Are as follows: 20 DEG C, 8,1%, 6~10.And the enzyme activity for measuring 4 plants of bacterium be respectively as follows: 2832.4576IU/mL, 185.2468IU/mL,
4.09IU/mL,26.98IU/mL.By experiment, obtains 4 plants of Mixed Microbes and 80% had been reached to the degradation rate of kitchen garbage in 25 days.
Although disclosing the embodiment of the present invention for the purpose of illustration, it will be appreciated by those skilled in the art that: not
Be detached from the present invention and spirit and scope of the appended claims in, various substitutions, changes and modifications be all it is possible, therefore, this
The range of invention is not limited to the embodiment and attached drawing disclosure of that.
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Claims (6)
1. a kind of kitchen garbage degradation bacteria, it is characterised in that: entitled D1, specific name are bacillus Bacillllus
Sp., deposit number are as follows: CGMCC No.17861, preservation date: on May 28th, 2019, BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. kitchen garbage degradation bacteria according to claim 1, it is characterised in that: the kitchen garbage bacterial isolation is eaten certainly
In the rubbish of kitchen.
3. kitchen garbage degradation bacteria according to claim 2, it is characterised in that: the basic physical and chemical of the kitchen garbage
Are as follows:
4. kitchen garbage degradation bacteria according to claim 1, it is characterised in that: the kitchen garbage degradation bacteria D1's is most suitable
Growth temperature, pH, salinity, best inoculation time are as follows: 30 DEG C, 6,1%, 9~12, enzyme activity are as follows: 2832.4576IU/mL.
5. as the described in any item kitchen garbage degradation bacterias of Claims 1-4 in terms of the kitchen garbage degradation in application.
6. as the described in any item kitchen garbage degradation bacterias of Claims 1-4 have the kitchen garbage of degradation capability micro- in preparation
Application in terms of bacteria agent.
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