CN108396000A - A kind of preparation method of low temperature resistant garbage disposal composite bacteria agent - Google Patents
A kind of preparation method of low temperature resistant garbage disposal composite bacteria agent Download PDFInfo
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- CN108396000A CN108396000A CN201810130093.8A CN201810130093A CN108396000A CN 108396000 A CN108396000 A CN 108396000A CN 201810130093 A CN201810130093 A CN 201810130093A CN 108396000 A CN108396000 A CN 108396000A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
Abstract
The invention belongs to microorganisms technical fields, and in particular to a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent includes the following steps：Strain acquisition, strain separating, strain enrichment and bacterial strain domestication, the preparation of bacterial strain primary dcreening operation, domestication by low temperature and composite bacteria agent；For the composite bacteria agent that method using the present invention is prepared under 10 DEG C of low-temperature conditions, the growing state of microbial inoculum is superior to commercially available common composite bacteria agent to ammonia nitrogen, the removal rate of COD and cellulose producing enzyme vigor in landfill leachate；It is also shown in outdoor simulating reactor test result, rubbish central temperature rises faster, and ammonia nitrogen, the concentration of COD are lower in landfill leachate, after reaction the sedimentation rate higher of rubbish.The solid refuse that the composite bacteria agent of the present invention is suitable for the Northwest is handled, and is had a good application prospect.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of preparation side of low temperature resistant garbage disposal composite bacteria agent
Method.
Background technology
The processing mode of northwest China area house refuse is based on sanitary landfills, recently as industrialization and urbanization
The problem of yield of the iterative method of process, house refuse rises year by year, refuse landfill also gradually highlights, such as landfill yard
The stabilisation time is long, persistently generate the high landfill leachate of pollution intensity, the long-term of foul gas distributes.The study found that
In the stabilization procedures of refuse landfill, the effect of microorganism is most important, is the main decomposition person of organic substance in field, but
Under the influence of landfill yard complex environment, the indigenous microorganism breakdown solid waste inefficiency in field is relied solely on, rubbish is caused
Degradation speed is slow, pollution concentration is high, Landfill Stabilization is fixed time length.The biological reinforced skill gradually developed by the eighties in last century
Art can enhance the degradation to various hard-degraded substances, improve original life that is, by microorganism of the introducing with specific degradation function
The efficiency of object system for handling.Using this technology, forefathers have carried out beneficial exploration in terms of the processing of solid refuse, by
The peculiar microorganism with degradation COD, cellulose is launched in refuse landfill, is improved the decomposition efficiency of solid refuse, is accelerated
The stabilisation process of landfill yard.But it finds under study for action, microorganism is closely related to the decomposition of rubbish and temperature, by simulation reaction
The temperature of device is controlled at 40 DEG C, and the efficiency of microorganism can achieve the effect that best, and the temperature at rubbish center is more up to 70
℃；But by temperature control at 20 DEG C, the decomposition rate of rubbish reduces by 50% or more compared with 40 DEG C of groups；And it is micro- at lower temperatures
Biology is then rarely reported the effect of solid refuse.The Northwest's located at inland area, dry cold, winter is very long, in 1 year
Average daily maximum temperature reaching in month 8 months less than 20 DEG C, wherein ice period is even more at 3 months or more, and microorganism is in low temperature shape
Slow-growing or even stagnation, will substantially reduce the decomposition efficiency of rubbish, and limit common micro-organisms microbial inoculum in this area under state
The application of solid refuse processing.
For this present situation, seminar carries out domestication by low temperature by screening efficient garbage decomposition microbial inoculum extensively, obtains one
The low temperature resistant garbage disposal composite bacteria agent of kind, under 10 DEG C of low-temperature conditions, the growing state of the microbial inoculum, to ammonia in landfill leachate
Nitrogen, the removal rate of COD and cellulose producing enzyme vigor are superior to commercially available common composite bacteria agent；In outdoor simulating reactor test result
It also shows, rubbish central temperature rises faster, and ammonia nitrogen, the concentration of COD are lower in landfill leachate, and rubbish is heavy after reaction
Drop rate higher.The composite bacteria agent is applicable to the solid refuse processing of the Northwest, has a good application prospect.
Invention content
To solve the above-mentioned problems, the present invention provides a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent, this hair
Bright low temperature resistant garbage disposal composite bacteria agent, under 10 DEG C of low-temperature conditions, the growing state of microbial inoculum, to ammonia in landfill leachate
Nitrogen, the removal rate of COD and cellulose producing enzyme vigor are superior to commercially available common composite bacteria agent；In outdoor simulating reactor test result
It also shows, rubbish central temperature rises faster, and ammonia nitrogen, the concentration of COD are lower in landfill leachate, and rubbish is heavy after reaction
Drop rate higher.
The technical solution that the present invention solves above-mentioned technical problem is as follows：A kind of preparation of low temperature resistant garbage disposal composite bacteria agent
Method includes the following steps：
(1) strain acquires
Rubbish sample is mixed with sterile water, obtains sample diluting liquid；
(2) strain separating
Above-mentioned sample diluting liquid 300~900 μ L in total are taken, three equal parts are divided, are seeded to the training of beef extract-peptone tablet respectively
Base, Gause I plating medium and Cha Shi plating mediums are supported, then by the beef extract-peptone plating medium after inoculation
It is positioned over growth cabinet, 37 DEG C are cultivated 1-2 days, and the Gause I training tablet after inoculation is supported base and Cha Shi plating mediums are put
It is placed in mold incubator, 28 DEG C are cultivated 3-4 days, and bacterium, actinomyces and mould are respectively obtained；Respectively to above-mentioned bacterium, actinomyces
And mould, bacterium colony is purified using plate streak, until obtaining single pure bacterium colony；
(3) strain enrichment and bacterial strain domestication
Bacterial liquid culture medium, actinomyces fluid nutrient medium and mould are seeded to by obtained single pure bacterium colony is corresponding respectively
Bacteria liquid culture medium, at 28 DEG C, 130r/min shaking table shake cultures will be above-mentioned after bacterium solution OD600nm reaches 0.7 ± 0.05
Bacterium solution is seeded in the fluid nutrient medium of the 20wt% containing landfill leachate and cultivates 4 days respectively, is then seeded to containing landfill leachate
It is cultivated 4 days in the fluid nutrient medium of 40wt%, inoculates into the fluid nutrient medium of the 60wt% containing landfill leachate and cultivate 4 days,
Then it is seeded in the fluid nutrient medium of the 80wt% containing landfill leachate and cultivates 4 days, be finally seeded to containing landfill leachate
It is cultivated 4 days in the fluid nutrient medium of 100wt%, obtains tolerable bacterium, actinomyces and mould；
(4) bacterial strain primary dcreening operation
Tolerable bacterium, actinomyces and mould after domestication is trained in bacterial liquid culture medium, unwrapping wire bacteria liquid respectively
It, respectively will be above-mentioned after enrichment culture to bacterium solution absorbance value (OD600nm) is 0.7 ± 0.05 in foster base and mould fluid nutrient medium
The bacterium solution of bacterium, actinomyces and mould is 1 by volume:20 ratio is seeded to landfill leachate, cultivates 48h, obtaining can
Remove bacterium, actinomyces and the mould of ammonia nitrogen and COD；
(6) domestication by low temperature
The above-mentioned bacterium that can remove ammonia nitrogen and COD, actinomyces and mould and cellulose-degrading bacteria are seeded to ox respectively
Meat extract peptone plating medium, Gause I plating medium, Cha Shi plating mediums and carboxymethyl cellulose tablet culture
Base carries out domestication by low temperature using the method for gradient cooling to bacterial strain, and 28 DEG C first are cultivated 2-3 days, and 3 DEG C of cultures of cultivation temperature are reduced
5-7 days, above-mentioned cooling culture operation is repeated, when temperature is reduced to 10 DEG C, obtains capable of removing the low temperature resistant of ammonia nitrogen and COD
Bacterium, the low temperature resistant actinomyces that ammonia nitrogen and COD can be removed, the low temperature resistant mould that ammonia nitrogen and COD can be removed and low temperature resistant
Cellulose-degrading bacteria；The low temperature resistant bacterium of ammonia nitrogen and COD will be removed with the bacterium in Low temperature resistant cellulose degradation bacteria by body
Product ratio 1:1 is mixed to get low temperature resistant bacterium；The low temperature resistant actinomyces of ammonia nitrogen and COD will be removed as low temperature resistant actinomyces；
It is 1 that the mould in the low temperature resistant mould and Low temperature resistant cellulose degradation bacteria of ammonia nitrogen and COD, which will be removed, by volume:1 mixing,
Obtain low temperature resistant mould；
(7) preparation of composite bacteria agent
Above-mentioned low temperature resistant bacterium, actinomyces and mould are seeded to bacterial liquid culture medium, actinomyces Liquid Culture respectively
Activation culture in base and mould fluid nutrient medium carries out compatibility by the volume fraction of bacterium 90%, actinomyces 3% and mould 7%,
Obtain composite bacteria agent.
On the basis of said program, the present invention can also be improved as follows.
Preferably, a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent, the rubbish sample described in step (1) are rubbish
Rubbish field winter freezes the following rubbish of layer, landfill leachate and by leachate contamination pedotheque.
Preferably, a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent, step (1) described sample diluting liquid it is dense
Degree can be 10-1、10-2、10-3、10-4、10-5、10-6。
Preferably, a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent, is counted in parts by weight, and step (2) is described
Beef extract-peptone plating medium is by 10.0 parts of peptone, 3.0 parts of beef extract, NaCl0.5 parts, 20 parts of agar and distilled water
1000 parts of compositions, pH value are adjusted to 7.0-7.2, and it is standby to be down flat plate in 121 DEG C, high pressure sterilization 30min, when being cooled to 60-70 DEG C
With；
The Gause I plating medium is by 20 parts of soluble starch, KN031 part, K2HP040.5 part, NaCI O.5
Part, MgS04·7H20 0.5 parts, FeS041000 parts of 0.0l parts, 20 parts of agar and distilled water, pH value is adjusted to 7.2-7.4, in
121 DEG C, high pressure sterilization 30min, it is spare to be down flat plate when being cooled to 60-70 DEG C；
The Cha Shi plating mediums are by 30 parts of glucose, NaNO32.0 parts, 0.5 part of KCl, K2HP041.0 parts,
MgS04·7H20 0.5 parts, FeS041000 parts of 0.01 part, 20 parts of agar and distilled water, pH value is adjusted to 7.2-7.4, in 121
DEG C, it is spare to be down flat plate when being cooled to 60-70 DEG C by high pressure sterilization 30min.
Preferably, a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent, step (2) described bacterium are bacillus
Category, Bifidobacterium, Curtobacterium, pseudomonas and molten Bacillus composition；Mould is cladosporium sp category, Trichoderma
The composition of category and Eurotium；Actinomyces are streptomyces.
Preferably, a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent, by weight score meter, step (4) and step
Suddenly (7) described bacterial liquid culture medium is by 10.0 parts of peptone, 3.0 parts of beef extract, 0.5 part of NaCl and distilled water 1000mL groups
At, pH value is adjusted to 7.0-7.2, in 121 DEG C, high pressure sterilization 30min, be cooled to 60-70 DEG C it is spare；
The actinomyces fluid nutrient medium is by 20 parts of soluble starch, KN031 part, K2HP040.5 part, NaCI O.5 part,
MgS04·7H20 0.5 parts, FeS040.0l parts and 1000 parts of distilled water, pH value is adjusted to 7.2-7.4, and in 121 DEG C, high pressure is gone out
Bacterium 30min, be cooled to 60-70 DEG C it is spare；
The mould fluid nutrient medium is by 30 parts of glucose, NaNO32.0 parts, 0.5 part of KCl, K2HP041.0 parts,
MgS04·7H20 0.5 parts, FeS040.01 part and 1000 parts of distilled water, pH value is adjusted to 7.2-7.4, and in 121 DEG C, high pressure is gone out
Bacterium 30min, be cooled to 60-70 DEG C it is spare.
Preferably, a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent, the cellulose-degrading bacteria described in step (6)
It is 1 by volume for pseudomonad, cladosporium sp and Trichoderma:1:1 mixes.
Preferably, a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent, the carboxymethyl cellulose described in step (6)
Plating medium is by 15 parts of CMC-Na, NH4N031 part, 1 part of yeast extract, MgS04·7H20 0.5 parts, KH2P041 part, fine jade
1000 parts of 20 parts of fat and distilled water, pH value is adjusted to 7.0-7.2, and in 121 DEG C, high pressure sterilization 30min is cooled to 60-70 DEG C
When to be down flat plate spare.
Beneficial effects of the present invention：The low temperature resistant garbage disposal composite bacteria agent of the present invention, under 10 DEG C of low-temperature conditions, bacterium
The growing state of agent is superior to ammonia nitrogen, the removal rate of COD and cellulose producing enzyme vigor in landfill leachate commercially available common compound
Microbial inoculum；It is also shown in outdoor simulating reactor test result, rubbish central temperature rises faster, ammonia nitrogen, COD in landfill leachate
Concentration it is lower, the sedimentation rate higher of rubbish after reaction.
Description of the drawings
The upgrowth situation that each microbial inoculums of Fig. 1 are cultivated at 10 DEG C；
Each microbial inoculums of Fig. 2 cultivated at 10 DEG C under to the treatment effect of garbage filter ammonia nitrogen；
Each microbial inoculums of Fig. 3 are in 10 DEG C for the treatment of effects to COD of waste leachate；
The yield of cellulase that each microbial inoculums of Fig. 4 are cultivated at 10 DEG C；
Fig. 5 analogue reactor garbage loading embeading central temperatures；
Effect of each microbial inoculums of Fig. 6 to garbage filter ammonia nitrogen in analogue reactor；
Effect of each microbial inoculums of Fig. 7 to COD of waste leachate in analogue reactor.
Specific implementation mode
The principles and features of the present invention are described below, and the given examples are served only to explain the present invention, is not intended to limit
Determine the scope of the present invention.
Culture medium used in the present embodiment is as follows：
Beef extract-peptone plating medium：Peptone 10.0g, beef extract 3.0g, NaCl 5g, agar 20g, distilled water
1000mL adjusts pH 7.0-7.2, and after above-mentioned culture medium prepares, in 121 DEG C, high pressure sterilization 30min is cooled to 60-70 DEG C
When to be down flat plate spare.
No. I training tablet of Gao Shi supports base：Soluble starch 20g, KN031g, K2HP040.5g, NaCI O.5g, MgS04·
7H20 0.5g, FeS040.0lg, agar 20g, distilled water 1000mL, pH value is adjusted to 7.2-7.4, after above-mentioned culture medium prepares,
It is spare it to be down flat plate in 121 DEG C, high pressure sterilization 30min, when being cooled to 60-70 DEG C.
Cha Shi trains tablet and supports base：Glucose 30g, NaNO32.0g, KCl 0.5g, K2HP041.0g, MgS04·7H20
0.5g, FeS040.01g, distilled water 1000mL, pH value are adjusted to 7.2-7.4.After above-mentioned culture medium prepares, in 121 DEG C, high pressure
Sterilize 30min, and it is spare to be down flat plate when being cooled to 60-70 DEG C.
Carboxymethyl cellulose plating medium is by CMC-Na 15g, NH4N031g, yeast extract 1g, MgS04·7H20
0.5g、KH2P041g, agar 20g and distilled water 1000ml, pH value are adjusted to 7.0-7.2, in 121 DEG C, high pressure sterilization 30min,
It is spare it to be down flat plate when being cooled to 60-70 DEG C.
Bacterial liquid culture medium：Peptone 10.0g, beef extract 3.0g, NaCl 0.5g and distilled water 1000mL compositions, pH
Value is adjusted to 7.0-7.2, in 121 DEG C, high pressure sterilization 30min, be cooled to 60-70 DEG C it is spare.
Actinomyces fluid nutrient medium：Soluble starch 20g, KN03 1g、K2HP04 0.5g、NaCI O.5g、MgS04·
7H20 0.5g、FeS040.0lg and distilled water 1000mL, pH value are adjusted to 7.2-7.4, and in 121 DEG C, high pressure sterilization 30min is cold
But spare to 60-70 DEG C.
Mould fluid nutrient medium：Glucose 30g, NaNO3 2.0g、KCl 0.5g、K2HP04 1.0g、MgS04·7H20
0.5g、FeS040.01g and distilled water 1000mL, pH value are adjusted to 7.2-7.4, and in 121 DEG C, high pressure sterilization 30min is cooled to
60-70 DEG C spare.
The commercially available microbial inoculum A used in the embodiment of the present invention is the EM bacteria agent of Ai Mule environment friendly biologicals Technology Co., Ltd.；This
Commercially available microbial inoculum B used in inventive embodiments is the garbage disposal microbial inoculum of Jiangsu green section's biology Co., Ltd.
Embodiment 1
A kind of preparation of low temperature resistant garbage disposal composite bacteria agent, includes the following steps：
(1) strain acquires
Acquiring Qinghai soot winter freezes the following rubbish of layer, landfill leachate and by totally 9 parts of leachate contamination pedotheque,
Every part of 500-1000g, takes 5g respectively from every part of sample, is transferred in the triangular flask equipped with 50mL sterile waters and vibrates 5min, system
At 10-1Sample diluting liquid reuses aqua sterilisa dilution, configures in order 10-2、10-3、10-4、10-5、10-6Concentration samples dilution,
Every part of sample configures above-mentioned 6 concentration, and 6 × 9 parts of dilutions are obtained；
(2) strain separating
Under sterile working, takes 600 μ L to divide three equal parts from above-mentioned every part of dilution, be seeded to beef extract respectively
Peptone plating medium, Gause I plating medium, Cha Shi plating mediums and carboxymethyl cellulose plating medium, and
One group of blank control is respectively set, to check whether to be contaminated；Above-mentioned beef extract-peptone plating medium is positioned over manually
Climate box, 37 DEG C are cultivated 1.5 days；Gause I plating medium and Cha Shi plating mediums are positioned over mold incubator, 28 DEG C
Culture 3 days；It is counted after tablet grows bacterium colony, and according to the form of bacterium colony, picking stalwartness, full bacterium colony are drawn using tablet
25 plants of bacterium, actinomyces 8 are obtained until obtaining the single pure bacterium colony of every class bacterium in collimation method purifying bacterial strain after being separately cultured
Strain, 10 plants of mould.
(4) strain enrichment and bacterial strain domestication
Isolated bacterium, actinomyces and mould are seeded to bacterial liquid culture medium, actinomyces Liquid Culture respectively
Base and mould fluid nutrient medium enrichment culture monitor strain growth situation using spectrophotometer, wait for bacterium solution absorbance value
(OD600nm) reaching 0.7 ± 0.05, strain growth to exponential phase is gone in the culture medium containing landfill leachate, first,
Above-mentioned bacterium solution is seeded to respectively in the fluid nutrient medium of the 20wt% containing landfill leachate and is cultivated 4 days, is then seeded to containing rubbish
It is cultivated 4 days in the fluid nutrient medium of percolate 40wt%, inoculates into the fluid nutrient medium of the 60wt% containing landfill leachate and train
It supports 4 days, is then seeded in the fluid nutrient medium of the 80wt% containing landfill leachate and cultivates 4 days, be finally seeded to containing garbage filter
It is cultivated 4 days in the fluid nutrient medium of liquid 100wt%, obtains tolerable bacterium, actinomyces and mould；
(5) strain primary dcreening operation
Bacterium, actinomyces and mould after domestication is cultivated in liquid medium to bacterium solution absorbance value (OD600nm)
After 0.7 ± 0.05, by above-mentioned bacterium solution by volume 1:20 ratio is inoculated in landfill leachate, after 48h measure ammonia nitrogen and
COD removal rates choose ammonia nitrogen removal frank in 20% or more bacterial strain as ammonia nitrogen removal bacterium, and COD removal rates are 20% or more
Bacterial strain is that COD goes degerming, is obtained while having 11 plants of the bacterium of ammonia nitrogen and COD removal effects, 3 plants of actinomyces, 8 plants of mould,
Individually 4 plants of the bacterium with ammonia nitrogen removal effect；
(6) domestication by low temperature
The above-mentioned bacterium that can remove ammonia nitrogen and COD, actinomyces and mould and cellulose-degrading bacteria are seeded to ox respectively
Meat extract peptone plating medium, Gause I plating medium, Cha Shi plating mediums and carboxymethyl cellulose tablet culture
Base carries out domestication by low temperature using the method for gradient cooling to bacterial strain, and 28 DEG C first are cultivated 2-3 days, and list is grown on tablet to be seeded
After bacterium colony, reduces by 3 DEG C of cultivation temperature and cultivate 5-7 days, daily to observe strain growth situation, tablet to be seeded grows macroscopic
Continue scribing line culture after single bacterium colony, above-mentioned cooling culture operation is repeated and chooses when temperature is reduced to 10 DEG C and remains to growth
Bacterial strain obtains that the low temperature resistant bacterium of ammonia nitrogen and COD can be removed, the low temperature resistant actinomyces that can remove ammonia nitrogen and COD, can go
Except the low temperature resistant mould and low temperature resistant cellulose-degrading bacteria of ammonia nitrogen and COD；Ammonia nitrogen and the low temperature resistant bacterium of COD will be removed
With the bacterium in Low temperature resistant cellulose degradation bacteria by volume 1:1 is mixed to get low temperature resistant bacterium；Ammonia nitrogen and COD will be removed
Low temperature resistant actinomyces as low temperature resistant actinomyces；Low temperature resistant mould and the Low temperature resistant cellulose of ammonia nitrogen and COD will be removed
Mould in degradation bacteria is 1 by volume:1 mixing, obtains low temperature resistant mould；10 plants of bacterium is obtained after domestication by low temperature, puts
1 plant of line bacterium, 6 plants of mould；
(7) preparation of composite bacteria agent
To make Mixed Microbes achieve the effect that mutually to cooperate with, the antagonism between bacterial strain is checked using Odontothrips loti, by 1:1
Ratio obtains bacterium without mutual antagonism, actinomyces and mould respectively by the respective compatibility of above-mentioned bacterium, actinomyces and mould
Bacterium.By all bacterial strains cultivate in liquid medium to bacterium solution absorbance value (OD600nm) be 0.7 ± 0.05 when, by 1:1 ratio
Example bacterium mixed bacteria liquid, actinomyces bacterium solution and mould mixed bacteria liquid is respectively prepared, using orthogonal test determine bacterium, actinomyces and
The best mixed proportion of mould.As a result the mixed proportion for being shown in bacterium, actinomyces and mould is 9:0.5:When 1, Mixed Microbes
The bacterium solution absorbance rising of agent is most fast, therefore takes bacterium 90%, actinomyces 3%, mould 7% that compound bacteria is made by volume fraction proportioning
Agent.
Embodiment 2
To landfill leachate treatment and cellulose decomposition effect under low-temperature condition：
1, under low-temperature condition composite bacteria agent upgrowth situation
To compare domestication by low temperature composite bacteria agent effect, it is respectively city that we, which have purchased two kinds of commercially available garbage disposal composite bacteria agents,
Microbial inoculum A and commercially available microbial inoculum B are sold, each microbial inoculum is inoculated in beef extract-peptone fluid nutrient medium, shaken cultivation to bacteria concentration is
3.11-3.78×108When a/ml, it is inoculated in the conical flask of the enriched medium containing 100ml by volume fraction 10%, in 10
DEG C, 130r/min shaking table shaken cultivations measure bacterium solution absorbance value per 4h, monitor composite bacteria agent growing state, and draw each group
The growth curve of composite bacteria agent.The result is shown in Figure 1.
The results show that under 10 DEG C of low-temperature condition, the composite bacteria agent growing state through domestication by low temperature is consistently higher than two kinds
Commercially available composite bacteria agent, domestication by low temperature group enters after 48h to be stablized growth period, and commercially available composite bacteria agent enters surely at the rears 96h
Determine growth period, and microbial inoculum maximum growth concentration is compared with low temperature group difference low 32.2% and 44.8%.
2, removal situation of the composite bacteria agent to garbage filter ammonia nitrogen and COD under low-temperature condition
Each microbial inoculum is inoculated in beef extract-peptone fluid nutrient medium, shaken cultivation to bacteria concentration be 3.23-3.97 ×
108When a/ml, it is inoculated in landfill leachate by volume fraction 10%, in 10 DEG C, 130r/min shaking table shaken cultivations, after 48h
Each group is measured to the removal rate of diafiltration ammonia nitrogen and COD, as a result sees Fig. 2 and Fig. 3.
The results show that under 10 DEG C of low-temperature condition, the composite bacteria agent through domestication by low temperature goes diafiltration ammonia nitrogen and COD
Except efficiency is above two kinds of commercially available composite bacteria agents, domestication by low temperature composite bacteria agent is to percolate ammonia nitrogen removal frank compared with two kinds of commercially available microbial inoculums
Difference high 25.6% and 20.8% distinguishes high 30.3% and 31.7% to the removal rate of COD.
3, under low-temperature condition composite bacteria agent cellulolytic enzyme yield
Each microbial inoculum is inoculated in beef extract-peptone fluid nutrient medium, in 10 DEG C, 130r/min shaking tables shaken cultivation 5
It, takes supernatant after taking bacterium solution 6000r/min to centrifuge, measures the yield of cellulase of each group, as a result see Fig. 4.
The results show that after being cultivated 5 days at 10 DEG C, the Filter paperlyase of domestication by low temperature composite bacteria agent, carboxymethylcelluloenzyme enzyme and micro-
Crystalline cellulose enzyme activity is above two kinds of 30% or more commercially available microbial inoculums.
Embodiment 3
Outdoor simulating tests the treatment effect of composite bacteria agent：
Using environment in bioreactor simulation refuse landfill, filling house refuse is in bioreactor, by each group
Composite bacteria agent culture to bacteria concentration is 3.55-5.36 × 108When a/ml, added in landfill waste by volume fraction 10%, it is right
According to group isometric aseptic culture medium of addition.Ammonia nitrogen and COD are dense in dynamic monitoring rubbish central temperature, percolate during the test
Degree and rubbish sedimentation rate.
It is tested November, is terminated May next year, minimum -2 DEG C of outdoor average daily temperature, up to 16 DEG C, average out to
5.8℃.As shown in Fig. 5 results, test the starting stage, the central temperature of each group rubbish is 15 DEG C or so, after be gradually increasing, add
The test group rubbish central temperature of composite bacteria agent has been added to be above control group, wherein being added to the upper of domestication by low temperature composite bacteria agent group
Increasing degree degree highest, rubbish central temperature has been more than 40 DEG C after testing 20 days, and reaches 51 DEG C of maximum temperature at the 42nd day；And
It adds commercially available composite bacteria agent group and reaches 39 DEG C of maximum temperature at 50 days rears of experiment, domestication by low temperature is consistently lower than during experiment
Group.At low ambient temperatures, the startup of domestication by low temperature group microbial inoculum faster, and can keep higher activity for prompt.
Fig. 6 and Fig. 7, each group ammonia nitrogen are shown in the monitoring result of ammonia nitrogen in landfill leachate and COD concentration during the test
The process for first rising and declining afterwards is experienced with COD concentration, domestication by low temperature group is superior to other to the removal effect of ammonia nitrogen and COD
Group, at the 18th day of experiment and the 24th day, the removal rate of ammonia nitrogen and COD was more than 50%, and commercially available microbial inoculum group at the 54th day and
The removal rate of 66 days ammonia nitrogens and COD just reach 50%, and are below domestication by low temperature group in the whole removal rate of reaction.Reason may be
At low ambient temperatures, more preferably due to the growing state of domestication by low temperature group microbial inoculum, therefore to the removal of ammonia nitrogen in landfill leachate and COD
Rate higher.
The monitoring of rubbish sedimentation rate is found in experiment, the sedimentation rate of domestication by low temperature microbial inoculum group rubbish faster, in experiment the
84 days, low temperature group reached the 80% of maximum settlement rate to the sedimentation rate of rubbish, and control group and two kinds of commercially available composite bacteria agent groups
Reach the 80% of maximum settlement rate the 126th day, 108 days and 114 days respectively.After the test, domestication by low temperature compound bacteria is added
The final settlement rate of agent group rubbish is high by 32.5%, 18.7% and 23.6% compared with control group, two kinds of commercially available composite bacteria agent group difference.It carries
Show that domestication by low temperature group becomes apparent the minimizing effect of rubbish.
By screening extensively and domestication by low temperature, obtained one kind be applicable under low temperature environment that there is solid refuse it is efficient
The composite bacteria agent for decomposing efficiency, shows, compared with common composite bacteria agent, which exists through laboratory and field test
There is good growing state under low temperature environment, to the decomposition efficiency higher of organic matter in garbage matter, be applicable to northwest drying
Cold weather conditions, have a good application prospect.
Claims (8)
1. a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent, which is characterized in that include the following steps：
(1) strain acquires
Rubbish sample is mixed with sterile water, obtains sample diluting liquid；
(2) strain separating
Take above-mentioned sample diluting liquid 300~900 μ L in total, divide three equal parts, be seeded to respectively beef extract-peptone plating medium,
Then beef extract-peptone plating medium after inoculation is positioned over by Gause I plating medium and Cha Shi plating mediums
Growth cabinet, 37 DEG C are cultivated 1-2 days, by after inoculation the foster base of Gause I training tablet and Cha Shi plating mediums be positioned over it is mould
Bacterium incubator, 28 DEG C are cultivated 3-4 days, and bacterium, actinomyces and mould are respectively obtained；Respectively to above-mentioned bacterium, actinomyces and mould,
Bacterium colony is purified using plate streak, until obtaining single pure bacterium colony；
(3) strain enrichment and bacterial strain domestication
Bacterial liquid culture medium, actinomyces fluid nutrient medium and mould liquid are seeded to by obtained single pure bacterium colony is corresponding respectively
Body culture medium, at 28 DEG C, 130r/min shaking table shake cultures, after bacterium solution OD600nm reaches 0.7 ± 0.05, by above-mentioned bacterium solution
It is seeded in the fluid nutrient medium of the 20wt% containing landfill leachate and cultivates 4 days respectively, be then seeded to containing landfill leachate
It is cultivated 4 days in the fluid nutrient medium of 40wt%, inoculates into the fluid nutrient medium of the 60wt% containing landfill leachate and cultivate 4 days,
Then it is seeded in the fluid nutrient medium of the 80wt% containing landfill leachate and cultivates 4 days, be finally seeded to containing landfill leachate
It is cultivated 4 days in the fluid nutrient medium of 100wt%, obtains tolerable bacterium, actinomyces and mould；
(4) bacterial strain primary dcreening operation
By tolerable bacterium, actinomyces and the mould after domestication respectively in bacterial liquid culture medium, actinomyces fluid nutrient medium
With enrichment culture in mould fluid nutrient medium to bacterium solution OD600nm be 0.7 ± 0.05 after, respectively by above-mentioned bacterium, actinomyces and
The bacterium solution of mould is 1 by volume:20 ratio is seeded to landfill leachate, cultivates 48h, obtains that ammonia nitrogen and COD can be removed
Bacterium, actinomyces and mould；
(6) domestication by low temperature
The above-mentioned bacterium that can remove ammonia nitrogen and COD, actinomyces and mould and cellulose-degrading bacteria are seeded to beef extract respectively
Peptone plating medium, Gause I plating medium, Cha Shi plating mediums and carboxymethyl cellulose plating medium, are adopted
Domestication by low temperature is carried out to bacterial strain with the method for gradient cooling, 28 DEG C first are cultivated 2-3 days, and 3 DEG C of culture 5-7 of cultivation temperature are reduced
It, is repeated above-mentioned cooling culture operation, when temperature is reduced to 10 DEG C, obtains capable of removing the low temperature resistant thin of ammonia nitrogen and COD
Bacterium, the low temperature resistant actinomyces that ammonia nitrogen and COD can be removed, the low temperature resistant mould that ammonia nitrogen and COD can be removed and low temperature resistant fibre
The plain degradation bacteria of dimension；The low temperature resistant bacterium of ammonia nitrogen and COD will be removed with the bacterium in Low temperature resistant cellulose degradation bacteria by volume
Than 1:1 is mixed to get low temperature resistant bacterium；The low temperature resistant actinomyces of ammonia nitrogen and COD will be removed as low temperature resistant actinomyces；It will
It is 1 that the mould in the low temperature resistant mould and Low temperature resistant cellulose degradation bacteria of ammonia nitrogen and COD, which can be removed, by volume:1 mixing, obtains
To low temperature resistant mould；
(7) preparation of composite bacteria agent
Respectively by above-mentioned low temperature resistant bacterium, actinomyces and mould be seeded to bacterial liquid culture medium, actinomyces fluid nutrient medium and
Activation culture in mould fluid nutrient medium carries out compatibility by the volume fraction of bacterium 90%, actinomyces 3% and mould 7%, obtains
Composite bacteria agent.
2. a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent according to claim 1, which is characterized in that step
(1) the rubbish sample described in is to freeze the following rubbish of layer, landfill leachate soot winter and by leachate contamination pedotheque.
3. a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent according to claim 1, which is characterized in that step
(1) concentration of the sample diluting liquid can be 10-1、10-2、10-3、10-4、10-5、10-6。
4. a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent according to claim 1, which is characterized in that by weight
Number meter is measured, the beef extract-peptone plating medium is by 10.0 parts of peptone, 3.0 parts of beef extract, 0.5 part of NaCl, agar
20 parts are adjusted to 7.0-7.2 with 1000 parts of compositions of distilled water, pH value, and in 121 DEG C, high pressure sterilization 30min is cooled to 60-70
DEG C when to be down flat plate spare；
The Gause I plating medium is by 20 parts of soluble starch, KN031 part, K2HP040.5 part, NaCI O.5 part,
MgS04·7H20 0.5 parts, FeS041000 parts of 0.0l parts, 20 parts of agar and distilled water, pH value is adjusted to 7.2-7.4, in 121
DEG C, it is spare to be down flat plate when being cooled to 60-70 DEG C by high pressure sterilization 30min；
The Cha Shi plating mediums are by 30 parts of glucose, NaNO32.0 parts, 0.5 part of KCl, K2HP041.0 parts, MgS04·
7H20 0.5 parts, FeS041000 parts of 0.01 part, 20 parts of agar and distilled water, pH value is adjusted to 7.2-7.4, in 121 DEG C, high pressure
Sterilize 30min, and it is spare to be down flat plate when being cooled to 60-70 DEG C.
5. a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent according to claim 1, which is characterized in that step
(2) bacterium is the composition of bacillus, Bifidobacterium, Curtobacterium, pseudomonas and molten Bacillus；It is mould
Bacterium is the composition of cladosporium sp category, trichoderma and Eurotium；Actinomyces are streptomyces.
6. a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent according to claim 1, which is characterized in that by weight
Score meter is measured, in step (4) and step (7), the bacterial liquid culture medium is by 10.0 parts of peptone, 3.0 parts of beef extract, NaCl
0.5 part and distilled water 1000mL forms, and pH value is adjusted to 7.0-7.2, and in 121 DEG C, high pressure sterilization 30min is cooled to 60-70 DEG C
It is spare；
The actinomyces fluid nutrient medium is by 20 parts of soluble starch, KN031 part, K2HP040.5 part, NaCI O.5 part,
MgS04·7H20 0.5 parts, FeS040.0l parts and 1000 parts of distilled water, pH value is adjusted to 7.2-7.4, and in 121 DEG C, high pressure is gone out
Bacterium 30min, be cooled to 60-70 DEG C it is spare；
The mould fluid nutrient medium is by 30 parts of glucose, NaNO32.0 parts, 0.5 part of KCl, K2HP041.0 parts, MgS04·
7H20 0.5 parts, FeS040.01 part and 1000 parts of distilled water, pH value is adjusted to 7.2-7.4, in 121 DEG C, high pressure sterilization 30min,
Be cooled to 60-70 DEG C it is spare.
7. a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent according to claim 1, which is characterized in that step
(6) cellulose-degrading bacteria described in is that pseudomonad, cladosporium sp and Trichoderma are 1 by volume:1:1 mixes.
8. a kind of preparation method of low temperature resistant garbage disposal composite bacteria agent according to claim 1, which is characterized in that step
(6) the carboxymethyl cellulose plating medium described in is by 15 parts of CMC-Na, 1 part of NH4N03,1 part of yeast extract, MgS04
1000 parts of 0.5 part of 7H20,1 part of KH2P04,20 parts of agar and distilled water, pH value is adjusted to 7.0-7.2, and in 121 DEG C, high pressure is gone out
It is spare to be down flat plate when being cooled to 60-70 DEG C by bacterium 30min.
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