CN106701617A - Method for domesticating auto-aggregation propionic acid methanogenesis microbial agent - Google Patents
Method for domesticating auto-aggregation propionic acid methanogenesis microbial agent Download PDFInfo
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Abstract
The invention discloses a method for domesticating an auto-aggregation propionic acid methanogenesis microbial agent. According to the method, an extracellular polymeric substance of biogas fermentative fluid serves as an inoculum, and propionic acid serves as a carbon source. The method comprises the following steps: taking biogas fermentative fluid capable of secreting the extracellular polymeric substance, separating to obtain the extracellular polymeric substance so as to be inoculated into a propionic acid methanogenesis selective medium, and culturing the substance in an anaerobic bottle table at the temperature of 37 DEG C for 3-4 weeks until pellet microbial aggregate occurs in the medium; and adding the cenobium into a fresh culture medium, culturing for 7-10 days, and domesticating, thereby obtaining the auto-aggregation propionic acid methanogenesis cenobium. The obtained auto-aggregation propionic acid methanogenesis microbial agent can degrade 3.0g/L of 95% propionic acid within one week, the gas production rate is 70% of the theoretical yield, the utilization rate of the propionic acid and acetic acid is higher than 90%, the microbial agent can be used for a gas production drop or rancidity fermentation system caused by propionic acid accumulation, propionic acid degradation and methanogenesis of degradation products of the propionic acid can be facilitated, and the biogas fermentation performance is improved.
Description
Technical field：
The present invention relates to microbial technology field, and in particular to a kind of acclimation method from aggegation propionic acid bactericide for generating methane.
Background technology：
Under the dual-pressure of energy shortage and environmental pollution, can process discarded object can produce the natural pond of regenerative resource again
Gas industry has obtained very fast development, and large-scale production has been moved towards from small-scale household biogas.According to statistics, national biogas user is about
43000000 families, at nationwide scaleization biogas engineering about 100,000, however, most methane-generating pits are in due to that cannot run steadily in the long term
Idle state, most of biogas engineering cannot get a profit so far.It is restriction by acid suppression problem caused by VFA accumulation
One of factor of biogas fermentation stability and economy.Acid suppresses or phenomenon of becoming sour has in kitchen garbage, agriculture and forestry organic waste material, city
Machine rubbish, energy crop etc. in the anaerobic fermentation process of raw material often to occur.Acid suppresses to be because is produced from anaerobic fermentation system upstream
Sour process is unbalance with downstream methane phase process, intermediate product volatile fatty acid accumulation (mainly propionic acid, acetic acid), causes fermentation
System pH reductions, it is suppressed that the activity of methanogen, ultimately result in fermentation system gas deliverability and decline or become sour.Generally
Solution be macroscopically to optimize operational factor, such as add alkali regulation pH, or reduce organic loading and fermentation concentration, make hair
Ferment runs under relatively low fermentation concentration, which limits fermentation efficiency.As biological reinforcing technology is led in wastewater treatment
The application in domain, researcher begins attempt to apply it in marsh gas fermenting system, i.e., micro- life of artificial addition specific function
Thing tackles sour suppression, improves the stability of fermentation system, improves fermentation concentration, or recover the system aerogenesis that becomes sour.
Although being related to biological reinforcing technology and having alleviated the report that acid suppresses, feeding function bacterium is generally purchase
The syntrophism propionic acid oxidizing bacteria for propionic acid degradation of pure culture, or for acetic acid and methane producing process methane phase Gu bacterium, also or
It is artificially to be combined several microorganisms to be made complex micro organism fungicide.There is following asking in the technology of preparing of these microbial inoculums
Topic：First, the microorganism propionic acid oxidizing bacteria or methanogen of pure culture are difficult to adapt to anaerobism hair as the strain that external source is added
Complicated environmental condition in ferment system, such as low pH, high-concentration ammonia-nitrogen etc.；Second, the microorganism of single type is used as biological reinforced
Microbial inoculum is added to after anaerobic fermentation system it is difficult to the biological volume of holding of the competition of resistance indigenous microorganism and chronically holding is to play
Invigoration effect.Therefore, it is acquisition bioactivity stabilization and the biological reinforced microbial inoculum of efficient degradation propionic acid, it is necessary to which providing one kind has
The acclimation method of effect.
The content of the invention：
It is an object of the invention to provide a kind of acclimation method from aggegation propionic acid bactericide for generating methane, obtain from aggegation propionic acid
Bactericide for generating methane has the advantages of impact resistance is strong, and bioaugmentation stability is high, also with the ability of aggegation agglomerate, can be
Cenobium inside thalline is protected under conditions of growing environment is unfavorable and activity is maintained, for improving biogas fermentation performance.
The present invention is achieved by the following technical programs：
A kind of acclimation method from aggegation propionic acid bactericide for generating methane, the method is with the extracellular polymeric of biogas fermentative liquid to connect
Thing is planted, with propionic acid as carbon source, the method is comprised the following steps：Take the biogas fermentative liquid of secretion extracellular polymeric, isolated born of the same parents
Outer polymer is inoculated into the selective medium of propionic acid methane phase, be put into anaerobism bottle shaking table in 37 DEG C carry out culture 3-4 week directly
Occurs the microorganism agglutination body of pelletizing shape into culture medium；Cenobium being taken again and adding fresh culture medium, culture is tamed for 7-10 days
Arrive from aggegation propionic acid methane phase cenobium；The selective medium compound method is：400mg NH are added in 1L water4Cl、
250mg MgSO4·6H2O、400mg KCl、120mg CaCl2·2H2O、80mg(NH4)2HPO4、55mg FeCl3·6H2O、
5000mg NaHCO3, it is additionally added CoCl2·6H2O、NiCl2·6H2O、MnCl2·4H2O、CuCl2·2H2O、AlCl3·6H2O、
ZnCl2、Na2WO4·2H2O、H3BO3、Na2SeO3、Ma2MoO4·2H2Each 0.0005mg of O, are additionally added sodium propionate 4200mg in addition,
PH is adjusted to 7.0.
The biogas fermentative liquid of the secretion extracellular polymeric, its determination methods are to be centrifuged under the rotating speed more than 10000rpm
After biogas fermentative liquid 10-15min, there is the phenomenon that zymotic fluid is divided into three layers, upper strata is as clear as crystal, the sticky milky in intermediate layer,
Bottom black containing solid impurity；Described isolated extracellular polymeric, refers to centrifugation secretion born of the same parents under the rotating speed more than 10000rpm
After the biogas fermentative liquid 10-15min of outer polymer, zymotic fluid is divided into three layers, removes the clear liquid and bottom on upper strata
Solids of sedimentation, collects the sticky milky white color substance in intermediate layer, and sticky milky thing is centrifuged under the rotating speed more than 10000rpm again
Matter 15min, collects the bacterium mud of precipitation as inoculum.
The present invention also protects the application from aggegation propionic acid methanogen, and what is obtained after above-mentioned domestication produces from aggegation propionic acid
Methane backeria is used for the propionic acid of degradable 95% 3.0g/L in biological reinforced biogas fermentation, 1 week, and its factor of created gase is theoretical yield
70%, the utilization rate of propionic acid and acetic acid is more than 90%.
The present invention also provides a kind of preparation method from aggegation propionic acid methanogen solid fungicide, will be obtained after above-mentioned domestication
The bacterium solution from aggegation propionic acid methanogen through centrifugation, discarding room temperature under be placed in after liquid portion anaerobic environment and air-drying, collect solid
Simultaneously vacuum sealing preservation obtains target product to body powder.
The present invention also provides a kind of preparation method of liquid concentration from aggegation propionic acid bactericide for generating methane, will be obtained after above-mentioned domestication
Arrive from aggegation propionic acid methanogen bacterium solution through centrifugation, whole solids of sedimentation and a little supernatant are collected, using the liquid of sterilizing
Body glycerine sealing liquid face is completely cutting off air, -20 DEG C of Cord bloods.
Beneficial effects of the present invention are as follows：
(1) present invention obtain from aggegation propionic acid bactericide for generating methane propionic acid capable of being fast degraded, 1 week interior degradable 95%
The propionic acid of 3.0g/L, its factor of created gase is the 70% of theoretical yield, and the utilization rate of propionic acid and acetic acid is more than 90%.Further, since bacterium
Agent can increase the material transmission between strain from the characteristic of aggegation, and protect internal thalline not to be hit, with impact resistance it is strong,
The advantages of bioaugmentation stability is high, can be used to, because aerogenesis declines or the fermentation system that becomes sour caused by propionic acid accumulation, promote
Propionic acid degradation and its catabolite methane phase, improve biogas fermentation performance.
(2) present invention obtain from aggegation propionic acid methanogen solid fungicide and liquid concentration from aggegation propionic acid methanogen
Agent is easy to transport and long-term preservation.
Brief description of the drawings：
Fig. 1 be embodiment 1 tame obtain from aggegation propionic acid methane phase cenobium.
Specific embodiment：
Further illustrated the following is to of the invention, rather than limitation of the present invention.
Embodiment 1：
The present embodiment inoculum is the extracellular polymeric of the biogas fermentative liquid for secreting extracellular polymeric, and preparation method is as follows：
Biogas fermentative liquid (kitchen garbage zymotic fluid or other biogas fermentations of secretion extracellular polymeric are centrifuged under the rotating speed of 13000rpm
Liquid) after 15min, zymotic fluid is divided into three layers, and upper strata is as clear as crystal, the sticky milky in intermediate layer, bottom black containing solid impurity, receive
Collect the sticky milky white color substance in intermediate layer, sticky milky white color substance 15min is centrifuged under the rotating speed of 13000rpm again, collect precipitation
Bacterium mud as inoculum.
Culture apparatus is 200ml anaerobism bottle.
The present embodiment is comprised the following steps from the acclimation method of aggegation propionic acid methanogen methane microbial inoculum：Take secretion extracellular
The extracellular polymeric that the biogas fermentative liquid of polymer is isolated is inoculated into the selective medium of propionic acid methane phase, is put into and is detested
37 DEG C carry out culture 3-4 weeks until there is the microorganism agglutination body of pelletizing shape in culture medium in oxygen bottle shaking table；Cenobium addition is taken again
Fresh culture medium, culture domestication in 7-10 days obtain from aggegation propionic acid methane phase cenobium, as shown in Figure 1.The selectivity training
Base is supported, compound method is：400mg NH are added in 1L water4Cl、250mg MgSO4·6H2O、400mg KCl、120mg
CaCl2·2H2O、80mg(NH4)2HPO4、55mg FeCl3·6H2O、5000mg NaHCO3, it is additionally added CoCl2·6H2O、
NiCl2·6H2O、MnCl2·4H2O、CuCl2·2H2O、AlCl3·6H2O、ZnCl2、Na2WO4·2H2O、H3BO3、Na2SeO3、
Ma2MoO4·2H2Each 0.0005mg of O, are additionally added sodium propionate 4200mg in addition, and pH is adjusted to 7.0.
Being used for from aggegation propionic acid methanogen for being obtained after above-mentioned domestication is degradable in biological reinforced biogas fermentation, 1 week
The propionic acid of 95% 3.0g/L, its factor of created gase is the 70% of theoretical yield, and the utilization rate of propionic acid and acetic acid is more than 90%.
5- is centrifuged with the rotating speed of 5000-8000rpm from aggegation propionic acid methanogen bacterium solution by what is obtained after above-mentioned domestication
10min, discarding room temperature under be placed in after liquid portion anaerobic environment (anaerobic operation case, anaerobism airbag) and air-drying, and collects solid powder
And vacuum sealing is preserved and obtained from aggegation propionic acid methanogen solid fungicide.
5- is centrifuged with the rotating speed of 5000-8000rpm from aggegation propionic acid methanogen bacterium solution by what is obtained after above-mentioned domestication
10min, collects whole solids of sedimentation and a little supernatant, using the liquid glycerol sealing liquid face of sterilizing completely cutting off air, -20 DEG C
Cord blood.
Embodiment 2：The present invention obtain from aggegation propionic acid bactericide for generating methane to energy grass anaerobic fermentation of biological invigoration effect
Fermentation raw material：The energy grass of marginal land plantation.
Inoculum：Swine manure treatment factory anaerobic fermented liquid.
Reaction unit：The on-line automatic gas producing devices of continuous CSTR of 2L.
Setup Experiments：2 semicontinuous anaerobic fermentation tanks of 2L, respectively R1, R2 are run altogether.R1 is blank, i.e. the energy
The independent anaerobic fermentation of grass, without microbial inoculum.R2 be daily addition embodiment 1 obtain from aggegation propionic acid methane phase solid fungicide
0.3g。
The running of 2 reactors is as follows：Hydraulic detention time (HRT) is 20 days, i.e. the fermentation of discharge 100ml daily
Liquid, injects the energy grass and aqueous mixtures material of 100ml.2 reactors co-continuous operation 120d, i.e., 6 HRT, each HTR's has
Machine rate of load condensate (OLR) is followed successively by 0.5g/L d, 0.75g/L d, 1.0g/L d, 1.5g/L d, 2.0g/L d and 2.5g/L d.
Result shows：Organic loading rate is not higher than in first 60 days of 1.0g/L d, and R1 and R2 can stablize aerogenesis, but addition
The average gas production of the R2 of microbial inoculum improves 10%, when organic loading is promoted to 1.5g/L d, fermentations of the R1 with microbial inoculum than R1
System aerogenesis is decreased obviously, and the fermentation system gas production of R2 addition microbial inoculums increases, two HRT hereafter, R2 aerogenesis stabilization, has
Machine rate of load condensate 2.5g/L d still can stable operation, in zymotic fluid propionate concentration be less than 1.0g/L.And the R1 for being not added with microbial inoculum is having
Machine rate of load condensate is carried to 2.0g/L d and is unable to aerogenesis completely, and propionate concentration reaches 4.0g/L.
As can be seen here, by the present invention obtain from aggegation propionic acid bactericide for generating methane can tackle anaerobic fermentation process in third
Acid suppresses, and improves the organic loading of energy grass anaerobic fermentation, promotes aerogenesis.
Claims (6)
1. a kind of acclimation method from aggegation propionic acid bactericide for generating methane, it is characterised in that the method is with the extracellular of biogas fermentative liquid
Polymer is inoculum, and with propionic acid as carbon source, the method is comprised the following steps：The biogas fermentative liquid of secretion extracellular polymeric is taken,
Isolated extracellular polymeric is inoculated into the selective medium of propionic acid methane phase, be put into anaerobism bottle shaking table in 37 DEG C trained
Support 3-4 weeks until there is the microorganism agglutination body of pelletizing shape in culture medium；Cenobium is taken again and adds fresh culture medium, cultivate 7-10
Its domestication obtain from aggegation propionic acid methane phase cenobium；The selective medium compound method is：400mg is added in 1L water
NH4Cl、250mg MgSO4·6H2O、400mg KCl、120mg CaCl2·2H2O、80mg(NH4)2HPO4、55mg FeCl3·
6H2O、5000mg NaHCO3, it is additionally added CoCl2·6H2O、NiCl2·6H2O、MnCl2·4H2O、CuCl2·2H2O、AlCl3·
6H2O、ZnCl2、Na2WO4·2H2O、H3BO3、Na2SeO3、Ma2MoO4·2H2Each 0.0005mg of O, are additionally added sodium propionate in addition
4200mg, pH are adjusted to 7.0.
2. the acclimation method from aggegation propionic acid bactericide for generating methane according to claim 1, it is characterised in that the secretion born of the same parents
The biogas fermentative liquid of outer polymer, its determination methods are that biogas fermentative liquid 10-15min is centrifuged under the rotating speed more than 10000rpm
Afterwards, there is the phenomenon that zymotic fluid is divided into three layers, upper strata is as clear as crystal, the sticky milky in intermediate layer, bottom black containing solid impurity.
3. the acclimation method from aggegation propionic acid bactericide for generating methane according to claim 1, it is characterised in that described separation
Extracellular polymeric is obtained, refers to the biogas fermentative liquid 10-15min of centrifugation secretion extracellular polymeric under the rotating speed more than 10000rpm
Afterwards, zymotic fluid is divided into three layers, removes the clear liquid on upper strata and the solids of sedimentation of bottom, collects the sticky milky white of intermediate layer
Color substance, is centrifuged sticky milky white color substance 15min again under the rotating speed more than 10000rpm, collect the bacterium mud of precipitation as inoculation
Thing.
4. a kind of application from aggegation propionic acid methanogen, it is characterised in that obtained after claim 1 domestication from aggegation propionic acid
Methanogen is used for biological reinforced biogas fermentation.
5. a kind of preparation method from aggegation propionic acid methanogen solid fungicide, it is characterised in that after claim 1 is tamed
The bacterium solution from aggegation propionic acid methanogen for arriving discarding room temperature under be placed in after liquid portion anaerobic environment and air-drying through centrifugation, collects
Simultaneously vacuum sealing preservation obtains target product to solid powder.
6. preparation method of a kind of liquid concentration from aggegation propionic acid bactericide for generating methane, it is characterised in that after claim 1 is tamed
Obtain from aggegation propionic acid methanogen bacterium solution through centrifugation, whole solids of sedimentation and a little supernatant are collected, using sterilizing
To completely cut off air, -20 DEG C preserve in liquid glycerol sealing liquid face.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111733190A (en) * | 2020-07-03 | 2020-10-02 | 中国科学院广州能源研究所 | Self-sufficient biological strengthening method for methane fermentation performance |
| CN112342250A (en) * | 2019-08-07 | 2021-02-09 | 首都师范大学 | Method for preparing granular sludge for producing methane by degrading propionic acid |
| CN113308496A (en) * | 2020-02-26 | 2021-08-27 | 南开大学 | Method for recovering methane production performance of anaerobic fermentation acid inhibition system by using photosynthetic bacteria |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112342250A (en) * | 2019-08-07 | 2021-02-09 | 首都师范大学 | Method for preparing granular sludge for producing methane by degrading propionic acid |
| CN112342250B (en) * | 2019-08-07 | 2022-06-21 | 首都师范大学 | Method for preparing granular sludge for producing methane by degrading propionic acid |
| CN113308496A (en) * | 2020-02-26 | 2021-08-27 | 南开大学 | Method for recovering methane production performance of anaerobic fermentation acid inhibition system by using photosynthetic bacteria |
| CN113308496B (en) * | 2020-02-26 | 2022-11-08 | 南开大学 | Method for recovering methane production performance of anaerobic fermentation acid inhibition system by using photosynthetic bacteria |
| CN111733190A (en) * | 2020-07-03 | 2020-10-02 | 中国科学院广州能源研究所 | Self-sufficient biological strengthening method for methane fermentation performance |
| CN111733190B (en) * | 2020-07-03 | 2022-02-25 | 中国科学院广州能源研究所 | Self-sufficient biological strengthening method for methane fermentation performance |
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