CN107523521A - A kind of tetrachloro-ethylene anaerobic degradation Mixed Microbes, the preparation method and application of microbial inoculum - Google Patents
A kind of tetrachloro-ethylene anaerobic degradation Mixed Microbes, the preparation method and application of microbial inoculum Download PDFInfo
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Abstract
The invention discloses the preparation method and application of a kind of tetrachloro-ethylene anaerobic degrading bacteria, microbial inoculum, it is main including anaerobism from be enriched with dominant bacteria by the place of chloroalkene pollution for a long time, screening and the domestication of mix bacterium agent, mixed degradation microbial inoculum are applied.The present invention is set under co-metabolic substance glucose existence condition, 25 DEG C, pH value 7.0 or so when, microbial inoculum addition 20%, Mixed Microbes reach 96.1% to the degradation rate of tetrachloro-ethylene.When adding mix bacterium agent, tetrachloro-ethylene degradation rate can reach 94.3%.The simple to operate, high treating effect of the present invention, cost are low.By using the tetrachloro-ethylene degraded Mixed Microbes of orientation domestication, select appropriate Co metabolism nutriment and made liquid bacterial agent, solve tetrachloro-ethylene and pollute that degradation rate existing for biological prosthetic process is low, degradation effect is undesirable, the problem of single-minded microorganism is vulnerable to changes in environmental conditions is relied on, it is significant in tetrachloro-ethylene pollution control.
Description
Technical field
The invention belongs to chloroalkene biodegradation technique, it is related to a kind of tetrachloro-ethylene anaerobic degradation Mixed Microbes, microbial inoculum
Prepare and apply.
Background technology
Chloroalkene the industry such as is widely used in printing, dry-cleaned as a kind of organic solvent.Due to storing improper, rubbish
Unreasonable disposal after the infiltration or use of percolate to waste liquid etc. so that a large amount of chloroalkene enter water body, air and soil
Earth simultaneously causes different degrees of pollution, wherein, tetrachloro-ethylene is one of pollutant most often detected in water environment, and the mankind are good for
Health and ecological environment, which are formed, to be seriously endangered.Tetrachloro-ethylene (PCE) is a kind of biodegradable organic compoundses, to the central nervous system of people
System has strong inhibitory action, and three with " carcinogenic, teratogenesis, mutagenesis " cause effect, and international tumor research center is arranged
Enter possible human carcinogen (B2Level).
Currently used tetrachloro-ethylene minimizing technology has Physical, chemical method and bioanalysis.Physical treatment method cost is low
It is honest and clean, it is capable of the fast and effective concentration for reducing pollutant, is adapted to the quick processing of high density pollution thing, but such method can not be from
Fundamentally eliminate pollution, it is possible to secondary pollution can be caused to environment.Chemical method processing tetrachloro-ethylene significant effect, but meeting
Introduce chemical agent and produce new pollution, the shortcomings that treating capacity is small, costly also be present.Bioanalysis is to utilize microorganism or plant
The metabolic activity of thing reaches the purpose of depollution, has low excellent of efficient, economic, harmless, non-secondary pollution, maintenance cost
Point, therefore bioanalysis is paid close attention to by domestic and international numerous researchers.The bioremediation of tetrachloro-ethylene will possess wide in future
Application prospect.
It is generally believed that biochemical reaction degraded occurs only in anaerobic environment for tetrachloro-ethylene generates low chloroalkene and second
Alkene, under certain condition will the effective dechlorination of ability by way of Co metabolism.Single degradation bacteria and the change for being vulnerable to external environment
The interference of change, influences biological removal effect.Therefore specific anaerobic degradation Mixed Microbes are cultivated, is favorably improved adaptive capacity to environment
And treatment effect.
The content of the invention
For problems of the prior art, the present invention provides a kind of anaerobism drop that can be used for degraded tetrachloro-ethylene pollution
Mixed Microbes, the preparation method of microbial inoculum are solved, in by the place soil of chloroalkene pollution, is enriched with, tame out can be with anaerobism from for a long time
The superior mixed flora of degraded tetrachloro-ethylene.
An object of the present invention is, there is provided a kind of preparation method of tetrachloro-ethylene anaerobic degradation Mixed Microbes.
The second object of the present invention is, there is provided a kind of tetrachloro-ethylene anaerobic degradation Mixed Microbes are removing tetrachloro-ethylene side
The application in face.
The third object of the present invention is, there is provided a kind of preparation method of tetrachloro-ethylene anaerobic degradation mix bacterium agent.
The fourth object of the present invention is, there is provided a kind of tetrachloro-ethylene anaerobic degradation mix bacterium agent is removing tetrachloro-ethylene side
The application in face.
Technical solution of the present invention is as follows：
First, in order to realize first purpose, there is provided a kind of preparation method of tetrachloro-ethylene anaerobic degradation Mixed Microbes, main bag
Include enrichment, domestication and culture.
(1) soil of 30~40cm under by the place earth's surface of tetrachloro-ethylene pollution for a long time is taken, removes deadwood weeds and stone
It is standby afterwards.
(2) glucose, dusty yeast, trace element and inorganic salts are added and prepare basal medium, are eventually adding step (1)
1~2kg of soil (water and soil mass ratioes 10::1~5:1) sealing culture afterwards；
(3) enrichment and domestication, the anaerobic degradation Mixed Microbes of tetrachloro-ethylene are obtained.
In the step (2), it is adding in 1L water that other contents are mentioned in inorganic salts, the content of trace element and text
Enter amount：
Inorganic salts (mg/L)：NH4Cl, 320；Sulphur, 667；KH2PO4, 34；Na2CO3, 132；CaCl2, 63.
Micro- (mg/L)：MgSO4, 2.5；H3BO3, 0.05；NiSO4·6H2O, 0.44；FeCl2·4H2O, 3；
ZnSO4·7H2O, 0.05；MnCl2·4H2O, 2.5；CoCl2·6H2O, 0.52；CuSO4·5H2O, 0.03；(NH4)6Mo7O24·
4H2O, 0.32.
In the step (2), glucose 1000mg/L, dusty yeast 99.5mg/L.
In the step (3), using 2~3d as an enrichment cycle, it is enriched with 2 weeks, using 3~4d as an acclimation period, tames and dociles
Change 4 weeks；Gradually increase the addition of tetrachloro-ethylene during domestication：By 0.2mg/L, 0.4mg/L, 0.6mg/L, 0.8mg/L,
1.0mg/L amounts gradually increase, the corresponding addition for reducing energy substance：By 1000mg/L, 800mg/L, 600mg/L, 400mg/
L, 200mg/L amounts gradually decrease.
2nd, in order to realize second purpose, the present invention applies obtained Mixed Microbes in the water containing tetrachloro-ethylene.
Mixed Microbes degradation condition applicatory is that μ g/L of tce concentration 0.0~900.0, nitrogen source are dusty yeast.
Mixed Microbes degradation condition applicatory is that glucose, sucrose, formates, acetate are used as carbon source, temperature 10
~30 DEG C, pH value 5.0~9.0, inoculation volume ratio 10%~40%, 100~120r/min of constant temperature oscillator rotating speed；The present invention is ground
Studied carefully under difficult environmental conditions Mixed Microbes to the degradation property of tetrachloro-ethylene.
Preferable degradation condition is that tetrachloro-ethylene anaerobic degradation Mixed Microbes are in glucose as co-metabolic substance, temperature 25
DEG C, pH value 7, inoculation 10~20%, 100~120r/min of constant temperature oscillator rotating speed.
3rd, in order to realize the 3rd purpose, a kind of preparation of tetrachloro-ethylene anaerobic degradation mix bacterium agent provided by the invention
Method is as follows：
(1) the anaerobic degradation Mixed Microbes bacterium solution tamed made from the invention described above 4 weeks is taken, by 1:101, 1:102, 1:
103, 1:104, 1:105, 1:106, 1:107, 1:108, i.e. bacterium solution and the ratio between total liquid volume is dilute step by step after adding Sterile dilution water
Release, obtain the dilution of 8 parts of different dilution ranks, take the serial bacteria suspension 1 of dilution respectively:105, 1:106, 1:107, 1:108
It is even to be coated on LB solid mediums, 0.4~0.8mg/L tetrachloro-ethylenes are added, are placed in 28~30 DEG C of biochemical cultivation cases continuous
Culture；When there is single bacterium colony, different shape feature is chosen, the vigorous a fairly large number of single bacterium colony of growth is re-seeded into containing four
On the LB solid mediums of vinyl chloride, cultivated in 28~35 DEG C of incubators；The a fairly large number of single bacterium colony of picking after purified
Be inoculated in fluid nutrient medium containing tetrachloro-ethylene, carry out enrichment culture again, after the completion of preserve bacterium solution, be made tetrachloro-ethylene anaerobism mix
Close bacterium；
(2) bacterium solution in above-mentioned steps after purification is taken to send sequencing company sequencing with the species of this determination bacterial strain；It is identified mixed
Close bacterium and mainly contain Pi Shi Rolstons bacterium (Ralstonia pickettii), P. myrsinacearum (Phyllobacterium
Myrsinacearum), sphingolipid monad (Sphingomonas aeria)；
(3) above-mentioned 3 kinds of bacterial solutions are taken as inoculum, by inoculation bacterium solution volume proportioning, Pi Shi Rolston bacterium：Zijin
Ox leaf bacillus：Sphingolipid monad=1:0.8~1:0.8~1 is added to the basis culture containing 0.5~1.0mg/L tetrachloro-ethylenes
In base, in cultivating 48~72h in 28~35 DEG C of incubators, liquid mix bacterium agent is obtained.
The compound method of LB culture mediums is NaCl 10.0g in above-mentioned steps (1), peptone 10.0g, dusty yeast 5.0g,
10~15.0g of agar, distilled water 1L, 6.8~7.2,121 DEG C of autoclaving 30min of pH value, sterile working.
4th, in order to realize the 4th purpose, the present invention applies obtained mix bacterium agent in the water containing tetrachloro-ethylene.
Tetrachloro-ethylene anaerobic degradation mix bacterium agent is in glucose as 25 DEG C of co-metabolic substance, temperature, pH value 7.0, inoculation
10~20%, applied under conditions of 100~120r/min of constant temperature oscillator rotating speed.
The simple to operate, high treating effect of the present invention, cost are low.Degraded and mixed by using the tetrachloro-ethylene of orientation domestication
Bacterium, select appropriate Co metabolism nutriment and made liquid bacterial agent, solve the biological prosthetic process of tetrachloro-ethylene pollution and deposit
Degradation rate is low, degradation effect is undesirable, the problem of single-minded microorganism is vulnerable to changes in environmental conditions is relied on, in tetrachloro
It is significant in ethene pollution control.
Brief description of the drawings
The degraded situation of Fig. 1 anaerobic degradations Mixed Microbes tetrachloro-ethylene under the conditions of 20%, pH value 7.0,25 DEG C of temperature；
Fig. 2 anaerobic degradations Mixed Microbes cell concentration under the conditions of 20%, pH value 7.0,25 DEG C of temperature changes.
Embodiment
The present invention is further detailed and explained by example in detail below, following examples are descriptive
, it is not intended to limit the present invention.
Embodiment 1
A kind of preparation method of tetrachloro-ethylene anaerobic degradation Mixed Microbes, it is main to include enrichment, domestication and culture.
(1) be derived from Tianjin Dongli District heavenly maiden chemical plant (being dry cleaning agent factory originally) nearby under earth's surface 30~40cm soil
Earth.
(2) energy substance and growth needed for the soil added to 10.0L incubator in 2.0kg (1) and growth because
Son, incubator is sealed after being sufficiently mixed, lucifuge, room temperature condition is continuously cultivated, and is enriched with and same technical scheme the step of domestication.
(3) at the end of each cultivation cycle, 2.0L culture mediums are taken out, add fresh culture and tolerance material tetrachloro
Ethene, it is standby after the completion of domestication, you can to obtain tetrachloro-ethylene anaerobic degradation Mixed Microbes.Culture medium forms and the condition of acclimating
It is shown in Table 1, the condition of nutrient media components and acclimating.
The condition of the nutrient media components of table 1 and acclimating
Embodiment 2
A kind of preparation method of tetrachloro-ethylene anaerobic degradation Mixed Microbes, it is main to include enrichment, domestication and culture.
(1) be derived from Tianjin Dongli District heavenly maiden chemical plant (being dry cleaning agent factory originally) nearby under earth's surface 30~40cm soil
Earth.
(2) soil added to 10.0L incubator in 1.5kg (1), i.e. water and soil mass ratio 7.5:1, and add growth
Required energy substance and growth factor, incubator is sealed after being sufficiently mixed, lucifuge, room temperature condition is continuously cultivated, enrichment with
The step of domestication same technical scheme.
(3) at the end of each cultivation cycle, 2.0L culture mediums are taken out, add fresh culture and tolerance material tetrachloro
Ethene, it is standby after the completion of domestication, you can to obtain tetrachloro-ethylene anaerobic degradation Mixed Microbes.Culture medium forms and the condition of acclimating
It is shown in Table 1, the condition of nutrient media components and acclimating.
Embodiment 3
A kind of preparation method of tetrachloro-ethylene anaerobic degradation Mixed Microbes, it is main to include enrichment, domestication and culture.
(1) be derived from Tianjin Dongli District heavenly maiden chemical plant (being dry cleaning agent factory originally) nearby under earth's surface 30~40cm soil
Earth.
(2) soil added to 10.0L incubator in 1.0kg (1), i.e. water and soil mass ratio 10:1, and add growth
Required energy substance and growth factor, incubator is sealed after being sufficiently mixed, lucifuge, room temperature condition is continuously cultivated, enrichment with
The step of domestication same technical scheme.
(3) at the end of each cultivation cycle, 2.0L culture mediums are taken out, add fresh culture and tolerance material tetrachloro
Ethene, it is standby after the completion of domestication, you can to obtain tetrachloro-ethylene anaerobic degradation Mixed Microbes.Culture medium forms and the condition of acclimating
It is shown in Table 1, the condition of nutrient media components and acclimating.
Embodiment 4
The application of tetrachloro-ethylene anaerobic degradation Mixed Microbes under difficult environmental conditions, obtain in the present embodiment Example 1
Mixed Microbes be object.
(1) influence of co-metabolic substance
Formates, acetate, dextrose and saccharose are separately added into the reactor of 20% bacteria suspension of inoculation, adds tetrachloro
Ethene initial mass concentration is 900.0 μ g/L, and regulation other conditions are consistent, and it is constant for 120r/min, temperature to be placed in rotating speed
To be cultivated in 30 DEG C of constant temperature oscillators.After 3 weeks the degradation rate of tetrachloro-ethylene be respectively 86.1%, 82.0%, 76.5%,
83.2%.Co-metabolic substance best results of the glucose as anaerobism Mixed Microbes.
(2) influence of tce concentration
20% Mixed Microbes are inoculated with the reactor of 250ml fluid nutrient mediums, regulation pH value is 7.0, adds different volumes
Tetrachloro-ethylene storing solution, final initial concentration are respectively 121.5 μ g/L, 237.9 μ g/L, 613.0 μ g/L, 933.1 μ g/L.Put
In rotating speed be 120r/min, temperature it is constant be 30 DEG C constant temperature oscillator in cultivated, the degradation rate of tetrachloro-ethylene point after 3 weeks
Wei 98.3%, 94.9%, 91.3%, 85.9%.Under different tce concentrations, Mixed Microbes keep good degraded energy
Power.
(3) influence of inoculum concentration
10%, 20%, 30%, 40% (V/V) bacteria suspension, tetrachloro-ethylene addition phase are separately added into reactor
Together, culture medium and total liquid volume are consistent, and regulation pH value is 7.0.Be placed in rotating speed be 120r/min, constant temperature be 30 DEG C
Constant temperature oscillator in cultivated, after 3 weeks the degradation rate of tetrachloro-ethylene be respectively 94.1%, 94.8%, 93.4%,
91.0%.Degraded of the inoculum concentration on tetrachloro-ethylene influences smaller, 10%~20% or so inoculum concentration.
(4) pH influence
Bacteria suspension 20%, culture medium are identical with the addition of tetrachloro-ethylene in each reactor, adjust pH value respectively and are
5.0th, 6.0,7.0,8.0,9.0, be placed in rotating speed be 120r/min, temperature it is constant be 30 DEG C constant temperature oscillator in cultivated, 3
The degradation rate of tetrachloro-ethylene is respectively 53.9%, 83.6%, 94.8%, 92.9%, 66.5% after week.PH on Mixed Microbes influence compared with
Greatly, action effect is optimal when pH value is 7.0.
(5) influence of temperature
It is 20% by inoculum concentration, pH value 7.0, the reactor that adds same volume tetrachloro-ethylene and nutritional ingredient put respectively
Continuously cultivated in (rotating speed 120r/min) in 10 DEG C, 20 DEG C, 25 DEG C and 30 DEG C thermostatic oscillators, tetrachloro-ethylene after 3 weeks
Degradation rate be respectively 75.5%, 88.6%, 96.2%, 95.1%.Mixed Microbes keep good degradability under different temperatures
Can, best results at 25 DEG C.
Embodiment 5
The application of tetrachloro-ethylene anaerobic degradation Mixed Microbes target contaminant in purified water under optimal conditions.
Using glucose as co-metabolic substance, the Mixed Microbes obtained in Example 1 are by inoculum concentration 20%, pH value 7.0, reaction
25 DEG C of temperature, after 400 μ g/L tetrachloro-ethylenes are added to reactor in air-tightness sample introduction needle, reactor is sealed, is placed in constant temperature
In oscillator.Liquid phase tetrachloro-ethylene content is measured at regular intervals.The final degradation rate of tetrachloro-ethylene is 96.1%, as a result as schemed
1, Fig. 1 represents the degraded situation of anaerobic degradation Mixed Microbes tetrachloro-ethylene under the conditions of 20%, pH value 7.0,25 DEG C of temperature, four chloroethenes
Alkene gradually decreases, and cell concentration change is shown in that Fig. 2, Fig. 2 represent anaerobic degradation Mixed Microbes in 25 DEG C of 20%, pH value 7.0, temperature conditions
Hypothallus change in concentration, bacterium can be to reach larger biomass in the short period.
Embodiment 6
The preparation method of tetrachloro-ethylene anaerobic degradation mix bacterium agent.
(1) the Mixed Microbes bacterium solution 1ml for taking domestication to complete, by 1:101, 1:102, 1:103, 1:104, 1:105, 1:106, 1:
107, 1:108Obtain being serially diluted bacteria suspension after diluting step by step.1:106, 1:107, 1:108Serial bacteria suspension it is equal with " rubbing method "
It is even to be coated on LB solid mediums, suitable 0.5mg/L tetrachloro-ethylenes are added, back-off is put into 30 DEG C of biochemical cultivation cases and connected after sealing
48~72h of continuous culture.When there is single bacterium colony, different shape feature is chosen, eugonic single bacterium colony is re-seeded into containing four
On vinyl chloride 0.5mg/L LB solid mediums, cultivated in 28 DEG C of incubators, the same technical scheme of other conditions.
(2) repeatedly after purification, the eugonic single bacterium colony of picking is inoculated in containing tetrachloro in pure strain cultures for switching
Ethylene Liquid culture medium, enrichment culture is carried out again, and saved backup in 4 DEG C of refrigerators.
(3) fresh bacterium solution is taken to send sequencing company to be sequenced, by known sequence in obtained sequence and GenBank databases
Homologous comparison is carried out with the Pseudomonas of this determination bacterial strain.Identified tetrachloro-ethylene anaerobism Mixed Microbes mainly include Pi Shi Rolston bacterium
(Ralstonia pickettii), P. myrsinacearum (Phyllobacterium myrsinacearum), sphingolipid monad
(Sphingomonas aeria)。
(4) using the pure bacterial strain bacterium solution after enrichment as inoculum, after repeatedly sterile water washing is resuspended, by bacterium solution volume
Than Pi Shi Rolston bacterium：P. myrsinacearum：Sphingolipid monad=1:1:1 ratio is inoculated in containing tetrachloro-ethylene respectively
In 0.6mg/L sterilized base culture medium, in cultivating 72h in 30 DEG C of incubators, be taken as liquid bacterial agent refrigerate it is standby, you can
To tetrachloro-ethylene anaerobic degradation mix bacterium agent.
Embodiment 7
The preparation method of tetrachloro-ethylene anaerobic degradation mix bacterium agent.
(1) the Mixed Microbes bacterium solution 1ml for taking domestication to complete, by 1:101, 1:102, 1:103, 1:104, 1:105, 1:106, 1:
107, 1:108Obtain being serially diluted bacteria suspension after diluting step by step.1:106, 1:107, 1:108Serial bacteria suspension it is equal with " rubbing method "
It is even to be coated on LB solid mediums, 0.5mg/L tetrachloro-ethylenes are added, back-off is put into 30 DEG C of biochemical cultivation cases continuously after sealing
Cultivate 48~72h.When there is single bacterium colony, different shape feature is chosen, eugonic single bacterium colony is re-seeded into containing tetrachloro
On ethene 0.5mg/L LB solid mediums, cultivated in 30 DEG C of incubators, the same technical scheme of other conditions.
(2) repeatedly after purification, the eugonic single bacterium colony of picking is inoculated in containing tetrachloro in pure strain cultures for switching
Ethylene Liquid culture medium, enrichment culture is carried out again, and saved backup in 4 DEG C of refrigerators.
(3) fresh bacterium solution is taken to send sequencing company to be sequenced, by known sequence in obtained sequence and GenBank databases
Homologous comparison is carried out with the Pseudomonas of this determination bacterial strain.Identified tetrachloro-ethylene anaerobism Mixed Microbes mainly include Pi Shi Rolston bacterium
(Ralstonia pickettii), P. myrsinacearum (Phyllobacterium myrsinacearum), sphingolipid monad
(Sphingomonas aeria)。
(4) using the pure bacterial strain bacterium solution after enrichment as inoculum, after repeatedly sterile water washing is resuspended, by bacterium solution volume
Than Pi Shi Rolston bacterium：P. myrsinacearum：Sphingolipid monad=1:0.8:0.8 ratio is inoculated in containing tetrachloro respectively
In ethene 0.6mg/L sterilized base culture medium, in cultivating 72h in 30 DEG C of incubators, be taken as liquid bacterial agent refrigerate it is standby, i.e.,
It can obtain tetrachloro-ethylene anaerobic degradation mix bacterium agent.
Embodiment 8
The preparation method of tetrachloro-ethylene anaerobic degradation mix bacterium agent.
(1) the Mixed Microbes bacterium solution 1ml for taking domestication to complete, by 1:101, 1:102, 1:103, 1:104, 1:105, 1:106, 1:
107, 1:108Obtain being serially diluted bacteria suspension after diluting step by step.1:106, 1:107, 1:108Serial bacteria suspension it is equal with " rubbing method "
It is even to be coated on LB solid mediums, 0.5mg/L tetrachloro-ethylenes are added, back-off is put into 30 DEG C of biochemical cultivation cases continuously after sealing
Cultivate 48~72h.When there is single bacterium colony, different shape feature is chosen, eugonic single bacterium colony is re-seeded into containing tetrachloro
On ethene 0.5mg/L LB solid mediums, cultivated in 30 DEG C of incubators, the same technical scheme of other conditions.
(2) repeatedly after purification, the eugonic single bacterium colony of picking is inoculated in containing tetrachloro in pure strain cultures for switching
Ethylene Liquid culture medium, enrichment culture is carried out again, and saved backup in 4 DEG C of refrigerators.
(3) fresh bacterium solution is taken to send sequencing company to be sequenced, by known sequence in obtained sequence and GenBank databases
Homologous comparison is carried out with the Pseudomonas of this determination bacterial strain.Identified tetrachloro-ethylene anaerobism Mixed Microbes mainly include Pi Shi Rolston bacterium
(Ralstonia pickettii), P. myrsinacearum (Phyllobacterium myrsinacearum), sphingolipid monad
(Sphingomonas aeria)。
(4) using the pure bacterial strain bacterium solution after enrichment as inoculum, after repeatedly sterile water washing is resuspended, by bacterium solution volume
Than Pi Shi Rolston bacterium：P. myrsinacearum：Sphingolipid monad=1:0.9:0.9 ratio is inoculated in containing tetrachloro respectively
In ethene 0.6mg/L sterilized base culture medium, in cultivating 72h in 30 DEG C of incubators, be taken as liquid bacterial agent refrigerate it is standby, i.e.,
It can obtain tetrachloro-ethylene anaerobic degradation mix bacterium agent.
Embodiment 9
The application of tetrachloro-ethylene anaerobic degradation mix bacterium agent.
The water sample of the μ g/L containing tetrachloro-ethylene 400 is added in reactor, glucose is co-metabolic substance, Example 6
Three kinds of ratio microbial inoculums of middle preparation, by inoculum concentration 20%, pH value 7.0,25 DEG C of temperature, it is placed in constant temperature oscillator.Tetrachloro-ethylene
Final degradation rate reaches 94.3%, and obtained mix bacterium agent can effectively remove tetrachloro-ethylene in embodiment 6.
Embodiment 10
The application of tetrachloro-ethylene anaerobic degradation mix bacterium agent.
The water sample of the μ g/L containing tetrachloro-ethylene 400 is added in reactor, glucose is co-metabolic substance, Example 7
Three kinds of ratio microbial inoculums of middle preparation, by inoculum concentration 20%, pH value 7.0,25 DEG C of temperature, it is placed in constant temperature oscillator.Tetrachloro-ethylene
Final degradation rate reaches 92.1%, and obtained mix bacterium agent can effectively remove tetrachloro-ethylene in embodiment 7.
Embodiment 11
The application of tetrachloro-ethylene anaerobic degradation mix bacterium agent.
The water sample of the μ g/L containing tetrachloro-ethylene 400 is added in reactor, glucose is co-metabolic substance, Example 8
Three kinds of ratio microbial inoculums of middle preparation, by inoculum concentration 20%, pH value 7.0,25 DEG C of temperature, it is placed in constant temperature oscillator.Tetrachloro-ethylene
Final degradation rate reaches 91.6%, and obtained mix bacterium agent can effectively remove tetrachloro-ethylene in embodiment 8.
Embodiment described above is merely to illustrate technical key point, it is impossible to limits the model of the invention implemented with this
Enclose, i.e., the equal change or modification done according to the scope of the claims of the present invention and description, still fall within the scope that the present invention covers.
Claims (9)
1. a kind of preparation method of tetrachloro-ethylene anaerobic degradation Mixed Microbes, including be enriched with, tame and cultivate；It is characterized in that step is such as
Under：
(1) soil of 30~40cm under by the place earth's surface of tetrachloro-ethylene pollution for a long time is taken, removes deadwood weeds and stone standby
With standby.
(2) add glucose, dusty yeast, trace element and inorganic salts to prepare in basal medium, after adding the soil of step (1)
Sealing culture, wherein water and soil mass ratio 10::1~5:1；
(3) enrichment and domestication, the anaerobic degradation Mixed Microbes of tetrachloro-ethylene are obtained.
2. the method as described in claim 1, it is characterized in that in the step (2), inorganic salt content is in every liter of water：Inorganic salts
(mg/L)：NH4Cl, 320；Sulphur, 667；KH2PO4, 34；Na2CO3, 132；CaCl2, 63.
3. the method as described in claim 1, it is characterized in that in the step (2), micronutrient levels is in every liter of water：It is micro
Element (mg/L)：MgSO4, 2.5；H3BO3, 0.05；NiSO4·6H2O, 0.44；FeCl2·4H2O, 3；ZnSO4·7H2O, 0.05；
MnCl2·4H2O, 2.5；CoCl2·6H2O, 0.52；CuSO4·5H2O, 0.03；(NH4)6Mo7O24·4H2O, 0.32.
4. the method as described in claim 1, it is characterized in that in the step (2), every liter of aqueous humor glucose 1000mg/L, yeast
Powder 99.5mg/L.
5. the method as described in claim 1, it is characterized in that in step (3), using 2~3d as an enrichment cycle, it is enriched with 2 weeks,
Using 3~4d as an acclimation period, tame 4 weeks；Gradually increase the addition of tetrachloro-ethylene during domestication：By 0.2mg/L,
0.4mg/L, 0.6mg/L, 0.8mg/L, 1.0mg/L amount gradually increase, the corresponding addition for reducing energy substance：By 1000mg/
L, 800mg/L, 600mg/L, 400mg/L, 200mg/L amount gradually decrease.
Degraded 6. the Mixed Microbes that claim 1 method obtains are applied in the water containing tetrachloro-ethylene；Degradation condition is tetrachloro-ethylene
μ g/L of concentration 0.0~900.0, nitrogen source are dusty yeast.
7. the method that the Mixed Microbes that claim 1 method obtains prepare tetrachloro-ethylene anaerobic degradation mix bacterium agent, it is characterized in that step
It is rapid as follows：
(1) the anaerobic degradation Mixed Microbes bacterium solution tamed 4 weeks is taken, by 1:101, 1:102, 1:103, 1:104, 1:105, 1:106, 1:
107, 1:108, the ratio between total liquid volume dilutes step by step after adding Sterile dilution water, obtains the dilutions of 8 parts of different dilution ranks,
The serial bacteria suspension 1 of dilution is taken respectively:105, 1:106, 1:107, 1:108It is uniformly coated on LB solid mediums, adds 0.4
~0.8mg/L tetrachloro-ethylenes, it is placed in 28~30 DEG C of biochemical cultivation cases and continuously cultivates；When there is single bacterium colony, choose different
Morphological feature, the vigorous a fairly large number of single bacterium colony of growth are re-seeded on the LB solid mediums containing tetrachloro-ethylene, in 28~
Cultivated in 35 DEG C of incubators；The a fairly large number of single bacterium colony of picking is inoculated in fluid nutrient medium containing tetrachloro-ethylene after purified, then
Secondary carry out enrichment culture, after the completion of preserve bacterium solution, tetrachloro-ethylene anaerobism Mixed Microbes are made；
(2) bacterium solution in above-mentioned steps after purification is taken to send sequencing company sequencing with the species of this determination bacterial strain；Identified Mixed Microbes
Mainly contain Pi Shi Rolstons bacterium, P. myrsinacearum and sphingolipid monad；
(3) above-mentioned 3 kinds of bacterial solutions are taken as inoculum, by inoculation bacterium solution volume proportioning Pi Shi Rolston bacterium：Japanese ardisia leaf
Bacillus：Sphingolipid monad=1:0.8~1:0.8~1 is added in the basal medium containing 0.5~1.0mg/L tetrachloro-ethylenes,
In cultivating 48~72h in 28~35 DEG C of incubators, liquid mix bacterium agent is obtained.
8. method as claimed in claim 7, it is characterized in that the compound method of LB culture mediums is NaCl 10.0g in step (1),
Peptone 10.0g, dusty yeast 5.0g, 10~15.0g of agar, distilled water 1L, 6.8~7.2,121 DEG C of autoclavings of pH value
30min, sterile working.
9. mix bacterium agent made from claim 7 method is applied in the water containing tetrachloro-ethylene；Tetrachloro-ethylene anaerobic degradation mixes
Microbial inoculum in glucose as 25 DEG C of co-metabolic substance, temperature, pH value 7.0, inoculation 10~20%, constant temperature oscillator rotating speed 100~
Applied under conditions of 120r/min.
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| CN108911120A (en) * | 2018-07-18 | 2018-11-30 | 山西龙盘微生物科技有限公司 | A kind of acclimation method for daily use chemicals cleaning product activated sludge of degrading |
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| CN111676147A (en) * | 2020-05-07 | 2020-09-18 | 中山大学 | Method for enriching and separating dechlorinating tetrachloroethylene bacteria and application |
| CN116396898A (en) * | 2023-03-10 | 2023-07-07 | 江苏诚冉环境修复工程有限公司 | 1, 2-trichloroethane degrading bacterium and application thereof |
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