CN109536417A - A kind of biology drop phenol microbial inoculum and its application method - Google Patents
A kind of biology drop phenol microbial inoculum and its application method Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, provide a kind of high-performance bio drop phenol microbial inoculum and its application method, the composite bacteria agent is made of two kinds of bacterium, the self-contained phenol sewage disposal system of strain isolation, it is respectively Alcaligenes and bacillus cereus by 16SrDNA identification, two bacterial strain deposit numbers are respectively CGMCC NO.16158 and CGMCC NO.16157, using when, according to the content for volatile phenol of intaking in sewerage, by Alcaligenes and bacillus cereus bacterium powder by weight 1-3:1 admixture activation, then it is inoculated in aerobic sludge according to the volume ratio of 1:100-1000, drop phenol ability in 12 hours is up to 99%.It is numerous that strain of the invention is easy to cultivate expansion, it is prepared as solid bacterium powder, it is readily transported and stores, when in use can be according to the amount and ratio of selection addition bacterium powder the case where influent quality, strain separating environment from sewage disposal system is adaptable, resistance to phenol ability is strong, degradation concentration is high, degradation speed is fast, does not easily cause secondary pollution.
Description
Technical field
The invention belongs to technical field of bioengineering, a kind of biology drop phenol microbial inoculum and its application method are provided.
Background technique
Phenol is widely used as the raw material of Chemical Manufacture, for manufacturing a variety of industrial products.Producing these industrial products
During the phenols wastewaters of different pollution concentrations can be discharged.Phenol is a kind of highly toxic substance, it can generate plasm very big
Bio-toxicity.The phenol of various concentration, the denaturation and precipitating that protein can be made different degrees of.Phenol can cause biological cell directly
Injury, and there is extremely strong epithelial cell corrosivity.Long-term contacting or drink will appear slow poisoning by its water polluted
Phenomenon；It also will affect the normal growth and breeding of aquatile, when concentration is big, or even will cause a large amount of dead of aquatile
It dies；When phenol wastewater is used for irrigated farmland simultaneously, crop production reduction and death are also resulted in.
The processing method of phenol wastewater includes: physical method, chemical method and bioanalysis.Wherein physics and method of chemical treatment are all deposited
It is at high cost, complicated for operation, be easy to cause the defects of secondary pollution.Bioanalysis is mainly using with efficient degradation phenol object
The decomposition and inversion ability of the microorganism of mass-energy power converts inorganic matter for the phenolic compound in water, is purified waste water, reaches
To national emission standard.For existing technology, bioanalysis has economical, efficient compared with physics, chemical method
Advantage, prior bioanalysis can be achieved on innoxious, without secondary pollution, and it is to study at present that water is big for processing, sludge output is low
More Treatment of Phenol Containing Water.
Biologic treating technique is confined to the phenol wastewater of processing low concentration, the height biology poison of high-concentration phenolic wastewater at present
Property, the application of this technology is constrained, and in the prior art substantially or based on single culture improvement phenol wastewater, it is difficult to reach
To preferably promotion, therefore separate has the bacterial strain or flora of high tolerance and degradation capability applied to biological reinforced with breeding
Technology, which seems, to be even more important.Looking for, which becomes those skilled in the art for the more applicable drop phenol microbial inoculum of phenol wastewater, is difficult to solve
The problem of.
Summary of the invention
The present invention is existing insufficient in view of the above technology, provides a kind of high-performance bio drop phenol microbial inoculum and its application method,
The composite bacteria agent is made of two kinds of bacterium, the self-contained phenol sewage disposal system of strain isolation, is respectively to produce by 16SrDNA identification
Alkali bacterium and bacillus cereus, two bacterial strain deposit numbers are respectively CGMCC NO.16158 and CGMCC NO.16157, application
When, according to the content for volatile phenol of intaking in sewerage, added according to the amount of 0.1-1g/L, by Alcaligenes and bacillus cereus
Bacterium powder after 1-3:1 admixture activation by weight using, and the drop phenol ability in 12 hours is up to 99%.Strain of the invention is easy to cultivate
Expand numerous, is prepared as solid bacterium powder, is readily transported and stores, it when in use can be according to selection addition bacterium powder the case where influent quality
Amount and ratio, strain separating environment from sewage disposal system is adaptable, resistance to phenol ability is strong, and maximum tolerance is reachable
3000mg/L, degradation concentration is high, degradation speed is fast, does not easily cause secondary pollution, and shows compound bacteria degrading phenol by experiment
The effect and treatment effeciency of compound are much larger than single culture.
The present inventor obtains two plants of microorganisms with preferable phenol decomposition effect first, is respectively Alcaligenes
(Alcaligenes spp) and bacillus cereus (Bacillus cereus),
Inventor is respectively designated as YJY18-10 and YJY18-09；
And it is preserved in Chinese microorganism strain preservation conservator's common micro-organisms center, deposit number: produce alkali
Bacterium is CGMCC No.16158, and bacillus cereus is CGMCC No.16157, and qualification result is survival.
The morphological feature Gram-negative bacteria of above-mentioned Alcaligenes, no pod membrane, rod-short, on ordinary nutrient agar culture medium
Circular petite is formed, is milky white or yellowish；The morphological feature Gram-positive of bacillus cereus, no pod membrane, bar
Shape, bacterium colony is big, round or approximate circle, non-pigment, slightly glossiness white colony；
Inventor has carried out 16SrDNA sequencing to it respectively, and nucleotide sequence is respectively Seq ID No:1 and Seq ID
Shown in No:2, which is the complete sequence of the 16SrDNA of bacterial strain.Measured 16SrDNA sequence carries out BLAST comparison, is dividing
It is determined as Alcaligenes and bacillus cereus in sub- level.
It is as follows that its method applied to phenols wastewater processing when is further provided in inventor after obtaining above-mentioned bacterial strains:
Bacterium powder is obtained first with above-mentioned bacterial strains, the preparation method of bacterium powder is as follows:
(1) Alcaligenes, bacillus cereus fermented and cultured: are inoculated in fermented and cultured in sterilizing post-fermentation culture medium respectively
Obtain respective bacterium solution；Under conditions of temperature is 28-30 DEG C, fermentation tank culture to dissolved oxygen rises, pH decline；
(2) it prepares bacterium powder: after fermentation liquid is centrifuged, adding auxiliary materials and mixing, obtain solid bacterium powder after drying, specifically
The step of it is as follows:
1) actication of culture: on aseptic operating platform, the Alcaligenes that take 1~5 μ L to freeze and bacillus cereus are respectively at containing
Have in the test tube of LB liquid medium, 28-32 DEG C, 130-180rpm cultivates 18-24h；
2) prepared by seed: on aseptic operating platform, the strain of activation being transferred to the triangle containing sterilized liquid LB culture medium
In bottle, the 200mL culture medium is dispensed in every triangular flask, the test tube strains of an activation are inoculated with a triangular flask, then 28-32
DEG C, 130~180rpm cultivates 18-24h；
3) inoculation fermentation tank: fermentation tank, pipeline, air filter are subjected to sky first and disappeared, 121 DEG C, sky disappears
30min injects culture medium after temperature reduction, then disappear in fact to culture medium, 121 DEG C, disappear 30min in fact；It is down to temperature
It is inoculated at 28-32 DEG C；
4) fermentation condition: inoculum concentration v/v 5-10%；28-32 DEG C of cultivation temperature；PH:6.8-7.0；Initial speed:
200rpm；Fermentor pressure: 0.05Mpa；Ventilatory capacity ratio: 1:1；Fermentation period: 16-24h；
5) prepared by bacterium powder: after fermentation, addition diatomite, cornstarch adsorb thallus, dry later, crush and can obtain
To corresponding bacterium powder.
The bacterium powder obtained by the above method, bacterium powder bacterium amount single bacterium can reach 9 × 109-5×1010A/g.
In above-mentioned steps, the formula of LB liquid medium are as follows: peptone 10g/L, NaCl 10g/L, yeast extract 5g/L,
PH 7.0-7.2, solvent are water；
For being formed for the fermentation medium of Alcaligenes in the culture medium in fermentor are as follows:
By weight are as follows: 1.0-1.5 parts of peptones, 0.5-1 parts of yeast extracts, 1-1.5 parts of sodium chloride, 96-97.5
Part softened water；
For the fermentation medium composition of bacillus cereus are as follows: by weight are as follows: 1-1.5 parts of glucose, 1-1.5 parts
Dregs of beans, 0.2-0.3 divide ammonium nitrate, 0.01-0.03 parts of manganese sulfates, 0.03-0.07 parts of magnesium sulfate, 0.02-0.05 parts of sodium chloride,
0.02-0.05 parts of calcium chloride, 0.003-0.005 parts of ferrous sulfate, 96.5-97.7 parts of softened waters；
(3) strain compounds: by experiment of single factor twice, screening degradation effect and most significantly combines.
After obtaining above-mentioned bacterium powder, when being applied to sewerage, ratio is added according to the weight of the dischargeable capacity of aerobic tank
Volume ratio (g/L) 1 ‰ -1% is added, which is used to drop phenol processing, the two bacterium powder for activation after need to being activated
Weight ratio Alcaligenes bacterium powder: bacillus cereus bacterium powder is 1-3:1, and both more preferably ratio is 1:1.
The sewage of sewerage in above-mentioned strain compound process is derived from two emersion water of petrochemical industry, pH6.8-7.1；
The activation method of above-mentioned bacterium powder when in use are as follows:
Bacterium powder is inoculated with according to the w/v of 1-5g/L, at 28-32 DEG C in nutrient salt solution, 130-180rpm
Shaking table culture 1-2h is activated, and wherein the composition of nutrient salt solution is as follows: by weight are as follows:
0.3-0.5 parts of dipotassium hydrogen phosphates；0.01-0.02 parts of ferrous sulfate；0.1-0.3 divides calcium chloride；0.3-0.5 parts of phosphoric acid
Potassium dihydrogen；0.1-0.5 divides magnesium sulfate；0.01-0.03 parts of manganese sulfates；0.1-0.2 parts of sodium chloride；0.5-1 parts of ammonium nitrate, 97-
98.6 part softened water；
In above-mentioned activation salting liquid, while the phenol for proportionally adding 300mg/L is activated as sole carbon source
Processing；
Phenol wastewater can be handled after above-mentioned microbial inoculum is added, but generally require wastewater pH when control processing
6.0-7.0,25-35 DEG C of temperature, the content that microbial inoculum provided by the present invention can handle water inlet phenol is lower than the sewage of 3000mg/L；On
Under the conditions of stating, microbial inoculum provided by the present invention can preferably work.
The microbial inoculum that the present invention is invented is the strain strong by the tolerance by screening obtained in sewage disposal system, and
Inventor uses the composite bacteria agent that Alcaligenes bacterium powder and bacillus cereus bacterium powder compound and handles, with conventional single culture
The microbial inoculum of composition is compared, and environmental suitability is stronger, while its composition is clear, quality controllable, is obtained after two strains compounding compound
It is up to 3500mg/L to the tolerance of phenol that it, which can be improved, in microbial inoculum, much higher than the tolerance of existing single culture；
In preparation process of the invention, microbial inoculum is prepared into the problem of bacterium powder is in order to avoid subsequent transportation, convenient for storage
And transport, facilitate and expands its use scope, and it, by activation, is being induced to drop the performance of phenol ability using preceding, inventor passes through
The ratio of two kinds of strains is compounded, weeds out wherein the combination that tolerance is poor, degradation effect is unstable, degradation capability is poor, most
The optimum proportioning for having obtained above two bacterium powder eventually constructs efficiently drop phenol microbial inoculum system, verifies through pilot scale, the composite bacteria agent pair
The maximum tolerance of phenol is in 3500mg/L or so, and stable drop phenol ability reaches 3000mg/L to simulated production device for 24 hours；It is dropped stablizing
While phenol ability, COD degradation 80%, ammonia nitrogen reduces by 40% or more；
In addition to this composite bacteria fermentation period of the invention is short, and single bacteria fermentation time 24-48h is sent out using pilot scale
Fermentation tank can satisfy production requirement, can produce in batches；Used fermentation medium is the agricultural and sideline product and nutritive salt of routine,
It is tamed without phenol etc., improves production environment, reduce the pollution to environment, the cost for reducing production and using；
In conclusion strain of the invention be it is purebred be easy to cultivate expand it is numerous, be prepared as solid bacterium powder, be readily transported and store up
It deposits, for strain separating from sewage disposal system, environmental suitability is strong, resistance to phenol ability is strong, degradation concentration is high, degradation speed is fast, is not easy
Cause secondary pollution.
Inventor has carried out biological deposits to two bacterial strains, and preservation information is as follows:
Preservation information
The preservation time: on July 25th, 2018
Depositary institution's title: Chinese microorganism strain preservation conservator's common micro-organisms center
Deposit number: CGMCC No.16157
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology, the Chinese Academy of Sciences
Classification naming: bacillus cereus (Bacillus cereus)
The preservation time: on July 25th, 2018
Depositary institution's title: Chinese microorganism strain preservation conservator's common micro-organisms center
Deposit number: CGMCC No.16158
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology, the Chinese Academy of Sciences
Classification naming: Alcaligenes (Alcaligenes spp)
Detailed description of the invention
Fig. 1 is that its volatile phenol degradation for 24 hours that the microbial inoculum in embodiment 1 measures under the volatile phenol concentration of 3000mg/L is bent
Line；
It can be seen that when the microbial inoculum provided in embodiment 1 handles sewage, in 12h under the basic linear state of volatile phenol content
Drop has all been degraded when to 12h, and effect is much higher than blank group.
Specific embodiment
The specific embodiment of form by the following examples does further specifically above content of the invention
It is bright, but the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention, and it is complete to be all made of conventional prior unless otherwise specified, in following embodiments
At.
The acquisition of 1 bacterial strain of embodiment
Soil sample near phenol-containing wastewater processing system, separation screening obtain totally 30 plants of single bacterium, wherein having degradation
The bacterial strain of volatile phenol ability has 14 plants；
In the bacterial strain that there is drop phenol ability from 14 plants, by improving phenol concentration, while to its genetic stability and being applicable in
The research such as range screens two plants of bacterial strains that volatile phenols are high-efficient, are easy to cultivate and can stablize heredity of degrading；
Inventor has carried out 16SrDNA sequencing to it respectively, and nucleotide sequence is respectively Seq ID No:1 and Seq ID
Shown in No:2, which is the complete sequence of the 16SrDNA of bacterial strain.Measured 16SrDNA sequence carries out BLAST comparison, is dividing
It is determined as respectively Alcaligenes (Alcaligenes spp) and bacillus cereus (Bacillus cereus) in sub- level,
Inventor is respectively designated as YJY18-10 and YJY18-09；And it is preserved in Chinese microorganism strain preservation
Conservator's common micro-organisms center, deposit number: Alcaligenes are CGMCC No.16158, bacillus cereus CGMCC
No.16157, qualification result are survival.
The morphological feature Gram-negative bacteria of above-mentioned Alcaligenes, no pod membrane, rod-short, on ordinary nutrient agar culture medium
Circular petite is formed, is milky white or yellowish；The morphological feature Gram-positive of bacillus cereus, no pod membrane, bar
Shape, bacterium colony is big, round or approximate circle, non-pigment, slightly glossiness white colony.
The acquisition of 2 bacterium powder of embodiment
Bacterium powder is obtained using above-mentioned bacterial strains, the preparation method of bacterium powder is as follows:
(1) Alcaligenes, bacillus cereus fermented and cultured: are inoculated in fermented and cultured in sterilizing post-fermentation culture medium respectively
Obtain respective bacterium solution；Under conditions of temperature is 28-30 DEG C, fermentation tank culture to dissolved oxygen rises, pH decline；
(2) it prepares bacterium powder: after fermentation liquid is centrifuged, adding auxiliary materials and mixing, obtain solid bacterium powder after drying,
More specifically steps are as follows for the preparation of two kinds of bacterium powder:
1) actication of culture: on aseptic operating platform, the Alcaligenes that take 1-5 μ L to freeze and bacillus cereus are in containing LB liquid
In the test tube of body culture medium, 28-32 DEG C, 130-180rpm cultivates 18-24h；
2) prepared by seed: on aseptic operating platform, the strain of activation being transferred to the triangle containing sterilized liquid LB culture medium
In bottle, the 200mL culture medium is dispensed in every triangular flask, the test tube strains of an activation are inoculated with a triangular flask, then 28-32
DEG C, 130-180rpm cultivates 18-24h；
3) inoculation fermentation tank: fermentation tank, pipeline, air filter are subjected to sky first and disappeared, 121 DEG C, sky disappears
30min injects culture medium after temperature reduction, then disappear in fact to culture medium, 121 DEG C, disappear 30min in fact；It is down to temperature
It is inoculated at 28-32 DEG C；
4) fermentation condition: inoculum concentration v/v 5-10%；28-32 DEG C of cultivation temperature；PH:6.8-7.0；Initial speed:
200rpm；Fermentor pressure: 0.05Mpa；Ventilatory capacity ratio: 1:1；Fermentation period: 16-24h；
5) prepared by bacterium powder: after fermentation, adding diatomite or cornstarch adsorbs thallus, dry, crush later
Obtain corresponding bacterium powder.
The bacterium powder obtained by the above method, bacterium powder bacterium amount single bacterium can reach 9 × 109-5×1010A/g.
In above-mentioned steps, the formula of LB liquid medium are as follows: peptone 10g/L, NaCl 10g/L, yeast extract 5g/L,
PH 7.0-7.2, solvent are water；
In fluid nutrient medium, formed for the fermentation medium of Alcaligenes are as follows:
By weight are as follows: 1.0-1.5 parts of peptones, 0.5-1 parts of yeast extracts, 1-1.5 parts of sodium chloride, 96-97.5
Part softened water；
For the fermentation medium composition of bacillus cereus are as follows: by weight are as follows: 1-1.5 parts of glucose, 1-1.5 parts
Dregs of beans, 0.2-0.3 divide ammonium nitrate, 0.01-0.03 parts of manganese sulfates, 0.03-0.07 parts of magnesium sulfate, 0.02-0.05 parts of sodium chloride,
0.02-0.05 parts of calcium chloride, 0.003-0.005 parts of ferrous sulfate, 96.5-97.7 parts of softened waters.
The utilization 1 of 3 composite bacteria agent of embodiment
The bacterium powder that embodiment 2 obtains needs to be activated in advance when in use, activation method are as follows:
Bacterium powder be inoculated in nutritive salt being activated according to the w/v of 1-5g/L, activation condition are as follows: 28-32
DEG C, 130-180rpm shaking table culture 1-2h is activated,
Wherein the composition of nutrient salt solution is as follows: by weight are as follows:
0.3-0.5 parts of dipotassium hydrogen phosphates；0.01-0.02 parts of ferrous sulfate；0.1-0.3 divides calcium chloride；0.3-0.5 parts of phosphoric acid
Potassium dihydrogen；0.1-0.5 divides magnesium sulfate；0.01-0.03 parts of manganese sulfates；0.1-0.2 parts of sodium chloride；0.5-1 parts of ammonium nitrate, 97-
98.6 part softened water.
In above-mentioned activation salting liquid, while the phenol for proportionally adding 300mg/L is activated as sole carbon source
Processing；
1. water quality treatment: certain two emersion water of sewage treatment plant, petro-chemical corporation, COD:3000-3200mg/L, volatile phenol: 400-
500mg/L, ammonia nitrogen: 80-100mg/L is reached in following tests by adding the volatile phenol concentration in industrial phenol adjusting water inlet
Requirement, it is used industry phenol in 99% or more phenol content；
2. water process amount: total 6m3
3. processing method and technique: composite bacteria agent technical treatment phenol wastewater is utilized, according to the dischargeable capacity of aerobic tank
W/v (g/L) 0.1-1% is added, Alcaligenes bacterium powder in composite bacteria agent: the weight ratio of bacillus cereus bacterium powder
Respectively 1:1,3:1,1:3；
Specific use process is as follows: a part of aerobic sludge is added in processing pond, microbial inoculum adds the bulking value according to 1%
It is added than (g/L), two kinds of microbial inoculums add after activating according to corresponding ratio, and aeration makes dissolved oxygen in 2mg/L or more, and bored exposure is two days later
The primary total 1m of daily Inlet and outlet water3, control processing when sewage condition meet: sewage pH6.0-7.0,25-35 DEG C of temperature simultaneously by
It walks and adds industrial phenol raising water inlet phenol content, not add the activated sludge for dropping phenol compound bacteria as control, phenol content of intaking
In 2000mg/L for 24 hours after, the experimental group of 1:1 and 3:1 volatilization Phenol degradation rate is the degradation rate of 99%, 1:3 experimental group 95%,
Phenol content of intaking in 3000mg/L for 24 hours after, the experimental group of 1:1 and 3:1 volatilization Phenol degradation rate can still maintain 99%, blank group drop
For phenol rate when volatile phenol content is improved to 1500mg/L, degradation rate is less than 20%；It can be seen that Alcaligenes bacterium powder: bacillus cereus
The ratio of bacterium powder is in 1-3:1 within the scope of this, the water inlet highest of amount containing phenol of tolerance, and degradation rate is best.
The utilization 2 of 4 composite bacteria agent of embodiment
Bacterium powder is activated according to the method for embodiment 3, verifies its degradation capability to other volatile phenol substances.
1. water quality treatment: certain sewage treatment plant, petro-chemical corporation sewage, COD:3000-3200mg/L, volatile phenol: 400-
500mg/L, ammonia nitrogen: the recycling phenol of 80-100mg/L, addition petrochemical industry phenol extraction device adjust influent concentration, recycle in phenol
Phenol content is about 90%, other are some creosotes；
2. water process amount: total 6m3
3. processing method and technique: composite bacteria agent technical treatment phenol wastewater is utilized, according to the dischargeable capacity of aerobic tank
W/v (g/L) 0.1-1% is added, Alcaligenes bacterium powder in composite bacteria agent: the weight ratio of bacillus cereus bacterium powder
For 1:1；
Specific use process is as follows: adding a part of aerobic sludge in processing pond, a part of aerobic dirt is added in processing pond
Mud, microbial inoculum add w/v (g/L) addition according to 0.5%, and aeration makes dissolved oxygen in 2mg/L or more, and bored exposure is every two days later
The primary total 1m of its Inlet and outlet water3, control processing when sewage condition meet: sewage pH6.0-7.0,25-35 DEG C of temperature simultaneously gradually
Improve water inlet phenol content, using do not add drop phenol compound bacteria activated sludge as control, water inlet phenol content in 3000mg/L for 24 hours
When volatilization Phenol degradation rate is 99%, 3200mg/L afterwards, for degradation rate in 95% or more, 3500mg/L, degradation rate is about 85%, empty
White group of drop phenol rate later period is less than 20%；
Its volatile phenol degradation curve for 24 hours is measured under the volatile phenol concentration of 3000mg/L simultaneously, every 4h sampling is primary, sees
Attached drawing 1, experimental group volatile phenol content in 12h basic linear state decline as can be seen from Figure, when to 12h all
Degradation.
The utilization 3 of 5 composite bacteria agent of embodiment
Bacterium powder is activated according to the method for embodiment 3, verifies its degradation capability to other volatile phenol substances.
1. water quality treatment: certain small-sized sewage treatment plant, Coking Company, water quality indicator: COD:2800-3000mg/L, volatilization
Phenol: 300-400mg/L, ammonia nitrogen: 150-180mg/L passes through addition to verify its degradation capability in the way of in embodiment 3
Industrial phenol is stepped up into water volatile phenol content；
2. water process amount: total 6m3
3. processing method and technique: composite bacteria agent technical treatment phenol wastewater is utilized, according to the dischargeable capacity of aerobic tank
W/v (g/L) 1% is added, Alcaligenes bacterium powder in composite bacteria agent: the weight proportion of bacillus cereus bacterium powder is
1:1。
Concrete processing procedure is as follows: the aerobic sludge of a part of sewage disposal system is added in processing pond, according to above-mentioned
Ratio addition drop phenol microbial inoculum, aeration make dissolved oxygen in 2mg/L or more, the bored exposure primary total 1m of daily Inlet and outlet water two days later3, pass through simultaneously
It adds industrial phenol to step up into water phenol content, while the activated sludge not add drop phenol compound bacteria is intake as control
Phenol content in 3100mg/L for 24 hours afterwards volatilization Phenol degradation rate be 99%, 3300mg/L when, degradation rate is in 95% or more, 3500mg/
When L, degradation rate is about 85%, and blank group water inlet volatile phenol content drops phenol rate in 800mg/L and is decreased obviously, less than 20%.It can
See that composite bacteria agent provided by the present invention, in 3500mg/L, is much higher than existing microbial inoculum to the maximum tolerance of phenol.
Sequence table
<110>Yellow River Delta Jingbo Chemical Research Institute Co., Ltd.
<120>a kind of biology drop phenol microbial inoculum and its application method
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 1432
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ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac gacagccatg 420
cagcacctgt gttccggttc tcttgcgagc acggccaaat ctcttcggct ttccagacat 480
gtcaagggta ggtaaggttt ttcgcgttgc atcgaattaa tccacatcat ccaccgcttg 540
tgcgggtccc cgtcaattcc tttgagtttt aatcttgcga ccgtactccc caggcggtca 600
acttcacgcg ttagctgcgc tactaaggcc taacggcccc aacagctagt tgacatcgtt 660
tagggcgtgg actaccaggg tatctaatcc tgtttgctcc ccacgctttc gtgtctgagc 720
gtcagtatta tcccaggggg ctgccttcgc catcggtatt cctccacata tctacgcatt 780
tcactgctac acgtggaatt ctacccccct ctgacatact ctagctcggc agttaaaaat 840
gcagttccaa ggttgagccc tgggatttca catctttctt tccgaaccgc ctacacacgc 900
tttacgccca gtaattccga ttaacgcttg caccctacgt attaccgcgg ctgctggcac 960
gtagttagcc ggtgcttatt ctgcagatac cgtcagcagc atcccgtatt aggggatgcc 1020
ttttcttctc tgccaaaagt actttacaac ccgaaggcct tcatcataca cgcgggatgg 1080
ctggatcagg gtttccccca ttgtccaaaa ttccccactg ctgcctcccg taggagtctg 1140
ggccgtgtct cagtcccagt gtggctggtc gtcctctcaa accagctacg gatcgttgcc 1200
ttggtgagcc tttaccccac caactagcta atccgatatc ggccgctcca atagtgagag 1260
gtcttgcgat cccccccttt cccccgtagg gcgtatgcgg tattagccac tctttcgagt 1320
agttatcccc cgctactggg cacgttccga tatattactc acccgtccgc cactcgccac 1380
caagagagca agctctctcg tgctgccgtt cgacttgcat gtgaaagctg cg 1432
<210> 2
<211> 1055
<212> DNA
<213>Bacillus cercus (Bacillus cereus)
<400> 2
ccatgggcgg gctatacatg caagtcgagc gaatggatta agagcttgct cttatgaagt 60
tagcggcgga cgggtgagta acacgtgggt aacctgccca taagactggg ataactccgg 120
gaaaccgggg ctaataccgg ataacatttt gaaccgcatg gttcgaaatt gaaaggcggc 180
ttcggctgtc acttatggat ggacccgcgt cgcattagct agttggtgag gtaacggctc 240
accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360
cggagcaacg ccgcgtgagt gatgaaggct ttcgggtcgt aaaactctgt tgttagggaa 420
gaacaagtgc tagttgaata agctggcacc ttgacggtac ctaaccagaa agccacggct 480
aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttatccgg aattattggg 540
cgtaaagcgc gcgcaggtgg tttcttaagt ctgatgtgaa agcccacggc tcaaccgtgg 600
agggtcattg gaaactggga gacttgagtg cagaagagga aagtggaatt ccatgtgtag 660
cggtgaaatg cgtagagata tggaggaaca ccagtggcga aggcgacttt ctggtctgta 720
actgacactg aggcgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac 780
gccgtaaacg atgagtgcta agtgttagag ggtttccgcc ctttagtgct gaagttaacg 840
cattaagcac tccgcctggg gagtacggcc gcaaggctga aactcaaagg aattgacggg 900
ggcccgcaca gcggtggagc atgtggttta ttcgagcacg cgagacctta ccaggtctga 960
catctctgac accctagaga taggctctct cggagcagag tgacaggtgg tgcatgtgtc 1020
gtcagctcgt gtcgtgagat gttgggtagt cgcac 1055
Claims (10)
1. a kind of biology drop phenol microbial inoculum, it is characterised in that: be made of the bacterium powder of two plants of bacterium, respectively Alcaligenes (Alcaligenes
Spp), bacterial strain code is YJY18-10, and deposit number is CGMCC NO.16158；Bacillus cereus (Bacillus
Cereus), bacterial strain code is YJY18-09；Its deposit number is CGMCC NO.16157.
2. biology drop phenol microbial inoculum according to claim 1, it is characterised in that: the bacterium amount of the bacterium powder is single bacterium 9 × 109-5
×1010A/g.
3. biology drop phenol microbial inoculum according to claim 1, it is characterised in that: the bacterium powder is by Alcaligenes bacterium powder and waxy bud
Spore bacillus bacterium powder is compounded according to the weight ratio of 1-3:1, is used after activation.
4. a kind of preparation method of biology drop phenol microbial inoculum, which is characterized in that including following operating procedure:
(1) fermented and cultured: Alcaligenes, bacillus cereus are inoculated in fermented and cultured in sterilizing post-fermentation culture medium respectively and obtained
Respective bacterium solution；Under conditions of temperature is 28-30 DEG C, fermentation tank culture to dissolved oxygen rises, pH decline；
(2) it prepares bacterium powder: after fermentation liquid is centrifuged, adding auxiliary materials and mixing, solid bacterium powder is obtained after drying.
5. the preparation method of high-performance bio drop phenol microbial inoculum according to claim 4, which is characterized in that specific step is as follows:
1) actication of culture: on aseptic operating platform, the Alcaligenes that take 1~5 μ L to freeze and bacillus cereus are in containing LB liquid
In the test tube of culture medium, 28-32 DEG C, 130-180rpm cultivates 18-24h；
2) prepared by seed: on aseptic operating platform, the strain of activation is transferred in the triangular flask containing sterilized liquid LB culture medium,
Packing 200mL culture medium in every triangular flask, test tube strains one triangular flask of inoculation of an activation, then 28-32 DEG C, 130-
180rpm cultivates 18-24h；
3) inoculation fermentation tank: carrying out fermentation tank, pipeline, air filter sky and disappear first, and 121 DEG C, sky disappears 30min, to
After temperature reduces, culture medium is injected, then culture medium disappear in fact, 121 DEG C, disappear 30min in fact；28-32 DEG C is down to temperature
Shi Jinhang inoculation；
4) fermentation condition: inoculum concentration v/v 5-10%；28-32 DEG C of cultivation temperature；PH:6.8-7.0；Initial speed: 200rpm；Hair
Fermentation tank pressure: 0.05Mpa；Ventilatory capacity ratio: 1:1；Fermentation period: 16-24h；
5) prepared by bacterium powder: after fermentation, addition diatomite, cornstarch adsorb thallus, dry later, phase can be obtained in crushing
Answer bacterium powder.
6. the preparation method of biology drop phenol microbial inoculum according to claim 5, it is characterised in that: described for strain isolation
Culture medium is LB culture medium, culture medium prescription are as follows: peptone 10g/L, NaCl 10g/L, yeast extract 5g/L, pH 7.0-7.2,
Solvent is water.
7. the preparation method of biology drop phenol microbial inoculum according to claim 5, which is characterized in that for cultivating two kinds of strains
In culture medium, formed for the fermentation medium of Alcaligenes are as follows:
By weight are as follows: 1.0~1.5 parts of peptones, 0.5-1 parts of yeast extracts, 1~1.5 part of sodium chloride, 96~97.5 parts
Softened water；
For the fermentation medium composition of bacillus cereus are as follows: by weight are as follows: 1-1.5 parts of glucose, 1-1.5 parts of beans
The dregs of rice, 0.2-0.3 divide ammonium nitrate, 0.01-0.03 parts of manganese sulfates, 0.03-0.07 parts of magnesium sulfate, 0.02-0.05 parts of sodium chloride,
0.02-0.05 parts of calcium chloride, 0.003-0.005 parts of ferrous sulfate, 96.5-97.7 parts of softened waters.
8. the application method of the drop phenol microbial inoculum of biology described in claim 1, it is characterised in that: use condition is wastewater pH 6.0-
7.0 25-35 DEG C of temperature, the content for phenol of intaking in phenol wastewater is lower than 3000mg/L.
9. the application method of biology drop phenol microbial inoculum according to claim 8, it is characterised in that:
It needs to activate bacterium powder before use, specific activation method are as follows:
Bacterium powder is inoculated with according to the w/v of 1-5g/L, at 28-32 DEG C in nutrient salt solution, 130-180rpm shaking table
Culture 1-2h is activated,
Wherein the composition of nutrient salt solution is as follows: by weight are as follows:
0.3-0.5 parts of dipotassium hydrogen phosphates；0.01-0.02 parts of ferrous sulfate；0.1-0.3 divides calcium chloride；0.3-0.5 parts of biphosphates
Potassium；0.1-0.5 divides magnesium sulfate；0.01-0.03 parts of manganese sulfates；0.1-0.2 parts of sodium chloride；0.5-1 parts of ammonium nitrate, 97-98.6 parts
Softened water；
In above-mentioned activation salting liquid, while the phenol for proportionally adding 300mg/L is activated as sole carbon source.
10. the application method of biology drop phenol microbial inoculum according to claim 8, it is characterised in that: when being applied to sewerage,
It adds ratio and is added according to the w/v 0.1-1g/L of the dischargeable capacity of aerobic tank.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109456906A (en) * | 2018-12-27 | 2019-03-12 | 黄河三角洲京博化工研究院有限公司 | A kind of alcohol-containing type organic sewage dedicated processes microbial inoculum and processing method |
| CN109576187A (en) * | 2018-12-27 | 2019-04-05 | 黄河三角洲京博化工研究院有限公司 | One plant of cyanide degradation bacterial strain and the method for utilizing the strains for degrading cyanide |
| CN109628345A (en) * | 2018-12-28 | 2019-04-16 | 天津开发区坤禾生物技术有限公司 | A kind of prevention and treatment succulent brown rot composite bacteria agent and its preparation method and application |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002100781A1 (en) * | 2001-06-08 | 2002-12-19 | Idratech S.R.L. | Plant and method for purification of water coming from a seulphuration kerosene plant |
| CN102154174A (en) * | 2011-01-24 | 2011-08-17 | 中国科学院广州地球化学研究所 | Bacillus GZT and use thereof in degrading tribromophenol brominated flame retardant |
| CN104611247A (en) * | 2014-07-18 | 2015-05-13 | 中国矿业大学(北京) | Application of Alcaligenes faecalis sp. (DQP3) in degradation of phenol and quinoline |
| CN105154366A (en) * | 2015-09-25 | 2015-12-16 | 哈尔滨理工大学 | Bacillus cereus capable of degrading phenol and having electrogenesis characteristic and application thereof |
| CN106478288A (en) * | 2016-09-18 | 2017-03-08 | 广西大学 | A kind of soil conditioner of utilization microbial degradation Pesticide Residue in Soil |
| CN108220209A (en) * | 2018-03-26 | 2018-06-29 | 宁波大学 | A kind of binary composite bacteria agent used for aquiculture |
| WO2018145294A1 (en) * | 2017-02-10 | 2018-08-16 | Perfect (China) Co., Ltd. | Novel probiotics bifidobacteria strains |
| CN109593663A (en) * | 2018-12-27 | 2019-04-09 | 黄河三角洲京博化工研究院有限公司 | A kind of high-performance bio desulfurization microbial inoculum and its application method |
| CN109868233A (en) * | 2018-12-27 | 2019-06-11 | 黄河三角洲京博化工研究院有限公司 | A kind of efficient acid water special bacteria agent and its application method |
-
2018
- 2018-12-27 CN CN201811616998.2A patent/CN109536417A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002100781A1 (en) * | 2001-06-08 | 2002-12-19 | Idratech S.R.L. | Plant and method for purification of water coming from a seulphuration kerosene plant |
| CN102154174A (en) * | 2011-01-24 | 2011-08-17 | 中国科学院广州地球化学研究所 | Bacillus GZT and use thereof in degrading tribromophenol brominated flame retardant |
| CN104611247A (en) * | 2014-07-18 | 2015-05-13 | 中国矿业大学(北京) | Application of Alcaligenes faecalis sp. (DQP3) in degradation of phenol and quinoline |
| CN105154366A (en) * | 2015-09-25 | 2015-12-16 | 哈尔滨理工大学 | Bacillus cereus capable of degrading phenol and having electrogenesis characteristic and application thereof |
| CN106478288A (en) * | 2016-09-18 | 2017-03-08 | 广西大学 | A kind of soil conditioner of utilization microbial degradation Pesticide Residue in Soil |
| WO2018145294A1 (en) * | 2017-02-10 | 2018-08-16 | Perfect (China) Co., Ltd. | Novel probiotics bifidobacteria strains |
| CN108220209A (en) * | 2018-03-26 | 2018-06-29 | 宁波大学 | A kind of binary composite bacteria agent used for aquiculture |
| CN109593663A (en) * | 2018-12-27 | 2019-04-09 | 黄河三角洲京博化工研究院有限公司 | A kind of high-performance bio desulfurization microbial inoculum and its application method |
| CN109868233A (en) * | 2018-12-27 | 2019-06-11 | 黄河三角洲京博化工研究院有限公司 | A kind of efficient acid water special bacteria agent and its application method |
Non-Patent Citations (4)
| Title |
|---|
| ANNEELA IQBAL等: "Biodegradation of phenol and benzene by endophytic bacterial strains isolated from refinery wastewater-fed Cannabis sativa", 《ENVIRON TECHNOL》 * |
| CHANDRA,R 等: "Biodegradation of pyridine raffinate by teo bacterial co-cultures of Bacillus cereus (DQ435020) and Alcaligenes faecalis", 《WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY》 * |
| 姜和: "高效复合菌群选育及其降解苯酚特性的研究", 《中国优秀硕士学位论文全文数据库(电子期刊)工程科技I辑》 * |
| 马海娟 等: "产碱菌株F-3-4降解2,6-二叔丁基苯酚机理研究", 《上海环境科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109456906A (en) * | 2018-12-27 | 2019-03-12 | 黄河三角洲京博化工研究院有限公司 | A kind of alcohol-containing type organic sewage dedicated processes microbial inoculum and processing method |
| CN109576187A (en) * | 2018-12-27 | 2019-04-05 | 黄河三角洲京博化工研究院有限公司 | One plant of cyanide degradation bacterial strain and the method for utilizing the strains for degrading cyanide |
| CN109628345A (en) * | 2018-12-28 | 2019-04-16 | 天津开发区坤禾生物技术有限公司 | A kind of prevention and treatment succulent brown rot composite bacteria agent and its preparation method and application |
| CN109628345B (en) * | 2018-12-28 | 2021-07-09 | 天津开发区坤禾生物技术有限公司 | Composite microbial inoculum for preventing and treating brown rot of succulent plants as well as preparation method and application of composite microbial inoculum |
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