CN105219684B - The complex microorganism preparations and preparation method of degrading phenol incretion interferent - Google Patents
The complex microorganism preparations and preparation method of degrading phenol incretion interferent Download PDFInfo
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Abstract
The present invention relates to a kind of complex microorganism preparations and preparation method of degrading phenol incretion interferent, it is related to microbial project and stain disease handling process, including Klebsiella (Klebsiella sp.), raw fat azospirillum (Azospirillum lipoferum), Rhodococcus erythropolis (Rhodococcus erythropolis) and bacillus licheniformis (Bacillus licheniformis)；The bacterium number of the Klebsiella accounts for the percentage 30%~50% of total bacteria count, the bacterium number of the raw fat azospirillum accounts for the percentage 30%~50% of total bacteria count, the bacterium number of the Rhodococcus erythropolis accounts for the percentage 10%~30% of total bacteria count, and the bacterium number of the bacillus licheniformis accounts for the percentage 10%~30% of total bacteria count.The present invention strengthens stain disease handling process by the way that complex micro organism fungicide is added in anaerobic pond and Aerobic Pond.The complex microbial community is resistant to high concentration phenols endocrine disruptors, is made full use of and is degraded using it as organic matter needed for growth, optimizes stain disease handling process, reduces water outlet and the secondary pollution of sludge, improves Sludge landfill and agricultural security.
Description
Technical field
The present invention relates to a kind of complex microorganism preparations and preparation method of degrading phenol incretion interferent, it is related to micro- life
Thing engineering and stain disease handling process, belong to field of environment protection.
Background technology
Incretion interferent (Endocrine Disrupting Chemicals, i.e. EDCs), also referred to as environmental hormone
(Environmental Hormone), is a kind of chemical substance of exogenous disturbance endocrine system, energy present in finger ring border
The interference mankind or all links of animal internal system simultaneously cause the material of anomalous effect, and they are by the various ways such as taking in, accumulating
Footpath, anomalous effects are not brought to organism directly as noxious material, but similar estrogen works to organism, even if
Quantity is few, can also allow the endocrine imbalance of organism, a variety of anomalies occurs.This kind of material can cause animal body and human body
Genitals obstacle, abnormal behavior, fecundity decline, the young is dead, even become extinct.
Phenols endocrine disruptors in environment are not naturally occurring, and caused by being due to human factor.Such as nonyl
The alkyl phenols (AP) such as base phenol (NP), octyl phenol (OP) and dodecylphenol (DP), are mainly derived from a variety of non-ionic surface actives
The industrial production of agent and APES (APEO) and use process；Bisphenol-A (BPA) be mainly derived from makrolon and
The industrial production of epoxy resin and use process.The discharge of sanitary sewage and related industries waste water is along with a large amount of phenols incretions
The release of chaff interference, nonyl phenol (NP), NPE (NPEO), octyl phenol (OP), OPEO
(OPEO), this 6 kinds of compounds such as dodecylphenol (DP), bisphenol-A (BPA) account for phenols endocrine disruptors total amount in stain disease
More than 98%, while most of phenols endocrine disruptors have very strong lipophilic hydrophobicity and refractory organicses, are easily adsorbed onto
In solid deposits and mud granule thing.
Activated sludge process is most widely used in stain disease handling process at present, but phenols endocrine disruptors therein are several
Do not degraded, small part is remained in water body with ug/L levels, most of to be transferred to residue with subsidence style to adsorb
In sludge.And the disposal options of excess sludge caused by sewage treatment plant with fill with it is agricultural based on, this results in a large amount of phenols
Incretion interferent enters environment with sludge.Phenols endocrine disruptors in soil and in water body can be by number of ways in people
Accumulated with animal body, easily cause the harm such as endocrine disturbance, sex dysfunction；If its too high levels in animal body, can be straight
Connecing causes the death of animal, therefore the degraded of phenols endocrine disruptors receives significant attention in stain disease.
For degrading phenol incretion interferent, existing research uses photocatalytic method more, but photocatalysis efficiency is low, artificial
Ultraviolet light energy consumption is big, and photochemical catalyst (TiO2Or BiOX) be not easy to be made, without economy.
Phenols endocrine disruptors bioremediation is rarely reported, and practical application effect is pessimistic.Such as middle promulgated by the State Council
Bright patent, application number 201210539590.6《A kind of method for octyl phenol of being degraded using laccase》One kind is disclosed to drop using laccase
The method of octyl phenol is solved, but the defects of enzyme preparation is expensive, enzyme reaction condition is harsh, enzyme reaction substrate is single be present, application
Property is poor.Chinese invention patent, application number 200910019223.1《The pseudomonad of a variety of phenolic compounds of one high-efficiency degradation
XQ23》The pseudomonad of one plant of energy degrading phenol compound is disclosed, but because single bacterial strain enzyme system is single, it is impossible to make phenol generalization
Compound is degradable, it is impossible to be used in industry is promoted.Chinese invention patent, Patent No. 201210057537.2《A kind of microorganism
Application of the microbial inoculum in phenol wastewater biological treatment》, disclose a kind of microbial bacterial agent and be used for answering for the Industrial Wastewater Treatment containing phenol
With method, but the domestication of its microbial inoculum is of long duration, and have ignored the safety detection of sludge secondary pollution.
The content of the invention
For in place of overcome the deficiencies in the prior art, the present invention is directed to a variety of phenols endocrine disruptors, there is provided a kind of efficient
Single-minded degrading microorganism compound formulation, symbiosis co-operative system is formed through compounding, strengthens stain disease handling process, can effectively remove
The phenols endocrine disruptors such as including NP, NPEO, OP, OPEO, DP, BPA, it is a further object of the present invention to provide the microorganism to answer
Close the preparation method and application of preparation.
The technical solution adopted in the present invention is：The complex microorganism preparations of degrading phenol incretion interferent, including gram
The primary Salmonella of thunder (Klebsiella sp.), raw fat azospirillum (Azospirillum lipoferum), Rhodococcus erythropolis
(Rhodococcus erythropolis) and bacillus licheniformis (Bacillus licheniformis)；With complex microorganism
Total bacteria count contained by preparation calculates, and the percentage that the bacterium number of the Klebsiella accounts for total bacteria count is 30%~50%, the raw fat
The percentage that the bacterium number of azospirillum accounts for total bacteria count is 30%~50%, and the bacterium number of described Rhodococcus erythropolis accounts for the hundred of total bacteria count
It is 10%~30% to divide than the percentage that total bacteria count is accounted for for the bacterium number of 10%~30%, the bacillus licheniformis；The Cray
Primary Salmonella is one kind in Klebsiella ACCC11779, ACCC11692, ACCC11624；The raw fat azospirillum is raw
Fat azospirillum ACCC10481；The Rhodococcus erythropolis is one kind in Rhodococcus erythropolis ACCC40323, ACCC10214；Institute
It is one in bacillus licheniformis ACCC02698, ACCC19372, CGMCC1.10314, CGMCC1.521 to state bacillus licheniformis
Kind.
Further, in the complex microorganism preparations of described degrading phenol incretion interferent, the total bacteria count of microorganism
More than or equal to 4.5 × 109cfu/mL。
Research shows that Klebsiella (Klebsiella sp.) has abundant enzyme system, and metabolic function is various, to long alkane
Base phenol degradation effect is obvious.In addition, it can also degrade, such as chrysanthemum esters, furfural class, phenols, ethers, nitrobenzene compounds are more
Kind toxic organic compound.
Raw fat azospirillum (Azospirillum lipoferum) can produce laccase, and it is more to be catalyzed aromatic amine and phenols etc.
The oxidation of kind aromatic compound, there is efficient degradation ability to BPA.
Rhodococcus erythropolis (Rhodococcus erythropolis) has stronger tolerance to a variety of organic solvents, produces ring
It is hydroxylated dioxygenase, desulfurase, alcohol dehydrogenase, energy efficient degradation polycyclic aromatic hydrocarbon, purifying water body.
Bacillus licheniformis (Bacillus licheniformis) strong stress resistance, contains abundant cellulase, fat
Enzyme and amylase can cooperate with above-mentioned three kinds of microorganisms effectively to degrade the organic matter in stain disease.
A kind of method for the complex microorganism preparations for preparing the degrading phenol incretion interferent, its technological process are：
Seed liquor preparation → mixed fermentation → protectant preparation and addition → product quality inspection and packing, detailed implementation steps are such as
Under：
The first step：It is prepared by seed liquor
By one kind in Klebsiella (Klebsiella sp.) ACCC 11779, ACCC 11692, ACCC 11624,
Raw fat azospirillum (Azospirillum lipoferum) ACCC 10481, Rhodococcus erythropolis (Rhodococcus
Erythropolis) ACCC 40323, one kind in ACCC 10214, bacillus licheniformis (Bacillus
Licheniformis) one kind in ACCC 02698, ACCC19372, CGMCC1.10314, CGMCC1.521, through slant activation
Afterwards, it is inoculated in respectively in the triangular flask of the fluid nutrient medium containing beef extract-peptone, Shaking culture, condition of culture is liquid amount 35%
~45%, 30~37 DEG C, 110~160rpm of shaking speed, 12~36h of incubation time of temperature；
Above-mentioned cultured shake-flask seed liquid is respectively connected into seeding tank to be fermented, inoculum concentration is 5%~10%, culture
Condition is：Throughput 1:1~1.5v/v/min, 30~37 DEG C, 110~160rpm of mixing speed of temperature, fermentation time 12~
36h, obtained fermentation seed liquid viable count are more than or equal to 4.5 × 109cfu/mL；
Second step：Mixed fermentation
Fermentation seed liquid obtained above is prepared into seed mixture liquid according to percentage by weight, configuration proportion is：Cray primary
It is 30%~50% that Salmonella seed liquor, which accounts for seed mixture liquid percentage by weight, and raw fat azospirillum seed liquor accounts for seed mixture liquid weight
It is 30%~50% to measure percentage, and it is lichens that the seed liquor of Rhodococcus erythropolis 10%~30%, which accounts for seed mixture liquid percentage by weight,
Bacillus seed liquor accounts for seed mixture liquid percentage by weight as 10%~30%；
Liquid fermentation matrix is added in fermentation tank, 115 DEG C of sterilizing 30min, treats that fermentation jar temperature is down to less than 40 DEG C, will be upper
Seed mixture liquid is stated to add by liquid fermentation matrix weight percentage 5%~10%；It is 1 ︰ 1 to control and throughput is cultivated in fermentation tank
~1.5v/v/min, 30~37 DEG C, 110~160rpm of mixing speed of temperature, incubation time are 12~36h；Obtained compound bacteria
Agent total viable count is more than or equal to 4.5 × 109cfu/mL；
3rd step：Protectant preparation and addition
Protect agent prescription：Skimmed milk power 1.0~1.5%, sweet potato starch 2.0~2.5%, glycerine 2.0~2.5%, distillation
Water 100mL, percentage are mass volume ratio；Skimmed milk power ultra violet lamp 45min, with 115 DEG C of height after other compositions mixing
Steam sterilizing 30min is pressed, then is mixed in an aseptic environment；
Protective agent adds：The protective agent to have sterilized is added to compound by the percentage by weight 2%~5% of above-mentioned composite bacteria agent
In microbial inoculum, stir.
4th step：Product quality inspection and packing
Through examining viable count in product to be more than or equal to 4.5 × 109Cfu/mL, qualified products are dispensed with Plastic Drum.
The mixed fermentive culture medium is calculated with quality volume percentage, and its formula is：Cornstarch 5%~10%, beans
Dregs of rice powder 15%~20%, yeast extract 0.1%~0.2%, dipotassium hydrogen phosphate 0.2%~0.4%, sodium dihydrogen phosphate 0.3%~
0.6%, magnesium sulfate 0.005%~0.01%, water 100mL.
The complex microorganism preparations of degrading phenol incretion interferent of the present invention, its application process are as follows：
Complex microorganism preparations of the present invention are put into anaerobic pond and Aerobic Pond respectively, dosage fills for processing first
The 0.2%~0.5% of volume is set effective, inputs sanitary sewage or industrial wastewater containing phenols endocrine disruptors immediately；It is described
2~12h of hydraulic detention time of anaerobic pond, PH5.0~9.0,18~40 DEG C of water temperature；The hydraulic detention time 5 of the Aerobic Pond
~50h, PH5.0~9.0,18~40 DEG C of water temperature, gas-water ratio 10~20:1, the dissolved oxygen 2mg/L in exit；Control system solid
Residence time is 6~72h；Waste water after Aerobic Process for Treatment is continuously discharged after sedimentation basin precipitation, by the anaerobic pond and is sunk
The concentrated rear discharge of excess sludge that shallow lake pond is collected.
Inventive principle：
The present invention relates to a kind of composite microbial of degrade sanitary sewage and related industries Phenol for Waste Water class incretion interferent
Thing preparation, its contain Klebsiella (Klebsiella sp.), raw fat azospirillum (Azospirillum lipoferum),
Rhodococcus erythropolis (Rhodococcus erythropolis) and bacillus licheniformis (Bacillus licheniformis).Institute
The microorganism formulation stated has the abundant enzyme system that can be broken chain alkyl, aoxidize polycyclic aromatic hydrocarbon, can make nonyl phenol (NP), nonyl phenol
APEO (NPEO), octyl phenol (OP), OPEO (OPEO), dodecylphenol (DP) and bisphenol-A (BPA)
It is degraded into organic acid and alcohols, and final degradable Cheng Shui and carbon dioxide.Described degrading phenol incretion interferent
Have between complex microbial community and act synergistically, a variety of phenols endocrine disruptors in energy efficient degradation stain disease, optimization
Stain disease handling process, ensure Sludge landfill and agricultural security.
Beneficial effect：The present invention strengthens stain disease by the way that complex micro organism fungicide is added in anaerobic pond and Aerobic Pond
Handling process.The complex microbial community is resistant to high concentration phenols endocrine disruptors, and organic matter needed for growth is used as using it
Made full use of and degraded, optimize stain disease handling process, reduce water outlet and the secondary pollution of sludge, improve Sludge landfill
With agricultural security, preparation of the invention and application process have the characteristics of positive effect, easy to operate, cost is low, suitable for production
Industry popularization and application.
Brief description of the drawings
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is that complex microorganism preparations of the present invention are used to degrade different carbon chain lengths during dodecylphenol (DP)
Alkyl phenol (AnP) changes of contents figure.
Fig. 2 is the stain disease processing technological flow figure of the present invention.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
In the description of the invention, it is to be understood that term " longitudinal direction ", " transverse direction ", " on ", " under ", "front", "rear",
The orientation or position relationship of the instruction such as "left", "right", " vertical ", " level ", " top ", " bottom ", " interior ", " outer " is based on accompanying drawing institutes
The orientation or position relationship shown, the description present invention is for only for ease of, rather than the device or element of instruction or hint meaning must
There must be specific orientation, with specific azimuth configuration and operation, therefore be not considered as limiting the invention.
The complex microorganism preparations of degrading phenol incretion interferent of the present invention, including Klebsiella
(Klebsiella sp.), raw fat azospirillum (Azospirillum lipoferum), Rhodococcus erythropolis (Rhodococcus
) and bacillus licheniformis (Bacillus licheniformis) erythropolis；The bacterium number of the Klebsiella accounts for always
The percentage 30%~50% of bacterium number, the bacterium number of the bacterium life fat azospirillum account for the percentage 30%~50% of total bacteria count, institute
The bacterium number for stating Rhodococcus erythropolis accounts for the percentage 10%~30% of total bacteria count, and the bacterium number of the bacillus licheniformis accounts for total bacteria count
Percentage 10%~30%；The Klebsiella is in Klebsiella ACCC11779, ACCC11692, ACCC11624
It is a kind of；The raw fat azospirillum is raw fat azospirillum ACCC10481；The Rhodococcus erythropolis is Rhodococcus erythropolis
One kind in ACCC40323, ACCC10214；The bacillus licheniformis be bacillus licheniformis ACCC02698,
One kind in ACCC19372, CGMCC1.10314, CGMCC1.521.
Further, in the complex microorganism preparations of described degrading phenol incretion interferent, the total bacteria count of microorganism
More than or equal to 4.5 × 109cfu/mL。
The microorganism formulation preparation technology flow of degrading phenol incretion interferent of the present invention：Seed liquor preparation →
Mixed fermentation → protectant preparation and addition → product quality inspection and packing.Its step is：
The first step：It is prepared by seed liquor
By one kind in Klebsiella (Klebsiella sp.) ACCC 11779, ACCC 11692, ACCC 11624,
Raw fat azospirillum (Azospirillum lipoferum) ACCC 10481, Rhodococcus erythropolis (Rhodococcus
Erythropolis) ACCC 40323, one kind in ACCC 10214 and bacillus licheniformis (Bacillus
Licheniformis) one kind in ACCC 02698, ACCC 19372, CGMCC 1.10314, CGMCC 1.521 (is public
Open the bacterial strain of preservation), after slant activation, it is inoculated in the triangular flask of the fluid nutrient medium containing beef extract-peptone, shakes respectively
Bottle culture.Condition of culture is liquid amount 35%~45%, 30~37 DEG C, 110~160rpm of shaking speed of temperature, incubation time 12
~36h.
Above-mentioned cultured shake-flask seed liquid is respectively connected into seeding tank to be fermented, inoculum concentration is 5%~10%, culture
Condition is：Throughput 1:1~1.5v/v/min, 30~37 DEG C, 110~160rpm of mixing speed of temperature, fermentation time 12~
36h.Obtained fermentation seed liquid viable count is more than or equal to 4.5 × 109cfu/mL。
Second step：Mixed fermentation
Fermentation seed liquid obtained above is prepared into seed mixture liquid according to percentage by weight, configuration proportion is：Cray primary
It is 30%~50% that Salmonella seed liquor, which accounts for seed mixture liquid percentage by weight, and raw fat azospirillum seed liquor accounts for seed mixture liquid weight
It is 30%~50% to measure percentage, and it is lichens that the seed liquor of Rhodococcus erythropolis 10%~30%, which accounts for seed mixture liquid percentage by weight,
Bacillus seed liquor accounts for seed mixture liquid percentage by weight as 10%~30%；
Liquid fermentation matrix is added in fermentation tank, 115 DEG C of sterilizing 30min, treats that fermentation jar temperature is down to less than 40 DEG C, will be upper
Seed mixture liquid is stated (to add by liquid fermentation matrix weight percentage 5%~10%；It is 1 ︰ to control and throughput is cultivated in fermentation tank
1~1.5v/v/min, 30~37 DEG C, 110~160rpm of mixing speed of temperature, incubation time are 12~36h.Obtained compound bacteria
Agent total viable count is more than or equal to 4.5 × 109cfu/mL。
Liquid fermentation matrix formulations are：Cornstarch 5%~10%, bean cake powder 15%~20%, yeast extract 0.1%~
0.2%, dipotassium hydrogen phosphate 0.2%~0.4%, sodium dihydrogen phosphate 0.3%~0.6%, magnesium sulfate 0.005%~0.01%, water
100mL, percentage are mass volume ratio.
3rd step：Protectant preparation and addition
Protect agent prescription：Skimmed milk power 1.0~1.5%, sweet potato starch 2.0~2.5%, glycerine 2.0~2.5%, distillation
Water 100mL, percentage are mass volume ratio；Skimmed milk power ultra violet lamp 45min, with 115 DEG C of height after other compositions mixing
Steam sterilizing 30min is pressed, then is mixed in an aseptic environment.
Protective agent adds：The protective agent to have sterilized is added to compound by the percentage by weight 2%~5% of above-mentioned composite bacteria agent
In microbial inoculum, stir.
4th step：Product quality inspection and packing
Through examining viable count in product to be more than or equal to 4.5 × 109Cfu/mL, qualified products are dispensed with Plastic Drum.
The application of the complex microorganism preparations of degrading phenol incretion interferent of the present invention, one built according to Fig. 2
Exemplified by covering pilot scale sewage disposal system model, step is as follows：
Complex microorganism preparations of the present invention are put into anaerobic pond and Aerobic Pond respectively, dosage fills for processing first
It is set effective the 0.2%~0.5% of volume.Immediately sanitary sewage or industrial wastewater of the input containing phenols endocrine disruptors.Control
Anaerobic pond parameter：Hydraulic detention time (HRT) 2~12h, PH5.0~9.0,18~40 DEG C of water temperature；Control Aerobic Pond parameter：Water
Power residence time (HRT) 5~50h, PH5.0~9.0,18~40 DEG C of water temperature, gas-water ratio 10~20:1, the dissolved oxygen in exit
2mg/L；Control system solid retention time (OSRT) is 6~72h.Waste water after Aerobic Process for Treatment is continuously arranged after sedimentation basin precipitates
Go out.The concentrated rear discharge of excess sludge collected by anaerobic pond and sedimentation basin.
NP, NPEOs, OP, OPEOs, DP, BPA of the present invention detection method use SPE-chromatography of gases
Method is used in conjunction in (SPE-GC).
In the present invention, described Klebsiella, raw fat azospirillum, Rhodococcus erythropolis and bacillus licheniformis are all existing
Some conventional bacterial classifications, for example, can from China General Microbiological culture presevation administrative center (CGMCC) orhttp:// www.cgmcc.netInquire；And Chinese agriculture Microbiological Culture Collection administrative center (ACCC) is commercially available, or
http://www.accc.org.cn is inquired.
Embodiment 1：It is prepared by degrading phenol incretion interferent complex microorganism preparations
The microorganism formulation preparation method of degrading phenol incretion interferent of the present invention, detailed implementation steps are：
The first step：It is prepared by seed liquor
By one kind in Klebsiella (Klebsiella sp.) ACCC 11779, ACCC 11692, ACCC 11624,
Raw fat azospirillum (Azospirillum lipoferum) ACCC 10481, Rhodococcus erythropolis (Rhodococcus
Erythropolis) ACCC 40323, one kind in ACCC 10214 and bacillus licheniformis (Bacillus
Licheniformis) one kind in ACCC 02698, ACCC 19372, CGMCC 1.10314, CGMCC 1.521 (is public
Open the bacterial strain of preservation), after slant activation, it is inoculated in the triangular flask of the fluid nutrient medium containing beef extract-peptone and cultivates respectively
Prepare shake-flask seed liquid.Shake flask culture conditions are：Liquid amount 40%, 33 DEG C, shaking speed 140rpm of temperature, incubation time
12h；
Above-mentioned cultured shake-flask seed liquid is respectively connected into seeding tank to be fermented, inoculum concentration 8%, condition of culture
For：Throughput 1:1.5v/v/min, 34 DEG C, mixing speed 130rpm, fermentation time 24h of temperature.Obtained fermentation seed liquid is lived
Bacterium number is more than or equal to 4.5 × 109cfu/mL。
Second step：Mixed fermentation
Fermentation seed liquid obtained above is prepared into seed mixture liquid according to percentage by weight, configuration proportion is：Cray primary
It is 35% that Salmonella seed liquor, which accounts for seed mixture liquid percentage by weight, and raw fat azospirillum seed liquor accounts for seed mixture liquid weight percent
Than for 35%, it is that bacillus licheniformis seed liquor accounts for mixed that the seed liquor of Rhodococcus erythropolis 15%, which accounts for seed mixture liquid percentage by weight,
It is 15% to close seed liquor percentage by weight；
Liquid fermentation matrix is added in fermentation tank, 115 DEG C of sterilizing 30min, treats that fermentation jar temperature is down to less than 40 DEG C, will be upper
Seed mixture liquid is stated to add by liquid fermentation matrix weight percentage 8%；It is 1 that throughput is cultivated in fermentation tank:1.5v/v/min
34 DEG C, mixing speed 130rpm, incubation time 24h of temperature.Obtained composite bacteria agent total viable count be more than or equal to 4.5 ×
109cfu/mL。
Described liquid fermentation matrix formulations are：Cornstarch 10%, bean cake powder 15%, yeast extract 0.2%, phosphoric acid hydrogen two
Potassium 0.3%, sodium dihydrogen phosphate 0.5%, magnesium sulfate 0.006%, water 100mL, percentage are mass volume ratio.
3rd step：Protectant preparation and addition
Protect agent prescription：Skimmed milk power 1.2%, sweet potato starch 2.4%, glycerine 2.4%, distilled water 100mL, percentage are
Mass volume ratio；Skimmed milk power ultra violet lamp 45min, with 115 DEG C of high pressure steam sterilization 30min after other compositions mixing,
Mix in an aseptic environment again.
Protective agent adds：The protective agent to have sterilized is added to composite bacteria agent by the percentage by weight 4% of above-mentioned composite bacteria agent
In, stir.
4th step：Product quality inspection and packing
Through examining viable count in product to be more than or equal to 4.5 × 109Cfu/mL, qualified products are dispensed with Plastic Drum.
It is prepared by the degrading phenol incretion interferent complex microorganism preparations of embodiment 2
Detailed step is：
The first step：It is prepared by seed liquor
By one kind in Klebsiella (Klebsiella sp.) ACCC 11779, ACCC 11692, ACCC 11624,
Raw fat azospirillum (Azospirillum lipoferum) ACCC 10481, Rhodococcus erythropolis (Rhodococcus
Erythropolis) ACCC 40323, one kind in ACCC 10214 and bacillus licheniformis (Bacillus
Licheniformis) one kind in ACCC 02698, ACCC 19372, CGMCC 1.10314, CGMCC 1.521 (is public
Open the bacterial strain of preservation), after slant activation, it is inoculated in the triangular flask of the fluid nutrient medium containing beef extract-peptone, shakes respectively
Bottle culture.Shake flask culture conditions are：Liquid amount 38%, 35 DEG C, shaking speed 120rpm, incubation time 18h of temperature；
Above-mentioned cultured shake-flask seed liquid is respectively connected into seeding tank to be fermented, inoculum concentration 10%, condition of culture
For：Throughput 1:1.2v/v/min, 36 DEG C, mixing speed 150rpm, fermentation time 30h of temperature.Obtained fermentation seed liquid is lived
Bacterium number is more than or equal to 4.5 × 109cfu/mL。
Second step：Mixed fermentation
Fermentation seed liquid obtained above is prepared into seed mixture liquid according to percentage by weight, configuration proportion is：Cray primary
It is 40% that Salmonella seed liquor, which accounts for seed mixture liquid percentage by weight, and raw fat azospirillum seed liquor accounts for seed mixture liquid weight percent
Than for 30%, it is that bacillus licheniformis seed liquor accounts for mixed that the seed liquor of Rhodococcus erythropolis 20%, which accounts for seed mixture liquid percentage by weight,
It is 10% to close seed liquor percentage by weight.
Liquid fermentation matrix is added in fermentation tank, 115 DEG C of sterilizing 30min, treats that fermentation jar temperature is down to less than 40 DEG C, will be upper
Seed mixture liquid is stated to add by liquid fermentation matrix weight percentage 6%；It is 1 that throughput is cultivated in fermentation tank:1.2v/v/min
36 DEG C, mixing speed 140rpm, incubation time 30h of temperature.Obtained composite bacteria agent total viable count be more than or equal to 4.5 ×
109cfu/mL。
Described liquid fermentation matrix formulations are：Cornstarch 5%, bean cake powder 13%, yeast extract 0.15%, phosphoric acid hydrogen two
Potassium 0.4%, sodium dihydrogen phosphate 0.3%, magnesium sulfate 0.008%, water 100mL, percentage are mass volume ratio.
3rd step：Protectant preparation and addition
Protect agent prescription：Skimmed milk power 1.0%, sweet potato starch 2.5%, glycerine 2.2%, distilled water 100mL, percentage are
Mass volume ratio；Skimmed milk power ultra violet lamp 45min, with 115 DEG C of high pressure steam sterilization 30min after other compositions mixing,
Mix in an aseptic environment again.
Protective agent adds：The protective agent to have sterilized is added to composite bacteria agent by the percentage by weight 3% of above-mentioned composite bacteria agent
In, stir.
4th step：Product quality inspection and packing
Through examining viable count in product to be more than or equal to 4.5 × 109Cfu/mL, qualified products are dispensed with Plastic Drum.
It is prepared by the degrading phenol incretion interferent complex microorganism preparations of embodiment 3
The microorganism formulation preparation technology flow of degrading phenol incretion interferent of the present invention：Seed liquor preparation →
Mixed fermentation → protectant preparation and addition → product quality inspection and packing.Wherein detailed implementation steps are：
The first step：It is prepared by seed liquor
By one kind in Klebsiella (Klebsiella sp.) ACCC 11779, ACCC 11692, ACCC 11624,
Raw fat azospirillum (Azospirillum lipoferum) ACCC 10481, Rhodococcus erythropolis (Rhodococcus
Erythropolis) ACCC 40323, one kind in ACCC 10214 and bacillus licheniformis (Bacillus
Licheniformis) one kind in ACCC 02698, ACCC 19372, CGMCC 1.10314, CGMCC 1.521 (is public
Open the bacterial strain of preservation), after slant activation, it is inoculated in the triangular flask of the fluid nutrient medium containing beef extract-peptone, shakes respectively
Bottle culture.Shake flask culture conditions are：Liquid amount 42%, 36 DEG C, shaking speed 130rpm, incubation time 12h of temperature；
Above-mentioned cultured shake-flask seed liquid is respectively connected into seeding tank to be fermented, inoculum concentration 6%, condition of culture
For：Throughput 1:1.0v/v/min, 35 DEG C, mixing speed 140rpm, fermentation time 36h of temperature.Obtained fermentation seed liquid is lived
Bacterium number is more than or equal to 4.5 × 109cfu/mL。
Second step：Mixed fermentation
Fermentation seed liquid obtained above is prepared into seed mixture liquid according to percentage by weight, configuration proportion is：Cray primary
It is 30% that Salmonella seed liquor, which accounts for seed mixture liquid percentage by weight, and raw fat azospirillum seed liquor accounts for seed mixture liquid weight percent
Than for 40%, Rhodococcus erythropolis seed liquor accounts for seed mixture liquid percentage by weight as 12%, and bacillus licheniformis seed liquor accounts for mixed
It is 18% to close seed liquor percentage by weight.
Liquid fermentation matrix is added in fermentation tank, 115 DEG C of sterilizing 30min, treats that fermentation jar temperature is down to less than 40 DEG C, will be upper
Seed mixture liquid is stated to add by liquid fermentation matrix weight percentage 10%.It is 1 that throughput is cultivated in fermentation tank:1.5v/v/
Min, 30 DEG C, mixing speed 160rpm, incubation time 24h of temperature.Obtained composite bacteria agent total viable count be more than or equal to 4.5 ×
109cfu/mL。
Described liquid fermentation matrix formulations are：Cornstarch 6%, bean cake powder 20%, yeast extract 0.16%, phosphoric acid hydrogen two
Potassium 0.25%, sodium dihydrogen phosphate 0.55%, magnesium sulfate 0.009%, water 100mL, percentage are mass volume ratio.
3rd step：Protectant preparation and addition
Protect agent prescription：Skimmed milk power 1.5%, sweet potato starch 2.0%, glycerine 2.0%, distilled water 100mL, percentage are
Mass volume ratio；Skimmed milk power ultra violet lamp 45min, with 115 DEG C of high pressure steam sterilization 30min after other compositions mixing,
Mix in an aseptic environment again.
Protective agent adds：The protective agent to have sterilized is added to composite bacteria agent by the percentage by weight 2% of above-mentioned composite bacteria agent
In, stir.
4th step：Product quality inspection and packing
Through examining viable count in product to be more than or equal to 4.5 × 109Cfu/mL, qualified products are dispensed with Plastic Drum.
Example effects 1：Research of the complex microorganism preparations for dodecylphenol (DP) that degrade
Take basal mediums of the 100mL containing DP (500mg/L) to load 250mL triangular flasks, 121 DEG C, 20min sterilizings, press
Complex microorganism preparations described in the 0.5% inoculum concentration access embodiment of the present invention 1, constant-temperature table, shaking table condition are put into by triangular flask
30 DEG C, 160rpm are set to, is sampled per 1d, method is used in conjunction to DP degradation processes using SPE-chromatography of gases (SPE-GC)
Middle carbon chain lengths are respectively 12,8,4, the 2 qualitative and quantitative detection of homologue (AnP) progress.As shown in figure 1, in DP degradeds
During, not only its concentration (c) is changing, and the homologue (AnP) that carbon chain lengths are 8,4,2 can be also produced, with culture
The extension of time, chain length distribution peaks are gradually dropped to 2 from 12, total concentration (cTotal) day drop from initial value 500.00mg/L to the 6th
Low is 61.40mg/L, degradation rate 87.72%.
Example effects 2：Complex microorganism preparations are used for sanitary sewage centralized treatment plant of Hefei City sewage pilot plant test
The sanitary sewage in Hefei City sanitary sewage in March centralized treatment plant's setting pot exit is gathered, is built according to Fig. 2
A set of pilot scale sewage disposal system model, application process are as follows：
Complex microorganism preparations described in the embodiment of the present invention 1 are put into anaerobic pond and Aerobic Pond respectively, first dosage
For the 0.2% of processing unit dischargeable capacity, the control group returned sludge equivalent substitution for being derived from the sewage treatment plant is immediately continuous
Input sewage.Control anaerobic pond parameter：Hydraulic detention time (HRT) 3h, PH7.0,20 DEG C of water temperature；Control Aerobic Pond parameter：Water
Power residence time (HRT) 6h, PH7.0,20 DEG C of water temperature, gas-water ratio 15:1, the dissolved oxygen 2mg/L in exit；System solid retention
Time (OSRT) control is 12h.Sewage after Aerobic Process for Treatment is continuously discharged after sedimentation basin precipitates.Received by anaerobic pond and sedimentation basin
The excess sludge of collection is concentrated, is discharged after dehydration.Device continuous and steady operation 5 days, the water outlet and concentration of collection the 2nd, 3,4 day are dirty
Method measure NP, NPEOs, OP, OPEOs, DP, BPA concentration level is used in conjunction in mud, SPE-chromatography of gases (SPE-GC).Such as
Shown in table 1, complex microorganism preparations of the present invention are notable to the degradation effect of phenols endocrine disruptors, in the total phenols of water outlet
Secrete chaff interference concentration be 1.18 μ g/L, than control group reduction by 96.31%, the total phenols endocrine disruptors of thickened sludge it is dense
Spend for 2.49mg/kg, 91.10% is reduced than control group.
Sanitary sewage centralized treatment plant of the Hefei City sewage complex microorganism preparations pilot plant test result table of comparisons of table 1
* LOD represents instrument detection limit
Example effects 3：Complex microorganism preparations are used for certain city economic and technological development zone stain disease centralized treatment plant stain disease
Processing.
Gather certain city economic and technological development zone stain disease in March centralized treatment plant grid pond.The sewage treatment plant, which collects, to be come
From the resident living sewage of the economic and technological development zone and include the Industry Waste of pharmaceuticals industry, daily chemical industry, lube oil industry etc.
Water.A set of pilot scale sewage disposal system model is built according to Fig. 2, its application process is as follows：
Complex microorganism preparations described in the embodiment of the present invention 2 are put into anaerobic pond and Aerobic Pond respectively, first dosage
For the 0.4% of processing unit dischargeable capacity, the control group returned sludge equivalent substitution for being derived from the sewage treatment plant is continuous in time
Input stain disease.Control anaerobic pond parameter：Hydraulic detention time (HRT) 5h, PH7.0,24 DEG C of water temperature；Control Aerobic Pond parameter：
Hydraulic detention time (HRT) 12h, PH7.0,24 DEG C of water temperature, gas-water ratio 18:1, the dissolved oxygen 2mg/L in exit；System solid stops
It is 24h to stay time (OSRT) control.Sewage after Aerobic Process for Treatment is continuously discharged after sedimentation basin precipitates.By anaerobic pond and sedimentation basin
The excess sludge of collection is concentrated, is discharged after dehydration.Device continuous and steady operation 7 days, the water outlet and concentration of collection the 2nd, 4,6 day
Method measure NP, NPEOs, OP, OPEOs, DP, BPA concentration level is used in conjunction in sludge, SPE-chromatography of gases (SPE-GC).
As shown in table 2, complex microorganism preparations of the present invention are notable to the degradation effect of phenols endocrine disruptors, the total phenols of water outlet
The concentration of incretion interferent is 2.63 μ g/L, and 92.99% is reduced than control group, the total phenols endocrine disruptors of thickened sludge
Concentration is 3.18mg/kg, and 92.65% is reduced than control group.
Certain the city economic and technological development zone stain disease centralized treatment plant stain disease complex microorganism preparations result control of table 2
Table
* LOD represents instrument detection limit
Example effects 4：Complex microorganism preparations are used for certain the first sewage treatment plant of city stain disease processing
Certain the first sewage treatment plant in November of city grid pond is gathered, the sewage treatment plant, which collects stain disease, includes the city city
Resident living sewage, a weaving dyeing and weaving factory and a petrochemical plant, a set of pilot scale sewage disposal system is built according to Fig. 2
Model, its application process are as follows：
Complex microorganism preparations described in the embodiment of the present invention 3 are put into anaerobic pond and Aerobic Pond respectively, first dosage
For the 0.5% of processing unit dischargeable capacity, the control group returned sludge equivalent substitution for being derived from the sewage treatment plant is continuous in time
Input stain disease.Control anaerobic pond parameter：Hydraulic detention time (HRT) 6h, PH7.5,28 DEG C of water temperature；Control Aerobic Pond parameter：
Hydraulic detention time (HRT) 36h, PH7.5,28 DEG C of water temperature, gas-water ratio 20:1, the dissolved oxygen 2mg/L in exit；System solid stops
It is 48h to stay time (OSRT) control.Sewage after Aerobic Process for Treatment is continuously discharged after sedimentation basin precipitates.By anaerobic pond and sedimentation basin
The excess sludge of collection is concentrated, is discharged after dehydration.Device continuous and steady operation 10 days, the collection water outlet of the 3rd, 6,9 day and dense
Method measure NP, NPEOs, OP, OPEOs, DP, BPA concentration water is used in conjunction in contracting sludge, SPE-chromatography of gases (SPE-GC)
It is flat.As shown in table 3, complex microorganism preparations of the present invention are notable to the degradation effect of phenols endocrine disruptors, and water outlet is total
The concentration of phenols endocrine disruptors is 20.88 μ g/L, reduces by 89% than control group, the total phenols endocrine disruptors of thickened sludge
Concentration be 22.83mg/kg, than control group reduce by 90%.
The complex microorganism preparations of table 3 are used for certain the first sewage treatment plant of city stain disease result table of comparisons
* LOD represents instrument detection limit
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of changes, modification can be carried out to these embodiments, replace without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (5)
1. the complex microorganism preparations of degrading phenol incretion interferent, it is characterised in that including Klebsiella
(Klebsiella sp.), raw fat azospirillum (Azospirillum lipoferum), Rhodococcus erythropolis (Rhodococcus
) and bacillus licheniformis (Bacillus licheniformis) erythropolis；With total bacterium contained by complex microorganism preparations
Number calculates, and the percentage that the bacterium number of the Klebsiella accounts for total bacteria count is 30%~50%, the bacterium of the raw fat azospirillum
The percentages that number accounts for total bacteria count are 30%~50%, the bacterium number of described Rhodococcus erythropolis account for the percentage of total bacteria count for 10%~
30%, the percentage that the bacterium number of the bacillus licheniformis accounts for total bacteria count is 10%~30%；The Klebsiella is Cray
One kind in primary Salmonella ACCC11779, ACCC11692, ACCC11624；The raw fat azospirillum is raw fat azospirillum
ACCC10481；The Rhodococcus erythropolis is one kind in Rhodococcus erythropolis ACCC40323, ACCC10214；The lichens gemma
Bacillus is one kind in bacillus licheniformis ACCC02698, ACCC19372, CGMCC1.10314, CGMCC1.521.
2. the complex microorganism preparations of degrading phenol incretion interferent according to claim 1, it is characterised in that described
In the complex microorganism preparations of degrading phenol incretion interferent, the total bacteria count of microorganism is more than or equal to 4.5 × 109cfu/mL。
3. a kind of method for preparing the complex microorganism preparations of degrading phenol incretion interferent described in claim 1, its feature
It is with following steps：
The first step：It is prepared by seed liquor
By one kind in Klebsiella (Klebsiella sp.) ACCC 11779, ACCC 11692, ACCC 11624, raw fat
Azospirillum (Azospirillum lipoferum) ACCC 10481, Rhodococcus erythropolis (Rhodococcus
Erythropolis) ACCC 40323, one kind in ACCC 10214, bacillus licheniformis (Bacillus
Licheniformis) one kind in ACCC 02698, ACCC19372, CGMCC1.10314, CGMCC1.521, through slant activation
Afterwards, it is inoculated in respectively in the triangular flask of the fluid nutrient medium containing beef extract-peptone, Shaking culture, condition of culture is liquid amount 35%
~45%, 30~37 DEG C, 110~160rpm of shaking speed, 12~36h of incubation time of temperature；
Above-mentioned cultured shake-flask seed liquid is respectively connected into seeding tank to be fermented, inoculum concentration is 5%~10%, condition of culture
For：Throughput 1:1~1.5v/v/min, 30~37 DEG C, 110~160rpm of mixing speed, 12~36h of fermentation time of temperature, system
The fermentation seed liquid viable count obtained is more than or equal to 4.5 × 109cfu/mL；
Second step：Mixed fermentation
Fermentation seed liquid obtained above is prepared into seed mixture liquid according to percentage by weight, configuration proportion is：Klebsiella
It is 30%~50% that seed liquor, which accounts for seed mixture liquid percentage by weight, and raw fat azospirillum seed liquor accounts for seed mixture liquid weight hundred
Divide than being 30%~50%, the seed liquor of Rhodococcus erythropolis 10%~30% accounts for seed mixture liquid percentage by weight and is, lichens gemma
Bacillus seed liquor accounts for seed mixture liquid percentage by weight as 10%~30%；
Liquid fermentation matrix is added in fermentation tank, 115 DEG C of sterilizing 30min, treats that fermentation jar temperature is down to less than 40 DEG C, will be above-mentioned mixed
Seed liquor is closed to add by liquid fermentation matrix weight percentage 5%~10%；Control cultivated in fermentation tank throughput be 1 ︰ 1~
1.5v/v/min, 30~37 DEG C, 110~160rpm of mixing speed of temperature, incubation time are 12~36h；Obtained composite bacteria agent
Total viable count is more than or equal to 4.5 × 109cfu/mL；
3rd step：Protectant preparation and addition
Protect agent prescription：Skimmed milk power 1.0~1.5%, sweet potato starch 2.0~2.5%, glycerine 2.0~2.5%, distilled water
100mL, percentage are mass volume ratio；Skimmed milk power ultra violet lamp 45min, with 115 DEG C of high pressures after other compositions mixing
Steam sterilizing 30min, then mix in an aseptic environment；
Protective agent adds：The protective agent to have sterilized is added to composite bacteria agent by the percentage by weight 2%~5% of above-mentioned composite bacteria agent
In, stir；
4th step：Product quality inspection and packing
Through examining viable count in product to be more than or equal to 4.5 × 109Cfu/mL, qualified products are dispensed with Plastic Drum.
4. a kind of method for the complex microorganism preparations for preparing degrading phenol incretion interferent according to claim 3, its
It is characterised by, the liquid fermentation matrix is calculated with quality volume percentage, is made up of following raw material：Cornstarch 5%~
10%, bean cake powder 15%~20%, yeast extract 0.1%~0.2%, dipotassium hydrogen phosphate 0.2%~0.4%, sodium dihydrogen phosphate
0.3%~0.6%, magnesium sulfate 0.005%~0.01%, water 100mL.
5. the application process of the complex microorganism preparations of degrading phenol incretion interferent described in a kind of claim 1, its feature
It is, the complex microorganism preparations is put into anaerobic pond and Aerobic Pond respectively, dosage is that processing unit is effectively held first
Long-pending 0.2%~0.5%, sanitary sewage or industrial wastewater containing phenols endocrine disruptors are inputted immediately；The anaerobic pond
2~12h of hydraulic detention time, PH5.0~9.0,18~40 DEG C of water temperature；5~50h of hydraulic detention time of the Aerobic Pond,
PH5.0~9.0,18~40 DEG C of water temperature, gas-water ratio 10~20:1, the dissolved oxygen 2mg/L in exit；During control system solid retention
Between be 6~72h；Waste water after Aerobic Process for Treatment is continuously discharged after sedimentation basin precipitation, is received by the anaerobic pond and sedimentation basin
The concentrated rear discharge of excess sludge of collection.
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| CN109133316A (en) * | 2017-06-19 | 2019-01-04 | 湖南大学 | A method of using calper calcium peroxide oxidation pre-treatment containing emerging pollutant waste water |
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| CN111977893B (en) * | 2020-07-28 | 2021-12-21 | 杭州电子科技大学 | Biochemical treatment method for wastewater generated in production of hydrogen peroxide by anthraquinone process based on composite microbial inoculum |
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