CN109650554A - A kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions - Google Patents
A kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions Download PDFInfo
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- CN109650554A CN109650554A CN201910012974.4A CN201910012974A CN109650554A CN 109650554 A CN109650554 A CN 109650554A CN 201910012974 A CN201910012974 A CN 201910012974A CN 109650554 A CN109650554 A CN 109650554A
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- 239000002351 wastewater Substances 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000012545 processing Methods 0.000 title claims abstract description 18
- 238000002474 experimental method Methods 0.000 claims abstract description 38
- 230000001580 bacterial effect Effects 0.000 claims abstract description 16
- 238000012360 testing method Methods 0.000 claims abstract description 14
- 241000894006 Bacteria Species 0.000 claims abstract description 13
- 108020004465 16S ribosomal RNA Proteins 0.000 claims abstract description 8
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 18
- 235000010413 sodium alginate Nutrition 0.000 claims description 18
- 239000000661 sodium alginate Substances 0.000 claims description 18
- 229940005550 sodium alginate Drugs 0.000 claims description 18
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 11
- 239000001110 calcium chloride Substances 0.000 claims description 11
- 235000011148 calcium chloride Nutrition 0.000 claims description 11
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 11
- 238000004132 cross linking Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 7
- 238000013461 design Methods 0.000 claims description 6
- 238000010186 staining Methods 0.000 claims description 4
- 241000194108 Bacillus licheniformis Species 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 239000003653 coastal water Substances 0.000 claims description 3
- 239000003431 cross linking reagent Substances 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 244000005700 microbiome Species 0.000 abstract description 6
- 239000002245 particle Substances 0.000 abstract description 5
- 239000000843 powder Substances 0.000 abstract description 4
- 238000005273 aeration Methods 0.000 abstract description 3
- 238000012216 screening Methods 0.000 abstract description 3
- 238000003794 Gram staining Methods 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 241000498991 Bacillus licheniformis DSM 13 = ATCC 14580 Species 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 208000035199 Tetraploidy Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 239000003245 coal Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The present invention provides a kind of methods of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions, it include: to fetch ooze from seashore first, carry out aeration culture and domestication, then efficient salt-enduring strain is isolated from wherein screening, the physicochemical property of bacterium is probed by gram staining experiment, the kind for determining bacterium is further sequenced by 16S rDNA, and the efficient dominant strain that screening is isolated then is subjected to extension culture, bacterium powder is made, carries out bacterial strain immobilization experiment using bacterium powder.Bacterial strain immobilization experiment includes experiment of single factor, Plackett-Burman experiment and response surface experiment, selects main influence factor by Plackett-Burman testing sieve.Then respective face experiment is carried out on the basis of the major influence factors that Plackett-Burman testing sieve is selected.A kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions provided by the invention, makes fixation support be not easy to be decomposed by the microorganisms, and intensity is high, and stability is good, while immobilization particle recoverable, reduces pollution, economical convenient.
Description
Technical field
The present invention relates to high-salt wastewater processing, will apply to the production such as organic chemical industry, coal chemical industry, textile printing and dyeing, oil exploitation
Raw high-salt wastewater, in particular to a kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions.
Background technique
High salinity waste water refers to always divides water to be not less than 1% waste water containing salt quality, not only can corrode building, industry is set
It is standby, and Aerobic biological process system treatment effeciency can be made to be remarkably decreased, cause water outlet that cannot get a desired effect.At present
The physical chemistry method investment operating cost for handling brine waste is higher, easily causes secondary pollution and is difficult to push away in the actual production process
Wide application.Biochemical method is because processing cost is lower, have a wide range of application and do not easily cause secondary pollution due to is widely used.
In traditional microbiological treatment system, often natural subsidence is difficult for thallus, be inevitably generated thallus be lost and
It is separated by solid-liquid separation the problems such as difficult, it is necessary to by means of centrifuge or chemical coagulant, increase processing cost, seriously affect it and producing
In popularization and application, to promote the research and development of immobilized microorganism technology.
Embedded-cross-linked method is more prominent as one of microorganism immobilization method advantage.It is solid using embedded-cross-linked method
During fixedization, microbial activity institute is suppressed lower, and has higher mechanical strength and stability.The characteristics of this method
Being is that microbial cell is directly wrapped in fixation support, and operation is relatively simple, and is influenced on microbial activity
It is small, this method being most widely used in current immobilized microorganism technology.
Using the notably selection of fixing condition when embedded-cross-linked fixation, to guarantee preferable mass-transfer performance, we
Method makes fixation support be not easy to be decomposed by the microorganisms by the optimization to fixing condition, and has intensity high, and stability is good,
The features such as economic and practical.
Summary of the invention
To solve the problems mentioned in the above background technology, the purpose of the present invention is to provide a kind of determining immobilization salt tolerants
The method that bacterium handles high-salt wastewater optimal conditions, method is simple, high-efficient, and identification is accurate, and the COD removal rate of waste water significantly mentions
It is high.
In order to solve the above technical problems, technical solution provided by the invention are as follows:
A kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions, which is characterized in that comprising steps of
Ooze is fetched from seashore first, and holding C:N:P is 100:5:1, aeration culture and domestication is carried out, then from wherein sieving
Efficient salt-enduring strain is isolated in choosing, and the physicochemical property of bacterium is probed by gram staining experiment, further passes through 16S
The kind for determining bacterium, convenient application later in practice is sequenced in rDNA.Then efficient dominant strain screening isolated
Extension culture is carried out, bacterium powder is made, carries out bacterial strain immobilization experiment using bacterium powder.
Bacterial strain immobilization experiment includes that experiment of single factor, Plackett-Burman experiment and response surface experiment, single factor test are real
Test main investigation sodium alginate concentration, CaCl2 concentration, influent COD, embedded material pH, embedding amount, water inlet salinity, crosslinking time
The influence of seven factors is determined by experiment the optimum level of each factor, and it is real to carry out Plackett-Burman on this basis
It tests.Take the optimum level that experiment of single factor determines as the central horizontal of Plackett-Burman experiment, and determines according to reality
Situation determine determines according to actual conditions low-level and high level, and using software to the relationship between each factor and response into
Row multilinear fitting selects main influence factor by Plackett-Burman testing sieve with this.
Then respective face experiment is carried out on the basis of the major influence factors that Plackett-Burman testing sieve is selected,
The experimental design table provided according to software is tested, and is carried out to the relationship between each significant factor and response polynary non-thread
Property fitting, and provide the contour map between each factor and response and 3D surface chart, determine that highest response is corresponding with this
The level of each factor, it is possible thereby to determine optimal experiment condition.
Specific implementation step is as follows:
Step 1: fetching ooze from seashore, holding C:N:P is 100:5:1, carries out aeration culture and tames from coastal waters bed mud
In isolate and purify out salt-enduring strain；
Step 2: bacterial strain is identified by Gram's staining, 16S rDNA；
Step 3: using sodium alginate as embedding medium, experiment is immobilized by crosslinking agent of CaCl2；
Step 4: the influence that each factor handles Facultative Halophiles high-salt wastewater is probed by experiment of single factor；
Step 5: carrying out Plackett-Burman experiment by Design Expert 8.0.4 on the basis of step 4；
Step 6: the conclusion based on Plackett-Burman experiment carries out response surface by Design Expert 8.0.4
Experiment, obtains significantly affecting the nonlinear multivariable relational expression between factor and response.
Further, the bacterial strain learns that the bacterial strain is bacillus licheniformis by identifying, is in Gram-positive.With the bacterium
The primer 16S-1492R of 16S rDNA carries out PCR amplification, obtains gene sequencing such as: SEQUENCE LISTING.
Further, each variable is sodium alginate concentration, CaCl2 concentration, influent COD, embedded material in the single factor test
PH, embedding amount, water inlet salinity and crosslinking time.
Further, the single factor test optimum level is sodium alginate and CaCl2 solution concentration is 2%, and influent COD is
8000mg/L, embedded material pH are 7, and embedding amount is 2.5g/L, and water inlet salinity is 3%, and crosslinking time is for 24 hours.
Further, the Plackett-Burman experiment conspicuousness influence factor is embedding amount, influent COD and seaweed
Sour na concn.
Further, immobilization experiment optimum condition is embedding amount 2.33g/L, influent COD 7957.45mg/L,
Sodium alginate concentration 1.94%.
The beneficial effects of the present invention are:
The present invention handles high-salt wastewater using the Facultative Halophiles under optimum experimental condition, prepares immobilization Facultative Halophiles particle,
Using COD removal rate as index, treatment effeciency reaches 72.45%.The determination of optimal fixing condition make fixation support be not easy by
Microorganism decomposition, and there is the features such as intensity is high, and stability is good, while immobilization particle recoverable, pollution is reduced, it is economical
It is convenient.
Detailed description of the invention
Fig. 1 is form observed under the microscope after Gram's staining of the present invention；
Fig. 2 is immobilization Facultative Halophiles particle of the present invention；
Fig. 3 is influence of the sodium alginate concentration of the present invention to COD removal rate；
Fig. 4 is influence of the CaCl2 concentration of the present invention to COD removal rate；
Fig. 5 is influence of the inlet COD concentration of the present invention to COD removal rate；
Fig. 6 is influence of the embedding medium pH of the present invention to COD removal rate；
Fig. 7 is influence of the embedding amount of the present invention to COD removal rate；
Fig. 8 is influence of the water inlet salinity of the present invention to COD removal rate；
Fig. 9 is influence of the crosslinking time of the present invention to COD removal rate；
Figure 10 is Pareto diagram of the present invention；
Figure 11 is the 3D surface chart of inlet COD concentration of the present invention and embedding amount to COD removal rate；
Figure 12 is the 3D surface chart of sodium alginate concentration of the present invention and embedding amount to COD removal rate；
Figure 13 is the 3D surface chart of sodium alginate concentration of the present invention and inlet COD concentration to COD removal rate.
Specific embodiment
Illustrate the present invention with specific embodiment below, is not limitation of the present invention.
A kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions, comprising the following steps:
Step 1: isolating and purifying out salt-enduring strain from the bed mud of coastal waters；
Step 2: bacterial strain is identified by Gram's staining, 16S rDNA；
Step 3: using sodium alginate as embedding medium, experiment is immobilized by crosslinking agent of CaCl2；
Step 4: the influence that each factor handles Facultative Halophiles high-salt wastewater is probed by experiment of single factor；
Step 5: carrying out Plackett-Burman experiment by Design Expert 8.0.4 on the basis of step 4；
Step 6: the conclusion based on Plackett-Burman experiment carries out response surface by Design Expert 8.0.4
Experiment, obtains significantly affecting the nonlinear multivariable relational expression between factor and response.
The bacterial strain learns that the bacterial strain is bacillus licheniformis by identifying, is in Gram-positive.With the bacterium 16S rDNA
Primer 16S-1492R carry out PCR amplification, gene sequencing result: SEQUENCE LISTING is obtained, by the 16S of bacterial strain
RDNA sequence inputting world GenBank database carry out tetraploid rice, as a result, it has been found that, the 16S rDNA sequence of bacterial strain with
For the homology matching degree of Bacillus licheniformis ATCC 14580 up to 96%, identified bacterial strain is lichens gemma bar
Bacterium ATCC 14580.
In the single factor test each variable be sodium alginate concentration, CaCl2 concentration, influent COD, embedded material pH, embedding amount,
Salinity of intaking and crosslinking time.
The single factor test optimum level is sodium alginate and CaCl2 solution concentration is 2%, influent COD 8000mg/L,
Embedded material pH is 7, and embedding amount is 2.5g/L, and water inlet salinity is 3%, and crosslinking time is for 24 hours.
Plackett-Burman experiment conspicuousness influence factor is embedding amount, influent COD and sodium alginate concentration,
And between this three factors
Reciprocal effect is obvious.
Immobilization experiment optimum condition is embedding amount 2.33g/L, influent COD 7957.45mg/L, and sodium alginate is dense
Degree 1.94%.
High-salt wastewater is handled using the Facultative Halophiles under optimum experimental condition, prepares immobilization Facultative Halophiles particle, processing effect
Rate reaches 72.45%.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Sequence table
<110>Qingdao University of Science and Technology
<120>a kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions
<130> 2018-12-27
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 600
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gtgacgggcc gtgtgtaagg cccgggaacg tattcaccgc ggcatgctga tccgcgatta 60
ctagcgattc cagcttcacg cagtcgagtt gcagactgcg atccgaactg agaacagatt 120
tgtgggattg gcttaccctc gcggcttcgc tgccctttgt tctgcccatt gtagcacgtg 180
tgtagcccag gtcataaggg gcatgatgat ttgacgtcat ccccaccttc ctccggtttg 240
tcaccggcag tcaccttaga gtgcccaact gaatgctggc aactaagatc aagggttgcg 300
ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacaacc atgcaccacc 360
tgtcactctg cccccgaagg ggaagcccta tctctagggt tgtcagagga tgtcaagacc 420
tggtaaggtt cttcgcgttg cttcgaatta aaccacatgc tccaccgctt gtgcgggccc 480
ccgtcagttc ctttgagttg cgggcgtgcg accgtactac cccaggcggg agtgctttaa 540
tgcgtttggc tgcagcacta aagggggcgg aagaggcttc tagcgcgtta gccactcatc 600
Claims (6)
1. a kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions, which comprises the following steps:
Step 1: isolating and purifying out salt-enduring strain from the bed mud of coastal waters；
Step 2: bacterial strain is identified by Gram's staining, 16S rDNA；
Step 3: using sodium alginate as embedding medium, experiment is immobilized by crosslinking agent of CaCl2；
Step 4: the influence that each factor handles Facultative Halophiles high-salt wastewater is probed by experiment of single factor；
Step 5: carrying out Plackett-Burman experiment by Design Expert 8.0.4 on the basis of step 4；
Step 6: it is real to carry out response surface by Design Expert 8.0.4 for the conclusion based on Plackett-Burman experiment
It tests, obtains significantly affecting the nonlinear multivariable relational expression between factor and response.
2. a kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions according to claim 1, special
Sign is that it is in Gram-positive that the bacterial strain, which learns that the bacterial strain is bacillus licheniformis by identifying,.With the bacterium 16S rDNA's
Primer 16S-1492R carries out PCR amplification, obtains gene sequencing such as: SEQ ID NO.1.
3. a kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions according to claim 1, special
Sign is, in the single factor test each variable be sodium alginate concentration, CaCl2 concentration, influent COD, embedded material pH, embedding amount,
Salinity of intaking and crosslinking time.
4. a kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions according to claim 3, special
Sign is that the single factor test optimum level is sodium alginate and CaCl2 solution concentration is 2%, influent COD 8000mg/L,
Embedded material pH is 7, and embedding amount is 2.5g/L, and water inlet salinity is 3%, and crosslinking time is for 24 hours.
5. a kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions according to claim 1, special
Sign is that the Plackett-Burman experiment conspicuousness influence factor is embedding amount, influent COD and sodium alginate concentration.
6. a kind of method of determining immobilization Facultative Halophiles processing high-salt wastewater optimal conditions according to claim 1, special
Sign is, the immobilization experiment optimum condition is embedding amount 2.33g/L, influent COD 7957.45mg/L, and sodium alginate is dense
Degree 1.94%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876347A (en) * | 2020-03-25 | 2020-11-03 | 山东晨彰生物科技有限公司 | Salt-tolerant bacillus licheniformis A-A2-10, application and application method thereof |
| CN112794454A (en) * | 2021-01-27 | 2021-05-14 | 上饶师范学院 | Optimization method for removing copper element in aquaculture wastewater by using microalgae |
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- 2019-01-07 CN CN201910012974.4A patent/CN109650554A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876347A (en) * | 2020-03-25 | 2020-11-03 | 山东晨彰生物科技有限公司 | Salt-tolerant bacillus licheniformis A-A2-10, application and application method thereof |
| CN111876347B (en) * | 2020-03-25 | 2021-08-06 | 山东晨彰生物科技有限公司 | Salt-tolerant bacillus licheniformis A-A2-10, application and application method thereof |
| CN112794454A (en) * | 2021-01-27 | 2021-05-14 | 上饶师范学院 | Optimization method for removing copper element in aquaculture wastewater by using microalgae |
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