CN106085935A - A kind of processing method of aquaculture wastewater - Google Patents
A kind of processing method of aquaculture wastewater Download PDFInfo
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- CN106085935A CN106085935A CN201610416354.3A CN201610416354A CN106085935A CN 106085935 A CN106085935 A CN 106085935A CN 201610416354 A CN201610416354 A CN 201610416354A CN 106085935 A CN106085935 A CN 106085935A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The present invention relates to the processing method of a kind of aquaculture wastewater, it includes adding in breeding water body the microbial inoculum that water body is processed, and the bacterium in this microbial inoculum is that bacterium is strangled in non-decarboxylation Rec.Described bacterium has the effect killing contaminated bacteria in multiple breeding water body, can also effectively remove the toxic algae in wawter bloom simultaneously, have good application prospect for aquaculture.
Description
Technical field
The present invention relates to microorganism field, more particularly, it relates to the processing method of a kind of aquaculture wastewater.
Background technology
As aquaculture intensive degree improves constantly, physicochemical environment and the ecological environment of breeding water body deteriorate increasingly；
Meanwhile, the excess emitters of human lives's sewage, various industrial wastewater, agricultural water-break etc., also exacerbates under breeding water body quality
Fall；These problems cause the farming disease harms to be on the rise.The abuse of antibiotic and chemicals not only makes the resistance of pathogenic microorganism
Property strengthen, and cause the hazard residue of aquatic products to exceed standard, the directly health of the threat mankind.The countries and regions such as USA and EU
In succession having put into effect relevant law and having limited application in aquaculture for the antibiotic, therefore, aquaculture must provide for safety, high-quality
The aquatic products of nuisanceless green be trend of the times.Existing result of study shows that probiotics can not only reduce having of water body
Machine pollutes, purifying water body, may also suppress, kills pathogenic microorganism, and as foodstuff additive, thus partly can substitute or complete
Substitute the use of chemicals and antibiotic.And compound micro-ecological preparation due to have simultaneously improve intestinal environment, increase into
Eating, suppress pernicious bacteria, improving many effects such as water quality, the application in aquaculture of aquatic animal is increasingly becoming research heat
Point.
The ratio of Ryukyu university of Japan was praised clear professor husband and successfully be have developed compound micro-ecological preparation EM, China the eighties in last century
Starting probiotics is applied to culture fishery the nineties in last century, with carrying out of research work, Related product is also
Come out one after another, for mainly having CM, MCB, life tonifying clean water composite bacteria, " sweet bacterium dew ", " blade is green ", immune in terms of aquaculture
The raw agent of profit, water conservation are peaceful.Although existing most of compound micro-ecological preparation is a certain at water quality improvement and prevention disease or some
Aspect achieves obvious effect, but domestic preparation is fitted owing to there is bacterial classification unstable properties, function singleness and environment
The problems such as answering property difference make it not adapt to water body environment complicated and changeable, and after imposing on water body, effect is unstable, thus limits micro-
Popularization and application in aquaculture for the ecological agent.
Disclosing the probio being isolatable from enteron aisle non-decarboxylation Rec in prior art and strangling bacterium, this bacterium can in aquaculture
For killing harmful bacteria, there is good application prospect, but the bactericidal effect of this bacterium is not also very high, awaits into one
The optimization of step.
Content of the invention
The technical problem to be solved in the present invention is, for the bacterium of above-mentioned prior art, provides having more of a kind of transformation
Add spectrum sterilization and kill algae characteristic microorganism formulation and application.
The technical solution adopted for the present invention to solve the technical problems is: construct a kind of micro-life processing city domestic sewage
Thing preparation, includes viable count 2-6*10 in every milliliter of described microbial bacterial agent9, wherein said viable bacteria contains improved non-de-
Bacterium is strangled in carboxylic Rec.
Bacterium in microorganism formulation of the present invention is that bacterium that deposit number is CGMCC No:7813 is by having imported spy
Obtain after fixed albumen.
The albumen importing in the present invention is that amino acid sequence that size is 571aa is as shown in SEQIDNo:1 or in sequence 1
On the basis of the following sudden change that carries out: Val29Ser, Gly46Lys, Lys79Gly, Ser106Val, Ser244Leu,
Glu311Ser, Ala345Gly, Asn356Lys, Ala371Met, Lys456Cys, Asp471Gly, Trp486Ala,
Gly490Ser, Asn500Ile, Asn516Cys, Ala533Ser, Ala545Trp, Leu552Ser or Pro566Ser.
Applicant has carried out 270 rite-directed mutagenesises altogether for SEQ ID No:1 sequence, and wherein only screening obtains 19 and dashes forward
Becoming the bactericidal activity with raising, and remaining all not having or activity significantly reduces, this also illustrates the screening of sudden change simultaneously
It is not readily available.
Non-decarboxylation Rec is strangled bacterium and is also used engineered mode to construct genetic engineering bacterium.DNAMAN software is utilized to divide
Analysis, the nucleotide sequence KST gene described in full genome synthesis according to amino acid sequence SEQID NO:1, by expanding
Acquisition purpose fragment, PCR amplification acquisition genes of interest KST (simultaneously by multiplex PCR by corresponding mutational site Val29Ser,
Gly46Lys, Lys79Gly, Ser106Val, Ser244Leu, Glu311Ser, Ala345Gly, Asn356Lys,
Ala371Met, Lys456Cys, Asp471Gly, Trp486Ala, Gly490Ser, Asn500Ile, Asn516Cys,
Ala533Ser, Ala545Trp, Leu552Ser or Pro566Ser are introduced between in gene order, thus obtain different prominent
Variant gene), carry out double digestion PCR primer with restriction enzyme, with the expression vector pSDK-1 phase also passing through double digestion
Even, first pSDK-1 is imported in Escherichia coli, by way of plasmid commonly used in the art shifts, (see " Escherichia coli phosphoric acid
Method described in clone in Thiobacillus thioxidans for the fructokinase gene, expression and the analysis to organic carbon metabolism thereof ",
This does not states one by one) recombinant plasmid being proved to be successful is converted from Escherichia coli enter into described non-decarboxylation Rec and strangle in bacterium,
Obtain described genetic engineering bacterium.This engineering bacteria has more preferable application prospect.
The non-decarboxylation Rec of the present invention is strangled the non-decarboxylation Rec that bacterium is CGMCC No:7813 and is strangled bacterium.
The microbial bacterial agent of the present invention, obvious to breeding water body treatment effect, there is extremely strong sterilization and algae killing effect, should
With having a extensive future.
Detailed description of the invention
The inspection of bacterium antibiotic property is strangled in embodiment 1 non-decarboxylation Rec
(1) antibiotic property detection
A) prepared by pathogen flat board: by Candida albicans, Klebsiella Pneumoniae, staphylococcus aureus LB Liquid Culture
Based on 37 DEG C, vibrio parahaemolytious, Harbin vibrios 2216E Liquid Culture are cultivated to OD 600=O.8-1.0 based on 28 DEG C, takes
100-200ul nutrient solution, 6000-8000rpm room temperature centrifuges 3-5min, abandons supernatant, and the aseptic PBS of precipitation 100-200ul is again
Suspend.By Candida albicans, Klebsiella Pneumoniae, staphylococcus aureus suspension at LB solid medium flat board in 37
DEG C, by vibrio parahaemolytious, Harbin vibrios suspension in 2216E solid culture based on 28 DEG C, just putting fixing 2-3h respectively.
B) dibbling: the non-decarboxylation Rec of CGMCC No:7813 is strangled bacterium and takes the cultivation of 2216E fluid nutrient medium extremely
OD600=O.8-1.0, aseptic PBS, each 10-30ul of sterilized water, put respectively on the above-mentioned flat board filling pathogen.28-30℃
Just put 2-3h, be cultured after base absorption until dibbling liquid, be placed in the incubator of 28 DEG C or 37 DEG C inversion incubated overnight 24-48h.
(2) detection of algae property is killed
By the microcystic aeruginosa algae kind bought in 2000LX, the illumination box under the conditions of 25 DEG C quiescent culture, every
3h manually shakes once (all aseptically carrying out), continues preculture one week, and microscopy has or not miscellaneous algae and algal grown feelings
Condition, as reached to synchronize well to grow, so that it may as the algae seedling solution of test.
Microcystic aeruginosa, according to the Algaculture method of this area routine, is cultivated and is obtained algae solution.Respectively by 150 μ L (1) b
Bacterium thalline suspension is strangled in the non-decarboxylation Rec cultivated, fermented supernatant fluid is inoculated in tetra-kinds of algae solutions of 30mL, and comparison Control is
Landy culture medium, carries out molten algae experiment, measures Chlorophyll-a Content, calculate algal control rate after 7d.
(3) result: compare the result of aseptic PBS and sterilized water, observes non-decarboxylation Rec and strangles bacterium on each pathogen flat board
Growth and the production of inhibition zone.Discovery all creates significantly press down for the bacterium being detected, non-decarboxylation Rec Le bacterium
Bacterium circle.Aseptic PBS and sterilized water then have no that obvious inhibition zone produces.Meanwhile, the algal control of fermented liquid is strangled in non-decarboxylation Rec
Rate is very low.Show that bacterium is strangled almost without algae-lysing in non-decarboxylation Rec.
Statistical effect table
Note: a diameter of 0.8-1.3cm of " +++ " inhibition zone, the antibacterial circle diameter of " ++ " is 0.6-0.8cm, "+" antibacterial
Loop diameter is 0.4-0.6cm, and "-" produces without inhibition zone.
The preparation of bacterium is strangled in embodiment 2 genetic engineering bacterium non-decarboxylation Rec
Utilize DNAMAN software analysis, the nucleotides described in full genome synthesis according to amino acid sequence SEQID NO:1
Sequence KST gene, obtains purpose fragment by carrying out amplification, and PCR amplification obtains genes of interest KST (simultaneously will by multiplex PCR
Mutational site Val29Ser, Gly46Lys, Lys79Gly, Ser106Val, Ser244Leu, Glu311Ser accordingly,
Ala345Gly, Asn356Lys, Ala371Met, Lys456Cys, Asp471Gly, Trp486Ala, Gly490Ser,
Asn500Ile, Asn516Cys, Ala533Ser, Ala545Trp, Leu552Ser or Pro566Ser are introduced between gene sequence
In row, thus obtain different mutants gene), carry out double digestion PCR primer with restriction enzyme, and also pass through double enzyme
The expression vector pSDK-1 cutting is connected, and first imports pSDK-1 in Escherichia coli, by the side of plasmid transfer commonly used in the art
The recombinant plasmid being proved to be successful is converted the non-decarboxylation Lay entering into described preserving number CGMCC No:7813 from Escherichia coli by formula
Gram strangle in bacterium, it is thus achieved that described genetic engineering bacterium.The bacterium of the KST gene importing sudden change is respectively designated as non-decarboxylation Rec and strangles
Bacterium-KST2, non-decarboxylation Rec Le bacterium-KST3, non-decarboxylation Rec Le bacterium-KST4, non-decarboxylation Lay are strangled in bacterium-KST1, non-decarboxylation Rec
Gram strangle bacterium-KST5, bacterium-KST6 is strangled in non-decarboxylation Rec, bacterium-KST7 is strangled in non-decarboxylation Rec, bacterium-KST8 is strangled in non-decarboxylation Rec, non-de-
Bacterium-KST9 is strangled in carboxylic Rec, bacterium-KST10 is strangled in non-decarboxylation Rec, bacterium-KST11 is strangled in non-decarboxylation Rec, bacterium is strangled in non-decarboxylation Rec-
Bacterium-KST13, non-decarboxylation Rec Le bacterium-KST14, non-decarboxylation Rec Le bacterium-KST15, non-decarboxylation Lay are strangled in KST12, non-decarboxylation Rec
Gram strangle bacterium-KST16, non-decarboxylation Rec strangle bacterium-KST17, non-decarboxylation Rec strangle bacterium-KST18, non-decarboxylation Rec strangle bacterium-KST19.
Correspond respectively to Val29Ser, Gly46Lys, Lys79Gly, Ser106Val, Ser244Leu, the Glu311Ser of quiding gene,
Ala345Gly, Asn356Lys, Ala371Met, Lys456Cys, Asp471Gly, Trp486Ala, Gly490Ser,
Asn500Ile, Asn516Cys, Ala533Ser, Ala545Trp, Leu552Ser or Pro566Ser suddenly change.
Embodiment 3: bacterium compliance test result is strangled in non-decarboxylation Rec
According to the method for embodiment 1, carrying out corresponding effect process experiment, result is as follows:
As can be seen from the above results, the non-decarboxylation Rec using genetic engineering to build is strangled bacterium and is had compared with original strain
Enhanced bactericidal effect, and, there is the effect killing algae, this has preferable application prospect in aquaculture.
Embodiment 4: breeding water body experimental verification
In cray breeding water body, add non-decarboxylation Rec and strangle microbial inoculum prepared by bacterium KST1 bacterium, the concentration of bacterium in water body
Reach 3*103/ml, after reacting 3 days, the content of various bacterium and algae in measurement water body, the sterilizing rate of vibrios in discovery water body
Reaching 95%, the sterilizing rate of algae reaches 86%, has preferable effect.Other non-decarboxylation Recs suddenling change are used to strangle bacterium
Any one bacterium of KST2-19, its bactericidal effect also all approximates.
Described bacterium preparation is become corresponding microbial inoculum, can large-area killing for bacterium in aquaculture and algae
In, there is great application prospect.
Sequence table
<110>Li Qian
<120>processing method of a kind of aquaculture wastewater
<210> 1
<211> 571
<212> PRT
<213>artificial sequence
<400> 1
Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
Thr Met Met Phe Ser Ala Ser Ser Phe Ala Tyr Asp Val Pro Asp Tyr
Ala Ala Ala Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn
Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile
Glu Gly Arg Ile Ser Glu Phe Lys Val Phe Gly Arg Cys Glu Leu Ala
Ala Ala Met Lys Arg His Gly Leu Asp Asn Tyr Arg Gly Tyr Ser Leu
Gly Asn Trp Val Cys Ala Ala Lys Phe Glu Ser Asn Phe Asn Thr Gln
Ala Thr Asn Arg Asn Thr Asp Gly Ser Thr Asp Tyr Gly Ile Leu Gln
Ile Asn Ser Arg Trp Trp Cys Asn Asp Gly Arg Thr Pro Gly Ser Arg
Asn Leu Cys Asn Ile Pro Cys Ser Ala Leu Leu Ser Ser Asp Ile Thr
Ala Ser Val Asn Cys Ala Lys Lys Ile Val Ser Asp Gly Asn Gly Met
Asn Ala Trp Val Ala Trp Arg Asn Arg Cys Lys Gly Thr Asp Val Gln
Ala Trp Ile Arg Gly Cys Arg Leu Gly Lys Pro Arg Pro Tyr Ser Pro
Arg Pro Thr Ser His Pro Arg Pro Ile Arg Val
Claims (3)
1. the processing method of an aquaculture wastewater, it is characterised in that add the microbial inoculum that water body is processed, its feature in breeding water body
It is: the described bacterium in microbial inoculum is that bacterium is strangled in non-decarboxylation Rec.
2. the processing method of an aquaculture wastewater, it is characterised in that add the microbial inoculum that water body is processed, its feature in breeding water body
It is: the described bacterium in microbial inoculum is that the preservation having imported encoding amino acid sequence gene of albumen as shown in SEQIDNo:1 is compiled
Number strangle bacterium for the non-decarboxylation Rec of CGMCC No:7813, or for having imported carry out on the basis of SEQIDNo:1 as follows
Any one sudden change: Val29Ser, Gly46Lys, Lys79Gly, Ser106Val, Ser244Leu, Glu311Ser,
Ala345Gly, Asn356Lys, Ala371Met, Lys456Cys, Asp471Gly, Trp486Ala, Gly490Ser,
The guarantor of the gene of the albumen of Asn500Ile, Asn516Cys, Ala533Ser, Ala545Trp, Leu552Ser or Pro566Ser
Bacterium is strangled in the non-decarboxylation Rec hiding numbered CGMCC No:7813.
3. a protein variant, it is characterised in that: its amino acid sequence is following any for only carrying out on the basis of SEQIDNo:1
One sudden change: Val29Ser, Gly46Lys, Lys79Gly, Ser106Val, Ser244Leu, Glu311Ser, Ala345Gly,
Asn356Lys, Ala371Met, Lys456Cys, Asp471Gly, Trp486Ala, Gly490Ser, Asn500Ile,
Asn516Cys, Ala533Ser, Ala545Trp, Leu552Ser or Pro566Ser.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779422A (en) * | 2017-11-29 | 2018-03-09 | 中国农业科学院油料作物研究所 | One plant height effect suppresses the Leclercia adecarboxylata biocontrol bacterial strain of aspergillus flavus synthesis aflatoxin |
| CN110760457A (en) * | 2019-09-05 | 2020-02-07 | 华南农业大学 | Degrading strain of pyrethroid pesticide and application thereof |
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| CN103525719A (en) * | 2013-09-06 | 2014-01-22 | 中国科学院烟台海岸带研究所 | Probiotic separated from intestinal tracts and application thereof |
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2016
- 2016-06-14 CN CN201610416354.3A patent/CN106085935A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103525719A (en) * | 2013-09-06 | 2014-01-22 | 中国科学院烟台海岸带研究所 | Probiotic separated from intestinal tracts and application thereof |
| CN104761621A (en) * | 2014-01-06 | 2015-07-08 | 长春新悦然科技有限公司 | Antibacterial proteins |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779422A (en) * | 2017-11-29 | 2018-03-09 | 中国农业科学院油料作物研究所 | One plant height effect suppresses the Leclercia adecarboxylata biocontrol bacterial strain of aspergillus flavus synthesis aflatoxin |
| CN110760457A (en) * | 2019-09-05 | 2020-02-07 | 华南农业大学 | Degrading strain of pyrethroid pesticide and application thereof |
| CN110760457B (en) * | 2019-09-05 | 2021-01-26 | 华南农业大学 | Degrading strain of pyrethroid pesticide and application thereof |
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Application publication date: 20161109 |