CN108728372A - The Sphingol single-cell LPN080 and its microorganism formulation of one plant of different oxygen ammonia assimilation and application - Google Patents
The Sphingol single-cell LPN080 and its microorganism formulation of one plant of different oxygen ammonia assimilation and application Download PDFInfo
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Abstract
The invention discloses the Sphingol single-cell LPN080 of one plant of different oxygen ammonia assimilation and its microorganism formulation and applications.The strain isolation is in the shrimp culture environment of Maoming City, ability with very strong degradation of ammonia nitrogen, it is Sphingomonas (Sphingomonas sp.) through Molecular Identification, Sphingol single-cell LPN080 can carry out quick absorption and assimilation by heterotrophism form to ammonia nitrogen, therefore it is different from from oxygen nitre bacterium and nitrification bacteria, Sphingol single-cell LPN080 can be with the vibrios of conspicuousness Competitive assays breeding water body in addition.Sphingol single-cell LPN080 is applied in breeding water body, the content of ammonia nitrogen in breeding water body obviously can be quickly reduced, to cultivated animals safety.
Description
Technical field：
The invention belongs to aquaculture technology and ecological environmental protection technical fields, and in particular to one plant of different oxygen ammonia assimilation
Sphingol single-cell LPN080 and its microorganism formulation and application.
Background technology：
In aquaculture, a large amount of residual bait in aquaculture pond, excrement and its metabolite are continuously increased, these substances are micro-
Biological decomposition generates a large amount of ammonia nitrogen and nitrite, and the two generates toxicity, excessive ammonia nitrogen and nitrite to cultivated animals
Also the algae in breeding water body can be made largely to break out, it is dead after alga eruption to generate toxin, it also can largely cause aquaculture dynamic
The death of object, while pathogenic microorganism also can largely be grown.Therefore, ammonia nitrogen and nitrous acid in breeding water body are controlled in aquaculture
The concentration of salt is to be only second to the important water quality index of dissolved oxygen.
Microorganism formulation is applied in aquaculture extensively, including nitrobacteria and denitrifying bacteria.
The widely used nitrobacteria overwhelming majority is Autotrophic nitrification bacterium in existing market, is nitrite by ammoxidation, then by Asia
Nitric acid is further oxidized to nitrate.Autotrophic nitrification bacterium lives in the water body environment of oligotrophic, and the process of Autotrophic nitrification more
Very slowly, this may be in high-density breeding environment, and ammonia nitrogen and nitrous acid content are easy exceeded reason.Therefore, autotrophy
Nitrobacteria can not actually fully meet the purpose for regulating and controlling water body ammonia nitrogen and nitrous acid content using microorganism formulation.
Nitrification bacteria has been applied in sewage disposal at present, recently as the emerging of biological flocculation cultural technique
It rises, nitrification bacteria is gradually paid attention in aquaculture field, and heterotrophic nitrification metabolism is fast, growth is fast, certain special heterotrophism
Bacterium, which can synchronize, carries out heterotrophic nitrification and aerobic denitrification, has extensive use in terms of breeding water body water correction.
However above-mentioned bacterium can be only done the conversion (NH of ammonia existence form4 +/NO2 -/NO3 -), it can not be by inorganic states
Ammonia or nitrous acid are converted into the composition of bacterial cell itself.
Heterotrophic ammonium assimilation bacterium is same using ammonia nitrogen as nitrogen source using glutamine route of synthesis using extraneous carbon source as the energy
The organic matter needed for itself is turned to, in vivo by ammonia nitrogen accumulation.The practicability of heterotrophic ammonium assimilation bacterium is stronger in contrast, will not produce
The accumulation of raw nitrite and nitrate, can more solve the problems, such as that water body ammonia nitrogen and nitrate are excessively high in breeding production.About different
Report and research are also lacked before supporting nitrous acid assimilation Zoopagales, the example that do not applied in aquaculture more.
Invention content：
The first purpose of the invention is to provide one plant of Sphingol single-cell (Sphingomonas sp.) LPN080, the bacterium
It is preserved in Guangdong Province's Culture Collection (GDMCC), address on April 25th, 2018：Xianlie Middle Road, Guangzhou City 100
5 building, the building of compound the 59th, deposit number are GDMCC No：60365.
The present invention is from prawn culturing water body and bed mud aggregate sample, by being added in enrichment and isolation medium (EIM)
Glucose, the carbon sources such as sucrose are added ammonium sulfate as nitrogen source, a small amount of yeast extract are added as growth factor, enrichment contains
The heterotrophism nitrogen assimilation bacterium for dropping ammonia nitrogen ability is purified by the agar plate of corresponding above-mentioned culture medium, chooses single bacterium again and fall on
In above-mentioned culture medium, picks out the speed of growth and drop the strongest bacterial strain of ammonia nitrogen ability soon.It is identified by 16SrDNA, is determined as sheath ammonia
The bacterium of alcohol zygosaccharomyces (Sphingomonas sp.) is named as Sphingol single-cell (Sphingomonas sp.)
LPN080。
Second object of the present invention is to provide a kind of microorganism formulation, including the Sphingol single-cell
(Sphingomonas sp.)LPN080。
Third object of the present invention is to provide the preparation methods of the microorganism formulation, by the Sphingomonas
Bacterium (Sphingomonas sp.) LPN080 is inoculated into fermented and cultured in fluid nutrient medium, after fermentation, every liter of addition 1.5g
Trehalose, mix well, obtain microorganism formulation.
The fluid nutrient medium is SPM culture mediums, and every liter of culture medium is prepared by the following method：In 1L water
Ammonium sulfate 1.5g, peptone 0.5g, wheat bran 0.6g, glucose 6g, sucrose 6g, corn juice 1.5g is added, adjusts pH to 7.0.
Fourth object of the present invention be to provide Sphingol single-cell (Sphingomonas sp.) LPN080 or
Application of the microorganism formulation in water body ammonia nitrogen of degrading.
The water body is preferably breeding water body.
Fifth object of the present invention is to provide the Sphingol single-cell (Sphingomonas sp.) LPN080 or
Application of the microorganism formulation in preparing antibacterials.
The antibacterials are preferably to inhibit the drug of vibrios.
Sixth object of the present invention is to provide the Sphingol single-cell (Sphingomonas sp.) LPN080 or
Application of the microorganism formulation in aquaculture.
Sphingol single-cell LPN080 preparations (i.e. microorganism formulation) press 5-10 kilograms of breeding water body (depth of water 1m is calculated) per acre
Amount uses.Sphingol single-cell LPN080 preparations are added in the cultivation water of 4-10 times of volume first, by every liter of sphingol list
Brown sugar is added in the amount that 50-100g brown sugar is added in born of the same parents' bacterium LPN080 preparations, after mixing, is placed at room temperature for 4-12 hours, full pool spilling head is
It can.
The present invention Sphingol single-cell LPN080 and its microorganism formulation has the following advantages or good effect：
(1) speed of degradation of ammonia nitrogen is fast compared to Autotrophic nitrification bacterium；(2) ammonia nitrogen will not be converted to nitrous acid by absorption and assimilation
Salt or nitrate, therefore nitrogen really can be removed quickly from breeding water body；(3) Sphingol single-cell LPN080 is in breeding water body
Middle Reverse transcriptase vibrios growth, therefore this bacterium has effects that inhibit aquatic pathogenic bacterium simultaneously.
Sphingol single-cell (Sphingomonas sp.) LPN080 of the present invention was preserved in Guangdong on April 25th, 2018
Save Culture Collection (GDMCC), address：5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, deposit number are
GDMCC No：60365.
Description of the drawings：
Fig. 1 is that ammonia nitrogen curve graph drops in Sphingol single-cell (Sphingomonas sp.) LPN080.
Specific implementation mode：
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1：Sphingol single-cell LPN080 concentration and separations
Enrichment and isolation medium (EIM)：Ammonium sulfate 0.5g, peptone 1g, glucose 6g, sucrose 6g, yeast extract
0.5g、NaH2PO4The clean seawater 1L of 0.5g, sand filtration adjusts pH to 7.0, high pressure steam sterilization.
Sample source：Maoming City Dianbai area prawn culturing water body and bottom of pond mud aggregate sample.
Specific implementation steps are as follows：Under aseptic condition, in the conical flask of filtered sample 135ml to sterilizing, addition 10 ×
(formula of 10 × EIM culture mediums is EIM culture mediums：Ammonium sulfate 5g, peptone 10g, glucose 60g, sucrose 60g, yeast extraction
Object 5g, NaH2PO4The clean seawater 1L of 5g, sand filtration adjusts pH to 7.0, high pressure steam sterilization) 15ml, 30 in the shaking table of 150rpm
DEG C culture 48h, obtain pregnant solution.It takes 100 μ l of pregnant solution to be coated in above-mentioned EIM solid mediums, is trained in 30 DEG C of insulating box
It supports for 24 hours, picking single bacterium colony 20, is seeded in EIM fluid nutrient mediums and cultivates for 24 hours, purify 2 times, obtain in the flat lining outs of EIM
The bacterial strain of 20 purifying, is seeded in EIM culture mediums and cultivates.
Each bacterium takes the 100 μ l of bacterium solution of equivalent, is linked into fresh EIM culture mediums, measures ammonia nitrogen in initial culture medium
Concentration, 30 DEG C culture 48h after measure final ammonia nitrogen concentration.Pick out one plant of best bacterium of drop ammonia nitrogen effect, bacterial strain LPN080.
Embodiment 2：Sphingol single-cell LPN080 Molecular Identifications
It takes bacterial strain LNP080 bacterium solutions to centrifuge, abandons supernatant, genomic DNA is extracted with bacterial sediment, using genomic DNA as mould
Plate carries out 16SrDNA amplifications (doi by primer of 27F/1492R:10.1128/AEM.02272-07), it and is sequenced, nucleotide
For sequence as shown in SEQ ID NO.1, sequence is accredited as Sphingomonas (Sphingomonas through Blast search comparisons
Sp. bacterium) is named as Sphingol single-cell (Sphingomonas sp.) LPN080.
Embodiment 3：Removal effects of Sphingol single-cell (Sphingomonas sp.) LPN080 to breeding water body ammonia nitrogen
It takes cultivation water sample to use ammonium sulfate tune ammonia nitrogen concentration for 8mg/L or so, glucose is added to final concentration 1g/L, sterilizing
It is dispensed in 150ml to the 250ml shaking flasks of sterilizing after processing.Experimental group (E1, E2, E3) is added 30 DEG C, the training of 200rpm incubator overnights
Foster LPN080 bacterium solutions (culture medium is EIM culture mediums) 500 μ l, the EIM culture mediums of control group (C1, C2, C3) addition equivalent, 30
DEG C shaking table culture (rotating speed 200rpm), in 0h, 3h, 6h, 9h, 12h, for 24 hours, 36h, 48h water sampling.It is divided light using nessler reagent
It spends meter method and measures different time points ammonia-nitrogen content, the result is shown in Figure 1.
As seen from Figure 1, when initial cultivation water sample tune ammonia nitrogen concentration is reached for 7.89mg/L, Sphingomonas in 48 hours
Bacterium LPN080 reaches 99.4% to the removal efficiency of ammonia nitrogen, is nearly no detectable the presence of ammonia nitrogen, therefore Sphingol single-cell
LPN080 has very strong removal ability for the ammonia nitrogen in the cultivation water of high-concentration ammonia-nitrogen.
In order to prove that ammonia nitrogen is rather than to be converted into NO as nitrification bacteria by absorption and assimilation2 -, while 0h, 12h,
For 24 hours, when 48h, the NO of experimental group and control group each sample is determined2 -Content, as a result, it has been found that each group sample is four time points
NO2 -Content is 0.Due in this water sample and in the culture medium of above-described embodiment, in addition to ammonium sulfate provides nitrogen source, having no it
Its substance largely provides nitrogen source, and as a result these are true, determines that Sphingol single-cell LPN080 is with the different of notable drop ammonia ability
Support ammonium assimilation bacteria.
Embodiment 4：Sphingol single-cell LPN080 Evaluation of Biocompatibility
Three glass jars of experimental group (SE1, SE2, SE3), three glass jars of control group (SC1, SC2, SC3) are added per cylinder
Filtering sea 4L, 8 tail of shrimp of being rivals in a contest at random per cylinder.Take 30 DEG C, (culture medium is for the LPN080 bacterium solutions of 200rpm incubator overnight cultures
EIM culture mediums), 8000rpm centrifuges 2min, is washed twice with sterilizing seawater, bacterial concentration OD is suspended into sterilizing seawater600nm=1
(about 8 × 108A bacterium/ml), take 100 μ l (8 × 107A bacterium) it is added in the glass jar of experimental group cultivation, control group is added
Equivalent sterilizing seawater, normally feeds feed, and amount of survival is recorded in 1d, 2d, 3d, 4d, 5d, 6d, 7d.As a result such as the following table 1.
Influences of the 1 Sphingol single-cell LPN080 of table to prawn survival rate
By table 1 as it can be seen that even if in the presence of the Sphingol single-cell LPN080 of high concentration, experimental group control group with
Without any difference in terms of prawn survival rate, therefore illustrate that Sphingol single-cell LPN080 pacifies with very high cultivated animals
Quan Xing, non-pathogenic bacteria.
Embodiment 5：Sphingol single-cell LPN080 inhibits vibrios
Cultivation water sample 2L is taken, 2g glucose is added, is dispensed in 150ml to the 250ml conical flasks of sterilizing.Experimental group (VE1,
VE2, VE3, VE4) 30 DEG C, LPN080 bacterium solutions (culture medium is EIM culture mediums) 500 μ l of 200rpm incubator overnight cultures are added,
The EIM culture mediums of equivalent are added in control group (VC1, VC2, VC3, VC4).30 DEG C are placed in, 48h is cultivated in 150rpm shaking tables, is respectively taken
Water sample dilutes 10 with PBS buffer solution5Times, 100 μ l apply TCBS 30 DEG C of insulating boxs of tablet and are incubated overnight, and record vibrios bacterium colony on tablet
Number, as a result see the table below 2.
Inhibiting rates of the 2 Sphingol single-cell LPN080 of table to vibrios
| Experimental group | Clump count (a) | Control group | Clump count (a) |
| VE1 | 0 | VC1 | 11 |
| VE 2 | 0 | VC 2 | 4 |
| VE 3 | 1 | VC 3 | 28 |
| VE 4 | 0 | VC 4 | 11 |
| It is average | 0.25 | It is average | 13.5 |
It can be seen that by data in table 2 after LPN080 bacterium solutions are added in water sample, by incubation in 48 hours, 105Times
In the case of releasing, for the TCBS tablets average colony of experimental group less than 1, control group TCBS tablet average colonies are 13.5, because
In the case that this shows that a certain amount of glucose is added in water body, Sphingol single-cell LPN080 or with significantly Reverse transcriptase
Vibrios shows that Sphingol single-cell LPN080 other than the nitrogen nitrogen that can degrade, also has the effect of consumingly Reverse transcriptase vibrios.
Embodiment 6：Sphingol single-cell LPN080 preparations
Fermentation medium (SPM) composition：Ammonium sulfate 1.5kg, peptone 0.5kg, wheat bran 0.6kg, glucose 6kg, sucrose
6kg, corn juice 1.5kg, 1 ton of tap water adjust pH to 7.0 with NaOH, and the sterilizing of normal fermentation tank is cooling.
Sphingol single-cell LPN080 culture solutions are inoculated into the ratio of 1% (V/V) in SPM culture mediums, using routine
Fermentation tank, carry out conventional liquid deep layer fermenting, 30 DEG C, fermentation time 36h of fermentation temperature, after fermentation, every liter of addition
The trehalose of 1.5g, mixes well, and obtains Sphingol single-cell LPN080 preparations (i.e. microorganism formulation), room temperature or 4 DEG C of preservations.
Embodiment 7：Sphingol single-cell LPN080 formulation applications
Sphingol single-cell LPN080 preparations are used by 6 kilograms of amounts of breeding water body per acre (depth of water 1m is calculated).First by sheath ammonia
Alcohol monad LPN080 preparations are added in the cultivation water of 4-10 times of volume, are added by every liter of Sphingol single-cell LPN080 preparation
The amount of 80g brown sugar is added brown sugar, after mixing, is placed at room temperature for 10 hours, full pool spilling head.
It should be noted that above-described embodiment is merely illustrative of the technical solution of the present invention, rather than limit the present invention.?
On the basis of the present invention, the technical staff in field can be improved appropriately or deform, and these improvement do not make accordingly
Technical solution be substantially detached from protection scope of the present invention.Protection scope of the present invention is limited by claim and its equivalent
It is fixed.
Sequence table
<110>Chinese Academy of Science Nanhai Ocean Research Institute Guangdong Microbes Inst（In Guangdong Province's microbiological analysis detection
The heart）
<120>The Sphingol single-cell LPN080 and its microorganism formulation of one plant of different oxygen ammonia assimilation and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1254
<212> DNA
<213>Sphingol single-cell LPN080 (Sphingomonas sp. LPN080)
<400> 1
aggcttcggc cttagtggcg cacgggtgcg taacgcgtgg gaatctgccc cttggttcgg 60
aataacagtt ggaaacgact gctaataccg gatgatgacg taagtccaaa gatttatcgc 120
cgagggatga gcccgcgtag gattaggtag ttggtgtggt aaaggcgcac caagccgacg 180
atccttagct ggtctgagag gatgatcagc cacactggga ctgagacacg gcccagactc 240
ctacgggagg cagcagtggg gaatattgga caatgggcga aagcctgatc cagcaatgcc 300
gcgtgagtga tgaaggcctt agggttgtaa agctctttta cccgggatga taatgacagt 360
accgggagaa taagctccgg ctaactccgt gccagcagcc gcggtaatac ggagggagct 420
agcgttattc ggaattactg ggcgtaaagc gcacgtaggc ggctttgtaa gtaagaggtg 480
aaagcccaga gctcaactct ggaattgcct tttagactgc atcgcttgaa tcatggagag 540
gtcagtggaa ttccgagtgt agaggtgaaa ttcgtagata ttcggaagaa caccagtggc 600
gaaggcggct gactggacat gtattgacgc tgaggtgcga aagcgtgggg agcaaacagg 660
attagatacc ctggtagtcc acgccgtaaa cgatgataac tagctgtccg gggacttggt 720
ctttgggtgg cgcagctaac gcattaagtt atccgcctgg ggagtacggc cgcaaggtta 780
aaactcaaat gaattgacgg gggcctgcac aagcggtgga gcatgtggtt taattcgaag 840
caacgcgcag aaccttacca gcgtttgaca tggcaggacg acttccagag atggatttct 900
tcccttcggg gacctgcaca caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg 960
ttgggttaag tcccgcaacg agcgcaaccc tcgcctttag ttgccatcat ttagttgggc 1020
actttaaagg aaccgccggt gataagccgg aggaaggtgg ggatgacgtc aagtcctcat 1080
ggcccttacg cgctgggcta cacacgtgct acaatggcgg tgacagtggg cagcaagcac 1140
gcgagtgtga gctaatctcc aaaagccgtc tcagttcgga ttgttctctg caactcgaga 1200
gcatgaatgc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgt 1254
Claims (9)
- Sphingol single-cell 1. (Sphingomonas sp.) LPN080, deposit number is GDMCC No：60365.
- 2. a kind of microorganism formulation, which is characterized in that include Sphingol single-cell (Sphingomonas described in claim 1 sp.)LPN080。
- 3. the preparation method of the microorganism formulation described in claim 2, which is characterized in that by sphingol described in claim 1 Monad (Sphingomonas sp.) LPN080 is inoculated into fermented and cultured in fluid nutrient medium, after fermentation, every liter of addition The trehalose of 1.5g, mixes well, and obtains microorganism formulation.
- 4. preparation method according to claim 3, which is characterized in that the fluid nutrient medium is SPM culture mediums, every Culture medium is risen to be prepared by the following method：Ammonium sulfate 1.5g, peptone 0.5g, wheat bran 0.6g, grape are added in 1L water Sugared 6g, sucrose 6g, corn juice 1.5g adjust pH to 7.0.
- 5. micro- described in Sphingol single-cell (Sphingomonas sp.) LPN080 described in claim 1 or claim 2 Application of the biological agent in water body ammonia nitrogen of degrading.
- 6. application according to claim 5, which is characterized in that the water body is breeding water body.
- 7. micro- described in Sphingol single-cell (Sphingomonas sp.) LPN080 described in claim 1 or claim 2 Application of the biological agent in preparing antibacterials.
- 8. application according to claim 7, which is characterized in that the antibacterials are to inhibit the drug of vibrios.
- 9. micro- described in Sphingol single-cell (Sphingomonas sp.) LPN080 described in claim 1 or claim 2 Application of the biological agent in aquaculture.
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| CN112280719A (en) * | 2020-11-19 | 2021-01-29 | 江苏海洋大学 | Sphingomonas echinocandis N-LY-1 and application thereof |
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