CN105886422A - Bacillus subtilis BY7 and application thereof in degrading residual feeds and ammonia nitrogen - Google Patents

Bacillus subtilis BY7 and application thereof in degrading residual feeds and ammonia nitrogen Download PDF

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CN105886422A
CN105886422A CN201510854901.1A CN201510854901A CN105886422A CN 105886422 A CN105886422 A CN 105886422A CN 201510854901 A CN201510854901 A CN 201510854901A CN 105886422 A CN105886422 A CN 105886422A
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bacillus subtilis
ammonia nitrogen
nitrogen
ammonia
water body
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CN105886422B (en
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季相山
王慧
王晓云
赵燕
陈红菊
刘洪军
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Shandong Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • 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
    • C02F3/341—Consortia of bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Abstract

The invention relates to bacillus subtilis BY7 and an application thereof in degrading residual feeds and ammonia nitrogen. The bacillus subtilis BY7 is preserved in China Center for Type Culture Collection on September 29, 2015, with preservation number of CCTCC M 2015585; and the 16s rNDA of the bacillus subtilis BY7 is as shown in SEQ ID NO.1. The bacillus subtilis BY7 or a BY7 suspension can degrade ammonia nitrogen in a contaminated water body or an aquaculture water body and residual feeds in fee. The bacillus subtilis provided by the invention is applicable to the following aspects: (1) the bacillus subtilis, with a strong ammonia nitrogen degrading capacity, can be used for improving an ammonia-nitrogen pollution problem in the water body and for reducing an ammonia-nitrogen content in an aquaculture pond; and (2) the bacillus subtilis, which is capable of degrading protein in the residual feeds of the feed, can be used for degrading the residual feeds in the aquaculture pond and organic matters in the bottom of the pond. The bacillus subtilis disclosed by the invention has a broad application prospect in aquaculture ponds at high temperature in the summer season.

Description

Bacillus subtilis BY7 and the application of degrade residual bait and ammonia nitrogen thereof
One, technical field
The present invention relates to bacillus subtilis BY7 and the application of degrade residual bait and ammonia nitrogen thereof, belong to microbial technology field.
Two, background technology
In aquaculture pond, the itrogenous organic substance such as residual bait, feces at the bottom of pond caused by a large amount of bait throwing in is difficult to completely by natural micro-life Thing oxidation Decomposition, remaining Organic substance accumulates over a long period and is piling up at the bottom of pond, pollutes substrate and the water quality in pond.Therefore, artificially Can degrade the in a large number microorganism fungus kind of residual bait etc. of applying is very important to accelerate at the bottom of pond organic decomposition.Additionally, due to it is residual The itrogenous organic substance such as bait, feces is many with ammonia nitrogen (NH after being decomposed4 +-N) form be released in water body, cause water body ammonia nitrogen to contain Measuring too high, affect the growth of Aquatic farming animals, even jeopardize the life of cultivated animals, therefore, screening can reduce water body ammonia nitrogen Microorganism fungus kind to be applied to aquaculture production significant.
Bacillus subtilis is one of probiotic bacteria that the Ministry of Agriculture of China allows to use, owing to its action effect is more obvious, by The substrate being widely used in aquaculture process and water quality improvement.Mostly currently used Bacillus subtilis strain is from naturally supporting Grow what water body separated.In hot spring breeding water body, directly take from underground hot spring, its microorganism fungus kind and nature due to breeding water Differing greatly in breeding water body.
Three, summary of the invention
The present invention relates to a bacillus subtilis BY7 and the application of degrade residual bait and ammonia nitrogen thereof.It is to divide from hot spring breeding water body From filtering out high proteinase yield, efficient degradation feedstuff and the bacillus subtilis of ammonia nitrogen under a plant height temperature, this bacterial strain is at summer high temperature The cultivating pool in season has a extensive future.
Bacillus subtilis (Bacillus subtilis) BY7, is preserved in Chinese Typical Representative on the 29th in JIUYUE in 2015 and cultivates CCTCC (is called for short, address is: No. 299 Wuhan Universitys of Wuchang District, Wuhan City, Hubei Province Bayi Road) in thing preservation center, and preserving number is CCTCC M 2015585；Its 16s rDNA is as shown in SEQ.ID.NO 1.
The invention still further relates to the fermentative medium formula of suitable bacillus subtilis BY7 growth.
The invention still further relates to the bacteria suspension of bacillus subtilis BY7.Described bacteria suspension can be with the resuspended bacterium of physiological saline solution or PBS Body obtains.Described bacteria suspension OD600Value can reach 2.0.
The invention still further relates to bacillus subtilis BY7 or BY7 bacteria suspension ammonia nitrogen in pollution degradation water body or aquaculture system Application, comprises the steps: appropriate bacillus subtilis BY7 or BY7 bacteria suspension to be applied according to water body ammonia and nitrogen pollution degree In aquaculture system or ammonia and nitrogen pollution water body, utilize the ability of the degradation of ammonia nitrogen that bacillus subtilis BY7 has to reduce water Ammonia nitrogen in body.The pH value of aquaculture system or polluted-water is preferably between 5.0-8.0；Aquaculture system or contaminant water Body to have certain carbon source and nitrogen source, C/N between 3-7, preferably 6.
The present invention also protects the application in the degraded residual bait of feedstuff of bacillus subtilis BY7 or BY7 bacteria suspension, comprises the steps: Appropriate bacillus subtilis BY7 or BY7 bacteria suspension are put in aquaculture system, utilizes bacillus subtilis BY7 to have The ability of some protein degradations is degraded the residual bait of feedstuff.The pH value of aquaculture system is preferably between 5.0-8.0；Aquatic product Breeding water body to have certain carbon source and nitrogen source, C/N between 3-7, preferably 6.
The bacillus subtilis that the present invention provides, cultural method is simple, fast growth, be resistant to the ammonia nitrogen of high concentration, environment Strong adaptability, safety height.The bacillus subtilis that the present invention provides can be applicable to following aspect: (1) has the strongest due to it Ammonia nitrogen degradation ability, can be used for improving the ammonia and nitrogen pollution problem of water body；Can be used for reducing the ammonia-nitrogen content in aquaculture pond； (2) due to the protein in its residual bait of decomposable asymmetric choice net feedstuff, can be used for the degrade residual bait in aquaculture pond and the organic matter at the bottom of pond. The present invention has preferable application prospect.
Four, accompanying drawing explanation
Fig. 1 is that bacillus subtilis (Bacillus subtilis) BY7 is straight with batch separating obtained bacterial strain decomposition casein transparent circle Footpath is compared.
Fig. 1 illustrates: coat in casein culture medium by BY7 and other bacterial strains, after 37 DEG C of cultivation 24h with slide gauge respectively Measure transparent circle diameter and colony diameter, calculate the ratio of transparent circle diameter and colony diameter, can determine whether each bacterium according to ratio size Plant protelytic ability.
Fig. 2 A is bacillus subtilis (Bacillus subtilis) BY7 bacterium colony photo, and B is that microscope hypothallus form is shone Sheet.
In Fig. 2, BY7 is classified identifying from morphology by A figure and B figure.
Fig. 3 A is bacillus subtilis BY7 16S rDNA electrophoretogram, B be based on isolated strains and Genbank are included its The systematic evolution tree of the 16S rDNA sequence construct of his related strain.
Fig. 3 illustrates: systematic evolution tree based on each bacterial strain 16S rDNA sequence construct can its taxonomy of Preliminary Identification.
Fig. 4 is culture medium C/N (A), pH value (B) and temperature (C) be to bacillus subtilis (Bacillus subtilis) The impact of BY7 growth.
The purpose of Fig. 4 determines that the suitable culture conditions of bacillus subtilis BY7.
Fig. 5 is the product protease activity of bacillus subtilis (Bacillus subtilis) BY7 and this Laboratories Accession bacterial strain BS2 Property compares.
The purpose of Fig. 5 is that accurately to compare bacillus subtilis BY7 white with laying eggs of the bacillus subtilis BS2 of this Laboratories Accession Enzymatic activity difference.
Fig. 6 is bacillus subtilis (Bacillus subtilis) BY7 degradation effect to forage protein.
The purpose of Fig. 6 is the ability of detection bacillus subtilis BY7 degraded forage protein.
Fig. 7 is that bacillus subtilis (Bacillus subtilis) BY7 is to the degradation effect of ammonia nitrogen in water body.
The purpose of Fig. 7 is that detection bacillus subtilis BY7 is to the degradation capability of ammonia nitrogen in water body.
Five, detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experimental technique in following embodiment, If no special instructions, it is conventional method.Test material used in following embodiment, if no special instructions, is from conventional Change what reagent shop was commercially available.Quantitative test in following embodiment, is respectively provided with three times and repeats experiment, results averaged. In following example, it is deionized water for preparing the water of each culture medium.In following instance, culture medium is equal after having prepared Moist heat sterilization 20min at a temperature of 121 DEG C.Following example are used OD600Value characterizes thalli growth amount.C/N i.e. carbon nitrogen quality Ratio, also known as carbon-nitrogen ratio.
Basal medium: peptone 10g, yeast powder 5g, sodium chloride 10g, solid separately adds the agar of 15g, water 1000mL, Adjust pH 6.0.
Fermentation medium: glucose 15g, yeast powder 5g, peptone 10g, potassium dihydrogen phosphate 4.5g, sodium carbonate 2g, Sodium chloride 5g, magnesium sulfate 0.2g, trace element solution (zinc sulfate 0.22g/L, copper sulfate 0.08g/L, manganese sulfate 2.03 G/L, ferrous sulfate 0.2g/L, boric acid 2.86g/L, ammonium molybdate 0.02g/L) 0.5mL, water 1000mL, regulate pH To 6.0.
Casein culture medium: preparation A liquid and B liquid respectively.A liquid: weigh disodium hydrogen phosphate 1.07g, casein 5g, add about 100mL water, heating for dissolving.B liquid: weigh potassium dihydrogen phosphate 0.36g, adds about 100mL water dissolution.After the mixing of A, B liquid, Add agar 20g, be finally settled to 1000mL with water, regulate pH to 6.0.
Colourless culture medium: sodium citrate is as sole carbon source, and ammonium chloride is as only nitrogen source, sodium chloride 5g, potassium dihydrogen phosphate 0.7g, magnesium sulfate 1g, trace element solution (zinc sulfate 0.22g/L, copper sulfate 0.08g/L, manganese sulfate 2.03g/L, Ferrous sulfate 0.2g/L, boric acid 2.86g/L, ammonium molybdate 0.02g/L) 0.5mL, water 1000mL, regulate pH to 6.0.
Trace element solution: zinc sulfate 0.22g, copper sulfate 0.08g, manganese sulfate 2.03g, ferrous sulfate 0.2g, boric acid 2.86g, ammonium molybdate 0.02g, water 1000mL, regulate pH to 6.0.
100 μ g/mL tyrosine solutions: accurately weigh at the 105 DEG C of tyrosine dried to constant weight 100 μ g, be gradually added 6mL The hydrochloric acid of 1mol/L make dissolving, be settled to 100mL with 0.2mol/L hydrochloric acid, its concentration is 1000 μ g/mL, then inhales Take this liquid 10mL, be settled to 100mL with 0.2mol/L hydrochloric acid, be i.e. made into 100 μ g/mL tyrosine solutions, protect in refrigerator Deposit.
2% casein solution: accurately weigh casein 2g, adds 0.1mol/L NaOH solution 10mL, and heating in water bath dissolves, Then it is settled to 100mL with the phosphate buffer of pH 7.2, preserves in refrigerator.
Ammonia nitrogen (NH4 +-N) content by ET99732 multi-parameter water quality Quick testing instrument measure.
Isolated and purified and the qualification of example 1 bacillus subtilis BY7
One, strain is isolated and purified
1, fall albumen and the enrichment of ammonia nitrogen bacterial strain
Tilapia culturing pool in hot spring Aquatic product culture experiment field, Daiyue District, Colossoma brachypomum culturing pool, Ophicephalus argus culturing pool, Cyprinus carpio are supported Grow each water sampling 100mL in pond, gold crucian carp culturing pool, take 5mL after being mixed by the water sample of each culturing pool and be inoculated in equipped with 100mL Enrichment medium (adds NH in basal medium4Cl, is adjusted to 50mg/L by ammonia nitrogen concentration and obtains enrichment medium) conical flask In, 37 DEG C, 200r/min shaken cultivation 1 day；Take the culture fluid 1mL after cultivating 1 day and be again inoculated in 100mL enrichment In culture medium, enrichment culture 6 generation the most repeatedly.
2, fall albumen and the screening of ammonia nitrogen bacterial strain, separation
Take enrichment culture to the culture fluid (OD in the 6th generation600Value is 1.5) 1mL is in the centrifuge tube of 1.5mL, and 80 DEG C are boiled 15min, Culture fluid sterilized water after process is diluted to 10 respectively-5、10-6、10-7、10-8Times, take the training after above-mentioned four kinds of dilutions respectively Nutrient solution 50 μ L coats LB flat board, cultivates observation colony morphology characteristic after 14h for 37 DEG C, similar hay bud in picking form Spore bacillus and the bacterium colony that growth is fast, bacterium colony is big, quantity is many, use trilinear method to be purified, after purification 3 generation, carry out 4 DEG C respectively Inclined-plane preserves.
Above-mentioned single bacterium colony is inoculated in basal medium respectively, 37 DEG C of shaken cultivation 12h, then takes and coat ferment in right amount In culture medium, cultivate 24h, measure transparent circle diameter and colony diameter with slide gauge respectively, calculate its ratio, root for 37 DEG C Evaluate the ability of its extracellular proteinase according to ratio, finally obtain and grow rapidly and 8 strain bacterium of high proteinase yield, be respectively designated as BY1 BY8 (i.e. BY1, BY2, BY3, BY4, BY5, BY7, BY7, BY8).Transparent circle diameter/the colony diameter of BY1 BY8 Result is shown in Fig. 1, and wherein the transparent circle diameter of BY7 is the highest with colony diameter ratio, has reached 5.4.
Isolated and purified BY1 BY8 is inoculated in basal medium respectively, 37 DEG C of shaken cultivation 12-16h, takes training respectively Support thing 4000rpm and be centrifuged 10min, abandoning supernatant, make bacteria suspension by the resuspended precipitation of PBS, then be inoculated in 100mL respectively Screening culture medium (on the basis of described colourless culture medium adjust NH4The concentration of Cl, regulation ammonia nitrogen concentration obtains to 300mg/L To screening culture medium) in, 200r/min shaken cultivation measures ammonia-nitrogen content after 1 day, result display degradation of ammonia nitrogen effect is best Bacterial strain be BY7.
Two, the qualification of strain
1, morphologic observation
By BY7 inoculation on LB solid medium flat board, observe colony morphology characteristic.Figure is shown in by the bacterium colony photo of BY7 bacterial strain 2A, 100 power microscope enlarged photographs are shown in Fig. 2 B.BY7 bacterium colony is point-like, and milky is translucent, the smooth of the edge, protruding, There is fold in late stage of culture.
2, biochemical identification
Using Biolog microplate that BY7 bacterial strain is carried out Physiology and biochemistry qualification, qualification result is Bacillus subtilis.
The biochemical reactions result of bacterial strain BY7 collects as shown in table 1:
Note: "+" represent growth or positive reaction；"-" represents and does not grows or negative reaction.
3, molecular biology identification
Expand with the PCR that BY7 bacterium solution carries out 16S rDNA partial sequence for template:
Forward primer: 5'-AGAGTTTGATCMTGGCTCAG-3'(is as shown in SEQ.ID.NO 2),
Reverse primer: 5'-TACGGYTACCTTGTTACGACTT-3'(is as shown in SEQ.ID.NO 3).
PCR amplification program: 94 DEG C of denaturations 4min, 94 DEG C of degeneration 30s, 56 DEG C of renaturation 45s, 72 DEG C extend 90s, 7min, 4 DEG C of preservations are extended in 72 DEG C after 35 circulations.By pcr amplification product electrophoresis detection on the agarose gel of 1%, Electrophoresis result is shown in Fig. 3 A.The PCR primer of purification is connected on PMD-18T carrier, is then transformed into E. coli competent Intracellular, PCR is accredited as the clone of the positive and send company to check order, and obtains BY7 16s rDNA sequence, and sequence results is shown in SEQ.NO. 1.Sequencing result is carried out Blast comparison, utilizes Megalign software building cladogram (Fig. 3 B), find BY7 and Bacillus It is 1 that subtilis gathers.
Comprehensive morphological qualification, biochemical identification and the result of molecular biology identification, BY7 bacterial strain is accredited as bacillus subtilis (Bacillus subtilis), named bacillus subtilis (Bacillus subtilis) BY7.
Bacillus subtilis (Bacillus subtilis) BY7, is preserved in Chinese Typical Representative on the 29th in JIUYUE in 2015 and cultivates Thing preservation center (be called for short CCTCC, address is: No. 299 Wuhan Universitys of Wuchang District, Wuhan City, Hubei Province Bayi Road in the school, Wuhan University's preservation center), preserving number is CCTCC M 2015585.
Three, the safety evaluatio of strain
Upgrowth situation is good, and body weight is that the 90 tail boltis of about 80g are divided into 9 groups (often organizing 10 tail fishes), point Not being set to: bacillus subtilis BY7 group 4, the bacillus subtilis BY7 concentration gavaged is respectively 106、107、108、109 cfu/mL；Streptococcus agalactiae group 4, the streptococcus agalactiae concentration gavaged is respectively 106、107、108、109cfu/mL；Raw Reason saline group 1, gavages physiological saline solution.Every tail fish gavages 1mL bacterium solution or physiological saline solution.Result is as shown in table 2, Finding after raising 10 days, the mortality rate gavaging bacillus subtilis and normal saline is 0, and gavages Buddhist nun sieve of streptococcus agalactiae Tilapia mortality rate is between 10-30%, and this illustrates that the bacillus subtilis BY7 that we separate is safe.
Table 2 strain safety evaluatio result
The research of example 2 bacillus subtilis BY7 growth conditions
One, the C/N impact on strain growth
1, picking bacillus subtilis BY7 monoclonal, is inoculated in 10mL basal medium, 37 DEG C, 200r/min shakes Swing cultivation 12h and (make bacterium solution OD in cultivating system600Value reaches 1.0), 4000rpm is centrifuged 10min, collects thalline, uses Physiological saline solution washs 3 times.
2, by resuspended for above-mentioned thalline physiological saline solution, respectively take 2mL bacteria suspension and be inoculated in 9 respectively to fill 100mL colourless In the conical flask of culture medium (pH is 6.0), adjust sodium citrate and the concentration of ammonium chloride in each conical flask culture medium respectively, make 9 C/N in individual conical flask culture medium is respectively 1,3,4,5,6,7,9 and 11, then 37 DEG C, 200r/min vibration Cultivating, sampling 1 time every 2h measures bacterium solution OD600Value, finds after cultivating 12h, when C/N is 6, and bacillus subtilis BY7 growth is best, and when C/N is higher or lower, the growth of bacillus subtilis BY7 is all by a certain degree of suppression (Fig. 4 A). Therefore, the C/N of suitable bacillus subtilis BY7 growth is 6.
Two, the pH impact on strain growth
1, picking bacillus subtilis BY7 monoclonal, is inoculated in 10mL basal medium, 37 DEG C, 200r/min shakes Swing cultivation 12h and (make bacterium solution OD in cultivating system600Value reaches 1.0), 4000rpm is centrifuged 10min, collects thalline, uses Physiological saline solution washs 3 times.
2, by resuspended for above-mentioned thalline physiological saline solution, respectively take 2mL bacteria suspension and be inoculated in 7 respectively to fill 100mL colourless In the conical flask of culture medium (C/N is 6), respectively the pH of culture medium in each conical flask is adjusted to 3.0,4.0,5.0,6.0, 7.0,9.0 and 11.0, then 37 DEG C, 200r/min shaken cultivation, sample 1 time every 3h, measure bacterium solution OD600Value, Finding after cultivating 15h, pH is in the range of 5.0-7.0, and bacillus subtilis BY7 can keep preferable growth conditions (Fig. 4 B), pH be grow when 6.0 best.
Result shows, bacillus subtilis BY7 has stronger environmental resistance, in its suitable C/N and pH and breeding water body Environment basically identical, be beneficial to its growth, there are in aquaculture system the potentiality of application.
Three, the temperature impact on strain growth
1, picking bacillus subtilis BY7 monoclonal, is inoculated in 10mL activation medium, 37 DEG C, 200r/min shakes Swing cultivation 12h and (make bacterium solution OD in cultivating system600Value reaches 1.0), 4000rpm is centrifuged 10min, uses sterile physiological salt Water washing precipitation 3 times, then makes bacteria suspension by resuspended for precipitation physiological saline solution.
2, respectively take 2mL bacteria suspension to be inoculated in respectively in 5 conical flasks filling the colourless culture medium of 100mL (pH 6.0), point At 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C and 40 DEG C, after 200r/min shaken cultivation 24h, do not measure bacterium solution OD600Value, result Display bacillus subtilis BY7 at 25-30 DEG C grows best (Fig. 4 C).
Example 3 bacillus subtilis BY7 protease production is analyzed
One, the drafting of standard curve
1, the tyrosine solution of various variable concentrations is prepared by table 3
The tyrosine solution of various variable concentrations prepared by table 3
Guan Hao 1 2 3 4 5 6
Distilled water (mL) 10 8 6 4 2 0
100 μ g/mL tyrosine (mL) 0 2 4 6 8 10
Tyrosine ultimate density (μ g/mL) 0 20 40 60 80 100
2, taking 6 test tubes, be designated as No. 1-6, being separately added into 0-10mL concentration is 100 μ g/mL tyrosine, is configured to The solution of tyrosine ultimate density described in table 3, respectively add sodium carbonate liquor 5mL and the Folin reagent that concentration is 0.4mol/L 1mL.After fully mixing with vortex mixer, put reaction 20min in 37 DEG C of waters bath with thermostatic control.
3, compare with No. 1 pipe, respectively colorimetric determination each pipe A680, survey three times, average.
4, with tyrosine concentration as abscissa, clean A680Value is vertical coordinate, draws standard curve, sets up regression equation calculation and goes out to work as Absorbance is tyrosine amount (μ g) when 1, is extinction constant K.
Two, protease activity determination
1, difference picking bacillus subtilis BY7 and the bacillus subtilis BS2 monoclonal of this Laboratories Accession, be inoculated in 10mL In basal medium, 37 DEG C, 200r/min shaken cultivation 12h (make bacterium solution OD600Value reaches 1.0), 4000rpm is centrifuged 10min, precipitates 3 times with physiological saline solution washing, then makes bacteria suspension by the resuspended precipitation of physiological saline solution.
2, the bacteria suspension of BY7 and BS2 is inoculated in the fermentation medium of 100mL respectively with the inoculum concentration of volume ratio 5%, Then 37 DEG C, take each 5mL of fermentation liquid after 200r/min shaken cultivation 48h, 12000rpm is centrifuged 5～10min, takes The buffer of clear liquid pH 7.5 is diluted to debita spissitudo, for test.
3, take 6,15 × 100mm test tube and number, being divided into BY7 and BS2 two groups, often three repetitions of group.Often add in pipe Supernatant 1mL after dilution, is placed in 37 DEG C of waters bath with thermostatic control preheating 5min.Each casein solution 1 adding same preheating again ML, shakes up, and is accurately incubated 10min.After the time, respectively add the trichloroacetic acid 2mL of 0.4mol/L, to terminate Reaction, continues to be placed in insulation 10min in water-bath, centrifugal or filtration after making residual protein precipitation.
4, separately take 6,15 × 150mm test tube and number, being divided into BY7 and BS2 two groups, often three repetitions of group.Often add in pipe Entering filtrate 1mL, then add the sodium carbonate 5mL and Folin reagent 1mL that concentration is 0.4mol/L, shake up, 37 DEG C of water-baths are protected A is carried out after temperature color development 20min680Measure.
5, blank assay also takes three test tubes, and numbering 1,2,3, assay method ibid, only first added before adding casein solution Concentration is the trichloroacetic acid 2mL of 0.4mol/L, makes enzyme inactivate, adds casein.
6, the calculating of enzyme activity
Prolease activity (U/mL)=(sample A680-comparison A680)×K×V/T×N
K: in standard curve, absorbance is tyrosine micrograms time " 1 "；
V: enzymatic reaction cumulative volume；
T: the enzyme digestion reaction time (min)；
N: the extension rate of enzyme liquid.
Result shows, the product prolease activity of BS2 bacterial strain is 16.61U/mL, and the product prolease activity of BY7 bacterial strain is 43.28 U/mL (Fig. 5).The product prolease activity of BY7 is significantly higher than BS2, BY7 and can play a role in terms of aminosal, can apply Residual bait in degraded aquaculture pond and the organic matter at the bottom of pond.
Example 4 bacillus subtilis BY7 degrades feedstuff capability analysis
1,2% shrimp feed culture medium is prepared: shrimp feed 2g, potassium dihydrogen phosphate 4.5g, sodium chloride 5g, magnesium sulfate 0.2g, Add water and regulate pH to 6.0 after being settled to 100mL.
2, picking bacillus subtilis BY7 monoclonal is inoculated in 10mL basal medium, 37 DEG C, 200r/min vibration Cultivate 12h and (make bacterium solution OD600Value reaches 1.0), 4000rpm is centrifuged 10min, precipitates 3 times with physiological saline solution washing, Then bacteria suspension is made by the resuspended precipitation of physiological saline solution.
3, with the inoculum concentration of volume ratio 2% by bacterial suspension inoculation to the triangular flask containing 300mL 2% shrimp feed culture medium, 37 DEG C, 200r/min continuous oscillation is cultivated 5 days, and then 5000rpm is centrifuged 10min and respectively obtains cleer and peaceful precipitation, pellet frozen After drying, upper cleer and peaceful cryodesiccated precipitation uses Kjeldahl nitrogen determination protein content respectively.Blank group does not the most inoculate bacteria suspension, Other are identical with the BY7 group operation adding bacteria suspension.According to formula: N=(V1-V2)×C×0.0140×6.25/m Calculate the protein content of each sample, wherein V1It is the amount (mL) consuming hydrochloric acid during the BY7 group titration adding bacteria suspension, V2 Being the blank hydrochloric acid content (mL) organized and consumed, C is normal hydrochloric acid liquid concentration (mol/L), and m is sample quality (g), and 6.25 are Nitrogen is scaled the mean coefficient of protein.
Result shows, compared with blank group, the protein content of the BY7 group solid state adding bacteria suspension substantially reduces, and liquid The protein content of body state is the most significantly raised (Fig. 6), i.e. protein degradation in solid feed can be liquid by BY7 effectively Form, degradation rate is up to 66%.Due to the protein in bacillus subtilis BY7 decomposable asymmetric choice net feedstuff, can be used for degraded Aquatic product and support Grow the residual bait in pond and the organic matter at the bottom of pond.
Example 5 bacillus subtilis BY7 degradation of ammonia nitrogen characteristic research
1, preparation culture medium first: adjusting carbon source and the concentration in nitrogen source on the basis of colourless culture medium, making ammonia nitrogen concentration is 300 Mg/L, C/N are 6, and pH is 6.0.
2, picking bacillus subtilis BY7 monoclonal, is inoculated in 10mL basal medium, 37 DEG C, 200r/min shakes Swing cultivation 12-16h and (make bacterium solution OD600Value reaches 1.0), 4000rpm is centrifuged 10min, by physiological saline solution washing precipitation 3 times, then make bacteria suspension by the resuspended precipitation of physiological saline solution.
4, by 1mL bacterial suspension inoculation in 100mL culture medium first, 37 DEG C, 200r/min shaken cultivation, every 24h Sampling once, ammonia nitrogen concentration calculate ammonia nitrogen degradation rate in the centrifugal wild Oryza species of detection.Ammonia nitrogen concentration is by ET99732 multiparameter water Matter analyzer measures and obtains.Blank group does not the most inoculate bacteria suspension, and other are identical with the BY7 group operation adding bacteria suspension.
Ammonia nitrogen degradation rate=(ammonia nitrogen concentration in system after the ammonia nitrogen concentration in initial time cultivating system-cultivation 24h)/initial Ammonia nitrogen concentration × 100% in moment cultivating system.
Result shows, compared with blank group, in adding BY7 group 24h of bacteria suspension, ammonia-nitrogen content substantially reduces, i.e. BY7 bacterial strain Can effectively decompose the ammonia nitrogen in culture medium, the degradation rate after 120h is up to 60% (Fig. 7).Owing to bacillus subtilis BY7 has There is the strongest ammonia nitrogen degradation ability, can be used for reducing the ammonia-nitrogen content in aquaculture pond.

Claims (3)

1. a strain preserving number is the bacillus subtilis BY7 of CCTCC M 2015585；Its 16s rDNA such as SEQ.ID.NO 1 Shown in.
2. bacillus subtilis BY7 as claimed in claim 1 answering in pollution degradation water body or aquaculture system ammonia nitrogen With.
3. the bacillus subtilis BY7 as claimed in claim 1 application in the degraded residual bait of aquaculture feed.
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CN109355223A (en) * 2018-11-08 2019-02-19 中国科学院南海海洋研究所 One plant of bacillus subtilis N2 and its application with ammonia nitrogen degradation function
CN109355223B (en) * 2018-11-08 2020-10-09 中国科学院南海海洋研究所 Bacillus subtilis N2 with ammonia nitrogen degradation function and application thereof
CN110791454A (en) * 2019-11-26 2020-02-14 中国科学院烟台海岸带研究所 Efficient ammonia nitrogen degrading strain and application thereof
CN111748497A (en) * 2020-07-07 2020-10-09 山东农业大学 Bacillus megaterium and application thereof in rapid degradation of nitrite
CN111748497B (en) * 2020-07-07 2022-02-08 山东农业大学 Bacillus megaterium and application thereof in rapid degradation of nitrite
CN112553112A (en) * 2020-12-19 2021-03-26 武汉水之国环保科技有限公司 Denitrifying bacillus subtilis and application thereof in strain detoxification
CN112877247A (en) * 2021-02-24 2021-06-01 华南理工大学 Method for treating high ammonia nitrogen wastewater by using bacillus

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