CN103695353A - Bacillus subtilis with capability of degrading ammonia nitrogen and application of bacillus subtilis - Google Patents
Bacillus subtilis with capability of degrading ammonia nitrogen and application of bacillus subtilis Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis with capability of degrading ammonia nitrogen, wherein the bacillus subtilis is screened out; the bacillus subtilis is classified and named bacillus subtilis, and collected in China Center for Type Culture Collection (CCTCC), the collection number of the bacillus subtilis is CCTCC No: M2013490, and the collection date is October 23, 2013. The invention also discloses application of the bacillus subtilis for degrading ammonia nitrogen in water and treating aquaculture water. The screened bacillus subtilis is capable of quickly and effectively reducing the content of nitrate and the content of nitrite in the water, solving the problem of muddy water caused by pollution, and changing the water from muddy to clear; the bacillus subtilis has a strong water purification function and relatively strong activities of protease, lipase and amylase, so that the bacillus subtilis is capable of promoting the degradation of nutrients in feed, thus aquatic animals can absorb and utilize the feed more fully.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to subtilis and application thereof that a strain has ammonia nitrogen degradation ability.
Background technology
At present, due to the continuous expansion of aquaculture scale, the self-cleaning in pond and regulating power can not meet the needs of the eutrophy factors such as removing residual bait, fishes and shrimps movement, ammonia nitrogen, nitrate, nitrite, sulfide severe overweight in aquaculture water
[1], pond water quality runs down, and even injures the hygienic safety of each fishery products.Excessive use antibiotic medicine not only can make the resistance of bacterium strengthen, and also can cause the imbalance of microorganism and breeding environment, and microbiotic is residual in vivo, after people is edible, can produce harm to human body.
From the beginning of the eighties in last century, China brings into use probiotics as improver of water quality in aquaculture production.Be mainly photosynthetic bacteria, also obtained better effects, but it be long to exist the production cycle, the defect such as the quality guaranteed period is short, and quality is unstable.And from Pond Silt separated subtilis long quality-guarantee period, constant product quality, safety, has no side effect, and is therefore more suitable for using in breeding production.Because its production cost is low, can be made into various formulation, so be easier to large-scale industrialization, produce.
Ni Chunzhi etc.
[2]photosynthetic bacteria to the research of aquaculture water ecological regulation and control in, mention photosynthetic bacterium and apply after culturing pool, due to the breeding of self, bacterium number is increased gradually, 8～10d peaks.Use after photosynthetic bacterium, due to the breeding of photosynthetic bacterium, unwanted bacteria-vibrios, photogenic bacterium in culturing pool have reduced, and reach and purify water, Optimal culture environment, the balance of maintenance culturing pool microecosystem.Because photosynthetic bacterium bacterium is counted peak and comes across 6th～8d after applying, therefore throw in the timed interval of photosynthetic bacterium and be advisable with 8d left and right.
Chou Li
[3]in the research of applying in cultivation subtilis, illustrated in shrimp pond and used after subtilis, the content of the ammonia nitrogen of aquaculture water, nursery stage declines 52.5%, and mature stage declines 50%, nitroso-group nitrogen content, nursery stage declines 50%, and mature stage declines 32.5%, reduces moisturizing 60%.Active bacteria formulation is than 191 yuan/mu of chemicals usage quantity cost savings, and the average specification of the South America prawn same period improves 1cm, and per mu yield increases 24.08kg, and mu is 118.26 yuan of pure benefits once; Yu Jian etc.
[4]in the research of photosynthetic bacterium antioxygenation, simulating microbial film lipid, protein composition and being rich in two important mensuration systems of brain homogenate of lipid, affirmed the restraining effect of photosynthetic bacterium to lipid peroxidation.
Subtilis is a kind of unicellular, without pod membrane, and endogenous spore, the gram-positive microorganism that can move.Its distribution is very extensive, at soil, animal and plant body etc., locates all can find.Subtilis can produce many extracellular enzymes, decomposes rapidly organism in water body, promotes the oxidation of sulfide and nitrite, and the ability that has Fast Growth breeding and conform
[5].Xiong Wei etc.
[6]in Hainan spot joint shrimp aquaculture pond, use after bacillus subtilis formulation, in culturing pool, objectionable impurities significantly reduces as nitrite, hydrogen sulfide, and COD contrasts significantly and declines.
Summary of the invention
Technical problem to be solved by this invention is to provide the subtilis that a strain has ammonia nitrogen degradation ability.
The technical problem that the present invention also will solve is to provide the application of above-mentioned subtilis.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The subtilis that a strain of the present invention has ammonia nitrogen degradation ability is the bacterial strain that contriver separated from the sewage draining exit mud of Nanjing in August, 2012, through morphological observation, Physiology and biochemistry experiment and PCR, identify, confirm as subtilis, its Classification And Nomenclature is subtilis (Bacillus subtilis), now be preserved in Chinese Typical Representative culture collection center, address: China. Wuhan. Wuhan University, postcode 430072, deposit number: CCTCC No:M 2013490, preservation date: on October 23rd, 2013.
The above-mentioned subtilis with ammonia nitrogen degradation ability of the present invention is carried out to the degradation analysis of ammonia nitrogen concentration, its result shows, subtilis can effectively reduce rapidly nitrate, the nitrite content in water body, can improve the water turbidity problem causing due to pollution, water quality is clear by muddy change, there is very strong Function For Purifying Water, there is stronger proteolytic enzyme, lipase, diastatic activity, promote nutrient substance degraded in feed, make aquatic product animal more abundant to absorbing of feed.
Therefore the above-mentioned subtilis with ammonia nitrogen degradation ability can be applied in ammonia nitrogen in degraded water body.
The above-mentioned subtilis with ammonia nitrogen degradation ability can be applied in administering aquaculture system.
Beneficial effect: the subtilis that the present invention's screening obtains can effectively reduce rapidly nitrate, the nitrite content in water body, can improve the water turbidity problem causing due to pollution, water quality is clear by muddy change, there is very strong Function For Purifying Water, there is stronger proteolytic enzyme, lipase, diastatic activity, promote nutrient substance degraded in feed, make aquatic product animal more abundant to absorbing of feed.
Accompanying drawing explanation
Fig. 1 subtilis.
Fig. 2 subtilis bacterium colony.
Fig. 3 PH is to OD
600value affect result.
The affect result of Fig. 4 temperature on OD600 value.
Fig. 5 ammonia nitrogen residual concentration is curve over time.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described content of embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1:
1 bacterial classification source
From Pond Silt, getting appropriate sewage takes back laboratory and carries out isolation identification.
2 experiment equipments and reagent
2.1 experiment material
Broth agar culture medium: extractum carnis 5g, peptone 10g, sodium-chlor 5g, agar 18g, distilled water 1000mL.Sterilizing 15min under 121 ℃ of conditions, 4 ℃ save backup.
Strain activation and culture base: beef extract-peptone liquid nutrient medium: extractum carnis 3.0g, peptone 10.0g, NaCl 5.0g, H
2o 1000mL, pH7.2-7.4 value, is sub-packed in 150mL triangular flask, every bottle of 100mL, sterilizing 15min under 121 ℃ of conditions, 4 ℃ save backup.
Ammonia nitrogen substratum (simulation water body): glucose 5.0g, (NH4)
2sO
40.25g, NaCl 1.0g, K
2hPO
40.5g, MgSO
47H
2o 0.25g, H
2o 1000mL, pH value 7.0, wherein ammonia-nitrogen content is about 50mg/L, then gets 5mL dilution and be sub-packed in 50mL and support in pipe, sterilizing 15min under 121 ℃ of conditions, 4 ℃ save backup.
LB liquid nutrient medium: tryptone 10g/L, sodium-chlor 10g/L, yeast extract 5g/L, pH=7,121 ℃ of sterilizing 20min, LB solid medium for adding the agar of 18g/L among LB liquid nutrient medium.
2.2 experiment equipment
Culture dish, microscope, test tube, spreading rod, slide glass, transfering loop, inoculating needle, test-tube stand, triangular flask, beaker, Autoclave, incubator, visible spectrophotometer: 10～30mm cuvette.
3 experimental techniques
The separation of 3.1 bacteriums
The sewage taking back is got to several and by plate streaking partition method, coat on bouillon agar flat board, and substratum is inverted in to 37 ℃ of thermostat containers cultivation 18～24h.Choose well-grown bacterium colony and repeatedly inoculate, through for several times, cultivate screening, select on substratum, be single, canescence, irregular, colony edge is irregular, shaggy drying type bacterium colony microscopy, observe and carry out purifying cultivation.
3.2 morphological examination
The single bacterium colony smear of picking, utilizes Gram staining, with oily mirror, observes.
3.3 Physiology and biochemistry experiments
Separated bacterium is carried out to conventional Physiology and biochemistry experiment.
Glucose fermentation produces that acid, xylose fermentation for producing are sour, pectinose fermentation and acid, mannose ferment produce acid, lactose fermentation is produced acid, utilized Citrate trianion, Starch Hydrolysis, decomposition casein, oxydase, nitrate reduction, catalase.
3.4 PCR identify
[7]
The design of primer is with synthetic: according to the subtilis 16S rRNA gene order application Primer Premier5.0 software designed, designed of having delivered in GenBank, synthesized the primer of 1 pair of 1 154bp gene fragment that can increase, sequence is P15 '-CACTGGGACTGAGACACGG-3 ', P25 '-GGCGGCTGGTCCTAAAA-3 '.
The extraction of subtilis genomic dna: the single colony inoculation of picking subtilis is in 5mL nutrient broth, and 37 ℃ shake overnight incubation.
PCR system and reaction conditions: the subtilis complete genome DNA extracting of take is template, with synthetic primer, carries out pcr amplification.Reaction system (25 μ L): DNA profiling 2 μ L, 10 * PCR Buffer2.5 μ L, each 0.5 μ L of upstream and downstream primer (25 μ mol/L), dNTP2 μ L, TaqDNA polysaccharase 0.25 μ L, ddH
2o17.25 μ L.The PCR reaction conditions of optimizing: 94 ℃ of 5min; 94 ℃ of 1min; 57 ℃ of 1min; 72 ℃ of 1min; Totally 30 circulations; After 72 ℃ of 10min.PCR finish, get 5 μ L PCR products and carry out electrophoresis, observations with 1% sepharose.
3.5 degradation function analyses
The activation of bacterial strain: with transfering loop picking bacillus subtilis strain, be inoculated in activation medium, 35 ℃, 120rpm, shaking table is cultivated after 18-20h, and 4 ℃ save backup.
Live bacterial count (pour plate method): use physiological saline stepwise dilution, choose last 2 suitable dilution gradients and carry out flat board and pour into, each gradient establish 3 parallel, carry out viable bacteria numeration after cultivating 24h.
The inoculation of bacterial strain
[8]: the subtilis of activation is pressed to 10
4-10
5the viable bacteria amount of CFU/mL adds in ammonia nitrogen substratum, and 35 ℃, 120rpm, adds once every 12h, from adding for the first time beginning timing, adds 11 times, altogether 120h.
Measuring method: use Whitfield's ointment spectrophotometry ammonia nitrogen after each sampling.
4 results and analysis
4.1 isolation identification
4.11 morphological examination
0.7～0.8 * 2～3 microns of individual cells, uniform coloring.Without pod membrane, peritrichous, can move.Gram-positive microorganism, 0.6～0.9 * 1.0～1.5 microns of gemma, oval to column, in pairs normal or be catenation, after sporulation, thalline does not expand.Bacterium colony surface irregularity is opaque, and dirty white or micro-yellow, while growing in liquid medium within, often form pleat.Aerophil.As depicted in figs. 1 and 2.
4.12 biochemical identification result
The interpretation of result of table 1 biochemical identification
Shown in upper table 1, result all meets the physio-biochemical characteristics of subtilis, can tentatively regard as subtilis.
4.13 PCR qualification result
[7]
4.2 degradation function analyses
4.21 pH values are for the impact of bacillus subtilis bacteria growing
[9]
Different strains all has optimum acid or alkali environment, and under optimum acid or alkali environment, bacterial strain is just able to grow the soonest.Regulating LB liquid nutrient medium initial ph value is 1,2,3,4,5,6,7,8,9,10.In temperature, be that 35 ℃, shaking speed are 120r/min, the LB bacterium liquid 1mL of same bottle is inoculated among 100mLLB liquid nutrient medium.24h shaking table is measured the OD of thalli growth after cultivating
600value.LB liquid nutrient medium pH value affects result as Fig. 3 for strain growth.
As seen from the figure, pH value is elevated at 7 o'clock by 3, and strain growth amount progressively improves, and when PH=7, bacterial strain bacterium amount is maximum, optical density(OD) OD
600maximum.When pH value is tending towards alkalescence, strain growth is restricted, can think that the variation of pH value can cause the change of enzymic activity, the too small, excessive of pH value can make zymoprotein sex change and inactivation, only when specific pH value is PH=7, they mutually combine the dissociated state optimum of enzyme, substrate and coenzyme, and katalysis occurs, thereby make enzyme reaction reach maximum value.Therefore, best PH is 7.
The impact of 4.22 temperature on bacillus subtilis bacteria growing
[9]
Regulating respectively shaking table culture temperature is 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃.At LB substratum PH=7, during shaking speed 120r/min, the LB bacterium liquid 1mL of same bottle is inoculated among 100mLLB liquid nutrient medium, 24h shaking table is measured the OD of thalli growth after cultivating
600value.Temperature affects result as Fig. 4 for strain growth.
As seen from the figure, bacterial strain is grown best in the time of 35 ℃, and microbial biomass is maximum.Along with the rising of temperature, bacterial strain enzymic activity is strengthened, and growth velocity is accelerated.After but temperature surpasses the optimal temperature of strain growth, optical density(OD) OD
600decline, the activity of enzyme declines on the one hand, even enzyme deactivation; On the other hand, along with the rising of temperature, among LB nutrient solution, the concentration of dissolved oxygen reduces, and has limited the metabolism of bacterial strain, and therefore, the growth velocity of bacterial strain is restricted.So selecting optimum culturing temperature is 35 ℃.
4.23 ammonia nitrogen residual concentrations over time
Fermented liquid by subtilis under optimal conditions of fermentation is seeded in 100mL degraded substratum with the inoculum size of 1mL, at 35 ℃, PH is 7, under the condition of 120r/min, cultivate, at set intervals, measure ammonia nitrogen residual concentration, ammonia nitrogen residual concentration is the following Fig. 5 of curve record over time.
In ammonia nitrogen degradation dynamic experiment, can find out, along with the prolongation of time, the residual concentration of ammonia nitrogen declines gradually.Between 12h-48h, degradation capability is the strongest, reaches after certain numerical value, and the variation of ammonia nitrogen residual concentration reduces gradually.This explanation is when starting, and the degradation capability of subtilis is stronger, and its reason may be that nutritive substance is abundant, As time goes on, the digestion of nutritive substance totally, until the ammonia nitrogen degradation ability of bacterial strain weakens gradually.
China aquaculture great majority are hydrostatic cultivation, and it is water-bed that therefore highdensity cultivation causes the organic depositions such as the movement of a large amount of fishes and shrimps and residual bait, make zone of oxidation attenuation in top layer at the bottom of pond, and the reducing zone thickening of lower floor, redox potential reduction.In the cultivation busy season, because temperature is high, cause anaerobion amount reproduction, organism is decomposed, and discharges the objectionable impuritiess such as a large amount of ammonia and hydrogen sulfide, thereby affects growth and the growth of fish and shrimp, causes fish and shrimp disease.How to purify water and become a difficult problem for cultural technique and study hotspot.
Subtilis is a kind of to the nontoxic gram positive organism of people and animals, and energy fast decoupled organism is strong to adaptive capacity to environment, fast growth.This experimental data has confirmed that subtilis has ammonia nitrogen degradation ability, and the temperature of its optimum growth is 35 ℃, and the PH that optimum is cultivated is 7, and under the condition of optimum growth, the ability of subtilis degradation of ammonia nitrogen is the strongest.By simulation water body is tested, can find out that bacillus subtilis formulation can regulate and control aquaculture water effectively, effective decomposing pollutant matter, purify and stabilizing water quality, the more important thing is the use that can reduce antibiotic etc, the objectionable impurities reducing in aquatic animal body is residual, improves aquatic products quality.
In recent years, there are many probioticses for regulating and controlling fishery water quality, and obtain good result of use, as photosynthetic bacteria, beautiful bacterium, compound EM bacterium, the bacillus cereus preparation etc. built.But some bacterial classification may be also conditioned pathogen as bacillus cereus, also some bacterial classification forms comparatively complicatedly, and the quality monitoring of product and stability are brought to larger difficulty.
Bacillus subtilis formulation is called as super Bacterium lacticum, not only water quality is had to good improving effect, is also widely used in probiotics medical, for animals and fodder additives.It is single that bacillus subtilis formulation has bacterial classification, and antibiotic etc is had no drug resistance, and is suitable for scale operation, low production cost, the advantage such as quality product is relatively stable.Comparing other kind microecological water quality improving agent is more worth of widely use.
Subtilis is a kind of probiotic bacterium, thereby this bacterium is as purifying agent for polluted water, has certain application prospect in aquaculture.
Reference
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Claims (3)
1. a strain has the subtilis of ammonia nitrogen degradation ability, its Classification And Nomenclature is subtilis (Bacillus subtilis), deposit number: CCTCC No:M 2013490, preservation date: on October 23rd, 2013 have now been contained in Chinese Typical Representative culture collection center.
2. the application with subtilis ammonia nitrogen in degraded water body of ammonia nitrogen degradation ability claimed in claim 1.
3. the application of the subtilis with ammonia nitrogen degradation ability claimed in claim 1 in administering aquaculture system.
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| CN113913353A (en) * | 2021-12-02 | 2022-01-11 | 中国水产科学研究院黑龙江水产研究所 | Ammonia nitrogen degrading bacterium suitable for saline-alkali aquaculture water and use method thereof |
| CN113913353B (en) * | 2021-12-02 | 2024-03-01 | 中国水产科学研究院黑龙江水产研究所 | Ammonia nitrogen degrading bacteria suitable for saline-alkali culture water body and application method thereof |
| CN117568221A (en) * | 2023-11-21 | 2024-02-20 | 中国海洋大学 | Bacillus subtilis for high yield of lipase and application thereof |
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