CN104498382A - Microbial preparation for improving aquatic product culture environment and preparation method thereof - Google Patents

Microbial preparation for improving aquatic product culture environment and preparation method thereof Download PDF

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CN104498382A
CN104498382A CN201410517092.0A CN201410517092A CN104498382A CN 104498382 A CN104498382 A CN 104498382A CN 201410517092 A CN201410517092 A CN 201410517092A CN 104498382 A CN104498382 A CN 104498382A
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bacterial strain
psb20
preparation
substratum
microbial preparation
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CN104498382B (en
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徐长安
唐旭
刘源森
林凌
方卫东
黄仕新
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Fujian Huisheng Biotechnology Co., Ltd.
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Third Institute of Oceanography SOA
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38Pseudomonas
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/34Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
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    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00Nature of the contaminant
    • C02F2101/10Inorganic compounds
    • C02F2101/16Nitrogen compounds, e.g. ammonia
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00Nature of the contaminant
    • C02F2101/10Inorganic compounds
    • C02F2101/16Nitrogen compounds, e.g. ammonia
    • C02F2101/166Nitrites
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07Bacillus
    • C12R2001/125Bacillus subtilis ; Hay bacillus; Grass bacillus

Abstract

The invention discloses a microbial preparation for improving an aquatic product culture environment and a preparation method thereof. A strain with optimal nitrite degradation effects is screened and separated from a mud sample in Tongan and Haicang ports of Xiamen city of the Fujian province, another strain with optimal nitrite degradation effects is screened and separated from a mud sample in the intertidal zone of Ningde city of the Fujian province, and the two strains respectively are Rhodopseudomonas palustris PSB20 and Bacillus subtilis ND04. The microbial preparation prepared from the two strains can degrade ammonia nitrogen and nitrous acid in an aquatic product culture environment, has good decomposition effects on residual bait organic matters, has a certain effect of inhibiting pathogenic vibrio bacterium growth in culture water, and has a good production application prospect. The microbial preparation has the characteristic of simple preparation processes.

Description

A kind of microbial preparation and preparation method thereof being applied to culture environment of aquatic products improvement
Technical field
The invention belongs to aquaculture water quality improving technology field, particularly relate to a kind of microbial preparation and preparation method thereof being applied to culture environment of aquatic products improvement.
Background technology
Along with the development of culture fishery, pond is aging year by year, and cultivation water, substrate are subject to severe contamination, causes the farming disease harms frequently to occur, one of water quality improvement major measure becoming healthy aquaculture.
In recent years, the research of improver of water quality is also in constantly developing.Traditional improver of water quality utilizes the principle of physics and chemistry, water quality is improved by adsorbing or increasing water body dissolved oxygen, efficiency is lower, the requirement of the purification of water quality under current high-density breeding pattern cannot be solved, it is therefore current that to use more in cultivation water improvement be biological water quality modifying agent, main component is: photosynthetic bacterium, genus bacillus, lactobacillus, nitrobacteria, denitrifying bacterium, yeast, pseudomonas, bifidus bacillus etc., wherein photosynthetic bacterium, genus bacillus, nitrobacteria, denitrifying bacterium is common Tiny ecosystem aquaculture water quality modifying agent preparation, if the patent No. is CN200910044291.3, name is called " a kind of improver of water quality or feed, fertilizer synergist and production method thereof " Chinese invention patent in just disclose with genus bacillus, photosynthetic bacterium, nitrobacteria, milk-acid bacteria, the microbial preparation of actinomyces and yeast composition, in this microbial preparation, the content of biological growth promotor is 50-100 hundred million/gram, effect is: 1, the growth and breeding speed of plankton can be promoted, improve plant plankton to the nitrogen in water body, phosphorus, the conversion receptivity of the materials such as potassium, thus raising effect of fertilizer, improve Fishery Benefit.2, the transformation efficiency of the materials such as nitrogen, phosphorus, potassium to plant plankton can be improved, promote phytoplankton growth, the generation of prevention algal bloom.
Generally believe that biological control may be solve one of comparatively effective approach of aquiculture disease at present in the world, but also do not find the biological antibiotic medicament of various disease in special anti-aquaculture both at home and abroad temporarily, so, develop a kind of microbiobacterial agent effectively can preventing and treating aquiculture disease be those skilled in the art institute urgently hopefully.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of microbial preparation being applied to culture environment of aquatic products improvement, and discloses the preparation method of this microbial preparation simultaneously.
The present invention solves the problems of the technologies described above by the following technical programs:
Be applied to a microbial preparation for culture environment of aquatic products improvement, it is made up of the component of following mass percent:
Through the bacterial strain PSB20 5-25% cultivating, concentrate and after solidification;
Bacterial strain ND04 10-25% after spreading cultivation, solidifying;
Surplus is carrier;
Wherein:
Described carrier is diatomite or zeolite powder, and the smashing fineness of diatomite, zeolite powder is 120-250 order;
Described bacterial strain PSB20 is Rhodopseudomonas palustris bacterial strain PSB20( rhodopseudomonas palustris), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 09 02nd, 2014, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No:9632;
Described bacterial strain ND04 is bacillus subtilis strain ND04( bacillus subtilis), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 08 25th, 2014, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No:9545.
Disclose the above-mentioned preparation method being applied to the microbial preparation of culture environment of aquatic products improvement, the concrete steps of this preparation method are simultaneously:
(1) cultivation of Rhodopseudomonas palustris bacterial strain PSB20: get described Rhodopseudomonas palustris bacterial strain PSB20 and be seeded in substratum I, and in 25-35 DEG C, illumination cultivation 72-144h under 2500-3500lx, as the OD of nutrient solution 660seed liquor is obtained when reaching 1.5-2.2; Then by 5-20% inoculum size, seed liquor is seeded to transparent plastics plastic sheeting for farm use, sunlight illumination is utilized to carry out plastics film cultivation in 30 ~ 35 DEG C, incubation time 3 ~ 5 days, and when cloudy day and night, adopt 60W/25L incandescent light to irradiate, when bacterium liquid presents dark red, cultivate;
(2) the concentrated and solidification of Rhodopseudomonas palustris bacterial strain PSB20: cultivate in the good bacterium liquid obtained toward step (1) and add flocculation agent, hold over night is flocculated, and removes supernatant; Then protective material, carrier according to claim 1 is added successively, then air-dry at 30-40 DEG C, obtain the Rhodopseudomonas palustris bacterial strain PSB20 after solidification, for subsequent use;
Wherein, the addition of flocculation agent is that step (1) cultivates the long-pending 5-15% of the good bacteria liquid obtained; Protectant addition is: add protective material 0.1-0.5g in the dense thick bacterium liquid that every 1L obtains after flocculation; The addition of carrier is: add carrier 50-2000g in the dense thick bacterium liquid that every 1L obtains after flocculation; And carrier is smashing fineness 120-250 object diatomite or zeolite powder;
(3) the spreading cultivation and solidify of bacillus subtilis strain ND04: get described bacillus subtilis strain ND04 and be seeded in substratum II, and in 30-40 DEG C, cultivate 24-48h, obtained seed liquor under 150-200rpm; Then gained seed liquor is seeded to fermention medium by 5-10% inoculum size, in 30-40 DEG C, pH6.5-8,50-200 rpm condition bottom fermentation cultivation 24-48h; Fermented liquid after fermentation ends is dried at 50-70 DEG C, obtains the bacillus subtilis strain ND04 after solidification, for subsequent use;
(4) composite: first to take component according to following masses percentage ratio: the bacillus subtilis strain ND04 after the solidification of Rhodopseudomonas palustris bacterial strain PSB20,10-25% step (3) gained after the solidification of 5-25% step (2) gained, surplus are smashing fineness 120-250 object diatomite or zeolite powder; Then each component taken mixed and stirs, carrying out point packing afterwards and namely obtain described microbial preparation.
Further, the aluminum potassium sulfate solution of described flocculation agent to be concentration be 0.5-1.0g/L, protective material is solid trehalose.
Further, the component of described substratum I is: sodium-acetate 3.3g, potassium primary phosphate 0.6g, ammonium chloride 1.0g, magnesium sulfate 0.3g, calcium chloride 0.05g, sodium-chlor 1.0g, yeast extract paste 1.0g, manganous sulfate 0.023g, ferrous sulfate 0.005g, water 1000mL, pH7.5 ~ 8.5; Substratum used cultivated by described plastics film is PSB substratum, and its component is: ammonium chloride 1.0g, sodium-acetate 3.5g, magnesium chloride 2.0g, calcium chloride 0.1g, potassium primary phosphate 0.6g, dipotassium hydrogen phosphate 0.4g, yeast extract paste 0.1g, water 1000mL, pH 7.2; Described substratum II is beef-protein medium, and its component is: extractum carnis 3.0g, peptone 10.0g, sodium-chlor 5.0g, water 1000mL, pH7.2-7.4; The component of described fermention medium is: molasses 1-3%, soybean meal 0.1-0.3%, Semen Maydis powder 0.1-0.3%, magnesium sulfate 0.05-0.15%, dipotassium hydrogen phosphate 0.03-0.06%, calcium carbonate 0.05-0.15%; And the per-cent in substratum is mass percent.
Beneficial effect of the present invention is:
By utilizing Rhodopseudomonas palustris bacterial strain PSB20, bacillus subtilis strain ND04( bacillus subtilis) prepare the microbial preparation of gained, ammonia nitrogen, the nitrous acid that can degrade in aquatic products cultivation water environment, and to bait residual organic matter, also there is good discomposing effect, the effect of Developing restraint can also be played simultaneously to a certain extent to vibrios of curing the disease in aquaculture water, namely there is good production application prospect; In addition, this biotechnological formulation also possesses the simple feature of preparation method.
Embodiment
Two bacterial strains and Rhodopseudomonas palustris bacterial strain PSB20 and Bacillus subtillis bacterial strain DN04 is related in the present invention; Wherein, Rhodopseudomonas palustris bacterial strain PSB20 is function stem improvement water body environment being had to promoter action obtained from screening and separating the mud sample picking up from the harbour such as deep blue, Tongan City, sea, Xiamen City, Fujian Province, and Bacillus subtillis bacterial strain DN04 is function stem improvement water body environment being had to promoter action that screening obtains from Fujian Province's Ningde City mudflat sludge sample; And adopt 16s rDNA method to carry out sequencing analysis to this two bacterial strain respectively, final qualification they be respectively Rhodopseudomonas palustris ( rhodopseudomonas palustris) a bacterial strain and subtilis ( bacillus subtilis) a bacterial strain.
1, the separation of first bacterial strain:
(1) primary dcreening operation: take 10g mudflat sludge sample, and add in 100mL 0.85%NaCl, gets the turbid liquid in upper strata and is placed in enrichment medium after stirring, and in 34 DEG C, 3000lx illumination enrichment culture reddens to bacterium liquid; Get after red bacterium liquid carries out gradient dilution and carry out lock out operation with double-layer agar technique on isolation medium, the red colonies that picking isolation medium has been grown, saves backup.
(2) multiple sieve: get different red colonies that primary dcreening operation obtains and be placed in enrichment medium, 34 DEG C, 3000lx illumination cultivation reddens and OD to bacterium liquid 660be 1.5, then being seeded to containing starting point concentration by 10% inoculum size is in the enrichment medium of 500mg/L Sodium Nitrite, 34 DEG C, 3000lx illumination cultivation 5 days, N-(1-naphthyl)-amine light method is used to measure its nitrite content, relatively its degradation effect and then filter out the bacterial strain of degrading nitrite best results, is labeled as bacterial strain PSB20 by this bacterial strain.
2, the qualification of first bacterial strain:
Screening obtained strains PSB20 is seeded to isolation medium, in 34 DEG C, 3000lx illumination cultivation, observes colonial morphology, and do the mensuration of some physiological-biochemical characteristics, result is as follows: red colonies, smooth surface, neat in edge, opaque, without gemma, Gram-negative.16S rDNA mensuration is carried out to this bacterial strain simultaneously, obtain its 16s rDNA sequence as shown in SEQ ID NO:1.
It should be noted that, the component of above-mentioned enrichment medium is: ammonium chloride 0.1g, sodium bicarbonate 0.1g, dipotassium hydrogen phosphate 0.02g, sodium-acetate 0.3g, magnesium sulfate 0.02g, sodium-chlor 0.2g, somatomedin 1mL, trace element solution 1mL, distilled water 97mL, pH7.0; The component of isolation medium is: ammonium chloride 1.0g, sodium-acetate 3.5g, magnesium sulfate 0.1g, calcium chloride 0.1g, potassium primary phosphate 0.6g, dipotassium hydrogen phosphate 0.4g, yeast extract paste 0.1g, agar 20g, distilled water 1000mL, pH7.0.
The 16s rDNA sequence inputting NCBI recorded is carried out homology search, find its similarity the highest for Rhodopseudomonas palustris ( rhodopseudomonas palustris); Then in conjunction with physiological and biochemical test result and 16s rDNA sequence library comparison result, determine this bacterial strain PSB20 be Rhodopseudomonas palustris ( rhodopseudomonas palustris) a bacterial strain.
3, the separation of second bacterial strain:
(1) primary dcreening operation: take 10g mudflat sludge sample, and add in 100mL 0.85%NaCl, get the turbid liquid in upper strata after stirring and be placed in LB substratum, and in 37 DEG C, enrichment culture under 180rpm, after enrichment culture 30h, bacterium liquid is placed in 75 DEG C of water-bath 20min, the bacterium liquid after water-bath carries out gradient dilution and spread plate carries out lock out operation, the genus bacillus of the different colonial morphologies afterwards picking LB flat board grown, saves backup.
(2) multiple sieve: the genus bacillus of the different colonial morphologies that picking primary dcreening operation obtains also is placed in LB substratum, in 37 DEG C, activate under 180rpm condition, after activation 6h, containing starting point concentration by 5% inoculum size access is in the minimal medium of 0.5g/L Sodium Nitrite, and in 37 DEG C, under 180rpm shaking table concussion cultivate 30h, N-(1-naphthyl)-amine light method is used to measure its nitrite content, relatively its degradation effect and then filter out the bacterial strain of degrading nitrite best results, is labeled as bacterial strain ND04 by this bacterial strain.
Wherein, the component of minimal medium is: potassium primary phosphate 0.5g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.2g, calcium chloride 0.1g, sodium-chlor 0.2g, manganous sulfate 0.005g, Sodium Nitrite 0.5g, water 1000mL, pH7.0.
4, the qualification of second bacterial strain
The bacterial strain ND04 of screening gained is seeded to LB solid medium, 37 DEG C of cultivations, observes colonial morphology, and do the mensuration of some physiological-biochemical characteristics, result is as follows: the micro-Huang of bacteria colony white, and surface irregularity is opaque, Gram-positive, produce gemma, glucose fermentation is positive, and hydrolyzed starch is positive.16S rDNA mensuration is carried out to this bacterial strain simultaneously, obtain its 16s rDNA sequence as shown in SEQ ID NO:2.
The 16s rDNA sequence inputting NCBI recorded is carried out homology search, find its similarity the highest for subtilis ( bacillus subtilis); Then in conjunction with physiological and biochemical test result and 16s rDNA sequence library comparison result, determine this bacterial strain ND04 be subtilis ( bacillus subtilis) a bacterial strain.
5. be applied to a microbial preparation for culture environment of aquatic products improvement, it is made up of the component of following mass percent:
Through the bacterial strain PSB20 5-25% cultivating, concentrate and after solidification;
Bacterial strain ND04 10-25% after spreading cultivation, solidifying;
Surplus is carrier;
Described carrier is diatomite or zeolite powder, and the smashing fineness of diatomite, zeolite powder is 120-250 order.
And the concrete operation step of this microbial preparation preparation method is as follows:
(1) cultivation of Rhodopseudomonas palustris bacterial strain PSB20: get Rhodopseudomonas palustris bacterial strain PSB20 and be seeded in substratum I, and in 25-35 DEG C, illumination cultivation 72-144h under 2500-3500lx, as the OD of nutrient solution 660seed liquor is obtained when reaching 1.5-2.2; Then by 5-20% inoculum size, seed liquor is seeded to transparent plastics plastic sheeting for farm use, utilizes sunlight illumination cultivation 3 ~ 5 days in 30 ~ 35 DEG C, and when cloudy day and night, adopt 60W/25L incandescent light to irradiate, when bacterium liquid presents dark red, cultivated;
(2) the concentrated and solidification of Rhodopseudomonas palustris bacterial strain PSB20: cultivate in the good bacterium liquid obtained toward step (1) and add flocculation agent, hold over night is flocculated, and removes supernatant; Then protective material, carrier according to claim 1 is added successively, then air-dry at 30-40 DEG C, obtain the Rhodopseudomonas palustris bacterial strain PSB20 after solidification, for subsequent use;
Wherein, the addition of flocculation agent is that step (1) cultivates the long-pending 5-15% of the good bacteria liquid obtained; Protectant addition is: add protective material 0.1-0.5g in the dense thick bacterium liquid that every 1L obtains after flocculation; The addition of carrier is: add carrier 50-2000g in the dense thick bacterium liquid that every 1L obtains after flocculation; And carrier is smashing fineness 120-250 object diatomite or zeolite powder;
(3) the spreading cultivation and solidify of bacillus subtilis strain ND04: get described bacillus subtilis strain ND04 and be seeded in substratum II, and in 30-40 DEG C, cultivate 24-48h, obtained seed liquor under 150-200rpm; Then gained seed liquor is seeded to fermention medium by 5-10% inoculum size, in 30-40 DEG C, pH6.5-8,50-200 rpm condition bottom fermentation cultivation 24-48h; Fermented liquid after fermentation ends is dried at 50-70 DEG C, obtains the bacillus subtilis strain ND04 after solidification, for subsequent use;
(4) composite: first to take component according to following masses percentage ratio: the bacillus subtilis strain ND04 after the solidification of Rhodopseudomonas palustris bacterial strain PSB20,10-25% step (3) gained after the solidification of 5-25% step (2) gained, surplus are smashing fineness 120-250 object diatomite or zeolite powder; Then each component taken mixed and stirs, carrying out point packing afterwards and namely obtain described microbial preparation.
Wherein, the component of described substratum I is: sodium-acetate 3.3g, potassium primary phosphate 0.6g, ammonium chloride 1.0g, magnesium sulfate 0.3g, calcium chloride 0.05g, sodium-chlor 1.0g, yeast extract paste 1.0g, manganous sulfate 0.023g, ferrous sulfate 0.005g, water 1000mL, pH7.5 ~ 8.5; Substratum used cultivated by described plastics film is PSB substratum, and its component is: ammonium chloride 1.0g, sodium-acetate 3.5g, magnesium chloride 2.0g, calcium chloride 0.1g, potassium primary phosphate 0.6g, dipotassium hydrogen phosphate 0.4g, yeast extract paste 0.1g, water 1000mL, pH 7.2; Described substratum II is beef-protein medium, and its component is: extractum carnis 3.0g, peptone 10.0g, sodium-chlor 5.0g, water 1000mL, pH7.2-7.4; The component of described fermention medium is: molasses 1-3%, soybean meal 0.1-0.3%, Semen Maydis powder 0.1-0.3%, magnesium sulfate 0.05-0.15%, dipotassium hydrogen phosphate 0.03-0.06%, calcium carbonate 0.05-0.15%; And the per-cent in substratum is mass percent.
It should be noted that, involved by the present invention: the component of LB substratum is: peptone 10g, yeast powder 5g, NaCl 10g, H 20 1000 mL; The component of solid LB media, SLB substratum is: peptone 10g, yeast powder 5g, NaCl 10g, agar 15g, H 20 1000 mL.Flocculation agent can also select chitosan or chitin; Protective material is optional gelatin or glycerine also.
In order to better be further elaborated explanation to microbial preparation in the present invention, applicant illustrates following embodiment.
Embodiment one
The cultivation of bacterial strain PSB20: get bacterial strain PSB20 and be seeded in substratum I, and in 27 DEG C, illumination cultivation 72h under 2500lx, as the OD of nutrient solution 660seed liquor is obtained when reaching 1.5; Then by 10% inoculum size, seed liquor is seeded to transparent plastics plastic sheeting for farm use, utilize sunlight illumination cultivation 3 days in 30 ~ 35 DEG C (then adopting water spray to lower the temperature when the same day, temperature was too high), and when cloudy day and night, adopt 60W/25L incandescent light to irradiate, when bacterium liquid presents dark red, cultivate.
Concentrated and the solidification of bacterial strain PSB20: cultivated toward bacterial strain PSB20 in the bacterium liquid of acquisition and added the aluminum potassium sulfate solution that concentration is 0.5g/L, hold over night is flocculated, and removes supernatant; Then solid trehalose, diatomite (smashing fineness 120 order) is added successively, then air-dry at 30 DEG C, obtain the Rhodopseudomonas palustris bacterial strain PSB20 after solidification, for subsequent use;
Wherein, the addition of aluminum potassium sulfate solution be bacterial strain PSB20 cultivated the bacteria liquid of acquisition long-pending 5%; The addition of solid trehalose is: add solid trehalose 0.1g in the dense thick bacterium liquid that every 1L obtains after flocculation; Diatomaceous addition is: add diatomite 1000g in the dense thick bacterium liquid that every 1L obtains after flocculation.
Bacterial strain ND04 spreads cultivation and solidifies: get bacterial strain ND04 and be seeded in substratum II, and in 30 DEG C, cultivate 24h under 150rpm, obtained seed liquor; Then gained seed liquor is seeded to fermention medium by 5% inoculum size, in 30 DEG C, pH6.5,100 rpm condition bottom fermentations cultivation 24h; Fermented liquid after fermentation ends is dried at 50 DEG C, obtains the bacillus subtilis strain ND04 after solidification, for subsequent use.
Composite: first to take component according to following masses percentage ratio: the bacillus subtilis strain ND04 after the Rhodopseudomonas palustris bacterial strain PSB20 after the solidification of 10% gained, the solidification of 15% gained, 75% smashing fineness 120 object diatomite; Then each component taken mixed and stirs, obtaining described microbial preparation.
Embodiment two
The cultivation of bacterial strain PSB20: get bacterial strain PSB20 and be seeded in substratum I, and in 30 DEG C, illumination cultivation 96h under 3000lx, as the OD of nutrient solution 660seed liquor is obtained when reaching 1.7; Then by 5% inoculum size, seed liquor is seeded to transparent plastics plastic sheeting for farm use, utilize sunlight illumination cultivation 5 days in 30 ~ 35 DEG C (then adopting water spray to lower the temperature when the same day, temperature was too high), and when cloudy day and night, adopt 60W/25L incandescent light to irradiate, when bacterium liquid presents dark red, cultivate.
Concentrated and the solidification of bacterial strain PSB20: cultivated toward bacterial strain PSB20 in the bacterium liquid of acquisition and added the aluminum potassium sulfate solution that concentration is 0.7g/L, hold over night is flocculated, and removes supernatant; Then solid trehalose, zeolite powder (smashing fineness 250 order) is added successively, then air-dry at 40 DEG C, obtain the Rhodopseudomonas palustris bacterial strain PSB20 after solidification, for subsequent use;
Wherein, the addition of aluminum potassium sulfate solution be bacterial strain PSB20 cultivated the bacteria liquid of acquisition long-pending 10%; The addition of solid trehalose is: add solid trehalose 0.3g in the dense thick bacterium liquid that every 1L obtains after flocculation; The addition of zeolite powder is: add zeolite powder 1000g in the dense thick bacterium liquid that every 1L obtains after flocculation.
Bacterial strain ND04 spreads cultivation and solidifies: get bacterial strain ND04 and be seeded in substratum II, and in 35 DEG C, cultivate 30h under 170rpm, obtained seed liquor; Then gained seed liquor is seeded to fermention medium by 7% inoculum size, in 35 DEG C, pH6.5,130 rpm condition bottom fermentations cultivation 36h; Fermented liquid after fermentation ends is dried at 60 DEG C, obtains the bacillus subtilis strain ND04 after solidification, for subsequent use.
Composite: first to take component according to following masses percentage ratio: the bacillus subtilis strain ND04 after the Rhodopseudomonas palustris bacterial strain PSB20 after the solidification of 15% gained, the solidification of 20% gained, 65% smashing fineness 250 object zeolite powder; Then each component taken mixed and stirs, obtaining described microbial preparation.
Embodiment three
The cultivation of bacterial strain PSB20: get bacterial strain PSB20 and be seeded in substratum I, and in 34 DEG C, illumination cultivation 120h under 3500lx, as the OD of nutrient solution 660seed liquor is obtained when reaching 1.9; Then by 15% inoculum size, seed liquor is seeded to transparent plastics plastic sheeting for farm use, utilize sunlight illumination cultivation 4 days in 30 ~ 35 DEG C (then adopting water spray to lower the temperature when the same day, temperature was too high), and when cloudy day and night, adopt 60W/25L incandescent light to irradiate, when bacterium liquid presents dark red, cultivate;
Concentrated and the solidification of bacterial strain PSB20: cultivated toward bacterial strain PSB20 in the bacterium liquid of acquisition and added the aluminum potassium sulfate solution that concentration is 1.0g/L, hold over night is flocculated, and removes supernatant; Then solid trehalose, diatomite (smashing fineness 1800 order) is added successively, then air-dry at 35 DEG C, obtain the Rhodopseudomonas palustris bacterial strain PSB20 after solidification, for subsequent use;
Wherein, the addition of aluminum potassium sulfate solution be bacterial strain PSB20 cultivated the bacteria liquid of acquisition long-pending 15%; The addition of solid trehalose is: add solid trehalose 0.5g in the dense thick bacterium liquid that every 1L obtains after flocculation; The addition of zeolite powder is: add zeolite powder 1500g in the dense thick bacterium liquid that every 1L obtains after flocculation.
Bacterial strain ND04 spreads cultivation and solidifies: get bacterial strain ND04 and be seeded in substratum II, and in 37 DEG C, cultivate 48h under 170rpm, obtained seed liquor; Then gained seed liquor is seeded to fermention medium by 5% inoculum size, in 35 DEG C, pH7.0,150 rpm condition bottom fermentations cultivation 48h; Fermented liquid after fermentation ends is dried at 70 DEG C, obtains the bacillus subtilis strain ND04 after solidification, for subsequent use;
Composite: first to take component according to following masses percentage ratio: the bacillus subtilis strain ND04 after the Rhodopseudomonas palustris bacterial strain PSB20 after the solidification of 20% gained, the solidification of 25% gained, 55% smashing fineness 180 object diatomite; Then each component taken mixed and stirs, obtaining described microbial preparation.
6. the improved test of pair culture environment of aquatic products
Test one: embodiment one prepares the improved test of gained microbial preparation to culture environment of aquatic products
(1) test method: Example one is prepared gained microbial preparation and used on the cultivation white leg Shrimp pool: design one group of control group, one group of experimental group; In experimental group, every cubic metre of water body uses 2g embodiment one to prepare gained microbial preparation at every turn, carries out full pool spilling head, and use weekly once during use after being watered dilution 10 times; Control group is blank, does not namely use embodiment one to prepare gained microbial preparation; The shrimp pool its ammonia nitrogen of water sample detection, nitrous acid, vibrios sum and organic content is got after one month.
(2) test-results: the control group ammonia nitrogen observed value not using embodiment one to prepare gained microbial preparation is 0.6mg/L, nitrous acid observed value is 0.1mg/L, vibrios adds up to 850/ml, organic content is 1.3%; And the ammonia nitrogen observed value of experimental group be 0.2mg/L, nitrous acid observed value is 0.01mg/L, vibrios adds up to 570/ml, organic content is 0.79%; Namely the water quality on the test group shrimp pool is obviously improved.
Test two: embodiment two prepares the improved test of gained microbial preparation to culture environment of aquatic products
(1) test method: Example two is prepared gained microbial preparation and used on the cultivation white leg Shrimp pool: design one group of control group, one group of experimental group; In experimental group, every cubic metre of water body uses 2g embodiment two to prepare gained microbial preparation at every turn, carries out full pool spilling head, and use weekly once during use after being watered dilution 10 times; Control group is blank, does not namely use embodiment three to prepare gained microbial preparation; The shrimp pool its ammonia nitrogen of water sample detection, nitrous acid, vibrios sum and organic content is got after one month.
(2) test-results: the control group ammonia nitrogen observed value not using embodiment two to prepare gained microbial preparation is 0.6mg/L, nitrous acid observed value is 0.1mg/L, vibrios adds up to 850/ml, organic content is 1.3%; And the ammonia nitrogen observed value < 0.2mg/L of experimental group, nitrous acid observed value be 0.01mg/L, vibrios adds up to 480/ml, organic content is 0.73%; Namely the water quality on the test group shrimp pool is obviously improved.
Test three: embodiment three prepares the improved test of gained microbial preparation to culture environment of aquatic products
(1) test method: Example three is prepared gained microbial preparation and used on the cultivation white leg Shrimp pool: design one group of control group, one group of experimental group; In experimental group, every cubic metre of water body uses 5g embodiment two to prepare gained microbial preparation at every turn, carries out full pool spilling head, and use weekly once during use after being watered dilution 10 times; Control group is blank, does not namely use embodiment three to prepare gained microbial preparation; The shrimp pool its ammonia nitrogen of water sample detection, nitrous acid, vibrios sum and organic content is got after one month.
(2) test-results: the ammonia nitrogen observed value not using embodiment three to prepare gained microbial preparation is 0.6mg/L, nitrous acid observed value is 0.1mg/L, vibrios adds up to 850/ml, organic content is 1.3%; And the ammonia nitrogen observed value of experimental group be < 0.2mg/L, nitrous acid observed value is 0.05mg/L, vibrios adds up to 440/ml, organic content is 0.67%; Namely the water quality on the test group shrimp pool is obviously improved.
In summary, the present invention utilizes Rhodopseudomonas palustris bacterial strain PSB20, bacillus subtilis strain ND04( bacillus subtilis) prepare the microbial preparation of gained, ammonia nitrogen, the nitrous acid that can degrade in aquatic products cultivation water environment, and to bait residual organic matter, also there is good discomposing effect, the effect of Developing restraint can also be played simultaneously to a certain extent to vibrios of curing the disease in aquaculture water, namely there is good production application prospect.
SEQUENCE LISTING
 
<110> State Oceanic Administration Bureau The Third Oceanography Institute
 
<120> mono-kind is applied to microbial preparation of culture environment of aquatic products improvement and preparation method thereof
 
<130> 10000
 
<160> 2
 
<170> PatentIn version 3.3
 
<210> 1
<211> 1424
<212> DNA
<213> Rhodopseudomonas palustris (Rhodopseudomonas palustris)
 
<400> 1
ggacgggcgg cagcttacac atgcaagtcg aacgggcgta gcaatacgtc agtggcagac 60
 
gggtgagtaa cgcgtgggaa cgtacctttt ggttcggaac aacacaggga aacttgtgct 120
 
aataccggat aagcccttac ggggaaagat ttatcgccga aagatcggcc cgcgtctgat 180
 
tagctagttg gtgaggtaat ggctcaccaa ggcgacgatc agtagctggt ctgagaggat 240
 
gatcagccac attgggactg agacacggcc caaactccta cgggaggcag cagtggggaa 300
 
tattggacaa tgggcgaaag cctgatccag ccatgccgcg tgagtgatga aggccctagg 360
 
gttgtaaagc tcttttgtgc gggaagataa tgacggtacc gcaagaataa gccccggcta 420
 
acttcgtgcc agcagccgcg gtaatacgaa gggggctagc gttgctcgga atcactgggc 480
 
gtaaagggtg cgtaggcggg tttctaagtc agaggtgaaa gcctggagct caactccaga 540
 
actgcctttg atactggaag tcttgagtat ggcagaggtg agtggaactg cgagtgtaga 600
 
ggtgaaattc gtagatattc gcaagaacac cagtggcgaa ggcggctcac tgggccatta 660
 
ctgacgctga ggcacgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 720
 
ccgtaaacga tgaatgccag ccgttagtgg gtttactcac tagtggcgca gctaacgctt 780
 
taagcattcc gcctggggag tacggtcgca agattaaaac tcaaaggaat tgacgggggc 840
 
ccgcacaagc ggtggagcat gtggtttaat tcgacgcaac gcgcagaacc ttaccagccc 900
 
ttgacatgtc caggaccggt cgcagagacg tgaccttctc ttcggagcct ggagcacagg 960
 
tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1020
 
caacccccgt ccttagttgc taccatttag ttgagcactc taaggagact gccggtgata 1080
 
agccgcgagg aaggtgggga tgacgtcaag tcctcatggc ccttacgggc tgggctacac 1140
 
acgtgctaca atggcggtga caatgggaag ctaaggggtg acccttcgca aatctcaaaa 1200
 
agccgtctca gttcggattg ggctctgcaa ctcgagccca tgaagttgga atcgctagta 1260
 
atcgtggatc agcatgccac ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1320
 
accatgggag ttggctttac ctgaagacgg tgcgctaacc cgcaaggggg gcagccggcc 1380
 
acggtagggt cagcgactgg gggaagtcga acaagtgcat gccc 1424
 
 
<210> 2
<211> 1475
<212> DNA
<213> subtilis (Bacillus subtilis)
 
<400> 2
aacctggcct cggactaata catgcaagtc gagcggacag atgggagctt gctccctgat 60
 
gttagcggcg gacgggtgag taacacgtgg gtaacctgcc tgtaagactg ggataactcc 120
 
gggaaaccgg ggctaatacc ggatggttgt ttgaaccgca tggttcagac ataaaaggtg 180
 
gcttcggcta ccacttacag atggacccgc ggcgcattag ctagttggtg aggtaacggc 240
 
tcaccaaggc gacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga 300
 
cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct 360
 
gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct gttgttaggg 420
 
aagaacaagt gccgttcaaa tagggcggca ccttgacggt acctaaccag aaagccacgg 480
 
ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggaattattg 540
 
ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg 600
 
ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa ttccacgtgt 660
 
agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact ctctggtctg 720
 
taactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc 780
 
acgccgtaaa cgatgagtgc taagtgttag ggggtttccg ccccttagtg ctgcagctaa 840
 
cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg 900
 
ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 960
 
aggtcttgac atcctctgac aatcctagag ataggacgtc cccttcgggg gcagagtgac 1020
 
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080
 
gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg actgccggtg 1140
 
acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1200
 
acacgtgcta caatggacag aacaaagggc agcgaaaccg cgaggttaag ccaatcccac 1260
 
aaatctgttc tcagttcgga tcgcagtctg caactcgact gcgtgaagct ggaatcgcta 1320
 
gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380
 
cacaccacga gagtttgtaa cacccgaagt cggtgaggta accttttagg agccagccgc 1440
 
cgaaggtggg acagatgatg ggggaagtcg atcaa 1475

Claims (4)

1. be applied to a microbial preparation for culture environment of aquatic products improvement, it is characterized in that: be made up of the component of following mass percent:
Through the bacterial strain PSB20 5-25% cultivating, concentrate and after solidification;
Bacterial strain ND04 10-25% after spreading cultivation, solidifying;
Surplus is carrier;
Wherein:
Described carrier is diatomite or zeolite powder, and the smashing fineness of diatomite, zeolite powder is 120-250 order;
Described bacterial strain PSB20 is Rhodopseudomonas palustris bacterial strain PSB20( rhodopseudomonas palustris), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 09 02nd, 2014, deposit number is CGMCC No:9632;
Described bacterial strain ND04 is bacillus subtilis strain ND04( bacillus subtilis), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 08 25th, 2014, deposit number is CGMCC No:9545.
2. be applied to a preparation method for the microbial preparation of culture environment of aquatic products improvement described in claim 1, it is characterized in that: concrete steps are:
(1) cultivation of Rhodopseudomonas palustris bacterial strain PSB20: get the Rhodopseudomonas palustris bacterial strain PSB20 described in claim 1 and be seeded in substratum I, and in 25-35 DEG C, illumination cultivation 72-144h under 2500-3500lx, as the OD of nutrient solution 660seed liquor is obtained when reaching 1.5-2.2; Then by 5-20% inoculum size, seed liquor is seeded to transparent plastics plastic sheeting for farm use, sunlight illumination is utilized to carry out plastics film cultivation in 30 ~ 35 DEG C, incubation time 3 ~ 5 days, and when cloudy day and night, adopt 60W/25L incandescent light to irradiate, when bacterium liquid presents dark red, cultivate;
(2) the concentrated and solidification of Rhodopseudomonas palustris bacterial strain PSB20: cultivate in the good bacterium liquid obtained toward step (1) and add flocculation agent, hold over night is flocculated, and removes supernatant; Then protective material, carrier according to claim 1 is added successively, then air-dry at 30-40 DEG C, obtain the Rhodopseudomonas palustris bacterial strain PSB20 after solidification, for subsequent use;
Wherein, the addition of flocculation agent is that step (1) cultivates the long-pending 5-15% of the good bacteria liquid obtained; Protectant addition is: add protective material 0.1-0.5g in the dense thick bacterium liquid that every 1L obtains after flocculation; The addition of carrier is: add carrier 50-2000g in the dense thick bacterium liquid that every 1L obtains after flocculation; And carrier is smashing fineness 120-250 object diatomite or zeolite powder;
(3) the spreading cultivation and solidify of bacillus subtilis strain ND04: get the bacillus subtilis strain ND04 described in claim 1 and be seeded in substratum II, and in 30-40 DEG C, cultivate 24-48h, obtained seed liquor under 150-200rpm; Then gained seed liquor is seeded to fermention medium by 5-10% inoculum size, in 30-40 DEG C, pH6.5-8,50-200 rpm condition bottom fermentation cultivation 24-48h; Fermented liquid after fermentation ends is dried at 50-70 DEG C, obtains the bacillus subtilis strain ND04 after solidification, for subsequent use;
(4) composite: first to take component according to following masses percentage ratio: the bacillus subtilis strain ND04 after the solidification of Rhodopseudomonas palustris bacterial strain PSB20,10-25% step (3) gained after the solidification of 5-25% step (2) gained, surplus are smashing fineness 120-250 object diatomite or zeolite powder; Then each component taken mixed and stirs, carrying out point packing afterwards and namely obtain described microbial preparation.
3. a kind of preparation method of microbial preparation being applied to culture environment of aquatic products improvement according to claim 2, is characterized in that: the aluminum potassium sulfate solution of described flocculation agent to be concentration be 0.5-1.0g/L, protective material is solid trehalose.
4. a kind of preparation method of microbial preparation being applied to culture environment of aquatic products improvement according to claim 2, it is characterized in that: the component of described substratum I is: sodium-acetate 3.3g, potassium primary phosphate 0.6g, ammonium chloride 1.0g, magnesium sulfate 0.3g, calcium chloride 0.05g, sodium-chlor 1.0g, yeast extract paste 1.0g, manganous sulfate 0.023g, ferrous sulfate 0.005g, water 1000mL, pH7.5 ~ 8.5; Substratum used cultivated by described plastics film is PSB substratum, and its component is: ammonium chloride 1.0g, sodium-acetate 3.5g, magnesium chloride 2.0g, calcium chloride 0.1g, potassium primary phosphate 0.6g, dipotassium hydrogen phosphate 0.4g, yeast extract paste 0.1g, water 1000mL, pH 7.2; Described substratum II is beef-protein medium, and its component is: extractum carnis 3.0g, peptone 10.0g, sodium-chlor 5.0g, water 1000mL, pH7.2-7.4; The component of described fermention medium is: molasses 1-3%, soybean meal 0.1-0.3%, Semen Maydis powder 0.1-0.3%, magnesium sulfate 0.05-0.15%, dipotassium hydrogen phosphate 0.03-0.06%, calcium carbonate 0.05-0.15%; And the per-cent in substratum is mass percent.
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CN106635928A (en) * 2017-02-13 2017-05-10 天津市农业生物技术研究中心 Culture method for continuous and scaled production of photosynthetic bacteria
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CN108094283A (en) * 2017-12-29 2018-06-01 海南中正水产科技有限公司 A kind of method of indoor industrially biological breeding Penaeus Vannmei shrimp seedling
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