CN108865940A - One plant of heterotrophic nitrification-aerobic denitrification bacillus and its composite bacteria preparation - Google Patents
One plant of heterotrophic nitrification-aerobic denitrification bacillus and its composite bacteria preparation Download PDFInfo
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Abstract
The invention patent belongs to microorganism and its application field more particularly to one plant of heterotrophic nitrification-aerobic denitrification bacillus BSXS-1602；It further relates to more than one and states heterotrophic nitrification-aerobic denitrification bacillus BSXS-1602 composite bacteria preparation as main component；The invention further relates to more than one to state heterotrophic nitrification-aerobic denitrification bacillus BSXS-1602 Mixed Microbes powder as main component.Heterotrophic nitrification-aerobic denitrification bacillus of the invention is bacillus BSXS-1602, has carried out preservation in China General Microbiological culture presevation administrative center on June 15th, 2018, deposit number is：CGMCC NO.15950.Bacillus BSXS-1602 of the invention is separated from sea water culture environment, is had efficient heterotrophic nitrification-aerobic denitrification capability, can be used to aquaculture water environment.
Description
Technical field
The invention patent belongs to microorganism and its application field more particularly to one plant of heterotrophic nitrification-aerobic denitrification gemma
Bacillus BSXS-1602；It further relates to more than one and states heterotrophic nitrification-aerobic denitrification bacillus BSXS-1602 be main component
Composite bacteria preparation；It is main for stating heterotrophic nitrification-aerobic denitrification bacillus BSXS-1602 the invention further relates to more than one
The Mixed Microbes powder of ingredient.
Background technique
Polluted by nitrogen is to seriously threaten the existence of aquatile one of an important factor for causing cultivation water to deteriorate.Biology is de-
Nitrogen because its have many advantages, such as it is economical, efficiently, environmental protection due to be increasingly becoming research hotspot in recent years.Traditional biological denitrificaion usually wraps
Containing two parts, the first step is nitrification, i.e., Autotrophic nitrification bacterium carries out nitrogen conversion under aerobic condition：NH4 +-N→NO2 --N
→NO3 --N；Second step is that allotrophic nitrobacteria carries out nitrogen conversion under anaerobic：NO3 --N→NO2 -- N → NO, N2O→N2。
The condition as required for the two stages is completely different, and traditional biological denitrification system must include two individual bodies
System causes denitrogenation cost to increase.Studies have shown that allotrophic nitrobacteria can will cultivate while being grown using carbon source
Nitrogenous compound in water body is converted into NH by nitrification2OH、NO2 -- N or NO3 -- N etc.；At the same time, most of thin
Bacterium can also carry out aerobic denitrification simultaneously, by NO2 -- N or NO3 -- N is converted into NO, N2O and N2Equal gases.Paracoccu
Denitrificans (being initially named as Thiosphaera pantotropha) be first strain be reported have aerobic denitrification
The bacterial strain of effect, can be by NO3 -- N or NO2 -- N denitrification is N2.Hereafter, more new aerobic denitrifying bacterias are separated
And research, such as Thiosphaerapantotropha, pseudomonas (Pseudomonas), Bacillus foecalis alkaligenes
(Alcaligenes faecalis), bacillus (Bacillus) etc..
Recent study shows that bacillus has stronger nitrification and denitrification performance, can effectively remove cultivation
NH in water body4 +- N and NO2 -- N reduces NH4 +- N and NO2 -Toxic action of the substances such as-N to aquaculture organism.That reports at present is big
Part heterotrophic nitrification-aerobic denitrification strain isolation is isolated from sea-farming from soil, sewage disposal system and biological filter membrane etc.
The rarely seen report of the bacterial strain of environment, the living environment difference of bacterial strain cause its adaptability and nitrogen removal performance there are biggish difference, because
There is this separation screening from sea water culture environment the bacterial strain of efficient denitrification ability to be particularly important.
Summary of the invention
An object of the present invention is that separation screening one kind has efficient heterotrophic nitrification-good from sea water culture environment
The bacillus of oxygen denitrification capability.
In order to solve the above-mentioned technical problem, the present invention uses following technical scheme, a kind of heterotrophic nitrification-aerobic denitrification bud
Spore bacillus, be bacillus BSXS-1602, on June 15th, 2018 China General Microbiological culture presevation administrative center into
Preservation is gone, deposit number is：CGMCC NO.15950.
The bacillus BSXS-1602 removal capacity mainly includes to NH4 +- N and NO2 -In terms of the removal of-N, the bacterial strain
It is separation screening from the denitrifying bacillocin of litopenaeus vannamei (Litopenaeus vannamei) cultivating pool bed mud, is named as
BSXS-1602 is identified as bacillus subtilis (Bacillus subtilis).
The invention also discloses a kind of above-mentioned bacillus BSXS-1602 to purify water, remove NH4 +- N and NO2 --N
And the application in aquaculture water environment conditioning.
One probiotics bacterial strain success be applied to production practices, not only with bacterial strain itself it is higher conversion and denitrogenation
Can be related, it is also most important to the adaptability of environment.Result of study shows that bacillus BSXS-1602 of the invention has
Higher heterotrophic nitrification-aerobic denitrification capability, wider to the adaptation range of C/N, salt tolerant range is wide, has stronger oxytolerant
Property, there is good potential using value in aquaculture water environment conditioning.
A kind of bacterium powder of above-mentioned bacillus BSXS-1602, preparation method are as follows：Bacillus BSXS-1602 is living
Change, expand culture, through 28 DEG C of liquid state fermentations, thalline were collected by centrifugation by 8000rpm, bentonite is added as carrier, by thallus with it is swollen
Profit soil stirs evenly, and the bacterium powder that bacterium amount is 100~50,000,000,000/g is made in 45 DEG C of drying.
The invention also discloses a kind of above-mentioned bacillus BSXS-1602 bacterium powder answering as aquatic animal feed additive
With, and regulation water quality application add when the bacillus BSXS-1602 bacterium powder is as aquatic animal feed additive
Dosage is the 0.05-0.2% of feeding quality.Bacillus BSXS-1602 bacterium powder of the invention facilitates storage and use, can promote
Into the appetite of aquatic livestock, when feeding litopenaeus vannamei as feed addictive, feed coefficient can be effectively reduced, is promoted all
The shore prawn that receives growth.
It is a further object of the present invention to provide one kind with bacillus BSXS-1602 composite bacteria preparation as main component,
The composite bacteria preparation is by bacillus BSXS-1602, white tail feather bacillus (Bacillus baekryungensis)
BBHS-01 is with 10~12:1 bacterial population ratio is combined.The white tail feather gemma bar BBHS-01, on December 7th, 2012
Preservation, preservation address have been carried out in China Committee for Culture Collection of Microorganisms's common micro-organisms collection：Court of Beijing
The road Yang Qu great Tun, Institute of Microorganism, Academia Sinica, deposit number are：CGMCC NO.6939.
More than one are stated bacillus BSXS-1602 composite bacteria preparation as main component and are purifying water, removing NH4 +-N
And NO2 -Application in-N and aquaculture water environment conditioning.
The experimental results showed that bacillus BSXS-1602 and Bai Ling bacillus BBHS-01 is without antagonism, by gemma bar
Bacterium BSXS-1602, white tail feather bacillus BBHS-01 are with 10~12:1 bacterial population ratio compound tense, bacillus BSXS-1602
It quickly can well be grown with white tail feather bacillus BBHS-01, it is bacillus BSXS-1602, BBHS-01 pairs of tail feather bacillus white
The removal effect aspect of nitrogen pollutant has synergistic effect, is embodied in：Bacillus BSXS-1602, white tail feather bacillus
BBHS-01 is with 10~12:The compound obtained composite bacteria preparation of 1 bacterial population ratio can effectively remove NH4 +- N and
NO2 -- N improves breeding environment, and under dose profile, complex enzyme formulation is to NH4 +- N and NO2 -The removal rate of-N is significantly higher than arbitrarily
A kind of removal efficiency of single strain.
A kind of Mixed Microbes powder as main component with bacillus BSXS-1602, preparation method are as follows：By gemma bar
Bacterium BSXS-1602 and Bai Ling bacillus BBHS-01 is activated respectively, is expanded culture, then respectively through 28 DEG C of liquid state fermentations, then will
Bacillus BSXS-1602 and Bai Ling bacillus BBHS-01 is according to 10~15:1 bacterial population ratio mixing, 8000rpm from
The heart collects thallus, and bentonite and xanthan gum is added as carrier, thallus and bentonite, xanthan gum are stirred evenly, 45 DEG C of drying,
The bacterium powder that total bacterium amount is 100~50,000,000,000/g is made.
Further, the carrier is bentonite and xanthan gum according to 5:1 mass ratio mixes.By using swelling
Soil and xanthan gum can load bacillus BSXS-1602 and Bai Ling bacillus BBHS-01 as carrier well, make this
The Mixed Microbes powder of invention is not easy to be broken up by water flow, and then is conducive to bacillus BSXS-1602 and Bai Ling bacillus BBHS-
The performance of 01 synergistic effect.
The invention also discloses more than one to state application of the Mixed Microbes powder as feed addictive, and regulates and controls answering for water quality
With when the Mixed Microbes powder is as feed addictive, additive amount is the 0.01~0.1% of feeding quality.
Mixed Microbes powder of the invention can promote the appetite of aquatic livestock, when feeding aquatic livestock as feed addictive, energy
Improve aquatic livestock enteron aisle structure of community, than as can improve stichopus japonicus and litopenaeus vannamei enteron aisle structure of community, can be effective
Reduction feed coefficient, promote Growth of Litopenaeus vannamei, furthermore Mixed Microbes powder of the invention facilitate storage and it is easy to use.
Compared with prior art, the beneficial effects of the invention are as follows：
(1) by being enriched with to litopenaeus vannamei cultivating pool bed mud, screening has efficient heterotrophic nitrification-for this research
The bacillus BSXS-1602 of aerobic denitrification capability, according to the morphological feature of bacterial strain, physio-biochemical characteristics and 16S rDNA
Sequence and specific sequence compare analysis, and bacterial strain BSXS-1602 is accredited as bacillus subtilis (Bacillus
subtilis).One probiotics bacterial strain success be applied to production practices, not only with the higher conversion of bacterial strain itself and denitrogenation
Performance is related, also most important to the adaptability of environment.The present invention passes through analysis the varying environment factor (carbon source, C/N (quality
Than), pH, salinity and revolving speed) influence to bacterial strain BSXS-1602 heterotrophic nitrification-aerobic denitrification characteristic, find at 28 DEG C of temperature
Under, bacterial strain BSXS-1602 is respectively glucose, C/N 18, pH value 7.5, salinity 30 ‰ and revolving speed 160rpm condition in carbon source
Under heterotrophic nitrification-aerobic denitrification capability with higher.On this basis, bacterial strain BSXS-1602 heterotrophism nitre is had studied respectively
Change and aerobic denitrifying optimum reaction condition.Orthogonal experiment results show when with NH4When Cl is nitrogen source, bacterial strain BSXS-
1602 best nitration condition is that C/N 10, pH value 9.0, salinity 10 ‰ and revolving speed are 160rpm respectively.When with NaNO2For nitrogen source
When, the best Denitrification Conditions of bacterial strain BSXS-1602 are that C/N 10, pH value 6.0, salinity 30 ‰ and revolving speed are 160rpm respectively.
To sum up, bacillus BSXS-1602 heterotrophic nitrification-aerobic denitrification capability with higher of the invention, to the adaptation model of C/N
It encloses relatively extensively, salt tolerant range is wide, has stronger oxygen resistence, has potential application in aquaculture water environment conditioning.
(2) bacillus BSXS-1602, white tail feather bacillus BBHS-01 are with 10~12:The compound institute of 1 bacterial population ratio
Composite bacteria preparation obtained can effectively remove NH4 +- N and NO2 -- N improves breeding environment, under dose profile, complex enzyme
Preparation is to NH4 +- N and NO2 -The removal rate of-N is significantly higher than the removal efficiency of any one single strain.
(3) the bacterium powder of bacillus BSXS-1602 and with bacillus BSXS-1602 Mixed Microbes powder as main component
The appetite that can promote aquatic livestock when feeding aquatic livestock as feed addictive, can effectively reduce feed coefficient, promote
Growth of Litopenaeus vannamei, furthermore Mixed Microbes powder of the invention facilitate storage and it is easy to use.
Detailed description of the invention
Fig. 1 constructs the phylogenetic tree of bacterial strain BSXS-1602 based on 16S rDNA sequence；
Fig. 2 is the phylogenetic tree based on 16S rDNA gene order of white tail feather bacillus BBHS-01 and related strain.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.
Sample source：The bed mud sample in prawn culturing pond used in this research picks up from Jiangsu Province in July, 2014
Three prawn bait throwing in of the Jia Xin aquatic products development corporation, Ltd. of the Agricultural Industrialized Parks Ganyu County (34 ° of 58 ' N, 119 ° of 20 ' E) cultivate
Pond (28 DEG C of temperature).
Main agents and instrument：The Primary Chemical that this research uses is purchased from the limited public affairs of Chinese medicines group chemical reagent
Department.It is that analysis is pure except specified otherwise；PCR reagent is purchased from complete biological (TransGen Biotech) company of scholar's gold in Beijing, bacterium
Universal primer and specific sequence amplimer are closed by raw work bioengineering (Sangon Biotech) Co., Ltd in Shanghai
At.Key instrument used includes：- 80 DEG C of ultra low temperature freezers (SANYO Electric Co., Ltd.Japan)；Electronic balance
(METTLED TOLEDO Corp.Shanghai,China)；AIR TECH superclean bench (ten thousand net safe and sound companies of group)；
SPX type Intelligent biochemistry incubator (Ningbo Jiangnan instrument plant)；(Chinese Harbin City east joins electronic technology to HZQ-Q constant-temperature table
Development corporation, Ltd.)；556 multi-parameter water quality measuring instrument of YSI (YSI Incorporated, USA)；DR2700 type spectrophotometric
It counts (HACH Incorporated, USA)；High speed freezing centrifuge (Hitachi Koki Co., Ltd.Japan).
Culture medium
Culture medium used in this research mainly includes：
(1) enriched medium：(NH4)2SO4 2.0g、MgSO4 0.10g、NaH2PO4.2H2O 0.26g、K2HPO4 0.5g、
CaCO3 1.0g、FeSO4.7H2O 0.183g, glucose 5g, KNO2 1.0g、NH4Cl 0.5g, yeast extract 0.02g, seawater
1000mL,pH 7.5；Separation plate is the agar that 2.0% (w/v) is added in above-mentioned enriched medium；
(2) 2216E culture medium：Tryptone 6.0g, yeast extract 2.0g, seawater 1000mL, pH 7.5.2216E points
It is the agar that 2.0% (w/v) is added in above-mentioned enriched medium from plate；
The nitrogen removal performance detection of bacterial strain uses the aquaculture simulated wastewater manually prepared, and mainly includes：Basic test liquid,
Heterotrophic nitrification culture medium (HNM) and nitrite denitrification culture medium (NDM).Wherein,
Basic test liquid：NH4Cl 0.1605g、NaNO20.05g, glucose 2.475g, seawater 1000mL, pH 7.5；
Heterotrophic nitrification culture medium (HNM)：NH4Cl 0.1605g, glucose 2.0808g, seawater 1000mL, pH 7.5；
Nitrite denitrification culture medium (NDM)：NaNO20.207g, glucose 2.0808g, seawater 1000mL, pH
7.5；
Above-mentioned enriched medium and manual simulation's aquiculture waste water are handled through high pressure sterilization using preceding.
The separation and identification of 1 bacterial strain bacillus BSXS-1602 of embodiment
1, the separation and screening of Bacillus strain
Bed mud sample after taking prawn culturing pond to premix carries out gradient dilution with antiseptic sea water, and sufficiently oscillation is quiet after mixing
It sets.Supernatant is taken in sterile centrifugation tube and places it in heating water bath 20min in 90 DEG C of water-baths.Water intaking bath treated supernatant
Liquid is seeded in the 250mL conical flask containing enriched medium.By one star of conical flask continuous oscillation culture after inoculation
Phase obtains initial enrichment bacterium solution.According to 1:Initial enrichment bacterium solution is seeded to and continues to train in enriched medium by 10 inoculative proportion
A week is supported, repeatedly domestication 6 periods of culture.Last time enrichment bacterium solution is taken, the separation and purifying of bacterial strain are carried out.
Bacterial strain after purification is subjected to Gram's staining and spore staining.Select Gram's staining and bacterium that spore staining is positive
Strain carries out the pathogenic detection of bacterial strain using blood plate method, chooses and further studied there is no pathogenic bacterial strain.Utilize base
Plinth test fluid further screens the above-mentioned bacterial strain filtered out, selects the bacterial strain with nitrification and denitrification ability.
Water-bath, Gram's staining and spore staining, preliminary screening, which are carried out, by the bacterial strain filtered out to acclimating obtains 5
Bacillus；Then by removing liquid NH to basis with bacterial strain4 +- N and NO2 -The removal ability of-N is foundation, is further screened
Obtaining 1 plant has high efficiency nitrification-denitrification capability bacterial strain, as BSXS-1602.
2, the identification of bacterial strain
The measurement of bacterial strain physio-biochemical characteristics：The project of identification mainly includes form, Gram's staining, the gemma dye of bacterial strain
Color, methyl red, V-P measurement, oxidizing ferment, catalase, amylolytic enzyme, lipase, lysozyme, nitrite reductase, Western-style lemon
Lemon hydrochlorate, glucose fermentation, mannitol, gelatin liquefaction, anaerobic growth, dynamic culture base and H2S。
The 16S rDNA gene order (1460bp) and NCBI database sequence of bacterial strain BSXS-1602 after sequencing amplification
Be compared, chosen based on 16S rDNA sequence homology related strain gene order phylogenetic tree construction (see Fig. 1,
Wherein BSXS-1602 is bacterial strain bacillus BSXS-1602 of the invention).In conjunction with bio-chemical characteristics result and 16S rDNA
Sequence Identification can determine that bacterial strain BSXS-1602 is bacillus subtilis.
3, the morphological observation of bacterial strain BSXS-1602
Under an optical microscope, bacterial strain BSXS-1602 is in rod-short, has flagellum, can move.By Gram's staining and
The bacterial strain of spore staining is under an optical microscope Gram-positive, and spore staining is uniform.By the bacterial strain BSXS- of purifying
1602 be placed in 28 DEG C of biochemical cultivation case under the conditions of cultivate 24 hours after, bacterium colony on 2216E plate is milky, flat, impermeable
It is bright, edge is irregular.
4, Physiology and biochemistry qualification result
The Physiology and biochemistry of bacterial strain BSXS-1602 and type strain bacillus subtilis CGMCC1.3358 the results are shown in Table 1.
The Physiology and biochemistry result of table 1 bacterial strain BSXS-1602 and bacillus subtilis CGMCC1.3358
Remarks："+" represents the positive；"-" represents feminine gender.
5, the influence of varying environment factor pair bacterial strain bacillus BSXS-1602
By the analysis varying environment factor (carbon source, C/N (mass ratio), pH, salinity and revolving speed) to bacterial strain BSXS-1602
The influence of heterotrophic nitrification-aerobic denitrification characteristic finds that at 28 DEG C of temperature, bacterial strain BSXS-1602 is respectively grape in carbon source
Heterotrophic nitrification-aerobic denitrification with higher under the conditions of sugar, C/N 18, pH value 7.5, salinity 30 ‰ and revolving speed 160rpm
Energy.Result of study shows bacterial strain heterotrophic nitrification-aerobic denitrification capability with higher, in aquaculture water environment conditioning
In have potential application.
The main feature of the white tail feather bacillus BBHS-01 of embodiment 2 and its removal of water pollutant is tested
1. the main feature of white tail feather bacillus BBHS-01 is as follows：
(1) morphological feature：
Form be it is rod-shaped, can move, can produce gemma, binary fission reproduction, thallus is individually, two-by-two or the gathering of short chain.
(2) physiological and biochemical property：
Bacterial strain Gram-positive, spore staining is positive, grease hydrolysis enzyme positive, Starch Hydrolysis enzyme positive, contacts enzyme positive,
V-P negative, glucose oxidative fermentation produce gas, the gelatin positive, Lactose-positive, the maltose positive, mannitol feminine gender, sweet dew
Sugared positive, sucrose feminine gender, arabinose is negative, xylose is negative.
(3) bacterial strain is identified
Bacterium is finally determined according to the phylogenetic tree of the 16S rDNA of white tail feather bacillus BBHS-01 and related strain building
Strain is the bacterium of white tail feather bacillus (see Fig. 2, wherein BBHS-01 is the white tail feather bacillus of bacterial strain of the invention).
2. white tail feather bacillus BBHS-01 tests the removal of water pollutant：
(1) prawn feed solid plate growing state detects：
White tail feather bacillus BBSH-01 is separated from the bed mud of prawn culturing pond, and (deposit number is： CGMCC
NO.6939), in 28 ± 1 DEG C of shrimp feed mediums in 20g/L, (commercially available prawn feed 20g is ground into fine powder to the bacterial strain, sterilizing
Chen Haishui 1000mL impregnates centrifuging and taking supernatant after 48h, adds 25 g of agar, adjusts 7.8,121 DEG C of pH, and sterilize 20min) on growing way
Good, growth rapidly, cultivates colony diameter for 24 hours up to 2mm or so.
(2) the removal effect detection of prawn feed leachate：
1) white tail feather bacillus BBHS-01 tests the removal effect of prawn feed leachate
Experiment removal liquid used：It is ground into fine powder for commercially available prawn feed 20g, sterilize Chen Haishui 1000mL, after impregnating 48h
Centrifuging and taking supernatant adds sodium nitrite 0.058g, adjusts pH 7.8, is distributed into 100mL/ bottles, and in 121 DEG C, sterilize 20min.
The object of the activation culture for 24 hours 8000rpm of white tail feather bacillus BBHS-01 is centrifuged 10min, is washed with sterile saline
Agent precipitates thallus, and is diluted to containing 1 × 106Then bacterium solution is added to the prawn of 100mL by the bacterium solution of cfu/mL in 1% ratio
Bait removes in liquid, and 28 ± 1 DEG C, shaken cultivation 5d under 160rpm, daily timing sampling measure the ammonia nitrogen (NH in sample liquid4 +-
N), cultured water (NO2 -- N) content, OD600, using be not added with bacterial strain as control treatment, each processing sets 3 repetitions.
2) experimental result
Experimental result is shown in Table 2 respectively.The experimental results showed that：White tail feather bacillus BBHS-01 is to prawn feed removal liquid tool
Have apparent removal effect, during the experiment with the growth of the bacterial strain, can significantly remove inorganic nitrogen in bait removal liquid at
Point, wherein to NH4 +-N、NO2 -The highest removal rate of-N is respectively 60.34%, 92.40%.
The white tail feather bacillus BBHS-01 of table 2 is in different incubation times to the removal effect of bait leachate
The antagonistic experiment of 3 bacillus BSXS-1602 of embodiment and white tail feather bacillus BBHS-01
It takes bacillus BSXS-1602, white tail feather bacillus BBHS-01 to cross in 2216E solid medium respectively, is placed in
After 28 DEG C of cultures for 24 hours, picks them separately single colonie and carry out cross scribing line on 2216E solid medium, by the plate of scribing line in 28
DEG C culture for 24 hours after, check strain growth situation, antagonistic experiment scribing line cultivation results show that antagonism is not present in two bacterial strains.
4 bacillus BSXS-1602 of embodiment tests the removal of water pollutant
(1) prawn feed solid plate growing state detects：
In 28 ± 1 DEG C of shrimp feed mediums in 20g/L, (commercially available prawn feed 20g is ground bacillus BSXS-1602
At fine powder, sterilize Chen Haishui 1000mL, impregnates centrifuging and taking supernatant after 48h, adds agar 25g, adjusts 7.8,121 DEG C of pH, sterilizing
Growing way is good on 20min), and growth rapidly, cultivates colony diameter for 24 hours up to 2mm or so.
(2) the removal effect detection of prawn feed leachate：
1) bacillus BSXS-1602 tests the removal effect of prawn feed leachate
Testing test fluid used is：Commercially available prawn feed 20g is ground into fine powder, and sterilize Chen Haishui 1000mL, impregnates 48h
Afterwards, centrifuging and taking supernatant adds sodium nitrite 0.058g, adjusts pH 7.8, is distributed into 100mL/ bottles, and in 121 DEG C, sterilize 20min.
The object of the activation culture for 24 hours 8000rpm of bacillus BSXS-1602 is centrifuged 10min, with sterile saline lotion
Thallus is precipitated, and is diluted to containing 1 × 106Then bacterium solution is added to the prawn bait of 100mL by the bacterium solution of cfu/mL in 1% ratio
Expect in leachate, 28 DEG C, 160rpm continuous oscillation culture 5d, daily timing sampling, measures the ammonia nitrogen (NH in sample liquid4 +-N)、
Cultured water (NO2 -- N) content, thalli growth amount, bacterium is not added as control treatment, each processing sets 3 repetitions.
2) experimental result
Experimental result is shown in Table 5 respectively.The experimental results showed that：Bacillus BSXS-1602 is in prawn feed leachate
NH4 +- N and NO2 -- N has apparent removal effect, during the experiment with the growth of the bacterial strain, can significantly remove bait leaching
Inorganic nitrogen component in liquid, wherein to NH4 +- N and NO2 -The highest removal rate of-N is respectively 52.35%, 97.23%.
5 bacillus BSXS-1602 of table is in different incubation times to the removal effect of bait leachate
5 composite bacteria preparation of embodiment tests the removal of water pollutant
The composite bacteria preparation of the present embodiment is by bacillus BSXS-1602, white tail feather bacillus BBHS-01 with 10:1
Bacterial population ratio be combined.
Testing test fluid used is：Commercially available prawn feed 20g is ground into fine powder, and sterilize Chen Haishui 1000mL, after impregnating 48h
Centrifuging and taking supernatant adds sodium nitrite 0.058g, adjusts pH 7.8, is distributed into 100mL/ bottles, and in 121 DEG C, sterilize 20min.
Bacillus BSXS-1602 and Bai Ling bacillus BBHS-01 is cultivated through activation culture for 24 hours, expansion respectively, then
Respectively through 28 DEG C of liquid state fermentations, then by bacillus BSXS-1602 and Bai Ling bacillus BBHS-01 according to 10:1 bacterium
Number ratio mixing, precipitates thallus with sterile saline lotion, will mix mycelium dilution, and it is 1 × 10 that concentration, which is made,6Cfu/mL's
Then bacterium solution is added to bacterium solution in the prawn feed leachate of 100 mL in 1% ratio, 28 DEG C, 160rpm continuous oscillation training
5d is supported, daily timing sampling measures the ammonia nitrogen (NH in sample liquid4 +- N), cultured water (NO2 -- N) content, thalli growth amount,
Bacterium is not added as control treatment, each processing sets 3 repetitions.Experimental result is shown in Table 6 respectively.The experimental results showed that：Gemma bar
Bacterium BSXS-1602 is to the NH in prawn feed leachate4 +- N and NO2 -- N have apparent removal effect, during the experiment with
The growth of the bacterial strain can significantly remove the inorganic nitrogen component in bait removal liquid, wherein to NH4 +- N and NO2 -The highest of-N removes
Rate is respectively 93.66%, 100%.
6 composite bacteria preparation of table is in different incubation times to the removal effect (1~5d) of bait removal liquid
Facilitation of the 6 bacillus BSXS-1602 bacterium powder of embodiment to Growth of Litopenaeus vannamei
The bacterium powder of the bacillus BSXS-1602 of the present embodiment, preparation method are as follows：Bacillus BSXS-1602 is living
Change, expand culture, through 28 DEG C of liquid state fermentations, thalline were collected by centrifugation by 8000rpm, bentonite is added as carrier, by thallus with it is swollen
Profit soil stirs evenly, and the bacterium powder that bacterium amount is 100~50,000,000,000/g is made in 45 DEG C of drying.
The bacillus BSXS-1602 bacterium powder of the present embodiment is pressed to 0.05%, 0.1%, 0.15%, 0.2% matter respectively
Amount percentage is respectively added in prawn mixed feed, and not add the processing group of bacterium powder as a control group, each processing is set 3 times
It repeats, continuous feeding litopenaeus vannamei (prawn original body mass is 0.6 ± 0.08g) 45 days, at the end of experiment, addition 0.05%
The processing group litopenaeus vannamei average growth rate of bacillus BSXS-1602 bacterium powder is 283.3%, feed coefficient 1.65；Add
The processing group litopenaeus vannamei average growth rate for adding 0.1% bacillus BSXS-1602 bacterium powder is 297.5%, and feed coefficient is
1.57；The processing group litopenaeus vannamei average growth rate for adding 0.15% bacillus BSXS-1602 bacterium powder is 318.4%, bait
Expect that coefficient is 1.51；The processing group litopenaeus vannamei average growth rate for adding 0.2% bacillus BSXS-1602 bacterium powder is
318.9%, feed coefficient 1.48；Control group litopenaeus vannamei average growth rate is 261.8%, feed coefficient 2.04.
Bacillus BSXS-1602 bacterium powder of the invention facilitates storage and use, can promote the appetite of aquatic livestock, as
When feed addictive feeds litopenaeus vannamei, feed coefficient can be effectively reduced, promotes Growth of Litopenaeus vannamei.
Embodiment 7 is with Mixed Microbes powder bacillus BSXS-1602 as main component to the rush of Growth of Litopenaeus vannamei
Into effect
The Mixed Microbes powder as main component with bacillus BSXS-1602 of the present embodiment, preparation method are as follows：It will
Bacillus BSXS-1602 and Bai Ling bacillus BBHS-01 is activated respectively, is expanded culture, then respectively through 28 DEG C of liquid state fermentations,
Then by bacillus BSXS-1602 and Bai Ling bacillus BBHS-01 according to 15:1 bacterial population ratio mixing, 8000rpm
Thalline were collected by centrifugation, and bentonite and xanthan gum is added as carrier, and (mass ratio of bentonite and xanthan gum is 5:1), by thallus with
Bentonite, xanthan gum stir evenly, and the bacterium powder that total bacterium amount is 100~50,000,000,000/g is made in 45 DEG C of drying.
The Mixed Microbes powder of the present embodiment is pressed to 0.01%, 0.05%, 0.08%, 0.1% mass percent difference respectively
It is added in prawn mixed feed, not add the processing group of Mixed Microbes powder as a control group, each processing sets 3 repetitions, even
Continuous feeding litopenaeus vannamei (prawn original body mass is 0.6 ± 0.08g) 45 days, at the end of experiment, adds 0.01% Mixed Microbes powder
Processing group litopenaeus vannamei average growth rate be 281.7%, feed coefficient 1.68；Add the processing of 0.05% Mixed Microbes powder
Group litopenaeus vannamei average growth rate is 299.3%, feed coefficient 1.59；The processing group for adding 0.08% Mixed Microbes powder all is received
Shore prawn average growth rate is 317.2%, feed coefficient 1.48；Add the processing group litopenaeus vannamei of 0.1% Mixed Microbes powder
Average growth rate is 320.2%, feed coefficient 1.45；Control group litopenaeus vannamei average growth rate is 261.8%, bait system
Number is 2.04.
Mixed Microbes powder of the invention facilitates storage and use, can promote the appetite of aquatic livestock, throws as feed addictive
When feeding litopenaeus vannamei, feed coefficient can be effectively reduced, promotes Growth of Litopenaeus vannamei.
The above is only a preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, without departing from the principle of the present invention, several improvement can also be made, these improvement should be regarded as guarantor of the invention
Protect range.
Claims (10)
1. a kind of heterotrophic nitrification-aerobic denitrification bacillus is bacillus BSXS-1602, on June 15th, 2018 in
State's General Microbiological Culture preservation administrative center has carried out preservation, and deposit number is：CGMCC NO.15950.
2. a kind of bacillus BSXS-1602 described in claim 1 is in removal NH4 +- N and NO2 -- N and aquaculture water ring
Application in the regulation of border.
3. a kind of bacterium powder made of bacillus BSXS-1602 described in claim 1.
4. a kind of preparation method of bacterium powder as claimed in claim 3, which is characterized in that include the following steps：By bacillus
BSXS-1602 activation expands culture, and through 28 DEG C of liquid state fermentations, 8000rpm is collected by centrifugation bacterium mud, bentonite is added as carrier,
Bacterium mud and bentonite are stirred evenly, the bacterium powder that bacterium amount is 100~50,000,000,000/g is made in 45 DEG C of drying.
5. a kind of application of bacterium powder as claimed in claim 3 as aquatic animal feed additive, and the application of regulation water quality.
6. one kind is with bacillus BSXS-1602 composite bacteria preparation as main component described in claim 1, feature exists
In the composite bacteria preparation is by bacillus BSXS-1602, white tail feather bacillus BBHS-01 with 10~12:1 bacterial population
Ratio is combined；The white tail feather bacillus BBHS-01 (Bacillus baekryungensis), on December 7th, 2012
Preservation is carried out in China Committee for Culture Collection of Microorganisms's common micro-organisms collection, deposit number is：CGMCC
NO.6939。
7. a kind of composite bacteria preparation as claimed in claim 6 is in degradation NH4 +- N and NO2 -In-N and aquaculture water environment conditioning
Application.
8. a kind of Mixed Microbes powder as main component with bacillus BSXS-1602 described in claim 1, feature exist
In preparation method is as follows：Bacillus BSXS-1602 and Bai Ling bacillus BBHS-01 is activated respectively, expands culture, then
Respectively through 28 DEG C of liquid state fermentations, then by bacillus BSXS-1602 and Bai Ling bacillus BBHS-01 according to 10~15:1
The mixing of bacterial population ratio, thalline were collected by centrifugation by 8000rpm, and bentonite and xanthan gum is added and is used as carrier, by thallus and bentonite,
Xanthan gum stirs evenly, and the bacterium powder that total bacterium amount is 100~50,000,000,000/g is made in 45 DEG C of drying.
9. Mixed Microbes powder according to claim 8, which is characterized in that the carrier is bentonite and xanthan gum according to 5:1
Mass ratio mix.
10. a kind of application of Mixed Microbes powder according to any one of claims 8 as feed addictive, and the application of regulation water quality.
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