CN103289933A - Bacillus amyloliquefaciens plant subspecies B-01 and application thereof - Google Patents
Bacillus amyloliquefaciens plant subspecies B-01 and application thereof Download PDFInfo
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Abstract
The invention relates to the field of microbes, and particularly relates to bacillus amyloliquefaciens and application thereof. The bacillus amyloliquefaciens is a bacillus amyloliquefaciens plant subspecies B-01, and the preservation number of the bacillus amyloliquefaciens plant subspecies B-01 is CCTCC: M2012191. The strain disclosed by the invention can be used as a water environment ecological purifying agent for purification and can keep the characteristic of higher enzyme activity under the condition of low temperature and play a better biological role in a majority of national regions with lower environmental temperature.
Description
Technical field
The present invention relates to microorganism field, be specifically related to a kind of bacillus amyloliquefaciens and application thereof.
Background technology
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens, BA) be that the present Ministry of Agriculture makes (2008 No. 1126) zymin class fodder additives allow the probiotic bacterium kind of using, but this bacterium make in the animal microorganism fodder additives column still unlisted in the Ministry of Agriculture.Studies show that this bacterium is as the adjustable animal digestive tract micro-ecological environment of probiotic bacterium, suppress harmful bacterium, improve the organism metabolism function, this type of bacterial classification suitable growth temperature that kind of center is protected by present country to be provided is generally about 28-35 ℃, under low temperature water body envrionment conditions, because the related enzyme systems vigor is lower, suppressed the performance of probiotic bacterium quasi-biology function greatly, this mushroom is mainly used in production of enzyme preparation at present, does not relate to as yet in purification of water quality.
Summary of the invention
In order to solve the existing B-01 bacillus amyloliquefaciens lower problem of enzymic activity at low temperatures, separation in the barnyard grass grass of the present invention from the cold environment of the northern area of China-35 ℃, purifying, cultivation are to a strain low temperature high activity bacterial strain and protect kind, protect the existing relevant bacterial classification in kind of center with present country and have different biological characteristicses, solved the deficiency that present technology exists effectively.
National preservation mechanism such as bacillus amyloliquefaciens bacterial strain of the present invention and Chinese industrial microbial strains preservation administrative center existing bacterial classification source and biological characteristics are different, have low temperature tolerance characteristics, well-grown under 18 ℃ of conditions.Carrying out bacterium classification in Chinese industrial microbial strains preservation administrative center identifies, identify that clearly B-01 is bacillus amyloliquefaciens plant subspecies (Bacillus amyloliquefaciens subsp.plantarum), administrative center has carried out the genetic stability test by the preservation of Chinese industrial microbial strains, but proves this bacterial strain proterties genetic stability.Its deposit number is CCTCC:M2012191
B-01 bacillus amyloliquefaciens plant subspecies of the present invention can be brought into play higher biologic activity in purification of water quality.This bacterium is well-grown under 18 ℃ of temperature conditions on the low side, secreted amylase, lipase, the proteolytic enzyme of this bacterium all has higher enzyme activity, in the low water temperature environment of eutrophy, larger molecular organics can be degraded to small-molecule substance, but one side back feeding probiotic bacterium, can directly be utilized by aquatic animal on the other hand, help the restoration of the ecosystem of water body environment.This bacterial strain is as water quality cleansing agent, can effectively degrade larger molecular organics (protein, carbohydrate, fat) in the water body, reduce chemical oxygen demand (COD) (COD), ammonia nitrogen, nitrite,, improve oxygen capacity (DO), improve indexs such as pH, turbidity, suspension content.
The B-01 bacillus amyloliquefaciens can be used as novel water-ring border probiotic bacterium ecological purificant and novel animal microorganism fodder additives is used.
Compare with existing bacillus amyloliquefaciens, bacterial strain of the present invention can keep higher enzyme activity characteristic under cold condition, can bring into play biological action preferably than lowland area in the most of envrionment temperatures of China.Can effectively the degrade organic substance of overnutritionization in the environment water of bacterial strain of the present invention is degraded to the small-molecule substance that aquatic animals and plants can utilize with larger molecular organics, turns waste into wealth, as the water surrounding ecological purificant.This purifies the restoration of the ecosystem of livestock industry aquaculture water and life fluvial-environment for alleviating the current water resource environment that worsens day by day, and life, the production water surrounding of building green safety have positive sociology meaning and economics and be worth.In addition, this bacterial strain also can be used as the animal microorganism fodder additives and is applied to livestock breeding industry, uses as the microbiotic substitute, can be to alleviate microbiotic abuse present situation performance significant role.
Description of drawings
Different condition bacterial count variation diagram in time in the orthogonal experimental design of Fig. 1 culture condition.
Fig. 2 probiotic bacterium liquid fermenting viable count is variation diagram in time.
Fig. 3 probiotic bacterium liquid fermenting pH is variation diagram in time.
Fig. 4 probiotic bacterium liquid fermenting dehydrogenase activity is variation diagram in time.
Fig. 5 is bacillus amyloliquefaciens plant subspecies B-01 bacterial strain field emission scanning electron microscope photo of the present invention.20,000 times of S4800 field emission scanning electron microscope photos, the long 0.8-3um of bacterium, wide 0.6um.
Fig. 6 is photo under the bacillus amyloliquefaciens plant subspecies B-01 bacterial strain oil mirror of the present invention
Annotate: the NA substratum, 37 ℃, cultivated thalli morphology 1 day: shaft-like, 0.5 μ m * 2.0～2.67 μ m, single, paired or paliform is arranged Gram-positive.
Fig. 7 is that bacillus amyloliquefaciens plant subspecies B-01 bacterial strain agar of the present invention is cultivated the bacterium colony photo
Annotate: the NA substratum, 37 ℃, cultivated colonial morphology 1 day: oyster white, irregular shape, surface ruffle, coarse, translucent, the edge is irregular.
18 ℃ of amylase of Fig. 8 (AMS) vigor is variation diagram in time.
18 ℃ of lipase of Fig. 9 (LPS) vigor is variation diagram in time.
18 ℃ of proteinase activities of Figure 10 are variation diagram in time.
18 ℃ of bacterial counts of Figure 11 are variation diagram in time.
Figure 12 laboratory probiotic bacterium effects of purification quality figure (before the experiment)
Annotate: the eutrophic water control bottle that does not add probiotic bacterium.
Figure 13 laboratory probiotic bacterium effects of purification quality figure (experiment back)
Annotate: the eutrophic water assay flask that adds the 2%B-01 bacillus amyloliquefaciens.
The a large amount of pond of Figure 14 sewage (210m
3) probiotic bacterium purification of water quality (before the experiment).
The a large amount of pond of Figure 15 sewage (210m
3) probiotic bacterium purification of water quality (experiment back).
Figure 16 B-01 bacterial strain is grown tree with relevant kind 16SrDNA sequential system.
Figure 17 B-01 and relevant gyrA gene order phylogenetic tree of planting.
Figure 18 B-01 bacterial strain (the 1st batch) and relevant gyrA gene order phylogenetic tree of planting.
Figure 19 B-01 bacterial strain (the 1st batch continuous passage 5 times after bacterial strain) and relevant kind gyrA gene order phylogenetic tree.
Bacillus amyloliquefaciens plant subspecies B-01 of the present invention (Bacillus amyloliquefaciens subsp.plantarum B-01), be preserved in the Chinese typical culture collection center that is positioned at Chinese Wuhan University on May 30th, 2012, its preserving number is CCTCC NO:M2012191.
Embodiment
The screening of embodiment one bacterial strain
1.1 the separation of bacterial strain, screening, purge process
Barnyard grass grass, fiber crops grass, each 1 gram of sheep's hay of choosing in the cold environment of the northern area of China-35 ℃ are inoculated in and soak 2h in the physiological saline, get the 2mL soak solution and are inoculated in 100mL and stimulate gemma grown cultures liquid (peptone 10g, yeast extract paste 3g, starch 3g, MgSO
40.1g, KH
2PO41.5g, Na
2HPO
42g distilled water 1000mL, pH7.8,121 ℃ of sterilization 20min) 18 ℃ be cultured to the adularescent mycelia and grow.
Get bacteria suspension and kill not spore production bacteria body in 80 ℃ of water-bath 15min(), choose 10 times of dilution plates of bacteria suspension and coat gemma isolation medium (peptone 5g, glucose 5g, yeast extract paste 5g, K
2HPO
44g, 3.08%MnSO41mL, agar 18g, distilled water 1000mL, pH nature) 18 ℃ be cultured to single bacterium colony and grown.
Select the good bacterium colony of growth conditions under the cold condition, dyeing is observed, and the bacterium colony cryogenic purincation is cultivated, called after B-01.
2.1 nutrition substrate positive quadraturing design test
Cultivate to guarantee optimal culture condition.Adopt viable count as the deliberated index of cultivation conditions.
Orthogonal design sees Table 1: inoculation B-01 bacterium liquid measure is 30%+70% nutrient solution (weight percent)
Table 1 orthogonal experimental design scheme
According to the different content of energy, protein and mineral substance, the application gold is herded software design and is gone out to fill a prescription as table 2:
Table 2 orthogonal test scheme (9 kinds of optimization processes)
According to the trophic level prescription of table design, the application gold is herded software design and is gone out composition of raw materials as showing:
Table 3.9 group raw material nutritional formula
Annotate: above-mentioned 9 assembly Fang Zhongjun press table 4 and add small powder
Table 4 adds little material formula
| Each prescription group is all added small powder: | MgSO 4 | MnSO 4 | K 2HPO 4 | Add up to |
| Weight percent (%) | 0.15 | 0.01 | 0.11 | 0.27 |
Comparison between the orthogonal design different affecting factors, each test combinations colony number sees Table 5.
Table 5 orthogonal experimental design measurement result
The single factor statistics of table 6 orthogonal experimental design
From table 6 intuitive analysis, the order of excellence of each level of factor is: A
1A
2A
3, B
1B
2B
3, C
2C
3C
1
Table 7 orthogonal experimental design The results of analysis of variance
| Soruces of variation | Sum of sguares of deviation from mean | Degree of freedom | All square | The F value | The P value |
| Energy (A) | 353.018 | 2 | 176.509 | 45.808 | 0.021 |
| Crude protein (B) | 995.704 | 2 | 497.852 | 129.205 | 0.008 |
| Mineral substance (C) | 23.341 | 2 | 11.670 | 3.029 | 0.248 |
| Error | 7.706 | 2 | 3.853 | ? | ? |
| Total variation | 1379.769 | 8 | ? | ? | ? |
From the The results of analysis of variance of table 7 as seen, crude protein (B) has utmost point significance influence (P＜0.001) to colony number; Energy (A) has significance influence (P＜0.005) to colony number; Mineral substance is less to the colony number influence, and difference is not remarkable, and each trophic factor primary and secondary is B in proper order〉A〉C, the best nutritional substrate of selected microbial culture is A
1B
1C
2, that is, and energy 2.0MC/kg, protein 15 %, mineral substance 3.5%(CaHPO
43%, NaCl0.5%)
In conjunction with orthogonal experiments, utilize gold to herd software (VF123-2006 version) and design nutrition substrate prescription as table 8:
Table 8 nutrition substrate optimum formula
| Title | Wheat bran | Dregs of beans | Corn | Rice husk | CaHPO 4 | NaCl | Small powder |
| Weight percent (%) | 29.8 | 15.18 | 35.8 | 15.45 | 3.0 | 0.5 | 0.27 |
2.2 culture condition orthogonal test
Table 9 orthogonal experimental design scheme
Carry out 4 factors, 3 horizontal quadrature design experiments by table 9, every 3h measures the viable count of bacterium, amounts to 48h.In the orthogonal experimental design of culture condition the different condition bacterial count in time variation diagram see Fig. 1. it is that bacterial count increases peak point that the different condition colony number changes as can be known about 24h in time, therefore with the 24h viable count as criterion.
Table 10 orthogonal experimental design The results of analysis of variance
| Soruces of variation | Sum of sguares of deviation from mean | Degree of freedom | All square | The F value | The P value |
| pH(A) | 3513.849 | 2 | 1756.924 | 184.608 | .000 |
| Temperature (B) | 721.529 | 2 | 360.764 | 37.907 | .000 |
| Inoculum size (C) | 4386.596 | 2 | 2193.298 | 230.460 | .000 |
| Substrate (D) | 264.427 | 2 | 132.213 | 13.892 | .000 |
| Error | 171.307 | 18 | 9.517. | ? | ? |
| Total variation | 9057.707 | 26 | ? | ? | ? |
The orthogonal experimental design The results of analysis of variance sees Table 10, from the The results of analysis of variance of table 10 as seen, each factor of ABCD all has extremely significance influence (P＜0.001) to viable count, and each factor primary and secondary is C in proper order〉A〉B〉D, the best nutritional substrate of selected microbial culture is A
2B
2C
3D
2
Draw the optimal culture condition of B-01: pH7.2,35 ℃ of temperature, inoculum size 15%, concentration of substrate 10%.
2.3B-01 the dynamic research of bacillus amyloliquefaciens strain liquid fermentating metabolism rule
Select the suitableeest above-mentioned breeding condition of B-01 bacterial strain, the fermentation capacity is 30L, every 3h dynamic measurement viable count, and pH value and desaturase total activity, 24h repeats 4 times altogether.Ferment colony number in time shown in the variation diagram as Fig. 2 .B-01 bacillus amyloliquefaciens strain liquid, the 1st batch, the 2nd batch, the 3rd batch, the 4th batch colony number expands numerous to 141 * 108cfu/mL by 8 * 108cfu/mL respectively, expand numerous to 149 * 108cfu/mL by 13 * 108cfu/mL, numerous to 168 * 108cfu/mL by 16 * 108cfu/mL expansion, expand numerous to 162 * 108cfu/mL by 17 * 108cfu/mL.0h compares with fermentation, and the 6-24h colony number is higher, and difference is (P＜0.01) extremely significantly; 3h, 6h compare with 9h with fermentation, and the 12-24h colony number is higher, and difference is (P＜0.01) extremely significantly; 12h, 15h compare with fermentation, and the 18-24h colony number is higher, and difference is (P＜0.01) extremely significantly; 18h compares with fermentation, and the 24h colony number is higher, and difference is (P＜0.01) extremely significantly.Constantly cultivation is in rising trend along with bacterium liquid for colony number, the beneficial flora increased activity; In time shown in the variation diagram, the 1st crowd, the 2nd crowd, the 3rd crowd, the 4th crowd pH drops to 6.2 by 6.76 respectively as Fig. 3 .B-01 bacillus amyloliquefaciens strain liquid fermentation pH number, drops to 6.01 by 6.62, drops to 5.94 by 6.78, drops to 5.99 by 6.76.0h, 3h compare with fermentation, and 6-24h pH reduces, and difference is (P＜0.01) extremely significantly; 6h compares with fermentation, and 18-24hpH reduces, and difference is (P＜0.01) extremely significantly; Fermentation 9h-21h compares, and 24h pH reduces, and difference is (P＜0.01) extremely significantly.The pH value changes in time and constantly reduces, and shows that bacterium is in constantly fermentation propagation; As Fig. 4, shown in the variation diagram, the 1st crowd, the 2nd crowd, the 3rd crowd, the 4th crowd TDHA brings up to 0.694 by 0.005 respectively to B-01 bacillus amyloliquefaciens strain liquid fermentation TDHA, brings up to 0.896 by 0.010 in time, bring up to 1.003 by 0.023, bring up to 0.997 by 0.029.0h, 3h compare with fermentation, and 12-24h TDHA is higher, and difference is (P＜0.01) extremely significantly; 6-12h compares with fermentation, and 18-24h TDHA is higher, and difference is (P＜0.01) extremely significantly; 15-21h compares with fermentation, and 24h TDHA is higher, and difference is (P＜0.01) extremely significantly.In the 24h fermentation period, TDHA increases trend gradually along with course of fermentation is, and shows that the bacterium enzyme activity of beneficial flora strengthens gradually.
3, low temperature modification high reactivity B-01 bacillus amyloliquefaciens morphology research
Above-mentioned screening obtained strains B-01 is at 20,000 times of S4800 field emission scanning electron microscope photos such as Fig. 5, the long 0.8-3um of bacterium, wide 0.6um.
With bacterial strain B-01 at the NA substratum, 37 ℃, cultivated 1 day, observe under the oily mirror then, photo such as Fig. 6, visible thalli morphology: shaft-like, 0.5 μ m * 2.0～2.67 μ m, single, in pairs or paliform arrange Gram-positive.
Bacterial strain B-01 at the NA substratum, 37 ℃, was cultivated colonial morphology 1 day: oyster white, irregular shape, surface ruffle, coarse, translucent, edge irregular (Fig. 7).
The evaluation of embodiment two bacterial strains
Bacterial strain B-01 send Chinese industrial microbial strains preservation administrative center (CICC) to carry out the microorganism identification analysis.
1.BA closing the 16S rDNA sequential system of planting, bacterial strain and phase and phase grow tree
Adopt MEGA4.1 software, the ortho position connection method shows B-01 and relevant 16S rDNA phylogenetic tree of planting, and carries out 1000 times similarity double counting, grow tree node among the figure and only show the Bootstrap value greater than 50% numerical value, (E., Escherichia).
By Figure 16. the 16S rDNA phylogenetic tree analysis revealed of bacterial strain B-01: a plurality of kinds are gathered in a phylogeny branch in bacterial strain B-01 and the bacillus subtilis flora (Bacillus subtilis group), and sequence homology is more than 99.2%.
B-01 bacterial strain dna sequence dna is shown in SEQ ID No.1.
2.B-01 the gyrA gene order phylogenetic tree with relevant kind
Adopt MEGA4.1 software, the ortho position connection method shows B-01 and relevant gyrA Phylogenetic Tree of planting, and carries out 1000 times similarity double counting, grows tree node among the figure and only shows that the Bootstrap value is greater than 50% numerical value.
Shown by the pheS gene order phylogenetic tree Phylogenetic Analysis of Figure 17 .B-01 bacillus amyloliquefaciens bacterial strain with relevant kind: bacterial strain B-01 and bacillus amyloliquefaciens plant subspecies (Bacillusamyloliquefaciens subsp.plantarum) are gathered in a phylogeny branch, and sequence homology is 98.7%.This bacterial strain can utilize glycan to produce acid, gives birth in the gemma, and these characteristics are consistent with bacillus amyloliquefaciens.
By 16S rDNA sequence and gyrA gene order Phylogenetic Analysis, bacterial strain B-01 is accredited as: bacillus amyloliquefaciens plant subspecies (Bacillus amyloliquefaciens subsp.plantarum).3.B-01 bacillus amyloliquefaciens bacterial classification genetic stability is analyzed
Bacterial strain bacillus amyloliquefaciens B-01 send Chinese industrial microbial strains preservation administrative center (CICC) to carry out the analysis of microbial strains genetic stability.Bacterial strain bacillus amyloliquefaciens B-01 (1 crowd) cultivates through continuous passage, and strain morphology is learned feature and do not found visible change, gyrA gene order no change, and physiological and biochemical property does not have considerable change, but bacterial strain proterties genetic stability.
3.1B-01 bacterial strain physiological and biochemical property (seeing Table 11)
Table 11B-01 bacterial strain physiological and biochemical property
Nomenclature: "+", the positive; "-", feminine gender; "+w ", the weak positive; ND does not survey.
3.2 sequential analysis
Sequence alignment: bacillus amyloliquefaciens B-01 (1 crowd)
98.4%Bacillus?amyloliquefaciens?subsp.plantarum?FZB42T(CP000560)
95.1%Bacillus?amyloliquefaciens?supsp.amyloliquefaciens?KCTC1660T(AF272015)
83.6%Bacillus?mojavensis?NRRL?B-14698T(EU138598)
83.4%Bacillus?subtilis?subsp.inaquosorum?NRRL?B-23052T(EU138605)
83.0%Bacillus?subtilis?subsp.spizizenii?NRRL?B-23049T(AF272020)
82.6%Bacillus?subtilis?subsp.subtilis?KCTC3135T(AF272021)
82.4%Bacillus?tequilensis?NRRL?B-41771T(EU138625)
82.3%Bacillus?atrophaeus?KCTC3701T(AF272016)
82.2%Bacillus?vallismortis?NRRL?B-14890T(AF272025)
80.3%Bacillus?sonorensis?NRRL?B-23154T(EU138611)
Bacterial strain bacillus amyloliquefaciens B-01 (1 crowd) and relevant gyrA gene order phylogenetic tree of planting (Figure 18 B-01 bacillus amyloliquefaciens bacterial strain (1 batch) and relevant gyrA gene order phylogenetic tree of planting)
Expert's conclusion: bacillus amyloliquefaciens plant subspecies B.amyloliquefaciens subsp.Plantarum
Sequence alignment: bacillus amyloliquefaciens B-01 (1 crowd continuous passage 5 times after bacterial strain)
98.4%Bacillus?amyloliquefaciens?subsp.plantarum?FZB42T(CP000560)
95.1%Bacillus?amyloliquefaciens?supsp.amyloliquefaciens?KCTC1660T(AF272015)
83.6%Bacillus?mojavensis?NRRL?B-14698T(EU138598)
83.4%Bacillus?subtilis?subsp.inaquosorum?NRRL?B-23052T(EU138605)
83.0%Bacillus?subtilis?subsp.spizizenii?NRRL?B-23049T(AF272020)
82.6%Bacillus?subtilis?subsp.subtilis?KCTC3135T(AF272021)
82.4%Bacillus?tequilensis?NRRL?B-41771T(EU138625)
82.3%Bacillus?atrophaeus?KCTC3701T(AF272016)
82.2%Bacillus?vallismortis?NRRL?B-14890T(AF272025)
80.3%Bacillus?sonorensis?NRRL?B-23154T(EU138611)
Bacterial strain bacillus amyloliquefaciens B-01 (1 crowd continuous passage 5 times after bacterial strain) and relevant kind gyrA gene order phylogenetic tree (seeing Figure 19 B-01 bacillus amyloliquefaciens bacterial strain (1 batch continuous passage 5 times after bacterial strain) and relevant kind gyrA gene order phylogenetic tree)
Expert's conclusion: bacillus amyloliquefaciens plant subspecies B.amyloliquefaciens subsp.Plantarum.
Embodiment three low temperature modification high reactivity B-01 bacillus amyloliquefaciens bacterial strain enzymic activity dynamic studies and bacterium are dynamically counted
Relevant fungus strain enzyme assay is all controlled under 18 ℃ of culture condition and is carried out, and every duplicate samples fermentation capacity is 200Ml, 3 parts of parallel repetition samples.The enzyme assay of existing market application product mushroom, because enzyme activity is relatively poor under the cold condition, usually measure high temperature conditions such as adopting 60 ℃ of amylase activities, 40 ℃ in lipase, proteolytic enzyme 40-55 ℃, the low temperature modification probiotic products will manifest special advantages in the lower purification of water quality process of actual environment temperature, can keep higher biologic activity, therefore have good market application prospect.
18 ℃ are carried out enzyme activity determination (build up lipase, the determination of amylase test kit that bio-engineering research provides with Nanjing, measure proteinase activity with the GB/T1803-1993 method)
The amylase activity unit definition: the AMS in the 100ml enzyme liquid, 18 ℃ with substrate-function 30 minutes, hydrolysis 10mg starch is 1 unit
Amylase AMSu/dl=(blank pipe absorbancy-mensuration pipe absorbancy)/the preceding extension rate of blank pipe absorbancy * (0.4*0.5/10) * (30 minutes/7.5 minutes) * (1/0.05) * test sample
=(blank pipe absorbancy-mensuration pipe absorbancy)/blank pipe absorbancy * 0.16
The lipase activity unit definition: under 18 ℃ of conditions, every person of outstanding talent rise enzyme liquid in this reaction system with substrate reactions 1 minute, every consumption 1umol substrate is an enzyme activity unit.
The LPS vigor=(A1-A2)/the extension rate * reaction times (10 minute) of As* standard pipe concentration (umol/l) * sample in reaction system
The proteinase activity definition: the 1ml liquid enzymes is at 18 ℃, and under the PH7.3 condition, it is an enzyme activity unit (u/ml) that the 1min hydrolyzed casein produces 1ug tyrosine
Proteinase activity=A*K*4/10*n
1.B-01 the amylase of bacterium, lipase, the research of proteolytic enzyme enzyme activity dynamic rule
By Fig. 8 .B-01 bacillus amyloliquefaciens bacterial strain amylase activity in time shown in the variation diagram, variation diagram is as can be known in time for 18 ℃ of amylase (AMS) vigor, amylase activity is rising always, amylase activity has significantly growth at 44h and 62h respectively, 62h-74h maintains higher level, and takadiastase vigor is 11.532u/dl.
By Fig. 9 .B-01 bacillus amyloliquefaciens bacterial strain lipase activity in time shown in the variation diagram, variation diagram is as can be known in time for 18 ℃ of lipase (LPS) vigor, lipase activity significantly rises when 44h, and reaching maximum when 56h is 1.56411u/l, and lipase activity descends again to some extent subsequently.
By Figure 10 .B-01 bacillus amyloliquefaciens strain protein enzyme activity in time shown in the variation diagram, variation diagram is as can be known in time for 18 ℃ of proteinase activities, proteinase activity changes in time and constantly raises, maintain higher level when 62h-74h, wherein reaching maximum when 62h is 8.836u/mL.
2.B-01 bacterium is at colony number dynamic rule research in time under the low temperature culture condition (under Figure 11 .B-01 bacillus amyloliquefaciens bacterial strain cold condition colony number variation diagram) in time
Measure colony number under the cold condition behind the 20h, as shown in figure 11, the 20h-44h bacterial growth is slower, and the 44h-68h thalli growth is vigorous, reaches the highest colony number 81 * 10 during 68h
8Cfu/mL, bacteria containing amount decreases subsequently.
Embodiment four B-01 bacillus amyloliquefaciens purification of water quality The Characteristic Study
1. laboratory probiotic bacterium purification of water quality
Under laboratory condition, adopt special eutrophic water (prescription: ground pork 50g+ bran powder 50g+ bean cake powder 50g+ Semen Maydis powder 50g+ water 1L, through 100 ℃ boil 1h after, get filtrate and add water to the 10L constant volume), add B-01 bacillus amyloliquefaciens bacterium liquid 2mL(viable count of the present invention by every 100mL nutritive water: 10,000,000,000/mL) mixings, leave standstill the apparent variation of water quality such as Figure 12,13 when observing 20 days.
It is muddy and give an offensive smell not add the eutrophic water control bottle water body of probiotic bacterium, and the eutrophic water assay flask water body that adds the 2%B-01 bacillus amyloliquefaciens is limpid bright, free from extraneous odour.
2. a large amount of pond sewage (210m3) probiotic bacterium purification of water qualitys contrasted before and after 20 days
In sewage lagoon, splash 1 ton of bacterium liquid (0.48% bacterium liquid) to the water surface, observe purifying water effect and measure the every water-quality guideline that contrasts before and after 20 days, see Table 12.The apparent variation of water quality such as Figure 14,15.
Every water-quality determination index all adopts the GB measuring method:
1. chemical oxygen demand (COD) (COD) adopts potassium permanganate process GB/T15456-2008,
2. oxygen capacity (DO) adopts iodimetry,iodometry GB7489-87
3. ammonia nitrogen Nessler's reagent spectrophotometry GB7479-87
4. nitrite spectrophotometry GBT7493-87
5.pH measure with pH meter
The correlation water index changed before and after table 12 employing B-01 bacillus amyloliquefaciens purified water
| The purification of water quality index | Water body before purifying | Purify the back water body |
| Chemical oxygen demand (COD) (COD) | 11.4mg/L | 5.8mg/L |
| Oxygen capacity (DO) | 1.72mg/L | 6.8mg/L |
| : ammonia nitrogen | 2.7mg/L | 1.83mg/L |
| Nitrite | 0.047mg/L | 0.023mg/L |
| pH | 6.4 | 7.2 |
Annotate: water body muddiness and the purification back water quality that gives an offensive smell are limpid before purifying, free from extraneous odour.
Claims (3)
1. bacillus amyloliquefaciens plant subspecies (Bacillus amyloliquefaciens subsp.plantarum) B-01 bacterial strain, its preserving number is CCTCC:M2012191.
2. the described bacillus amyloliquefaciens plant of claim 1 subspecies B-01 bacterial strain is as the application of water surrounding ecological purificant.
3. the described bacillus amyloliquefaciens plant of claim 1 subspecies B-01 bacterial strain is as the application of animal microorganism fodder additives.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195072A (en) * | 2014-08-06 | 2014-12-10 | 江苏农林职业技术学院 | Bacillus amyloliquefaciens subsp.plantarum B232 and application thereof |
| CN105132323A (en) * | 2015-09-08 | 2015-12-09 | 常州大学 | Salt-tolerance bacillus and application thereof in high-salinity wastewater treatment |
| CN105820980A (en) * | 2016-04-25 | 2016-08-03 | 湖北工业大学 | Bacillus for generating lipase and application of bacillus |
| CN106987536A (en) * | 2017-03-01 | 2017-07-28 | 沃邦环保有限公司 | Complex micro organism fungicide for river regulation and its preparation method and application |
| CN106987535A (en) * | 2017-03-01 | 2017-07-28 | 沃邦环保有限公司 | It is a kind of for complex micro organism fungicide of river regulation and preparation method thereof, river sewage processing method |
| CN107043718A (en) * | 2017-03-01 | 2017-08-15 | 沃邦环保有限公司 | It is a kind of for complex micro organism fungicide of river regulation and its preparation method and application |
| CN108238681A (en) * | 2017-12-22 | 2018-07-03 | 江苏世邦生物工程科技有限公司 | Composite biological agent for low-temperature wastewater treatment and its preparation method and application |
| CN111454861A (en) * | 2020-04-10 | 2020-07-28 | 北京盛合正泰环保工程有限公司 | Bacillus amyloliquefaciens for efficiently purifying sewage, microbial agent and application |
| CN112481153A (en) * | 2020-11-17 | 2021-03-12 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | Rhodoblast sphagnola coupling complex microbial inoculum and application thereof |
| CN116144556A (en) * | 2023-03-23 | 2023-05-23 | 内蒙古农业大学 | Bacillus for treating monosodium glutamate wastewater, treating agent, and preparation method and application thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195072A (en) * | 2014-08-06 | 2014-12-10 | 江苏农林职业技术学院 | Bacillus amyloliquefaciens subsp.plantarum B232 and application thereof |
| CN105132323A (en) * | 2015-09-08 | 2015-12-09 | 常州大学 | Salt-tolerance bacillus and application thereof in high-salinity wastewater treatment |
| CN105132323B (en) * | 2015-09-08 | 2018-10-16 | 常州大学 | One plant of salt tolerant bacillus and its application in high-salt wastewater processing |
| CN105820980B (en) * | 2016-04-25 | 2019-04-05 | 湖北工业大学 | A kind of bacillus of yielding lipase and its application |
| CN105820980A (en) * | 2016-04-25 | 2016-08-03 | 湖北工业大学 | Bacillus for generating lipase and application of bacillus |
| CN106987536A (en) * | 2017-03-01 | 2017-07-28 | 沃邦环保有限公司 | Complex micro organism fungicide for river regulation and its preparation method and application |
| CN106987535A (en) * | 2017-03-01 | 2017-07-28 | 沃邦环保有限公司 | It is a kind of for complex micro organism fungicide of river regulation and preparation method thereof, river sewage processing method |
| CN107043718A (en) * | 2017-03-01 | 2017-08-15 | 沃邦环保有限公司 | It is a kind of for complex micro organism fungicide of river regulation and its preparation method and application |
| CN108238681A (en) * | 2017-12-22 | 2018-07-03 | 江苏世邦生物工程科技有限公司 | Composite biological agent for low-temperature wastewater treatment and its preparation method and application |
| CN111454861A (en) * | 2020-04-10 | 2020-07-28 | 北京盛合正泰环保工程有限公司 | Bacillus amyloliquefaciens for efficiently purifying sewage, microbial agent and application |
| CN112481153A (en) * | 2020-11-17 | 2021-03-12 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | Rhodoblast sphagnola coupling complex microbial inoculum and application thereof |
| CN112481153B (en) * | 2020-11-17 | 2023-11-07 | 新疆晋芳农业科技有限公司 | Rhodoblastus sphagnicola coupled composite microbial agent and application thereof |
| CN116144556A (en) * | 2023-03-23 | 2023-05-23 | 内蒙古农业大学 | Bacillus for treating monosodium glutamate wastewater, treating agent, and preparation method and application thereof |
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