CN114621884B - Bacillus subtilis and application thereof in water quality purification - Google Patents
Bacillus subtilis and application thereof in water quality purification Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of functional microorganism screening and application, in particular to a novel bacillus subtilis (Bacillus subtilis) and application thereof in water quality purification. The bacillus subtilis sieve is selected from the waste water of the shrimp culture pond of Guangdong river, and the preservation number of the bacillus subtilis sieve is CCCTCC NO: m2020356. The strain can efficiently remove ammoniacal nitrogen and nitrite nitrogen in the culture water body, realizes water quality purification, has high safety, and can be widely applied to the field of freshwater and mariculture.
Description
Technical Field
The invention relates to the technical field of functional microorganism screening, in particular to bacillus subtilis and application thereof in water quality purification.
Background
The aquaculture industry in China is vigorously developed, and becomes a support of agricultural economy. However, when aquaculture rapidly develops, the ageing and degradation of the cultivated species, the high-density cultivation of the pond, the random discharge of untreated wastewater and the ageing of the pond are carried out, and especially in the existing cultivation mode mainly based on feeding, the rich nutrient factors such as residual bait, feces, nitrogen, phosphorus and the like are discharged into the water body, so that the water area environment is deteriorated, the fish and shrimp diseases frequently occur, and the method has become a bottleneck affecting the aquaculture in China.
The microecological preparation is a preparation containing a large amount of beneficial bacteria, which is formed by culturing and amplifying microorganisms extracted and separated from natural environment, and the strictly defined microecological preparation can be classified into 3 types of Probiotics (Probiotics), chemical Probiotics (Prebiotics) and synbiotics (Synbiotics). The two most important species in current research are probiotics (probiotics) and chemical probiotics (Prebiotics). Probiotics (Probiotics) are also called Probiotics, probiotic bacteria and live bacteria preparation.
The catalogue of agricultural rural feed additives breeds, 2013, publication No. 2045, approved 30 species of microbial species that can be used on aquaculture animals including photosynthetic bacteria, bacillus, lactobacillus, bifidobacteria, and yeast. At present, the application in the aquaculture industry mainly comprises feed additives, water quality improvers and the like.
The photosynthetic bacteria contains rich nutrition, the protein content is more than 60%, and meanwhile, the photosynthetic bacteria also contains nutrient substances such as coenzyme Q, vitamin B, folic acid and the like, so that the growth of aquatic animals can be promoted, and the immunity of the aquatic animals can be improved. Jiang Song and the like, by adding photosynthetic bacteria into the holothuria nobilis selenka seedling cultivation water body, not only are the seedling quality, the seedling survival rate and the digestive enzyme activity remarkably improved, but also the water quality of the seedling cultivation water body is improved.
The bacillus has simple nutrition requirement, high metabolism speed, easy separation, culture and preservation and no harsh requirement on industrial production technical conditions. Shenqian and the like are added into the feed to study the influence of the bacillus subtilis on the growth, the digestive enzyme activity and the fish body composition of the black carp, and the result shows that the addition of the bacillus subtilis into the black carp feed can obviously improve the weight gain ratio and the intestinal protease activity of the black carp and obviously reduce the bait coefficient. The shrimp pond of the spilled bacillus subtilis and the un-spilled bacillus subtilis are compared immediately, and the spilled bacillus subtilis can obviously reduce the COD, nitrite nitrogen and hydrogen sulfide concentration of the shrimp pond and improve the total alkalinity, so that the bacillus subtilis has the effect of purifying water quality.
Lactobacillus is an important class of antibacterial compounds that can produce lactic acid, H2O2. The lactobacillus acts on intestinal tracts, can regulate the microbial distribution balance, strengthen the immunity and the resistance of organisms and promote the growth and the development of the intestinal tracts. Liu Wenshu in the research of the anti-infection risk mechanism of lactobacillus, the lactobacillus adhered to the feed can change the structure of the adhered flora of the intestinal tract of tilapia, and the high dosage level has remarkable protection effect on the toxicity of aeromonas hydrophila NJ-1.
Bifidobacteria are key microorganisms in the intestinal tracts of humans and animals, are also well known as intestinal probiotics, and have the effects of regulating intestinal flora, reducing cholesterol, preventing intestinal lesions, delaying aging and the like. Gui Yuanming and the like show that the bifidobacterium preparation has good effects in the aspects of treating the carp fulminant hepatitis and adjusting intestinal flora and has positive significance in the aspect of promoting growth in the preliminary research report on the effect of treating the carp fulminant hepatitis.
Yeast has good effects in purifying water quality, inhibiting water bloom, promoting organism growth, and enhancing organism immunity. She Qiu and the like find that in the research on the inhibition effect of different additives on the water bloom algae, the effective microbiota (EM group) can destroy the chlorophyll structure of algae cells through extracellular secretion, thereby realizing the inhibition of the algae and preventing the occurrence of the water bloom.
The microecological preparation has no toxic or side effect, no pollution, can improve the water quality, can improve the immunity of aquatic animals and promote the growth of the aquatic animals, plays an increasingly important role in the current advocating the green and scientific cultivation concept, and still needs to continuously develop the specific and efficient microecological preparation to enhance the action effect.
Disclosure of Invention
The invention aims to provide a novel bacillus subtilis (Bacillus subtilis) and provides application thereof in water quality purification. The bacillus subtilis can efficiently remove ammoniacal nitrogen and nitrite nitrogen in the culture water body, realizes water body purification, and has wide application prospect.
In one aspect, the present invention provides a bacillus subtilis, named bacillus subtilis VSB091 (Bacillus subtilis VSB 091), which has been preserved in the China center for type culture collection of university of armed Han and Wuhan in China, 7 months and 27 days in 2020, and has a preservation number of cctccc NO: m2020355.
In one aspect, the invention provides application of the bacillus subtilis in water quality purification.
The invention also provides a microbial preparation comprising the bacillus subtilis VSB091.
The microbial preparation also comprises any one or a combination of two or more of bacillus, lactobacillus, photosynthetic bacteria, clostridium butyricum, halomonas, bacillus and pseudoalteromonas.
The bacillus is preferably bacillus licheniformis, bacillus pumilus, bacillus megaterium, bacillus coagulans, bacillus laterosporus, bacillus methylotrophicus or bacillus siamensis.
The lactobacillus is preferably enterococcus faecium, lactobacillus plantarum, lactobacillus reuteri or pediococcus pentosaceus.
The viable bacteria amount of the bacillus subtilis VSB091 in the microbial preparation is at least 10 8 CFU/g.
The invention also provides application of the microbial preparation in water quality purification.
The bacillus subtilis VSB091 obtained by screening of the invention has good denitrification performance under different salinity, especially under the condition of 30 per mill salinity, the removal rate of ammoniacal nitrogen and nitrite nitrogen reaches 100% and 99.87%, and unexpected effects are obtained. The strain also has higher protease and cellulase production capacity, and the diameter of hydrolysis circles is 150mm and 192mm respectively; the protease activity in the fermentation supernatant is up to 120U/ml, and the cellulase activity is up to 58.5U/ml, so that the strain is beneficial to effectively decomposing and utilizing residual baits, plant protein sources and plant fibers in the culture water body.
In addition, the bacillus subtilis VSB091 has broad-spectrum antibacterial capability and has a certain inhibition effect on vibrio harveyi, vibrio parahaemolyticus, vibrio alginolyticus, aeromonas hydrophila and flavobacterium columnificus; the plant has certain tolerance to low temperature, can grow normally at 15 ℃, and accords with the proper growth temperature range of main cold water-borne aquatic economic species such as sea cucumbers, rainbow trout and the like; the safety is good, the aquatic product is free from hemolysis, is sensitive to common antibiotics for aquaculture, and has no drug resistance, so that the aquatic product can be widely applied to the fields of fresh water and mariculture.
Drawings
FIG. 1 is a colony chart of a VSB091 strain;
FIG. 2 is a mass spectrum peak diagram of VSB091 strain protein;
FIG. 3 is a gene fingerprint of VSB091 strain;
FIG. 4 is a screening chart of extracellular enzyme producing plates of the VSB091 strain.
Detailed Description
The invention is further illustrated below in connection with specific examples. With respect to the specific methods or materials used in the embodiments, those skilled in the art may perform conventional alternatives based on the technical idea of the present invention and are not limited to the specific descriptions of the embodiments of the present invention.
The equipment and reagents selected for use in the present invention may be selected from any of those commercially available. The formula of the culture medium related to the invention is as follows:
①NH3 Enrichment medium: the glucose 0.5g,CH3COONa 0.5g,(NH4)2SO4 0.1g,K2HPO4.·H2O 1.2g,MgSO4·7H2O 0.5g is sterilized by passing through a 0.45um microporous membrane to obtain seawater with constant volume of 1L, pH7.5, and sterilizing with high pressure steam at 121deg.C for 20min. Wherein (NH 4)2SO4, adding (NH 4)2SO4 solution after filtering with 0.22um microporous membrane to sterilize) into the sterilized culture medium (N20 mg/L, C/N10)
②NO2 The enrichment medium ：CH3COONa 0.5g,NaNO2 0.1g,K2HPO4·H2O 1.2g,Fe3PO4·H2O 0.01g,MgSO4·7H2O 0.5g is sterilized by passing through a 0.45um microporous membrane to obtain seawater with constant volume of 1L, pH of 7.5, and sterilizing with high pressure steam at 121deg.C for 20min. (N20 mg/L, C/N10)
③NH4 + -N primary screening medium: the glucose 0.25g,(NH4)2SO4 0.05g,K2HPO4·3H2O 1.2g,MgSO4·7H2O 0.5g, is filtered by a microporous membrane with 0.45um to remove bacteria, the seawater is fixed to 1L, the pH is 7.5, and the sterilization is carried out by high-pressure steam at 121 ℃ for 20min. Wherein (NH 4)2SO4, after the solution of (NH 4)2SO4) was filtered through a 0.22um microporous filter membrane and sterilized, the solution was added to the sterilized medium, and 2% agar was added to the selection medium (N10 mg/L, C/N10).
④NO2 The N primary screening culture medium ：CH3COONa 0.25g,NaNO2 0.05g,K2HPO4·3H2O 1.2g,Fe3PO4·H2O 0.01g,MgSO4·7H2O 0.5g, is subjected to filtration sterilization by a microporous membrane with 0.45um to obtain seawater with constant volume of 1L, pH of 7.5, and high pressure steam sterilization at 121 ℃ for 20min. Screening medium was supplemented with 2% agar. (N10 mg/L, C/N10).
⑤NH4 + -N re-screening medium: glucose 0.05g,(NH4)2SO4 0.01g,K2HPO4·3H2O 1g,KH2PO40.3g,MgSO4·7H2O 0.25g,FeSO4·7H2O 0.05g,MnSO4·4H2O 0.01g,NaCl 30g. was fixed to a volume of 1L, pH 8.0, and autoclaved at 121℃for 20min. Wherein (NH 4)2SO4 is not added before sterilization, and (NH 4)2SO4 solution is added into the sterilized culture medium after filtration sterilization by a 0.22um microporous filter membrane.
⑥NO2 -N re-screening medium: glucose 0.125g,NaNO2 0.025g,K2HPO4·3H2O 1g,KH2PO40.3g,MgSO4·7H2O 0.25g,FeSO4·7H2O 0.05g,MnSO4·4H2O 0.01g,NaCl 30g. was fixed to a volume of 1L, pH 8.0, and autoclaved at 121℃for 20min.
⑦ Composite nitrogen source culture medium: glucose 0.1g,(NH4)2SO4 0.01g,NaNO2 0.01g,K2HPO4·3H2O 1g,KH2PO40.3g,MgSO4·7H2O 0.25g,FeSO4·7H2O 0.05g,MnSO4·4H2O 0.01g,NaCl 30g. was fixed to a volume of 1L, pH 8.0, and autoclaved at 121℃for 20min. Wherein (NH 4)2SO4 is not added before sterilization, and (NH 4)2SO4 solution is added into the sterilized culture medium after filtration sterilization by a 0.22um microporous filter membrane.
⑧ 2216E medium: purchased from Qingdao sea Bo biotechnology Co., ltd, sterilized at 121℃for 15min.
Example 1 isolation, screening and identification of strains
1.1 Isolation and screening of strains
10ML of culture wastewater (collected from a Guangdong river shrimp culture pond) is collected into a 250mL conical flask containing 100mL of enrichment medium, and enrichment culture is carried out for three days at 28 ℃ and 150 r/min. Wherein half of the filter sterilized enrichment medium was changed daily.
The enrichment solution is subjected to gradient dilution, 100ul of three gradient dilutions of 10 -4、10-5、10-6 are respectively coated on NH 4 + -N and NO 2 -N primary screening media, and the culture is carried out at the constant temperature of 28 ℃ for 24-48 hours until colonies grow out. And (3) obtaining inconsistent bacterial colonies according to the size, the color and the morphology, continuing to separate and purify on a primary screening culture medium until single bacteria are obtained, numbering the bacterial strains, and streaking and purifying on a 2216E culture medium to perform liquid glycerol seed conservation.
The single colony obtained by primary screening is picked and inoculated into 800ul of NH 4 + -N and NO 2 -N secondary screening culture mediums respectively, the corresponding culture mediums without inoculating the strain are used as blank control, the culture is carried out for 48 hours at 28 ℃ in a shaking table at 150rpm, and then the centrifugation is carried out for 5 minutes at 4500rpm, and the supernatant is taken. The method described by national standard GB 17378.4-2007 is referred to for measuring the content of ammoniacal nitrogen and nitrite nitrogen in the supernatant respectively, and the removal rate of the bacterial strain to the ammoniacal nitrogen and nitrite nitrogen is calculated according to the following formula,
Ammonia nitrogen removal (%) = (X1-X2)/X1.
Nitrite nitrogen removal (%) = (Y1-Y2)/Y1.
X1 is the ammonia nitrogen content in the blank control after 48 hours of culture, and X2 is the ammonia nitrogen content in the test group culture medium after 48 hours of culture; y1 is the nitrite nitrogen content in the blank control after 48h of culture, and Y2 is the nitrite nitrogen content in the test group medium after 48h of culture.
The applicant obtains a strain with excellent denitrification performance through preliminary screening and secondary screening, which is named as VSB091, and the removal rate of ammonia nitrogen and nitrite nitrogen reaches 100 percent.
1.2 Identification of strains
1. Colony morphology identification
The VSB091 strain was inoculated on nutrient agar medium, and after culturing at 37℃for 24 hours, the colony morphology was observed. As shown in FIG. 1, the colony of the VSB091 strain has a diameter of about 0.4-1mm, and is round, yellowish, opaque, slightly convex in the middle, rough in the surface, irregular in the edge, and free from mucus.
2. 16S rRNA molecular identification
Genomic DNA of strain VSB091 was extracted using the kit. The 16S rRNA was then amplified using the genomic DNA as a template with specific primers 27F (5'-AGAGTTTGATCATGGCTCAG-3') and 1492R (5'-TAGGGTTACCTTACGACTT-3'). The PCR system comprises: 0.7. Mu.l 27F, 0.7. Mu.l 14992R, 4. Mu.l template DNA, 17.5. Mu. l SuperMiX and 12.1. Mu.l water. The PCR reaction conditions were set as follows: (1) 94 ℃ for 5min; (2) pre-denaturation at 94℃for 30s; (3) 55 ℃ for 30s; (4) 72 ℃ for 1min; performing the loop of steps (2) to (4) 35; (5) at 72℃for 10min. The amplified PCR product is subjected to 1% agarose gel electrophoresis detection, and the result shows that the size of the PCR product is about 1500bp, thereby meeting the requirements. The nucleotide sequence of the PCR product is SEQ ID NO. 1 after sequencing. The sequence was subjected to BLAST alignment in NCBI database, which showed the highest similarity to Bacillus subtilis (Bacillus subtilis). Thus, the VSB091 strain was preliminarily determined to be Bacillus subtilis (Bacillus subtilis).
3. MALDI-TOF-MS protein mass spectrum identification
A small amount of VSB091 single colony is coated on a target plate in a film form; adding 1 mu L of lysate in the mass spectrum sample pretreatment kit, and naturally airing at room temperature; adding 1 mu L of matrix solution in the mass spectrum sample pretreatment kit to cover the sample, and naturally airing at room temperature; and (5) placing the sample target into a mass spectrometer for identification. The identification result shows that the VSB091 strain is bacillus subtilis (Bacillus subtilis), and the protein mass spectrum peak diagram is shown in figure 2.
4. RiboPrinter full-automatic microorganism gene fingerprint identification
The strain VSB091 was identified on-line according to the full-automatic microorganism gene fingerprint identification system operation instructions, and rRNA gene fingerprint patterns thereof were obtained as shown in FIG. 3. By comparison with the fingerprint patterns of known standard strain libraries, the similarity of the strain VSB091 and bacillus subtilis is up to more than 90%, so that the strain is identified as bacillus subtilis (Bacillus subtilis).
The bacterial strain VSB091 is identified by utilizing three molecular biological means of 16S rRNA sequencing, MALDI-TOF-MS protein mass spectrum and RiboPrinter full-automatic microorganism gene fingerprint identification system, the identification result is consistent, the applicant determines that the bacterial strain is bacillus subtilis (Bacillus Subtilis), is named bacillus subtilis VSB091 (Bacillus subtilis VSB 091), and is preserved in China center for type culture collection of university of Wuhan in China, with the preservation number of CCTCC M2020355, 7 months and 27 days in 2020.
EXAMPLE 2 evaluation of safety of Bacillus subtilis VSB091
1. Hemolysis:
The activated bacillus subtilis VSB091 was inoculated on a blood plate, and after incubation at 28℃for 24 hours, it was observed whether a transparent hydrolytic ring was produced around the colony. The results showed no hydrolytic circle, indicating that bacillus subtilis VSB091 does not possess hemolysis and is applicable to aquaculture.
2. Drug resistance:
The drug resistance of the strain may cause potential safety hazards in the production and application process. To prevent the bacillus subtilis VSB091 from having drug resistance, its antibiotic susceptibility was studied. The minimum inhibitory concentration (MIC value) of bacillus subtilis VSB091 among 9 common antibiotics, which are Sigma standards, was determined according to CLSI antibiotic susceptibility test gradient dilution method, and the susceptibility of VSB091 to antibiotics was evaluated with reference to EFSA (2012) standard. See table 1 for specific results.
Table 19 minimum inhibitory concentration (MIC value) of antibiotics against Bacillus subtilis VSB091
Note that: unit ug/ml, S represents sensitivity according to the strain sensitivity to antibiotics evaluation standard EFSA (2012); r represents drug resistance.
From the results in Table 1, it is clear that Bacillus subtilis VSB091 is sensitive to all 9 antibiotics, has no drug resistance, and can be used in aquaculture.
3. Animal test:
To further verify the safety of bacillus subtilis VSB091, it was evaluated whether it was safe for farmed animals, animal tests were performed in the aquaculture circulatory system, and the test animal was penaeus vannamei.
The dipping method is adopted. Before the test, the penaeus vannamei boone with similar specifications is selected and temporarily cultured for 4 days, then 20 penaeus vannamei boone after temporary culture is randomly selected and placed in a 60L glass water tank, the dip dyeing concentration of bacillus subtilis VSB091 in the test group is set to be 10 5 CFU/ml, and the control group is not treated. The experimental and control groups were each set in three replicates for 7d. Observing the living state of the prawns every day during the experimental period, recording the survival condition, and calculating the survival rate; experiments 0d and 7d respectively weigh the total weight of the prawns in each water vat, and calculate the weight gain rate. The experimental results are shown in Table 2.
TABLE 2 influence of Bacillus subtilis VSB091 on growth and survival of Penaeus vannamei Boone
| Survival rate of prawn | Weight gain rate of prawn | |
| Control group | 96.5％±2.4％a | 8.2％±1.2％b |
| Experimental group | 98.3％±2.3％a | 8.4％±0.6％b |
As can be seen from the results of Table 2, compared with the control group, the survival rate and the weight gain rate of the experimental group of prawns fed with the bacillus subtilis VSB091 are both obviously improved, so that the bacillus subtilis VSB098 provided by the invention has good safety, has no influence on the growth and survival of the penaeus vannamei boone, and can also effectively improve the intestinal environment of the penaeus vannamei boone and promote the growth of the penaeus vannamei boone.
EXAMPLE 3 Bacillus subtilis VSB091 enzyme producing Properties
The aquatic culture water body is rich in organic impurities such as residual bait, excrement and the like and plant fibers which are difficult to utilize, and in order to evaluate the capability of bacillus subtilis VSB091 in utilizing the substances, a dibbling method is adopted to evaluate the protease production capability and the cellulose production capability of the bacillus subtilis VSB091, and the fermentation enzyme activity of the bacillus subtilis is measured.
1. Protease activity assay:
Bacillus subtilis VSB091 was inoculated into protease enzyme-producing liquid medium (casein 0.8g, na 2HPO4 0.2g,MgSO4 0.05.05 g, naCl 0.5g, beef extract 0.3g, agar 1.5g, ultrapure water 100mL, pH 7.4) at a ratio of 1%, and cultured at 28℃for 24 hours. The fermentation broth was then centrifuged at 4500rpm for 20min and the supernatant was used for protease activity determination. The enzyme activity is determined by the Fulin method, which is referred to national standard SB/T10317-1999.
(1) Protease activity definition: the enzyme amount required for decomposing bovine serum albumin to produce 1 mu mol tryptophan per minute at 37 ℃ is one enzyme activity unit.
(2) The enzyme activity determination method comprises the following steps: mu.L of Bovine Serum Albumin (BSA) at a concentration of 1% (w/v) was mixed with 450. Mu.L of an enzyme solution, 1.5mL of sodium acetate buffer at a concentration of 0.1mol/L and pH 7.0, and incubated at 37℃for 5min. The reaction was terminated with 0.5mL of 10% strength trichloroacetic acid and the absorbance was measured at 280 nm.
Blank control: the same conditions as above were applied to distilled water instead of the enzyme solution.
The enzyme activity formula: enzyme activity (U/mL) = (k×w) (/ v×t), where K is the dilution of enzyme solution; w is the amount of tryptophan (. Mu. Mol) produced; v is the volume (mL) of the reaction enzyme solution; t is the reaction time (min).
2. Cellulase activity determination:
Bacillus subtilis VSB091 was inoculated into a cellulase liquid medium (sodium carboxymethylcellulose 1g, peptone 1g, yeast extract 0.5g, KH 2PO4 0.1g,MgSO4 0.02.02 g, naCl 1g, glucose 0.2g, agar 1.5g, ultrapure water 100mL. PH 7.2. Developer: congo red (1 mg/ml)) at 1%, and cultured at 28℃and 150rpm for 24 hours. Then, the fermentation broth was centrifuged at 4500rpm for 20 minutes, and the supernatant was collected and the enzyme activity was measured by the 3, 5-dinitrosalicylic acid method (DNS method).
As can be seen from FIG. 4, bacillus subtilis VSB091 has high protease and cellulase production capacities, and the hydrolysis circles thereof have diameters of 150mm and 192mm, respectively; the protease activity in the fermentation supernatant is up to 120U/ml, and the cellulase activity is up to 58.5U/ml, so that the strain is beneficial to effectively decomposing and utilizing residual baits, plant protein sources and plant fibers in the culture water body.
Example 4 bacteriostatic ability of Bacillus subtilis VSB091 against common pathogenic bacteria for aquaculture
Several common pathogenic bacteria for aquaculture are selected: vibrio harveyi (Vibrio harveyi), vibrio parahaemolyticus (Vibrio parahemolyticus), vibrio alginolyticus (Vibrio alginolyticus), aeromonas hydrophila (Aeromonas hydrophila) and Flavobacterium columniform (Flavobacterium columnare) are cultured in corresponding liquid culture medium at 28 ℃ and 150rpm for 16 hours, then the concentration is diluted to 108CFU/mL by using a Mitsubishi turbidimeter, and 100ul of the diluted solution is uniformly coated on a nutrient agar plate. By adopting a punching method, 3 round holes with the diameter of 0.9mm are reserved on each nutrient agar plate, 100ul of bacillus subtilis VSB091 with the same concentration is inoculated into the round holes, the bacillus subtilis is cultured in a biochemical incubator at 37 ℃ for 24 hours, and the bacteriostasis capacity of the bacillus subtilis VSB091 on the pathogenic bacteria is evaluated by measuring the diameter (cm) of a bacteriostasis ring. The results are shown in Table 3.
TABLE 3 diameter of the zone of inhibition (units; cm) of Bacillus subtilis VSB091 against common pathogenic bacteria
| Vibrio harveyi | Vibrio parahaemolyticus | Vibrio alginolyticus | Aeromonas hydrophila | Flavobacterium columnar |
| 1.3 | 1.1 | 1.2 | 1.5 | 1.2 |
As can be seen from the results of Table 3, the Bacillus subtilis VSB091 provided by the invention has a certain inhibition effect on Vibrio harveyi, vibrio parahaemolyticus, vibrio alginolyticus, aeromonas hydrophila and Flavobacterium columnificus. The broad-spectrum antibacterial capability of the strain is beneficial to the wide application of the strain in aquaculture.
EXAMPLE 5 tolerance of Bacillus subtilis VSB091 to Low temperatures
Inoculating Bacillus subtilis VSB091 into nutrient agar plates by dibbling method, culturing in incubator with different temperature gradients (5 deg.C, 10 deg.C, 15 deg.C, 20 deg.C) for 48 hr, and observing growth condition. The result shows that the bacillus subtilis VSB091 has no obvious colony appearance at 5 ℃ and hardly grows; the colony is small and transparent at 10 ℃, and is large and obvious at 15 ℃ and 20 ℃ and can grow normally. Therefore, the bacillus subtilis VSB091 has a certain tolerance to low temperature, accords with the proper growth temperature range of main cold water-borne aquaculture economic species of aquatic products such as sea cucumbers, rainbow trout and the like, and has wide application prospect.
EXAMPLE 6 Bacillus subtilis VSB091 Effect on removal of ammoniacal nitrogen and nitrite nitrogen at different salinity
Bacillus subtilis VSB091 was inoculated into 2216E medium and activated at 28℃and 160rpm for 16 hours. 1ml of the activated bacterial liquid was centrifuged at 4000rpm for 5min. Washing the precipitate with 0.9% physiological saline for three times, inoculating into 50ml of compound nitrogen source culture medium, performing shake culture at 28deg.C and 150rpm for 48 hr without treatment, centrifuging at 4500rpm for 5min to obtain supernatant, and respectively measuring the contents of ammoniacal nitrogen and nitrite nitrogen in the supernatant, wherein the measurement method is described in national standard GB17378.4-2007. The specific results are shown in Table 4.
Ammonia nitrogen removal (%) = (X1-X2)/X1.
Nitrite nitrogen removal (%) = (Y1-Y2)/Y1.
X1 is the ammonia nitrogen content in the blank control after 48 hours of culture, and X2 is the ammonia nitrogen content in the test group culture medium after 48 hours of culture; y1 is the nitrite nitrogen content in the blank control after 48h of culture, and Y2 is the nitrite nitrogen content in the test group medium after 48h of culture.
TABLE 4 removal of ammoniacal nitrogen and nitrite nitrogen by Bacillus subtilis VSB091 at different salinity
| Salinity of | Ammonia nitrogen removal rate | Nitrite nitrogen removal rate |
| 0‰NaCl | 78.52％ | 96.31％ |
| 10‰NaCl | 79.73％ | 96.68％ |
| 20‰NaCl | 78.94％ | 95.82％ |
| 30‰NaCl | 100％ | 99.87％ |
As can be seen from the data in Table 4, the bacillus subtilis VSB091 provided by the invention has good denitrification performance under different salinity, especially under the condition of 30 per mill salinity, the removal rate of ammoniacal nitrogen and nitrite nitrogen reaches 100% and 99.87%, and unexpected effects are obtained. The bacillus subtilis VSB091 can be widely applied to freshwater and mariculture.
In conclusion, the bacillus subtilis VSB091 obtained by separation and screening can efficiently remove ammoniacal nitrogen and nitrite nitrogen in water, the removal rate can reach 100%, and the bacillus subtilis VSB091 has strong tolerance to low temperature and salinity. The strain has no hemolysis, does not generate drug resistance and has good safety, so the strain can be widely applied to freshwater and mariculture.
Sequence listing
<110> Shandong Uygur biotechnology Co., ltd
<120> Bacillus subtilis and application thereof in water purification
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1390
<212> DNA
<213> Bacillus subtilis (Bacillus subtilis)
<400> 1
caccgacttc gggtgttaca aactctcgtg gtgtgacggg cggtgtgtac aaggcccggg 60
aacgtattca ccgcggcatg ctgatccgcg attactagcg attccagctt cacgcagtcg 120
agttgcagac tgcgatccga actgagaaca gatttgtggg attggcttaa cctcgcggtt 180
tcgctgccct ttgttctgtc cattgtagca cgtgtgtagc ccaggtcata aggggcatga 240
tgatttgacg tcatccccac cttcctccgg tttgtcaccg gcagtcacct tagagtgccc 300
aactgaatgc tggcaactaa gatcaagggt tgcgctcgtt gcgggactta acccaacatc 360
tcacgacacg agctgacgac aaccatgcac cacctgtcac tctgcccccg aaggggacgt 420
cctatctcta ggattgtcag aggatgtcaa gacctggtaa ggttcttcgc gttgcttcga 480
attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga gtttcagtct 540
tgcgaccgta ctccccaggc ggagtgctta atgcgttagc tgcagcacta aggggcggaa 600
accccctaac acttagcact catcgtttac ggcgtggact accagggtat ctaatcctgt 660
tcgctcccca cgctttcgct cctcagcgtc agttacagac cagagagtcg ccttcgccac 720
tggtgttcct ccacatctct acgcatttca ccgctacacg tggaattcca cccctcctct 780
tctgcactca agttccccca gtttccaatg accctccccg gttgagccgg gggctttcac 840
atcagactta agaaaccgcc tgcgagccct ttacgcccaa taattccgga caacgcttgc 900
cacctacgta ttaccgcggc tgctggcacg tagttagccg tggctttctg gttaggtacc 960
gtcaaggtac cgccctattc gaacggtact tgttcttccc taacaacaga gctttacgat 1020
ccgaaaacct tcatcactca cgcggcgttg ctccgtcaga ctttcgtcca ttgcggaaga 1080
ttccctactg ctgcctcccg taggagtctg ggccgtgtct cagtcccagt gtggccgatc 1140
accctctcag gtcggctacg catcgttgcc ttggtgagcc gttacctcac caactagcta 1200
atgcgccgcg ggtccatctg taagtggtag ccgaagccac cttttatgtt tgaaccatgc 1260
ggttcaaaca accatccggt attagccccg gtttcccgga gttatcccag tcttacaggc 1320
aggttaccca cgtgttactc acccgtccgc cgctaacatc agggagcaag ctcccatctg 1380
tccgctcgac 1390
Claims (8)
1. The bacillus subtilis is characterized in that the collection number of the bacillus subtilis is CCTCC NO: m2020355.
2. The use of the bacillus subtilis according to claim 1 for purifying water.
3. A microbial preparation comprising the bacillus subtilis of claim 1.
4. The microbial preparation according to claim 3, further comprising any one or a combination of two or more of bacillus, lactobacillus, photosynthetic bacteria, clostridium butyricum, halomonas, bacillus, pseudoalteromonas.
5. The microbial preparation of claim 4, wherein the bacillus is any one or a combination of two or more of bacillus licheniformis, bacillus pumilus, bacillus megaterium, bacillus coagulans, bacillus laterosporus, bacillus methylotrophicus or bacillus siamensis.
6. The microbial preparation according to claim 4 or 5, wherein the lactic acid bacteria are any one or a combination of two or more of enterococcus faecium, lactobacillus plantarum, lactobacillus reuteri and pediococcus pentosaceus.
7. The microbial preparation according to any one of claims 3 to 6, wherein the viable count of bacillus subtilis in the microbial preparation is at least 10 8 CFU/g.
8. Use of the microbial preparation according to any one of claims 3-6 for water purification.
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| CN111690558A (en) * | 2020-05-31 | 2020-09-22 | 青岛玛斯特生物技术有限公司 | Bacillus subtilis strain and application thereof in aquaculture |
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