CN112875872B - Application of Bacillus belgii in improvement of phosphorus pollution of water body - Google Patents

Application of Bacillus belgii in improvement of phosphorus pollution of water body Download PDF

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CN112875872B
CN112875872B CN202110018321.4A CN202110018321A CN112875872B CN 112875872 B CN112875872 B CN 112875872B CN 202110018321 A CN202110018321 A CN 202110018321A CN 112875872 B CN112875872 B CN 112875872B
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bacillus belgii
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CN112875872A (en
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王磊
郭燕锋
姜仁旭
黄捷
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Shenzhen Weixin Biotechnology Co ltd
Guangdong Weixin Biotechnology Co ltd
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    • 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
    • C02F2101/105—Phosphorus compounds
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry

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Abstract

The invention relates to an application of Bacillus belgii in improving phosphorus pollution of water, wherein the Bacillus belgii is Bacillus velezensis H18 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020557. Through the identification of phosphorus accumulation activity and the detection of phosphorus accumulation capacity, the Bacillus belgii is applied to the improvement of phosphorus pollution of a water body, the phosphorus removal rate of a water body sample is as high as 92.6%, the phosphorus removal speed is high, and the phosphorus removal maintenance time is long.

Description

Application of Bacillus belgii in improvement of phosphorus pollution of water body
Technical Field
The invention relates to the technical field of sewage treatment, in particular to application of bacillus beilesensis in improving phosphorus pollution in a water body.
Background
Currently, aquaculture, especially high-density intensive freshwater aquaculture, faces the trouble of water Eutrophication, and water Eutrophication (Eutrophication) affects all aquatic ecosystems, which is the most common global problem in recent decades. The water eutrophication refers to the phenomenon of water pollution caused by excessive content of nutrient salts such as N, P in the water. The method is characterized in that the excessive input of nutrient salt leads to the overgrowth of single species, so that the species distribution of the aquatic ecosystem is unbalanced, the flow of substances and energy of the system is damaged, and the whole aquatic ecosystem gradually goes to death.
At present, the physical and chemical method is a main way for reducing the ecological environmental pollution of water bodies. In the aspect of water body phosphorus removal, the traditional phosphorus removal modes comprise a physical adsorption method, a chemical precipitation method, an electrolyte method, an electrodialysis method, an artificial wetland method and the like, and the methods have high cost and various harmful effects, can not achieve the purpose of radical treatment and are easy to cause secondary pollution to the water body.
The biological phosphorus removal mainly comprises algae phosphorus removal, microorganism phosphorus removal and aquatic plant phosphorus removal. Compared with the traditional phosphorus removal method, the biological phosphorus removal method achieves the purpose of phosphorus removal by utilizing the absorption and adsorption effects of organisms on phosphorus in the growth and metabolism process, has low cost, high automation level and outstanding effect, and becomes a research hotspot. Microbial phosphorus removal technology based on microbial diversity is one of the important emerging technologies for water body restoration. The phosphorus accumulating bacteria are microorganisms capable of excessively absorbing phosphorus and storing phosphorus, and comprise acinetobacter, aeromonas, corynebacterium, microfilaria and the like, and are often used for biological phosphorus removal.
Mangrove is an ecological system which is not completely developed, contains a wide variety of microorganisms and has multiple functions, and is a treasure house for discovering and mining functional microorganisms of industry, agriculture, medicine and the like.
Disclosure of Invention
Based on the above, the invention aims to provide the application of bacillus beilesensis in improving the phosphorus pollution of the water body, the strain can absorb copper in the water body and gather in a body of the strain under an aerobic condition, and the phosphorus absorption capacity of the strain is durable, so that the content of phosphorus in the water body can be effectively reduced, and the problem of serious phosphorus pollution of the water body is solved.
The technical scheme adopted by the invention is as follows:
the application of Bacillus belgii in improving phosphorus pollution of water is disclosed, wherein the Bacillus belgii is Bacillus velezensis H18 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020557.
The application of the Bacillus belgii in improving the phosphorus pollution of the water body has the advantages that the phosphorus removal rate of the water body sample is as high as 92.6%, the phosphorus removal speed is high, and the phosphorus removal maintenance time is long.
Further, the method for culturing the bacillus belgii comprises the following steps:
s1 inoculation: the Bacillus velezensis H18 of Bacillus belgii is 1.0 × 105～5.0×108Inoculating the strain in a culture medium at an inoculation concentration of 1: 25-1: 100 in a volume ratio of a bacterial liquid to the culture medium;
s2 culture: and (2) oscillating the culture medium inoculated with the Bacillus velezensis H18 at the speed of 180-220 rpm at the temperature of 25-37 ℃. Culturing for 16-36 hours at constant temperature.
Further, the culture medium is a DTE liquid culture medium, and the DTE liquid culture medium comprises the following raw materials in parts by weight: 5 parts of tryptone, 5 parts of yeast extract powder, 15 parts of brown sugar, 10 parts of sodium chloride and 1000 parts of sterile water.
Further, the inoculation concentration in step S1 was 2.5X 106one/mL.
Further, the inoculation amount of the bacterial liquid to the culture medium in the step S1 is 1:50 in volume ratio.
Further, the culture temperature in step S2 was 30 ℃.
Further, the incubation period in step S2 is 24 hours.
For a better understanding and practice, the present invention is described in detail below with reference to the following examples.
Detailed Description
The Bacillus beiLeisi strain adopted by the invention is named as Bacillus beiLeisi H18, and is preserved in China center for type culture Collection (address: China center for type culture Collection in Wuhan university, Wuhan City, Hubei, China) in 9-29 th 2020 with the preservation number of CCTCC NO: M2020557.
The following examples are provided to facilitate understanding of the present invention, but are not intended to limit the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The first embodiment is as follows: preparation of Bacillus beilesiensis fermentation broth
The Bacillus belgii uses DTE liquid culture medium, and the process is as follows:
activation before culture: glycerol seed solution of Bacillus velezensis (Bacillus velezensis H18, CCTCC NO: M2020557, provided by southern China agricultural university) stored at-20 ℃ is mixed with 1: inoculating the strain with the inoculation amount of 100 volume ratio into a DTE liquid culture medium, and placing the DTE liquid culture medium in a constant temperature shaking table to perform constant temperature shaking culture at 37 ℃ for 28h to obtain activated bacterial liquid.
S1 inoculation: taking activated bacterial liquid according to the proportion of 1: inoculating 100 volumes of bacterial liquid and culture medium into 500mL of DTE liquid culture medium;
s2 culture: and (3) placing the culture bottle inoculated with the Bacillus velezensis H18 in a constant temperature shaking table at 37 ℃ and constant temperature shaking culture at the speed of 200rpm for 20 hours to obtain the fermentation liquor of the strain.
The DTE liquid culture medium comprises the following raw materials in parts by weight: 5 parts of tryptone, 5 parts of yeast extract powder, 15 parts of brown sugar, 10 parts of sodium chloride and 1000 parts of sterile water. The manufacturing method comprises the following steps: accurately adding the components, diluting to desired volume, packaging into conical flask, sterilizing with high pressure steam at 121 deg.C and 101KPa for 20min in a sterilizing pan, cooling to below 70 deg.C, recovering pressure to 0KPa, and storing.
Example two: identification of phosphorus accumulation activity of bacillus belgii thallus
S1: the bacterial density of the fermentation broth of Bacillus belgii prepared in example one and prepared in the invention is adjusted to OD600＝1.0；
S2: taking OD600Bacterial suspension 100. mu.L (blank CK group with 100. mu.L ddH added thereto)20) Inoculating into sample application holes of a 96-well plate containing 900 mu L of PAM-TBO culture medium, and uniformly mixing, wherein each strain is divided into three parallel groups;
the PAM-TBO culture medium comprises the following raw materials in parts by weight: 4 parts of sodium citrate, 0.5 part of NaCl0, (NH)4)2SO42.5 parts of CaCl20.25 part of MgSO 240.05 part of Na2HPO412.8 parts of KH2PO43 parts of malt0.01 part of sugar, 0.025 part of toluidine blue-O and 1000 parts of distilled water. The manufacturing method comprises the following steps: accurately adding the components, diluting to desired volume, packaging into conical flask, sterilizing with high pressure steam at 121 deg.C and 101KPa for 20min, cooling to below 70 deg.C, recovering pressure to 0KPa, and collecting CaCl2，MgSO4Maltose is added separately to prepare mother liquor, and the mother liquor is filtered by a 0.22 mu m sterile filter and added in proportion, and is stored for later use.
S3: standing and culturing at 28 deg.C for 48 hr in biochemical incubator;
s4: the 96-well plate was centrifuged while determining OD by a microplate reader625And calculating the phosphorus accumulation rate and determining the phosphorus accumulation capacity of the strain. The phosphorus accumulation rate calculation formula is as follows: phosphorus accumulation rate (blank control OD)625Experimental group OD625) Idle control OD625X 100%. If the bacterial strain has phosphorus-accumulating capacity, polyphosphate particles can be formed in the bacterial strain, toluidine blue in the culture solution can enter thalli to react with the thalli to be colored, so that the toluidine blue content in the culture solution is reduced, the color of the culture solution is lightened, the color fading effect is better, and the higher the phosphorus-accumulating capacity of the bacterial strain is. The results are shown in Table 1. Under the condition of standing at a constant temperature of 28 ℃, the phosphorus accumulation rate measured in a micropore plate for 48 hours reaches 83.2 percent.
TABLE 1 evaluation results of phosphorus-accumulating activity of Bacillus belgii
Figure BDA0002887474350000041
Example three: detection of phosphorus accumulation capacity of bacillus belgii
S1: taking the fermentation liquor of the Bacillus belgii prepared in the first embodiment of the invention, and centrifuging at 12000r/min for 10min to obtain thalli;
s2: washing the thalli obtained in the step S1 with sterile water, centrifuging at 12000r/min for 5min, and repeating the step for three times to obtain thalli for removing phosphorus and phosphorus from sewage;
s3: and (4) adding the thalli obtained in the step (S2) into the sewage of the urban lake according to the proportion of 0.1% (m/v) to perform anaerobic-aerobic process treatment.
S4 the concrete operation of the anaerobic-aerobic process: adding the bacteria into a sealed glass container containing 40L of sewage sample at a ratio of 0.1% (m/v), uniformly stirring, introducing nitrogen into the container for 6min, preserving the sealed state of the container, opening the stirring function of the container, and keeping the rotating speed at 30r/min for treatment for 12h, namely anaerobic treatment. Then the container is opened, the container is sealed by 400 meshes of gauze, and the stirring speed is increased to 60r/min for treatment for 36h, namely aerobic treatment. After aerobic treatment, the sewage sample is kept still for 150 hours. Taking water samples at different processing time points, centrifuging the water samples (12000r/min, 10min) to take supernatant, measuring the concentration of TP (total phosphorus) in the water samples at the moment, and calculating the removal rate of the TP.
S5 sewage sample source and pretreatment: the sewage sample in the step S4 is collected from Yanghu in university of south China agriculture in Guangzhou city, the water quality indexes are pH 6.4 and TP 4.68mg/L, which are far more than the phosphorus content standard of water body by 0.3mg/ml, and simultaneously, the sludge at the bottom of the water body is collected; and placing the collected water sample in a glass container, and filling sludge with the height of 10cm at the bottom of a glass cylinder to build a water body environment.
S6: and the TP determination method adopts a molybdenum-antimony anti-spectrophotometer method to determine the content of the phosphorus TP in the water body.
a. Sample pretreatment: putting 25ml of water sample into a 50ml centrifuge tube, adding 1ml of potassium persulfate, uniformly mixing, sealing, putting into a high-pressure steam sterilization pot, heating (101KPa, 30min) for digestion, taking out when the temperature of the sterilization pot is reduced to 60 ℃ after heating is finished, and cooling at room temperature.
b. Color development: adding 5ml of digested water sample into a 50ml colorimetric tube, adding deionized water to 50ml, adding 1ml of 10% ascorbic acid solution into the colorimetric tube, mixing uniformly for 20s, adding 2ml of molybdate solution, mixing uniformly, and standing at room temperature for 15 min.
c. Measurement: adding 3ml of developed solution into a colorimetric cylinder, and measuring the absorbance (OD)700) Then, the phosphorus content is calculated according to an ammonium molybdate standard curve.
d. Standard curve of ammonium molybdate: taking several 50ml colorimetric tubes, adding phosphate standard solution 0, 0.5ml, 1ml, 3ml and 5ml respectively10ml and 15ml, adding water to 50 ml; adding 1ml of 10% ascorbic acid solution into the colorimetric tube, mixing for 20s, adding 2ml of molybdate solution, mixing, and standing at room temperature for 15 min; adding 3ml of developed solution into a colorimetric cylinder, and measuring the absorbance (OD)700)。
e. Phosphate standard use solution: drying monopotassium phosphate at 110 ℃ for 2h, cooling, weighing 0.2197g, dissolving in water, adding 5ml of hydrochloric acid solution (1+1), and fixing the volume to 1L to obtain a phosphate standard solution; then 10ml of phosphate standard solution is diluted by water to be constant volume to 250ml, and the phosphate standard use solution is obtained.
f. Phosphate calculation formula:
phosphate (P, mg/L) ═ m/v
In the formula: m is the phosphorus content, ug, found from the standard curve; v-volume of water sample, ml
As can be seen from the results in Table 2, when the anaerobic treatment was carried out for 12 hours, the Total Phosphorus (TP) concentration in the wastewater sample continued to increase and reached a high concentration of 5.56mg/L at 12 hours, indicating that the Bacillus belgii of the present invention was in the process of phosphorus release at this stage; after the anaerobic treatment is changed into the aerobic treatment, the phosphorus concentration in the sewage sample is linearly reduced, and when the sewage is treated for 48 hours, the total phosphorus concentration in the sewage is only 0.27mg/L, and the phosphorus removal rate is up to 94.23 percent, which indicates that the Bacillus belgii is in a phosphorus accumulation state at the treatment stage, has good phosphorus removal effect and high phosphorus removal speed; after the aerobic treatment is finished, the phosphorus concentration is still maintained at a very low level, which shows that the Bacillus belief phosphate removing effect has a certain long-term property and great sewage treatment potential;
TABLE 2 dephosphorization Effect of Bacillus belgii Bacillus velezensis H18
Figure BDA0002887474350000061
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims (5)

1. The application of Bacillus belgii in improving phosphorus pollution of water is characterized in that: the Bacillus belgii is Bacillus velezensis H18 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020557; the method for culturing the Bacillus belgii comprises the following steps:
s1 inoculation: the Bacillus velezensis H18 of Bacillus belgii is 1.0 × 105～5.0×108Inoculating the bacterial solution with the inoculation concentration of 1: 25-1: 100 and the volume ratio of the bacterial solution to the culture medium into the culture medium;
s2 culture: culturing a culture medium inoculated with the Bacillus velezensis H18 at 25-37 ℃ and under the speed of 180-220 rpm for 16-36 hours at constant temperature;
the culture medium is a DTE liquid culture medium, and the DTE liquid culture medium comprises the following raw materials in parts by weight: 5 parts of tryptone, 5 parts of yeast extract powder, 15 parts of brown sugar, 10 parts of sodium chloride and 1000 parts of sterile water.
2. The use of bacillus belgii according to claim 1 for ameliorating phosphorus pollution in a water body, wherein: the inoculation concentration in step S1 was 2.5X 106one/mL.
3. The use of bacillus belgii according to claim 1 for ameliorating phosphorus pollution in a water body, wherein: the volume ratio inoculation amount of the bacterial liquid and the culture medium in the step S1 is 1: 50.
4. The use of bacillus belgii according to claim 1 for ameliorating phosphorus pollution in a water body, wherein: the culture temperature in step S2 was 30 ℃.
5. The use of bacillus belgii according to claim 1 for ameliorating phosphorus pollution in a water body, wherein: the incubation period in step S2 was 24 hours.
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