CN113773982B - Stenotrophomonas strain KT48, algicidal bacteria liquid, preparation method and application thereof - Google Patents
Stenotrophomonas strain KT48, algicidal bacteria liquid, preparation method and application thereof Download PDFInfo
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Abstract
The present disclosure provides an stenotrophomonas strain KT48, algicidal bacteria liquid, and preparation methods and applications thereof, the preservation number of the stenotrophomonas strain KT48 is CGMCC NO.22722, and the algicidal bacteria solution is prepared from the stenotrophomonas strain KT48 with the preservation number of CGMCC NO. 22722; the stenotrophomonas KT48 has the algae removal effect, but not the metabolites of the stenotrophomonas KT48 have the algae removal effect, so that the stenotrophomonas KT is more convenient for practical application; inoculating the stenotrophomonas strain KT48 to a culture medium when preparing the algicidal bacteria solution, and culturing in a constant-temperature shaking box until the temperature is stable to obtain the algicidal bacteria solution, wherein the preparation method is simple; the stenotrophomonas KT48 can break the cell membrane of the microcystis aeruginosa and destroy the cell structure; the algicidal bacteria solution prepared by the stenotrophomonas KT48 can reduce the chlorophyll content of blue algae, effectively control the outbreak of water bloom, has high efficiency, biological specificity and environmental friendliness, and is biodegradable. The algae-dissolving bacteria liquid is used for controlling cyanobacteria bloom, and has the advantages of low cost, simple operation, strong safety and no secondary pollution.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an stenotrophomonas strain KT48, algicidal bacteria liquid, and a preparation method and application thereof.
Background
In recent years, the phenomenon of eutrophication of water body has attracted a great deal of attention worldwide, and the frequency of algal bloom outbreaks is also significantly increased, especially in shallow water type lakes. The use of physical and chemical methods to control the onset of algal bloom in a freshwater system for a short period of time is most common. In practice, however, the endogenous pollution of the water is not reduced, the algal bloom is not effectively controlled for a long time, and the repeated cycle of 'outbreak-treatment-outbreak' is involved. The clay is utilized to induce the water bloom flocculation, which is the only effective algae control measure at present, but algae settled into the water body can be converted into endogenous pollution, and the risk of secondary pollution exists.
Therefore, there is a need for an algae removal method that is highly efficient, specific and eco-friendly.
Disclosure of Invention
In view of the above, the present disclosure aims to provide an stenotrophomonas strain KT48, an algicidal solution, and a preparation method and application thereof.
Based on the above object, the first aspect of the present disclosure provides an stenotrophomonas strain KT48 with a preservation number of CGMCC No.22722.
Based on the same purpose, the second aspect of the disclosure provides an algicidal solution, which is prepared from an stenotrophomonas strain KT48 with a preservation number of CGMCC No.22722.
Based on the same object, a third aspect of the present disclosure provides a preparation method of an algicidal bacteria solution, including the steps of:
taking a stenotrophomonas strain KT48 with the preservation number of CGMCC No.22722 which is frozen and preserved, sterilizing at high temperature, and inoculating the stenotrophomonas strain KT48 into a first culture medium;
placing the inoculated first culture medium into a constant temperature shake incubator with the temperature of 25-40 ℃ and the rotating speed of 130-230 rmp for culturing for 24-72 hours to obtain an activated strain;
transferring the activated strain to a second culture medium after high-temperature sterilization according to the inoculation amount of 1%, and culturing for 24-72 h in a constant-temperature shake incubator with the temperature of 25-40 ℃ and the rotating speed of 130-230 rmp to obtain a stock solution.
Optionally, the method further comprises: centrifuging the original bacterial liquid at the rotation speed of 5000-10000 rmp for 5-15 min, collecting supernatant after centrifugation, and re-suspending the supernatant with a third culture medium to obtain a bacterial suspension.
Optionally, the first culture medium is an LB liquid culture medium or a beef extract peptone liquid culture medium.
Optionally, the second culture medium is prepared by adding glucose or starch into LB liquid culture medium or beef extract peptone liquid culture medium which is diluted by 6-8 times.
Optionally, the addition amount of the glucose or the starch is 0.2-0.5%.
Optionally, the third culture medium is prepared by adding 0.2-0.5% of glucose or starch into LB liquid culture medium or beef extract peptone liquid culture medium which is diluted by 6-8 times.
Based on the same purpose, a fourth aspect of the disclosure provides an application of the algicidal solution in controlling cyanobacterial bloom.
Based on the same purpose, a fifth aspect of the disclosure provides an application of the algicidal solution in controlling cyanobacterial bloom taking microcystis aeruginosa as a dominant species.
From the above, the stenotrophomonas strain KT48, the algicidal bacteria solution, the preparation method and the application thereof provided by the invention have the algae removal effect, and the stenotrophomonas strain KT48 but not the metabolites of the strain have the algae removal effect, so that the invention is more convenient for practical application; inoculating the stenotrophomonas strain KT48 to a culture medium when preparing the algicidal bacteria solution, and culturing in a constant-temperature shaking box until the temperature is stable to obtain the algicidal bacteria solution, wherein the preparation method is simple; the stenotrophomonas KT48 can break the cell membrane of the microcystis aeruginosa and destroy the cell structure; the algicidal bacteria solution prepared by the stenotrophomonas KT48 can reduce the chlorophyll content of blue algae, effectively control the outbreak of water bloom, has high efficiency, biological specificity and environmental friendliness, and is biodegradable. The algae-dissolving bacteria liquid is used for controlling cyanobacteria bloom, has low cost, simple operation, strong safety and no secondary pollution, and is beneficial to field application.
Drawings
FIG. 1a is a bacterial community morphology diagram provided by an embodiment of the present disclosure;
FIG. 1b is a gram stain provided by an embodiment of the present disclosure;
FIG. 2 is a comparison of yellowing after addition of bacterial suspension with no addition of algicidal bacterial liquid provided by embodiments of the present disclosure;
FIG. 3a is a scanning electron microscope image at day 2 of bacterial suspension addition provided by an embodiment of the present disclosure;
FIG. 3b is a scanning electron microscope image at day 14 of bacterial suspension addition provided by an embodiment of the present disclosure;
fig. 4 is a graph of algicidal effect provided by embodiments of the present disclosure.
Detailed Description
For the purposes of promoting an understanding of the principles and advantages of the disclosure, reference will now be made in detail to the following specific examples.
It should be noted that unless otherwise defined, technical or scientific terms used in the embodiments of the present invention should be given the ordinary meaning as understood by one of ordinary skill in the art to which the present disclosure pertains.
In recent years, the phenomenon of eutrophication of water body has attracted a great deal of attention worldwide, and the frequency of algal bloom outbreaks is also significantly increased, especially in shallow water type lakes. This will directly lead to local ecological environmental deterioration, seriously threaten the safety of drinking water near the water body, interfere with the normal operation of aquaculture, and hinder the development of peripheral economy to a certain extent. Part of blue algae (such as microcystis, tremella and the like) can also release algae toxins, and long-term exposure to air of toxic algae bloom can form a potential threat to human health.
With the development of socioeconomic performance, the emission of a large amount of greenhouse gases promotes global warming, thereby further exacerbating the difficulty in controlling eutrophication of water bodies and outbreaks of algal bloom. Temperature is the primary driving force for algae growth, followed by Total Phosphorus (TP) content. The phosphorus element in the algae cells not only can maintain the growth of algae, but also can resist the inhibition effect of ultraviolet rays on the algae cells, thereby providing possibility for further outbreak of cyanobacteria bloom.
Therefore, it is necessary to control the increase of the concentration of nutrients in the water body by reducing the pollution of the point source and the non-point source, but due to the hysteresis of the effect produced by the method, when the algal bloom is outbreak, physical, chemical and other methods are generally adopted to reduce the endogenous pollution. The physical algae control method mainly comprises mechanical algae removal, dredging and scheduling, sediment dredging, shading algae removal, ultrasonic algae removal and the like. The chemical algae control method mainly comprises chemical agents (copper sulfate), synthetic compounds (Zn-Fe layered double hydroxide), flocculation method (modified soil) and the like.
In engineering applications, it is most common to employ physical and chemical methods for short-term control of algal bloom outbreaks in freshwater systems. In practice, however, the endogenous pollution of the water is not reduced, the algal bloom is not effectively controlled for a long time, and the repeated cycle of 'outbreak-treatment-outbreak' is involved. The clay is utilized to induce the water bloom flocculation, which is the only effective algae control measure at present, but algae settled into the water body can be converted into endogenous pollution, and the risk of secondary pollution exists.
Therefore, there is a need for an algae removal method that is highly efficient, specific and eco-friendly.
In order to solve the problems, the present disclosure provides an stenotrophomonas strain KT48 with a preservation number of CGMCC No.22722.
Description of biological preservation
Stenotrophomonas KT48: the classification is named: stenotrophomonas KT48 (stenotrophomonassp. Kt48) was deposited in the China general microbiological culture collection center (CGMCC) for 6 months and 16 days of 2021, and the deposit center address is: the national institute of science and microbiology of hospital 1, north chen xi lu, the korean region of beijing; the preservation number is CGMCC NO.22722.
The nucleotide sequence of the stenotrophomonas KT48 is shown as SEQ ID NO: 1.
The stenotrophomonas strain KT48 has the following physiological and biochemical characteristics:
(1) The stenotrophomonas KT48 belongs to gram-negative bacteria;
(2) The community of the stenotrophomonas KT48 is in a milky yellow color on a solid culture medium, is convex in a dot shape, and has regular edges and high viscosity.
(3) The stenotrophomonas KT48 has stronger viability and can grow in LB culture medium with 5 percent NaCl, 4-45 ℃ and pH value of 5-10 respectively.
(4) The stenotrophomonas KT48 strain has negative effects on starch hydrolysis, indole test, acetyl methyl alcohol (V-P), methyl red (M.R.), fluorescent pigment and hydrogen sulfide production, positive effects on oxidase and nitrite reduction, and glucose fermentation.
The disclosure also provides an algicidal bacteria solution prepared from stenotrophomonas strain KT48 with a preservation number of CGMCC No.22722.
The disclosure also provides a preparation method of the algicidal bacteria solution, which can comprise the following steps:
s1, taking a cryopreserved stenotrophomonas strain KT48 with a preservation number of CGMCC No.22722, sterilizing at high temperature, and inoculating into a first culture medium;
s2, placing the inoculated first culture medium into a constant temperature shake incubator with the temperature of 25-40 ℃ and the rotating speed of 130-230 rmp for culturing for 24-72 hours to obtain an activated strain; wherein the temperature of the constant temperature shake incubator can be 25 ℃,30 ℃, 35 ℃,37 ℃ or 40 ℃ and the like, and the temperature is not particularly limited; the rotation speed of the constant-temperature shake incubator can be 130rmo, 150rmp, 170rmp, 180rmp, 200rmp, 215rmp or 230rmp, etc., and the rotation speed is not particularly limited; the culture time can be 24h, 30h, 36h, 42h, 48h, 55h, 60h, 66h, 70h or 72h, etc., and is not particularly limited;
s3, transferring the activated strain into a second culture medium after high-temperature sterilization according to the inoculation amount of 1%, and culturing for 24-72 hours in a constant-temperature shake incubator with the temperature of 25-40 ℃ and the rotating speed of 130-230 rmp to obtain a stock solution; wherein the temperature of the constant temperature shake incubator can be 25 ℃,30 ℃, 33 ℃, 35 ℃, 39 ℃ or 40 ℃ and the like, and the temperature is not particularly limited; the rotation speed of the constant-temperature shake incubator can be 130rmo, 145rmp, 170rmp, 185rmp, 200rmp, 215rmp or 230rmp, etc., and the rotation speed is not particularly limited; the culture time may be 24h, 36h, 42h, 48h, 55h, 60h, 66h, 70h or 72h, etc., and is not particularly limited.
In some possible embodiments, the method may further comprise:
centrifuging the original bacterial liquid at the rotation speed of 5000-10000 rmp for 5-15 min, collecting supernatant after centrifugation, and re-suspending the supernatant with a third culture medium to obtain bacterial suspension; the rotation speed can be 5000rmp, 5300rmp, 5500rmp, 6000rmp, 6500rmp, 7000rmp, 7500rmp, 8000rmp, 8500rmp, 9000rmp, 9500rmp or 10000rmp, and the like, and the rotation speed is not particularly limited; the centrifugation time may be 5min, 7min, 9min, 12min or 15min, etc., and is not particularly limited.
In some possible embodiments, the method may further comprise:
centrifuging the stock solution at the rotation speed of 5000-10000 rmp for 5-15 min, collecting supernatant after centrifugation, and filtering with a filter membrane with the diameter of 0.22 mu m to obtain supernatant.
In some possible embodiments, the first medium may be LB liquid medium or beef extract peptone liquid medium. Preferably, the first medium is an LB liquid medium.
In some possible embodiments, the second medium may be prepared by adding glucose or starch to LB liquid medium or beef extract peptone liquid medium after 6-8 times dilution. For example, the LB liquid medium or beef extract peptone liquid medium may be diluted 6-fold, 6.5-fold, 7-fold, 7.5-fold or 8-fold, without being particularly limited; preferably, the second medium is prepared by diluting the LB liquid medium 8 times and then adding glucose.
In some possible embodiments, glucose or starch may be added in an amount of 0.2 to 0.5%; 0.2 to 0.5 percent refers to the volume percentage of the LB liquid culture medium or the beef extract peptone liquid culture medium with the addition of glucose or starch accounting for 6 to 8 times of the dilution; the addition amount may be 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5%, etc., and is not particularly limited; preferably, 0.5% glucose is added.
In some possible embodiments, the third medium may be prepared by adding glucose or starch to LB liquid medium or beef extract peptone liquid medium after dilution 6 to 8 times. For example, the LB liquid medium or beef extract peptone liquid medium may be diluted 6-fold, 6.5-fold, 7-fold, 7.5-fold or 8-fold, without being particularly limited; preferably, the third medium is prepared by diluting the LB liquid medium 8 times and then adding glucose.
The disclosure also provides application of the algicidal solution in controlling cyanobacterial bloom.
Further, the present disclosure provides the use of algicidal bacteria solutions in controlling cyanobacterial bloom with microcystis aeruginosa as a dominant species.
The algae-dissolving bacterial liquid can control cyanobacteria bloom by inhibiting cyanobacteria proliferation, and the inhibition of cyanobacteria proliferation can be realized by the following steps: destroying the blue algae cell structure and cracking the blue algae cell; or/and, the photosynthetic efficiency of blue algae is reduced, and the growth of blue algae is inhibited; or/and, inducing the generation of excessive active oxygen and aggravating membrane lipid peroxidation, and inhibiting the generation of antioxidant enzyme; or/and, the community structure of the ecological system where the blue algae is positioned is changed, so that the blue algae is removed.
The present disclosure is described in detail below with reference to specific examples and figures.
EXAMPLE isolation and identification of the Stenotrophomonas strain KT48 (Stenotrophomonas sp.KT48)
1. Isolation and screening of bacterial strain KT48
(1) The blue algae bloom in the pit of the water area upstream of the rock-mouth reservoir in Yiwu city of Zhejiang province is frequently outbreaked, the water sample after the water bloom outbreak in the water area is collected, and a 1.2 mu m filter membrane is firstly selected for filtering to remove large-sized particles and impurities in the water; then pumping and filtering the membrane by using a 0.45 mu m filter membrane, shearing the 0.45 mu m filter membrane, putting the sheared membrane into a beef extract peptone liquid culture medium after autoclaving, and culturing the beef extract peptone liquid culture medium in a 180rmp constant-temperature shaking incubator for 36 hours;
(2) Diluting the sample obtained in step (1) by 10 4 Doubling, coating on a nutrient agar solid plate, and culturing in a 35 ℃ incubator for 36h;
(3) Selecting different types of single colonies, scribing on a nutrient agar solid plate, and culturing in a 30 ℃ incubator for 48 hours;
(4) Repeating the step (3) until the separated bacteria are single bacteria;
(5) Inoculating the separated single bacteria to a sterilized LB liquid culture medium, and culturing in a 150rmp constant-temperature shaking incubator for 48 hours to obtain a bacterial culture solution;
(6) Centrifuging the bacterial culture solution at 6000rmp for 10min, collecting the centrifuged thalli, and re-suspending the thalli with sterilized 0.9% physiological saline to obtain a bacterial body weight suspension;
(7) Adding the bacterial body weight suspension into a microcystis aeruginosa culture solution in a stationary phase, culturing for one week at (25+/-1) DEG C, illumination intensity 2000lx and light-dark ratio of 12h to 12h, respectively setting 3 parallel bacteria by taking 0.9% physiological saline as a control, and observing whether the microcystis aeruginosa is yellow or subsided, wherein if the microcystis aeruginosa is yellow or subsided, the single bacteria have algae inhibiting performance.
The strain with the best algae inhibiting effect is selected and named KT48.
2. Physiological and biochemical characterization of strain KT48
2.1 morphological observations
Culturing purified strain KT48 at 30deg.C for 24h by using nutrient agar solid culture medium, wherein the morphology of the obtained bacterial community is shown in figure 1a, observing the shape of the bacterial community by using a microscope, and carrying out gram staining, wherein the staining method is carried out by referring to the method of common bacteria identification handbook (authors: dongxiu bead, cai Miaoying, etc., publishers: science publishers, publication time: 2 months in 2001), and the experimental result is shown in figure 1b, so that the strain is gram-negative bacteria, has a stick-ball shape and no flagellum.
2.2 salt tolerance
The purified strain KT48 was inoculated into LB liquid medium containing 2%, 5%, 7% and 10% NaCl, and cultured in a shaking incubator at 150rmp for one week, and the growth was observed. The results show that: the strain can grow under the condition of containing 2% and 5% of NaCl, and the strain can not grow when the NaCl concentration is as high as 7% or more.
2.3 heat resistance
The purified strain KT48 was inoculated into different test tubes and cultured in an environment of 4℃ 20℃ 30℃37℃45℃65℃water bath for 48 hours. And the seed transfer is carried out for three times at the temperature of more than 37 ℃. The results show that: the strain can grow at 4 ℃, 20 ℃,30 ℃,37 ℃, 45 ℃ and can not grow at 65 ℃.
2.4 acid resistance
Purified strain KT48 was inoculated into LB liquid medium having ph=5.0, 7.0 and 10.0, and cultured in a shaking incubator having 150rmp for one week, and growth was observed. The results show that: the strain has better acid and alkali resistance and can grow under the conditions of pH=5.0, 7.0 and 10.0.
2.5 starch hydrolysis
Adding 0.2% soluble starch into nutrient agar medium, sterilizing at high temperature under high pressure, and pouring into flat plate. The strain KT48 is streaked and inoculated on the plate, and after culturing for 48 hours in a 37 ℃ incubator, a small amount of iodine solution is dripped to evenly spread the whole plate. If a colorless transparent zone appears around the colony, this indicates that the starch has been hydrolyzed. The results show that: the strain KT48 is negative for starch hydrolysis.
2.6 Acetylmethyl alcohol (V-P)
Subpackaging a liquid culture medium prepared from peptone, glucose, dipotassium hydrogen phosphate and water into test tubes, and performing high-temperature high-pressure sterilization for standby. The strain KT48 was inoculated to the above medium, and a blank group was set and cultured in an incubator at 37℃for 24 hours. Then, 10 drops of 40% sodium hydroxide solution are added into the culture solution, then an equal amount of 5% naphthol solution is added, the culture solution is stirred forcefully and then is put into an incubator for heat preservation for 20min, and if the culture solution is red, the culture solution is positive. The results show that: strain KT48 was negative for acetylmethyl alcohol (V-P).
2.7 methyl red (M.R.)
Strain KT48 was inoculated into 2.6 of the medium, and a blank group was set and cultured in an incubator at 37℃for 24 hours. Then, dripping 4-6 drops of methyl red reagent into the culture solution along the pipe wall, wherein the bright red is positive, and the orange is negative. The results show that: strain KT48 methyl red (m.r.) was negative.
2.8 fluorescent pigments
Culture medium prepared from peptone 2g, agar 1.5g, glycerol 1g, distilled water 100mL, dipotassium hydrogen phosphate 0.15g and magnesium sulfate heptahydrate 0.15g is subpackaged in test tubes, sterilized at 121deg.C for 20min, and placed on inclined plane. Young cultures were inoculated on a slant, incubated at 30℃for 1, 3, 5 days, and then observed for fluorescent reaction under UV light or the like. The results show that: strain KT48 contained no fluorescent pigment.
2.9 glucose Oxidation fermentation
The culture medium prepared from 2g of peptone, 5g of NaCl, 0.2g of dipotassium hydrogen phosphate, 10g of glucose, 6g of agar, 3mL of 1% bromothymol blue aqueous solution and 1000mL of distilled water is subpackaged into test tubes, and sterilized for later use. Puncture inoculation young seeds, four per plant, two blank. Wherein, two inoculation are covered by vaseline paraffin oil to form a closed tube, and the other two inoculation are not covered by vaseline paraffin oil to form an open tube. Only the open-pipe acid-producing yellowing person is oxidative, and both the open-pipe and closed-pipe acid-producing yellowing person is fermentation type. The results show that: strain KT48 is of the fermentation type.
2.10 Hydrogen sulfide production
100ml of nutrient agar and 0.5ml of 10% ferrous sulfate, respectively carrying out high-temperature high-pressure filtration sterilization for standby, and subpackaging with a sterile test tube with the height of 4-5 cm. Puncture inoculation, culturing at 30 ℃ for 1, 3 and 5 days, and turning black to positive and no change to negative. The results show that: strain KT48 was negative.
2.11 oxidase
Single colonies were picked with a disposable inoculating needle and smeared onto a test paper. The color change to blue or bluish purple within 30 seconds is positive, and no color change or slow color change is negative. The results show that: strain KT48 was negative.
2.12 indole experiments
1g of peptone, 0.5g of sodium chloride, 0.2g of disodium hydrogen phosphate, 0.2g of dipotassium hydrogen phosphate, 0.5g of glucose and 100mL of water, and packaging the prepared culture medium into test tubes, and sterilizing at high temperature for later use. The strain KT48 was inoculated into the above medium and cultured at 37℃for 24 hours. Adding reagent prepared from 0.8g of p-dimethylaminobenzaldehyde, 76ml of 95% ethanol and 16ml of concentrated HCl into the wall of a tank along the height of 3-5mm, wherein the interface of the liquid layer is red, namely positive reaction. If the color is not obvious, 4-5 drops of diethyl ether can be added into the culture solution, the culture solution is vibrated, and the reagent is added after floating up, so that whether the culture solution is red or not is observed. The results show that: strain KT48 was negative.
3. Molecular characterization
Extracting DNA of a strain KT48, amplifying a PCR product which is a coding gene of 16S-rRNA by using a bacterial 16S rRNA universal primer through PCR, performing electrophoresis detection on the PCR product to obtain a product, and carrying out sequencing on the product, wherein the nucleotide sequence of the strain KT48 is shown as SEQ ID NO: 1.
After sequence alignment, the 16S-rRNA encoding gene of the strain KT48 has 100% similarity with the 16S-rRNA encoding gene of the genus Stenotrophomonas (stenotophomonas sp.); therefore, the strain KT48 is of the genus stenotrophomonas and has been preserved in China general microbiological culture Collection center (CGMCC) for culture Collection of microorganisms, which is 6 and 16 days of 2021, and the preservation center address is: the collection number of the North Chen West Lu 1 institute of China science microorganisms (CGMCC No. 22722) in the Korean region of Beijing is named as Stenotrophomonas KT48 (Stenotrophomonas sp.KT48).
Application example alga inhibition application of Stenotrophomonas strain KT48 (Stenotrophomonas sp.KT48)
1. Algae control application and effect
(1) Preparation of algicidal bacteria liquid
1) Taking isolated, purified and identified stenotrophomonas strain KT48 which is frozen and stored in a refrigerator, sterilizing at high temperature, and inoculating in LB liquid medium;
2) Placing the inoculated culture medium into a constant-temperature shake incubator with the temperature of 35 ℃ and the rotating speed of 180rmp for culturing for 36 hours to obtain an activated strain;
3) Transferring the activated strain to a 1/8LB culture medium+0.5% glucose after high-temperature sterilization according to the inoculation amount of 1%, and culturing for 36h in a constant-temperature shake incubator with the temperature of 35 ℃ and the rotating speed of 180rmp to obtain bacterial culture solution, namely raw bacterial solution;
4) Centrifuging the original bacterial liquid at 6000rmp for 10min, collecting the centrifuged bacterial body, and re-suspending the bacterial body by using 1/8LB culture medium to obtain bacterial suspension;
5) Centrifuging the stock solution at 6000rmp for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain supernatant.
(2) Culturing Microcystis aeruginosa PCC7820 in BG11 culture medium, inoculating into conical flask under aseptic condition, and culturing in illumination incubator with (25+ -1deg.C, illumination intensity of 2000lx and light-dark ratio of 12h to stable period;
(3) Taking 10mL of the prepared original bacterial liquid, supernatant and bacterial suspension respectively, adding 100mL of microcystis aeruginosa culture liquid, setting an equivalent 1/8LB culture medium as a control experiment, co-culturing under the conditions of (25+/-1) DEG C, illumination intensity 2000lx and light-dark ratio of 12 hours to 12 hours, sampling and detecting chlorophyll a content of the culture liquid on days 5 and 8, setting three parallel samples, and taking an average value; yellowing phenomenon after adding algicidal bacteria solution for 10 days was observed, and scanning electron microscope observation was performed after adding algicidal bacteria solution for 2 nd and 14 th days.
FIG. 2 is a graph comparing yellowing after addition of bacterial suspension with that after no addition of bacterial suspension, wherein the left side is a water sample of a treatment group, and the right side is a water sample of a control group, and as can be seen from FIG. 2, after 10 days of treatment by adopting bacterial suspension, the algae control effect is remarkable; the treated water sample is light yellow, has high transparency, and can be almost completely removed; the control group algae liquid is dark green, and the microcystis aeruginosa grows better.
Fig. 3a is a scanning electron microscope image of the day 2 of the bacterial suspension addition, fig. 3b is a scanning electron microscope image of the day 14 of the bacterial suspension addition, and comparing fig. 3a and 3b shows that when the bacterial suspension does not exert the algae inhibiting effect, the microcystis aeruginosa is spherical, well-defined and smooth in surface, and after a period of time of the bacterial suspension treatment, the algae cells are obviously broken, and it is presumed that the stenotrophomonas oligotrophica strain KT48 in the bacterial suspension can directly attack the algae cells, so as to realize the algae inhibiting effect.
2. Determination step of chlorophyll a
(1) 10mL of the co-culture water sample is taken, and is frozen at-80 ℃ for 24 hours after being subjected to a 0.45 mu m mixed cellulose membrane.
(2) The blue cap bottle is filled with a proper amount of 90% ethanol and heated in a water bath kettle at 80-85 ℃.
(3) After the sample was taken out, 10mL of 90% hot ethanol was added to the centrifuge tube and the tube was water-bathed at 80-85℃for 5min.
(4) After the samples were extracted at room temperature in the dark for 6h, 6000rmp was centrifuged for 10min.
(5) After extraction, the sample is subjected to spectrophotometry, and is subjected to 10mm colorimetric with 90% ethanol as a reference
Dish
Colorimetric determination was performed to measure absorbance at 665nm and 750nm wavelengths, and E665-E750 was recorded; then 1 drop of 1+9 hydrochloric acid is added into the sample cuvette for acidification, the mixture is covered and shaken uniformly, absorbance is measured again at 665nm and 750nm after 1.0min, and A665-A750 is recorded.
(6) The calculation formula is as follows:
Chl-a(μg/L)＝27.9V ethanol {(E665-E750)-(A665-A750)}/V Sample of
Wherein V is Ethanol Representing the volume of ethanol added to the sample; v (V) Sample of Representing the volume of the filtered water sample;
3. the calculation formula of the algae inhibition rate is as follows: algae inhibition rate = (ρ2- ρ1)/ρ2x100%
Wherein ρ1 represents the chlorophyll a content of the sample in mg/L on the same day; ρ2 represents chlorophyll a content in mg/L on the day of control.
Fig. 4 is a diagram showing the algae-lysing effect, and as can be seen from fig. 4, the bacterial suspension in the algae-lysing solution has the best effect, and the bacterial suspension is the original bacterial solution. The supernatant, namely the cell metabolite of the stenotrophomonas KT48, has a slightly promoting effect on the growth of microcystis aeruginosa; on the 8 th day of treatment, the algae inhibition rates of the original bacterial liquid, the supernatant and the bacterial suspension are 89.04%, 16.44% and 97.26%, respectively, which shows that the stenotrophomonas strain KT48 in the algicidal bacterial liquid mainly realizes algae control through the direct action of thalli and algae cells, but not cell metabolites.
In summary, the stenotrophomonas strain KT48, the algicidal bacteria solution, the preparation method and the application thereof provided by the disclosure have the algae removal effect, but not the metabolites of the stenotrophomonas strain KT48 have the algae removal effect, so that the practical application is more convenient; inoculating the stenotrophomonas strain KT48 to a culture medium when preparing the algicidal bacteria solution, and culturing in a constant-temperature shaking box until the temperature is stable to obtain the algicidal bacteria solution, wherein the preparation method is simple; the stenotrophomonas KT48 can break the cell membrane of the microcystis aeruginosa and destroy the cell structure; the algicidal bacteria solution prepared by the stenotrophomonas KT48 can reduce the chlorophyll content of blue algae, effectively control the outbreak of water bloom, has high efficiency, biological specificity and environmental friendliness, and is biodegradable. The algae-dissolving bacteria liquid is used for controlling cyanobacteria bloom, has low cost, simple operation, strong safety and no secondary pollution, and is beneficial to field application.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples; combinations of features of the above embodiments or in different embodiments are also possible within the spirit of the present disclosure, steps may be implemented in any order, and there are many other variations of the different aspects of one or more embodiments described above which are not provided in detail for the sake of brevity.
SEQUENCE LISTING
<110> national academy of sciences ecological Environment research center
<120> Oenomonas strain KT48, algicidal bacteria liquid, and preparation method and application thereof
<130> FI210923
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 880
<212> DNA
<213> Methoxymonas strain KT48
<400> 1
cgccctcccg aaggttaagc tacctgcttc tggtgcaaca aactcccatg gtgtgacggg 60
cggtgtgtac aaggcccggg aacgtattca ccgcagcaat gctgatctgc gattactagc 120
gattccgact tcatggagtc gagttgcaga ctccaatccg gactgagata gggtttctgg 180
gattggctta ccgtcgccgg cttgcagccc tctgtcccta ccattgtagt acgtgtgtag 240
ccctggccgt aagggccatg atgacttgac gtcatcccca ccttcctccg gtttgtcacc 300
ggcggtctcc ttagagttcc caccattacg tgctggcaac taaggacaag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacagccatg cagcacctgt 420
gttcgagttc ccgaaggcac caatccatct ctggaaagtt ctcgacatgt caaggccagg 480
taaggttctt cgcgttgcat cgaattaaac cacatactcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcag tcttgcgacc gtactcccca ggcggcgaac ttaacgcgtt 600
agcttcgata ctgcgtgcca aattgcaccc aacatccagt tcgcatcgtt tagggcgtgg 660
actaccaggg tatctaatcc tgtttgctcc ccacgctttc gtgcctcagt gtcaatgttg 720
gtccaggtag ctgccttcgc catggatgtt cctcctgatc tctacgcatt tcactgctac 780
accaggaatt ccgctaccct ctaccacatt ctagtcgccc agtatccact gcagttccca 840
ggttgagccc agggctttca caacggactt aaacgaccac 880
Claims (9)
1. Stenotrophomonas with preservation number of CGMCC No.22722(Stenotrophomonas sp.)Strain KT48.
2. The algicidal bacteria solution is characterized by being prepared from stenotrophomonas strain KT48 with a preservation number of CGMCC No.22722.
3. A method for preparing the algicidal solution according to claim 2, comprising the steps of:
taking a stenotrophomonas strain KT48 with the preservation number of CGMCC No.22722 which is frozen and preserved, and inoculating the stenotrophomonas strain KT48 into a first culture medium after high-temperature sterilization;
placing the inoculated first culture medium into a constant temperature shake incubator with the temperature of 25-40 ℃ and the rotating speed of 130-230 rmp for culturing for 24-72 hours to obtain an activated strain;
transferring the activated strain to a second culture medium after high-temperature sterilization according to the inoculation amount of 1%, and culturing for 24-72 h in a constant-temperature shake incubator with the temperature of 25-40 ℃ and the rotating speed of 130-230 rmp to obtain a stock solution.
4. The method for preparing algicidal solution according to claim 3, further comprising:
centrifuging the original bacterial liquid at the rotation speed of 5000-10000 rmp for 5-15 min, collecting the centrifuged bacterial body, and re-suspending the bacterial body by using a third culture medium to obtain a bacterial suspension.
5. The method for preparing algicidal solution according to claim 3, wherein the first medium is LB liquid medium or beef extract peptone liquid medium.
6. The method for preparing algicidal bacteria solution according to claim 3, wherein the second culture medium is prepared by adding glucose or starch into LB liquid culture medium or beef extract peptone liquid culture medium after dilution by 6-8 times.
7. The method for preparing algicidal bacteria solution according to claim 6, wherein the glucose or starch is added in an amount of 0.2-0.5%.
8. The method according to claim 4, wherein the third medium is prepared by adding 0.2 to 0.5% glucose or starch to LB liquid medium or beef extract peptone liquid medium diluted 6 to 8 times.
9. The use of the algicidal solution of claim 2 for controlling cyanobacterial bloom with microcystis aeruginosa as a dominant species.
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