Application of pseudomonas orientalis to simultaneous inhibition of algae growth and promotion of carbon dioxide fixation

Abstract

The invention discloses application of Pseudomonas orientalis to inhibition of algae growth and promotion of carbon dioxide fixation at the same time, and relates to the technical field of environmental microorganisms and ecological management. The invention provides application of pseudomonas orientalis to inhibition of algae growth and promotion of carbon dioxide fixation at the same time. The inhibition rate of the Pseudomonas orientalis to algae is as high as 97.57%, and the fixation rate of the Pseudomonas orientalis to dioxide is 0.43 g / L.d. The invention also provides a pseudomonas orientalis microbial agent and a preparation method thereof. According to the invention, synchronous regulation and control of algae inhibition and carbon sequestration are realized for the first time, and the problem of mutual restriction of algae control and carbon sequestration processes in the prior art is effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental microorganisms and ecological management, and in particular to the application of Pseudomonas orientalis in simultaneously inhibiting algae growth and promoting carbon dioxide fixation. Background Art

[0002] With the rapid development of industry and agriculture and the accelerating pace of urbanization, the problem of eutrophication of water bodies is becoming increasingly serious. This leads to the frequent and abnormal proliferation of algae (such as green algae and blue algae), which seriously disrupts the balance of aquatic ecosystems. At the same time, global carbon emissions continue to increase, and the fixation of carbon dioxide has become a key technology for mitigating the greenhouse effect.

[0003] Existing algae control technologies have significant limitations. At present, the means of controlling excessive algae growth mainly include physical methods (such as mechanical salvage, ultraviolet killing), chemical methods (such as the introduction of algaecides such as copper sulfate and hydrogen peroxide, etc.) and biological methods (such as the introduction of competitive aquatic plants or algae-lytic bacteria). Although physical methods can quickly produce results, technical limitations and cost issues limit their implementation in a wider area. Chemical methods are prone to secondary pollution. Although traditional biological methods are relatively environmentally friendly, there are problems such as the risk of species invasion or unstable effects. Algae-inhibiting bacteria have become one of the research hotspots of microbial algae control. They inhibit algae growth or dissolve algae cells through bacteria in direct or indirect ways. This method is not only low-cost and easy to operate, but also environmentally friendly, representing the future development direction of algal bloom control technology. In the field of carbon dioxide fixation, existing technologies mostly focus on chemical adsorption, mineral carbonization or microalgae carbon fixation, but have limitations such as high energy consumption, low carbon fixation efficiency or large area occupation.

[0004] In recent years, the rise of environmental microbial technology has provided new insights into these issues. Studies have shown that some functional microorganisms (e.g., Bacillus and Pseudomonas) can inhibit algal growth by secreting algae-inhibiting substances (e.g., antibiotics and algae-lyzing enzymes), while some autotrophic or heterotrophic microorganisms (e.g., nitrogen-fixing bacteria and cyanobacteria) can convert carbon dioxide into organic matter or stable carbon forms through metabolic pathways. However, existing research has largely been limited to single-function studies (e.g., algae inhibition or carbon fixation).

[0005] Therefore, developing an environmentally friendly and sustainable microbial technology that combines algae inhibition and carbon dioxide fixation functions is of great significance to water ecological restoration and carbon reduction. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the present invention provides the application of Pseudomonas orientalis in simultaneously inhibiting algae growth and promoting carbon dioxide fixation, realizing for the first time the simultaneous regulation of algae inhibition and carbon fixation, and effectively solving the problem of mutual restraint between algae control and carbon fixation processes in traditional technologies.

[0007] To achieve the above-mentioned object, the technical solution adopted by the present invention to solve the technical problem is: to provide the use of Pseudomonas orientalis in simultaneously inhibiting algae growth and promoting carbon dioxide fixation.

[0008] Furthermore, Pseudomonas orientalis inhibits algae growth by secreting anti-algae active substances.

[0009] Furthermore, the algae is Scenedesmus obliquus and / or Chlorella vulgaris.

[0010] The method of using the above-mentioned Pseudomonas orientalis to inhibit algae growth and promote carbon dioxide fixation includes the following steps: culturing Pseudomonas orientalis in beef extract peptone liquid culture medium to obtain bacterial liquid, and inoculating the bacterial liquid into algae liquid in the logarithmic growth phase.

[0011] Furthermore, the bacteria-algae ratio of the bacterial liquid to the algae liquid is 1:4-6.

[0012] Furthermore, the ratio of bacteria to algae in the bacterial solution is 1:4.

[0013] A Pseudomonas orientalis agent comprises the above-mentioned Pseudomonas orientalis.

[0014] The preparation method of the above-mentioned Pseudomonas orientalis agent comprises the following steps:

[0015] S1, sterilizing and drying the porous calcium-based material, and then adding it to a Pseudomonas orientalis bacterial solution for cultivation;

[0016] S2. Filter the cultured porous calcium-based material to remove the unadsorbed bacterial solution, and then freeze-dry the porous calcium-based material that has adsorbed the bacterial solution to obtain a Pseudomonas orientalis agent.

[0017] Furthermore, in step S1, the porous calcium-based material is porous calcium carbonate.

[0018] The beneficial effect of adopting the above further solution is: using porous calcium-based materials as carriers.

[0019] Furthermore, in step S1, the mixture is sterilized twice at 121° C. for 20 min under high temperature and high pressure.

[0020] Furthermore, in step S1, the product is sterilized 2-3 times at 120-122° C. for 19-21 minutes under high temperature and high pressure.

[0021] Furthermore, in step S1, the mixture is dried in an oven at 55-65°C.

[0022] Furthermore, in step S1, the product is dried in an oven at 60°C.

[0023] Furthermore, in step S1, the culture is carried out for 23-25 ​​hours.

[0024] Furthermore, in step S1, the culture is carried out for 24 hours.

[0025] Furthermore, in step S1, the amount of the porous calcium-based material added is 14-16% of the mass of the Pseudomonas orientalis bacterial solution.

[0026] Furthermore, in step S1, the amount of the porous calcium-based material added is 15% of the mass of the Pseudomonas orientalis bacterial solution.

[0027] Furthermore, in step S2, the mixture is pre-frozen at -79°C to -81°C for 23-25 ​​hours and then freeze-dried for 47-49 hours.

[0028] Furthermore, in step S2, the mixture is pre-frozen at -80°C for 24 hours and then freeze-dried for 48 hours.

[0029] The present invention has the following beneficial effects:

[0030] 1. This invention achieves the simultaneous regulation of algae control and carbon fixation for the first time. The Pseudomonas orientalis provided by this invention exhibits a strong algae-control effect against Scenedesmus obliquus and promotes carbon dioxide fixation. Under optimal conditions, the algae-control rate against Scenedesmus obliquus reaches 97.57%, and the carbon dioxide fixation rate is 0.43 g / L·d. This solves the problem of the mutual constraints between algae control and carbon fixation in traditional technologies. It provides a green solution for water eutrophication control that combines algae control with carbon sequestration.

[0031] 2. This invention discloses a microbial technology that simultaneously inhibits algae growth and fixes carbon dioxide, demonstrating its environmentally friendly and sustainable development potential. This technology is of great significance for restoring aquatic ecosystems and reducing carbon emissions. By screening microbial strains with specific functions and establishing a synergistic system, it effectively overcomes the limitations of traditional technologies with single functions, providing an integrated solution for eutrophic water treatment and carbon reduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the growth curve of Pseudomonas orientalis;

[0033] Figure 2 This is a Gram-stained microscope observation of Pseudomonas orientalis;

[0034] Figure 3 This is the result diagram of the effect of different bacteria-algae ratios on the algae inhibition effect of Scenedesmus obliquus;

[0035] Figure 4 The results of the effects of different treatments on the algae inhibition rate are shown in the figure;

[0036] Figure 5 This is the effect of Pseudomonas orientalis on CaCO3 production;

[0037] Figure 6 This is a picture of the finished product of the algaecide prepared by the present invention. DETAILED DESCRIPTION

[0038] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0039] Example 1 Cultivation of Pseudomonas orientalis and algae

[0040] (1) Cultivation of Pseudomonas orientalis

[0041] Pseudomonas orientalis DSM 17489 purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) was inoculated into beef extract peptone liquid medium for activation, and then transferred to a solid slant medium. After constant temperature culture at 15°C for 48 hours, it was stored at 4°C for subsequent use.

[0042] Under sterile conditions, a single colony was picked from the solid slant culture medium and placed in 250 mL of beef extract peptone liquid medium, and cultured at 15°C and 120 rpm until the exponential growth phase (OD600: 1.2-1.3) to obtain a primary culture of Pseudomonas orientalis. The components of the beef extract peptone liquid medium include: 5 g beef extract, 10 g peptone, 10 g sodium chloride and 1 L distilled water, adjusted to pH 7.0-7.2, and sterilized at 121°C for 20 min. The components of the solid slant culture medium include: 20 g agar powder, 5 g beef extract, 10 g peptone, 10 g sodium chloride and 1 L distilled water, adjusted to pH 7.0-7.2, and sterilized at 121°C for 20 min.

[0043] (2) Cultivation of algae

[0044] Inoculate algae into BG11 medium and, after growth reaches the exponential phase, use a pipette to transfer the glycerol and bacterial culture into cryovials and store at -20°C. BG11 medium contains: 1.5g sodium nitrate, 0.04g potassium phosphate dihydrate, 0.075g magnesium sulfate heptahydrate, 0.036g calcium chloride dihydrate, 0.006g citric acid, 0.006g ammonium ferric citrate, 0.001g EDTA, 0.02g sodium carbonate, 0.00286g boric acid, 0.00181g manganese chloride monohydrate, 0.000222g zinc sulfate heptahydrate, 0.000079g copper sulfate pentahydrate, 0.00039g sodium molybdate dihydrate, and 0.000049g cobalt nitrate hexahydrate. Adjust the pH to 7.0-7.2 and sterilize at 121°C for 20 minutes.

[0045] Example 2 Biological characteristics experiment of Pseudomonas orientalis

[0046] (1) Determination of the growth curve of Pseudomonas orientalis

[0047] The OD600 value of the bacterial solution was measured every 2 hours using an ultraviolet spectrophotometer. The blank control used sterile beef extract peptone liquid culture medium. The growth curve of Pseudomonas orientalis is shown in the figure below. Figure 1 shown.

[0048] (2) Gram staining of Pseudomonas orientalis

[0049] The Gram staining method includes the following steps: ① Smear: drop a drop of water on a clean glass slide, pick up the bacteria with an inoculating loop and evenly spread it in the water; ② Fixation: place the glass slide close to the flame of an alcohol burner to evaporate the water, but do not burn it; ③ Primary staining: stain the bacterial solution smear with alkaline pigment crystal violet for 1 minute and then wash with water; ④ Mordanting: mordanting with iodine solution for 1 minute and then wash with water and dry the water; ⑤ Decolorization: The key step is to decolorize with 95% ethanol for 30 seconds, shake it evenly to make the ethanol decolorize completely, then wash with water and dry it; ⑥ Counterstaining: counterstain with safranin for 30 seconds, wash with water, dry it, dry it, and then dry it for microscopic examination. The results are as follows Figure 2 shown.

[0050] Depend on Figure 2 It can be seen that the red staining result of Pseudomonas orientalis indicates Gram-negative bacteria.

[0051] Example 3: Algae-inhibiting effect of Pseudomonas orientalis on algae

[0052] (1) Effects of different bacteria-algae ratios on the algae inhibition effect of Pseudomonas orientalis

[0053] The effect of different bacteria-algae ratios (volume ratios) on the algae inhibition effect of Pseudomonas orientalis was investigated. Taking Scenedesmus obliquus as an example, the primary culture solution of Pseudomonas orientalis in Example 1 was inoculated into 30 mL of Scenedesmus obliquus in the logarithmic growth phase at bacteria-algae ratios of 1:4, 1:5, 1:6, 1:7 and 1:8, respectively. The blank culture medium with the same volume was used as the control group. After culture in a light incubator at 15 ° C, a light intensity of 1000 lux, a light and dark cycle of 12h:12h, and an oscillation speed of 80 rpm for 1 week, the chlorophyll a content was determined, and the algae inhibition rate was calculated.

[0054] The calculation formula of algae inhibition rate is: Algae inhibition rate (%) = (1-chlorophyll a content of algae cells in the experimental group / chlorophyll a content of algae cells in the control group) × 100

[0055] The results of the effects of different bacteria-algae ratios on the algae inhibition effect are as follows Figure 3 shown.

[0056] Depend on Figure 3It can be seen that when the bacteria-algae ratio is 1:5, 1:6, 1:7 and 1:8, the algae inhibition rates are 86.57%, 72.15%, 45.4%, 40.69% and 22.72% respectively. When the bacteria-algae ratio is 1:4, the algae inhibition rate is the highest, which is 97.57%.

[0057] (2) The anti-algae effect of Pseudomonas orientalis on Chlorella

[0058] The primary culture solution in Example 1 was inoculated into 30 mL of Scenedesmus obliquus in the logarithmic phase at a bacteria-algae ratio of 1:6. A blank culture medium of the same volume was used as a control group. After culturing for one week in a light incubator at 15°C, a light intensity of 1000 lux, a light:dark cycle of 12 h:12 h, and a shaking speed of 80 rpm, the chlorophyll a content was measured, and the algae inhibition rate was calculated as 84.56% according to the above method.

[0059] (3) The anti-algae effect of Pseudomonas orientalis on Scenedesmus obliquus

[0060] The primary culture solution from Example 1 was inoculated into 30 mL of logarithmic-phase Chlorella solution at a bacteria-algae ratio of 1:6. A blank culture medium of the same volume served as a control group. The culture was incubated in a light incubator at 15°C, 1000 lux light intensity, a 12 h:12 h light-dark cycle, and an oscillation speed of 80 rpm for one week. The chlorophyll a content was measured, and the algae inhibition rate was calculated as 88.69% according to the above method.

[0061] (4) The anti-algae effect of Pseudomonas orientalis on natural lake water

[0062] Water from the central lake at Southwest University of Science and Technology, where algae outbreaks are severe, was inoculated at a bacteria-to-water ratio of 1:6 (volume ratio). An equal volume of lake water served as a control group. After culturing for one week in a light incubator at 15°C, 1000 lux illumination, a 12h:12h light:dark cycle, and an oscillation speed of 80 rpm, the chlorophyll a content was measured. The algae inhibition rate, calculated using the above method, was 44.54%.

[0063] Experimental Example 4: Algae Inhibition Methods of Pseudomonas orientalis

[0064] To explore the anti-algae effect of Pseudomonas orientalis, Pseudomonas orientalis was inoculated into beef extract peptone liquid culture medium and cultured for 48 hours to obtain bacterial liquid. The bacterial liquid was divided into three groups for different treatments: (1) Untreated bacterial liquid group: 50 mL of bacterial liquid was taken without any treatment and set aside; (2) Sterile filtered liquid group: 50 mL of bacterial liquid was taken and centrifuged at 8000 rpm for 5 minutes, the supernatant was aspirated, and filtered with a 0.22m Millipore filter membrane to obtain filtrate. At the same time, 100 L of filtrate was spread on beef extract peptone agar plate and cultured in a constant temperature incubator at 28℃ for 48 hours. The filtrate was checked for complete sterility and set aside; (3) Washed bacterial cell group: The bacterial cells after centrifugation in (2) were washed twice with sterile liquid beef extract peptone and resuspended in 50 mL of sterile liquid beef extract peptone culture medium and set aside.

[0065] The three groups of treatment solutions were added to 30 mL of Scenedesmus obliquus solution in the logarithmic growth phase at a volume ratio of 1:6. Three parallel experiments were conducted in each group. The algae inhibition rate was measured after one week of treatment. The results are shown in the figure. Figure 4 shown.

[0066] Depend on Figure 4 It can be seen that the algae inhibition rates of the untreated bacterial liquid group and the sterile filtered liquid group were 87.64% and 91.1% respectively, while the algae inhibition rate of the washed bacterial group was 60.02%, which was significantly different from the former two. This shows that Pseudomonas orientalis inhibits the growth of Scenedesmus obliquus by secreting algae-inhibiting active substances, and its algae inhibition method is indirect algae inhibition.

[0067] Example 5 Effects of Pseudomonas orientalis on inhibiting algae and promoting carbon dioxide fixation

[0068] The sedimentation system was constructed with a volume of 300 mL. Combined with the results of Huanglong water quality analysis and considering the objectivity of the results under experimental conditions, the Ca 2+ and HCO3 - The concentrations were set at 0.1 mol / L and 0.2 mol / L, respectively.

[0069] 30 mL of calcium chloride solution and 60 mL of sodium bicarbonate solution were added to the sedimentation system. 40 mL of Pseudomonas orientalis bacterial culture and 160 mL of Scenedesmus obliquus in its logarithmic growth phase were added as the experimental group. A control group consisted of 40 mL of ultrapure water and 160 mL of Scenedesmus obliquus in its logarithmic growth phase. Any volume less than 300 mL in each system was made up with ultrapure water. Three replicates were run in each group. Each system was placed in a constant temperature shaker at 15°C and 120 rpm for 7 days. After 7 days, the algae inhibition rate in the experimental group was measured and calculated, using the aforementioned method, to be 92.22%.

[0070] After the experiment, the sediments obtained from the experimental and control groups were centrifuged at 6000 rpm for 10 minutes, the supernatant was removed, and the resulting precipitate was washed three times with ultrapure water, centrifuged under the same conditions after each wash. The washed precipitate was placed in an oven until the water was completely dried. The weight of the dried precipitate was weighed to obtain the amount of calcium carbonate (CaCO3). The results are as follows: Figure 5 shown.

[0071] Depend on Figure 5 It can be seen that the amount of calcium carbonate produced in the experimental group was 3.68 g, which was 2.78 g higher than that in the control group. From the above results, it can be seen that Pseudomonas orientalis can promote carbon dioxide fixation.

[0072] The carbon dioxide fixation rate was measured according to the calculation formula of carbon dioxide fixation rate.

[0073] The formula for calculating the carbon dioxide fixation rate is: V 二氧化碳 = (calcium carbonate production in the experimental group - calcium carbonate production in the control group) / (deposition system volume·time)

[0074] Calculation showed that the carbon dioxide fixation rate in the experimental group was 0.43 g / L·d.

[0075] In summary, Pseudomonas orientalis can promote carbon dioxide fixation while inhibiting algal growth.

[0076] The Ca content in the sediment system was measured daily using an automatic biochemical analyzer. 2+ The content was measured continuously for 7 days, and the kinetic process of mineralization was fitted by an exponential decay equation.

[0077] The kinetic equation of mineralization fitted by the exponential decay equation is [Ca 2+ ]=ekt+b, and take the logarithm In[Ca 2+ ]=kt+b.

[0078] Calculation showed that the fitting equations for Pseudomonas orientalis were y=-0.1352x+0.1158.

[0079] Example 6 Preparation of Pseudomonas orientalis Agent

[0080] After the porous calcium carbonate is sterilized twice at 121°C for 20 minutes under high temperature and high pressure, it is dried in an oven at 60°C to obtain a carrier. The carrier is added to the Pseudomonas Orientalis bacterial solution. The amount of the carrier added is 15% of the mass of the Pseudomonas Orientalis bacterial solution and cultured for 24 hours. The porous calcium carbonate that has been fully adsorbed is filtered to remove the excess unadsorbed bacterial solution. The porous calcium carbonate that has adsorbed the bacterial solution is placed on a glass culture dish, pre-frozen at -80°C for 24 hours, and then freeze-dried for 48 hours to obtain the Pseudomonas Orientalis bacterial agent. It is stored at 4°C. The finished product is as follows: Figure 6 shown.

[0081] 5 g of Pseudomonas orientalis inoculum was added to 30 mL of Scenedesmus obliquus in the logarithmic growth phase. After 7 days, the algae inhibition rate was measured to be 72.35%.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of Pseudomonas orientalis in simultaneously inhibiting algal growth and promoting carbon dioxide fixation.

2. The use according to claim 1, characterized in that The Pseudomonas orientalis inhibits the growth of algae by secreting algae-inhibiting active substances.

3. The use according to claim 1 or 2, characterized in that The algae are Scenedesmus obliquus and / or Chlorella vulgaris.

4. A method for inhibiting algae growth and promoting carbon dioxide fixation using the Pseudomonas orientalis of claim 1, characterized in that: The following steps are involved: Pseudomonas orientalis is cultured in a beef extract peptone liquid culture medium to obtain a bacterial liquid, which is then inoculated into an algae liquid in a logarithmic growth phase.

5. The method for inhibiting algae growth and promoting carbon dioxide fixation using Pseudomonas orientalis according to claim 4, wherein: The bacteria-algae ratio of the bacterial liquid to the algae liquid is 1:4-6.

6. A Pseudomonas orientalis agent, characterized in that The invention comprises the Pseudomonas orientalis described in claim 1.

7. The method for preparing the Pseudomonas orientalis agent according to claim 6, characterized in that: The following steps are involved: S1, sterilizing and drying the porous calcium-based material, and then adding it to a Pseudomonas orientalis bacterial solution for cultivation; S2. Filter the cultured porous calcium-based material to remove the unadsorbed bacterial solution, and then freeze-dry the porous calcium-based material that has adsorbed the bacterial solution to obtain a Pseudomonas orientalis agent.

8. The method for preparing the Pseudomonas orientalis agent according to claim 7, wherein: In step S1, the porous calcium-based material is porous calcium carbonate.

9. The method for preparing the Pseudomonas orientalis agent according to claim 7, wherein: In step S1, the amount of the porous calcium-based material added is 14-16% of the mass of the Pseudomonas orientalis bacterial solution.

10. The method for preparing the Pseudomonas orientalis agent according to claim 7, wherein: In step S2, the product is pre-frozen at -79°C to -81°C for 23-25 ​​hours and then freeze-dried for 47-49 hours.

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