Use of pseudomonas orientalis in simultaneous inhibition of algal growth and promotion of carbon dioxide fixation

Abstract

This invention discloses the application of *Pseudomonas orientalis* in simultaneously inhibiting algal growth and promoting carbon dioxide fixation, relating to the fields of environmental microbiology and ecological governance. The invention provides the application of *Pseudomonas orientalis* in simultaneously inhibiting algal growth and promoting carbon dioxide fixation. *Pseudomonas orientalis* exhibits an algal inhibition rate as high as 97.57%, and its carbon dioxide fixation rate is 0.43 g / L·d. This invention also provides a *Pseudomonas orientalis* inoculum and its preparation method. This invention achieves, for the first time, the simultaneous regulation of algal inhibition and carbon fixation, effectively solving the problem of mutual constraint between algal control and carbon fixation processes in traditional technologies.

Description

Technical Field

[0001] This invention relates to the fields of environmental microbiology and ecological governance technology, specifically to the application of *Pseudomonas orientalis* in simultaneously inhibiting algal growth and promoting carbon dioxide fixation. Background Technology

[0002] With the rapid development of industry and agriculture and the acceleration of urbanization, the problem of eutrophication in water bodies is becoming increasingly serious, leading to frequent abnormal proliferations of algae (such as green algae and cyanobacteria), which severely disrupts the balance of aquatic ecosystems. Meanwhile, global carbon emissions continue to increase, making carbon dioxide sequestration a key technology for mitigating the greenhouse effect.

[0003] Existing algae control technologies have significant limitations. Currently, control methods for excessive algal growth mainly include physical methods (such as mechanical harvesting and ultraviolet sterilization), chemical methods (such as the application of algaecides like copper sulfate and hydrogen peroxide), and biological methods (such as the introduction of competitive aquatic plants or algicidal bacteria). Although physical methods can produce rapid results, their implementation in wider areas is limited by technological constraints and cost issues. Chemical methods are prone to causing secondary pollution. Traditional biological methods, while relatively environmentally friendly, pose risks such as species invasion or unstable effectiveness. Algicidal bacteria have become a research hotspot in microbial algae control; they inhibit algal growth or dissolve algal cells directly or indirectly. This method is not only low-cost and easy to operate but also environmentally friendly, representing the future direction of algal bloom control technology. In the field of carbon dioxide fixation, existing technologies mostly focus on chemical adsorption, mineral carbonization, or microalgal carbon fixation, but these have limitations such as high energy consumption, low carbon fixation efficiency, or large land area requirements.

[0004] In recent years, the rise of environmental microbiology technology has provided new insights into the aforementioned problems. Studies have shown that some functional microorganisms (such as Bacillus and Pseudomonas) can inhibit algal growth by secreting algicidal active substances (such as antibiotics and algicidal enzymes), while certain autotrophic or heterotrophic microorganisms (such as nitrogen-fixing bacteria and cyanobacteria) can convert carbon dioxide into organic matter or stable carbon forms through metabolic pathways. However, existing research is mostly limited to a single function (algal inhibition only or carbon fixation only).

[0005] Therefore, developing a microbial technology that combines algae suppression and carbon dioxide fixation capabilities, while being environmentally friendly and sustainable, is of great significance for aquatic ecological restoration and carbon reduction. Summary of the Invention

[0006] To address the aforementioned shortcomings in existing technologies, this invention provides the application of *Pseudomonas orientalis* in simultaneously inhibiting algal growth and promoting carbon dioxide fixation. This invention achieves, for the first time, the simultaneous regulation of algal inhibition and carbon fixation, effectively solving the problem of mutual constraints between algal control and carbon fixation processes in traditional technologies.

[0007] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is: to provide the application of Pseudomonas orientalis in simultaneously inhibiting algal growth and promoting carbon dioxide fixation.

[0008] Furthermore, *Pseudomonas orientalis* inhibits algal growth by secreting algae-inhibiting substances.

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

[0010] The method for inhibiting algal growth and promoting carbon dioxide fixation using the above-mentioned Pseudomonas orientalis includes the following steps: culturing Pseudomonas orientalis in beef extract peptone liquid medium to obtain a bacterial suspension, and inoculating the bacterial suspension into an algal suspension in the logarithmic growth phase.

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

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

[0013] An agent containing *Pseudomonas orientalis*, comprising the aforementioned *Pseudomonas orientalis*.

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

[0015] S1. Sterilize and dry the porous calcium-based material, then add it to the culture of Pseudomonas orientalis bacterial solution for incubation.

[0016] S2. The cultured porous calcium-based material is filtered to remove the unadsorbed bacterial solution, and then the porous calcium-based material with the adsorbed bacterial solution is freeze-dried to obtain Pseudomonas orientalis inoculum.

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

[0018] The beneficial effect of adopting the above-mentioned further solutions is that porous calcium-based materials are used as carriers.

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

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

[0021] Furthermore, in step S1, the product 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 porous calcium-based material added is 14-16% of the mass of the Pseudomonas orientalis bacterial culture.

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

[0027] Furthermore, in step S2, the product is pre-frozen at -79℃ to -81℃ 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, for the first time, the simultaneous regulation of algae suppression and carbon fixation. The *Pseudomonas orientalis* strain provided by this invention exhibits a strong algae-suppressing effect on *Scenedesmus obliquus* and promotes carbon dioxide fixation. Under optimal conditions, the algae suppression rate against *Scenedesmus obliquus* reaches 97.57%, and the carbon dioxide fixation rate is 0.43 g / L·d. This solves the problem of mutual constraints between algae control and carbon fixation processes in traditional technologies. It provides a green solution for eutrophication treatment that combines algae suppression and carbon sequestration enhancement.

[0031] 2. This invention discloses a microbial technology that simultaneously inhibits algal growth and fixes carbon dioxide, exhibiting environmentally friendly and sustainable development characteristics. This technology is significant for restoring aquatic ecosystems and reducing carbon emissions. By screening microbial strains with specific functions, a synergistic system is constructed, effectively overcoming the limitations of traditional single-function technologies and providing an integrated solution for eutrophic water treatment and carbon emission reduction. Attached Figure Description

[0032] Figure 1 A growth curve of *Pseudomonas orientalis*;

[0033] Figure 2 Microscopic observation of Gram staining of Pseudomonas orientalis;

[0034] Figure 3 Figure showing the effect of different bacteria-to-algae ratios on the algae-suppressing effect of Scenedesmus obliquus;

[0035] Figure 4 Figure 1 shows the effect of different treatments on algae inhibition rate.

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

[0037] Figure 6 This is a picture of the finished product of the algae-inhibiting agent prepared according to the present invention. Detailed Implementation

[0038] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0039] Example 1: Culture of Pseudomonas orientalis and algae

[0040] (1) Culture of Pseudomonas orientalis

[0041] The *Pseudomonas orientalis* DSM 17489, purchased from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), was activated by inoculating it into beef extract peptone liquid medium, then transferred it to solid slant medium, incubated at 15°C for 48 hours, and then stored at 4°C for later use.

[0042] Under aseptic conditions, single colonies were picked from solid agar slant culture medium and added to 250 mL of beef extract peptone liquid medium. The medium was incubated 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 beef extract peptone liquid medium consisted of 5 g beef extract, 10 g peptone, 10 g sodium chloride, and 1 L distilled water. The pH was adjusted to 7.0-7.2, and the medium was sterilized at 121°C for 20 min. The solid agar slant culture medium consisted of 20 g agar powder, 5 g beef extract, 10 g peptone, 10 g sodium chloride, and 1 L distilled water. The pH was adjusted to 7.0-7.2, and the medium was sterilized at 121°C for 20 min.

[0043] (2) Algae cultivation

[0044] Algae were inoculated into BG11 medium. Once the algae reached the exponential growth phase, glycerol and the bacterial culture were transferred to cryovials using a pipette and stored at -20°C. The BG11 medium consisted of: 1.5g sodium nitrate, 0.04g dipotassium hydrogen phosphate trihydrate, 0.075g magnesium sulfate heptahydrate, 0.036g calcium chloride dihydrate, 0.006g citric acid, 0.006g ferric ammonium 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. The pH was adjusted to 7.0-7.2, and the medium was sterilized at 121°C for 20 minutes.

[0045] Example 2: Experiment on the biological characteristics of Pseudomonas orientalis

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

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

[0048] (2) Gram staining of Pseudomonas orientalis

[0049] Gram staining includes the following steps: ① Smear: Place a drop of water on a clean glass slide, pick up the bacterial cells with an inoculation loop, and spread them evenly in the water; ② Fixation: Place the slide near an alcohol lamp flame to evaporate the water, but do not scorch it; ③ Primary stain: Primary stain the bacterial smear with the basic pigment crystal violet for 1 minute, then wash with water; ④ Mordanting: Mordant with iodine solution for 1 minute, then wash with water and blot dry; ⑤ Destaining: The key step, destain with 95% ethanol for 30 seconds, shake gently to ensure complete ethanol destaining, then wash with water and blot dry; ⑥ Counterstain: Counterstain with safranin for 30 seconds, wash with water, blot dry, and then dry for microscopic examination. The results are as follows. Figure 2 As shown.

[0050] Depend on Figure 2 It can be seen that red staining of Pseudomonas orientalis indicates that it is a Gram-negative bacterium.

[0051] Example 3: Algal-inhibiting effect of *Pseudomonas orientalis* on algae.

[0052] (1) Effect of different bacterial-to-algae ratios on the algicidal effect of Pseudomonas orientalis

[0053] To investigate the effect of different bacterial-to-algae ratios (volume ratios) on the algicidal effect of *Pseudomonas orientalis*, taking *Scenedesmus obliquus* as an example, the primary culture of *Pseudomonas orientalis* from Example 1 was inoculated into 30 mL of *Scenedesmus obliquus* culture in the logarithmic growth phase at bacterial-to-algae ratios of 1:4, 1:5, 1:6, 1:7, and 1:8, respectively. A blank culture medium of the same volume was used as a control group. After one week of cultivation in a light incubator at 15℃, light intensity of 1000 lux, light-dark cycle of 12h:12h, and shaking speed of 80 rpm, the chlorophyll a content was measured, and the algicidal rate was calculated.

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

[0055] The effects of different bacteria-to-algae ratios on algae suppression are as follows: Figure 3 As shown.

[0056] Depend on Figure 3It can be seen that when the bacteria-to-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. The algae inhibition rate is the highest when the bacteria-to-algae ratio is 1:4, which is 97.57%.

[0057] (2) The algicidal effect of Pseudomonas orientalis on Chlorella

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

[0059] (3) The algicidal effect of Pseudomonas orientalis on Scenedesmus obliquus

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

[0061] (4) Algae-inhibiting effect of Pseudomonas orientalis on natural lake water

[0062] Water from the central lake at Southwest University of Science and Technology, which was severely affected by algal blooms, was inoculated at a bacterial-to-water ratio of 1:6 (by volume). A control group of the same volume of lake water was used. The cells were incubated for one week in a light incubator at 15°C, with 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 then measured, and the algal inhibition rate was calculated to be 44.54% using the above method.

[0063] Experiment Example 4: Algal Inhibition Mechanism of Pseudomonas orientalis

[0064] To investigate the algae-inhibiting mechanism of *Pseudomonas orientalis*, *Pseudomonas orientalis* was inoculated into beef extract peptone liquid medium and cultured for 48 h to obtain bacterial suspension. The bacterial suspension was divided into three groups for different treatments: (1) Untreated bacterial suspension group: 50 mL of bacterial suspension was taken without any treatment and kept for later use; (2) Sterile filtrate group: 50 mL of bacterial suspension was taken, centrifuged at 8000 rpm for 5 min, the supernatant was collected, and filtered through a 0.22 μm Millpore filter membrane to obtain filtrate. At the same time, 100 L of filtrate was spread on beef extract peptone agar plates and cultured at 28 °C for 48 h in a constant temperature incubator to check whether the filtrate was completely sterile and kept for later use; (3) Washed bacterial cells 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 medium and kept for later use.

[0065] The above 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 set up for each group. The algae inhibition rate was measured after one week of treatment. The results are as follows: Figure 4 As shown.

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

[0067] Example 5: Effects of Pseudomonas orientalis on algae inhibition and carbon dioxide fixation

[0068] A sedimentation system with a volume of 300 mL was constructed. Based on the water quality analysis results from Huanglong, and considering the objectivity of the results under the experimental conditions, the Ca content was adjusted accordingly. 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 suspension and 160 mL of *Scenedesmus obliquus* suspension in the logarithmic growth phase were added as the experimental group. 40 mL of ultrapure water and 160 mL of *Scenedesmus obliquus* suspension in the logarithmic growth phase were used as the control group. Any remaining volume in each system less than 300 mL was made up with ultrapure water. Three parallel experiments were set up for each group. All systems were placed in a constant temperature shaking incubator and sedimented continuously at 15℃ and 120 rpm for 7 days. After 7 days, the algae inhibition rate in the experimental group was measured, and the algae inhibition rate was calculated to be 92.22% using the above method.

[0070] After the experiment, the sediments obtained from the experimental and control groups were centrifuged at 6000 rpm for 10 min to remove the supernatant. 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 completely dry, and the weight of the dried precipitate was measured to obtain the amount of calcium carbonate (CaCO3) produced. The results are as follows: Figure 5 As shown.

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

[0072] The carbon dioxide fixation rate was measured using the formula for calculating the carbon dioxide fixation rate.

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

[0074] The calculated carbon dioxide fixation rate in the experimental group was 0.43 g / L·d.

[0075] In conclusion, *Pseudomonas orientalis* can inhibit algal growth while promoting carbon dioxide fixation.

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

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

[0078] The calculated fitting equations for *Pseudomonas orientalis* are y = -0.1352x + 0.1158.

[0079] Example 6: Preparation of *Pseudomonas orientalis* inoculum

[0080] Porous calcium carbonate was sterilized twice under high temperature and high pressure at 121℃ for 20 min each time, and then dried in an oven at 60℃ to obtain a carrier. This carrier was added to *Pseudomonas orientalis* bacterial culture at 15% of the bacterial culture mass, and cultured for 24 h. The fully adsorbed porous calcium carbonate was then filtered to remove excess unadsorbed bacterial culture. The adsorbed porous calcium carbonate was placed on a glass petri dish, pre-frozen at -80℃ for 24 h, and then freeze-dried for 48 h to obtain *Pseudomonas orientalis* inoculum. This inoculum was stored at 4℃. The finished product is shown below. Figure 6 As shown.

[0081] Adding 5g of Pseudomonas orientalis inoculum to 30mL of Scenedesmus obliquus in the logarithmic growth phase resulted in an algae inhibition rate of 72.35% after 7 days.

[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 within the protection scope of the present invention.

Claims

1. The application of *Pseudomonas orientalis* in simultaneously inhibiting algal growth and promoting carbon dioxide fixation, wherein the *Pseudomonas orientalis* is designated DSM 17489 and the algae is *Scenedesmus obliquus*.

2. The application as described in claim 1, characterized in that, The *Pseudomonas orientalis* inhibits algal growth by secreting algae-inhibiting substances.

3. A method for simultaneously inhibiting algal growth and promoting carbon dioxide fixation using *Pseudomonas orientalis*, characterized in that... Includes the following steps: The bacterium *Pseudomonas orientalis* was cultured in beef extract peptone liquid medium to obtain a bacterial suspension. The bacterial suspension was then inoculated into an algal suspension in the logarithmic growth phase. The *Pseudomonas orientalis* strain was designated DSM 17489, and the algae was *Scenedesmus obliquus*.

4. The method as described in claim 3, characterized in that, The ratio of bacteria to algae in the bacterial solution to the algae solution is 1:4-6.

5. The application of *Pseudomonas orientalis* inoculant in simultaneously inhibiting algal growth and promoting carbon dioxide fixation, characterized in that... The *Pseudomonas orientalis* strain is designated DSM 17489, and the algae is *Scenedesmus obliquus*.

6. The application as described in claim 5, characterized in that, The preparation method of the *Pseudomonas orientalis* inoculum includes the following steps: S1. Sterilize and dry the porous calcium-based material, then add it to the culture of Pseudomonas orientalis bacterial solution for incubation. S2. The cultured porous calcium-based material is filtered to remove the unadsorbed bacterial solution, and then the porous calcium-based material with the adsorbed bacterial solution is freeze-dried to obtain Pseudomonas orientalis inoculum.

7. The application as described in claim 6, characterized in that, In step S1, the porous calcium-based material is porous calcium carbonate.

8. The application as described in claim 6, characterized in that, In step S1, the amount of porous calcium-based material added is 14-16% of the mass of the Pseudomonas orientalis bacterial culture.

9. The application as described in claim 6, characterized in that, In step S2, the product is pre-frozen at -79℃ to -81℃ for 23-25 ​​hours and then freeze-dried for 47-49 hours.

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