A type of Pseudomonas R90 promotes the growth of red oligotrophic bacteria in sewage (lm1). T Applications in growth

Abstract

This invention provides a method for promoting the growth of red oligotrophic bacteria (lm1) in wastewater using Pseudomonas R90. T In its application within the field of microbial technology, *Pseudomonas bariensis* R90 can promote the growth of red oligotrophic bacteria in wastewater in MS inorganic salt medium with glucose as the carbon source. T The growth, and lm1 T It can recruit R90 to grow around it and can also promote the growth of R90 to a certain extent. There is a synergistic effect between the two, which is of great significance for studying the biological characteristics of oligotrophic bacteria and understanding the interaction of microorganisms in the ecosystem.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a method for promoting the growth of red oligotrophic bacteria (lm1) in wastewater using Pseudomonas R90. T Applications during growth. Background Technology

[0002] Oligotrophic bacteria are a class of bacteria that can grow on culture media with an organic matter content of 1-15 mg / L. They are widely distributed in oceans, rivers, soils, and lakes, playing an important role in biogeochemical cycles. In recent years, human exploitation of natural resources has made oligotrophic environments increasingly common on Earth. Therefore, studying the status and ecological role of oligotrophic bacteria in ecosystems is of great significance. As of 2023, Chinese scientists have isolated thousands of oligotrophic bacteria from forest soils, and studies have shown that they play an important ecological role in the carbon budget of forest ecosystems. However, due to the difficulty in isolating and culturing oligotrophic bacteria and their slow growth, there are currently few reports on them.

[0003] Red oligotrophic bacteria in sewage ( Rhodoligotrophos defluvii )lm1 T This is a new bacterium screened from activated sludge of propylene oxide saponification wastewater in our laboratory (Resources and Environmental Microbiology Laboratory, Jinan University). Phylogenetic analysis revealed that it is related to lm1. T The type strain of the same genus belongs to oligotrophic bacteria; through previous experiments, we have demonstrated that lm1 T It is a facultative oligotrophic bacterium, and 1m1 was found. T Unable to utilize glucose as a carbon source to maintain its growth and development; further investigation of lm1 T of phaC Bioinformatics analysis of the gene revealed lm1 T It has the ability to synthesize polyhydroxyalkanoates (PHAs), and this has been experimentally demonstrated in 1m1. T It is indeed possible to synthesize PHA using short-chain organic acids.

[0004] In barren environments, bacteria face starvation. On the one hand, they need to increase the speed at which they utilize nutrients from the surrounding environment. On the other hand, the lack of energy will slow down the metabolic rate, making it impossible for cells to quickly cope with the ever-changing environmental pressures and thus unable to make the corresponding physiological responses. In this emergency, bacteria can degrade their own stored substances and intracellular components in a short period of time to cope with starvation. However, in natural environments, the amount of PHA stored in the cells of microorganisms is limited, and the carbon source required to maintain long-term survival may come from the degradation of hexoses by other symbiotic microorganisms.

[0005] In bacterial co-culture systems, symbiotic microorganisms often reside in a relatively compact space to facilitate the exchange of metabolites. Experiments have demonstrated that bacteria possess sensitive molecular sensing mechanisms, enabling them to detect the presence of metabolites and quorum sensing factors, thereby recognizing interacting partners and exhibiting chemotaxis and self-aggregation behaviors. Pseudomonas is a Gram-negative bacterium that is easily cultured in vitro and widely distributed, making it a focus of scientific research. While studies have reported on the interaction mechanisms between Pseudomonas and plants such as Arabidopsis thaliana, research on the interactions between Pseudomonas and other bacteria is scarce.

[0006] In the field of microbiology, "recruitment" generally refers to the behavior of a bacterium releasing chemical substances into its environment to attract or induce other microorganisms or cells. These chemical substances include signaling molecules, enzymes, hormones, etc., which can promote the growth and division of microorganisms, thereby enhancing their adaptability and competitive advantage in the environment. During the "recruitment" process, bacteria can release chemical substances such as signaling molecules or hormones to induce the movement of other microorganisms, attracting them to move to a specific location, thereby achieving cooperation between microorganisms.

[0007] Red oligotrophic bacteria in sewage ( Rhodoligotrophos defluvii )lm1 T As an oligotrophic bacterium, it is difficult to cultivate and grows slowly because it cannot utilize glucose as a carbon source to sustain its growth and development. The search is for a method that can promote lm1... T Methods for studying bacterial growth are of great significance for researching the biological characteristics and ecological status of oligotrophic bacteria. Summary of the Invention

[0008] To address the above problems, this invention provides a method for promoting the growth of red oligotrophic bacteria (lm1) in wastewater using Pseudomonas R90. T The application in growth involves combining *Pseudomonas bariensis* R90 with *L. 1m*, a red oligotrophic bacterium found in wastewater. T When co-cultured on MS medium containing glucose and inorganic salts, R90 can promote 1 m1 T The growth, and lm1 T It can recruit R90s to gather around it.

[0009] The technical solution of this invention is as follows:

[0010] Baliaris ( Pseudomonas balearica R90 promotes the growth of red oligotrophic bacteria in wastewater ( Rhodoligotrophos defluvii )lm1 T Applications during growth;

[0011] The strain of *Pseudomonas bariensis* R90 has the accession number CCTCC NO: M 2022947.

[0012] The wastewater red oligotrophic bacteria lm1 T The strain has the preservation number CCTCC AB 2019071.

[0013] Preferably, the application method involves mixing *Pseudomonas bariensis* R90 with *Red Oligotrophic Bacteria lm1* from wastewater. T Co-cultured on MS inorganic salt medium containing 0.04–0.06 g / L glucose.

[0014] Preferably, the application method involves mixing *Pseudomonas bariensis* R90 with *Red Oligotrophic Bacteria lm1* from wastewater. T Co-cultured on MS medium containing 0.05 g / L glucose.

[0015] Preferably, the MS inorganic salt culture medium comprises the following components: glucose 0.04~0.06 g / L, Na2HPO4•12H2O 9.0 g / L, KH2PO4 1.5 g / L, (NH4)2SO4 1.0 g / L, MgSO4•7H2O 0.4 g / L, trace elements 1.0 mL / L, agar 20 g / L, sterilized at 121℃ for 20 min;

[0016] The trace elements include the following components: H3BO3 0.3 g / L, CaCl2•6H2O 0.2 g / L, ZnSO4•7H2O 0.1 g / L, MnCl2•4H2O 30 mg / L, NaMoO4•2H2O 30 mg / L, NiCl2•6H2O 20 mg / L, and CuSO4•5H2O 10 mg / L.

[0017] Preferably, the specific steps include:

[0018] (1) Bacillus bariensis R90 and red oligotrophic bacteria lm1 from sewage were respectively added. T Streaking was performed in three zones on LB solid medium, and the culture was inverted at 37°C to obtain activated strains of *Pseudomonas bariensis* R90 and 1 m1 of red oligotrophic bacteria from sewage. T Activated strains;

[0019] (2) Select the activated strain of Pseudomonas bariensis R90 and the red oligotrophic bacteria lm1 from the sewage in step (1). T Single colonies of the activated strain were inoculated into LB liquid medium and cultured overnight at 37±1℃ and 180~200rpm. After incubation, the colonies were inoculated into fresh LB liquid medium at a volume ratio of 3% and cultured until the logarithmic growth phase, yielding *Pseudomonas bariensis* R90 seed culture and 1m1 of red oligotrophic bacteria from sewage. T Seed liquid;

[0020] (3) Under aseptic conditions, place the Oxford cup into an empty plate, pour MS inorganic salt medium containing 0.04~0.06 g / L glucose into the plate, and use it as the bottom medium after solidification; centrifuge the Pseudomonas bariensis R90 seed culture from step (2), retain the bacterial cells, wash with deionized water, centrifuge again, and resuspend the centrifuged bacterial cells with deionized water to obtain OD. 600 The resuspension of Pseudomonas bariensis R90 was prepared at a concentration of 0.8-1.2. The resuspension of Pseudomonas bariensis R90 was inoculated at a volume ratio of 1% into MS inorganic salt medium containing 0.04-0.06 g / L glucose, which was cooled to 40-45°C. The medium was poured into the plate and solidified to form the upper culture medium. The Oxford cup was then removed, and the round holes left by the Oxford cup would appear on the plate.

[0021] (4) Add 1 m1 of red oligotrophic bacteria from the wastewater in step (2). T After centrifuging the seed culture, retain the bacterial cells, wash with deionized water, centrifuge again, and then resuspend the centrifuged bacterial cells in deionized water to obtain the OD. 600 0.8~1.2 μL of red oligotrophic bacteria in wastewater T Resuspend the bacterial solution and add 1 m1 of red oligotrophic bacteria from the sewage. T Add 100 μL of the resuspended bacterial solution to the well on the plate in step (3), and then place the plate in a constant temperature environment of 37°C for incubation.

[0022] Preferably, the centrifugation conditions in steps (3) and (4) are: 4°C, 8000~10000rpm for 5~10min.

[0023] The wastewater red oligotrophic bacteria lm1 T Application in promoting the growth of Pseudomonas bariensis R90.

[0024] The wastewater red oligotrophic bacteria lm1 T Application in recruiting Pseudomonas bariarius R90.

[0025] Preferably, the application method involves mixing *Pseudomonas bariensis* R90 with *Red Oligotrophic Bacteria lm1* from wastewater. T Co-cultured on MS inorganic salt medium containing 0.04–0.06 g / L glucose.

[0026] Beneficial effects:

[0027] This invention has discovered that *Pseudomonas bariensis* R90 can promote the growth of red oligotrophic bacteria in sewage in MS inorganic salt medium with glucose as the carbon source. T The growth, and lm1 TIt can recruit R90 to grow around it and promote the growth of R90 to a certain extent. There is a synergistic effect between the two, which is of great significance for studying the biological characteristics of oligotrophic bacteria and understanding the interaction of microorganisms in the ecosystem. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the co-culture of bacterial strains using the double-layer plate method.

[0029] Figure 2 For R79+R90+wg1 T Three strains of bacteria and lm1 T Growth status of co-cultured organisms;

[0030] Figure 3 For R79, R90, wg1 T The three strains were combined in pairs with lm1 T Growth status of co-cultured organisms;

[0031] Figure 4 For R90 and lm1 T Growth status of co-cultured organisms;

[0032] Figure 5 For R90, wg1 T R79 and lm1 respectively T Growth status of co-cultured organisms;

[0033] Figure 6 for Figure 3 A magnified view of Figure B in the middle;

[0034] Figure 7 for Figure 5 A magnified view of Figure B in the middle;

[0035] Figure 8 for Figure 5 A magnified view of Figure C. Detailed Implementation

[0036] The following description is based on specific embodiments:

[0037] The following examples involve *Pseudomonas bariensis* R90 and *Oligotrophic bacterium lm1* from wastewater. T , Defective shortwave monoclonal bacteria ( Brevundimonas diminuta R79, Binzhou Paracoccus ( Paracoccus binzhouense wg1 T All four strains were screened from activated sludge in our laboratory and have been deposited at the China Center for Type Culture Collection; their accession numbers are as follows:

[0038] Pseudomonas bariairis R90: Preservation number CCTCC NO: M 2022947;

[0039] Red oligotrophic bacteria in sewage lm1 T The accession number is CCTCC AB 2019071;

[0040] Defective shortwave monoclonal antibody R79: accession number CCTCC NO: M2022946;

[0041] Binzhou Paracoccus wg1 T The accession number is CCTCC AB 2019400.

[0042] The aforementioned bacterial species, *Pseudomonas bariensis* R90 and *Bacillus malariae* R79, have been disclosed in patent "CN115322924A," and *Paragonimus binzhouensis* wg1... T The red oligotrophic bacteria lm1 in wastewater has been disclosed in patent "CN116854262A". T Disclosed through non-patented means.

[0043] Example 1:

[0044] Defective shortwave monoclonal bacteria R79, Pseudomonas bariensis R90, and Paracoccus binzhouensis wg1 were included. T The three strains were mixed with 1m1 of red oligotrophic bacteria from sewage. T Co-cultivation, the specific steps are as follows:

[0045] (1) Activation of strains: R79, R90, and wg1 strains stored at -70℃ were taken respectively. T and lm1 T Streaking was performed in three zones on LB solid medium, followed by inverted culture at 37°C to obtain activated strains R79, R90, and wg1. T Activated strains and lm1 T Activated strains;

[0046] (2) Preparation of seed culture: Select the R79 activated strain, R90 activated strain, and wg1 from step (1). T Activated strains and lm1 T Single colonies of the activated strain were inoculated into 30 mL of LB liquid medium and incubated overnight at 37°C and 200 rpm. After incubation, 3 mL of the bacterial culture was inoculated into 100 mL of fresh LB liquid medium at a volume ratio of 3% and incubated until the logarithmic growth phase to obtain R79 seed culture, R90 seed culture, and wg1. T Seed liquid and lm1 T Seed liquid;

[0047] (3) Preparation of double-layer plates: Under aseptic conditions, take three Oxford cups and place them into the same empty plate. Pour MS inorganic salt medium containing 0.05 g / L glucose into the plate and let it solidify to serve as the bottom layer medium. Add the R79 seed culture, R90 seed culture, and wg1 from step (2) to the plate respectively. T After centrifuging the seed culture, the bacterial cells were retained, washed with deionized water, centrifuged again, and then resuspended in deionized water to obtain OD values. 600 To obtain a 1.0 resuspension of bacteria, the resuspensions were mixed at a volume ratio of 1:1:1 to form a mixed bacterial solution. The mixed bacterial solution (base culture) was added at an inoculum volume of 1% to MS inorganic salt medium containing 0.05 g / L glucose at 40°C. The mixture was then poured into the aforementioned plates, and after solidification, it served as the upper culture medium. A schematic diagram of the double-layer plate is shown below. Figure 1 As shown; remove the Oxford cup, and the round hole left by the Oxford cup will appear on the flat surface;

[0048] Control group setup: The upper culture medium was MS inorganic salt medium containing 0.05 g / L glucose without substrate bacteria;

[0049] (4) Add 1 m1 of red oligotrophic bacteria from the wastewater in step (2). T After centrifuging the seed culture, retain the bacterial cells, wash with deionized water, centrifuge again, and then resuspend the centrifuged bacterial cells in deionized water to obtain the OD. 600 lm1 is 1.0 T Resuspend the bacterial culture, add 1m1 T Add 100 μL of the resuspended bacterial solution to the round well in step (3), and then incubate at 37°C to observe the growth of the strain.

[0050] The MS inorganic salt culture medium comprises the following components: 0.05 g / L glucose, 9.0 g / L Na2HPO4•12H2O, 1.5 g / L KH2PO4, 1.0 g / L (NH4)2SO4, 0.4 g / L MgSO4•7H2O, 1.0 mL / L trace elements, 20 g / L agar, sterilized at 121℃ for 20 min;

[0051] The trace elements include the following components: H3BO3 0.3 g / L, CaCl2•6H2O 0.2 g / L, ZnSO4•7H2O 0.1 g / L, MnCl2•4H2O 30 mg / L, NaMoO4•2H2O 30 mg / L, NiCl2•6H2O 20 mg / L, CuSO4•5H2O 10 mg / L;

[0052] All of the above centrifugation conditions were 4℃, 9000rpm for 8min.

[0053] R79+R90+wg1 T Three strains of bacteria and lm1 T The growth of co-cultured organisms is as follows: Figure 2 As shown, Figure A is lm1 T Growth in the absence of substrate bacteria (no mixed bacterial solution added), Figure B shows 1m1. T With a base culture (add R79+R90+wg1) T Growth status at that time; by Figure 2 It can be seen that when there are no substrate bacteria in the upper culture medium of the control group, 1 m1 T No growth; after adding substrate bacteria, 1m1 T It can grow normally, and the basal bacteria also grow, which indicates that one or more strains of the three bacteria are related to lm1. T A symbiotic relationship exists.

[0054] Example 2:

[0055] Unlike Example 1, the substrate bacteria added to the upper culture medium in this example are R79, R90, and wg1, respectively. T After combining the bacterial cultures in pairs, the remaining steps are the same as in Example 1, and the growth of the bacterial strains is observed.

[0056] R79, R90, wg1 T The three strains were combined in pairs with lm1 T The growth of co-cultured organisms is as follows: Figure 3 As shown, by Figure 3 As shown in Figures A and C, the substrate bacteria containing R90 can promote 1m1 T The growth, and the basal bacteria all surround strain lm1 T Growing around; by Figure 3 As shown in Figure B, the basal bacteria without R90 grow in a scattered distribution, and strain lm1 T It also does not grow; the above results confirm that strain R90 and strain lm1 T A symbiotic relationship exists; R90 can promote lm1. T Normal growth.

[0057] Example 3:

[0058] Unlike Example 1, this example uses lm1 T Co-culture with R90 was performed, and a control group was set up (specifically as follows). Figure 4 As shown in the figure, the interaction between the two was further verified. The remaining steps were the same as in Example 1, and the growth of the strain was observed.

[0059] lm1 T Growth of co-cultured with R90 as follows Figure 4As shown in Figures A and B, without R90, lm1 T Neither R90 nor basal bacteria can grow independently; as shown in Figure C, when basal bacteria R90 are added to the upper culture medium, 1 m1 T It can grow normally, and R90 is around 1m1. T Surrounding growth; as shown in Figure D, in the absence of lm1 T Under these conditions, the basal bacteria R90 grows randomly; as shown in Figures E-F, adding R90 to the round wells produces an R90 growth zone, while adding 1 m1 of basal bacteria to the upper culture medium results in a different growth pattern. T Subsequently, the growth zone of R90 increased in size. This indicates that R90 can not only promote lm1 growth. T Growth, lm1 T It can also promote the growth of R90 to some extent, and lm1 T The ability to attract and recruit R90 to grow around it suggests that strain lm1 is the source of this phenomenon. T There is a mutually beneficial symbiotic relationship between it and strain R90.

[0060] Comparative Example 1:

[0061] Unlike Example 1, this comparative example uses R90 and wg1. T R79 and lm1 respectively T Perform co-culture of the two bacteria, and follow the same steps as in Example 1, and observe the growth of the strains.

[0062] R90, wg1 T R79 and lm1 respectively T The growth of co-cultured organisms is as follows: Figure 5 As shown, by Figure 5 From Figure A, we can see that lm1 T It can grow and R90 grows around it, by Figure 5 From diagrams B and C, we can see that wg1 T Neither R79 nor LM1 can promote LM1 T Growth, and lm1 T For wg1 T Neither strain R79 nor strain R79 exhibited recruitment phenomena.

Claims

1. *Pseudomonas bariensis* ( Pseudomonas balearica R90 promotes the growth of red oligotrophic bacteria in wastewater ( Rhodoligotrophos defluvii lm1 T Applications during growth; The strain of *Pseudomonas bariensis* R90 has the accession number CCTCC NO: M 2022947. The wastewater red oligotrophic bacteria lm1 T The strain has the preservation number CCTCC AB 2019071.

2. The application as described in claim 1, characterized in that, The application method involves mixing *Pseudomonas bariensis* R90 with *L. 1m* of red oligotrophic bacteria from sewage. T Co-cultured on MS inorganic salt medium containing 0.04–0.06 g / L glucose.

3. The application as described in claim 1, characterized in that, The application method involves mixing *Pseudomonas bariensis* R90 with *L. 1m* of red oligotrophic bacteria from sewage. T Co-cultured on MS medium containing 0.05 g / L glucose.

4. The application as described in claim 2, characterized in that, The MS inorganic salt culture medium comprises the following components: glucose 0.04~0.06 g / L, Na2HPO4•12H2O 9.0 g / L, KH2PO4 1.5 g / L, (NH4)2SO4 1.0 g / L, MgSO4•7H2O 0.4 g / L, trace elements 1.0 mL / L, agar 20 g / L, sterilized at 121℃ for 20 min; The trace elements include the following components: H3BO3 0.3g / L, CaCl2•6H2O 0.2g / L, ZnSO4•7H2O 0.1g / L, MnCl2•4H2O 30mg / L, NaMoO4•2H2O 30mg / L, NiCl2•6H2O 20mg / L, and CuSO4•5H2O 10mg / L.

5. The wastewater red oligotrophic bacteria lm1 as described in claim 1 T Application in promoting the growth of the aforementioned Pseudomonas bariensis R90.

6. The application as described in claim 5, characterized in that, The application method involves mixing *Pseudomonas bariensis* R90 with *L. 1m* of red oligotrophic bacteria from sewage. T Co-cultured on MS inorganic salt medium containing 0.04–0.06 g / L glucose.

7. The wastewater red oligotrophic bacteria lm1 as described in claim 1 T Application in recruiting the aforementioned Pseudomonas bariensis R90.

8. The application as described in claim 7, characterized in that, The application method involves mixing *Pseudomonas bariensis* R90 with *L. 1m* of red oligotrophic bacteria from sewage. T Co-cultured on MS inorganic salt medium containing 0.04–0.06 g / L glucose.

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