Enterobacter ludwigii and application thereof in preparation of microbial flocculant

Abstract

The invention discloses Enterobacter ludwigii and an application of the Enterobacter ludwigii in preparation of a microbial flocculant. The strain is classified and named as Enterobacter ludwigii, and is preserved in the China General Microbiological Culture Collection Center on February 21, 2025, the preservation number is CGMCC No.33608, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. A high-yield extracellular product is obtained after fermentation culture conditions are optimized, the extracellular product is used as a microbial flocculant (RCG2P), and the flocculation activity of the extracellular product is compared and investigated. The flocculation efficiency of the RCG2P measured by a kaolin suspension flocculation method is 69.00% (1 mg / mL), when the concentration of the kaolin suspension is increased, the flocculation efficiency of the RCG2P is not obviously reduced when the concentration of the RCG2P is 1 mg / mL, the RCG2P is relatively stable, and the RCG2P has the potential of serving as an efficient and stable flocculant.

Description

Technical Field

[0001] The present invention belongs to the technical field of sewage treatment microbial flocculants, and specifically relates to an Enterobacter ludwigii and its application in the preparation of microbial flocculants, and relates to the application of microbial extracellular products in sewage flocculation. Background Art

[0002] China is facing problems such as uncoordinated distribution of water resources, serious water pollution, and water resource waste. The problem of water pollution has received increasing attention from the country. How to effectively and scientifically treat wastewater is an urgent problem to be solved today. Flocculants are widely used in the separation in sewage treatment, such as drinking water purification, wastewater treatment, activated sludge dewatering, etc. They usually carry different charges and can adsorb insoluble impurities in water and substances with charges opposite to those carried by the flocculant itself, so as to make them settle and achieve the purpose of water purification. The common industrial water treatment flocculants we see are high molecular polymers, such as polyacrylamide types, polyaluminum chloride types, and also include the flocculants commonly used in life before, such as alum (potassium alum). The types of flocculants are roughly divided into the following four categories: organic flocculants, such as the polyacrylamide types mentioned above; inorganic flocculants, such as alum; composite flocculants, such as polysilicate metal salts; microbial flocculants. The application methods mainly include directly using cells for treatment, or applying extracellular products of cells, extracting and purifying them and then applying them to water treatment. Traditional flocculants will introduce other harmful substances while treating water, making subsequent treatment difficult and causing harm to the environment and humans. Therefore, microbial flocculants have received extensive attention due to their basically harmless treatment and high treatment efficiency.

[0003] At present, the activated sludge method is a commonly used method in wastewater treatment, mainly relying on microorganisms in the sludge to produce effective flocculating components to flocculate some adsorbable impurities in the sewage, so as to achieve the purpose of water purification. In wastewater treatment, using microbial flocculants to treat wastewater instead of the activated sludge method has the advantages of low cost, convenient treatment, no secondary pollution, high safety, and non-toxicity. Although many microorganisms have been found to be able to produce bioflocculants, and bioflocculants are considered to be able to effectively replace chemical flocculants in wastewater treatment, there are few reports on bioflocculants of safe strains. The research and search for efficient flocculants have received extensive attention around the world. Microbial flocculants can not only adsorb large particulate suspensions in water, but also have good adsorption and sedimentation effects on some soluble impurities. Therefore, the present invention aims to develop a microbial flocculant with high flocculation activity, which is of great significance for the harmless treatment of wastewater. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides an Enterobacter ludwigii and its application in the preparation of a microbial flocculant for highly efficient and low-cost sewage flocculation.

[0005] To solve the above problems, the technical solutions adopted in the present invention are as follows: In the first aspect of the present invention, an Enterobacter ludwigii RGC2 is provided. This strain was deposited on February 21, 2025, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 33608, and the deposit address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The Enterobacter ludwigii RGC2 was screened and purified from activated sludge. The specific method is as follows: After gradient dilution of the activated sludge suspension, it was evenly coated on a solid medium plate using a dilution spreader and cultured in a constant temperature incubator at 37 °C for 24 - 48 h. Observe the state of the colonies on the medium. Those with a moist and highly viscous surface are strains with flocculation potential. These colonies were picked out with an inoculation needle and then subjected to plate streaking and purification culture for 2 - 3 d. Repeat the above process multiple times until a purified strain was obtained through biochemical analysis and named RCG2.

[0007] In some specific embodiments of the present invention, gene sequence determination and Gram staining were performed on the RCG2 strain, and it was determined that the strain belongs to the phylum Proteobacteria, genus Enterobacter, and is a Gram-positive bacterium.

[0008] In the second aspect of the present invention, the above Enterobacter ludwigii RGC2 is provided for use in the preparation of a microbial flocculant.

[0009] Specifically, the preparation method of the above microbial flocculant includes: Taking an appropriate amount of the obtained RCG2 strain and inoculating it into an LB basal medium with an inoculation loop, and performing proliferation culture in a constant temperature shaker at 28 °C and 140 rpm. After 1 d, 5 mL of the cultured bacterial liquid was taken from it and subjected to seed culture in an LB medium under the same conditions. The bacterial liquid in the logarithmic growth phase (OD600 = 0.6 - 1.4) was used as the seed liquid; The seed liquid was inoculated into a fermentation medium, the fermentation temperature was 22 - 40 °C, the fermentation time was 5 - 11 d, and the supernatant was collected by centrifugation; The supernatant was mixed with a pre-cooled ethanol solution for alcohol precipitation extraction, and the precipitate was freeze-dried to obtain the microbial flocculant.

[0010] In the above technical solution, the composition of the fermentation medium includes 20 - 30 g / L of glucose and 3 g / L of potassium nitrate. Further, the fermentation medium also includes an HTM basal medium, and the composition of the HTM basal medium is 1.3 g / L of NaH2PO4·2H2O; 0.115 g / L of MgSO4; 0.0228 g / L of FeSO4·7H2O; 0.07 g / L of CaCl2; 0.0075 g / L of ZnCl2; 0.0033 g / L of MnSO4·H2O.

[0011] In some specific embodiments of the present invention, glucose and potassium nitrate are added to the HTM basal medium and dissolved thoroughly, then sealed and sterilized to obtain the fermentation medium.

[0012] In the above technical solution, the centrifugation is carried out at 14 °C and 5000 rpm for 10 min.

[0013] In the above technical solution, the ethanol solution is 95% ethanol.

[0014] In the above technical solution, after the supernatant is mixed with the pre-cooled ethanol solution, the final concentration (volume fraction) of ethanol is 50 - 80%.

[0015] In a specific embodiment of the present invention, the microbial flocculant RCG2P obtained by ethanol precipitation of the RCG2 fermentation culture product with 80% ethanol by final concentration is detected, and the polysaccharide content in the RCG2P component is 36.2%, the protein content is 28.86%, and the inorganic content is 28.04%.

[0016] The third aspect of the present invention provides a composite flocculant, including the above microbial flocculant.

[0017] The fourth aspect of the present invention provides the application of the above RCG2 strain, microbial flocculant or composite flocculant in sewage treatment flocculation.

[0018] The microbial flocculant prepared from Enterobacter ludwigii RGC2 provided by the present invention has higher flocculation activity and flocculation efficiency compared with the existing flocculant potassium alum dodecahydrate, and the dosage of this microbial flocculant is small, having the potential to be an efficient and stable flocculant. Description of the Drawings

[0019] Figure 1 It is the colony morphology of the RCG2 strain on the plate medium.

[0020] Figure 2 It is the phylogenetic tree of the RCG2 strain.

[0021] Figure 3 It is the growth curve of strain RCG2.

[0022] Figure 4 They are the (A) Gram staining results of strain RCG2 (magnification: 2000), (B) RCG2P powder, and (C) the morphology of RCG2P solution.

[0023] Figure 5 It is the comparison of the flocculation activities of microbial flocculant RCG2P and potassium alum dodecahydrate in kaolin suspension. Detailed implementation manners

[0024] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention will be given in conjunction with specific embodiments.

[0025] The culture media and their preparation methods used in the following examples are as follows: Preparation of HTM basal medium: Weigh 13 g of NaH2PO4·2H2O, 1.15 g of MgSO4, 0.228 g of FeSO4·7H2O, 0.7 g of CaCl2, 0.075 g of ZnCl2, and 0.033 g of MnSO4·H2O accurately with an analytical balance. Measure 100 mL of distilled water accurately with a measuring cylinder, pour it into a beaker to completely dissolve the above reagents, and then transfer it to a 1000 ml volumetric flask for volume fixation. Take 100 mL of the prepared 10×HTM medium and place it in a 1000 mL conical flask, add 900 mL of distilled water to prepare 1×HTM basal medium, and seal it.

[0026] Preparation of solid medium: Weigh 2 g of agar powder and 6 g of glucose and 0.6 g of potassium nitrate accurately. Put the above weighed reagents into a conical flask to be sterilized, add 200 mL of 1×HTM medium, seal it well and perform high-pressure steam sterilization under the conditions of 121 °C for 20 min. After sterilization is completed, transfer the medium to the workbench, turn on the ultraviolet lamp of the ultra-clean workbench in advance to irradiate and sterilize its space for 15 - 30 min. After the medium cools to a non-hot temperature, turn off the ultraviolet lamp, normally turn on the workbench, and perform the plate pouring operation. After it solidifies, invert it to form a smooth plate medium.

[0027] Preparation of fermentation medium: Weigh 30 g of glucose and 3 g of potassium nitrate accurately, add 1000 mL of 1×HTM basal medium to complete the preparation. Divide 1000 mL equally into 10 250 mL conical flasks, each being 100 mL. After sealing and sterilizing, it is reserved for use.

[0028] Preparation of LB medium: Weigh 10 g of peptone, 5 g of yeast extract, and 10 g of sodium chloride accurately using an electronic balance. Dissolve them in 1000 mL of distilled water and stir until completely dissolved. Seal and sterilize it for later use. Example 1

[0029] The activated sludge used in the present invention was taken from the secondary sedimentation tank in Changzhou Wastewater Treatment Plant. Take a small amount of sludge, add 25 mL of sterile water, place it in a 50 mL conical flask, and let it stand for half an hour. Then take the supernatant, which is the activated sludge suspension.

[0030] After preparing the solid medium, take 1 mL of the activated sludge suspension and put it into a 10 mL centrifuge tube. Add 9 mL of sterile water for gradient dilution. Then the dilution factor of this centrifuge tube is 10 times. Make a label on it. Repeat the above operation 2 times to obtain gradient dilution solutions with dilution factors of 10 -2 , 10 -3 respectively. Take 1 mL from each of them and spread it evenly on the plate medium using a spreader. Place it in a constant temperature incubator at 37 °C for 24 - 48 h. After obtaining the results of dilution coating separation, observe the state of the colonies on the medium. Those with a moist surface and relatively high viscosity are strains with flocculation potential. Pick out these colonies with an inoculation needle and perform plate streaking purification culture for 2 - 3 d (repeat the above process multiple times until it is a pure strain through biochemical analysis). Name the strain with the potential to produce extracellular flocculant as RCG2 ( Figure 1 ). Preserve the strain at low temperature for later use.

[0031] Perform gene sequence determination on the RCG2 strain. After obtaining the results, use BLAST for sequence alignment on the NCBI official website. After successful alignment, select some strains with relatively high alignment rates and use MEGA software to construct a phylogenetic tree to finally obtain the genus and phylum of the strain to be tested.

[0032] After genome sequencing of the strain, input its gene sequence into the National Centre for Biotechnology Information database for sequence alignment. After completion, input multiple groups of microbial genomes with relatively high alignment rates into MEGA software to construct the phylogenetic tree of the RCG2 strain. After completion, as Figure 2 shown. The part marked in red in the figure is the branch where the RCG2 strain is located. By consulting the literature, it is known that it belongs to the phylum Proteobacteria and the genus Enterobacter. This type of bacteria is widely present in soil, wastewater, and domestic waste. Its characteristics are facultative anaerobic, with low nutritional requirements, capable of growing diffusely on a simple solid medium, and having a relatively fast growth rate.

[0033] The RCG2 strain is classified and named as Enterobacter ludwigii. It was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 21, 2025, with the deposit number CGMCC No. 33608, and the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Example 2

[0034] Determination of the strain growth curve: In a laminar flow hood, an appropriate amount of the obtained RCG2 strain was inoculated into the LB basal medium with an inoculation loop and cultured for proliferation in a constant temperature shaker at 28 °C and 140 rpm. After 1 day, 5 mL of the cultured bacterial liquid was taken therefrom and used for seed culture in the LB medium under the same conditions. Every 6 h, 4 mL was taken therefrom and the absorbance was measured at 600 nm with an ultraviolet spectrophotometer. Sampling was carried out at least 6 times. A growth curve was plotted with the abscissa as time t and the ordinate as the absorbance OD600 to obtain the final result. The strain grew slowly within the first 6 h and was in the lag phase. It was in the logarithmic growth phase from 6 h to 30 h and in the stationary phase from 30 h to 36 h, basically stopping growing. Subsequently, when cultured further, the strain entered the decline phase and the absorbance decreased (as Figure 3 ). Generally speaking, the growth rate of this strain is fast and it is suitable for use in fermentation. Example 3

[0035] In this example, the bacterial liquid with an OD 600 of 0.7 cultured in the LB medium was used as the seed liquid to optimize the fermentation conditions.

[0036] Effect of time on fermentation: The above-inoculated fermentation medium was fermented for 5 d, 6 d, 7 d, 8 d, 9 d, 10 d, and 11 d respectively under the same conditions. Finally, after steps such as alcohol precipitation extraction and freeze-drying of the product, the product was obtained, and the final quality of the product was analyzed to determine the optimal fermentation duration among the above times. When the fermentation times were 5 d, 6 d, 7 d, 8 d, 9 d, 10 d, and 11 d respectively, the quality of the product increased with the increase of the fermentation time. Accordingly, among these 7 fermentation times, the product quality was the highest when fermented for 9 d (as shown in Table 1).

[0037] Effect of temperature on fermentation: The above-inoculated fermentation medium was fermented at 28 °C, 31 °C, 34 °C, 37 °C, and 40 °C respectively while keeping the other conditions unchanged. After product extraction, quality comparison was carried out to determine the optimal fermentation temperature. When the fermentation temperatures were 22 °C, 25 °C, 28 °C, 31 °C, 34 °C, 37 °C, and 40 °C respectively, the quality of the product did not show a linear change with the increase of fermentation temperature, but reached the maximum value at 28 °C. Therefore, among these 7 fermentation temperatures, 28 °C is the optimal fermentation temperature (as shown in Table 2).

[0038] Effect of medium components on fermentation: Accurately weigh 2 g, 3 g, 4 g, 5 g, and 6 g of glucose respectively on an electronic balance into different conical flasks, add an equal amount of 0.3 g of potassium nitrate and 100 mL of the above 1×HTM basal medium. In a laminar flow hood, inoculate 5 mL of the seed liquid into the fermentation broth, and let it ferment for 7 days at 28 °C and 140 rpm in a constant temperature shaker. Finally, the fermentation broth was centrifuged, precipitated with alcohol, extracted and freeze-dried to obtain the product, and the quality of the product was compared to determine which component was more conducive to fermentation. The results showed that when the glucose concentrations were 2%, 3%, 4%, 5%, and 6% respectively, the quality of the product increased with the increase of the carbon source concentration. Therefore, among these 5 concentrations, 6% glucose concentration is the optimal concentration. In the results of the single-factor experiment, the optimal fermentation time is 9 days, the optimal fermentation temperature is 28 °C, the optimal glucose concentration is 6%, and the product concentration is 1.553 g / L (as shown in Table 3).

[0039] Table 1 Product quality at different fermentation times

[0040] Table 2 Product quality at different fermentation temperatures

[0041] Table 3 Product quality under different medium components Example 4

[0042] Effect of extraction conditions on yield: Four final concentrations of ethanol (50%, 60%, 70%, 80%) were used to extract the same product respectively, and the quality was measured after freeze-drying to determine the optimal alcohol precipitation concentration.

[0043] According to the optimal fermentation conditions, the selected strains were inoculated in seed culture medium (same as fermentation medium) for expansion culture for 1 day. After completion, 5 mL of seed solution was inoculated in 100 mL of fermentation medium and fermented for 7 days in a constant temperature shaker at 28 ℃ and 140 rpm. The fermentation broth of each strain was centrifuged at 14 ℃ and 5000 rpm for 10 min to take the supernatant, discard the bacteria, mix the fermentation broth with a pre-cooled 95% ethanol solution, and use ethanol with a final concentration of 50%, 60%, 70%, and 80% to extract the same product by alcohol precipitation. After standing for 3 minutes, the precipitate was removed and squeezed dry, and freeze-dried in a freeze dryer at -80 ℃. After weighing its mass, it was ground into powder for use.

[0044] Table 4 Product quality under different concentrations of alcohol precipitation

[0045] The above 80% alcohol precipitation product was named RCG2P. The total sugar content was determined by sulfuric acid phenol method, the protein content was determined by BCA method, the ash content was determined, and the bacterial species were observed by Gram staining. The components of RCG2P were analyzed. The polysaccharide content was 36.20% by phenol sulfuric acid method, the ash content was 28.04% after calcination in muffle furnace, and the protein content was 28.86% by BCA method. It was observed that the strain RCG2 screened was blue-purple in Gram staining and was short rod-shaped, belonging to Gram-positive bacteria ( Figure 4 A); The extracellular product of the bacteria was freeze-dried and ground into an off-white powder ( Figure 4 B), soluble in water, into a gel ( Figure 4 C). Example 5

[0046] Determination of flocculation activity of composite flocculant: prepare a kaolin suspension with a concentration of 5 g / L, accurately weigh 5 g of kaolin on an electronic balance with weighing paper, add 950 mL of distilled water and a calcium chloride solution with a concentration of 10 g / L, add a buffer solution to adjust the pH to 8, and finally prepare a 5 g / L kaolin suspension. Shake well before use (kaolin suspension will precipitate in the natural state). Take 100 mL of the above suspension, add 1 mL of a flocculant solution with a concentration of 1 mg / mL, place it on a magnetic stirrer at 250 rpm, centrifuge for 5 min, and then let it stand for 10 min. Then measure the light absorption value at 550 nm on a UV spectrophotometer. Use the kaolin suspension without flocculant as a control, and use the absorbance value A measured by the control group minus the absorbance value B measured by adding flocculant, and then divide the result by A and multiply by 100% to obtain the flocculation efficiency (repeat three times and take the average value), so as to calculate the final result.

[0047] From Figure 5 It can be found that, compared with potassium alum, a commonly used flocculant in daily life, RCG2P has better flocculation activity. Moreover, with the change of the concentration of kaolin suspension (5 g / L, 10 g / L, 15 g / L, 20 g / L), the flocculation efficiency is maintained within a certain range, showing the potential to be an efficient and stable flocculant.

[0048] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A Enterobacter ludwigii, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 21, 2025, with a deposit number of CGMCC No.33608, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial flocculant, characterized in that: The method is prepared from the fermentation product of Enterobacter Ludwigii according to claim 1.

3. The method for preparing the microbial flocculant according to claim 2, characterized in that: The method comprises the following steps: performing seed culture of the Enterobacter Ludwigii in LB medium to obtain seed liquid; The seed liquid is inoculated into a fermentation medium for fermentation and cultivation, and the supernatant containing the fermentation product is obtained by centrifugation; the supernatant is subjected to alcohol precipitation with an ethanol solution to extract a precipitate, and the precipitate is freeze-dried to obtain the microbial flocculant.

4. The method for preparing a microbial flocculant according to claim 3, characterized in that: The fermentation medium composition includes 20-60 g / L of glucose and 3 g / L of potassium nitrate; the pH of the fermentation medium is neutral.

5. The method for preparing a microbial flocculant according to claim 3, characterized in that: The fermentation culture temperature is 22-40°C, and the fermentation time is 5-11 days.

6. The method for preparing a microbial flocculant according to claim 3, characterized in that: The volume fraction of the ethanol solution is 95%, and the volume fraction of ethanol after the supernatant is mixed with the ethanol solution is 50%-80%.

7. A composite flocculant, characterized in that: The invention comprises the microbial flocculant according to claim 2 or the microbial flocculant prepared by the method according to any one of claims 3 to 6.

8. Use of the Enterobacter ludwigii according to claim 1, the microbial flocculant according to claim 2, the microbial flocculant prepared by the method according to any one of claims 3 to 6, or the composite flocculant according to claim 7 in sewage treatment flocculation.

Images