Stenotrophomonas maltophilia with flocculation activity, biological flocculant and application of stenotrophomonas maltophilia
By using the Stenotrophomonas maltophilia XN01 strain to produce extracellular polymers, a bioflocculant was prepared, which solved the problems of secondary pollution in chemical flocculation and low efficiency in physical sedimentation, achieving efficient and safe wastewater treatment.
Patent Information
- Application Number
- CN202511147955.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-16
- Publication Date
- 2025-11-14
AI Technical Summary
Existing chemical flocculation methods pose a risk of secondary pollution, while physical sedimentation methods are inefficient and costly, making it difficult to effectively remove heavy metals and organic pollutants from wastewater. Microbial flocculants are also rare in terms of having both high flocculation activity and safety.
Stenotrophomonas sp. XN01 strain was used to produce extracellular polymers to achieve efficient flocculation and prepare a bioflocculant for wastewater treatment. Combined with coagulants such as CaCl2, pregelatinized starch, and diatomaceous earth, stable flocs were formed.
It achieves a high flocculation rate (up to 57.43%), is highly safe, produces no secondary pollution, can quickly adsorb and degrade organic pollutants in wastewater, reduces treatment costs, and is suitable for large-scale production.
Smart Images
Figure CN120944766A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial and sewage sludge treatment technology, specifically relating to a Stenotrophomonas maltophilia with flocculation activity, a bioflocculant and its application. Background Technology
[0002] Traditional wastewater treatment methods primarily address the difficulty in settling colloidal suspensions in wastewater by employing chemical flocculation and physical sedimentation. Chemical flocculation typically uses inorganic flocculants (such as polyaluminum chloride and polyferric sulfate) or organic flocculants (such as polyacrylamide) to cause particle flocculation and sedimentation through charge neutralization and bridging. However, chemical flocculants may remain in the water, causing secondary pollution, and the environmental risks and ecological costs of long-term use cannot be ignored. In particular, the residues of heavy metal ions and organic matter may pose long-term hazards to the environment and human health. Chemical flocculation requires precise control of pH, temperature, and flocculant dosage, making it complex to operate and demanding on advanced equipment. While physical sedimentation is simpler to operate, it has lower treatment efficiency and may generate large amounts of sludge, further increasing treatment difficulty and cost. Furthermore, physical methods are inconsistent in their effectiveness against dissolved pollutants (such as heavy metal ions and organic pollutants), making complete removal of pollutants difficult.
[0003] In contrast, microbial agents exhibit low-carbon and green advantages in wastewater treatment: First, they are environmentally friendly, produce no secondary pollution, and are biodegradable; second, they efficiently remove pollutants, capable of degrading various pollutants such as organic matter, heavy metals, nitrogen, and phosphorus, with high treatment efficiency; third, they have low operating costs and simple equipment requirements, reducing treatment costs. Therefore, microbial flocculants are one of the main directions for promoting the environmental protection industry towards the policy of "synergistic efficiency improvement in pollution reduction and carbon reduction." Although many microorganisms have been found to have flocculation effects, there are few microbial strains that combine high flocculation activity with safety. Therefore, screening and discovering microbial strains that combine high flocculation activity with safety is of great significance. Summary of the Invention
[0004] To address the aforementioned problems, this invention provides a Stenotrophomonas maltophilia with flocculation activity, a bioflocculator, and its applications. The Stenotrophomonas maltophilia XN01 strain provided by this invention can produce a large amount of extracellular polymers, exhibits strong bioflocculation activity, and achieves a flocculation rate of up to 57.43% for kaolin. It also has the advantages of stable performance, no pollution, and high safety, and can be used to prepare bioflocculators or purify wastewater.
[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides a Stenotrophomonas maltophilia with flocculation activity, the taxonomic name of which is... Stenotrophomonassp. is deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO:65427.
[0006] A second aspect of the present invention provides a fermentation broth, which is obtained by the following preparation method: The Stenotrophomonas maltophilia described above was inoculated into a liquid culture medium and cultured at 26℃~30℃ for 1 to 5 days to obtain the fermentation broth.
[0007] Furthermore, the liquid culture medium is LB liquid culture medium.
[0008] A third aspect of the present invention provides the application of the above-described Stenotrophomonas maltophilia or fermentation broth in the preparation of bioflocculants.
[0009] A fourth aspect of the present invention provides a bioflocculant containing the above-described Stenotrophomonas maltophilia or fermentation broth, and a carrier or coagulant aid.
[0010] Furthermore, the biological coagulant is CaCl2, pregelatinized starch, diatomaceous earth, quicklime, polyaluminum chloride, polyferric sulfate, or polyacrylamide.
[0011] Furthermore, the flocculant is a liquid formulation or a powder.
[0012] The fifth aspect of the present invention provides the application of the above-described Stenotrophomonas maltophilia, fermentation broth, or bioflocculant in the removal of heavy metal ions or the settling of suspended solids.
[0013] Furthermore, the solid suspension is particulate organic carbon or organic nitrogen.
[0014] The sixth aspect of the present invention provides an application of the above-mentioned Stenotrophomonas maltophilia, fermentation broth, or bioflocculant in wastewater treatment. Stenotrophomonas maltophilia can produce a large number of extracellular polymers, which can rapidly and effectively adsorb and degrade organic pollutants in wastewater, and are used for water purification of polluted water bodies.
[0015] The seventh aspect of the present invention provides a method for purifying wastewater, wherein the above-mentioned Stenotrophomonas maltophilia, fermentation broth, or bioflocculant is added to the wastewater and reacted for 1 to 8 days.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses a Stenotrophomonas maltophilia with flocculation activity, the taxonomic name of which is [missing information]. StenotrophomonasThe strain *Stenotrophomonas maltophilia* sp., deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC NO:65427, provides the following: *Stenotrophomonas maltophilia* can produce a large amount of extracellular polymers, enabling rapid and effective adsorption and degradation of organic pollutants in wastewater. It exhibits strong bioflocculation activity, achieving a flocculation rate of up to 57.43% for kaolin. *Stenotrophomonas maltophilia* and its fermentation broth can be used to prepare bioflocculators or directly purify wastewater. The *Stenotrophomonas maltophilia* strain, fermentation broth, or microbial flocculant of this invention also possesses advantages such as stable performance, no pollution, high safety, and scalability, showing promising application prospects.
[0017] Instructions for the Preservation of Biological Materials Stenotrophomonas sp. XN01, referred to as strain XN01 in this invention, was deposited on November 6, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 65427). The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China. The taxonomic name is [not provided in the original text]. Stenotrophomonas sp., Latin name is Stenotrophomonas sp. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1 This is a statistical chart comparing the flocculation performance of different flocculants. Strain EX01 represents Microbacterium (…). Microbacterium Fermentation broth of strain EX01, strain WN2 represents Trichomonas vaginalis (sp.) Comamonas Fermentation broth of strain WN2, strain XN02 represents Acinetobacter (sp.) WN2. Acinetobacter Fermentation broth of strain XN02, strain XN01 represents Stenotrophomonas maltophilia (sp.) Stenotrophomonas strain XN01 (sp.)
[0020] Figure 2 OD of XN01 bacterial culture 600 The relationship between the value and the flocculation rate.
[0021] Figure 3 Correlation analysis was conducted on the inoculation time of strain XN01 with total organic carbon (TOC) and total nitrogen (TNb) content. Figure 3 Figure A in the figure shows the correlation analysis between the inoculation time and TOC content of strain XN01. Figure 3 Figure B in the figure shows the correlation analysis between the inoculation time of strain XN01 and the TNb content.
[0022] Figure 4 The morphology and structure of flocs. Figure 4 The scale bar for Figure A in the image is 10 μm. Figure 4 The scale bar of Figure B in the image is 5 μm. Detailed Implementation
[0023] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.
[0024] The *Trichomonas vaginalis* in this invention ( Comamonas The preservation number of strain WN2 is GDMCC 64336, and the microbacterium (sp.) Microbacterium The EX01 strain was donated by the Fuzhou Institute of Marine Science and Technology, and it contains Acinetobacter (sp.) Acinetobacter The XN02 strain (sp.) was donated by the Fuzhou Institute of Oceanography.
[0025] Microbial agents have shown advantages in wastewater treatment due to their low carbon and green properties. Although many microorganisms have been found to have flocculation effects, there are few microbial strains that combine high flocculation activity with safety. Therefore, screening and discovering microbial strains that combine high flocculation activity with safety is of great significance.
[0026] This invention provides a *Stenotrophomonas maltophilia* with flocculation activity, a bioflocculating agent, and its applications. The taxonomic name of this *Stenotrophomonas maltophilia* is... Stenotrophomonas This strain, *Stenotrophomonas maltophilia*, is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 65427). The *Stenotrophomonas maltophilia* provided by this invention can produce a large amount of extracellular polymers, enabling rapid and effective adsorption and degradation of organic pollutants in wastewater. It exhibits strong bioflocculation activity, achieving a flocculation rate of up to 57.43% for kaolin. *Stenotrophomonas maltophilia* and its fermentation broth can be used to prepare bioflocculators or directly purify wastewater.
[0027] Example 1: Screening of strains with flocculation ability Kaolin particles are typically very small (usually less than 2 μm in diameter) and have a negatively charged surface, making them prone to forming stable suspensions that are difficult to separate by natural sedimentation. The particle size and surface properties of kaolin particles are similar to suspended solids in wastewater, effectively simulating wastewater turbidity. Furthermore, kaolin suspensions exhibit good stability over short periods, and kaolin is environmentally friendly and harmless to humans. The resulting kaolin suspensions are also easy to settle and treat, making them suitable for flocculation experiments under laboratory conditions. Therefore, this invention utilizes kaolin to screen for bacterial strains with flocculation capabilities.
[0028] Isolation, culture and identification of strain XN01: The XN01 strain of this invention was isolated from marine tidal flat sediments.
[0029] Separation: Place the sediment sample to be separated in a test tube, add sterile water and perform multiple serial dilutions, spread it on LB solid medium and incubate at 28°C.
[0030] Purification: Once a single colony has formed, pick it with an inoculation needle and purify the strain by streak plating for 5 generations. After purification, the strain is obtained and named strain XN01.
[0031] Identification: The purified XN01 strain was cultured on a large scale, and bacterial DNA was extracted and sequenced. The 16S rDNA gene sequence of the XN01 strain is shown in SEQ ID NO.1: SEQ ID NO.1:
[0032] Preparation of kaolin suspension: Prepare a clean, impurity-free beaker, add 40g of kaolin powder to the beaker, and dilute to 1L with deionized water to prepare a 4w / v kaolin suspension stock solution. Mix the kaolin suspension stock solution evenly with a magnetic stirrer for 3 hours, and adjust the pH to 4.5.
[0033] Experimental groups: based on OD 600 Fermentation broth of Stenotrophomonas maltophilia XN01 strain with a value of 0.8, and Trichophyton spp. ( Comamonas Fermentation broth of strain WN2, Microbes (sp.) Microbacterium Fermentation broth of strain EX01 and Acinetobacter (sp.) EX01 and Acinetob Acinetobacter Fermentation broth of strain XN02 was used as the experimental group, and 1 wt% potato starch solution, 0.5 wt% pregelatinized starch solution, and 0.1 wt% polyacrylamide (PAM) solution were used as the control groups.
[0034] 2 mL of experimental or control group sample and 2 mL of 1 wt% CaCl2 solution were added to 46 mL of kaolin suspension. The absorbance at 540 nm was measured and the flocculation rate was calculated according to the flocculation activity formula.
[0035] According to the flocculation activity formula: flocculation rate E (%) = (AB) / A × 100%, where A is the absorbance of the control group and B is the absorbance of the experimental group.
[0036] Experimental results Figure 1 As shown, calculations revealed that the XN01 strain fermentation broth exhibited the highest flocculation rate, reaching 57.43%, significantly higher than other strains (all with flocculation rates below 10%), indicating that the XN01 strain demonstrated the best flocculation effect on kaolin. In comparison, the flocculation rate of the XN01 strain was comparable to that of a 1wt% potato starch solution and a 0.5wt% pregelatinized starch solution. Although the flocculation rate of the XN01 strain fermentation broth was lower than that of a 0.1wt% polyacrylamide (PAM) solution, PAM, as a synthetic organic polymer flocculant, suffers from high cost, complex operation, and potential secondary pollution, resulting in significant limitations in its application. The XN01 strain effectively overcomes these issues, demonstrating broad application prospects in wastewater treatment and water purification.
[0037] Example 2: Flocculation Performance Test of Strain XN01 Take 1L of cooled and sterilized LB liquid culture medium, add 4g of kaolin and mix well to prepare a 0.4wt% kaolin-LB suspension. In a clean bench, inoculate 100μL of XN01 bacterial culture and place in a shaking incubator at 200rpm for fermentation at 28℃. During the incubation period, samples are continuously taken, and the OD of the fermentation broth cells is measured using an ultraviolet spectrophotometer. 600 The flocculation rate of the fermentation broth was also measured. The flocculation rate was measured as follows: 2 mL of fermentation broth (strain culture medium) and 2 mL of 1 wt% CaCl2 were added to 46 mL of kaolin suspension. The mixture was stirred at 200 rpm for 1 min, then at 100 rpm for 3 min. After standing for 5 min, the supernatant was collected and the absorbance at 540 nm was measured using a visible spectrophotometer. Pure water was used as a blank control.
[0038] Experimental results are as follows Figure 2 As shown, with the OD of the fermentation broth of strain XN01... 600 With the increase of the OD value, the flocculation rate of strain XN01 showed a trend of first increasing and then decreasing. When the culture time was 2 days, the OD value of the fermentation broth was... 600 The highest flocculation rate, reaching 57.43%, was observed at a value of 0.819. Furthermore, this indirectly suggests that the improved flocculation performance of strain XN01 is not entirely determined by cell concentration. The underlying principle may be that the extracellular polymeric substances (EPS) produced by strain XN01 possess both aggregation and sedimentation potential. 600 With a value of 0.819, strain XN01 exhibited the best EPS flocculation ability.
[0039] Example 3: EPS flocculation ability test of strain XN01 To further verify whether the flocculation ability of strain XN01 is directly related to EPS production, as shown in Example 2, an experiment was designed to test the EPS flocculation ability of strain XN01. 1000 mL of distilled water and 10 g of LB liquid medium were added to an Erlenmeyer flask, which was then sterilized at 120°C. After cooling to room temperature, 1 g of kaolin was added, followed by 1000 μL of XN01 bacterial culture. The flask was incubated at 28°C and 100 rpm. Samples were taken every two days during the fermentation process.
[0040] Cell concentration in fermentation broth: OD of the sample was measured using an ultraviolet spectrophotometer. 600 value.
[0041] EPS determination: Centrifuge the above fermentation broth sample at 3200 rpm for 30 min, remove the supernatant, transfer to a glass test tube, add 5 mL of 9wt% NaCl solution, mix well, and heat at 100℃ for 1 h. After heating, cool to room temperature, transfer to a centrifuge tube again, centrifuge at 3200 rpm for 30 min, retain the supernatant, and measure the total organic carbon (TOC) and total nitrogen (TNb) content of the supernatant to reflect the EPS produced by the experimental strain.
[0042] TOC and TNb refer to the total amount of carbon and nitrogen in all organic compounds in water or solution. TOC includes dissolved organic carbon (DOC) and particulate organic carbon (POC). Polysaccharides, proteins, nucleic acids, and lipids in secreted EPS are important sources of organic carbon; therefore, there is a direct positive correlation between EPS production levels and TOC and TNb levels.
[0043] Experimental results are as follows Figure 3 As shown, the concentration of XN01 cells increased with the increase of inoculation and culture time. At the same time, the TOC and TNb contents of EPS secreted by XN01 also gradually increased. When the culture time was 8 days, the contents of TOC and TNb reached the peak, indicating that the level of EPS secretion reached the highest at this time, the flocculation ability of the bioflocculator was the strongest, and it could effectively adsorb and degrade organic pollutants in wastewater.
[0044] Example 4: Analysis of the flocculation morphology and structure of XN01 bacterial cells To further investigate the flocculation mechanism of strain XN01, flocs were collected and their morphological structure was analyzed using scanning electron microscopy (SEM). This SEM analysis employed a Hitachi SU8000 field emission scanning electron microscope (FE-SEM) at an accelerating voltage of 5.0 kV to observe the floc samples of XN01.
[0045] like Figure 4 As shown in the figure, electron microscopy clearly reveals that, under static conditions, a large amount of kaolinite is adsorbed onto the spherical surface of the XN01 bacteria and aggregates into a granular structure with a diameter exceeding 10µm, exhibiting both loose porous and dense structural morphologies. This indicates that the flocculation mechanism of the XN01 strain primarily involves the formation of bioflocs through the production of EPS micelles by microorganisms, resulting in a flocculent granular structure similar to alum flocs, thus achieving effective and rapid sedimentation.
[0046] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.
[0047] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A Stenotrophomonas maltophilia with flocculation activity, characterized in that, The taxonomic name of *Stenotrophomonas maltophilia* is: Stenotrophomonas sp. is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNO:65427.
2. A fermentation broth, characterized in that, The fermentation broth is obtained by the following preparation method: The Stenotrophomonas maltophiliae of claim 1 was inoculated into a liquid culture medium and cultured at 26℃~30℃ for 1 to 5 days to obtain the fermentation broth.
3. The use of Stenotrophomonas maltophiliae according to claim 1 or the fermentation broth according to claim 2 in the preparation of a bioflocculant.
4. A bio-flocculating agent, characterized in that, The bioflocculent contains Stenotrophomonas maltophilia as described in claim 1 or the fermentation broth as described in claim 2, as well as a carrier or coagulant.
5. The bioflocculator according to claim 4, characterized in that, The coagulant is CaCl2, pregelatinized starch, diatomaceous earth, quicklime, polyaluminum chloride, polyferric sulfate, or polyacrylamide.
6. The bioflocculator according to claim 4, characterized in that, The bioflocculant is a liquid formulation or a powder.
7. The application of Stenotrophomonas maltophiliae according to claim 1, the fermentation broth according to claim 2, or the bioflocculant according to claim 4 in the removal of heavy metal ions or the settling of suspended solids.
8. The application of Stenotrophomonas maltophilia according to claim 1, the fermentation broth according to claim 2, or the bioflocculant according to claim 4 in wastewater treatment.
9. The application according to claim 8, characterized in that, The Stenotrophomonas maltophilia, fermentation broth, or bioflocculant are used for water purification of polluted water bodies.
10. A method for purifying wastewater, characterized in that, The Stenotrophomonas maltophiliae of claim 1, the fermentation broth of claim 2, or the bioflocculant of claim 4 are added to the wastewater and reacted for 1 to 8 days.