Microbial flocculant and method for producing the same

Abstract

The application discloses a kind of microbial flocculants and preparation method thereof, the preparation method includes the following steps: camellia cake meal is mixed with water, ultrasonic, hydrothermal treatment, filtration, filtrate is collected, and camellia cake meal extraction solution is obtained, configuration is camellia cake meal culture medium, inoculation flocculation function bacteria seed liquid is cultivated, and the preparation of microbial flocculants is completed.In the application, the way of ultrasonic extraction and hydrothermal extraction is combined, and camellia cake meal extraction solution with rich nutritional ingredients and good adaptability of various nutrients can be prepared, which can promote the rapid growth of flocculation function bacteria when used for culturing flocculation function bacteria, increase biomass, and induce more extracellular polymeric substance, thereby obtaining microbial flocculants with high yield and high flocculation rate.In the application, camellia cake meal is used as raw material, which can greatly reduce production cost and simplify production steps, promote large-scale use of microbial flocculants, and realize resource utilization of camellia cake meal.

Description

Technical Field

[0001] This invention belongs to the field of microbial flocculant preparation technology, and relates to a microbial flocculant and its preparation method. Background Technology

[0002] Flocculants, a commonly used agent in water treatment, are mainly classified into inorganic flocculants, synthetic organic polymer flocculants, and natural polymer flocculants. Inorganic flocculants typically contain metal ions, and the residual metal ions after addition are difficult to remove, easily causing secondary pollution to water bodies. Although synthetic organic polymer flocculants have low self-toxicity, they are difficult to degrade, and residual monomers can cause birth defects, cancers, and mutations, posing potential health hazards. Natural organic polymer flocculants are generally harmless to humans, easily biodegradable, and safer than inorganic and synthetic organic polymer flocculants.

[0003] Microbial flocculants are natural polymeric flocculants, mostly derived from microorganisms isolated from activated sludge, soil, and water bodies, or their secreted metabolites, such as extracellular polymers, which are high-molecular-weight compounds like glycoproteins, polysaccharides, proteins, and DNA. Unlike traditional flocculants, the components of microbial flocculants are easily decomposed and do not cause secondary pollution to the environment. They are green, safe, and non-toxic flocculants, and are of great significance to environmental protection and human health. To date, microbial flocculants have been used in applications such as heavy metal wastewater treatment, dye wastewater treatment, and sludge dewatering. However, the culture media used in the preparation of existing microbial flocculants mainly consist of sucrose, yeast extract, and various inorganic salts, resulting in complex production processes and high production costs for large-scale applications. To reduce production costs, researchers have proposed using organic wastewater or waste residue to cultivate microorganisms and prepare microbial flocculants. However, many organic wastewaters or waste residues have complex compositions, unpredictable key nutrients, and may contain toxic and harmful substances. This results in the prepared microbial flocculants having defects such as low yield, low flocculation rate, and high toxicity, making it difficult to achieve large-scale production and application of microbial flocculants.

[0004] Camellia oil cake meal is a major byproduct of camellia oil production, rich in nutrients. Besides lignocellulose, it contains bioactive components such as protein, polysaccharides, polyphenols, and tea saponins, making it a potential raw material for preparing microbial alternative culture media. However, in existing technologies, when using camellia oil cake meal extract with extracted tea saponins as a culture medium, the highest flocculation rate of the prepared microbial flocculant on kaolin suspension is only 86.1%, indicating a low flocculation rate. Furthermore, the methods for preparing microbial flocculants using camellia oil cake meal extract require the addition of nutrients such as sucrose, yeast extract, and inorganic salts, further hindering cost reduction. Therefore, developing a high-yield, high-flocculation-rate microbial flocculant from camellia oil cake meal is crucial for reducing production costs and simultaneously achieving high-value utilization of camellia oil cake meal. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the shortcomings of the existing technology by providing a microbial flocculant with high production efficiency, low production cost, high output, and high flocculation rate, as well as its preparation method.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a microbial flocculant includes the following steps:

[0008] S1. Mix camellia seed cake with water, sonicate, hydrothermally treat, filter, collect the filtrate to obtain camellia seed cake extract;

[0009] S2. Prepare a camellia oil cake extract obtained in step S1 into a camellia oil cake culture medium.

[0010] S3. Inoculate the seed liquid of flocculating bacteria into the tea oil cake medium obtained in step S2 for cultivation to complete the preparation of microbial flocculant.

[0011] In a further improvement to the above preparation method, in step S1, the ultrasound is performed at a temperature of 30℃~40℃; the ultrasound time is 20min~50min; the hydrothermal treatment is performed at a temperature of 111℃~126℃; the hydrothermal treatment time is 10min~50min.

[0012] In a further improvement to the above preparation method, in step S1, the ratio of camellia oil cake meal to water is 30g-200g:1L; the camellia oil cake meal also includes the following treatment before use: drying the camellia oil cake meal, crushing it, and passing it through a 40-60 mesh sieve to obtain camellia oil cake meal granules.

[0013] In a further improvement to the above preparation method, in step S1, the ratio of camellia seed cake to water is 50g to 150g: 1L.

[0014] In a further improvement to the above preparation method, in step S1, the polysaccharide content in the camellia oil cake extract is 1800 mg / L to 15000 mg / L, the protein content is 3300 mg / L to 24000 mg / L, and the SCOD content is 18000 mg / L to 110000 mg / L.

[0015] In a further improvement to the above preparation method, in step S2, the preparation method of the camellia oil cake meal culture medium is as follows: Camellia oil cake meal extract, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate are mixed to obtain the camellia oil cake meal culture medium; the concentration of potassium dihydrogen phosphate in the camellia oil cake meal culture medium is 1.0 g / L to 2.0 g / L, and the concentration of dipotassium hydrogen phosphate is 3.0 g / L to 5.0 g / L; the pH value of the camellia oil cake meal culture medium is 5 to 9.

[0016] In a further improvement to the above preparation method, in step S3, the inoculation amount of the flocculating functional bacteria seed liquid is 4% to 5% of the volume of the camellia oil cake culture medium.

[0017] In a further improvement to the above preparation method, step S3 further includes the following treatment of the flocculating functional bacteria seed solution before inoculation:

[0018] (1) The flocculating functional bacteria strain was inoculated into a solid activation medium and cultured at 30℃~35℃ for 12.0h~24.0h to obtain primary seed colonies; the formula of the solid activation medium was: 10.0g / L peptone, 3.0g / L beef extract, 5.0g / L sodium chloride, 20.0g / L agar powder, and pH value of 7~8; the flocculating functional bacteria strain was one of Bacillus polymyxa, lactic acid bacteria, Clostridium butyricum and Halomonas; the Bacillus polymyxa was GA1;

[0019] (2) The primary seed colonies obtained in step (1) are inoculated into liquid proliferation medium and cultured at a temperature of 25℃~30℃ and a rotation speed of 130rpm~150rpm for 12.0h~24.0h to obtain flocculation functional bacteria seed liquid; the formula of the liquid proliferation medium is: 10.0g / L peptone, 3.0g / L beef extract, 5.0g / L sodium chloride, and pH value of 7~8.

[0020] In a further improvement to the above preparation method, step S3 involves culturing for 12.0h to 24.0h at a temperature of 30℃ to 35℃ and a rotation speed of 130rpm to 150rpm, and then culturing for 24.0h to 72.0h at a temperature of 20℃ to 25℃ and a rotation speed of 100rpm to 120rpm. After the culturing is completed, the following treatment is also included: centrifuging the cultured product to remove bacterial cells; the centrifugation speed is 8000rpm to 10000rpm; and the centrifugation time is 8min to 10min.

[0021] As a general technical concept, the present invention also provides a microbial flocculant prepared by the above-described preparation method.

[0022] Compared with the prior art, the advantages of the present invention are as follows:

[0023] (1) To address the problems of low production efficiency, high production cost, low yield, and low flocculation rate in existing microbial flocculant preparation methods, this invention creatively proposes a method for preparing microbial flocculants. First, camellia oil cake is mixed with water and subjected to ultrasonic and hydrothermal treatment. A combination of ultrasonic and hydrothermal extraction is used to fully dissolve the effective components in the camellia oil cake, thereby preparing a camellia oil cake extract rich in nutrients and with good compatibility among the various nutrients. On the one hand, the camellia oil cake extract obtained after the combined ultrasonic and hydrothermal extraction process contains a high content of polysaccharides and proteins, which provides sufficient and balanced nutrients for the subsequent growth of flocculating bacteria (such as Bacillus polymyxa GA1), thus facilitating the acquisition of a higher biomass. On the other hand, after treatment using a combination of ultrasonic and hydrothermal extraction, antibacterial components such as tea saponins and tannins contained in camellia oil cake can be transferred to the camellia oil cake extract. These appropriate amounts of antibacterial components can induce flocculating bacteria to secrete more extracellular polymers. At the same time, the increased extracellular polymers will coat the surface of the flocculating bacteria to form a protective layer, thereby changing the surface characteristics of the bacteria and improving their resistance and survival ability. Based on this, the camellia oil cake extract can be prepared into a camellia oil cake culture medium and inoculated with seed liquid of flocculating bacteria for cultivation. This can promote the rapid growth of flocculating bacteria and produce more extracellular polymers, thereby obtaining a high-yield, high-flocculation-rate microbial flocculant with extracellular polymers as the main component. In addition, in the preparation method of this invention, using camellia oil cake as the main nutrient component of the microbial culture medium can significantly reduce the production cost of microbial flocculants, simplify the production steps of microbial flocculants, and realize the resource utilization of camellia oil by-products. Compared with conventional methods for preparing microbial flocculants, the preparation method of this invention has the following advantages: (a) Low production cost: Using camellia oil cake as raw material to provide the main nutrients required for cultivation can significantly reduce the production cost of microbial flocculants. The raw material cost per unit of flocculant yield is approximately RMB 12,000 / ton, while the raw material cost per unit of flocculant yield is approximately RMB 365,000 / ton when using traditional culture media to prepare microbial flocculants. Therefore, the raw material cost of this invention is reduced by approximately 96.7%; (b) High production efficiency: Compared with traditional culture media, this invention uses camellia oil cake extract, which is rich in nutrients and has good compatibility with various nutrients, as raw material to prepare the culture medium. This results in a higher growth rate and proliferation density of the flocculating bacteria, which is beneficial for the fermentation system to quickly enter the flocculant production stage. The biomass of the flocculating bacteria is increased by at least one order of magnitude, from 0.55 × 10⁻⁶. 7 CFU / mL increased to 1.79 × 10⁻⁶ 8(c) High yield and high quality: In this invention, under the controllable concentration range of components such as tea saponin and tannin, the yield and quality of microbial flocculant products mainly composed of extracellular metabolites of microorganisms can be improved. This can increase the yield of microbial flocculants and enhance their flocculation activity. Specifically, under the condition of a material-to-liquid ratio of 50 g / L for camellia seed cake extract, the yield of microbial flocculants is 6.61 g / L, corresponding to a flocculant yield of 0.13 g / g COC per unit mass of camellia seed cake, which is 20.3% higher than that of traditional culture media. The flocculation rate of kaolin suspension reaches 98.9%, which is 1.02% higher than that of flocculants obtained from traditional culture media.

[0024] (2) In this invention, by optimizing the conditions of ultrasound, specifically by ultrasound at a temperature of 30℃~40℃ for 20min~50min, under the condition of ensuring energy saving, the thermal effect and cavitation effect can be used to enable the camellia oil cake meal to dissolve as much soluble matter as possible in a short time and transfer it into the solution. In particular, it can promote the dissolution of polysaccharides and proteins, which is beneficial to improving the utilization rate of nutrients in the camellia oil cake meal. At the same time, by optimizing the temperature to 30℃~40℃, it is beneficial to control the dissolution of tea saponins and tannins, and to facilitate the regulation of the content of tea saponins, tannins and other components in the extract. Furthermore, under the combined effect of hydrothermal treatment, it is more beneficial to prepare a camellia oil cake meal extract with rich nutrients and good compatibility of various nutrients.

[0025] (3) In this invention, by optimizing the ratio of camellia seed cake to water, specifically optimizing the ratio of the two to 30g~200g∶1L, and especially optimizing the ratio of the two to 50g~150g∶1L, the content of nutrients in the extract can be effectively increased. When it is used to cultivate flocculating bacteria, it is not only conducive to the formation of microbial flocculants with high flocculation rate as the main component of extracellular polymers, but also significantly increases the unit yield of microbial flocculants, which is conducive to improving the utilization rate of camellia seed cake and making the preparation cost lower.

[0026] (4) In this invention, by optimizing the culture conditions, specifically by inoculating the seed liquid of flocculating functional bacteria into the culture medium of camellia cake, first culturing it at a temperature of 30℃~35℃ and a rotation speed of 130rpm~150rpm for 12.0h~24.0h, and then culturing it at a temperature of 20℃~25℃ and a rotation speed of 100rpm~120rpm for 24.0h~72.0h, the microorganisms grow rapidly in the first stage of fermentation and produce a large amount of flocs in the second stage of fermentation. Thus, while reducing the fermentation time, a high-yield and high-flocculation-rate microbial flocculant can be obtained, which is conducive to large-scale preparation and convenient for industrial application. Attached Figure Description

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0028] Figure 1 This is a flowchart of the preparation process of the microbial flocculant in Example 1 of the present invention.

[0029] Figure 2 This is a comparison chart showing the content of polysaccharides, proteins, and SCOD in the extract of camellia seed cake under different material-to-liquid ratios in Examples 1-4 of the present invention.

[0030] Figure 3 This is a comparison diagram showing the growth of bacteria in the culture medium for preparing camellia seed cake under different material-to-liquid ratios in Examples 1-4 of the present invention.

[0031] Figure 4 This is a comparison chart of the bacterial dry weight in the microbial fermentation products under different material-to-liquid ratios in Examples 1-4 of the present invention.

[0032] Figure 5 This is a comparison chart of the yield of microbial flocculants under different feed-liquid ratios in Examples 1-4 of the present invention.

[0033] Figure 6 This is a comparison chart showing the yield of microbial flocculants prepared in camellia seed cake culture media at different pH values ​​in Example 5 of the present invention.

[0034] Figure 7 This is a graph showing the yield of microbial flocculant prepared under different culture time conditions in Example 5 of the present invention.

[0035] Figure 8 This is a comparison chart of the flocculation rates of microbial flocculants prepared under different culture time conditions in Example 5 of the present invention. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0037] The materials and instruments used in the following examples are all commercially available.

[0038] Example 1:

[0039] A method for preparing a microbial flocculant, the process flow diagram of which is shown below. Figure 1 As shown, it includes the following steps:

[0040] (1) Preparation of camellia seed cake extract

[0041] (1.1) Place the camellia oil cake and dry it continuously at 60℃ for 5.0h until constant weight. Then crush it with a pulverizer and use a steel screen to take 40-60 mesh camellia oil cake particles for later use.

[0042] (1.2) Mix 50g of sieved camellia oil cake granules with water per 1000mL of water, resulting in a material-to-liquid ratio of 50g / L. First, place the mixture in an ultrasonic device and sonicate at 40℃ for 30min. Then, place it in a high-pressure steam sterilizer and hydrothermally heat it at 121℃ for 30min. Vacuum filter the hydrothermally heated solution using a 300-500 mesh filter cloth. The resulting filtrate is the camellia oil cake extract. The polysaccharide content in this extract is 1800mg / L, the protein content is 3300mg / L, and the SCOD content is 18000mg / L.

[0043] If the ultrasonic treatment is omitted in this step, the content of nutrients dissolved in the extract will be significantly reduced, making it difficult to meet the growth requirements. If other nutrients are added, the cost of raw materials will inevitably increase. This is a technical problem that needs to be overcome in this invention.

[0044] (2) Preparation of Camellia oleifera cake meal culture medium

[0045] Take the extract of camellia seed cake, add potassium dihydrogen phosphate and dipotassium hydrogen phosphate solution to make the concentration of potassium dihydrogen phosphate in the mixed solution 2.0 g / L and the concentration of dipotassium hydrogen phosphate 5.0 g / L, and adjust the pH value to 8. Then place it in a high pressure steam sterilizer and sterilize it at 121℃ for 30 min to obtain camellia seed cake culture medium.

[0046] (3) Preparation of microbial flocculants

[0047] (3.1) Weigh 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride and 20.0 g / L agar powder, and adjust the pH to 7. Put the prepared culture medium into autoclave and sterilize at 121°C for 30 min. After sterilization, pour the culture medium into a clean culture dish in a sterile operating table, wait for the culture medium to cool and solidify, and then invert it to obtain a solid activated culture medium.

[0048] Weigh out 10.0 g / L peptone, 3.0 g / L beef extract and 5.0 g / L sodium chloride, and adjust the pH to 7. Place the prepared culture medium in an autoclave and sterilize at 121°C for 30 min to obtain liquid proliferation culture medium.

[0049] (3.2) Take the bacterial strain (Bacillus polymyxa GA1) stored in glycerol tubes and inoculate it into solid activation medium. Incubate at 35℃ for 24.0h to activate the bacterial strain and form primary seed colonies.

[0050] (3.3) The primary seed colonies were inoculated into liquid proliferation medium and cultured in a shaker incubator at 30℃ and 150rpm for 24.0h to proliferate the microorganisms and obtain the secondary seed liquid, which was named Bacillus polymyxa GA1 seed liquid.

[0051] (3.4) The seed liquid was inoculated at 5% of the volume of the Camellia oleifera cake culture medium. The above-mentioned Bacillus polymyxa GA1 seed liquid was added to the Camellia oleifera cake culture medium and transferred to a shaker incubator. It was first cultured at 30℃ and 150 rpm for 24.0 h, and then cultured at 25℃ and 120 rpm for 48.0 h. The fermentation product was taken out and placed in a centrifuge. It was centrifuged at 10000 rpm for 10.0 min to remove the bacterial cells. The fermentation product with the bacterial cells removed can be used as a microbial flocculant.

[0052] Example 2

[0053] A method for preparing a microbial flocculant includes the following steps:

[0054] (1) Preparation of camellia seed cake extract

[0055] (1.1) Place the camellia oil cake and dry it continuously at 60℃ for 5.0h until constant weight. Then crush it with a pulverizer and use a steel screen to take 40-60 mesh camellia oil cake particles for later use.

[0056] (1.2) Mix 100g of sieved camellia oil cake granules with 1000mL of water, resulting in a material-to-liquid ratio of 100g / L. First, place the mixture in an ultrasonic device and sonicate at 40℃ for 30min. Then, place it in a high-pressure steam sterilizer and hydrothermally heat at 121℃ for 30min. Vacuum filter the hydrothermally heated solution using a 300-500 mesh filter cloth. The resulting filtrate is the camellia oil cake extract. The polysaccharide content in this extract is 5600mg / L, the protein content is 10500mg / L, and the SCOD content is 42000mg / L.

[0057] (2) Preparation of Camellia oleifera cake meal culture medium

[0058] Take the extract of camellia seed cake, add potassium dihydrogen phosphate and dipotassium hydrogen phosphate solution to make the concentration of potassium dihydrogen phosphate in the mixed solution 2.0 g / L and the concentration of dipotassium hydrogen phosphate 5.0 g / L, and adjust the pH value to 8. Then place it in a high pressure steam sterilizer and sterilize it at 121℃ for 30 min to obtain camellia seed cake culture medium.

[0059] (3) Preparation of microbial flocculants

[0060] (3.1) Weigh 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride and 20.0 g / L agar powder, and adjust the pH to 7. Put the prepared culture medium into autoclave and sterilize at 121°C for 30 min. After sterilization, pour the culture medium into a clean culture dish in a sterile operating table, wait for the culture medium to cool and solidify, and then invert it to obtain a solid activated culture medium.

[0061] Weigh out 10.0 g / L peptone, 3.0 g / L beef extract and 5.0 g / L sodium chloride, and adjust the pH to 7. Place the prepared culture medium in an autoclave and sterilize at 121°C for 30 min to obtain liquid proliferation culture medium.

[0062] (3.2) Take the bacterial strain (Bacillus polymyxa GA1) stored in glycerol tubes and inoculate it into solid activation medium. Incubate at 35℃ for 24.0h to activate the bacterial strain and form primary seed colonies.

[0063] (3.3) The primary seed colonies were inoculated into liquid proliferation medium and cultured in a shaker incubator at 30℃ and 150rpm for 24.0h to proliferate the microorganisms and obtain the secondary seed liquid, which was named Bacillus polymyxa GA1 seed liquid.

[0064] (3.4) The seed liquid was inoculated at 5% of the volume of the Camellia oleifera cake culture medium. The above-mentioned Bacillus polymyxa GA1 seed liquid was added to the Camellia oleifera cake culture medium and transferred to a shaker incubator. It was first cultured at 30℃ and 150 rpm for 24.0 h, and then cultured at 25℃ and 120 rpm for 48.0 h. The fermentation product was taken out and placed in a centrifuge. It was centrifuged at 10000 rpm for 10.0 min to remove the bacterial cells. The fermentation product with the bacterial cells removed can be used as a microbial flocculant.

[0065] Example 3

[0066] A method for preparing a microbial flocculant includes the following steps:

[0067] (1) Preparation of camellia seed cake extract

[0068] (1.1) Place the camellia oil cake and dry it continuously at 60℃ for 5.0h until constant weight. Then crush it with a pulverizer and use a steel screen to take 40-60 mesh camellia oil cake particles for later use.

[0069] (1.2) Mix 150g of sieved camellia oil cake granules with water at a ratio of 150g / L (material-to-liquid ratio). First, place the mixture in an ultrasonic device and sonicate at 40℃ for 30min. Then, place it in a high-pressure steam sterilizer and hydrothermally heat at 121℃ for 30min. Vacuum filter the hydrothermally heated solution using a 300-500 mesh filter cloth. The resulting filtrate is the camellia oil cake extract. The polysaccharide content of this extract is 8300mg / L, the protein content is 14000mg / L, and the SCOD content is 61000mg / L.

[0070] (2) Preparation of Camellia oleifera cake meal culture medium

[0071] Take the extract of camellia seed cake, add potassium dihydrogen phosphate and dipotassium hydrogen phosphate solution to make the concentration of potassium dihydrogen phosphate in the mixed solution 2.0 g / L and the concentration of dipotassium hydrogen phosphate 5.0 g / L, and adjust the pH value to 8. Then place it in a high pressure steam sterilizer and sterilize it at 121℃ for 30 min to obtain camellia seed cake culture medium.

[0072] (3) Preparation of microbial flocculants

[0073] (3.1) Weigh 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride and 20.0 g / L agar powder, and adjust the pH to 8. Put the prepared culture medium into high pressure steam sterilization and sterilize at 121℃ for 30 min. After sterilization, pour the culture medium into a clean culture dish in a sterile operating table, wait for the culture medium to cool and solidify, and then invert it to obtain solid activated culture medium.

[0074] Weigh out 10.0 g / L peptone, 3.0 g / L beef extract and 5.0 g / L sodium chloride, and adjust the pH to 8. Place the prepared culture medium in an autoclave and sterilize at 121°C for 30 min to obtain liquid proliferation culture medium.

[0075] (3.2) Take the bacterial strain (Bacillus polymyxa GA1) stored in glycerol tubes and inoculate it into solid activation medium. Incubate at 35℃ for 24.0h to activate the bacterial strain and form primary seed colonies.

[0076] (3.3) The primary seed colonies were inoculated into liquid proliferation medium and cultured in a shaker incubator at 30℃ and 150rpm for 24.0h to proliferate the microorganisms and obtain the secondary seed liquid, which was named Bacillus polymyxa GA1 seed liquid.

[0077] (3.4) The seed liquid was inoculated at 5% of the volume of the Camellia oleifera cake culture medium. The above-mentioned Bacillus polymyxa GA1 seed liquid was added to the Camellia oleifera cake culture medium and transferred to a shaker incubator. It was first cultured at 30℃ and 150 rpm for 24.0 h, and then cultured at 25℃ and 120 rpm for 48.0 h. The fermentation product was taken out and placed in a centrifuge. It was centrifuged at 10000 rpm for 10.0 min to remove the bacterial cells. The fermentation product with the bacterial cells removed can be used as a microbial flocculant.

[0078] Example 4

[0079] A method for preparing a microbial flocculant includes the following steps:

[0080] (1) Preparation of camellia seed cake extract

[0081] (1.1) Place the camellia oil cake and dry it continuously at 60℃ for 5.0h until constant weight. Then crush it with a pulverizer and use a steel screen to take 40-60 mesh camellia oil cake particles for later use.

[0082] (1.2) Mix 200g of sieved camellia oil cake granules with water per 1000mL of water, resulting in a material-to-liquid ratio of 200g / L. First, place the mixture in an ultrasonic device and sonicate at 40℃ for 30min. Then, place it in a high-pressure steam sterilizer and hydrothermally heat it at 121℃ for 30min. Vacuum filter the hydrothermally heated solution using a 300-500 mesh filter cloth. The resulting filtrate is the camellia oil cake extract. This extract contains 15000mg / L of polysaccharide, 240000mg / L of protein, and 110000mg / L of SCOD.

[0083] (2) Preparation of Camellia oleifera cake meal culture medium

[0084] Take the extract of camellia seed cake, add potassium dihydrogen phosphate and dipotassium hydrogen phosphate solution to make the concentration of potassium dihydrogen phosphate in the mixed solution 2.0 g / L and the concentration of dipotassium hydrogen phosphate 5.0 g / L, and adjust the pH value to 8. Then place it in a high pressure steam sterilizer and sterilize it at 121℃ for 30 min to obtain camellia seed cake culture medium.

[0085] (3) Preparation of microbial flocculants

[0086] (3.1) Weigh 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride and 20.0 g / L agar powder, and adjust the pH to 7. Put the prepared culture medium into autoclave and sterilize at 121°C for 30 min. After sterilization, pour the culture medium into a clean culture dish in a sterile operating table, wait for the culture medium to cool and solidify, and then invert it to obtain a solid activated culture medium.

[0087] Weigh out 10.0 g / L peptone, 3.0 g / L beef extract and 5.0 g / L sodium chloride, and adjust the pH to 7. Place the prepared culture medium in an autoclave and sterilize at 121°C for 30 min to obtain liquid proliferation culture medium.

[0088] (3.2) Take the bacterial strain (Bacillus polymyxa GA1) stored in glycerol tubes and inoculate it into solid activation medium. Incubate at 35℃ for 24.0h to activate the bacterial strain and form primary seed colonies.

[0089] (3.3) The primary seed colonies were inoculated into liquid proliferation medium and cultured in a shaker incubator at 30℃ and 150rpm for 24.0h to proliferate the microorganisms and obtain the secondary seed liquid, which was named Bacillus polymyxa GA1 seed liquid.

[0090] (3.4) The seed liquid was inoculated at 5% of the volume of the Camellia oleifera cake culture medium. The above-mentioned Bacillus polymyxa GA1 seed liquid was added to the Camellia oleifera cake culture medium and transferred to a shaker incubator. It was first cultured at 30℃ and 150 rpm for 24.0 h, and then cultured at 25℃ and 120 rpm for 48.0 h. The fermentation product was taken out and placed in a centrifuge. It was centrifuged at 10000 rpm for 10.0 min to remove the bacterial cells. The fermentation product with the bacterial cells removed can be used as a microbial flocculant.

[0091] Figure 2 This is a comparison chart showing the polysaccharide, protein, and SCOD contents in the extract of camellia seed cake under different material-to-liquid ratios in Examples 1-4 of this invention. Figure 2 It can be seen that as the ratio of tea seed cake to liquid increases, the content of polysaccharides, proteins and SCOD in the extract increases.

[0092] Figure 3 This is a comparison chart showing the bacterial growth in the culture medium for preparing camellia oil cake meal under different material-to-liquid ratios in Examples 1-4 of this invention. Figure 3It was found that the colony count of GA1 in the camellia oil cake medium was more than an order of magnitude higher than that in the traditional fermentation medium. Furthermore, the highest colony count of GA1 was observed when the solid-liquid ratio of camellia oil cake was 150 g / L. When the solid-liquid ratio reached 200 g / L, the logarithmic growth phase of GA1 was delayed. This is because as the solid-liquid ratio of camellia oil cake increased, the concentration of antibacterial substances tannins and tea saponins increased, reaching a certain value that inhibited the growth of GA1 or prolonged its adaptation period.

[0093] Figure 4 This is a comparison chart showing the bacterial dry weight in the microbial fermentation products under different feed-to-liquid ratios in Examples 1-4 of the present invention. Figure 4 It can be seen that the biomass of GA1 in the camellia oil cake medium is higher than that in the traditional fermentation medium.

[0094] Figure 5 This is a comparison chart showing the yield of microbial flocculants under different feed-to-liquid ratios in Examples 1-4 of the present invention. Figure 5 It can be seen that a large amount of microbial flocculant can be obtained when the material-to-liquid ratio is between 50 g / L and 200 g / L. Furthermore, the yield of microbial flocculant increases continuously with the increase of the material-to-liquid ratio. However, when the material-to-liquid ratio is between 50 g / L and 100 g / L, the unit yield of microbial flocculant is even higher. This indicates that under these conditions, the utilization rate of camellia oil cake is higher, and more microbial flocculant can be obtained at a lower cost. Meanwhile, from... Figure 5 It can be seen that the highest yield of microbial flocculant in Examples 1-4 exceeds 18 g / L.

[0095] In addition, the flocculation rate of the microbial flocculants prepared in Examples 1-4 was tested. The test method was as follows: 500 mL of 4 g / L kaolin suspension was prepared, 10 mL of 10% calcium chloride solution was added, 10 mL of microbial flocculant was added, and the pH of the solution was adjusted to 8-8.5. A kaolin suspension without flocculant was used as a control. The mixture was stirred rapidly for 3 min, followed by slow stirring for 3 min, and then allowed to settle for 5 min. Liquid from 1 cm below the surface was collected, and the absorbance was measured at 550 nm using a UV spectrophotometer. The flocculation rate of the microbial flocculant on the kaolin suspension was calculated. The test results are shown in Table 1.

[0096] Formula for calculating flocculation rate:

[0097] Flocculation rate / % = (AB) / A * 100%

[0098] Where: A—OD500 of the original kaolin suspension;

[0099] B – OD500 of kaolin suspension after adding flocculant.

[0100] Comparative Example 1

[0101] Streaking the preserved Aspergillus saccharina var. soysarum onto LB agar and incubating at 30°C for 3 days. Once the agar surface is covered with Aspergillus saccharina var. soysarum, gently scrape spores with an inoculation loop, wash with sterile water into a conical flask, and shake at room temperature for 10 minutes to fully disperse the spores. Remove hyphae and clumps of spores with absorbent cotton. Calculate the spore concentration using a hemocytometer and adjust the final concentration of the spore suspension to 1 × 10⁻⁶. 7 CFU / mL. The above Aspergillus spore suspension was inoculated into 1L of fermentation medium at an inoculation rate of 4%, and cultured at 28℃ and 130rpm for 3 days with shaking. The culture broth was collected. The bacterial cells in the culture broth were removed by filtration with gauze to obtain the fermentation broth. The broth was then centrifuged at 10000r / min for 15min. 3mL of the supernatant was taken as the microbial flocculant solution and added to 96mL of 0.5% kaolin suspension. 1% CaCl2 was used as a coagulant aid, and the pH was adjusted to 6.5. The mixture was stirred at 300rpm for 15min and allowed to stand for 10min. The liquid was taken from 1cm below the surface, and the absorbance was measured at 550nm using a UV spectrophotometer. The flocculation rate of the microbial flocculant on the kaolin suspension was calculated. The results are shown in Table 1.

[0102] Comparative Example 2

[0103] A single colony of *Goldenella pulmonale* was selected as the pure culture, and then inoculated into a sterilized Erlenmeyer flask containing 50 mL of liquid (LB 100 mL) using an inoculation loop. Fermentation was carried out on a shaker. The temperature was set to 30℃, and after 72 h of incubation, the fermentation broth was centrifuged at 6000 rpm for 15 min, and the supernatant was used as the microbial flocculant. A 1000 mL beaker was prepared, containing a 4 g / L kaolin suspension. 25 mL of 1% CaCl2 solution was added, and the kaolin suspension was added to a final volume of 500 mL. The pH was adjusted to 8, and 10 mL of the microbial flocculant was added. The mixture was stirred rapidly at 180 rpm for 30 s, then slowly at 8 rpm for 5 min, and allowed to stand for 5 min. The liquid was collected from 1 cm below the surface, and the absorbance was measured at 550 nm using a UV spectrophotometer. The flocculation rate of the microbial flocculant on the kaolin suspension was calculated, and the results are shown in Table 1.

[0104] Comparative Example 3

[0105] A single colony of *Goldenella pulmonale* was selected as the pure culture. The colony was then inoculated into a sterilized Erlenmeyer flask containing 50 mL of liquid (LB 100 mL) using an inoculation loop and fermented in a shaker. The temperature was set to 30℃, and after 72 hours of incubation, the fermentation broth was used as a microbial flocculant. A 1000 mL beaker was prepared and a 4 g / L kaolin suspension was poured in. 25 mL of 1% CaCl2 solution was added, and the kaolin suspension was added to a final volume of 500 mL. The pH was adjusted to 8, and 10 mL of microbial flocculant was added. The mixture was stirred rapidly at 180 rpm for 30 seconds, then slowly at 8 rpm for 5 minutes, and allowed to stand for 5 minutes. A sample of the liquid 1 cm below the surface was taken, and the absorbance was measured at 550 nm using a UV spectrophotometer. The flocculation rate of the microbial flocculant on the kaolin suspension was calculated. The results are shown in Table 1.

[0106] Comparative Example 4

[0107] A single colony of *Gordonella pulmonale* was selected as the pure culture. The colony was then inoculated into a sterilized Erlenmeyer flask containing 50 mL of liquid (LB 100 mL) using an inoculation loop and fermented in a shaker. The temperature was set to 30℃, and after 72 h of incubation, the fermentation broth was centrifuged at 6000 rpm for 15 min. The bacterial cells were collected, washed, and prepared into a suspension as a microbial flocculant. A 1000 mL beaker was prepared and a 4 g / L kaolin suspension was poured in. 25 mL of 1% CaCl2 solution was added, and the kaolin suspension was added to a final volume of 500 mL. The pH was adjusted to 8, and 10 mL of the microbial flocculant was added. The mixture was stirred rapidly at 180 rpm for 30 s, then slowly at 8 rpm for 5 min, and allowed to stand for 5 min. A sample of liquid from 1 cm below the surface was taken, and the absorbance was measured at 550 nm using a UV spectrophotometer. The flocculation rate of the microbial flocculant on the kaolin suspension was calculated. The results are shown in Table 1.

[0108] Table 1. Flocculation Rate Detection Results

[0109] Testing items Example 1 Example 2 Example 3 Example 4 Flocculation rate (%) 98.63％ 96.84％ 93.35％ 90.46％ Testing items Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Flocculation rate (%) 90.00％ 87.58％ 93.88％ 38.68％

[0110] As shown in Table 1, the microbial flocculant prepared in this invention achieves a flocculation rate of up to 98.63% when used to treat 4 g / L kaolin suspension, demonstrating better flocculation effect. It also enables the resource utilization of camellia oil cake and reduces the production cost of microbial flocculants, which is of great significance for promoting the widespread application of microbial flocculants.

[0111] Example 5

[0112] In this embodiment, the effects of different culture conditions on the yield of microbial flocculants were also investigated. Except for the pH and culture time of the camellia seed cake culture medium, the other conditions were the same as in Example 1.

[0113] Figure 6This is a comparison chart showing the yield of microbial flocculants prepared in camellia seed cake culture media at different pH values ​​in Example 5 of the present invention. Figure 6 It can be seen that when the pH value of the camellia oil cake medium is ≥4, a high yield can be obtained. In particular, when the pH value of the camellia oil cake medium is 5-9, the yield of microbial flocculant is significantly increased.

[0114] Figure 7 This is a graph showing the yield of microbial flocculant prepared under different culture time conditions in Example 5 of the present invention. Figure 7 It can be seen that the yield of microbial flocculant first decreases within 24 hours, and then increases. This is because the polysaccharides, proteins, and other substances contained in the camellia oil cake itself are extracted along with the extracellular polymers of the microorganisms, resulting in a high initial yield of microbial flocculant. Subsequently, the polysaccharides, proteins, and other substances in the camellia oil cake are utilized by the growth of microorganisms, and the content of microbial flocculant decreases again. Finally, under the environment of 25℃ and 100rpm, the microorganisms produce a large amount of extracellular polymers, and the content of microbial flocculant increases, reaching a peak at the 72nd hour of fermentation.

[0115] In this embodiment, the flocculation rate of the microbial flocculant prepared under different culture time conditions was also investigated, and the results are as follows: Figure 8 As shown.

[0116] Figure 8 This is a comparison chart of the flocculation rates of the microbial flocculants prepared under different culture time conditions in Example 5 of the present invention. Figure 8 It can be seen that in the initial stage of cultivation, although the initial yield of microbial flocculant is relatively high, it is mainly composed of polysaccharides, proteins, and other substances contained in the camellia oil cake itself, resulting in low flocculation capacity. However, in the second stage of cultivation at 25℃ and 100rpm, the microorganisms produce a large amount of extracellular polymers, leading to an increase in the content of microbial flocculant and thus a significant improvement in flocculation capacity. The highest flocculation rate of 98.63% was obtained at 72 hours of fermentation, which is consistent with... Figure 7 The results are consistent with those in the previous section.

[0117] Depend on Figure 7 and Figure 8 It is understood that in this invention, by optimizing the cultivation conditions, microorganisms grow rapidly in the early stage of fermentation and produce a large amount of flocs in the later stage of fermentation. This allows for the reduction of fermentation time while obtaining high-yield, high-flocculation-rate microbial flocculants, which is beneficial for large-scale preparation and industrial application.

[0118] The results above show that, compared with conventional methods for preparing microbial flocculants, the preparation method of this invention has the following advantages: (a) Low production cost: Using camellia oil cake as raw material to provide the main nutrients required for cultivation can significantly reduce the production cost of microbial flocculants. The raw material cost per unit of flocculant yield is approximately RMB 12,000 / ton, while the raw material cost per unit of flocculant yield is approximately RMB 365,000 / ton when using traditional culture media to prepare microbial flocculants. Therefore, the raw material cost of this invention is reduced by approximately 96.7%; (b) High production efficiency: Compared with traditional culture media, this invention uses camellia oil cake extract, which is rich in nutrients and has good compatibility with various nutrients, as raw material to prepare the culture medium. This results in a higher growth rate and proliferation density of the flocculating bacteria, which is beneficial for the fermentation system to quickly enter the flocculant production stage. The biomass of the flocculating bacteria is increased by at least one order of magnitude, from 0.55 × 10⁻⁶. 7 CFU / mL increased to 1.79 × 10⁻⁶ 8 (c) High yield and high quality: In this invention, under controllable concentration ranges of components such as tea saponins and tannins, the yield and quality of microbial flocculants, mainly composed of extracellular metabolites of microorganisms, can be improved. This increases the yield of microbial flocculants and enhances their flocculation activity. Specifically, under the condition of a material-to-liquid ratio of 50 g / L for camellia oil cake extract, the yield of microbial flocculants is 6.61 g / L, corresponding to a flocculant yield of 0.13 g / g COC per unit mass of camellia oil cake, which is 20.3% higher than that of traditional culture media. The flocculation rate of kaolin suspension reaches 98.9%, which is 1.02% higher than that of flocculants obtained from traditional culture media. Therefore, the preparation method of this invention can prepare microbial flocculants with high yield and high flocculation rate, and has the advantages of low production cost and high production efficiency, while also realizing the resource utilization of camellia oil by-products.

[0119] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for producing a microbial flocculant, characterized by, The method comprises the following steps: S1, mix the oil tea cake meal with water, ultrasonic, hydrothermal treatment, filter, collect the filtrate, and obtain the oil tea cake meal extraction liquid; the ultrasonic is carried out at a temperature of 30-40 DEG C; the ultrasonic time is 20-50 min; the hydrothermal treatment is carried out at a temperature of 111-126 DEG C; the hydrothermal treatment time is 10-50 min; the ratio of the oil tea cake meal to water is 50-100 g: 1 L; S2, the oil tea cake meal extraction liquid obtained in step S1 is configured into an oil tea cake meal culture medium; the preparation method of the oil tea cake meal culture medium is as follows: the oil tea cake meal extraction liquid, potassium dihydrogen phosphate and dipotassium hydrogen phosphate are mixed to obtain the oil tea cake meal culture medium; the concentration of potassium dihydrogen phosphate in the oil tea cake meal culture medium is 1.0-2.0 g / L, and the concentration of dipotassium hydrogen phosphate is 3.0-5.0 g / L; S3, inoculate the flocculation functional bacteria seed liquid into the oil tea cake meal culture medium obtained in step S2 for culture to complete the preparation of the microbial flocculant; the bacterial strain in the flocculation functional bacteria seed liquid is Paenibacillus polymyxa.

2. The production method according to claim 1, characterized by, In step S1, the oil tea cake meal further comprises the following treatment before use: drying the oil tea cake meal, crushing, passing through a 40-60 mesh sieve, and obtaining the oil tea cake meal particles.

3. The production method according to claim 1, characterized by, In step S1, the content of polysaccharide in the oil tea cake meal extraction liquid is 1800-15000 mg / L, the content of protein is 3300-24000 mg / L, and the content of SCOD is 18000-110000 mg / L.

4. The production method according to any one of claims 1 to 3, characterized by, In step S2, the pH value of the oil tea cake meal culture medium is 5-9.

5. The production method according to any one of claims 1 to 3, characterized by, In step S3, the inoculation amount of the flocculation functional bacteria seed liquid is 4-5% of the volume of the oil tea cake meal culture medium.

6. The production method according to claim 5, wherein In step S3, the flocculation functional bacteria seed liquid further comprises the following treatment before inoculation: (1) inoculate the flocculation functional bacteria strain into a solid activation culture medium, culture at a temperature of 30-35 DEG C for 12.0-24.0 h to obtain a primary seed colony; the formula of the solid activation culture medium is as follows: 10.0 g / L of peptone, 3.0 g / L of beef extract, 5.0 g / L of sodium chloride, 20.0 g / L of agar powder, and the pH value is 7-8; the Paenibacillus polymyxa is GA1; (2) inoculate the primary seed colony obtained in step (1) into a liquid proliferation culture medium, and culture at a temperature of 25-30 DEG C and a rotation speed of 130-150 rpm for 12.0-24.0 h to obtain the flocculation functional bacteria seed liquid; the formula of the liquid proliferation culture medium is as follows: 10.0 g / L of peptone, 3.0 g / L of beef extract, 5.0 g / L of sodium chloride, and the pH value is 7-8. ​ 7. The production method according to any one of claims 1 to 3, characterized by, In step S3, the culturing is: culturing under the condition that the temperature is 30-35 DEG C and the rotating speed is 130-150 rpm for 12.0-24.0 hours, and culturing under the condition that the temperature is 20-25 DEG C and the rotating speed is 100-120 rpm for 24.0-72.0 hours; after the culturing is completed, the following treatment is further included: centrifuging the product after culturing to remove the bacterial bodies; the rotating speed of the centrifuging is 8000-10000 rpm; the centrifuging time is 8-10 minutes.

8. A microbial flocculant, characterized by, The microbial flocculant is prepared by the preparation method in any one of claims 1-7.

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