Microbial flocculant and preparation method thereof

Abstract

The preparation method comprises the following steps: mixing oil-tea cake with water, carrying out ultrasonic treatment, carrying out hydrothermal treatment, filtering, collecting filtrate to obtain an oil-tea cake extract, preparing an oil-tea cake culture medium, inoculating a flocculation functional bacterium seed solution, and culturing to complete the preparation of the microbial flocculant. According to the method, the mode of combining ultrasonic extraction and hydrothermal extraction is adopted, the camellia oleifera cake extract liquid which is rich in nutritional ingredients and good in adaptability of all nutrient substances can be prepared, when the camellia oleifera cake extract liquid is used for culturing flocculation functional bacteria, rapid growth of the flocculation functional bacteria can be promoted, more extracellular polymeric substances can be induced to be generated while the biomass is increased, and the flocculation functional bacteria can be cultured. Therefore, the microbial flocculant with high yield and high flocculation rate is obtained. According to the method, the oil-tea cake is used as the raw material, so that the production cost can be greatly reduced, the production steps are simplified, and the resource utilization of the oil-tea cake can be realized while the large-scale use of the microbial flocculant is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial flocculant preparation and relates to a microbial flocculant and a preparation method thereof. Background Art

[0002] Flocculants, a widely used agent in water treatment, are primarily categorized as inorganic, synthetic organic polymer, and natural polymer flocculants. Inorganic flocculants often contain metal ions, and residual metal ions after addition are difficult to remove, potentially causing secondary water pollution. While synthetic organic polymer flocculants have low toxicity, they are difficult to degrade, and residual monomers can be teratogenic, carcinogenic, and mutagenic, posing potential health risks. Natural organic polymer flocculants are generally harmless to humans and easily biodegrade, making them safer than inorganic and synthetic organic polymer flocculants.

[0003] Microbial flocculants are naturally occurring high-molecular-weight flocculants, mostly derived from microorganisms isolated from activated sludge, soil, and water, or from their secreted metabolites, such as extracellular polymers (EPs), which are high-molecular-weight compounds such as glycoproteins, polysaccharides, proteins, and DNA. Unlike traditional flocculants, their components are easily degradable and do not cause secondary environmental pollution. They are green, safe, and non-toxic, with significant implications for 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 medium used to prepare existing microbial flocculants primarily consists of sucrose, yeast extract, and various inorganic salts, resulting in a complex production process and high production costs for large-scale application. To reduce production costs, some 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, key nutrients vary unpredictably, and toxic and harmful substances may be present. 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 oleifera cake is a major byproduct of camellia oil production. It contains a wealth of nutrients, including bioactive ingredients such as proteins, polysaccharides, polyphenols, and tea saponins, in addition to lignocellulose. It is a potential raw material for making alternative microbial culture media. However, in the prior art, when a tea saponin-extracted camellia oleifera cake extract is used as a culture medium, the resulting microbial flocculant achieves a maximum flocculation rate of only 86.1% on a kaolin suspension, resulting in a relatively low flocculation rate. Furthermore, the aforementioned methods for preparing microbial flocculants using camellia oleifera cake extracts require the addition of additional nutrients such as sucrose, yeast extract, and inorganic salts, making it difficult to effectively reduce production costs. Therefore, the development of high-yield, high-flocculation-rate microbial flocculants using camellia oleifera cake is crucial for reducing production costs and simultaneously achieving high-value utilization of camellia oleifera cake. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a microbial flocculant with high production efficiency, low production cost, high output and high flocculation rate and a preparation method thereof in view of the deficiencies in the prior art.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

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

[0008] S1. Mixing camellia oil cake with water, performing ultrasonic treatment, hydrothermal treatment, filtering, and collecting the filtrate to obtain a camellia oil cake extract;

[0009] S2, preparing a camellia oil cake culture medium by preparing the camellia oil cake extract obtained in step S1;

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

[0011] The above preparation method is further improved. In step S1, the ultrasound is performed at a temperature of 30°C to 40°C; the ultrasound time is 20min to 50min; the hydrothermal treatment is performed at a temperature of 111°C to 126°C; and the hydrothermal treatment time is 10min to 50min.

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

[0013] The above preparation method is further improved in that in step S1, the ratio of the camellia oil cake to water is 50g-150g:1L.

[0014] The above preparation method is further improved. In step S1, the polysaccharide content in the camellia oleifera 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] The above preparation method is further improved. In step S2, the preparation method of the camellia oleifera cake culture medium is: mixing the camellia oleifera cake extract, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate to obtain the camellia oleifera cake culture medium; the concentration of potassium dihydrogen phosphate in the camellia oleifera cake 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 oleifera cake culture medium is 5 to 9.

[0016] The above preparation method is further improved in that, 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] The above preparation method is further improved in that, in step S3, the flocculating functional bacteria seed liquid further comprises the following treatment before inoculation:

[0018] (1) Inoculating a flocculating functional bacterial strain into a solid activation culture medium, culturing at a temperature of 30° C. to 35° C. for 12.0 h to 24.0 h to obtain a primary seed colony; the solid activation culture medium has a formula of: 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, 20.0 g / L agar powder, and a pH value of 7 to 8; the flocculating functional bacterial strain is one of Paenibacillus polymyxa, lactic acid bacteria, Clostridium butyricum, and Halomonas; the Paenibacillus polymyxa is GA1;

[0019] (2) inoculating the primary seed colony obtained in step (1) into a liquid proliferation culture medium, and culturing the culture medium for 12.0 hours to 24.0 hours at a temperature of 25° C. to 30° C. and a rotation speed of 130 rpm to 150 rpm to obtain a flocculating functional bacteria seed solution; the formula of the liquid proliferation culture medium is: 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, and a pH value of 7 to 8.

[0020] The above preparation method is further improved, in step S3, the culturing is: culturing for 12.0h to 24.0h at a temperature of 30°C to 35°C and a rotation speed of 130rpm to 150rpm, and culturing for 24.0h to 72.0h at a temperature of 20°C to 25°C 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 the bacteria; the centrifugal speed is 8000rpm to 10000rpm; the centrifugal time is 8min to 10min.

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

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

[0023] (1) In view of the problems existing in the existing methods for preparing microbial flocculants, such as low production efficiency, high production cost, low yield, and low flocculation rate, the present invention creatively proposes a method for preparing a microbial flocculant. First, the oil-tea cake is mixed with water and subjected to ultrasonic and hydrothermal treatment. The effective components in the oil-tea cake are fully dissolved by combining ultrasonic extraction and hydrothermal extraction, thereby preparing an oil-tea cake extract with rich nutritional components and good adaptability of various nutrients. On the one hand, after being treated by combining ultrasonic extraction and hydrothermal extraction, the obtained oil-tea cake extract contains a high content of nutrients such as polysaccharides and proteins, thereby providing sufficient and balanced nutrients for the growth of subsequent flocculating functional bacteria (such as Bacillus polymyxa GA1), which is conducive to obtaining a higher biomass. On the other hand, after a combination of ultrasonic and hydrothermal extraction, the antibacterial components contained in tea cake, such as tea saponins and tannins, can be transferred to the tea 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 the stress resistance and survival ability of the flocculating bacteria. On this basis, the tea cake extract is configured into a tea cake culture medium and inoculated with flocculating bacteria seed liquid 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 the present invention, using tea cake as the main nutrient component of the microbial culture medium can significantly reduce the production cost of the microbial flocculant, simplify the production steps of the microbial flocculant, and realize the resource utilization of tea cake by-products. Compared with conventional microbial flocculant preparation methods, the preparation method of the present invention has the following advantages: (a) low production cost. The oil-tea cake is used as a raw material to provide the main nutrients required for cultivation, which can significantly reduce the production cost of microbial flocculants. The raw material cost per unit flocculant output is about RMB 12,000 / ton, while when the traditional culture medium is used to prepare microbial flocculants, the raw material cost per unit flocculant output is about RMB 365,000 / ton. It can be seen that the raw material cost of the present invention is reduced by about 96.7%; (b) high production efficiency. Compared with the traditional culture medium, the oil-tea cake extract of the present invention is used as a raw material to prepare the culture medium, which is rich in nutrients and has good adaptability of various nutrients. This makes the flocculating functional bacteria have a higher growth rate and proliferation density, which is conducive to the fermentation system to quickly enter the flocculent production stage. The biomass of the flocculating functional bacteria is increased by at least one order of magnitude, from 0.55×10 7 The CFU / mL increased to 1.79×10 8CFU / mL; (c) high yield and high quality. In the present 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 microbial extracellular metabolites can be promoted and improved, thereby increasing the yield of microbial flocculants and enhancing flocculation activity. Among them, under the condition of a camellia oil cake extract with a solid-liquid ratio of 50 g / L, the yield of the microbial flocculant is 6.61 g / L, and the flocculant yield per unit mass of camellia oil cake is 0.13 g / g COC, which is 20.3% higher than that of traditional culture medium; the flocculation rate of kaolin suspension reaches 98.9%, which is 1.02% higher than that of flocculants obtained from traditional culture medium.

[0024] (2) In the present invention, by optimizing the ultrasonic conditions, specifically ultrasonicating at a temperature of 30°C to 40°C for 20min to 50min, while ensuring energy saving, the thermal effect and cavitation effect can be utilized to enable the oil-tea cake to dissolve as much soluble matter as possible in a relatively short time and transfer it to 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 oil-tea cake. At the same time, by optimizing the temperature to 30°C to 40°C, it is beneficial to control the dissolution amount of tea saponin and tannin, and it is convenient to regulate the content of tea saponin, tannin and other components in the extract. Moreover, under the joint action of hydrothermal treatment, it is more conducive to preparing the oil-tea cake extract with rich nutrients and good adaptability of various nutrients.

[0025] (3) In the present invention, by optimizing the ratio of camellia oleifera cake to water, specifically optimizing the ratio of the two to 30g-200g:1L, and in particular 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 functional bacteria, it is not only conducive to the formation of a microbial flocculant with a high flocculation rate with extracellular polymers as the main component, but also can significantly increase the unit yield of the microbial flocculant, which is conducive to improving the utilization rate of camellia oleifera cake and making the preparation cost lower.

[0026] (4) In the present invention, by optimizing the culture conditions, specifically, the flocculating functional bacteria seed liquid is inoculated into the oil-tea camellia cake culture medium, firstly cultured at a temperature of 30°C to 35°C and a rotation speed of 130rpm to 150rpm for 12.0h to 24.0h, and then cultured at a temperature of 20°C to 25°C and a rotation speed of 100rpm to 120rpm for 24.0h to 72.0h. During the fermentation process in the former stage, the microorganisms grow rapidly, and during the fermentation process in the latter stage, a large amount of flocculants are produced. Thus, a microbial flocculant with high yield and high flocculation rate can be obtained while reducing the fermentation time, which is conducive to large-scale preparation and convenient for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

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

[0029] Figure 2 This is a comparison chart of the polysaccharide, protein, and SCOD contents in the oil-tea camellia cake extract under different material-liquid ratios in Examples 1-4 of the present invention.

[0030] Figure 3 This is a comparison chart of the growth of bacteria in the tea cake culture medium prepared under different material-to-liquid ratios in Examples 1-4 of the present invention.

[0031] Figure 4 This is a comparison chart of the dry weight of bacteria 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 material-liquid ratios in Examples 1-4 of the present invention.

[0033] Figure 6 This is a comparison chart of the yields of microbial flocculants prepared in camellia oil 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 flocculants 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 the microbial flocculants prepared under different culture time conditions in Example 5 of the present invention. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the scope of protection of the present invention is not limited thereby.

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

[0038] Example 1:

[0039] A preparation method of a microbial flocculant, the preparation process flow chart is as follows Figure 1 As shown, the following steps are included:

[0040] (1) Preparation of Camellia oleifera cake extract

[0041] (1.1) Place the oil-tea camellia cake in an oven and dry it at 60°C for 5.0 hours until it reaches a constant weight. Then, crush it in a grinder and use a steel sieve to select 40-60 mesh oil-tea camellia cake particles for later use.

[0042] (1.2) 50 g of sieved camellia oleifera cake particles were mixed with 1000 mL of water at a material-to-liquid ratio of 50 g / L. The particles were then ultrasonically treated at 40°C for 30 minutes in an ultrasonic device. The mixture was then hydrothermally treated at 121°C for 30 minutes in a high-pressure steam autoclave. The hydrothermal solution was vacuum filtered through a 300-500 mesh filter cloth to obtain the filtrate, which was the camellia oleifera cake extract. The camellia oleifera cake extract contained 1800 mg / L of polysaccharides, 3300 mg / L of protein, and 18,000 mg / L of SCOD.

[0043] In this step, if ultrasonic treatment is omitted, the content of nutrients dissolved in the extract will be significantly reduced, making it difficult to meet 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 the present invention.

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

[0045] Take the oil-tea camellia cake extract, add potassium dihydrogen phosphate and dipotassium hydrogen phosphate solutions to make the concentration of potassium dihydrogen phosphate in the mixed solution be 2.0 g / L and the concentration of dipotassium hydrogen phosphate be 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°C for 30 minutes to obtain the oil-tea camellia 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. Sterilize the prepared culture medium in an autoclave at 121°C for 30 min. Pour the sterilized culture medium into a clean Petri dish on a sterile workbench. After the culture medium cools and solidifies, invert it to prepare a solid activated culture medium.

[0048] Weigh 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 a high-pressure steam sterilizer and sterilize at 121°C for 30 minutes to prepare a liquid proliferation medium.

[0049] (3.2) The strain (Bacillus polymyxa GA1) stored in the glycerol tube was inoculated into a solid activation medium and incubated at 35°C for 24 hours to activate the strain and form a primary seed colony.

[0050] (3.3) The primary seed colony was inoculated into a liquid proliferation medium and cultured in a shaking incubator at 30°C and 150 rpm for 24 h to proliferate the microorganisms and produce a secondary seed solution, designated as Paenibacillus polymyxa GA1 seed solution.

[0051] (3.4) The inoculation amount of the seed liquid is 5% of the volume of the camellia oleifera cake culture medium. The above-mentioned Bacillus polymyxa GA1 seed liquid is added to the camellia oleifera cake culture medium and transferred to a shaking incubator. It is first cultured at a temperature of 30°C and a rotation speed of 150 rpm for 24.0 hours, and then cultured at a temperature of 25°C and a rotation speed of 120 rpm for 48.0 hours. The obtained fermentation product is taken out and placed in a centrifuge. It is centrifuged at a speed of 10,000 rpm for 10.0 minutes to remove the bacteria. The fermentation product after removing the bacteria can be used as a microbial flocculant.

[0052] Example 2

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

[0054] (1) Preparation of Camellia oleifera cake extract

[0055] (1.1) Place the oil-tea camellia cake in an oven and dry it at 60°C for 5.0 hours until it reaches a constant weight. Then, crush it in a grinder and use a steel sieve to select 40-60 mesh oil-tea camellia cake particles for later use.

[0056] (1.2) 100 g of sieved camellia oleifera cake particles were mixed with 1000 mL of water at a material-to-liquid ratio of 100 g / L. The particles were then ultrasonically treated at 40°C for 30 minutes in an ultrasonic device. The mixture was then hydrothermally treated at 121°C for 30 minutes in a high-pressure steam autoclave. The hydrothermal solution was vacuum filtered through a 300-500 mesh filter cloth to obtain the filtrate, which was the camellia oleifera cake extract. This camellia oleifera cake extract contained 5600 mg / L of polysaccharides, 10500 mg / L of protein, and 42000 mg / L of SCOD.

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

[0058] Take the oil-tea camellia cake extract, add potassium dihydrogen phosphate and dipotassium hydrogen phosphate solutions to make the concentration of potassium dihydrogen phosphate in the mixed solution be 2.0 g / L and the concentration of dipotassium hydrogen phosphate be 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°C for 30 minutes to obtain the oil-tea camellia 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. Sterilize the prepared culture medium in an autoclave at 121°C for 30 min. Pour the sterilized culture medium into a clean Petri dish on a sterile workbench. After the culture medium cools and solidifies, invert it to prepare a solid activated culture medium.

[0061] Weigh 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 a high-pressure steam sterilizer and sterilize at 121°C for 30 minutes to prepare a liquid proliferation medium.

[0062] (3.2) The strain (Bacillus polymyxa GA1) stored in the glycerol tube was inoculated into a solid activation medium and incubated at 35°C for 24 hours to activate the strain and form a primary seed colony.

[0063] (3.3) The primary seed colony was inoculated into a liquid proliferation medium and cultured in a shaking incubator at 30°C and 150 rpm for 24 h to proliferate the microorganisms and produce a secondary seed solution, designated as Paenibacillus polymyxa GA1 seed solution.

[0064] (3.4) The inoculation amount of the seed liquid is 5% of the volume of the camellia oleifera cake culture medium. The above-mentioned Bacillus polymyxa GA1 seed liquid is added to the camellia oleifera cake culture medium and transferred to a shaking incubator. It is first cultured at a temperature of 30°C and a rotation speed of 150 rpm for 24.0 hours, and then cultured at a temperature of 25°C and a rotation speed of 120 rpm for 48.0 hours. The obtained fermentation product is taken out and placed in a centrifuge. It is centrifuged at a speed of 10,000 rpm for 10.0 minutes to remove the bacteria. The fermentation product after removing the bacteria can be used as a microbial flocculant.

[0065] Example 3

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

[0067] (1) Preparation of Camellia oleifera cake extract

[0068] (1.1) Place the oil-tea camellia cake in an oven and dry it at 60°C for 5.0 hours until it reaches a constant weight. Then, crush it in a grinder and use a steel sieve to select 40-60 mesh oil-tea camellia cake particles for later use.

[0069] (1.2) 150 g of sieved camellia oleifera cake particles were mixed with 1000 mL of water at a material-to-liquid ratio of 150 g / L. The camellia oleifera cake particles were mixed with water and ultrasonicated at 40°C for 30 minutes. The mixture was then hydrothermally heated at 121°C for 30 minutes in a high-pressure steam autoclave. The hydrothermal solution was vacuum filtered through a 300-500 mesh filter cloth to obtain the filtrate, which was the camellia oleifera cake extract. The camellia oleifera cake extract contained 8300 mg / L of polysaccharides, 14000 mg / L of protein, and 61000 mg / L of SCOD.

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

[0071] Take the oil-tea camellia cake extract, add potassium dihydrogen phosphate and dipotassium hydrogen phosphate solutions to make the concentration of potassium dihydrogen phosphate in the mixed solution be 2.0 g / L and the concentration of dipotassium hydrogen phosphate be 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°C for 30 minutes to obtain the oil-tea camellia 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. Sterilize the prepared culture medium in an autoclave at 121°C for 30 min. Pour the sterilized culture medium into a clean Petri dish on a sterile workbench. After the culture medium cools and solidifies, invert it to prepare a solid activated culture medium.

[0074] Weigh 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 a high-pressure steam sterilizer and sterilize at 121°C for 30 minutes to prepare a liquid proliferation medium.

[0075] (3.2) The strain (Bacillus polymyxa GA1) stored in the glycerol tube was inoculated into a solid activation medium and incubated at 35°C for 24 hours to activate the strain and form a primary seed colony.

[0076] (3.3) The primary seed colony was inoculated into a liquid proliferation medium and cultured in a shaking incubator at 30°C and 150 rpm for 24 h to proliferate the microorganisms and produce a secondary seed solution, designated as Paenibacillus polymyxa GA1 seed solution.

[0077] (3.4) The inoculation amount of the seed liquid is 5% of the volume of the camellia oleifera cake culture medium. The above-mentioned Bacillus polymyxa GA1 seed liquid is added to the camellia oleifera cake culture medium and transferred to a shaking incubator. It is first cultured at a temperature of 30°C and a rotation speed of 150 rpm for 24.0 hours, and then cultured at a temperature of 25°C and a rotation speed of 120 rpm for 48.0 hours. The obtained fermentation product is taken out and placed in a centrifuge. It is centrifuged at a speed of 10,000 rpm for 10.0 minutes to remove the bacteria. The fermentation product after removing the bacteria can be used as a microbial flocculant.

[0078] Example 4

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

[0080] (1) Preparation of Camellia oleifera cake extract

[0081] (1.1) Place the oil-tea camellia cake in an oven and dry it at 60°C for 5.0 hours until it reaches a constant weight. Then, crush it in a grinder and use a steel sieve to select 40-60 mesh oil-tea camellia cake particles for later use.

[0082] (1.2) 200 g of sieved camellia oleifera cake particles were mixed with 1000 mL of water at a material-to-liquid ratio of 200 g / L. The particles were then ultrasonically treated at 40°C for 30 minutes in an ultrasonic device. The mixture was then hydrothermally treated at 121°C for 30 minutes in a high-pressure steam autoclave. The hydrothermal solution was vacuum filtered through a 300-500 mesh filter cloth to obtain the filtrate, which was the camellia oleifera cake extract. The camellia oleifera cake extract contained 15,000 mg / L of polysaccharides, 240,000 mg / L of protein, and 110,000 mg / L of SCOD.

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

[0084] Take the oil-tea camellia cake extract, add potassium dihydrogen phosphate and dipotassium hydrogen phosphate solutions to make the concentration of potassium dihydrogen phosphate in the mixed solution be 2.0 g / L and the concentration of dipotassium hydrogen phosphate be 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°C for 30 minutes to obtain the oil-tea camellia 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. Sterilize the prepared culture medium in an autoclave at 121°C for 30 min. Pour the sterilized culture medium into a clean Petri dish on a sterile workbench. After the culture medium cools and solidifies, invert it to prepare a solid activated culture medium.

[0087] Weigh 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 a high-pressure steam sterilizer and sterilize at 121°C for 30 minutes to prepare a liquid proliferation medium.

[0088] (3.2) The strain (Bacillus polymyxa GA1) stored in the glycerol tube was inoculated into a solid activation medium and incubated at 35°C for 24 hours to activate the strain and form a primary seed colony.

[0089] (3.3) The primary seed colony was inoculated into a liquid proliferation medium and cultured in a shaking incubator at 30°C and 150 rpm for 24 h to proliferate the microorganisms and produce a secondary seed solution, designated as Paenibacillus polymyxa GA1 seed solution.

[0090] (3.4) The inoculation amount of the seed liquid is 5% of the volume of the camellia oleifera cake culture medium. The above-mentioned Bacillus polymyxa GA1 seed liquid is added to the camellia oleifera cake culture medium and transferred to a shaking incubator. It is first cultured at a temperature of 30°C and a rotation speed of 150 rpm for 24.0 hours, and then cultured at a temperature of 25°C and a rotation speed of 120 rpm for 48.0 hours. The obtained fermentation product is taken out and placed in a centrifuge. It is centrifuged at a speed of 10,000 rpm for 10.0 minutes to remove the bacteria. The fermentation product after removing the bacteria can be used as a microbial flocculant.

[0091] Figure 2 This is a comparison chart of the polysaccharide, protein, and SCOD contents in the tea cake extract under different material-liquid ratios in Examples 1-4 of the present invention. Figure 2 It can be seen that with the increase of the material-liquid ratio of camellia oil cake, the contents of polysaccharides, protein and SCOD in the extract increased.

[0092] Figure 3 The following is a comparison chart of the bacterial growth in the oil-tea cake culture medium prepared under different material-liquid ratios in Examples 1-4 of the present invention. Figure 3The colony count of GA1 in tea cake medium was over an order of magnitude higher than that in traditional fermentation medium. Furthermore, GA1 achieved its highest colony count when the tea cake-to-liquid ratio was 150 g / L. When the tea cake-to-liquid ratio reached 200 g / L, GA1's logarithmic growth phase was delayed. This is because as the tea cake-to-liquid ratio increases, the concentrations of antibacterial substances such as tannins and saponins increase, ultimately inhibiting GA1's growth or prolonging its adaptation period.

[0093] Figure 4 The figure is a comparison chart of the dry weight of bacteria in the microbial fermentation products under different material-liquid ratio conditions in Examples 1-4 of the present invention. Figure 4 It can be seen that the biomass of GA1 in camellia oil cake medium is higher than that in traditional fermentation medium.

[0094] Figure 5 The graph is a comparison of the yield of microbial flocculants under different material-liquid ratios in Examples 1-4 of the present invention. Figure 5 It can be seen that when the material-liquid ratio is 50g / L-200g / L, a lot of microbial flocculants can be obtained, and with the increase of the material-liquid ratio, the yield of microbial flocculants continues to increase. However, when the material-liquid ratio is 50g / L-100g / L, the unit yield of microbial flocculants is higher, which shows that under this condition, the utilization rate of camellia oil cake is higher, and more microbial flocculants can be obtained at a lower cost. At the same time, Figure 5 It can be seen that the highest yield of the microbial flocculant in Examples 1-4 exceeds 18 g / L.

[0095] In addition, the flocculation efficiency of the microbial flocculants prepared in Examples 1-4 was tested using the following method: 500 mL of a 4 g / L kaolin suspension was prepared, 10 mL of a 10% calcium chloride solution was added, and 10 mL of the microbial flocculant was added. The pH of the solution was adjusted to 8-8.5. A kaolin suspension without flocculant was used as a control. The suspension was stirred rapidly for 3 minutes, then slowly for 3 minutes, and then allowed to settle for 5 minutes. The liquid was aspirated 1 cm below the liquid surface, and the absorbance was measured at a wavelength of 550 nm using an ultraviolet spectrophotometer. The flocculation efficiency of the microbial flocculant on the kaolin suspension was calculated. The test results are shown in Table 1.

[0096] Flocculation rate calculation formula:

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

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

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

[0100] Comparative Example 1

[0101] Take the Aspergillus sojae preserved on the slant and streak inoculate it on LB solid medium, culture it at 30℃ for 3 days, and when the surface of the plate is covered with Aspergillus sojae, use an inoculation loop to gently scrape the spores, wash them with sterile water into a conical flask, shake at room temperature for 10 minutes to fully disperse the spores, remove the hyphae and clumped spores with absorbent cotton, calculate the Aspergillus sojae spore concentration by hemocytometer, and adjust the final concentration of the spore suspension to 1×10 7 The above-mentioned Aspergillus sojae spore suspension was inoculated into 1L fermentation medium at a 4% inoculum size and cultured at 28°C, 130rpm, and shaking for 3 days, and the culture fluid was collected. The bacterial cells in the culture fluid were filtered out with gauze to obtain the fermentation fluid, which was then refrigerated and centrifuged at 10,000 rpm for 15 minutes. 3mL of the supernatant was taken as a microbial flocculant solution and added to 96mL of a 0.5% kaolin suspension with 1% CaCl2 as a coagulant aid, the pH was adjusted to 6.5, and the mixture was stirred at 300rpm for 15 minutes and allowed to stand for 10 minutes. The liquid 1 cm below the liquid level was drawn and the absorbance was measured at a wavelength of 550nm using an ultraviolet spectrophotometer to calculate the flocculation rate of the microbial flocculant on the kaolin suspension. The test results are shown in Table 1.

[0102] Comparative Example 2

[0103] A single colony of Gordonia spp. was selected as a pure strain. Using an inoculating loop, the single colony was inoculated into a sterilized Erlenmeyer flask containing 50 mL of liquid LB and 100 mL of LB. Fermentation was performed in a shaker. The temperature was set at 30°C. After 72 hours of incubation, the fermentation broth was centrifuged at 6000 rpm for 15 minutes, and the supernatant was collected as a microbial flocculant. A 1000 mL beaker was prepared, filled with a 4 g / L kaolin suspension, and 25 mL of a 1% CaCl2 solution was added. The kaolin suspension was then brought to 500 mL and the pH adjusted to 8. 10 mL of microbial flocculant was added, and the mixture was stirred rapidly at 180 rpm for 30 seconds, then slowly at 8 rpm for 5 minutes. The mixture was then allowed to stand for 5 minutes. The liquid was aspirated 1 cm below the liquid surface, and the absorbance was measured at 550 nm using a UV spectrophotometer. The flocculation rate of the kaolin suspension by the microbial flocculant was calculated. The test results are shown in Table 1.

[0104] Comparative Example 3

[0105] A single colony of Gordonia spp. was selected as a pure strain. Using an inoculating loop, the single colony was inoculated into a sterilized Erlenmeyer flask containing 50 mL of liquid LB and 100 mL of LB. Fermentation was performed in a shaker. The temperature was set at 30°C. After 72 hours of incubation, the fermentation broth was collected and used as a microbial flocculant. A 1000 mL beaker was prepared, and a 4 g / L kaolin suspension was prepared. 25 mL of 1% CaCl₂ solution was added. The kaolin suspension was then filled to 500 mL and the pH was adjusted to 8. 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. The mixture was allowed to stand for 5 minutes. The liquid was aspirated 1 cm below the liquid surface and the absorbance was measured at 550 nm using a UV spectrophotometer. The flocculation rate of the kaolin suspension by the microbial flocculant was calculated. The test results are shown in Table 1.

[0106] Comparative Example 4

[0107] A single colony of Gordonia spp. was selected as a pure strain. Using an inoculating loop, the single colony was inoculated into a sterilized Erlenmeyer flask containing 50 mL of liquid LB and 100 mL of LB. Fermentation was carried out in a shaker. The temperature was set at 30°C. After 72 hours of incubation, the fermentation broth was centrifuged at 6000 rpm for 15 minutes. The cells were collected, washed, and then prepared into a suspension for use as a microbial flocculant. A 1000 mL beaker was prepared, filled with a 4 g / L kaolin suspension, and 25 mL of 1% CaCl₂ solution was added. The kaolin suspension was then filled to 500 mL and the pH adjusted to 8. 10 mL of microbial flocculant was added, and the mixture was stirred rapidly at 180 rpm for 30 seconds, then slowly at 8 rpm for 5 minutes. The mixture was then allowed to stand for 5 minutes. The liquid 1 cm below the surface was aspirated 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.

[0108] Table 1 Flocculation rate test results

[0109] Test items Example 1 Example 2 Example 3 Example 4 Flocculation rate (%) 98.63％ 96.84％ 93.35％ 90.46％ Test 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 by the present invention has a flocculation rate of up to 98.63% when used to treat a 4 g / L kaolin suspension, which has a better flocculation effect. At the same time, it can realize the resource utilization of camellia oil cake, reduce the production cost of the microbial flocculant, and is of great significance for promoting the widespread application of microbial flocculants.

[0111] Example 5

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

[0113] Figure 6This is a comparison chart of the yield of microbial flocculants prepared in tea cake culture medium with 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 culture medium is ≥4, a higher yield can be obtained. In particular, when the pH value of the camellia oil cake culture medium is 5-9, the yield of the microbial flocculant is significantly improved.

[0114] Figure 7 This is a graph showing the yield of microbial flocculants prepared under different culture time conditions in Example 5 of the present invention. Figure 7 As can be seen, the production of microbial flocculants initially decreased within 24 hours before increasing. This is because polysaccharides, proteins, and other substances contained in the camellia oil cake were extracted along with the microbial extracellular polymers (ECPs), resulting in a high initial production of microbial flocculants. Subsequently, the polysaccharides, proteins, and other substances in the camellia oil cake were utilized by the microorganisms, causing the content of microbial flocculants to decrease again. Finally, at 25°C and 100 rpm, the microorganisms produced a large amount of ECPs, and the content of microbial flocculants increased, reaching a peak at the 72nd hour of fermentation.

[0115] In this example, the flocculation rate of the microbial flocculant prepared under different culture time conditions was also investigated. Figure 8 shown.

[0116] Figure 8 The figure 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 early stage of cultivation, although the initial production of microbial flocculants was high, it was mainly composed of polysaccharides, proteins and other substances contained in the camellia oil cake itself, and the flocculation ability was low. However, in the second stage of cultivation at 25°C and 100rpm, the microorganisms produced a large amount of extracellular polymers, resulting in an increase in the content of microbial flocculants, which in turn led to a significant improvement in flocculation ability. The highest flocculation rate of 98.63% was obtained at the 72nd hour of fermentation, which is consistent with the Figure 7 The results in are consistent.

[0117] Depend on Figure 7 and Figure 8 It can be seen that in the present invention, by optimizing the culture conditions, the microorganisms grow rapidly in the early stage of fermentation and produce a large amount of flocculants in the latter stage of fermentation. This can reduce the fermentation time while also obtaining a microbial flocculant with high yield and high flocculation rate, which is conducive to large-scale preparation and convenient for industrial application.

[0118] The above results show that, compared with the conventional microbial flocculant preparation method, the preparation method of the present invention has the following advantages: (a) low production cost. The oil-tea cake is used as the raw material to provide the main nutrients required for the culture, which can significantly reduce the production cost of the microbial flocculant. The raw material cost per unit flocculant output is about RMB 12,000 / ton. When the traditional culture medium is used to prepare the microbial flocculant, the raw material cost per unit flocculant output is about RMB 365,000 / ton. It can be seen that the raw material cost of the present invention is reduced by about 96.7%; (b) high production efficiency. Compared with the traditional culture medium, the oil-tea cake extract of the present invention is used as the raw material to prepare the culture medium, which is rich in nutrients and has good adaptability of various nutrients. This makes the flocculating functional bacteria have a higher growth rate and proliferation density, which is conducive to the fermentation system to quickly enter the flocculent production stage. The biomass of the flocculating functional bacteria is increased by at least one order of magnitude, from 0.55×10 7 The CFU / mL increased to 1.79×10 8 CFU / mL; (c) high yield and high quality. In the present invention, under the controllable concentration range of tea saponin, tannin and other ingredients, the yield and quality of microbial flocculant products with microbial extracellular metabolites as the main body can be promoted, thereby increasing the yield of microbial flocculants and enhancing the flocculation activity function. Among them, under the condition of a camellia oil cake extract with a solid-liquid ratio of 50g / L, the yield of microbial flocculant is 6.61g / L, and the flocculant yield per unit mass of camellia oil cake is 0.13g / g COC, which is 20.3% higher than that of traditional culture medium; the flocculation rate of kaolin suspension reaches 98.9%, which is 1.02% higher than that of flocculants obtained from traditional culture medium. Therefore, the preparation method of the present invention can prepare microbial flocculants with high yield and high flocculation rate, and has the advantages of low production cost and high production efficiency, and can also realize 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 that fall within the scope of protection of the present invention are within the scope of protection of the present invention. It should be noted that improvements and modifications that can be made by a person skilled in the art without departing from the principles of the present invention are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a microbial flocculant, characterized in that: The following steps are involved: S1, mixing camellia oleifera cake with water, ultrasonicating, hydrothermally treating, filtering, collecting the filtrate, and obtaining a camellia oleifera cake extract; S2, preparing a tea cake culture medium by using the tea cake extract obtained in step S1; S3, inoculating the flocculating functional bacteria seed solution into the camellia oleifera cake culture medium obtained in step S2 for culturing, thereby completing the preparation of the microbial flocculant.

2. The preparation method according to claim 1, characterized in that: In step S1, the ultrasonic treatment is performed at a temperature of 30°C to 40°C; the ultrasonic treatment time is 20min to 50min; the hydrothermal treatment is performed at a temperature of 111°C to 126°C; the hydrothermal treatment time is 10min to 50min.

3. The preparation method according to claim 2, characterized in that: In step S1, the ratio of the camellia oleifera cake to water is 30g-200g:1L; the camellia oleifera cake is further processed as follows before use: drying the camellia oleifera cake, crushing the camellia oleifera cake, and passing the cake through a 40-mesh to 60-mesh sieve to obtain camellia oleifera cake particles.

4. The preparation method according to claim 3, characterized in that: In step S1, the ratio of the oil-tea camellia cake to water is 50g-150g:1L.

5. The preparation method according to claim 1, characterized in that: In step S1, the content of polysaccharide in the oil-tea camellia cake extract is 1800mg / L-15000mg / L, the content of protein is 3300mg / L-24000mg / L, and the content of SCOD is 18000mg / L-110000mg / L.

6. The preparation method according to any one of claims 1 to 5, characterized in that In step S2, the preparation method of the camellia oleifera cake culture medium is: mixing camellia oleifera cake extract, potassium dihydrogen phosphate and dipotassium hydrogen phosphate to obtain a camellia oleifera cake culture medium; the concentration of potassium dihydrogen phosphate in the camellia oleifera cake 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 oleifera cake culture medium is 5 to 9.

7. The preparation method according to any one of claims 1 to 5, characterized in that In step S3, the inoculation amount of the flocculation functional bacteria seed solution is 4% to 5% of the volume of the camellia oleifera cake culture medium.

8. The preparation method according to claim 7, characterized in that: In step S3, the flocculation functional bacteria seed solution further includes the following treatments before inoculation: (1) Inoculate the flocculating functional bacteria into a solid activation culture medium, and culture at a temperature of 30° C. to 35° C. for 12.0 h to 24.0 h to obtain a primary seed colony; the formula of the solid activation culture medium is: 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 a pH value of 7-8; the flocculating functional bacteria is one of Paenibacillus polymyxa, lactic acid bacteria, Clostridium butyricum and Halomonas; the Paenibacillus polymyxa is GA1; (2) inoculating the primary seed colony obtained in step (1) into a liquid proliferation culture medium, culturing for 12.0 h to 24.0 h at a temperature of 25° C. to 30° C. and a rotation speed of 130 rpm to 150 rpm to obtain a flocculation functional bacteria seed solution; the formula of the liquid proliferation culture medium is: 10.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, and a pH value of 7 to 8.

9. The preparation method according to any one of claims 1 to 5, characterized in that In step S3, the culturing is: culturing for 12.0h to 24.0h at a temperature of 30°C to 35°C and a rotation speed of 130rpm to 150rpm, and culturing for 24.0h to 72.0h at a temperature of 20°C to 25°C 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 the bacteria; the centrifugal speed is 8000rpm to 10000rpm; the centrifugal time is 8min to 10min.

10. A microbial flocculant, characterized in that: The microbial flocculant is prepared by the preparation method according to any one of claims 1 to 9.

Images