Method for forming rhizopus arrhizus biofilm

Abstract

The invention provides a method for forming a rhizopus arrhizus biological membrane, and aims to realize efficient and stable formation of the biological membrane by optimizing a culture medium formula and controlling culture conditions. The method comprises the following steps: preparing a culture medium with specific components, adding a carbon source, a nitrogen source, monopotassium phosphate, magnesium sulfate and various trace elements, and culturing by inoculating rhizopus arrhizus strains. In the culture process, the secretion of extracellular polymeric substances is promoted and the formation of biological membranes is accelerated by continuously supplying oxygen and adding a calcium chloride inducer. And culturing in a constant-temperature environment of 28-32 DEG C for 48-72 hours to obtain a stable rhizopus arrhizus biological membrane. Compared with the prior art, the culture method provided by the invention can effectively improve the thickness and stability of the biological membrane and shorten the formation period, and is suitable for industrial application requirements.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial biofilm formation methods, specifically a method for forming a Rhizopus microsporus biofilm. BACKGROUND

[0002] Rhizopus microsporus is an important filamentous fungus widely used in food fermentation, pharmaceutical production, biodegradation, and other fields. In recent years, microbial biofilm research has gained increasing attention in industrial and environmental applications because biofilms provide a stable growth environment for microorganisms, enhancing their survival ability and metabolic efficiency under different environmental conditions. Biofilm formation involves microbial attachment, growth on solid surfaces, and maintenance of cell population stability through the secretion of extracellular polymeric substances (EPS). In practical applications, by controlling culture conditions and adding external inducers, microbial biofilm formation can be regulated to optimize biofilm structure and function. These characteristics make microbial biofilms have great application potential in wastewater treatment, biodegradation, food industry, pharmaceutical development, and other fields.

[0003] Currently, the industrial application of Rhizopus microsporus mainly focuses on the production of fermented foods such as traditional soy sauce, miso, yogurt, and other products. During these fermentation processes, Rhizopus microsporus can degrade complex polysaccharides into simple sugars for use by fermenting microorganisms through its unique enzyme system. However, the application potential of Rhizopus microsporus goes far beyond this. In recent years, with the in-depth study of microbial biofilms, the biofilm formation characteristics of Rhizopus microsporus have been gradually revealed, showing potential application value in environmental protection, bioremediation, and biomedicine.

[0004] The formation of Rhizopus microsporus biofilm has important significance in microbial ecosystems. Compared with free-floating cells, microbial populations forming biofilms have higher stress resistance and survival ability. Cells in biofilms can form a tight network structure through the secretion of extracellular polymeric substances, thereby resisting external adverse conditions such as pH fluctuations, temperature changes, anoxic environments, and attacks by antibacterial substances. This characteristic is of great significance in wastewater treatment and soil remediation applications, as microbial biofilms can continuously degrade and treat pollutants in harsh environments. In addition, the metabolic activity of Rhizopus microsporus in the biofilm state is usually more active, enabling more efficient sugar conversion and enzyme secretion, thus showing good application prospects in the fermentation industry.

[0005] Although Rhizopus arrhizus biofilm formation has good application prospects, it still faces some technical challenges in actual operation. First, the formation of biofilm depends on various environmental factors, such as culture medium composition, temperature, pH value, dissolved oxygen concentration, etc. Changes in these factors will directly affect the stability and function of the biofilm. Second, in order to effectively utilize the biofilm characteristics of Rhizopus arrhizus in industrial production, a controllable and stable biofilm formation method is needed to ensure the stability of the production process and the efficient output of the product. However, the existing biofilm formation methods are mostly developed under laboratory conditions, and their adaptability to large-scale industrial production still needs to be further optimized. For example, the traditional culture medium may not provide enough nutrients to support long-term biofilm growth, and the lack of dissolved oxygen during cultivation may lead to the disintegration of the biofilm.

[0006] The present application aims to solve the above problems by providing an optimized Rhizopus arrhizus biofilm formation method. By formulating a culture medium with specific components and controlling key parameters such as temperature, dissolved oxygen concentration and the use of external inducers during cultivation, a stable and efficient Rhizopus arrhizus biofilm can be formed in a short time. This method not only improves the efficiency of biofilm formation, but also enhances the stability and thickness of the biofilm by regulating the secretion of extracellular polymers, thereby meeting the needs of Rhizopus arrhizus biofilm in different application scenarios. In addition, calcium chloride (CaCl2) used in this application as an inducer can effectively promote the secretion of extracellular polymers, thereby shortening the formation period of the biofilm. These technical improvements not only improve the efficiency of Rhizopus arrhizus biofilm formation, but also provide a feasible solution for its large-scale application in industrial production.

[0007] In summary, the development of Rhizopus arrhizus biofilm formation technology will provide a new breakthrough for microbial fermentation industry and environmental remediation technology. By improving the culture medium formula and cultivation conditions, the present application successfully solves some of the deficiencies in the prior art, opening up a new path for the application of microbial biofilms in industrial production. This technology not only improves the stability and metabolic efficiency of the biofilm, but also adapts to diverse industrial production needs, and has broad application prospects.

[0008] Therefore, we designed a Rhizopus arrhizus biofilm formation method to solve the above problems. SUMMARY

[0009] The present application aims to solve the above problems by providing an optimized Rhizopus arrhizus biofilm formation method. By formulating a culture medium with specific components and controlling key parameters such as temperature, dissolved oxygen concentration and the use of external inducers during cultivation, a stable and efficient Rhizopus arrhizus biofilm can be formed in a short time. This method not only improves the efficiency of biofilm formation, but also enhances the stability and thickness of the biofilm by regulating the secretion of extracellular polymers, thereby meeting the needs of Rhizopus arrhizus biofilm in different application scenarios. In addition, calcium chloride (CaCl2) used in this application as an inducer can effectively promote the secretion of extracellular polymers, thereby shortening the formation period of the biofilm. These technical improvements not only improve the efficiency of Rhizopus arrhizus biofilm formation, but also provide a feasible solution for its large-scale application in industrial production.

[0010] To achieve the above-mentioned purpose, the present application provides the following technical solution: a Rhizopus arrhizus biofilm formation method, comprising the following steps:

[0011] The culture medium comprises 10-30 g / L of carbon source, 1-3 g / L of nitrogen source, 0.5-1 g / L of potassium dihydrogen phosphate, 0.1-0.2 g / L of magnesium sulfate, 0.01-0.02 g / L of ferrous sulfate, 0.01-0.02 g / L of zinc sulfate, and 0.01-0.02 g / L of manganese sulfate;

[0012] The Rhizopus arrhizus spore is inoculated into the culture medium at an inoculation amount of 10 6 -10 7 CFU / mL;

[0013] The reactor containing the culture medium and the spore is placed in a constant temperature environment of 28°C-32°C, preferably 30°C;

[0014] Continuous oxygen supply is performed by a gas pump or a stirring device to maintain the dissolved oxygen concentration at 3-5 mg / L;

[0015] Calcium chloride (CaCl2) is added as an inducer during the culture process at a concentration of 0.01-0.02 g / L to promote the secretion of extracellular polymers and the formation of biofilm;

[0016] After 48-72 hours of culture, a stable Rhizopus arrhizus biofilm is obtained.

[0017] As a preferred technical solution of the present application, the carbon source in the culture medium is glucose, and the concentration range is 10-20 g / L, preferably 15 g / L, to ensure the energy needs of Rhizopus arrhizus during the culture process and promote the synthesis of extracellular polymers.

[0018] As a preferred technical solution of the present application, the carbon source in the culture medium is fructose, and the concentration range is 10-20 g / L, preferably 15 g / L.

[0019] As a preferred technical solution of the present application, the nitrogen source in the culture medium is ammonium nitrate, and the concentration range is 1-2 g / L, preferably 1.5 g / L, to provide the nitrogen elements required for the metabolic activity of Rhizopus arrhizus, thereby supporting its rapid growth and the stability of the biofilm.

[0020] As a preferred technical solution of the present application, the nitrogen source in the culture medium is urea, and the concentration range is 1-2 g / L, preferably 1.5 g / L.

[0021] As a preferred technical solution of the present application, the concentration of potassium dihydrogen phosphate in the culture medium is 0.5-1 g / L, preferably 0.8 g / L. Potassium dihydrogen phosphate serves as a source of phosphorus elements, which can promote the synthesis of ATP, thereby enhancing the energy metabolism efficiency of Rhizopus arrhizus.

[0022] As a preferred technical scheme of the present application, the concentration of magnesium sulfate in the culture medium is 0.1-0.2 g / L, preferably 0.15 g / L, and magnesium element plays an important auxiliary role in cell function and extracellular polymer formation of Rhizopus arrhizus.

[0023] As a preferred technical scheme of the present application, the trace elements added to the culture medium include ferrous sulfate 0.015 g / L, zinc sulfate 0.015 g / L, and manganese sulfate 0.015 g / L, which play an important catalytic role in the metabolism of Rhizopus arrhizus biofilm and help promote the secretion of extracellular polymers, thereby improving the stability and density of the biofilm.

[0024] As a preferred technical scheme of the present application, the initial pH value of the culture medium is adjusted to 5.0-6.0, preferably 5.5, by adding sodium hydroxide or hydrochloric acid, which can promote the optimal growth environment of Rhizopus arrhizus and enhance the production of extracellular polymers.

[0025] As a preferred technical scheme of the present application, the concentration of the inducer calcium chloride (CaCl2) is controlled at 0.015 g / L, and by adding calcium chloride during the culture process, the secretion of extracellular polymers can be effectively promoted, the formation of biofilm can be accelerated, the formation period can be shortened to 48-72 hours, and the thickness and stability of the biofilm can be improved, making it suitable for industrial application requirements.

[0026] Compared with the prior art, the present application has the following advantages: by optimizing the culture medium composition and controlling the culture conditions, the biofilm of Rhizopus arrhizus can be formed efficiently in a short time. With the combination of specific carbon source, nitrogen source and trace elements, the biofilm can be stably generated within 48-72 hours, significantly shortening the time period of traditional biofilm culture, improving the efficiency and controllability of operation, and meeting the application requirements of industrialization.

[0027] The present application adds calcium chloride as an inducer, which significantly promotes the secretion of extracellular polymers, thereby improving the thickness and density of the biofilm, making it more stable in industrial environment. The biofilm structure is compact, has good stress resistance, and can maintain stability under various environmental conditions, which helps to cope with external disturbances such as pH fluctuation and temperature change.

[0028] By precisely controlling the culture parameters, the Rhizopus arrhizus biofilm generated by the present method is suitable for industrial fermentation, wastewater treatment and biodegradation, etc. This technology provides a new way for the industrial application of Rhizopus arrhizus, especially in the fields of environmental protection and biomedicine, and solves the problems of complex operation and low stability in traditional culture techniques. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be described clearly and completely below in connection with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0030] The specific embodiments of the present application will be described in detail below in connection with a plurality of embodiments.

[0031] The present application provides a method for quickly and stably forming Rhizopus arrhizus biofilm by regulating medium components and culture conditions. In order to verify the effect of the method of the present application, a plurality of specific embodiments are listed below to show different operation methods and experimental data.

[0032] Example 1: Biofilm formation with glucose as carbon source

[0033] Experimental steps

[0034] Prepare the culture medium, which includes the following components:

[0035] 1. Carbon source: glucose 15 g / L

[0036] 2. Nitrogen source: ammonium nitrate 1.5 g / L

[0037] 3. Potassium dihydrogen phosphate: 0.8 g / L

[0038] 4. Magnesium sulfate: 0.15 g / L

[0039] 5. Ferrous sulfate: 0.015 g / L

[0040] 6. Zinc sulfate: 0.015 g / L

[0041] 7. Manganese sulfate: 0.015 g / L

[0042] Adjust the initial pH value of the culture medium to 5.5.

[0043] Inoculate the Rhizopus arrhizus spores into the culture medium at an inoculation amount of 10 6 CFU / mL.

[0044] Control the temperature in the culture reactor at 30°C, continuously supply oxygen, and maintain the dissolved oxygen concentration at 4 mg / L.

[0045] Add calcium chloride (CaCl2) 0.015 g / L in the culture medium to promote the secretion of extracellular polymers.

[0046] After 48 hours of culture, detect the thickness and biomass of the biofilm.

[0047] Experimental results

[0048] Culture time (hours) Biofilm thickness (mm) Biomass (g / L) 24 0.5 2.1 48 1.2 4.8 72 1.5 6.3

[0049] The experimental results show that under the above conditions, the Rhizopus arrhizus can quickly form a stable biofilm, and the biomass reaches a high level after 48 hours.

[0050] Example 2: Biofilm formation with fructose as carbon source

[0051] Experimental procedure

[0052] The culture medium was prepared, which included the following components:

[0053] 1. Carbon source: fructose 15 g / L

[0054] 2. Nitrogen source: urea 1.5 g / L

[0055] 3. Potassium dihydrogen phosphate: 0.8 g / L

[0056] 4. Magnesium sulfate: 0.15 g / L

[0057] 5. Ferrous sulfate: 0.015 g / L

[0058] 6. Zinc sulfate: 0.015 g / L

[0059] 7. Manganese sulfate: 0.015 g / L

[0060] The initial pH value of the culture medium was adjusted to 5.5.

[0061] The Rhizopus arrhizus strain was inoculated into the culture medium at an inoculation amount of 10 7 CFU / mL.

[0062] The temperature in the reactor was controlled at 28°C, and the dissolved oxygen concentration was maintained at 3 mg / L.

[0063] Calcium chloride (CaCl2) 0.01 g / L was added to the culture medium.

[0064] After 72 hours of cultivation, the thickness and biomass of the biofilm were detected.

[0065] Experimental results

[0066] Culture time (hours) Biofilm thickness (mm) Biomass (g / L) 24 0.4 1.8 48 1.0 3.5 72 1.6 5.0

[0067] The results show that when using fructose as a carbon source, the biofilm formation speed is slightly slower than that of glucose, but the thickness and biomass of the biofilm are relatively stable after 72 hours.

[0068] Example 3: Effect of different nitrogen sources on biofilm formation

[0069] Experimental procedure

[0070] Two groups of media were prepared, using ammonium nitrate and urea as nitrogen sources, respectively, with the following specific compositions:

[0071] Medium A (ammonium nitrate):

[0072] 1. Carbon source: glucose 20 g / L

[0073] 2. Nitrogen source: ammonium nitrate 2 g / L

[0074] 3. Potassium dihydrogen phosphate: 0.8 g / L

[0075] 4. Other components same as Example 1

[0076] 2. Medium B (urea):

[0077] 1. Carbon source: glucose 20 g / L

[0078] 2. Nitrogen source: urea 2 g / L

[0079] 3. Potassium dihydrogen phosphate: 0.8 g / L

[0080] 4. Other components same as Example 1

[0081] Each group of medium was inoculated with 10 6 CFU / mL of inoculum.

[0082] The reactor temperature was controlled at 30°C, with continuous oxygen supply, maintaining the dissolved oxygen concentration at 4 mg / L.

[0083] Calcium chloride (CaCl2) was added to the medium at 0.015 g / L.

[0084] After 48 hours, the thickness and biomass of the biofilm were detected.

[0085] Experimental results

[0086] Nitrogen source Biofilm thickness (mm) Biomass (g / L) Ammonium nitrate 1.3 5.2 Urea 1.0 4.1

[0087] The results showed that when using ammonium nitrate as the nitrogen source, the thickness and biomass of the biofilm were higher than when using urea as the nitrogen source, indicating that ammonium nitrate was more effective in the formation of Rhizopus arrhizus biofilm.

[0088] Example 4: Effect of different CaCl2 concentrations on biofilm

[0089] Experimental procedure

[0090] A medium was prepared with the same components as Example 1, but with different concentrations of calcium chloride added:

[0091] 1. Medium A: CaCl2 concentration of 0.01 g / L

[0092] 2. Medium B: CaCl2 concentration of 0.015 g / L

[0093] 3. Medium C: CaCl2 concentration of 0.02 g / L

[0094] Rhizopus arrhizus spores were inoculated into the medium, with an inoculation amount of 10 6 CFU / mL.

[0095] The temperature in the reactor was controlled at 30°C, oxygen was continuously supplied, and the dissolved oxygen concentration was maintained at 4 mg / L.

[0096] After 48 hours, the thickness and biomass of the biofilm were detected.

[0097] Experimental results

[0098] CaCl2 concentration (g / L) Biofilm thickness (mm) Biomass (g / L) 0.01 1.0 4.0 0.015 1.3 5.2 0.02 1.5 5.6

[0099] As can be seen from the experimental results, with the increase of CaCl2 concentration, the thickness and biomass of Rhizopus arrhizus biofilm are both improved, but the improvement rate slows down after 0.015 g / L. To balance the cost and effect, 0.015 g / L is a relatively optimal concentration.

[0100] The above examples show that by optimizing the carbon source, nitrogen source, and CaCl2 concentration, the formation speed, thickness, and stability of Rhizopus arrhizus biofilm can be significantly improved. The method of the present application can form stable and high-density Rhizopus arrhizus biofilm in a short time, and is suitable for various application scenarios in industrial production.

[0101] The contents not described in detail in the description belong to the prior art known to those skilled in the art. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments or make equivalent replacements to part of the technical features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for forming a biofilm of Rhizopus arrhizus, characterized in that The following steps are involved: Prepare culture medium: the culture medium includes 10-30 g / L carbon source, 1-3 g / L nitrogen source, 0.5-1 g / L potassium dihydrogen phosphate, 0.1-0.2 g / L magnesium sulfate, 0.01-0.02 g / L ferrous sulfate, 0.01-0.02 g / L zinc sulfate, and 0.01-0.02 g / L manganese sulfate; The Rhizopus arrhizus strain was inoculated into the above culture medium at an inoculum size of 10 6 CFU / mL-10 7 CFU / mL; The reactor containing the culture medium and bacterial strain obtained above was placed in a constant temperature environment of 28°C-32°C; Continuously supply oxygen to the reactor obtained in step 3 by an air pump or a stirring device to maintain the dissolved oxygen concentration at 3-5 mg / L; During the culture process, the inducer calcium chloride was added at a concentration of 0.01-0.02 g / L to promote the secretion of extracellular polymers and the formation of biofilms; After 48-72 hours of incubation, stable R. arrhizus biofilms were obtained.

2. The method for forming a Rhizopus arrhizus biofilm according to claim 1, wherein The carbon source in the culture medium is glucose, and the concentration range is 10-20 g / L.

3. The method for forming a Rhizopus arrhizus biofilm according to claim 1, wherein The carbon source in the culture medium is fructose, and the concentration range is 10-20 g / L.

4. The method for forming a Rhizopus arrhizus biofilm according to claim 1, wherein The nitrogen source in the culture medium is ammonium nitrate with a concentration range of 1-2 g / L.

5. The method for forming a Rhizopus arrhizus biofilm according to claim 1, wherein The nitrogen source in the culture medium is urea, and the concentration range is 1-2 g / L.

6. The method for forming a Rhizopus arrhizus biofilm according to claim 1, wherein The concentration of potassium dihydrogen phosphate in the culture medium is 0.5-1 g / L, and potassium dihydrogen phosphate serves as a source of phosphorus.

7. The method for forming a Rhizopus arrhizus biofilm according to claim 1, wherein The concentration of magnesium sulfate in the culture medium is 0.1-0.2 g / L.

8. The method for forming a Rhizopus arrhizus biofilm according to claim 1, wherein The trace elements added to the culture medium include 0.015 g / L of ferrous sulfate, 0.015 g / L of zinc sulfate and 0.015 g / L of manganese sulfate.

9. The method for forming a Rhizopus arrhizus biofilm according to claim 1, wherein The initial pH value of the culture medium is adjusted to 5.0-6.0 by adding sodium hydroxide or hydrochloric acid. The appropriate pH value can promote the optimal growth environment of Rhizopus arrhizus and enhance the generation of extracellular polymers.

10. The method for forming a Rhizopus arrhizus biofilm according to claim 1, wherein The concentration of the inducer calcium chloride is controlled at 0.015 g / L. By adding calcium chloride during the culture process, the secretion of extracellular polymers is promoted, the formation of biofilm is accelerated, and the formation period is shortened to 48-72 hours.