Microbial growth activation accelerant based on rare earth elements, preparation method and application

Abstract

The invention relates to a microbial growth activation accelerant based on rare earth elements as well as a preparation method and application thereof, and belongs to the field of resources and environments. The microbial growth activation accelerant based on the rare earth elements is prepared from the following components: 1 to 150g of a rare earth compound, 0.2 to 2g of pantothenic acid, 0.2 to 2g of nicotinic acid, 0.02 to 0.1 g of nicotinamide and 1000g of deionized water, the rare earth compound is one or combination of more of chlorides of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, scandium and yttrium, nitric acid compounds and sulfuric acid compounds. A large number of cheap light rare earth elements are used as raw materials of the microbial accelerator, a new utilization approach is provided for the light rare earth elements, the light rare earth elements are converted into high-added-value products, pollution of the light rare earth elements to the environment is reduced, and meanwhile new elements are introduced into the field of microbial accelerators. More importantly, the preparation method of the microbial growth activation accelerant based on the rare earth elements provided by the invention is simple in production process and convenient for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of resources and environment, and particularly relates to a microbial growth activator based on rare earth elements, its preparation method and application. Background Technology

[0002] Microbial fermentation and synthesis technologies have gradually become key technologies for the production of large quantities of chemical raw materials and pharmaceuticals. However, due to their relatively low production rates, they are difficult to meet the demands of today's industrial production. Therefore, improving the growth and metabolic rates of industrial microorganisms has become a major challenge in the field of biomanufacturing.

[0003] Rare earth elements consist of 17 elements, including 15 lanthanide elements (lanthanum, cerium, praseodymium, neodymium, samarium, samarium, gadolinium, europium, dysprosium, holmium, erbium, thallium, yttrium, and lutetium), as well as scandium and yttrium. Today, medium and heavy rare earth elements, due to their unique chemical, electrical, magnetic, and optical properties, are gradually becoming one of the most important strategic resources of the 21st century, especially widely used in industrial production. With a deeper understanding of rare earth elements, a considerable amount of research considers them a new type of "life element," playing a crucial regulatory role in microorganisms, plants, and even animals. Appropriate concentrations of rare earth elements can significantly promote the growth and metabolism of organisms. However, research on the application of rare earth elements as microbial growth promoters is rarely reported. Summary of the Invention

[0004] To meet the current demand for industrial microbial growth promoters and address the issues of low fermentation efficiency and excessive redundancy of light rare earth elements, this invention provides a microbial growth promoter based on light rare earth elements, its preparation method, and its applications. This microbial promoter can provide excellent growth promotion effects on a wide range of microorganisms, showing significant promotion effects on the growth and metabolism of many representative strains with industrial production benefits. Furthermore, the production process is simple and suitable for large-scale production.

[0005] The technical problem to be solved by this invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a rare earth element-based microbial growth activation promoter, its preparation method, and its application. To solve the above technical problem, the technical solution proposed by this invention is as follows: A rare earth element-based microbial growth activator, wherein the rare earth element-based microbial growth activator comprises: 1-150 g of rare earth compound, 0.2-2 g of pantothenic acid, 0.2-2 g of nicotinic acid, 0.02-0.1 g of nicotinamide and 1000 g of deionized water.

[0006] In some embodiments, the rare earth compound is one or more combinations of chlorides and sulfates of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, scandium, and yttrium.

[0007] This invention also provides a method for preparing the above-mentioned rare earth element-based microbial growth activator, the preparation method comprising the following steps: S1 uses 1L of water as a solvent. Weigh out 200-2000mg of pantothenic acid, 200-2000mg of nicotinic acid, and 20-100mg of nicotinamide, and dissolve them in water. S2 Weigh 1000-10000 mg of rare earth chloride and add it to the solvent in step S1; S3 is prepared by adjusting the pH to 3-4 with hydrochloric acid to ensure that there is no precipitate in the solution.

[0008] This invention also provides the application of the above-mentioned rare earth element-based microbial growth activator in microbial fermentation, microbial breeding, microbial screening, and microbial modification. The specific method for its application in microbial fermentation is as follows: The specific method for application in microbial breeding is as follows: During the microbial breeding process, microbial growth activation promoters can be added to the culture medium inoculated with microorganisms at a ratio of 1:100 to 1:1000, according to the tolerance of different microorganisms. This can significantly shorten the lag phase of microorganisms, improve metabolic efficiency, and thus improve breeding efficiency. The specific method for application in microbial screening is as follows: During the screening of functional microorganisms, microbial growth activators can be added to the culture medium inoculated with the microorganisms to be screened at a ratio of 1:100 to 1:1000, depending on the tolerance of different microorganisms. This can significantly increase the survival rate of the microorganisms to be screened and improve the screening efficiency. The specific method for microbial modification is as follows: During the modification process of genetically engineered bacteria, microbial growth activation promoters can be added to the culture medium inoculated with genetically engineered bacteria at a ratio of 1:100 to 1:1000, depending on the tolerance of different microorganisms. This can significantly increase the survival rate of genetically engineered bacteria and improve the modification efficiency.

[0009] Compared with the prior art, the present invention has the following advantages: (1) This invention uses rare earth elements as raw materials for microbial promoters, which not only provides a new way to utilize large quantities of inexpensive rare earth elements, transforming them into high-value-added products and reducing their environmental pollution, but also introduces new elements into the field of microbial promoters. (2) The rare earth element-based microbial growth activator provided by the present invention can effectively enhance the growth and metabolic functions of microorganisms, thereby effectively improving the production functions related to the microorganisms. (3) The microbial growth activation promoter based on rare earth elements provided by the present invention is prepared using inexpensive and abundant light rare earth elements, and the production process is simple and easy to produce on a large scale. Attached Figure Description

[0010] Figure 1 Example 1 shows the statistical chart of the concentration of viable bacteria of *Glucobacterium oxysporum* at different times using microbial growth activators prepared with different rare earth elements, diluted by 1:1000. Figure 2 Example 1 shows the statistical chart of the concentration of viable bacteria of *Glucobacterium oxysporum* at different times promoted by microbial growth activators prepared with different rare earth elements, with a dilution factor of 1:500. Figure 3 Example 1 shows the statistical chart of the concentration of viable bacteria of *Glucobacterium oxysporum* at different times using microbial growth activators prepared with different rare earth elements, diluted by 1:100. Figure 4 Example 2: Experiment on the promotion of yeast growth by the lanthanum microbial growth activator prepared in Example 2; Figure 5 Example 2: Experiment on the promotion of lactic acid bacteria growth by the lanthanum microbial growth activator prepared in Example 2; Figure 6 Example 3: Experiment on the recovery of preserved seeds of *Gluconobacterium oxysporum* using lanthanum microbial growth activator prepared in Example 3; Figure 7 Example 4: Experiment on improving the survival rate of genetically engineered strains and increasing the efficiency of strain screening using the lanthanum microbial growth activator prepared in this study. Detailed Implementation

[0011] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, technical process steps, specific implementation conditions and materials. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0012] Example 1 Experiments on the promotion of growth of glucosamine oxidans by different rare earth elements.

[0013] Prepare microbial growth promoters with different rare earth elements as the core: Weigh 0.2 g pantothenic acid, 0.2 g nicotinic acid, and 0.02 g nicotinamide and add them sequentially to 1000 g deionized water to form a homogeneous and stable mixed solution. Prepare 18 identical mixed solutions. Then weigh out 1.765 g lanthanum chloride, 1.759 g cerium chloride, 1.755 g praseodymium chloride, 1.738 g neodymium chloride, 1.733 g promethium chloride, 1.707 g samarium chloride, 1.700 g europium chloride, 1.676 g gadolinium chloride, 1.669 g terbium chloride, 1.655 g dysprosium chloride, 1.645 g holmium chloride, 1.636 g erbium chloride, 1.630 g thulium chloride, 1.615 g ytterbium chloride, 1.608 g lutetium chloride, 3.366 g scandium chloride, and 2.196 g lanthanum chloride. g of yttrium chloride was added to 17 portions of a mixed solution, and the pH of the mixed solution was adjusted to 3-4 by hydrochloric acid to obtain 17 microbial growth promoters with different rare earth elements and a blank control group without rare earth ions.

[0014] The prepared growth promoter and blank control group were sterilized by filtration through a 0.22 μm organic filter membrane. A growth medium for *Glucobacterium oxysporum* was prepared: 100 g / L sorbitol, 3 g / L yeast extract, and 2 g / L urea, and the medium was autoclaved. Microbial growth promoters were added to the media requiring them at volume ratios of 1:100, 1:500, and 1:1000, respectively. The activated *Glucobacterium oxysporum* strain was washed with sterile deionized water and then added to a final concentration of 10... 6 The cells / ml ratio was used for inoculation. The culture was continued at 200 r / min and 30 ℃ for 36 h, with samples taken periodically. Microbial cells were counted using a microscopic live cell counting method to observe the promoting effect of different rare earth element microbial growth promoters on the growth of *Glucosamine oxidans*. Results are as follows: Figure 1 , Figure 2 and Figure 3 As shown, compared with the blank control group, all rare earth element-based microbial growth promoters have the effect of promoting the growth of Gluconobacterium oxidans. However, the degree of promotion varies among different rare earth ions, and the higher the dilution factor of rare earth ions, the lower the promotion efficiency.

[0015] Example 2 Experiments on promoting the growth of yeast and lactic acid bacteria using lanthanum, a rare earth element, as a promoter.

[0016] The prepared growth promoter and blank control group were prepared using a lanthanum-based microbial growth promoter: 0.2 g of pantothenic acid, 0.2 g of nicotinic acid, and 0.02 g of nicotinamide were weighed and added sequentially to 1000 g of deionized water to form a homogeneous and stable mixed solution. Two identical mixed solutions were prepared. Then, 1.765 g of lanthanum chloride was weighed into the mixed solution, and the pH of the mixed solution was adjusted to 3-4 with hydrochloric acid to obtain the lanthanum-based microbial growth promoter and the blank control group without rare earth ions.

[0017] The growth promoter was sterilized by filtration through a 0.22 μm organic filter membrane. Yeast growth medium was prepared with 20 g / L glucose, 1 g / L yeast extract, and 2 g / L peptone; lactic acid bacteria growth medium was prepared with 20 g / L glucose, 1 g / L yeast extract, 2 g / L sodium chloride, and 2 g / L peptone. Both media were then autoclaved. Microbial growth promoters were added to the media requiring them at volume ratios of 1:100, 1:500, and 1:1000, respectively. The activated yeast and lactic acid bacteria cultures were washed with sterile deionized water and then added to a final concentration of 10... 6 The culture was carried out at a ratio of cells / ml. The culture was continued at 200 rpm and 30℃ for 36 h, with samples taken periodically. Microbial cells were counted using a microscopic live cell counting method to observe the effects of growth promoters on the growth of yeast and lactic acid bacteria. Results are as follows: Figure 4 and Figure 5 As shown, rare earth ion lanthanum has a growth-promoting effect on both yeast and lactic acid bacteria. Especially in the early stage of growth, it can effectively shorten the adaptation period of bacteria, enabling them to enter the logarithmic growth phase more quickly and significantly increasing the growth rate of bacteria. However, different concentrations of rare earth ions have different growth-promoting effects; the higher the dilution factor, the lower the promotion efficiency.

[0018] Example 3 Experiment on the recovery of seeds preserved by *Gluconobacter oxidans* using a catalytic agent mainly composed of rare earth element lanthanum. The prepared growth promoter and blank control group were prepared using a lanthanum-based microbial growth promoter: 0.2 g of pantothenic acid, 0.2 g of nicotinic acid, and 0.02 g of nicotinamide were weighed and added sequentially to 1000 g of deionized water to form a homogeneous and stable mixed solution. Two identical mixed solutions were prepared. Then, 1.765 g of lanthanum chloride was weighed into the mixed solution, and the pH of the mixed solution was adjusted to 3-4 with hydrochloric acid to obtain the lanthanum-based microbial growth promoter and the blank control group without rare earth ions.

[0019] The growth promoter was sterilized by filtration through a 0.22 μm organic filter membrane. A resuscitation medium for *Glucobacterium oxygenase* was prepared: 50 g / L sorbitol and 1 g / L yeast extract. The microbial growth promoter was added to the medium at a volume ratio of 1:100. 0.0001 g of lyophilized *Glucobacterium oxygenase* inoculum was inoculated. 0.001 ml of the *Glucobacterium oxygenase* -80℃ cryopreservation solution was inoculated. The culture was continued at 200 r / min and 30℃ for 24 h. Samples were taken periodically, and microbial cells were counted using a live cell counting method under a microscope to observe the promoting effect of the microbial growth promoter on the resuscitation process of *Glucobacterium oxygenase*. Results are as follows: Figure 6 As shown, compared with the blank control group, whether it is freeze-dried bacterial powder or bacterial liquid stored at -80℃, rare earth element-based microbial growth promoter can effectively promote the growth of microorganisms, significantly shorten the growth lag period, and accelerate the seed recovery process.

[0020] Example 4 Experiments using lanthanum, a rare earth element, as a promoter to improve the survival rate and screening efficiency of genetically engineered bacterial strains. The prepared growth promoter and blank control group were prepared using a lanthanum-based microbial growth promoter: 0.2 g of pantothenic acid, 0.2 g of nicotinic acid, and 0.02 g of nicotinamide were weighed and added sequentially to 1000 g of deionized water to form a homogeneous and stable mixed solution. Two identical mixed solutions were prepared. Then, 1.765 g of lanthanum chloride was weighed into the mixed solution, and the pH of the mixed solution was adjusted to 3-4 with hydrochloric acid to obtain the lanthanum-based microbial growth promoter and the blank control group without rare earth ions.

[0021] The growth promoter was sterilized by filtration through a 0.22 μm organic filter membrane. A screening medium for the genetically engineered bacteria was prepared: 50 g / L sorbitol, 10 g / L peptone, 10 g / L NaCl, and 1 g / L yeast extract. Microbial growth promoters were added to the medium at volume ratios of 1:100, 1:500, and 1:1000, respectively, to prepare solid media containing the growth promoters. The genetically engineered *Gluconobacter oxytetracycline* bacterial suspension was diluted and plated onto solid media containing different concentrations of growth promoters. The plates were incubated at 30°C for 24 hours, and the colony counts were performed to observe the effect of the growth promoters on the survival rate of the genetically engineered bacteria.

[0022] The growth promoter was sterilized by filtration through a 0.22 μm organic filter membrane. A natural microbial screening medium was prepared consisting of 10 g / L glucose, 10 g / L peptone, 10 g / L NaCl, and 1 g / L yeast extract. Microbial growth promoters were added to the medium at volume ratios of 1:100, 1:500, and 1:1000, respectively, to prepare solid media containing the growth promoters. The natural microbial bacterial suspensions were diluted and spread onto solid media containing different concentrations of growth promoters. The media were incubated at 30°C for 24 hours, and the colony counts were performed to observe the effect of the growth promoters on the survival rate of the natural microbial strains.

[0023] The results are as follows Figure 7 As shown, compared with the blank control group, rare earth element-based microbial growth promoters can effectively promote the survival rate of genetically engineered strains and natural strains, greatly improving breeding and screening efficiency. However, the degree of promotion varies with different concentrations of rare earth ions.

[0024] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

Claims

1. A rare earth element-based microbial growth activation promoter, characterized by, The rare earth element-based microbial growth activation promoter has the following composition: 1-150 g of rare earth compound, 0.2-2 g of pantothenic acid, 0.2-2 g of nicotinic acid, 0.02-0.1 g of nicotinamide and 1000 g of deionized water; the rare earth compound is one or more of chlorides and sulfate compounds of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, scandium and yttrium.

2. The method for producing a rare earth element-based microbial growth activation promoter according to claim 1, characterized by, The preparation method comprises the following steps: S1 dissolving pantothenic acid, nicotinic acid and nicotinamide in water according to the formula to form a uniform and stable mixed solution; S2 weighing the rare earth compound according to the formula and adding it into the mixed solution prepared in step S1; S3 adjusting the pH value of the mixed solution to 3-4 with hydrochloric acid so that the solution has no precipitate, and obtaining the rare earth element-based microbial growth activation promoter.

3. The rare earth element-based microbial growth activation promoter according to claim 1 is applied to microbial fermentation, microbial breeding and microbial screening, wherein the application to microbial fermentation comprises the following specific method: in the microbial fermentation production process, the microbial growth activation promoter can be added into the inoculated seed culture medium of the microorganism to be industrially fermented according to the tolerance of different microorganisms at a volume ratio of 1:100-1:1000. The application to microbial screening comprises the following specific method: in the functional microbial screening process, the microbial growth activation promoter can be added into the culture medium inoculated with the microorganism to be screened according to the tolerance of different microorganisms at a volume ratio of 1:100-1:1000. The application to microbial modification comprises the following specific method: in the modification process of the genetically engineered bacteria, the microbial growth activation promoter can be added into the culture medium inoculated with the genetically engineered bacteria according to the tolerance of different microorganisms at a volume ratio of 1:100-1:1000. ​

Images