Use of rhodosporidium sphaerocarpum or its progeny in enriching rare earth elements and aquaculture

Abstract

The application provides application of Rhodosporidium sphaerocarpum or its offspring in enrichment of rare earth elements and aquaculture of aquatic animals, and the Rhodosporidium sphaerocarpum has a strain number CICC 32489 in China Industrial Microbial Strain Preservation and Management Center. The Rhodosporidium sphaerocarpum or its offspring provided by the application can convert inorganic rare earth elements into microbial rare earth elements which are easy to be absorbed and utilized by animals, and the microbial rare earth elements are enriched in the Rhodosporidium sphaerocarpum cell bodies; the Rhodosporidium sphaerocarpum enriched with rare earth elements is safer and easier to be absorbed by aquatic animals, and can improve the growth of the aquatic animals; experiments prove that the Rhodosporidium sphaerocarpum enriched with rare earth elements can improve the survival rate of zebra fish under the conditions of challenge stress test and heat stress test, and is helpful to promote the healthy aquaculture of the aquatic animals.

Description

[0001] This application claims priority to Chinese patent application No. 202611042433.2, filed on July 14, 2026, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of microbial technology, and in particular to the application of Rhodotorula buergerianum or its progeny in the enrichment of rare earth elements and aquatic animal farming. Background Technology

[0003] Studies have shown that low doses of rare earth elements have biological effects such as regulating cell activity and promoting tissue repair. Lanthanum can act as a cofactor for methanol dehydrogenase, replacing calcium (Ca) in methanogenic bacteria to participate in catalytic reactions, a classic example of the clearly defined biochemical functions of rare earth elements. Cerium, such as cerium nanoparticles, has the ability to mimic antioxidant enzymes (such as SOD and CAT), scavenging reactive oxygen species and protecting cells. Neodymium, like lanthanum and cerium, is a rare earth element with similar functions. Although research on its activity mechanisms is relatively limited, existing biological functional studies have shown that neodymium has the potential to promote growth, fight bacteria, and fight tumors. In the aquaculture industry, these three rare earth elements can enhance the immunity of aquatic animals and effectively prevent infections by common pathogens under modern intensive farming conditions.

[0004] Inorganic rare earth elements, such as lanthanum chloride, cerium chloride, and neodymium chloride, can be toxic to fish if used in even slight excess. Moreover, under certain conditions, such as when exposed to alkali, inorganic rare earth elements can form hydroxide precipitates and are acidic in aqueous solutions, resulting in poor stability and practicality in aquaculture applications.

[0005] In recent years, various yeasts have been gradually developed into novel green feed additives due to their low fermentation production costs and rich protein content. Against this backdrop, identifying yeast strains capable of accumulating rare earth elements, establishing their cultivation methods, and evaluating their biological efficacy in aquaculture and other fields holds promise for integrating the dual advantages of rare earth elements and yeast, thereby further improving aquaculture efficiency.

[0006] After being absorbed and metabolized by yeast, rare earth elements (REEs) bind with intracellular organic matter, forming a more stable form. This stable form remains stable under specific processing conditions (such as a wide pH range) in various feed additives and preparations, and is less prone to decomposition or loss of activity. Compared to chloride forms, the cellular structure of yeast-derived REEs protects and slows their release, allowing for their gradual release and absorption within the digestive tract of aquatic animals, thus improving bioavailability. More importantly, the biotoxicity of REEs is significantly reduced after binding with yeast cells, minimizing potential harm to aquatic animals and further enhancing their application value and safety in the feed industry. Summary of the Invention

[0007] This invention provides an application of Rhodotorula buergerianum or its progeny in the enrichment of rare earth elements and aquatic animal farming.

[0008] In a first aspect, the present invention provides the application of *Rhodotorula cyclohexane* or its descendants in the enrichment of rare earth elements, wherein *Rhodotorula cyclohexane* (… Rhodosporidium toruloides The strain number at the China Industrial Microbial Culture Collection Center is CICC 32489.

[0009] In the applications described above, the rare earth element includes at least one of lanthanum, cerium, and neodymium.

[0010] As described above, the enrichment refers to Rhodotorula buergerianum as a carrier for enriching rare earth elements. Through microbial transformation, inorganic rare earth elements (lanthanum chloride, cerium chloride, neodymium chloride) are converted into microbial rare earth elements that are easily absorbed and utilized by objects, and then enriched in the Rhodotorula buergerianum cells.

[0011] As described above, the progeny of Rhodotorula circophylla refers to daughter cells produced through the growth of microorganisms (e.g., cultured in a culture medium). It is understood that the progeny of Rhodotorula circophylla retain the functions of the original Rhodotorula circophylla.

[0012] Secondly, the present invention provides a method for culturing Rhodotorula buergerianum, comprising: culturing the Rhodotorula buergerianum or its progeny in a culture medium containing rare earth elements, and enriching the rare earth elements in the culture medium into the Rhodotorula buergerianum cells; the strain number of the Rhodotorula buergerianum is CICC32489 at the China Industrial Microbial Culture Collection Center.

[0013] In the method described above, the rare earth elements in the culture medium exist in an inorganic form; in one specific embodiment, the inorganic form of the rare earth elements includes salts of rare earth elements; further, the inorganic rare earth elements in the culture medium are added in the form of chlorides of the corresponding elements, such as at least one of lanthanum chloride, cerium chloride, and neodymium chloride.

[0014] As described above, the culture medium can be a solid culture medium to isolate yeast cells and cultures. In one specific embodiment, the solid culture medium can be YPD solid culture medium or a culture medium suitable for the growth of *Rhodotorula glutinis*.

[0015] As described above, the culture temperature can be 30°C and the time can be 72 hours.

[0016] Thirdly, the present invention provides *Rhodotorula buergerianum* cultured according to any of the methods described above.

[0017] The *Rhodotorula circophylla* strain described above contains 7.1 × 10⁻⁶ cerium. 3 mg / kg; and / or, the lanthanum content in the *Rhodotorula circophylla* is 6.4 × 10 mg / kg. 3 mg / kg; and / or, the neodymium content in the *Rhodotorula circophylla* is 7.0 × 10 mg / kg. 3 mg / kg.

[0018] It is understandable that the types and amounts of rare earth elements enriched in Rhodotorula buergerianum are affected by culture conditions and are determined based on the actual situation.

[0019] Fourthly, the present invention provides a composition comprising *Rhodotorula buergerianum* cultured according to any of the methods described above.

[0020] The composition described above is a microbial agent. A microbial agent refers to a live microbial preparation made by amplifying target microorganisms and then using a carrier as an adsorbent to adsorb the microbial cells, or the fermentation broth or solid fermentation products of the microorganisms. Furthermore, the microbial agent can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

[0021] The composition described above may further include other biological or non-biological components. Non-biological components may include, for example, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc.

[0022] The composition described above can also be used as feed. In addition to the aforementioned *Rhodotorula circophylla* or its progeny, it may also include nitrogen, phosphorus, potassium, calcium, sulfur, magnesium, iron, manganese, zinc, copper, and other elements beneficial to animal growth and metabolism.

[0023] The composition described above can also be a medicine used to improve the survival rate of aquatic animals in adverse growth environments.

[0024] Fifthly, the present invention provides the application of Rhodotorula buergerianum cultured according to any of the above methods in aquaculture.

[0025] In a sixth aspect, the present invention provides a method for raising aquatic animals, comprising feeding the aquatic animals with Rhodotorula buergerianum cultured according to any of the methods described above.

[0026] In the method described above, the aquatic animal is a fish.

[0027] The Rhodotorula buergerianum or its progeny provided by this invention can convert inorganic rare earth elements into microbial rare earth elements that are easily absorbed and utilized by aquatic animals, and enrich them in the Rhodotorula buergerianum cells. The Rhodotorula buergerianum enriched with rare earth elements is safer and easier for aquatic animals to absorb, and can improve the growth of aquatic animals. Experiments have confirmed that under the conditions of toxic stress test and heat stress test, the Rhodotorula buergerianum enriched with rare earth elements can improve the survival rate of zebrafish, which helps to promote the healthy breeding of aquatic animals. Attached Figure Description

[0028] Figure 1 The images show the morphological comparison of Rhodotorula circophylla before and after enrichment with trace elements. Among them, A is the morphological image of Rhodotorula circophylla without rare earth element enrichment; B is the morphological image of Rhodotorula circophylla after enrichment with lanthanum; C is the morphological image of Rhodotorula circophylla after enrichment with cerium; and D is the morphological image of Rhodotorula circophylla after enrichment with neodymium. Figure 2 The images show a comparison of the rare earth element content in *Rhodotorula circophylla* before and after rare earth element enrichment. A represents a comparison of lanthanum content in *Rhodotorula circophylla* and *Rhodotorula circophylla* enriched with lanthanum; B represents a comparison of cerium content in *Rhodotorula circophylla* and *Rhodotorula circophylla* enriched with cerium; and C represents a comparison of neodymium content in *Rhodotorula circophylla* and *Rhodotorula circophylla* enriched with neodymium. Figure 3 The results of the growth indicators in the evaluation experiment of zebrafish culture enriched with lanthanum-enriched Rhodotorula buergerianum are as follows: A represents the statistical results of zebrafish initial weight (IBM); B represents the statistical results of zebrafish final weight (FBW); C represents the statistical results of zebrafish weight gain rate (WGR); D represents the statistical results of feed efficiency (FCR); and E represents the statistical results of survival rate (SR). Figure 4 To determine the survival rate of zebrafish challenge experiments with lanthanum-enriched Rhodotorula buergerianum yeast; Figure 5 The results of the growth indicators in the evaluation experiment of zebrafish culture with cerium-enriched Rhodotorula buergerianum are as follows: A represents the statistical results of zebrafish initial weight (IBM); B represents the statistical results of zebrafish final weight (FBW); C represents the statistical results of zebrafish weight gain rate (WGR); D represents the statistical results of feed efficiency (FCR); and E represents the statistical results of survival rate (SR). Figure 6 Survival rate of zebrafish challenge test with cerium-enriched Rhodotorula buergerianum; Figure 7 The results of the growth indicators in the evaluation experiment of zebrafish culture enriched with neodymium-enriched Rhodotorula buergerianum are as follows: A represents the statistical results of initial weight (IBM) of zebrafish; B represents the statistical results of final weight (FBW) of zebrafish; C represents the statistical results of weight gain rate (WGR) of zebrafish; D represents the statistical results of feed efficiency (FCR); and E represents the statistical results of survival rate (SR). Figure 8 To determine the survival rate of zebrafish subjected to heat stress by neodymium-enriched Rhodotorula buergerianum. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] Example 1: Screening of yeasts tolerant to rare earth elements (lanthanum, cerium, and neodymium) YPD solid medium (containing 20% ​​peptone, 20% glucose, 10% yeast extract, and 1% agar) was prepared and sterilized at 115°C for 20 min. Simultaneously, lanthanum chloride, cerium chloride, and neodymium chloride solutions with concentrations of 500 mg / ml, 500 mg / ml, and 500 mg / ml were prepared and sterilized by filtration. The sterilized lanthanum chloride, cerium chloride, and neodymium chloride solutions were added to the unsolidified YPD solid medium to prepare YPD solid medium containing different rare earth elements at working concentrations of 500 μg / ml, 1000 μg / ml, and 2000 μg / ml, respectively.

[0032] Yeast strains to be screened were inoculated into YPD solid medium containing three different rare earth elements and cultured. Yeast strains capable of growing on YPD medium with a rare earth element concentration of 500 μg / ml were screened. The selected yeast strains were then screened for higher tolerance concentrations, with the working concentrations of lanthanum chloride, cerium chloride, and neodymium chloride gradually increased, including 1000 μg / ml and 2000 μg / ml, until the yeast strain with the best tolerance to the three rare earth elements was selected.

[0033] The yeast selected was *Rhodotorula buergerianum* (Saccharomyces cerevisiae). Rhodosporidium toruloides (This is a strain numbered CICC 32489 at the China Industrial Microbial Culture Collection Center.)

[0034] Example 2: Enrichment experiment of three rare earth elements by Rhodotorula circophylla and determination of enrichment content. Because the addition of lanthanum chloride, cerium chloride, and neodymium chloride to liquid YPD medium produces precipitation, making it impossible to accurately separate bacterial cells and precipitates, solid culture media were used to prepare enriched products in all examples. YPD solid culture media with a working concentration of 2 g / L for lanthanum chloride, cerium chloride, and neodymium chloride were prepared separately.

[0035] After activating the cryopreserved Rhodotorula buergerianum in YPD solid medium, single colonies were picked and inoculated into YPD liquid medium. The culture was carried out at 30 °C until the late logarithmic phase, and the OD value was adjusted to about 0.8 to prepare a seed culture for later use.

[0036] 100 μl of seed culture was spread onto YPD solid medium containing the corresponding rare earth elements and cultured at 30 ℃ for 72 h. After the culture was completed, the bacterial cells were scraped off with a cell scraper, avoiding scraping off the solid medium. The cells were washed 2-3 times with deionized water (to remove unconverted chlorides) to collect the enriched cells. After freeze-drying, the enriched bacterial cell powder was obtained. The morphology of *Rhodotorula glutinis* before and after rare earth element enrichment was compared. Figure 1 As shown.

[0037] The lyophilized bacterial powder was directly sampled and analyzed by inductively coupled plasma (ICP) to determine the contents of lanthanum, cerium, and neodymium. The results are as follows: Figure 2 As shown, the lanthanum content of *Rhodotorula buergerianum* is 6.4 × 10⁻⁶. 3 The cerium content of *Rhodotorula buergerianum* was 7.1 × 10 mg / kg. 3 The neodymium content of *Rhodotorula buergerianum* is 7.0 × 10 mg / kg. 3 mg / kg.

[0038] Example 3: Lanthanum-enriched Rhodotorula buergerianum, Lanthanum-enriched Rhodotorula buergerianum, and Lanthanum-enriched Rhodotorula buergerianum were added as feed additives to the basal diet of zebrafish for aquaculture evaluation. Zebrafish are typical freshwater cyprinid fish, and their physiology, development, and metabolism are highly similar to those of mammals. The National Institutes of Health (NIH) lists zebrafish as the third most important vertebrate model organism. Zebrafish are currently the only vertebrates suitable for high-throughput drug screening. They are small in size, have a short developmental cycle, short experimental cycle, low cost, can be fertilized in vitro, are transparent, have a high number of eggs laid at one time, and require small amounts of drugs in experiments. Therefore, zebrafish are used in this embodiment as a typical representative of other fish species.

[0039] 1. The lanthanum-enriched Rhodotorula buergerianum was added as a feed additive to the basal diet of zebrafish for aquaculture evaluation.

[0040] The lanthanum-enriched freeze-dried *Rhodotorula circophylla* powder obtained in Example 2 was used as a feed additive in an aquaculture evaluation experiment. A negative control group without trace element addition was used; a positive control group containing rare earth organic rare earth elements (hereinafter referred to as rare earth) was used; and an experimental treatment group was used with lanthanum-enriched freeze-dried *Rhodotorula circophylla* powder. The effective lanthanum content was set at 25, 50, and 75 g / kg (hereinafter referred to as M-lanthanum 25, M-lanthanum 50, and M-lanthanum 75, respectively), resulting in a total of 5 groups. Table 1 shows the zebrafish aquaculture evaluation experiment formulation. Healthy 2-month-old zebrafish of uniform size (initial average weight approximately 47 mg) were selected as the experimental subjects and randomly divided into 5 experimental groups, with 7 replicates in each group and 10 zebrafish in each replicate, for a total of 350 zebrafish.

[0041] Table 1. Evaluation formula for zebrafish culture using lanthanum-enriched Rhodotorula buergerianum yeast

[0042] 2. Cerium-enriched Rhodotorula buergerianum was added as a feed additive to the basal diet of zebrafish for aquaculture evaluation.

[0043] The cerium-enriched *Rhodotorula circophylla* freeze-dried powder obtained in Example 2 was used as a feed additive in an aquaculture evaluation experiment. A group without added trace elements served as a negative control, while a group with added rare earth feed concentrate containing organic rare earth elements (hereinafter referred to as rare earth) served as a positive control. The group with added cerium-enriched *Rhodotorula circophylla* freeze-dried powder was the experimental treatment group, with effective cerium content set at 20, 40, and 60 g / kg (hereinafter referred to as M-cerium20, M-cerium40, and M-cerium60, respectively), resulting in a total of 5 groups. Table 2 shows the zebrafish aquaculture evaluation experiment formulation. Healthy 2-month-old zebrafish of uniform size (initial average weight approximately 65 mg) were selected as the experimental subjects and randomly divided into 5 experimental groups, with 7 replicates in each group, 10 fish per replicate, for a total of 350 fish.

[0044] Table 2. Evaluation test formula for cerium-enriched Rhodotorula buergerianum zebrafish culture

[0045] 3. Neodymium-enriched Rhodotorula buergerianum was added as a feed additive to the basal diet of zebrafish for aquaculture evaluation.

[0046] The neodymium-enriched *Rhodotorula scoparia* freeze-dried powder obtained in Example 2 was used as a feed additive in an aquaculture evaluation experiment. A negative control group without added trace elements was used; a positive control group containing rare earth organic rare earth elements (hereinafter referred to as rare earth) was used; and an experimental treatment group containing neodymium-enriched *Rhodotorula scoparia* freeze-dried powder was used. The effective neodymium content was set at 15, 30, and 60 g / kg (hereinafter referred to as M-Nd15, M-Nd30, and M-Nd60, respectively), resulting in a total of 5 groups. Table 3 shows the zebrafish aquaculture evaluation experiment formulation. Healthy 2-month-old zebrafish of uniform size (initial average weight approximately 47 mg) were selected as the experimental subjects and randomly divided into 5 experimental groups, with 7 replicates in each group and 10 zebrafish in each replicate, for a total of 350 zebrafish.

[0047] Table 3. Evaluation test formula for neodymium-enriched Rhodotorula buergerianum zebrafish culture

[0048] The growth performance of zebrafish was evaluated after 4 weeks of near-full feeding: Initial weight (IBW, g); Last weight (FBW, g); Weight gain rate (WGR, %) = 100% × (final average weight - initial average weight) / initial average weight; Feed efficiency (FCR) = (Final total body weight + Total body weight of dead fish - Initial total body weight) / Feed intake; Survival rate (SR, %) = 100% × (initial number of fish - final number of fish) / initial number of fish; The results are as follows: Figure 3 , 5 As shown in Figures 7 and 8.

[0049] 4. Zebrafish challenge stress experiment A challenge experiment was conducted on zebrafish cultured for 4 weeks after a 12-hour fasting period, using a combination of Aeromonas vesiculosus Hm091 and Aeromonas hydrophila NJ-1 at a challenge concentration of 1.7 x 10⁻⁶. 7 cfu / mL. Three replicates per group; 10 zebrafish per replicate. The number of zebrafish deaths was recorded every 1-5 hours and dead fish were removed promptly, continuing for 40 hours. The survival rate was calculated at the end. No feeding was performed during the challenge stress period to assess the zebrafish's tolerance.

[0050] Survival rate (%) = 100% × number of surviving fish at the time point recorded in the challenge stress test / number of fish at the start of the challenge stress test; The results of the challenge stress tests of lanthanum-enriched and cerium-enriched Rhodotorula buergerianum are as follows: Figure 4 , 6 As shown.

[0051] 5. Heat stress test on zebrafish A heat stress experiment was conducted on zebrafish cultured for 4 weeks, with a 12-hour fast. Five groups were included, with four replicates per group; each replicate contained 10 zebrafish. The zebrafish were placed in pre-cooled water to 40°C. The number of dead zebrafish was recorded every 1-5 hours, and dead fish were promptly removed. This process continued for 12 hours, and the survival rate was calculated. No feeding was provided during the heat stress period to assess the zebrafish's heat stress tolerance.

[0052] Survival rate (%) = 100% × number of surviving fish at the recorded time point of the heat stress test / number of fish at the start of the heat stress test; The results of the heat stress test of neodymium-enriched Rhodotorula buergerianum are as follows: Figure 8 As shown.

[0053] like Figure 3 As shown, compared to the rare earth group, lanthanum-enriched Rhodotorula buergerianum with an effective addition of 50 mg / kg significantly improved the growth of zebrafish; Figure 4 As shown, the survival rate of the M-lanthanum 50 group was the highest, exceeding that of the rare earth group and the control group. This aquaculture evaluation result demonstrates that appropriate addition of lanthanum-enriched Rhodotorula buergerianum can improve zebrafish growth and, under toxic stress conditions, increase their survival rate.

[0054] like Figure 5 As shown, compared to the rare earth group, cerium-enriched Rhodotorula buergerianum with an effective cerium addition of 40 mg / kg showed a tendency to promote zebrafish growth; such as Figure 6 As shown, the survival rate of the M-cerium 40 group was the highest, exceeding that of the rare earth group and the control group. This aquaculture evaluation result demonstrates that appropriate addition of cerium-enriched Rhodotorula buergerianum can improve zebrafish growth and increase their survival rate under toxic stress conditions.

[0055] like Figure 7 As shown, compared to the rare earth group, the neodymium-enriched Rhodotorula glutinis with an effective addition of 30 mg / kg had no negative impact on zebrafish growth; Figure 8 As shown, the survival rate of the M-Nd30 group was the highest and significantly higher than that of the rare earth group and the control group. This aquaculture evaluation result demonstrates that the appropriate amount of Nd:rhodotorula glutinis added has no negative effect on the growth of zebrafish and can improve the survival rate of zebrafish under heat stress test conditions.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Rhodotorula buergerianum or its progeny in the enrichment of rare earth elements, wherein the strain number of Rhodotorula buergerianum is CICC 32489 at the China Industrial Microbial Culture Collection Center.

2. The application according to claim 1, characterized in that, The rare earth elements include at least one of lanthanum, cerium, and neodymium.

3. A method for culturing *Rhodotorula circinata* or its progeny, characterized in that, include: The *Rhodotorula circophylla* or its progeny were cultured in a medium containing rare earth elements, thereby enriching the rare earth elements in the *Rhodotorula circophylla* cells. The strain number of the *Rhodotorula circophylla* at the China Industrial Microbial Culture Collection Center is CICC32489.

4. The method according to claim 3, characterized in that, The rare earth elements in the culture medium exist in an inorganic form.

5. The Rhodotorula buergerianum cultured according to claim 3 or 4.

6. The *Rhodotorula circophylla* according to claim 5, characterized in that, The cerium content in the *Rhodotorula circophylla* was 7.1 × 10⁻⁶. 3 mg / kg; and / or, the lanthanum content in the *Rhodotorula circophylla* is 6.4 × 10 mg / kg. 3 mg / kg; and / or, the neodymium content in the *Rhodotorula circophylla* is 7.0 × 10 mg / kg. 3 mg / kg.

7. The composition, characterized in that, This includes *Rhodotorula buergerianum* or its progeny cultured according to the method of claim 3 or 4.

8. The application of *Rhodotorula buergerianum* cultured according to claim 3 or 4 in aquaculture.

9. A method for raising aquatic animals, characterized in that, This includes feeding aquatic animals with *Rhodotorula buergerianum* cultured according to the method described in claim 3 or 4.

10. The method according to claim 9, characterized in that, The aquatic animals mentioned are fish.

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