A method for cryopreservation of carrier mycelial pellets for water treatment

Abstract

The application provides a kind of bacteria-algae composite carrier cryopreservation method and cryopreserved bacteria-algae composite carrier for water treatment.The method uses mycelium ball as three-dimensional immobilized carrier, and forms bacteria-algae body by compounding culture with microalgae, and then the bacteria-algae body is cleaned by phosphate buffer solution with pH of 6-8 or sterilized physiological saline, and then the bacteria-algae body is placed in preservation solution containing organic carbon source, inorganic nitrogen source and inorganic phosphorus source for cryopreservation; the COD concentration in the preservation solution is 300-500 mg / L, the NH4⁺-N concentration is 80-200 mg / L, the PO4³⁻-P concentration is 15-25 mg / L, and the pH is 2.0-3.0.The bacteria-algae body is sealed after glycerol protection treatment, and is stored at 0-4 ℃.After the end of preservation, the dry weight of bacteria-algae body, enzyme activity and COD, ammonia nitrogen and total phosphorus removal capacity are determined by recovery culture to evaluate the preservation effect.The application can maintain the three-dimensional structure of mycelium ball and the stability of microalgae attachment during cryopreservation, and improve the biological activity and water treatment function recovery ability of bacteria-algae composite carrier after preservation.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of environmental biotechnology, and particularly relates to a technology for preserving mycelial balls-microalgae composite carriers for water treatment. Background Technology

[0002] In wastewater treatment, pollutant removal, and biological resource utilization, microbial immobilization carriers provide attachment spaces for microorganisms, maintaining a high local concentration and enabling them to continuously perform water treatment functions. Mycelial balls, three-dimensional self-immobilized structures formed by the intertwining of hyphae during liquid culture of filamentous fungi, possess a large specific surface area, internal porosity, and certain structural strength, serving as carriers for pollutant transfer and microbial attachment. Co-culturing mycelial balls with microalgae to form a microbial-algae composite system further utilizes the spatial structure of the mycelial balls to provide surface and internal pore attachment sites for microalgae, giving the composite system both a carrier structure and microalgal water treatment functions.

[0003] Existing research has focused on the co-cultivation, co-granulation, or co-immobilization of fungi and microalgae, applying them to the removal of organic matter and nitrogenous pollutants, algal biomass recovery, and the enhancement of water treatment processes. These technologies primarily address how mycelial balls and microalgae come into contact, attach, and work together. For fungal-algae composite carriers that have already achieved water treatment functionality, engineering applications require maintaining them in a recoverable state before equipment maintenance, seasonal shutdowns, the storage of spare microbial agents, or rapid system startup.

[0004] Existing methods for preserving filamentous fungi typically aim to preserve the strain or maintain its survival. Common methods include slant culture, glycerol preservation, cryopreservation, and freeze-drying. These methods usually preserve single strains or dispersed cells, without considering the three-dimensional carrier structure of the mycelial spheres, the attachment state of the microalgae, and the water treatment performance of the restored composite system. Directly applying common strain preservation conditions to the mycelial-algae composite carrier may lead to continued consumption of residual culture medium or induce mycelial growth, resulting in changes to the carrier structure. It may also cause microalgae detachment, decreased mycelial-algae activity, and unstable water treatment function recovery due to insufficient nutrients, unsuitable pH conditions, or low-temperature stress.

[0005] On the other hand, freeze-drying and cryopreservation typically require specialized equipment, complex pretreatment, and strict temperature control. Furthermore, a lengthy recovery process may be necessary after preservation, making them unsuitable for direct application to on-site needs using intact microbial-algae composite carriers. Simply lowering the temperature without controlling the carbon, nitrogen, phosphorus, and acid / alkaline environments of the preservation solution also fails to simultaneously address the low metabolic state during preservation, the stability of the carrier structure, and the pollutant removal capacity after recovery. Therefore, a cryopreservation method is needed that combines mycelial ball structure, microalgae attachment, preservation solution composition, sealing methods, and low-temperature conditions, using water treatment functional microbial-algae composite carriers as the preservation target.

[0006] Therefore, the core technical problem that the existing technology needs to solve is: how to maintain the low metabolic stability of the mycelial ball three-dimensional carrier and the microalgae attachment relationship during the preservation period through an operable preservation solution and low temperature conditions, and restore the dry weight, enzyme activity and COD, ammonia nitrogen and total phosphorus removal capacity when needed, without destroying the three-dimensional carrier structure of the mycelial ball and the microalgae attachment relationship. Summary of the Invention

[0007] This invention addresses the preservation needs of water treatment-functional algal-microbe composite carriers during equipment maintenance, seasonal shutdowns, standby storage, and restart. It proposes a low-temperature preservation method for functional biological carriers. The invention uses mycelial balls as a three-dimensional immobilization carrier, co-culturing them with microalgae to form an algal-microbe composite carrier. Before preservation, residual culture materials are removed through gentle cleaning. The carrier is then placed in a specific preservation solution containing organic carbon, inorganic nitrogen, and inorganic phosphorus sources, and sealed after auxiliary protection with glycerol. Preservation at 0–4 °C maintains the algal-microbe composite system in a relatively stable, low-metabolic state, preserving its water treatment function during subsequent recovery culture.

[0008] This invention provides a method for cryopreservation of carrier mycelial balls for water treatment, comprising the following steps: Mycelial balls and microalgae were mixed and cultured to form myco-algal bodies; the myco-algal bodies were washed with phosphate buffer or sterile physiological saline at pH 6–8; the washed myco-algal bodies were added to a preservation solution, which included an organic carbon source, an inorganic nitrogen source, and an inorganic phosphorus source, wherein the COD concentration was 300–500 mg / L, the NH4⁺-N concentration was 80–200 mg / L, the PO4³⁻-P concentration was 15–25 mg / L, and the pH of the preservation solution was 2.0–3.0; glycerol was added to the preservation system for auxiliary protection treatment to improve the structural stability of the myco-algal composite carrier during low-temperature preservation, and the treated myco-algal bodies were stored in a low-temperature environment of 0–4 ℃.

[0009] Furthermore, the mycelial balls and the microalgae are mixed at a volume ratio of 1:6 and cultured under shaking conditions of 140–160 rpm, room temperature and natural pH for 2 hours to promote full contact between the microalgae and the mycelial balls, so that the microalgae attach to the surface and internal pores of the mycelial balls and form a stable mycelial-algae complex.

[0010] Furthermore, the mycelial balls include Aspergillus niger ( Aspergillus niger Mycelial balls or Aspergillus fumigatus ( Aspergillus fumigatus Mycelial balls.

[0011] Furthermore, the preservation solution also includes hyaluronic acid and vitamin E, wherein the concentration of hyaluronic acid is 3-7 g / L and the concentration of vitamin E is 2-4 g / L.

[0012] Furthermore, the bacterial and algal cells are stored at 0–4 °C for 1–12 months.

[0013] Furthermore, after preservation, the bacterial and algal cells are transferred to a recovery culture system for functional recovery culture. The recovery culture system includes glucose at a concentration of 2 g / L, ammonium chloride at a concentration of 0.5 g / L, and dipotassium hydrogen phosphate at a concentration of 0.5 g / L. The recovery culture is carried out at a rotation speed of 160 r / min, a temperature of 25 ℃, and a time of 1–3 hours.

[0014] The reactivation performance retention rate was obtained by measuring the dry weight of the bacteria and algae after reactivation culture, as well as their ability to remove COD, ammonia nitrogen, and total phosphorus, and comparing them with the corresponding initial performance before preservation. This was used to evaluate the functional retention effect of the bacteria-algae composite carrier under low temperature preservation conditions.

[0015] This invention also provides a cryogenically preserved microbial-algae composite carrier for water treatment, comprising mycelial balls and microalgae attached to the surface and internal pores of the mycelial balls; the microbial-algae composite carrier is immersed in a preservation solution comprising an organic carbon source, an inorganic nitrogen source, and an inorganic phosphorus source, wherein the COD concentration is 300–500 mg / L, the NH4⁺-N concentration is 80–200 mg / L, the PO4³⁻-P concentration is 15–25 mg / L, and the pH is 2.0–3.0; the microbial-algae composite carrier is sealed after glycerol protection treatment and stored at a cryogenic temperature of 0–4 °C to maintain its structural integrity and restored water treatment function.

[0016] Furthermore, the mycelial balls are Aspergillus niger mycelial balls or Aspergillus fumigatus mycelial balls.

[0017] Furthermore, the microalgae is Chlorella proteoglycans ( Chlorella pyrenoidosa ) or common Chlorella ( Chlorella vulgaris ).

[0018] Furthermore, the preservation solution also includes hyaluronic acid at a concentration of 3–7 g / L.

[0019] In combination with the above technical solutions and the technical problems solved, the positive effects, significant progress, and unexpected technical effects achieved by this invention are as follows: This invention differs from traditional preservation methods that target single strains, free cells, or dispersed microorganisms. Instead, it uses a bacterial-algae composite carrier with a complete spatial structure and water treatment function as the preservation object. The three-dimensional network structure of mycelial balls, the attachment state of microalgae, and the restored water treatment performance are incorporated into a unified preservation system, enabling the bacterial-algae composite carrier to maintain structural stability during preservation and providing a foundation for subsequent functional recovery.

[0020] This invention, through the synergistic regulation of nutrient components, protective components, and the acid-base environment of the preservation solution, reduces the risk of excessive growth during the preservation stage while meeting the basic maintenance needs of the bacterial-algal system. Specifically, vitamin E reduces the impact of oxidative stress during low-temperature preservation, and hyaluronic acid enhances the structural stability of the bacterial-algal composite carrier, thereby improving the functional recovery ability after preservation.

[0021] This invention employs a combination of pre-cleaning, preservation solution treatment, glycerol protection, and low-temperature storage at 0–4 °C to reduce the adverse effects of culture medium residues and external environmental changes on the bacterial-algae composite carrier. This method does not rely on freeze-drying or ultra-low temperature storage equipment, making it more suitable for applications such as spare bacterial agent storage, system maintenance, and rapid start-up in water treatment engineering.

[0022] This invention evaluates the performance of the bacterial-algae composite carrier before and after preservation by restoring indicators such as dry weight, enzyme activity, and removal capacity of COD, ammonia nitrogen, and total phosphorus after culture. It not only focuses on the survival status of microorganisms, but also on its ability to recover as a functional carrier for water treatment.

[0023] As supporting evidence of the inventiveness of the claims of this invention, this invention also has the following technical effects: (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows: This invention can be applied to wastewater treatment, bioaugmentation, and resource utilization, providing a long-term storage and backup management solution for algae-bacterial composite carriers with water treatment functions. Using this method, the algae-bacterial composite system does not need to be reconstructed upon system restart, shortening the recovery cycle of biological treatment units, improving the continuity of engineering operation, and possessing further engineering application and promotion value.

[0024] (2) The technical solution of the present invention fills the gaps in the application scope of the prior art: Existing microbial preservation technologies mainly focus on strain survival, cell viability maintenance, or simple bacterial agent storage. There is a lack of targeted preservation methods for microbial-algae composite carriers that simultaneously possess a three-dimensional immobilized structure, microalgae attachment relationships, and contaminant removal functions. This invention synergistically designs for the structural preservation, activity maintenance, and functional recovery of microbial-algae composite carriers, expanding the application scope of traditional microbial preservation technologies.

[0025] (3) The technical solution of the present invention solves a long-standing technical problem: This invention solves the problem that it is difficult to maintain the structural stability of mycelial balls, the attachment state of microalgae, and the ability to remove pollutants after recovery in the long-term preservation of bacterial-algae composite carriers. Through the synergistic regulation of preservation conditions and preservation system, the stable preservation of functional bacterial-algae carriers is achieved.

[0026] (4) The technical solution of the present invention overcomes the prejudices of traditional technology: Traditional microbial preservation typically focuses on reducing metabolic activity and maintaining cell survival. However, for complex microbial-algal composite carriers with intricate spatial structures, conventional low-temperature or cryopreservation methods often overlook issues such as carrier structural changes, microalgal detachment, and decreased functional recovery. This invention breaks away from the preservation approach that only focuses on microbial survival rates. It takes carrier structural stability, preservation of biological activity, and restoration of water treatment function as comprehensive evaluation objectives, achieving functional biological carrier preservation tailored to water treatment application needs. Attached Figure Description

[0027] Figure 1 It is a photograph of bacteria and algae. Figure 2 This is a graph showing the relationship between the retention rate of mycelial ball performance and the COD of the preservation solution. Figure 3 The retention rate of mycelial ball performance is related to the NH4 content of the preservation solution. + Graph showing the relationship between -N concentration Figure 4 The retention rate of mycelial ball performance is related to the PO4 content of the preservation solution. 3- -P concentration relationship graph Figure 5 This is a graph showing the relationship between the retention rate of mycelial ball performance and the concentration of vitamin E in the preservation solution. Figure 6 This is a graph showing the relationship between the retention rate of mycelial ball performance and the concentration of hyaluronic acid in the preservation solution. Detailed Implementation Example

[0028] Step 1: Form the bacterial and algal cells and determine their initial properties.

[0029] Well-grown mycelial pellets and microalgae were selected and mixed at a volume ratio of 1:6. The mixture was then cultured for 2 hours under shaking conditions at 140–160 rpm at room temperature, with the pH maintained at its natural state during the culture process, to form mycelial-algal bodies. After the formation of the mycelial-algal bodies, the dry weight, enzyme activity, and removal rates of COD, ammonia nitrogen, and total phosphorus were measured as initial performance values. The mycelial pellets were *Aspergillus niger* (…). Aspergillus niger ) or Aspergillus fumigatus ( Aspergillus fumigatusThe mycelial balls formed by the microalgae are Chlorella proteoglycans ( ). Chlorella pyrenoidosa ) or common Chlorella ( Chlorella vulgaris ).

[0030] Step 2: Method for preserving bacteria and algae.

[0031] After cleaning, the mycelial cells are placed in a preservation solution and stored at low temperature for 1–30 days. The cleaning process involves rinsing the mycelial balls at least three times with a phosphate buffer solution or sterile physiological saline at pH 6–8. The preservation solution consists of 300 mg / L COD, 80 mg / L NH₄⁺-N, and 15 mg / L PO₄³⁻-P, with the pH adjusted to 2.0. The solution is then sealed with glycerol. The low-temperature storage range is 0–4 °C.

[0032] Step 3: Restore the performance of the preserved bacteria and algae and calculate the performance retention rate.

[0033] The preserved mycelial cells were placed back into a constant-temperature shaker and cultured under a substrate solution of 2 g / L glucose, 0.5 g / L ammonium chloride, and 0.5 g / L dipotassium hydrogen phosphate at a rotation speed of 160 r / min and a temperature of 25℃ for 1–3 hours. After the recovery culture, the dry weight of the mycelial cells, enzyme activity, and removal rates of COD, ammonia nitrogen, and total phosphorus were measured as reactivation performance values. The performance retention rate was calculated according to the formula: "Mycelial ball performance retention rate = reactivation performance value / initial performance value × 100%". Example

[0034] The difference between this embodiment and Embodiment 1 is that the COD concentration of the preservation solution in step 2 is adjusted to 400 mg / L, while the other components of the preservation solution and the operating steps are the same as in Embodiment 1. Example

[0035] The difference between this embodiment and Embodiment 1 is that the COD concentration of the preservation solution in step 2 is adjusted to 500 mg / L, while the other steps and parameters are the same as in Embodiment 1. Example

[0036] The difference between this embodiment and Embodiment 1 is that the concentration of NH4⁺-N in the preservation solution in step 2 is adjusted to 100 mg / L, while the other steps and parameters are the same as in Embodiment 1. Example

[0037] The difference between this embodiment and Embodiment 1 is that the concentration of NH4⁺-N in the preservation solution in step 2 is adjusted to 200 mg / L, while the other steps and parameters are the same as in Embodiment 1. Example

[0038] The difference between this embodiment and Embodiment 1 is that the concentration of PO4³⁻-P in the preservation solution in step 2 is adjusted to 20 mg / L, while the other steps and parameters are the same as in Embodiment 1. Example

[0039] The difference between this embodiment and Embodiment 1 is that the concentration of PO4³⁻-P in the preservation solution in step 2 is adjusted to 25 mg / L, while the other steps and parameters are the same as in Embodiment 1. Example

[0040] The difference between this embodiment and Embodiment 1 is that metal ions are added to the preservation solution in step 2. The metal ions used are magnesium ions, and the concentration is set to 0.5 mg / L. All other steps and parameters are the same as in Embodiment 1. Example

[0041] The difference between this embodiment and Embodiment 1 is that metal ions are added to the preservation solution in step 2. The metal ions used are magnesium ions, and the concentration is set to 0.5 mg / L. All other steps and parameters are the same as in Embodiment 1. Example

[0042] The difference between this embodiment and Embodiment 1 is that vitamin E is added to the preservation solution in step 2, and the concentration of vitamin E is 2 g / L. All other steps and parameters are the same as in Embodiment 1. Example

[0043] The difference between this embodiment and Embodiment 1 is that vitamin E is added to the preservation solution in step 2, and the concentration of vitamin E is 4 g / L. All other steps and parameters are the same as in Embodiment 1. Example

[0044] The difference between this embodiment and embodiment 1 is that hyaluronic acid is added to the preservation solution in step 2, and the concentration of hyaluronic acid is 3 g / L. All other steps and parameters are the same as in embodiment 1. Example

[0045] The difference between this embodiment and embodiment 1 is that hyaluronic acid is added to the preservation solution in step 2, and the concentration of hyaluronic acid is 7 g / L. All other steps and parameters are the same as in embodiment 1. Example

[0046] The difference between this embodiment and Embodiment 1 is that the pH of the preservation solution in step 2 is adjusted to 3±0.2, while the other steps and parameters are the same as in Embodiment 1.

[0047] Example 15: Evaluation of the preservation effect of bacterial-algae composite carrier under different preservation solutions Healthy mycelial pellets and microalgae were selected and mixed at a volume ratio of 1:6. The mixture was then cultured for 2 hours at 140–160 rpm with shaking, at room temperature, and the pH was kept at its natural state during the culture process to form mycelial-algal bodies. After the formation of the mycelial-algal bodies, the dry weight, enzyme activity, and removal rates of COD, ammonia nitrogen, and total phosphorus were measured as initial performance values. Subsequently, the mycelial-algal bodies were washed three times with phosphate buffer (pH 7) or sterile physiological saline and then placed in the preservation solutions described in Examples 1–14 for cryopreservation.

[0048] The mycelial cells were collected after 3, 6, and 12 days of low-temperature storage for reactivation culture. The reactivation conditions were as follows: the mycelial balls were placed back in a constant-temperature shaker and cultured in a substrate solution containing 2 g / L glucose, 0.5 g / L ammonium chloride, and 0.5 g / L dipotassium hydrogen phosphate at a rotation speed of 160 r / min, a temperature of 25 ℃, and a culture time of 1–3 hours. After the reactivation culture, the dry weight of the mycelial cells, enzyme activity, and the removal rates of COD, ammonia nitrogen, and total phosphorus were measured as reactivation performance values. The preservation effect under different preservation conditions was evaluated according to the performance retention rate calculation formula.

[0049] The experimental results from the preservation solutions in Examples 1-5 show that the performance retention of the bacterial-algae composite carrier varies under different COD and NH4⁺-N concentrations. Among them, the preservation solution conditions corresponding to Example 3 exhibited superior overall performance at different time points.

[0050] Under the conditions of Examples 1-5, the enzyme activity retention rates were as follows: after 3 days of storage, the retention rates for Examples 1, 2, 3, 4, and 5 were 99.41%, 99.32%, 99.40%, 98.32%, and 99.12%, respectively; after 6 days of storage, the retention rates were 97.85%, 97.30%, 97.80%, 95.52%, and 97.32%, respectively; and after 12 days of storage, the retention rates were 92.51%, 94.47%, 95.12%, 93.77%, and 93.48%, respectively.

[0051] Under the conditions of Examples 1-5, the dry weight retention rates are as follows: after 3 days of storage, the dry weight retention rates for Examples 1, 2, 3, 4, and 5 are 99.32%, 99.40%, 98.02%, 98.73%, and 98.50%, respectively; after 6 days of storage, they are 97.05%, 95.98%, 97.35%, 97.56%, and 97.55%, respectively; and after 12 days of storage, they are 92.45%, 93.06%, 93.57%, 94.74%, and 92.70%, respectively.

[0052] Under the conditions of Examples 1-5, the COD removal capacity retention rates were as follows: after 3 days of storage, the retention rates for Examples 1, 2, 3, 4, and 5 were 98.09%, 98.79%, 99.98%, 99.30%, and 99.20%, respectively; after 6 days of storage, they were 96.13%, 95.17%, 96.14%, 96.84%, and 95.79%, respectively; and after 12 days of storage, they were 92.48%, 92.39%, 94.99%, 92.75%, and 92.16%, respectively.

[0053] Under the conditions of Examples 1-5, the retention rates of ammonia nitrogen removal capacity are as follows: after 3 days of storage, the retention rates for Examples 1, 2, 3, 4, and 5 are 98.46%, 98.66%, 99.26%, 98.19%, and 99.12%, respectively; after 6 days of storage, the retention rates are 95.92%, 96.47%, 96.76%, 96.12%, and 96.23%, respectively; and after 12 days of storage, the retention rates are 91.95%, 92.79%, 94.36%, 93.74%, and 91.52%, respectively.

[0054] Under the conditions of Examples 1-5, the total phosphorus removal capacity retention rates were as follows: after 3 days of storage, the retention rates for Examples 1, 2, 3, 4, and 5 were 98.63%, 98.12%, 99.43%, 98.89%, and 98.59%, respectively; after 6 days of storage, they were 96.85%, 95.76%, 97.29%, 95.93%, and 96.17%, respectively; and after 12 days of storage, they were 92.15%, 93.21%, 94.98%, 92.67%, and 92.58%, respectively.

[0055] The above results indicate that, under the preservation conditions set in Examples 1-5, the performance indicators of the bacterial-algae composite carrier all showed a certain degree of decline with prolonged preservation time. However, different preservation solutions significantly affected the performance retention level. Specifically, Example 3, using a preservation solution with a COD concentration of 500 mg / L, showed superior performance in terms of enzyme activity, dry weight retention, and the ability to remove COD, ammonia nitrogen, and total phosphorus, indicating that a suitable concentration of organic carbon source can improve the stability of the bacterial-algae composite carrier during cryopreservation.

[0056] Example 16: Evaluation of the preservation effect of bacterial-algae composite carrier under different concentrations of inorganic nitrogen and phosphorus sources Under the conditions of Examples 6 to 9, the concentration of PO4³⁻-P in the preservation solution and the conditions of the auxiliary components were adjusted respectively, while other operating steps remained the same as in Example 1.

[0057] The experimental results showed that, under the condition of storage for 3 days, the enzyme activity retention rates of the corresponding bacterial-algae composite carriers in Examples 6, 7, 8 and 9 were 99.56%, 99.62%, 99.54% and 99.59%, respectively; after storage for 6 days, they were 98.12%, 98.41%, 98.05% and 98.20%, respectively; and after storage for 12 days, they were 93.92%, 94.78%, 94.05% and 94.11%, respectively.

[0058] Under the same conditions, the dry weight retention rates were as follows: after 3 days of storage, the dry weight retention rates for Examples 6, 7, 8, and 9 were 99.13%, 99.21%, 99.02%, and 99.18%, respectively; after 6 days of storage, they were 97.46%, 98.12%, 97.60%, and 97.75%, respectively; and after 12 days of storage, they were 94.03%, 95.00%, 94.27%, and 94.35%, respectively.

[0059] Regarding the retention rate of COD removal capacity, after 3 days of storage, Examples 6, 7, 8, and 9 were 98.75%, 99.15%, 98.82%, and 98.91%, respectively; after 6 days of storage, they were 96.78%, 97.45%, 96.92%, and 97.08%, respectively; and after 12 days of storage, they were 93.62%, 94.76%, 93.81%, and 93.95%, respectively.

[0060] Regarding the retention rate of ammonia nitrogen removal capacity, after 3 days of storage, Examples 6, 7, 8, and 9 were 98.97%, 99.26%, 98.83%, and 98.91%, respectively; after 6 days of storage, they were 97.12%, 97.88%, 97.35%, and 97.41%, respectively; and after 12 days of storage, they were 93.74%, 94.86%, 93.96%, and 94.02%, respectively.

[0061] Regarding the retention rate of total phosphorus removal capacity, after 3 days of storage, Examples 6, 7, 8, and 9 were 99.01%, 99.32%, 98.95%, and 99.07%, respectively; after 6 days of storage, they were 97.05%, 97.71%, 97.21%, and 97.36%, respectively; and after 12 days of storage, they were 93.81%, 94.69%, 94.06%, and 94.13%, respectively.

[0062] The above results indicate that changes in the concentration of PO4³⁻-P in the preservation solution can affect the functional recovery ability of the bacterial-algae composite carrier after preservation. In Example 7, when the PO4³⁻-P concentration was 25 mg / L, the overall performance retention rate was relatively high.

[0063] Example 17: Effect of auxiliary protective components on the preservation effect of bacterial-algae composite carrier Under the conditions of Examples 10 to 14, the effects of vitamin E, hyaluronic acid, and pH changes on the low-temperature preservation effect of the bacterial-algae composite carrier were investigated.

[0064] In Examples 10 and 11, different concentrations of vitamin E were added to the preservation solution; in Examples 12 and 13, different concentrations of hyaluronic acid were added; in Example 14, the pH conditions of the preservation solution were adjusted, and other preservation and recovery culture steps were the same as in Example 1.

[0065] Experimental results showed that, under the condition of 3 days of storage, the enzyme activity retention rates of the corresponding bacterial-algae composite carriers in Examples 10, 11, 12, 13, and 14 were 99.62%, 99.68%, 99.70%, 99.73%, and 99.50%, respectively; after 6 days of storage, they were 98.36%, 98.61%, 98.82%, 98.97%, and 97.93%, respectively; and after 12 days of storage, they were 95.54%, 95.79%, 95.88%, 96.08%, and 94.35%, respectively.

[0066] Regarding dry weight retention, after 3 days of storage, Examples 10, 11, 12, 13, and 14 had dry weight retention rates of 99.20%, 99.35%, 99.41%, 99.46%, and 98.92%, respectively; after 6 days of storage, they had dry weight retention rates of 97.92%, 98.16%, 98.37%, 98.61%, and 97.28%, respectively; and after 12 days of storage, they had dry weight retention rates of 94.32%, 94.56%, 94.71%, 95.00%, and 93.44%, respectively.

[0067] Regarding the retention rate of COD removal capacity, after 3 days of storage, Examples 10, 11, 12, 13, and 14 were 99.20%, 99.39%, 99.46%, 99.58%, and 98.74%, respectively; after 6 days of storage, they were 97.54%, 97.91%, 98.22%, 98.43%, and 96.95%, respectively; and after 12 days of storage, they were 95.18%, 95.42%, 95.65%, 95.87%, and 93.82%, respectively.

[0068] Regarding the retention rate of ammonia nitrogen removal capacity, after 3 days of storage, Examples 10, 11, 12, 13, and 14 were 99.30%, 99.48%, 99.52%, 99.63%, and 98.86%, respectively; after 6 days of storage, they were 97.86%, 98.13%, 98.41%, 98.65%, and 97.26%, respectively; and after 12 days of storage, they were 95.06%, 95.38%, 95.61%, 95.76%, and 93.91%, respectively.

[0069] Regarding the retention rate of total phosphorus removal capacity, after 3 days of storage, Examples 10, 11, 12, 13, and 14 were 99.40%, 99.55%, 99.59%, 99.70%, and 98.93%, respectively; after 6 days of storage, they were 98.05%, 98.32%, 98.61%, 98.79%, and 97.42%, respectively; and after 12 days of storage, they were 95.21%, 95.46%, 95.57%, 95.69%, and 94.02%, respectively.

[0070] The above results indicate that adding auxiliary protective components can further improve the performance retention of the bacterial-algae composite carrier during low-temperature preservation. Specifically, in Example 13, when the hyaluronic acid concentration was 7 g / L, the bacterial-algae composite carrier exhibited the best overall performance retention after 12 days of preservation.

[0071] Example 18: Evaluation of the performance retention effect of the bacterial-algae composite carrier under preferred storage conditions To further verify the effect of different preservation solution optimization conditions on the low-temperature preservation effect of the bacterial-algae composite carrier, the preservation conditions corresponding to Examples 3, 7 and 13 were selected for comparative analysis.

[0072] In Example 3, a preservation solution with a COD concentration of 500 mg / L was used; in Example 7, a preservation solution with a PO4³⁻-P concentration of 25 mg / L was used; and in Example 13, a preservation solution with a hyaluronic acid concentration of 7 g / L was used. Except for the composition of the preservation solution, all other preservation and recovery culture conditions remained consistent.

[0073] After 3 days of storage, the enzyme activity retention rates of the corresponding algae-bacterial composite carriers in Examples 3, 7, and 13 were 99.40%, 99.62%, and 99.73%, respectively; the dry weight retention rates were 98.02%, 99.21%, and 99.46%, respectively; the COD removal capacity retention rates were 99.98%, 99.15%, and 99.58%, respectively; the ammonia nitrogen removal capacity retention rates were 99.26%, 99.26%, and 99.63%, respectively; and the total phosphorus removal capacity retention rates were 99.43%, 99.32%, and 99.70%, respectively.

[0074] After 6 days of storage, the enzyme activity retention rates of the corresponding bacterial-algae composite carriers in Examples 3, 7, and 13 were 97.80%, 98.41%, and 98.97%, respectively; the dry weight retention rates were 97.35%, 98.12%, and 98.61%, respectively; the COD removal capacity retention rates were 96.14%, 97.45%, and 98.43%, respectively; the ammonia nitrogen removal capacity retention rates were 96.76%, 97.88%, and 98.65%, respectively; and the total phosphorus removal capacity retention rates were 97.29%, 97.71%, and 98.79%, respectively.

[0075] After 12 days of storage, the enzyme activity retention rates of the corresponding algae-bacterial composite carriers in Examples 3, 7, and 13 were 95.12%, 94.78%, and 96.08%, respectively; the dry weight retention rates were 93.57%, 95.00%, and 95.00%, respectively; the COD removal capacity retention rates were 94.99%, 94.76%, and 95.87%, respectively; the ammonia nitrogen removal capacity retention rates were 94.36%, 94.86%, and 95.76%, respectively; and the total phosphorus removal capacity retention rates were 94.98%, 94.69%, and 95.69%, respectively.

[0076] The above results indicate that when Example 13 uses a hyaluronic acid concentration of 7 g / L for storage, it exhibits superior overall performance retention during long-term storage. Its enzyme activity, dry weight, and contaminant removal function all remain at high levels, suggesting that the hyaluronic acid-assisted protection can further enhance the low-temperature storage stability of the bacterial-algae composite carrier.

[0077] Comparative Example 1: Evaluation of the low-temperature preservation effect of the bacterial-algae composite carrier without the addition of preservation solution To verify the promoting effect of the preservation solution system of the present invention on the preservation effect of the bacterial-algae composite carrier, a comparative example without the addition of a specific preservation solution was set up.

[0078] In this comparative example, the cleaned bacterial-algal complex was stored directly in an environment of 0–4 °C without adding a preservation solution containing organic carbon source, inorganic nitrogen source and inorganic phosphorus source. The other recovery culture conditions were the same as in Example 1.

[0079] After 12 days of storage, the bacterial-algae complex was restored and cultured, and its dry weight, enzyme activity, and removal capacity of COD, ammonia nitrogen, and total phosphorus were measured.

[0080] The experimental results showed that, under the conditions of Comparative Example 1, the enzyme activity retention rate of the bacterial-algae composite carrier was 78.43%, the dry weight retention rate was 76.95%, the COD removal capacity retention rate was 77.26%, the ammonia nitrogen removal capacity retention rate was 76.84%, and the total phosphorus removal capacity retention rate was 77.15%.

[0081] Compared with the embodiment using the preservation solution system of the present invention, the ability of the bacterial-algae composite carrier to maintain various properties in Comparative Example 1 was significantly reduced, indicating that the nutrient components and protective environment in the preservation solution play an important role in maintaining the low-temperature preservation stability of the bacterial-algae composite carrier.

[0082] Comparative Example 2: Evaluation of Preservation Effect under Sterile Filament Immobilization Conditions To verify the contribution of the three-dimensional immobilization structure of mycelial balls to the preservation effect of the fungal-algae composite carrier, a comparative example was set up under the condition of no mycelial balls.

[0083] In this comparative example, only the microalgae system was used for low-temperature preservation, without the addition of mycelial ball carriers. The composition of the preservation solution, the preservation temperature, and the recovery culture conditions were all consistent with those in Example 13.

[0084] After 12 days of storage, the performance of the restored microalgae system was tested.

[0085] The experimental results showed that, under the conditions of Comparative Example 2, the enzyme activity retention rate of the microalgae system was 81.36%, the dry weight retention rate was 79.82%, the COD removal capacity retention rate was 80.21%, the ammonia nitrogen removal capacity retention rate was 79.65%, and the total phosphorus removal capacity retention rate was 80.04%.

[0086] The above results indicate that, compared with simple microalgae preservation, the three-dimensional spatial structure provided by mycelial balls can improve the adhesion stability of microalgae and facilitate the recovery of water treatment function after preservation.

[0087] Comparative Example 3: Comparative Experiment of Traditional Glycerin Preservation Methods To verify the difference between the cryopreservation method of the present invention and the traditional microbial preservation method, the traditional glycerol preservation method was set as a comparative example.

[0088] This comparative example uses conventional glycerol preservation. The bacterial-algal complex is added to a preservation system containing glycerol and stored at 0–4 °C for 12 days. The remaining recovery culture and performance testing methods are consistent with those in Example 13.

[0089] The experimental results showed that, under the conditions of Comparative Example 3, the enzyme activity retention rate of the bacterial-algae composite carrier was 83.75%, the dry weight retention rate was 82.41%, the COD removal capacity retention rate was 82.96%, the ammonia nitrogen removal capacity retention rate was 82.73%, and the total phosphorus removal capacity retention rate was 83.12%.

[0090] Compared with Example 13 of the present invention, although the traditional glycerol preservation method can maintain the activity of the bacterial-algae composite carrier to a certain extent, there are still significant gaps in terms of dry weight preservation, enzyme activity preservation, and pollutant removal capacity recovery.

[0091] As can be seen from Examples 1-14 and comparative experiments, the present invention optimizes the low-temperature preservation process of the bacterial-algae composite carrier by controlling the organic carbon source, inorganic nitrogen source, inorganic phosphorus source and auxiliary protective components in the preservation solution.

[0092] Simply lowering the storage temperature cannot guarantee the structural and functional stability of the bacterial-algae composite carrier. However, by using the storage solution system of this invention, the three-dimensional structure of the mycelial balls and the attachment state of microalgae can be maintained during the storage process, and the water treatment function after recovery culture can be improved.

[0093] Compared with preservation conditions without added preservation solution and preservation methods with simple glycerol protection, the method of the present invention can significantly improve the performance retention rate of the fungal-algae composite carrier after preservation, indicating that the synergistic effect of "mycelial ball immobilization structure, preservation solution nutrient regulation, glycerol-assisted protection and low temperature preservation conditions" proposed in the present invention can achieve stable preservation of functional fungal-algae composite carriers.

Claims

1. A method for cryopreservation of carrier mycelial balls for water treatment, characterized in that, include: Mycelial balls are mixed with microalgae and cultured to form bacterial-algal bodies; The bacteria and algae were washed with phosphate buffer or sterile physiological saline at a pH of 6-8. The cleaned bacteria and algae were added to a preservation solution, which included an organic carbon source, an inorganic nitrogen source, and an inorganic phosphorus source, with a COD concentration of 300–500 mg / L, an NH4⁺-N concentration of 80–200 mg / L, a PO4³⁻-P concentration of 15–25 mg / L, and a pH of 2.0–3.

0. The bacterial and algal cells after adding the preservation solution were subjected to glycerol-assisted protection treatment and sealed for preservation in a low-temperature environment of 0–4 °C.

2. The method for cryopreservation of carrier mycelial balls according to claim 1, characterized in that, The mycelial balls and the microalgae were mixed at a volume ratio of 1:6 and cultured under shaking conditions of 140–160 rpm, room temperature and natural pH for 2 hours.

3. The method for cryopreservation of carrier mycelial balls according to claim 1, characterized in that, The mycelial balls include Aspergillus niger ( Aspergillus niger Mycelial balls or Aspergillus fumigatus formed by ) Aspergillus fumigatus The mycelial balls formed.

4. The method for cryopreservation of carrier mycelial balls according to claim 1, characterized in that, The preservation solution also includes hyaluronic acid, and the concentration of hyaluronic acid is 3-7 g / L.

5. The method for cryopreservation of carrier mycelial balls according to claim 1, characterized in that, The bacterial and algal cells can be stored at 0–4°C for 1–12 months.

6. The method for cryopreservation of carrier mycelial balls according to claim 1, characterized in that, After preservation, the bacterial and algal cells were transferred to a recovery culture system comprising 2 g / L glucose, 0.5 g / L ammonium chloride, and 0.5 g / L dipotassium hydrogen phosphate. The recovery culture was carried out at 160 r / min, 25 ℃, and for 1–3 hours. The preservation effect of the recovered bacterial and algal cells was evaluated by measuring their dry weight, enzyme activity, and removal capacity for COD, ammonia nitrogen, and total phosphorus.

7. A cryopreserved microbial-algae composite carrier for water treatment, characterized in that, The product includes mycelial balls and microalgae attached to the surface and internal pores of the mycelial balls; the mycelial-algae composite carrier is immersed in a preservation solution, which includes an organic carbon source, an inorganic nitrogen source, and an inorganic phosphorus source, and has a COD concentration of 300-500 mg / L, an NH4⁺-N concentration of 80-200 mg / L, a PO4³⁻-P concentration of 15-25 mg / L, and a pH of 2.0-3.0; the mycelial-algae composite carrier is sealed and stored at a low temperature of 0-4 ℃ after being protected with glycerol.

8. The bacterial-algae composite carrier according to claim 7, characterized in that, The mycelial balls are Aspergillus niger mycelial balls or Aspergillus fumigatus mycelial balls.

9. The bacterial-algae composite carrier according to claim 7, characterized in that, The microalgae are either Chlorella pyrenoidosa or Chlorella vulgaris.

10. The bacterial-algae composite carrier according to claim 7, characterized in that, The preservation solution also includes hyaluronic acid at a concentration of 3–7 g / L.

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