Method for room temperature preservation of a microalgal suspension and use thereof

Abstract

The application belongs to the technical field of microalgae preservation, and discloses a room temperature preservation method of microalgae suspension and application thereof. The preservation method is that microalgae suspension is placed in a closed container, deoxygenating agent and bacteriostatic agent are added, pH is adjusted to 7.5-8.5, and microalgae preservation solution is obtained; a vacuum-pumping and air-filling system is used, nitrogen is introduced for headspace replacement, an anaerobic environment is established, and the dissolved oxygen concentration in the microalgae preservation solution is reduced, and the microalgae preservation solution is preserved at room temperature in the dark. The application realizes long-term maintenance of microalgae activity at room temperature and effective inhibition of bacterial proliferation, and the survival rate of microalgae can reach more than 69% in 90 days, which provides economic and practical technical support for the large-scale application of microalgae in the fields of food, feed, energy and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microalgae preservation, and particularly relates to a room temperature preservation method of microalgae suspension and application thereof. BACKGROUND

[0002] As a biological resource with great application potential, microalgae shows a wide application prospect in many fields such as food, feed, wastewater treatment and energy. In the industrial production, microalgae are usually cultured in large scale by open pond culture or photobioreactor. However, due to the spatial and temporal distance between the production site and the consumption site of microalgae, the microalgae culture solution must be able to maintain sufficient microalgae activity within a certain period of time. In addition, during the industrial large-scale culture of microalgae, there is inevitably a certain degree of bacterial contamination. This makes the microalgae culture solution face many challenges during transportation and storage, especially the problem of bacterial contamination. How to maintain the activity of microalgae and inhibit the growth of bacteria during transportation and storage is the key to realize the economic effect of microalgae products. Although reducing the preservation temperature of the algal solution can achieve this effect, the low-temperature preservation environment will increase the preservation and transportation cost, which restricts the application range of microalgae products. Therefore, it is of great significance to develop an effective room temperature preservation method for microalgae suspension for promoting the large-scale application of microalgae products. SUMMARY

[0003] The present application aims to improve the application efficiency and economic value of microalgae in actual production environment, and provides a room temperature preservation method of microalgae suspension.

[0004] Another object of the present application is to provide the application of the above-mentioned room temperature preservation method of microalgae suspension.

[0005] The object of the present application is achieved by the following technical solutions:

[0006] The present application provides a room temperature preservation method of microalgae suspension, which comprises the following steps: placing the microalgae suspension in a sealed container, adding an oxygen scavenger and a bacterial inhibitor, adjusting the pH to 7.5-8.5 to obtain a microalgae preservation solution; using a vacuum-gas filling system, nitrogen gas is introduced for headspace replacement to establish an anaerobic environment and reduce the dissolved oxygen concentration in the microalgae preservation solution, and the microalgae preservation solution is stored at room temperature in the dark.

[0007] Preferably, the microalgae is Chlorella.

[0008] The microalgae suspension is a microalgae suspension obtained by industrial production culture, such as a microalgae suspension obtained by outdoor photobioreactor culture, which is a mixture of algae and bacteria.

[0009] The concentration of algal cells in the microalgae preservation solution is 10 6 -10 7 individuals / mL.

[0010] The oxygen scavenger is preferably sodium sulfite (Na2SO3).

[0011] The concentration of the oxygen scavenger in the microalgae preservation solution is preferably 0-2500 mg / L; preferably 250-2500 mg / L; more preferably 500-1500 mg / L; more preferably 500-1000 mg / L; more preferably 500-626 mg / L.

[0012] The bacterial inhibitor is preferably sodium nitrite (NaNO2).

[0013] The concentration of the bacterial inhibitor in the microalgae preservation solution is preferably 0-400 mg / L; preferably 25-400 mg / L; more preferably 50-100 mg / L; most preferably 50-69 mg / L.

[0014] The pH is preferably 8.0.

[0015] The closed container is preferably a Schlenk flask or a silicon bottle.

[0016] The number of times of headspace replacement is preferably more than 3; more preferably 4-8 times; most preferably 5-6 times.

[0017] The room temperature refers to 10-35℃; preferably 20-35℃; more preferably 25-27℃.

[0018] The room temperature preservation method of the microalgae suspension is applied in the storage and transportation of the microalgae suspension.

[0019] The present application has the following advantages and effects relative to the prior art:

[0020] The present application realizes effective inhibition of bacterial growth and efficient maintenance of microalgae activity through the synergistic effect of multiple means. The core of the system is to construct a comprehensive preservation environment that is anaerobic, light-avoiding, weakly alkaline and contains specific bacteriostatic components, which specifically includes the following aspects:

[0021] (1) Initial oxygen removal and anaerobic environment maintenance: the initial oxygen is completely removed by replacing the headspace air of the culture solution with pure nitrogen, and the photosynthetic oxygen production of microalgae is inhibited under light-avoiding conditions, thereby maintaining the continuous anaerobic state of the system;

[0022] (2) Compound bacteriostatic strategy: sodium sulfite (Na2SO3) is added as an oxygen scavenger to continuously consume residual oxygen and strengthen the anaerobic environment; sodium nitrite (NaNO2) is added as a bacterial inhibitor to directly inhibit the reproduction of miscellaneous bacteria;

[0023] (3) pH optimization and regulation: the pH of the system is accurately adjusted to 8.0, which not only meets the optimal growth pH of microalgae, but also inhibits the growth of most bacteria.

[0024] (4) Light-protected storage: The entire process is kept in the dark to prevent microalgae from producing oxygen through photosynthesis and to further maintain the anaerobic state of the system.

[0025] This invention constructs a comprehensive preservation system that enables long-term maintenance of microalgae activity at room temperature and effectively inhibits bacterial proliferation. The microalgae survival rate can reach over 69% after 90 days, providing economical and practical technical support for the large-scale application of microalgae in food, feed, energy and other fields. Attached Figure Description

[0026] Figure 1 This figure shows the changes in cell number of pure Chlorella ZJ under different preservation conditions.

[0027] Figure 2 This figure shows the changes in cell number of microalgae and other bacteria under different preservation conditions.

[0028] Figure 3 The graph shows the combined effect of NaNO2 and Na2SO3 addition on microalgae survival rate (a) and the preservation results of microalgae suspension under optimized culture conditions (b). Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0030] Example 1

[0031] Prepare BG-11 medium and inoculate pre-cultured pure Chlorella ZJ at a volume percentage of 5%. Chlorella sp. ZJ, deposited at the Guangdong Provincial Microbial Culture Collection Center (accession number GDMCC No. 63704, disclosed in Chinese invention patent CN117402741B, entitled "A Chlorella and Its Applications"), was cultured at 30℃ and 8000 Lux for 6 days in a light incubator. The Chlorella culture medium was collected and plated on BG-11 solid medium (BG-11 medium containing 2% agar) to determine the Chlorella cell concentration. The concentration of Chlorella in the culture medium was determined to be 1.02 × 10⁻⁶. 7 per mL.

[0032] Take 50 mL of Chlorella algae liquid, place it in a 125 mL sterilized Erlenmeyer flask, use the vacuum-gas system to introduce sterile high-purity (purity 99.999%) nitrogen or dry air for 6 rounds of headspace replacement, and establish an anaerobic (nitrogen) and aerobic (sterile air) environment, respectively. Then place the Erlenmeyer flask at 26°C and store it in the dark or under weak light (200 lux) conditions. Test the effect of different preservation conditions on the preservation of pure microalgae culture by using the number of viable microalgae cells as an indicator.

[0033] The results are shown in Figure 1. After 90 days of preservation under weak light + aerobic, weak light + anaerobic, dark + aerobic, and dark + anaerobic conditions, the cell count of Chlorella was 107.08 x 10 7 , 98.15 x 10 7 , 94.26 x 10 7 , and 90.66 x 10 7 cells / mL, respectively. These results show that pure microalgae culture can achieve good preservation results under different preservation conditions, and the preservation effect under weak light conditions is significantly better than that in the dark; the preservation effect under aerobic conditions is better than that under anaerobic conditions. The best preservation condition is weak light + aerobic, under which the cell count of Chlorella even increases.

[0034] The composition of BG-11 liquid medium is as follows: NaNO3 1.00 g / L, K2HPO4 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, EDTA 0.001 g / L, Na2CO3 0.02 g / L, H3BO4 0.00286 g / L, MnCl2·H2O 0.00181 g / L, ZnSO4·7H2O 0.000222 g / L, CuSO4·5H2O 0.000079 g / L, Na2MoO4·2H2O 0.00039 g / L, Co(NO3)2·6H2O 0.000049 g / L; the solvent is deionized water.

[0035] Example 2

[0036] A transparent PVC pipe was selected to build a pipeline photobioreactor. The culture medium was BG-11 medium, which was inoculated with pre-cultured Chlorella ZJ at a volume percentage of 10%, placed in a greenhouse, and subjected to natural light, with the temperature maintained at 20-28°C. A circulating water pump was used to circulate the algal liquid at a flow rate of 0.5 m / s, while air was introduced at a flow rate of 0.2-0.4 Nm 3 / min, and cultured for 8 days. The Chlorella culture solution was collected and plated for dilution to determine the Chlorella cell concentration by BG-11 solid medium (BG-11 medium containing 2% agar) and the number of bacteria by LB solid medium. The Chlorella concentration in the algal solution was determined to be 1.45 x 10 7 cells / mL, and the bacteria concentration was 0.45 x 10 6 cells / mL.

[0037] The composition of the BG-11 liquid medium was the same as above.

[0038] The composition of the LB solid medium was as follows: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 20 g / L, and deionized water as the solvent.

[0039] The effects of weak light (200 lux) / dark and headspace air (aerobic) or nitrogen (anaerobic) preservation conditions on the activity of microalgae suspension were tested by taking the number of microalgae cells and the number of bacteria cells as indicators. The method for replacing the headspace air was the same as above. The survival of microalgae is shown in Fig. 2a. Under the four different storage conditions, the number of microalgae cells decreased significantly with the increase of storage days. The survival rate of microalgae under anaerobic conditions was better than that under aerobic conditions, and the survival rate under dark conditions was better than that under weak light conditions. If the headspace air was not replaced, the survival rate of microalgae after 90 days of preservation was very low, only 0.04-0.05%, indicating that the direct preservation of untreated microalgae culture solution in a Westlin bottle had poor effect. After the headspace air was replaced with nitrogen, the survival rate of microalgae increased significantly to 3.79-14.49%, with an increase of 94 to 275 times, proving that the oxygen removal of the system can greatly promote the survival of microalgae. Under the best condition of dark and anaerobic, the survival rate of microalgae after 90 days of preservation was 14.49%. Although it has been greatly improved compared with direct preservation, this survival rate still shows that the preservation strategy has room for further optimization. The results of monitoring the change of bacteria content are shown in Fig. 2b. With the extension of preservation time, the number of bacteria increased. The proliferation rate of bacteria under aerobic conditions was significantly higher than that under anaerobic conditions, and the growth of bacteria in the light environment was also faster than that in the dark environment. Microalgae can produce oxygen through photosynthesis under light, which may be one of the reasons for the accelerated proliferation of bacteria under light. In addition, the proliferation of bacteria was negatively correlated with the survival rate of microalgae. Under dark and anaerobic conditions, the growth of bacteria was effectively inhibited, and the survival rate of microalgae was relatively high, indicating that the excessive proliferation of bacteria is a key factor leading to the death of microalgae and the decrease of survival rate. Figure 2

[0040] ​Compared with pure culture microalgae, the mixed bacteria-containing microalgae suspension showed a completely different trend in preservation: the optimal condition in pure culture (weak light + aerobic) was the worst in the mixed bacteria-containing system, while the dark + anaerobic condition which was poor in pure culture, was the best in this system. This indicates that for the actual mixed bacteria-containing microalgae suspension, effectively inhibiting the growth of mixed bacteria and maintaining the activity of microalgae are the core to improve the preservation efficiency.

[0041] Example 3

[0042] Transparent PVC pipes were selected to build a pipeline photobioreactor. The culture medium was BG-11 medium, and the pre-cultured Chlorella ZJ was inoculated at a volume percentage of 10%, and placed in a greenhouse under natural light, with the temperature maintained at 20-28 ℃. A circulating water pump was used to circulate the algal liquid at a flow rate of 0.5 m / s, while air was introduced at a flow rate of 0.2-0.4 Nm 3 / min. The Chlorella culture solution was collected and the Chlorella cell concentration was determined by BG-11 medium plate dilution and coating, and the number of mixed bacteria was detected by LB medium plate dilution and coating. It was determined that the Chlorella concentration in the algal liquid was 1.41×10 7 / mL, and the mixed bacteria concentration was 0.25×10 6 / mL.

[0043] In order to improve the preservation effect of microalgae suspension, the main goal is to inhibit bacterial growth and maintain microalgae activity, and six variables are selected for Plackett-Burman design. The experimental level combination of the six selected variables is shown in Table 2. The basic preservation conditions of the microalgae suspension are as follows: in a 125 mL test tube system, 50 mL of microalgae suspension is taken, 75 mL of headspace is left, a vacuum-air system is used, and high-purity nitrogen gas is introduced for 6 rounds of headspace replacement. The headspace gas is replaced with nitrogen to reduce the dissolved oxygen concentration of the microalgae suspension, and the preservation is carried out in the dark for 90 days. The Plackett-Burman experiment results show that the number of microalgae changes from 0.41×10 6 / mL to 7.64×10 6 / mL, and the number of bacteria changes from 3.24×10 6 / mL to 127.69×10 6 / mL (Table 1). There is a significant negative correlation between the number of bacteria and the number of microalgae.

[0044] The Plackett-Burman experiment was analyzed by ANOVA with microalgae survival rate as the response value, and then a first-order polynomial model was fitted, with a determination coefficient (R²) of 95.57%, a p-value of the overall model of 0.0143, less than 0.05, indicating a good fitting effect (Table 3). It was found that six variables, sodium sulfite (Na2SO3), sodium alginate, Tris-HCl, sodium nitrite (NaNO2), pH and temperature, had a significant effect on the survival rate of microalgae. Among them, Na2SO3, NaNO2 and pH were positively correlated with the survival rate of microalgae, while sodium alginate, Tris-HCl and temperature were negatively correlated (Table 2). The addition of Na2SO3 can significantly improve the survival rate of microalgae, which is mainly due to its dual bacteriostatic effect: on the one hand, as an oxygen scavenger to maintain the anaerobic state of the system and inhibit the growth of aerobic bacteria; on the other hand, as a bacteriostatic agent, it can directly inhibit bacterial activity. The addition of NaNO2, which has broad-spectrum bacteriostatic properties, may also enhance the survival of microalgae by inhibiting bacterial growth. The survival rate of microalgae at pH=8 is higher than that at pH=7, which may be related to the different pH growth environments preferred by microalgae and bacteria. Sodium alginate, as a commonly used microbial preservation additive, can improve the tolerance of microorganisms and prolong the shelf life of microorganisms. However, due to the presence of bacterial contamination in the microalgae suspension, the addition of sodium alginate as an additional organic carbon source may lead to rapid bacterial reproduction, thereby reducing the preservation effect of microalgae. Similarly, the addition of Tris-HCl may also be due to the same reason. Lowering the temperature is beneficial to the survival of microalgae. On the one hand, reducing the temperature can reduce the metabolic activity of microorganisms, thereby improving the preservation effect of microorganisms; on the other hand, reducing the temperature can also slow down the growth rate of bacteria, further improving the survival rate of microalgae. Since the purpose of this invention is to explore the preservation conditions of microalgae suspension at room temperature, the temperature cannot be set too low, and the subsequent experiments will use a temperature of 26°C.

[0045] Table 1 P-B experimental design and results of microalgae suspension preservation conditions

[0046]

[0047] Note: A is Na2SO3 (g / L), B is sodium alginate (g / L), C is Tris-HCl (g / L), D is NaNO2 (mg / L), E is A5 element liquid (mL / L), F is pH, G is temperature (℃);

[0048] The A5 element liquid component is as follows: H3BO32.86 g / L, MnCl2·4H2O 1.81 g / L, ZnSO4·7H2O 0.222 g / L, Na2MoO4·2H2O 0.39 g / L, CuSO4·5H2O 0.079 g / L, Co(NO3)2·6H2O 0.0494 g / L.

[0049] Table 2 Analysis of P-B design according to microalgae survival rate

[0050]

[0051] R 2 =95.57%

[0052] Example 4

[0053] A transparent PVC pipe is selected to build a pipeline type photobioreactor. The culture medium is BG-11 medium, and the pre-cultured Chlorella ZJ is inoculated at a volume percentage of 10%, placed in a greenhouse, and subjected to natural light, with the temperature maintained at 20-28 ℃. A circulating water pump is used to circulate the algal liquid at a flow rate of 0.5 m / s, while air is introduced at a flow rate of 0.2-0.4 Nm 3 / min. The Chlorella culture solution is collected, and the Chlorella cell concentration is determined by dilution and coating on a BG-11 medium plate, and the number of miscellaneous bacteria is detected by dilution and coating on an LB medium plate. It is determined that the Chlorella concentration in the algal liquid is 1.4×10 7 / mL, and the miscellaneous bacteria concentration is 0.33×10 6 / mL.

[0054] Based on the results of the Plackett-Burman experiment, the steepest ascent method is used to further study the effects of Na2SO3 and NaNO2 on the preservation of microalgae suspension. The basic preservation conditions of the microalgae suspension are as follows: in a system of 125 mL silicon bottle, 50 mL of microalgae suspension is taken, 75 mL of headspace is left, a vacuum-gas system is used, high-purity nitrogen gas is introduced for 6 rounds of headspace replacement, the headspace gas is replaced with nitrogen to reduce the dissolved oxygen concentration of the microalgae suspension, the pH is adjusted to 8.0, and the microalgae suspension is preserved at 26 ℃ in the dark for 90 days. The results of the steepest ascent method are shown in Table 3. Under the preservation conditions of 500 mg / L Na2SO3 and 50 mg / L NaNO2, the survival rate of the microalgae suspension is the highest, reaching 67.9%. Based on this condition, a central composite design is designed to further optimize the preservation conditions of the microalgae suspension.

[0055] Table 3 Steepest ascent method design and results

[0056]

[0057] Example 5

[0058] Transparent PVC tubes were selected to build the pipeline photobioreactor. The medium was BG-11 medium, and the pre-cultured Chlorella ZJ was inoculated at a volume percentage of 10%. The Chlorella ZJ was placed in a greenhouse, and the natural light was used. The temperature was maintained at 20-28°C. The algae liquid was circulated at a flow rate of 0.5 m / s by using a circulating water pump, and air was introduced at a flow rate of 0.2-0.4 Nm 3 / min. The Chlorella ZJ was cultured for 8 days. The Chlorella ZJ culture solution was collected, and the Chlorella ZJ cell concentration was determined by dilution and coating on a BG-11 medium plate. The number of miscellaneous bacteria was detected by dilution and coating on an LB medium plate. It was determined that the Chlorella ZJ concentration in the algae liquid was 1.18 x 10 7 / mL, and the miscellaneous bacteria concentration was 0.11 x 10 6 / mL.

[0059] Based on the results of the steepest ascent experiment, a central composite design was designed to further optimize the concentrations of Na2SO3 and NaNO2. The basic preservation conditions of the microalgae suspension were as follows: in a system of 125 mL silicon bottles, 50 mL of microalgae suspension was taken, 75 mL of headspace was left, a vacuum-air system was used, high-purity nitrogen was introduced for 6 rounds of headspace replacement, the headspace gas was replaced with nitrogen to reduce the dissolved oxygen concentration of the microalgae suspension, the pH was adjusted to 8.0, and the microalgae suspension was stored at 26°C in the dark for 90 days.

[0060] The central composite design of the Design-Expert V13 software was used to optimize the preservation conditions of the microalgae, and 13 groups of experiments were carried out. The survival rate of the microalgae was between 37.97% and 78.98% (see Table 4). The regression model was highly significant (p<0.01) by variance analysis, and the R 2 value reached 91.17% (see Table 5). The software analysis predicted that when the concentrations of Na2SO3 and NaNO2 were set to 626.3 mg / L and 68.82 mg / L, respectively, the survival rate reached 77.41%.

[0061] The optimized preservation conditions of the microalgae suspension were as follows: in a system of 125 mL silicon bottles, 50 mL of microalgae suspension was taken, 75 mL of headspace was left, a vacuum-air system was used, high-purity nitrogen was introduced for 6 rounds of headspace replacement, the headspace gas was replaced with nitrogen to reduce the dissolved oxygen concentration of the microalgae suspension, the pH was adjusted to 8.0, 626 mg / L of Na2SO3 and 69 mg / L of NaNO2 were added, and the microalgae suspension was stored at 26°C in the dark for 90 days.

[0062] It was verified that the actual survival rate was 69.15% (69.15%). Figure 3), the unexpected 77.41% may be due to the difference in initial conditions of microalgae suspension in different batches of preservation experiments. In the verification experiment, the microalgae suspension contained 1.28x10 6 6 6 6 In the central composite experiment, the microalgae suspension contained 1.18x10

[0063] Table 4 Central composite design and results of microalgae suspension preservation

[0064]

[0065] Table 5 Variance analysis of central composite design

[0066]

[0067] R 2 =91.17%

[0068] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.​​​

Claims

1. A method for preserving microalgae suspensions at room temperature, characterized in that... The process includes the following steps: placing the microalgae suspension in a sealed container, adding an oxygen absorber and a bacterial inhibitor, adjusting the pH to 7.5–8.5 to obtain a microalgae preservation solution; using a vacuum-gas system, introducing nitrogen gas for headspace displacement to establish an anaerobic environment and reduce the dissolved oxygen concentration in the microalgae preservation solution, and storing it at room temperature in the dark. The microalgae mentioned is Chlorella; The oxygen scavenger is sodium sulfite; The bacterial inhibitor mentioned is sodium nitrite; The concentration of the oxygen scavenger in the microalgae preservation solution is 250–2500 mg / L; The concentration of the bacterial inhibitor in the microalgae preservation solution is 25–400 mg / L; The room temperature mentioned refers to 26–30 °C.

2. The method for room temperature preservation of microalgae suspension according to claim 1, characterized in that: The concentration of algal cells in the microalgae preservation solution is 10. 6 ~10 7 per mL.

3. The method for room temperature preservation of microalgae suspension according to claim 1, characterized in that: The concentration of the oxygen scavenger in the microalgae preservation solution is 500–1000 mg / L; The concentration of the bacterial inhibitor in the microalgae preservation solution is 50–100 mg / L.

4. The method for room temperature preservation of microalgae suspension according to claim 3, characterized in that: The concentration of the oxygen scavenger in the microalgae preservation solution is 500–626 mg / L; The concentration of the bacterial inhibitor in the microalgae preservation solution is 50–69 mg / L.

5. The method for room temperature preservation of microalgae suspension according to claim 1, characterized in that: The pH value is 8.

0.

6. The method for room temperature preservation of microalgae suspension according to claim 1, characterized in that: The sealed container is a vial or a silicone vial.

7. The method for room temperature preservation of microalgae suspension according to claim 1, characterized in that: The headspace displacement is performed three or more times.

8. The application of the room temperature preservation method for microalgae suspension according to any one of claims 1 to 7 in the storage and transportation of microalgae suspension.

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