Adaptive chlorella domesticated by fish meal processing wastewater, domestication method and application

Abstract

The invention discloses adaptive chlorella domesticated by fish meal processing wastewater, a domestication method and application, and belongs to the technical field of microalgae culture and wastewater treatment. The method comprises the following specific steps: 1) inoculating wild chlorella into fish meal processing wastewater, and carrying out first tolerance culture for at least two months to obtain a domesticated chlorella solution; and 2) inoculating the domesticated chlorella solution into a sterilized mixed domesticated culture medium, and carrying out second tolerance culture for 6-10 days to obtain the adaptive chlorella. The mixed domestication culture medium comprises fish meal processing wastewater and an N8 culture medium, wherein the volume ratio of the fish meal processing wastewater to the N8 culture medium is (6-14): 6. According to the method, the chlorella strain capable of tolerating high-concentration phosphate and ammonium salt is successfully domesticated, resource utilization of fish meal processing wastewater is realized, meanwhile, the microalgae culture cost is reduced, and the method has dual benefits of environmental protection and economy.

Description

Technical Field

[0001] This invention belongs to the field of microalgae cultivation and wastewater treatment technology, specifically relating to an adaptive Chlorella culture acclimated using fishmeal processing wastewater, the acclimation method, and its application. Background Technology

[0002] Microalgae cultivation, as an emerging wastewater treatment technology, has attracted widespread attention due to its high efficiency, environmental friendliness, and sustainability. Microalgae can utilize nutrients such as nitrogen and phosphorus in wastewater for growth, while simultaneously adsorbing heavy metals and organic pollutants, achieving wastewater purification and resource recovery. Furthermore, microalgae are rich in protein, lipids, and carbohydrates, making them valuable as antioxidants, feed, or food additives. Several successful cases of cultivating Chlorella using agricultural or industrial wastewater have been documented. For example, patent application CN120136599A discloses "An apparatus and method for producing microalgae liquid fertilizer using monosodium glutamate (MSG) wastewater." This method uses treated MSG wastewater instead of ordinary culture medium, successfully cultivating Chlorella and obtaining an algal solution that promotes wheat seedling growth and root development while reducing cultivation costs.

[0003] However, in practical applications, due to the complex composition of wastewater, the high concentration of toxic substances (such as heavy metals and organic pollutants), and the imbalanced nutrient ratio, common wild-type microalgae have poor adaptability to wastewater, exhibiting slow growth and insufficient biomass accumulation, which severely limits the large-scale application of microalgae in wastewater treatment. Therefore, how to improve the adaptability of microalgae to specific wastewater and optimize cultivation conditions to increase biomass yield and protein content has become a key research issue.

[0004] Most acclimatization methods employ a single strategy of gradually increasing wastewater concentration, resulting in long acclimatization cycles and limited stability and biomass improvement in subsequent high-proportion wastewater cultivation. Currently, research strategies for improving microalgal wastewater tolerance mainly include acclimatization cultivation, genetic modification, and optimization of culture medium ratios. Among these, acclimatization cultivation has attracted widespread attention due to its low cost and high efficiency. This method involves gradually increasing the wastewater exposure ratio to selectively screen and cultivate more adaptable algal strains. Simultaneously, using mixed culture media (such as mixing wastewater with synthetic media in a specific ratio) is also an effective approach, reducing the initial toxicity of the wastewater while supplementing essential nutrients, thereby promoting microalgal growth. However, for different types of wastewater with varying compositions, further research is needed to determine the optimal acclimatization strategy and suitable culture medium mixing ratios.

[0005] Of particular concern is the high-ammonia nitrogen wastewater generated during fishmeal processing. Direct discharge of this wastewater can easily lead to environmental problems such as eutrophication. Microalgae show great potential in treating this type of wastewater: microalgae can efficiently absorb and utilize nutrients such as nitrogen and phosphorus in the wastewater, achieving the dual goals of pollution control and resource recovery, and producing economically valuable algal biomass (such as protein-rich algae). However, the nutrient composition of fishmeal processing wastewater is extremely unbalanced, especially lacking many trace elements essential for microalgae growth. Directly introducing microalgae (especially unadapted strains) into this type of wastewater for cultivation will result in significant growth inhibition. Therefore, obtaining an adaptable Chlorella species suitable for treating high-ammonia nitrogen wastewater is urgently needed. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an optimized method for the domestication and cultivation of microalgae to improve their growth efficiency and protein accumulation capacity in fishmeal processing wastewater with high nitrogen and phosphorus content. Specifically, it provides an adaptive Chlorella that can be domesticated using fishmeal processing wastewater, a domestication method, and its application.

[0007] The specific technical solution adopted in this invention is as follows:

[0008] In a first aspect, the present invention provides a method for acclimating and adapting Chlorella using fishmeal processing wastewater, the specific steps of which are as follows:

[0009] S1: Wild-type Chlorella was inoculated into fishmeal processing wastewater for a first tolerance culture of at least 2 months, during which it was transferred every 6-12 days to obtain acclimatized algae solution;

[0010] S2: The acclimatized algae solution is inoculated into a sterilized mixed acclimatization medium for a second tolerance culture for 6 to 10 days to obtain adaptive Chlorella; the mixed acclimatization medium includes the fishmeal processing wastewater and N8 medium mentioned in step S1, wherein the volume ratio of fishmeal processing wastewater in the mixed acclimatization medium is 50% to 70%.

[0011] Preferably, the phosphate content in the fishmeal processing wastewater is 56~145 mg / L, and the ammonium salt content is 157~335 mg / L.

[0012] Preferably, the fishmeal processing wastewater is the washing wastewater or condensate wastewater from the fishmeal production process.

[0013] Preferably, the wild-type Chlorella is Chlorella from the CCAP algal gene bank, numbered CCAP211 / 52.

[0014] As a preferred embodiment, the first tolerance culture in step S1 is specifically as follows: wild-type Chlorella is directly inoculated into fishmeal processing wastewater and kept at a constant temperature of 20-25℃ with continuous light intensity of 50-100 μmol / m². -2 s -1 Under suitable conditions, the algae culture was acclimatized and cultured, and the algal solution was transferred to new fishmeal processing wastewater every 6-12 days, with the initial concentration controlled at 0.5-1×10⁻⁶. 7 The algae were cultured at cells / mL for 2-3 months to obtain the acclimatized algae solution.

[0015] Preferably, the N8 culture medium comprises: 1000 mg / L KNO3, 740 mg / L KH2PO4, 260 mg / L Na2HPO4·2H2O, 13 mg / L CaCl2·2H2O, 10 mg / L Fe EDTA, 50 mg / L MgSO4·7H2O, and 1 mL / L of trace element stock solution.

[0016] Furthermore, the trace element mother liquor comprises 3.58 g / L Al2(SO4)3·18H2O, 12.98 g / L LmnCl2·4H2O, 1.83 g / L CuSO4·5H2O, and 3.2 g / L ZnSO4·7H2O.

[0017] Preferably, the second tolerance culture in step S2 is as follows: the acclimatization algal solution in step S1 is controlled at an initial concentration of 0.5~1×10⁻⁶. 7 Cells / mL were inoculated into sterile mixed acclimatization medium and kept at a constant temperature of 20-25℃ under continuous light intensity of 50-100 μmol / mL. -2 s -1 Adapted Chlorella were obtained through domestication and cultivation under light conditions.

[0018] Secondly, the present invention provides an adaptive Chlorella powder obtained by the method of domesticating and adapting Chlorella described in the first aspect, wherein the adaptive Chlorella is freeze-dried to obtain the adaptive Chlorella powder.

[0019] Thirdly, the present invention provides an application of the adaptive Chlorella powder described in the second aspect in fish feed production, wherein the adaptive Chlorella powder described in claim 9 is added to fish feed as algal biomass.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] This invention involves inoculating wild-type Chlorella strains into a mixed acclimatization medium containing fishmeal processing wastewater, resulting in adaptive Chlorella with improved growth efficiency and protein content. Experimental results show that the soluble protein content in the adaptive Chlorella cultured using this method reaches 40%–50% of its dry weight, the cell density is up to four times that of the control group cultured alone in N8 medium, and the biomass yield is increased to 7.5 times that of the control group.

[0022] The method provided by this invention simultaneously purifies fishmeal processing wastewater and produces high-value-added microalgae biomass, combining environmental benefits with resource value. The obtained microalgae biomass can be used in the development of feed, food additives, or other high-value-added algae-based products. This method has advantages such as low cost, high efficiency, and environmental friendliness, showing broad application prospects in multiple fields such as food, environmental protection, and energy. Attached Figure Description

[0023] Figure 1 The growth curves of the adaptive Chlorella obtained in Examples 1-3 and Comparative Example 1 on fishmeal processing wastewater culture medium are shown.

[0024] Figure 2 The relative biomass yields of the adaptive Chlorella obtained in Examples 1-3 and Comparative Example 1;

[0025] Figure 3 The change in soluble protein content of the adaptive Chlorella obtained in Example 2;

[0026] Figure 4 The changes in the quantum yield (QY) of Chlorella before and after the experiments in Comparative Examples 2 and 3 are shown.

[0027] Figure 5 The change in specific growth rate of Chlorella before and after Comparative Examples 2 and 3 is shown. Detailed Implementation

[0028] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0029] The wild-type Chlorella used in the following examples is Chlorella from the CCAP algal gene bank, numbered CCAP211 / 52.

[0030] The N8 culture medium used in the following examples consists of the following components: 1000 mg / L KNO3, 740 mg / L KH2PO4, 260 mg / L Na2HPO4·2H2O, 13 mg / L CaCl2·2H2O, 10 mg / L Fe EDTA, 50 mg / L MgSO4·7H2O, and 1 mL / L of trace element stock solution; wherein the trace element stock solution includes 3.58 g / L Al2(SO4)3·18H2O, 12.98 g / L MnCl2·4H2O, 1.83 g / L CuSO4·5H2O, and 3.2 g / L ZnSO4·7H2O.

[0031] Example 1

[0032] This embodiment provides a method for acclimating and adapting Chlorella using condensate wastewater from fishmeal processing wastewater. The condensate wastewater is the condensate produced after cooling the water evaporated during the fishmeal drying process. The specific steps are as follows:

[0033] (1) Wild-type Chlorella was inoculated into the condensate wastewater of fishmeal processing wastewater and kept at a constant temperature of 25℃ and continuous light intensity of 80 μmol m. -2 s -1 The first acclimatization culture was conducted under irradiation conditions. Every 12 days, the algal solution was transferred to fresh condensate wastewater, ensuring that the initial concentration of algae in the culture medium was 0.69 × 10⁻⁶ each time. 7 The algae were cultured at cells / mL for 2 months to obtain the acclimatized algae solution B.

[0034] In this embodiment, the phosphate concentration in the condensate wastewater was 145.81 mg / L and the ammonium salt concentration was 335.35 mg / L.

[0035] (2) Preparation of mixed acclimatization culture medium: Mix the condensate wastewater and N8 culture medium at a volume ratio of 1:1 and then sterilize them, controlling the initial pH value at 6~7.

[0036] (3) Inoculate the acclimatized algae solution B into a sterilized mixed acclimatization medium (so that the initial concentration of algae is 0.69 × 10⁻⁶). 7 Adapted Chlorella was obtained by acclimatizing the algae at 25°C under constant temperature and continuous light for 8 days (cells / mL). This example also measured the changes in soluble protein content of the adapted Chlorella, and the results are as follows: Figure 3 As shown.

[0037] Example 2

[0038] This embodiment provides a method for acclimating and adapting Chlorella using condensate wastewater from fishmeal processing wastewater. The specific steps are as follows:

[0039] (1) Wild-type Chlorella was inoculated into the condensate wastewater of fishmeal processing wastewater and kept at a constant temperature of 25℃ and continuous light intensity of 80 μmol m. -2 s -1 The first acclimatization culture was conducted under irradiation conditions. Every 12 days, the algal solution was transferred to fresh condensate wastewater, ensuring that the initial concentration of algae in the culture medium was 0.69 × 10⁻⁶ each time. 7 The algae were cultured at cells / mL for 2 months to obtain the acclimatized algae solution B.

[0040] In this embodiment, the phosphate concentration in the condensate wastewater was 145.81 mg / L and the ammonium salt concentration was 335.35 mg / L.

[0041] (2) Preparation of mixed acclimatization culture medium: Mix the condensate wastewater and N8 culture medium at a volume ratio of 3:2 (60% condensate wastewater and 40% N8 culture medium) and sterilize them, and control the initial pH value at 6~7.

[0042] (3) Inoculate the acclimatized algae solution B into a sterilized mixed acclimatization medium (so that the initial concentration of algae is 0.69 × 10⁻⁶). 7 Adapted Chlorella was obtained by acclimatizing and culturing the cells / mL at 25℃ under constant temperature and continuous light conditions for 8 days.

[0043] Example 3

[0044] This embodiment provides a method for acclimating and adapting Chlorella using condensate wastewater from fishmeal processing wastewater. The specific steps are as follows:

[0045] (1) Wild-type Chlorella was inoculated into the condensate wastewater of fishmeal processing wastewater and kept at a constant temperature of 25℃ and continuous light intensity of 80 μmol m. -2 s -1 The first acclimatization culture was conducted under irradiation conditions. Every 12 days, the algal solution was transferred to fresh condensate wastewater, ensuring that the initial concentration of algae in the culture medium was 0.69 × 10⁻⁶ each time. 7 The algae were cultured at cells / mL for 2 months to obtain the acclimatized algae solution B.

[0046] In this embodiment, the phosphate concentration in the condensate wastewater was 145.81 mg / L and the ammonium salt concentration was 335.35 mg / L.

[0047] (2) Preparation of mixed acclimatization culture medium: Mix the condensate wastewater and N8 culture medium at a volume ratio of 7:3 (70% condensate wastewater and 30% N8 culture medium) and sterilize them, and control the initial pH value at 6~7.

[0048] (3) Inoculate the acclimatized algae solution B into a sterilized mixed acclimatization medium (so that the initial concentration of algae is 0.69 × 10⁻⁶). 7Adapted Chlorella was obtained by acclimatizing and culturing the cells / mL at 25℃ under constant temperature and continuous light conditions for 8 days.

[0049] Comparative Example 1

[0050] In this comparative example, wild-type Chlorella was cultured alone in N8 medium, as follows: Wild-type Chlorella was inoculated into sterile N8 medium, resulting in an initial algal concentration of 0.69 × 10⁻⁶. 7 / mL, kept at 25℃ under constant temperature and continuous light intensity of 80μmol / mL. -2 s -1 After culturing under irradiation conditions for 8 days, algal solution C was obtained.

[0051] Comparative Example 2

[0052] This comparative example uses washing wastewater from fishmeal processing wastewater to cultivate wild-type Chlorella separately. The washing wastewater is the wastewater generated from cleaning fishmeal processing equipment and the ground. The specific steps are as follows:

[0053] Wild-type Chlorella was inoculated into the washing wastewater of fishmeal processing wastewater and subjected to constant temperature of 25℃ and continuous light intensity of 80 μmol / m². -2 s -1 The first acclimatization culture was conducted under irradiation conditions, with the algal solution transferred to fresh washing wastewater every 6 days, ensuring an initial algal concentration of 0.69 × 10⁻⁶ in each culture. 7 The algae were cultured at cells / mL for two months to obtain acclimatized algae solution A. The changes in the soluble protein content of acclimatized algae solution A were also measured, and the results are as follows: Figure 3 As shown.

[0054] In this embodiment, the phosphate concentration in the washing wastewater was approximately 56.13 mg / L, and the ammonium salt concentration was approximately 157.33 mg / L.

[0055] Comparative Example 3

[0056] In this comparative example, wild-type Chlorella was cultured separately using condensate wastewater from fishmeal processing wastewater. The condensate wastewater is the condensate produced after the water evaporated during the fishmeal drying process is cooled. The specific steps are as follows:

[0057] Wild-type Chlorella was inoculated into the condensate wastewater from fishmeal processing and subjected to constant temperature at 25℃ and continuous light intensity of 80 μmol / m². -2 s -1 The first acclimatization culture was conducted under irradiation conditions. Every 12 days, the algal solution was transferred to fresh condensate wastewater, ensuring that the initial concentration of algae in the culture medium was 0.69 × 10⁻⁶ each time. 7 The algae were cultured at cells / mL for 2 months to obtain the acclimatized algae solution B.

[0058] like Figure 1 and Figure 2 As shown, the adapted Chlorella obtained through the method of this invention exhibits excellent adaptability and growth efficiency in the high-concentration phosphate and ammonium environment of fishmeal wastewater. Compared with wild-type algae cultured in standard N8 medium (Comparative Example 1), the adapted Chlorella cultured in 60% condensate wastewater + 40% N8 medium (Example 2) achieved a cell density four times that of Comparative Example 1, and its maximum biomass yield (g / L / day) increased to 7.5 times that of Comparative Example 1. This indicates that the adapted Chlorella cultured using this invention significantly improves both biomass yield and wastewater treatment efficiency.

[0059] like Figure 4 and Figure 5 As shown, although the algal solution obtained by directly culturing wild-type Chlorella using washing wastewater (Comparative Example 2) or condensation wastewater (Comparative Example 3) can also achieve a certain degree of performance improvement, it is far inferior to the results of Examples 1-3.

[0060] according to Figure 3 It is evident that the soluble protein content of the adapted Chlorella strain acclimated using the method provided by this invention can reach 40%-50% (wt%) of its dry weight, significantly higher than that of the wild-type algae. This indicates that the obtained biomass can be used for the development of feed, food additives, or other high-value-added microalgae-based products.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A method for acclimating and adapting Chlorella using fishmeal processing wastewater, characterized in that, The specific steps are as follows: S1: Wild-type Chlorella was inoculated into fishmeal processing wastewater for a first tolerance culture of at least 2 months to obtain acclimatized algal solution; S2: The acclimatized algae solution is inoculated into a sterilized mixed acclimatization medium for a second tolerance culture for 6 to 10 days to obtain adaptive Chlorella; the mixed acclimatization medium includes the fishmeal processing wastewater and N8 medium mentioned in step S1, wherein the volume ratio of fishmeal processing wastewater in the mixed acclimatization medium is 50% to 70%.

2. The method for acclimatizing and adapting Chlorella using fishmeal processing wastewater according to claim 1, characterized in that, The phosphate content in the fishmeal processing wastewater is 56~145 mg / L, and the ammonium content is 157~335 mg / L.

3. The method for acclimatizing and adapting Chlorella using fishmeal processing wastewater according to claim 1, characterized in that, The fishmeal processing wastewater is the washing wastewater or condensation wastewater from the fishmeal production process.

4. The method for acclimatizing and adapting Chlorella using fishmeal processing wastewater according to claim 1, characterized in that, The wild-type Chlorella used was Chlorella from the CCAP algal strain bank, numbered CCAP211 / 52.

5. The method for acclimatizing and adapting Chlorella using fishmeal processing wastewater according to claim 1, characterized in that, The first tolerance culture described in step S1 is as follows: Wild-type Chlorella is directly inoculated into fishmeal processing wastewater and kept at a constant temperature of 20-25℃ with continuous light intensity of 50-100 μmol / m². -2 s -1 Under suitable conditions, the algae culture was acclimatized and cultured, and the algal solution was transferred to new fishmeal processing wastewater every 6-12 days, with the initial concentration controlled at 0.5-1×10⁻⁶. 7 The algae were cultured at cells / mL for 2-3 months to obtain the acclimatized algae solution.

6. The method for acclimatizing and adapting Chlorella using fishmeal processing wastewater according to claim 1, characterized in that, The N8 culture medium comprises: 1000 mg / L KNO3, 740 mg / L KH2PO4, 260 mg / L Na2HPO4·2H2O, 13 mg / L CaCl2·2H2O, 10 mg / L Fe EDTA, 50 mg / L MgSO4·7H2O, and 1 mL / L of trace element stock solution.

7. The method for acclimatizing and adapting Chlorella using fishmeal processing wastewater according to claim 6, characterized in that, The trace element mother liquor includes 3.58 g / L Al2(SO4)3·18H2O, 12.98 g / L MnCl2·4H2O, 1.83 g / L CuSO4·5H2O and 3.2 g / L ZnSO4·7H2O.

8. The method for acclimatizing and adapting Chlorella using fishmeal processing wastewater according to claim 1, characterized in that, The second tolerance cultivation in step S2 is as follows: the acclimatization algae solution from step S1 is controlled at an initial concentration of 0.5~1×10⁻⁶. 7 Cells / mL were inoculated into sterile mixed acclimatization medium and kept at a constant temperature of 20-25℃ under continuous light intensity of 50-100 μmol / mL. -2 s -1 Adapted Chlorella were obtained through domestication and cultivation under light conditions.

9. An adaptive Chlorella powder obtained using the method for domesticating and adapting Chlorella according to any one of claims 1 to 8, characterized in that, The adaptive Chlorella was freeze-dried to obtain adaptive Chlorella powder.

10. The application of the adaptive Chlorella powder according to claim 9 in fish feed production, characterized in that, The adaptive Chlorella powder described in claim 9 is added to fish feed as algal biomass.

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