A method for enhancing production of chlorella protein by heterotrophic fermentation

Abstract

This invention relates to a method for enhancing Chlorella protein production through heterotrophic fermentation, comprising two stages: cell growth and protein accumulation. During the cell growth stage, a fed-batch culture medium with a low concentration of ammonium nitrogen (high C / N ratio medium) is used as the nitrogen source to achieve high cell density in Chlorella. During protein accumulation, the culture is switched to a fed-batch culture medium with a high concentration of ammonium nitrogen (low C / N ratio medium) to promote protein accumulation in the cells. Simultaneously, acetate solution is fed to counteract the cytotoxicity of high concentrations of ammonium to Chlorella under low C / N ratio induction conditions. This method promotes efficient protein accumulation in the algal cells while maintaining or minimizing cell biomass loss, and even allowing it to continue to grow, ultimately improving the quality (protein content) of Chlorella protein products. This invention is beneficial for reducing the production cost of Chlorella protein and improving the economic viability of the industry.

Description

Technical Field

[0001] This invention belongs to the field of microalgae technology, specifically relating to a method for enhancing the production of Chlorella protein through heterotrophic fermentation. Background Technology

[0002] Microalgae-derived single-celled Chlorella are considered an ideal high-quality protein source due to their rapid growth, high protein content, and balanced amino acid composition. Currently, commercial Chlorella biomass is primarily cultivated in traditional open raceway ponds. This photoautotrophic cultivation method is limited by seasonal and environmental conditions, resulting in low Chlorella biomass concentration, unstable product quality, high production costs, and low biomass productivity. Heterotrophic cultivation using mature and controllable fermentation equipment can achieve stable and efficient Chlorella biomass production, proving to be a more economical and efficient approach. Protein content and protein yield are two important indicators for evaluating Chlorella protein production. The achievable Chlorella protein content under heterotrophic fermentation determines the quality, price, and application areas of the Chlorella protein; commercially available Chlorella products generally require a protein content greater than 50%. Conversely, the protein yield determines the production cost and economic viability of Chlorella protein.

[0003] However, under heterotrophic culture, Chlorella cells typically tend to accumulate high levels of lipids or carbohydrates (>50%), while protein content is low (<40%), which significantly limits the application of heterotrophic Chlorella biomass as a protein source in the food and feed industries. Existing literature indicates that regulating nitrogen nutrient supply during Chlorella heterotrophic culture (increasing nitrogen concentration or decreasing the carbon-to-nitrogen ratio) can increase intracellular protein content. Furthermore, the type of nitrogen source in the culture medium also affects protein content and cell growth; compared to nitrate nitrogen (NO3),... - ) and urea nitrogen, ammonium nitrogen (NH4) of the same molar concentration +Because of its minimal energy consumption during utilization, it is considered more conducive to the accumulation of intracellular proteins in Chlorella. For example, the prior art of Chinese patent application CN115161203B discloses a two-stage culture mode to improve the protein yield of Chlorella under heterotrophic fermentation. This scheme divides the heterotrophic protein production process of Chlorella into two stages: biomass production and induced protein production. In the biomass production stage, ammonium chloride is used as the nitrogen source, and a high carbon-to-nitrogen ratio (C / N = 320-400:1) feed medium is used. When the cells reach the mid-to-late logarithmic growth stage, ammonium chloride or urea is used as the nitrogen source, and the feed medium is switched to a low carbon-to-nitrogen ratio (C / N = 3-12:1) feed medium to induce protein production. Experiments have shown that using ammonium chloride as the nitrogen source under low C / N ratio induction results in a higher protein content at the same time compared to using urea as the nitrogen source. However, the cell biomass concentration is reduced due to the toxic effects of high ammonium on cells under low C / N induction conditions, resulting in a loss of some protein yield. When urea is used instead of ammonium salt during the induction phase, cell concentration does not decrease significantly, which is beneficial for maintaining cell concentration. However, urea as a nitrogen source is less effective than ammonium salt in increasing intracellular protein content in Chlorella. Since protein yield = cell biomass (dry weight) × protein content, the final protein yield is directly related to both cell biomass and protein content. Therefore, it is evident that neither urea nor ammonium can maximize protein yield during the protein production induction phase.

[0004] In order to solve the technical problems such as the decline in cell biomass caused by the use of ammonium nitrogen and the poor effect of urea on increasing cell protein content, the inventors believe that it is necessary to propose an improvement scheme for the existing culture technology for chlorella protein production. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for enhancing the production of Chlorella protein through heterotrophic fermentation. This method mainly involves adding a low concentration of exogenous acetic acid during the stage of inducing intracellular protein accumulation in Chlorella to alleviate the cytotoxicity of high ammonium to Chlorella cells. This achieves the goal of increasing the protein content of Chlorella under high ammonium culture while maintaining or minimizing the decrease in cell biomass, or even allowing it to continue to grow. This solves the problem of high ammonium concentrations hindering cell growth and affecting protein yield in the prior art, and helps to reduce the production cost of Chlorella protein, improve economic efficiency, and provide technical support for promoting the large-scale production of Chlorella as a cheap and high-quality protein source.

[0007] (II) Technical Solution

[0008] In a first aspect, the present invention provides a method for enhancing the production of Chlorella protein through heterotrophic fermentation, comprising: inoculating Chlorella seed liquid into a basic culture medium in a fermenter for fermentation culture; during the entire fermentation process: the culture temperature is 22-30℃; the stirring speed and dissolved oxygen are coupled and controlled during the culture process; the dissolved oxygen is controlled at 5-80%; and the pH of the fermentation culture medium is 6.0-7.0; the fermentation process includes two stages: cell growth and protein accumulation.

[0009] The fermentation conditions for the cell growth stage are as follows: monitor the glucose concentration in the fermentation broth, and when the glucose concentration drops below 5 g / L, start fed-batch culture using a peristaltic pump until the cell dry weight reaches 200 g / L or above. At this point, the cell growth stage ends and the protein accumulation stage begins immediately.

[0010] The fermentation conditions for the protein accumulation stage are as follows: the growth feed medium used in the cell growth stage is switched to the induction feed medium for continued fed-batch culture, while acetate solution is added to the fermenter. During the culture, glucose concentration is monitored in real time, and the feed rate of the induction feed medium is adjusted accordingly to maintain a glucose concentration of 1-20 g / L in the culture medium. Simultaneously, the acetate concentration in the culture medium in the fermenter is monitored, and the feed rate of the acetate solution is adjusted accordingly to maintain an acetate concentration of 1-50 mM. The C / N ratio of the induction feed medium is lower than that of the growth feed medium.

[0011] Sampling is performed every 2-24 hours. When two consecutive sampling tests show that the cell dry weight remains unchanged or begins to decrease, the culture is terminated and the cells are harvested.

[0012] According to a preferred embodiment of the present invention, the basal culture medium is a modified Endo medium, and the growth feed medium and the induction feed medium are both concentrated modified Endo mediums; the carbon source in the basal culture medium is glucose with a concentration of 25-35 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 9-24; the carbon source in the growth feed medium is glucose with a concentration of 720-780 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 320-400:1; the carbon source in the induction feed medium is glucose with a concentration of 720-780 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 3-20:1.

[0013] According to a preferred embodiment of the present invention, the basal culture medium contains 20 g / L glucose and 1.5-4 g / L NH4Cl; the growth feed medium contains 750 g / L glucose and 3.34-4.18 g / L NH4Cl; and the induction feed medium contains 750 g / L glucose and 66.875-445.8 g / L NH4Cl.

[0014] Preferably, during the protein accumulation stage, the acetate concentration in the fermentation broth is controlled at 5-50 mM, more preferably 5-10 mM.

[0015] According to a preferred embodiment of the present invention, the acetate solution is acetic acid, an acetate salt, or a mixture of acetic acid and an acetate salt, wherein the acetate salt is at least one of sodium acetate and potassium acetate; the acetate concentration in the acetate solution is 0.9-1.1 M.

[0016] According to a preferred embodiment of the present invention, the Chlorella is wild-type Chlorella sorokinina or a Chlorella mutant with enhanced protein expression.

[0017] According to a preferred embodiment of the present invention, during the cell growth and protein accumulation stage, the fermentation temperature is maintained at 30°C, and the stirring speed and dissolved oxygen are coupled and controlled, with dissolved oxygen at 20%.

[0018] According to a preferred embodiment of the present invention, during the cell growth and protein accumulation stage, ammonia or 2-4M NaOH solution is used to control the pH of the fermentation culture medium to 6.0-7.0, preferably 6.5±0.2.

[0019] According to a preferred embodiment of the present invention, the seed culture is prepared in a shake flask, and the preparation process is as follows: Under sterile conditions, *Chlorella vulgaris* cells activated by shaking are inoculated into a 1000 mL shake flask at a volume percentage of 2-10%. The culture medium in the shake flask is 300-400 mL, and the culture is carried out in the dark with shaking at a temperature of 22-30°C and a rotation speed of 120-220 rpm for 2-5 days. The carbon source in the shake flask culture medium is glucose at a concentration of 30 g / L, and the nitrogen source is KNO3 at a concentration of 2 g / L.

[0020] In this application, C / N is the molar ratio of C atoms to N atoms in the culture medium.

[0021] (III) Beneficial Effects

[0022] (1) This invention employs heterotrophic fermentation to cultivate Chlorella to increase its protein yield. Ammonium nitrogen is used throughout the cultivation process, which is beneficial for the accumulation of intracellular protein in Chlorella. The fermentation process includes two stages: cell growth and protein accumulation. During the cell growth stage, a high C / N ratio basal medium is used, with a high C / N ratio fed-batch culture medium to increase the cell dry weight in the culture medium to 200 g / L or higher, thus obtaining a high-density cell culture medium. This provides a large biomass base for protein production. During the protein accumulation stage, a low C / N ratio induction fed-batch culture medium is used, with a low concentration of acetate solution added simultaneously. The acetate concentration in the culture medium during the protein accumulation stage is controlled to not exceed 50 mM to counteract the cytotoxicity of high concentrations of ammonium to Chlorella under low C / N ratio induction conditions. This promotes efficient protein accumulation in the algal cells while maintaining or minimizing cell biomass decline, ultimately achieving a significant increase in Chlorella protein yield and the quality of protein-containing Chlorella. This invention helps to reduce the production cost of Chlorella protein, improve its economic efficiency, and provide technical support for promoting the large-scale production of Chlorella as an inexpensive and high-quality protein source.

[0023] (2) This invention is the first to discover that low concentrations of acetic acid (root) can reduce the toxicity of ammonium to Chlorella cells under high ammonium stress, enabling Chlorella cells to significantly increase intracellular protein content under high ammonium conditions while maintaining or minimizing cell biomass decline, and even continuing to grow, ultimately achieving a simultaneous increase in protein content and yield. The solution of this invention is expected to serve as a novel method for mitigating ammonium toxicity and can be applied to other microalgae high ammonium culture processes. Attached Figure Description

[0024] Figure 1 The curves showing the change in dry weight (g / L) of Chlorella during the 0-240h fermentation culture of experimental group 1 and control groups 1-3 are shown.

[0025] Figure 2 The curves show the changes in ammonium concentration (mM) in the culture medium during the 0-240 h of Chlorella fermentation culture in experimental group 1 and control groups 1-3.

[0026] Figure 3 The curves showing the change in protein content (%) of Chlorella during the 0-240h fermentation culture of experimental group 1 and control groups 1-3 are shown.

[0027] Figure 4 The curves show the changes in protein yield (g / L) of Chlorella during the 0-240 h fermentation culture of experimental group 1 and control groups 1-3. Detailed Implementation

[0028] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] This invention provides a method for enhancing the production of Chlorella protein through heterotrophic fermentation, comprising: inoculating Chlorella seed culture into the basic culture medium of a fermenter for fermentation culture; during the entire fermentation process: the culture temperature is 22-30℃; the stirring speed and dissolved oxygen are coupled and controlled during the culture process, the dissolved oxygen is controlled at 5-80%, and the pH of the fermentation culture medium is 6.0-7.0; the fermentation process includes two stages: cell growth and protein accumulation.

[0030] The fermentation conditions for the cell growth stage are as follows: monitor the glucose concentration in the fermentation broth, and when the glucose concentration drops below 5 g / L, start fed-batch culture using a peristaltic pump until the cell dry weight reaches 200 g / L or above. At this point, the cell growth stage ends and the protein accumulation stage begins immediately.

[0031] The fermentation conditions for the protein accumulation stage are as follows: the growth feed medium used in the cell growth stage is switched to the induction feed medium for continued fed-batch culture, while acetate solution is added to the fermenter. During the culture, glucose concentration is monitored in real time, and the feed rate of the induction feed medium is adjusted accordingly to maintain a glucose concentration of 1-20 g / L in the culture medium. Simultaneously, the acetate concentration in the culture medium in the fermenter is monitored, and the feed rate of the acetate solution is adjusted accordingly to maintain an acetate concentration of 1-50 mM. The C / N ratio of the induction feed medium is lower than that of the growth feed medium.

[0032] Sampling is performed every 2-24 hours. When two consecutive sampling tests show that the cell dry weight remains unchanged or begins to decrease, the culture is terminated and the cells are harvested.

[0033] The culture conditions for the cell growth stage can be referenced from existing technologies CN115161203B or CN110195019A. The main purpose of this stage is to obtain a large number of cellular biomass units, providing a biomass basis for subsequently increasing the protein content of Chlorella. The culture conditions for this stage can be appropriately adjusted, for example, according to environmental temperature, climate, Chlorella growth, and initial inoculum concentration. There are no strict requirements, as long as Chlorella cells in the logarithmic growth phase with a dry weight of 200 g / L or higher can be obtained within a short time. This helps to shorten the culture cycle and improve production efficiency. If the initial inoculum is 10% (v / v) and fermentation is carried out according to the culture conditions described in CN115161203B or this invention, the dry weight of Chlorella can generally reach 200 g / L or higher within 120 hours.

[0034] After obtaining a high-density biomass culture medium, fermentation culture for protein accumulation is carried out. The main technical features of this process are: (1) using a culture medium with ammonium nitrogen and a low carbon-to-nitrogen ratio (C / N) for induced fed-batch culture, utilizing the lowest energy consumption during ammonium nitrogen utilization to promote the accumulation of intracellular proteins in Chlorella; (2) during the induced fed-batch culture, a low concentration of acetate is added (controlling the acetate concentration in the culture medium at 1-50 mM) to resist the cytotoxicity of high concentrations of ammonium to Chlorella under low C / N ratio induced culture conditions, promoting efficient protein accumulation in algal cells while maintaining cell biomass without or with minimal decrease, or even continuing to grow, ultimately achieving the technical effect of improving Chlorella protein products and Chlorella quality (protein content). Among them, the acetate solution is acetic acid, acetate salt, or a mixture of acetic acid and acetate salt, wherein the acetate salt is at least one of sodium acetate and potassium acetate; the total acetate concentration in the acetate solution is 0.9-1.1 M.

[0035] The basal culture medium was a modified Endo medium, and both the growth feed medium and the induction feed medium were concentrated modified Endo media. In the basal culture medium, glucose was the carbon source at a concentration of 25-35 g / L, ammonium chloride was the nitrogen source, and the C / N ratio was 9-24. In the growth feed medium, glucose was the carbon source at a concentration of 720-780 g / L, ammonium chloride was the nitrogen source, and the C / N ratio was 320-400:1. In the induction feed medium, glucose was the carbon source at a concentration of 720-780 g / L, ammonium chloride was the nitrogen source, and the C / N ratio was 3-20:1. The acetate ions provided by the acetate solution could also be utilized by Chlorella as a carbon source.

[0036] Preferably, the basal culture medium contains 20 g / L glucose and 1.5-4 g / L NH4Cl; the growth feed medium contains 750 g / L glucose and 3.34-4.18 g / L NH4Cl; and the induction feed medium contains 750 g / L glucose and 66.875-445.8 g / L NH4Cl.

[0037] In addition to carbon and nitrogen sources, the basal culture medium, growth feed medium, and induction feed medium also contain small amounts of inorganic salts, namely KH₂PO₄, MgSO₄, trisodium citrate, FeSO₄, EDTA stock solution, and trace element stock solution. The basal culture medium is a modified Endo medium, and the growth feed medium and induction feed medium are concentrated modified Endo media; therefore, the inorganic salt content is several times that of the basal culture medium, and the concentration factor of the carbon source is roughly consistent. However, those skilled in the art should know that inorganic salts do not have a decisive effect on the growth of Chlorella, and their addition amount and adjustment range are all conventional methods. Therefore, the specific content values ​​of inorganic salts in each culture medium in the examples should not be considered as limiting conditions of the scope of this invention.

[0038] More preferably, the compositions of the basal culture medium, the growth feed medium, and the induction feed medium are as follows:

[0039] Basic culture medium: glucose 20 g / L; NH4Cl 1.5-4 g / L; KH2PO4 1.2 g / L; MgSO4·7H2O 1.2 g / L; trisodium citrate 0.2 g / L; FeSO4 and EDTA stock solution 1 mL / L; trace element stock solution 1 mL / L, adjusted to pH 6.0. The FeSO4 and EDTA stock solution consisted of: FeSO4·7H2O 16 g / L, EDTA 2.1 g / L; the trace element stock solution consisted of: H3BO3 2.86 g / L, ZnSO4·7H2O 0.222 g / L, MnCl2·4H2O 1.81 g / L, NaMoO4 0.021 g / L, CuSO4·5H2O 0.07 g / L.

[0040] The growth feed medium consisted of: glucose 750 g / L; NH4Cl 3.34-4.18 g / L; KH2PO4 60 g / L; MgSO4·7H2O 60 g / L; trisodium citrate 5 g / L; FeSO4 and EDTA stock solution 50 mL / L; and trace element stock solution 50 mL / L. The FeSO4 and EDTA stock solution consisted of: FeSO4·7H2O 16 g / L and EDTA 2.1 g / L. The trace element stock solution consisted of: H3BO3 2.86 g / L, ZnSO4·7H2O 0.222 g / L, MnCl2·4H2O 1.81 g / L, NaMoO4 0.021 g / L, and CuSO4·5H2O 0.07 g / L.

[0041] Induction feeding medium: glucose 750 g / L; NH4Cl 66.875-445.8 g / L; KH2PO4 60 g / L; MgSO4·7H2O 60 g / L; trisodium citrate 5 g / L; FeSO4 and EDTA stock solution 50 mL / L; trace element stock solution 50 mL / L. The FeSO4 and EDTA stock solution consisted of: FeSO4·7H2O 16 g / L, EDTA 2.1 g / L; the trace element stock solution consisted of: H3BO3 2.86 g / L, ZnSO4·7H2O 0.222 g / L, MnCl2·4H2O 1.81 g / L, NaMoO4 0.021 g / L, CuSO4·5H2O 0.07 g / L.

[0042] To illustrate the technical effects of the present invention, the following description is provided in conjunction with specific embodiments and comparative examples. It should be noted that the seed culture preparation methods used in the embodiments and comparative examples are the same, and the preparation process is as follows: Under sterile conditions, *Chlorella vulgaris* cells activated by shaking were inoculated into a 1000 mL shaking flask at a volume percentage of 10%. The culture medium in the shaking flask was 350 mL, and the flask was incubated in the dark with shaking at 30°C and 200 rpm for 4 days. The composition of the shaking flask culture medium was: glucose 30 g / L; KNO3 2 g / L; KH2PO4 1.2 g / L; MgSO4·7H2O 1.2 g / L; trisodium citrate 0.2 g / L; FeSO4 and EDTA stock solution 1 mL / L; trace element stock solution 1 mL / L, adjusted to pH 6.0. The FeSO4 and EDTA mother liquor consisted of: FeSO4·7H2O 16 g / L, EDTA 2.1 g / L; the trace element mother liquor consisted of: H3BO3 2.86 g / L, ZnSO4·7H2O 0.222 g / L, MnCl2·4H2O 1.81 g / L, NaMoO4 0.021 g / L, CuSO4·5H2O 0.07 g / L. The activation process for *Chlorella sorokinica* cells can be found in existing techniques, such as CN110195019A, which involves two-stage seed activation followed by inoculation into shake flasks to prepare the seed culture.

[0043] Example 1

[0044] The Chlorella culture method in this embodiment is divided into two stages: cell growth and protein accumulation. The specific process is as follows: the seed culture of wild-type Chlorella sorokin is inoculated into the basic culture medium of a 7.5L fermenter at 10% (v / v) for fermentation culture. The culture temperature is 30℃ throughout the fermentation process. The stirring speed and dissolved oxygen are coupled and controlled during the culture process, and the dissolved oxygen is controlled at 20%. Ammonia water is used to control the pH of the culture medium at 6.5±0.2 throughout the fermentation period.

[0045] The basal culture medium consisted of: 20 g / L glucose; 2.8 g / L NH4Cl; the contents of other components are as described above.

[0046] During the cell growth phase: Monitor the glucose concentration in the fermentation broth. When the glucose concentration drops below 5 g / L, start fed-batch culture using a peristaltic pump. After 120 h of culture (when the cell dry weight reaches 200.0 g / L), the cell growth phase ends and the protein accumulation phase begins immediately.

[0047] The composition of the growth feed medium is: glucose 750 g / L; NH4Cl 3.4 g / L; the contents of other components are as described above.

[0048] Protein accumulation stage: The growth feed medium from the cell growth stage is switched to the induction feed medium for continued fed-batch culture. At the same time, a 1M acetate solution (acetic acid and sodium acetate molar ratio 1:1) is fed into the fermenter. During the culture, the glucose concentration is monitored in real time, and the feed rate of the induction feed medium is adjusted as needed to maintain the glucose concentration in the culture medium at 1-20 g / L. Simultaneously, the acetate concentration in the culture medium in the fermenter is monitored, and the feed rate of the acetate solution is adjusted as needed to maintain the acetate concentration at 5-10 mM.

[0049] The composition of the induction feeding medium is: glucose 750 g / L; NH4Cl 67.5 g / L; the contents of other components are as described above.

[0050] Samples were taken every 24 hours. When two consecutive samples showed that the cell dry weight remained unchanged or began to decrease, the culture was terminated and the cells were harvested. The total culture period was 240 hours.

[0051] Compare with Example 1

[0052] The *Chlorella* culture process in this comparative example was not divided into stages. The method was as follows: wild-type *Chlorella sorokinense* seed culture was inoculated at 10% (v / v) into the basal medium of a 7.5L fermenter for fermentation. The culture temperature was maintained at 30°C throughout the fermentation process. The stirring speed and dissolved oxygen were coupled and controlled, with dissolved oxygen maintained at 20%. Ammonia was used to maintain the pH of the culture medium at 6.5 ± 0.2 throughout the fermentation period. The glucose concentration in the fermentation medium was monitored during the culture period. When the glucose concentration dropped below 5 g / L, fed-batch culture was initiated using a peristaltic pump and ended after 240 hours. The composition of both the basal medium and the fed-batch culture medium is the same as in Example 1.

[0053] Compare with Example 2

[0054] The Chlorella culture process in this control example is the same as that in Example 1, except that acetate solution is not added during the "protein accumulation stage", and the cell growth stage is cultured for 120 hours, with a total culture period of 240 hours.

[0055] Compare with Example 3

[0056] The Chlorella culture process in this control example was the same as that in Control Example 2, except that 112.5 g / L urea was used as the nitrogen source to prepare the induction feeding medium during the "protein accumulation phase," and fed-batch culture was performed. The cell growth phase lasted for 120 h, with a total culture period of 240 h, and acetate solution was not added during the "protein accumulation phase" as well.

[0057] Example 2

[0058] The Chlorella culture process in this embodiment is the same as in Example 1, except that during the "protein accumulation stage," a 1M acetate solution (acetic acid and sodium acetate molar ratio 1:10) is added to maintain the acetate concentration in the culture medium at 40-50 mM. The cell growth stage is cultured for 120 hours, with a total culture period of 240 hours.

[0059] Example 3

[0060] The Chlorella culture process in this embodiment is the same as in Example 1, except that during the "protein accumulation phase," a 1M acetate solution (acetic acid and sodium acetate molar ratio 10:1) is added to maintain the acetate concentration in the culture medium at 90-100 mM. The cell growth phase is cultured for 120 hours, with a total culture period of 240 hours.

[0061] Table 1 compares the production performance indicators at the end of fermentation of Examples 1-3 and Control Examples 1-3, including cell dry weight (g / L), protein content (%), and protein yield (g / L):

[0062] Table 1: Groups

[0063]

[0064] Curves were plotted for cell dry weight, ammonium concentration, protein content, and protein yield during 240 hours of fermentation culture for Examples 1 and Controls 1-3, as shown in Figure 1. Figure 1-4 As shown.

[0065] From Table 1 and Figure 1-4It can be seen that in Control Example 1, the cell dry weight reached its maximum at the end of fermentation, reaching 240 g / L, but the protein content was low at 36.7%, and the final protein yield was only 88.1 g / L. Although this culture mode can obtain a large amount of cell biomass, the quality of Chlorella is low, the protein content is low, and the economic efficiency is poor. However, Control Example 1 also shows that using a low concentration of ammonium nitrogen as the nitrogen source throughout the entire feeding culture (the C / N ratio of the growth feed medium is 393) as the feeding medium throughout the entire process is beneficial for maintaining cell proliferation and obtaining high-density cell biomass, but it is not conducive to the accumulation of protein in the cells.

[0066] From Table 1 and Figure 1-4 It can be seen that although Control Example 2 was cultured according to two stages—cell growth and protein accumulation—the use of a fed-batch culture medium with a high concentration of ammonium chloride as the nitrogen source (C / N ratio of 19.8) for protein accumulation, coupled with the continuous supply of high-concentration ammonium nitrogen, led to the accumulation of ammonium nitrogen toxicity to Chlorella cells. The cell dry weight decreased by half from 200 g / L at 120 h of fermentation to 120.5 g / L at the culture endpoint of 240 h. This demonstrates that ammonium nitrogen has significant cytotoxicity to Chlorella cells, leading to cell cessation and even partial apoptosis. Although the protein content (%) reached 58.5% at the fermentation endpoint in Control Example 2, the protein yield was only 70.5 g / L. However, Control Example 2 also illustrates that high concentrations of ammonium nitrogen do indeed promote protein accumulation in Chlorella cells.

[0067] From Table 1 and Figure 1-4 It can be seen that Control Example 3 was cultured in two stages: cell growth and protein accumulation. During protein accumulation, a fed-batch culture medium with a high concentration of urea as the nitrogen source (C / N ratio of the induction feed medium was 13.3) was used. At the end of fermentation (240 h), the cell dry weight (g / L) was 196.2 g / L, a decrease of only 3.8 g / L compared to 200 g / L at 120 h, which was not significant. The protein content (%) at the end of fermentation was 50.6%, and the final protein yield was 99.28 g / L. Therefore, using urea instead of ammonium nitrogen during protein accumulation can reduce the toxicity of ammonium to Chlorella cells and thus maintain cell biomass. However, the protein accumulation in the cells was lower than that in Control Example 2 when ammonium nitrogen was used.

[0068] From Table 1 and Figure 1-4As can be seen, in Example 1, the culture was carried out in two stages: cell growth and protein accumulation. During protein accumulation, a fed-batch culture was conducted using a high-concentration ammonium nitrogen as the nitrogen source (C / N ratio of the induction feed medium was 19.8). Simultaneously, acetate was added to the fermenter to maintain the acetate concentration in the culture medium at 5-10 mM to counteract the toxicity of ammonium nitrogen to Chlorella cells. At the end of fermentation (240 h), the cell dry weight was 212.0 g / L, an increase of 12.0 g / L compared to 120 h. The protein content (%) at the end of fermentation was 57.6%, and the final protein yield was 122.1 g / L. Therefore, adding an appropriate amount of acetate during high-concentration ammonium nitrogen induction for protein accumulation can maintain a double increase in cell dry weight and protein content, thereby significantly improving the quality and protein yield of Chlorella and enhancing the economics of Chlorella protein production.

[0069] Depend on Figure 2 As shown, during the 120-240 h culture period of Control Example 1 and Example 1, the concentration of ammonium ions increased almost synchronously. This indicates that, compared with Control Example 1, the ammonium nitrogen itself did not decrease during the protein accumulation culture period in Example 1. It was only that the use of acetate in Example 1 reduced the toxic effect of ammonium nitrogen on Chlorella cells.

[0070] As shown in Table 1, a comparison of Examples 1 and 2-3 reveals that during the induction culture for protein accumulation, the acetate concentration in the fermentation broth needs to be controlled within a low range; otherwise, it will also inhibit cell growth. For example, in Example 3, the acetate concentration in the culture broth during the protein accumulation phase was maintained at 90-100 mM, and the cell dry weight (g / L) at the fermentation endpoint was only 125.4 g / L. Although the protein content in the cells reached 57.8%, the final protein yield was only 72.48 g / L, which was close to that of Control Example 2, but far lower than that of Example 1. Furthermore, in Example 2, the acetate concentration in the culture broth during the protein accumulation phase was maintained at 40-50 mM, and the cell dry weight (g / L) at the fermentation endpoint of 240 h was 195.5 g / L, a decrease of only 4.5 g / L from 200 g / L at 120 h, which was not significant. This indicates that maintaining the acetate concentration in the culture medium at 40-50 mM during the protein accumulation phase can significantly resist the toxic effects of ammonium ions on Chlorella cells. At the end of fermentation, the protein yield of Chlorella was 111.24 g / L, which was higher than that of the urea group in Control Example 3 among all examples and control examples, and second only to Example 1.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for enhancing the production of Chlorella protein through heterotrophic fermentation, characterized in that, It includes: The Chlorella seed liquid is inoculated into the base medium of the fermenter for fermentation culture, and the Chlorella is wild type Chlorella sorokiniana Chlorella sorokiniana ) ， During the whole fermentation process, the culture temperature is 30℃, the stirring speed and the dissolved oxygen are coupled control during the culture process, the dissolved oxygen is controlled at 20%, and the pH of the fermentation culture liquid is controlled at 6.5±0.2 by using ammonia water or 2-4M NaOH solution; the fermentation process includes two stages of cell growth and protein accumulation; The fermentation conditions for the cell growth stage are as follows: monitor the glucose concentration in the fermentation broth, and when the glucose concentration drops below 5 g / L, start fed-batch culture using a peristaltic pump until the cell dry weight reaches above 200 g / L. At this point, the cell growth stage ends and the protein accumulation stage begins immediately. The fermentation conditions for the protein accumulation stage are as follows: the growth feed medium used in the cell growth stage is switched to the induction feed medium for continued fed-batch culture. Simultaneously, an acetate solution (a 1:1 molar ratio of acetic acid to acetate salt) is added to the fermenter. During culture, the glucose concentration is monitored in real-time, and the feed rate of the induction feed medium is adjusted accordingly to maintain a glucose concentration of 1-20 g / L. The acetate concentration in the fermenter is also monitored, and the feed rate of the acetate solution is adjusted accordingly to maintain an acetate concentration of 5-10 mM. The C / N ratio of the induction feed medium is lower than that of the growth feed medium. Sampling was performed every 2-24 hours. When two consecutive sampling tests showed that the cell dry weight remained unchanged or began to decrease, the culture was ended and the cells were harvested. The total culture period was 240 hours. The basal culture medium is a modified Endo medium, in which glucose is the carbon source, ammonium chloride is the nitrogen source, the glucose content is 20 g / L, and the NH4Cl content is 2.8 g / L. Both the growth feed medium and the induction feed medium are concentrated modified Endo medium; the growth feed medium contains 750 g / L glucose and 3.4 g / L NH4Cl; the induction feed medium contains 750 g / L glucose and 67.5 g / L NH4Cl.

2. The method according to claim 1, characterized in that, The acetate is at least one of sodium acetate and potassium acetate; the acetate concentration in the acetate solution is 0.9-1.1 M.

3. The method according to claim 1, characterized in that, The seed culture was prepared in a shake flask as follows: Under sterile conditions, activated Chlorella cells were inoculated into a 1000 mL shake flask at a volume percentage of 2-10%. The culture medium in the shake flask was 300-400 mL. The culture was carried out in the dark with shaking at a temperature of 22-30℃ and a rotation speed of 120-220 rpm for 2-5 days. The carbon source in the shake flask culture medium was glucose at a concentration of 30 g / L, and the nitrogen source was KNO3 at a concentration of 2 g / L.

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