High-density heterotrophic fed-batch culture method of cell wall defect type chlamydomonas reinhardtii

Abstract

The invention relates to the field of heterotrophic culture of cell wall defect type chlamydomonas reinhardtii. In order to culture cell wall defect type chlamydomonas reinhardtii on a large scale, the invention provides a ZET culture medium which does not contain Tris and ammonium chloride, provides a carbon source through acetate, provides a nitrogen source through ammonium, provides a phosphorus source through hydrogen phosphate, and controls pH through ammonia water. The selected components can reduce the salinity of the culture medium, are cheap and easy to obtain, have better culture effect and are widely applied. Meanwhile, the invention provides a heterotrophic fed-batch culture process of the wall-deficient chlamydomonas reinhardtii. The ZET culture medium in a tank is sterilized and then matched with a fed-batch culture medium and ammonia water to realize fed-batch control and pH range control. In the process provided by the invention, the non-wall chlamydomonas reinhardtii is relatively good in adaptability, relatively high in growth speed and excellent in cell dry weight increase condition, and is suitable for large-scale commercial application in the fields of foods and transgenic biological products.

Description

Technical Field

[0001] The invention relates to the field of microalgae biotechnology, in particular to a heterotrophic culture process of cell wall-defective Chlamydomonas reinhardtii. Background Art

[0002] Chlamydomonas reinhardtii is a unicellular eukaryotic green algae belonging to the genus Chlamydomonas in the family Chlamydomonas. Chlamydomonas reinhardtii is an important model organism among microalgae and a natural algae edible by humans. Chlamydomonas reinhardtii can grow under light or dark conditions and is widely distributed in freshwater environments around the world. Wild-type Chlamydomonas reinhardtii cells have a diameter of 7-10 μm and are spherical or elliptical in shape.

[0003] Chlamydomonas reinhardtii is rich in proteins, polysaccharides, vitamins, etc., has high nutritional value, and has been approved as a new food ingredient in my country in 2022. Therefore, it has broad application prospects in the fields of food and health. Chlamydomonas reinhardtii is recognized as one of the most promising cell factory chassis cells in microalgae, and can achieve the synthesis of high-value biological products such as functional proteins, terpenes, vaccines, etc. through genetic technology. Because Chlamydomonas reinhardtii cells contain complex post-translational folding and modification systems, using Chlamydomonas reinhardtii as a chassis cell for exogenous gene expression can ensure the biological activity of the product; in addition, it also has the advantages of low culture cost, good biosafety performance, and easy product extraction and purification.

[0004] Chlamydomonas reinhardtii has many mutants, among which the discovery of cell wall deficient C. reinhardtii is of great significance for the establishment of an efficient transgenic system of Chlamydomonas reinhardtii. Cell wall deficient C. reinhardtii was first isolated by Davies and Plaskitt in 1970 and was used to study the cell wall structure and assembly mechanism of Chlamydomonas reinhardtii. Using cell wall deficient C. reinhardtii as chassis cells, gene expression efficiency is higher, and the product is easier to separate and extract. When used as a food raw material, compared with Chlorella, Spirulina and walled Chlamydomonas reinhardtii with cell walls, cell wall-deficient C. reinhardtii has the advantage of being easier to digest and absorb by the human body.

[0005] At present, the research on the cultivation of cell wall-deficient Chlamydomonas reinhardtii mainly focuses on genetic transformation, and the cultivation of the algae and transformed strains involved is basically carried out in 50-250mL shake flasks. Zedler et al. used TAP medium in a 100L PBR to cultivate cell wall-deficient Chlamydomonas reinhardtii CW15 mixotrophically for 7 days to reach the highest cell dry weight of 0.45g / L, which is the largest scale of cell wall-deficient Chlamydomonas reinhardtii cultivation at present; Freudenberg et al. designed 6XP photoautotrophic medium, at 700μmol / m 2 / s of light, the cell wall-deficient Chlamydomonas reinhardtii CW15 can be cultured in 2L, 6L, and 20L PBR for 9d, 12d, and 21d, respectively, with the highest cell dry weights of 6.5g / L, 5g / L, and 3g / L, respectively, which are the highest cell densities reported so far for the cultivation of cell wall-deficient Chlamydomonas reinhardtii. Obviously, the cultivation level that can be achieved through the current mixotrophic and photoautotrophic culture modes cannot meet the large-scale production of cell wall-deficient Chlamydomonas reinhardtii.

[0006] Heterotrophy is a nutritional method in which microalgae absorb organic carbon sources in the environment under dark conditions to grow and reproduce. It requires fermentation tanks and fermentation technology to support the cultivation by providing organic carbon sources, nitrogen sources, phosphorus sources and other inorganic nutrients. The culture temperature, pH and dissolved oxygen and other environmental conditions in the fermentation tank are controllable, which can keep the microalgae cells in a rapid growth stage for a long time, and can obtain a higher cell density and a faster cell growth rate. In addition, heterotrophic culture also has the advantages of easy scale-up, low heavy metal content, and small footprint. It is currently the most effective means of large-scale cultivation of microalgae.

[0007] Chlamydomonas reinhardtii can use acetate as the sole carbon source for growth and reproduction in a dark environment. The pH changes caused by the consumption of acetate in its heterotrophic culture process may even lead to the death of algal cells. Fed-batch culture refers to a culture method that maintains cell growth by feeding during batch culture, which is of great significance for the heterotrophic culture of Chlamydomonas reinhardtii. Through fed-batch culture based on pH feedback control, the acetic acid in the algal liquid is in a relative balance of consumption and replenishment, and the timely replenishment of other nutrients can enable the algal cells to continue to grow, thereby achieving a higher cell density. Based on this strategy, the laboratory of the inventor of the present application has achieved high-density heterotrophic cultivation of Chlamydomonas reinhardtii CC1690 (with cell walls). The highest cell dry weight of 23.80 g / L was obtained in 5L fermenter after heterotrophic cultivation of Chlamydomonas reinhardtii CC1690 for 250 h, and the highest cell dry weight of 25.44 g / L was obtained in 50L tank after 237 h of cultivation (see Zhang Z, Tan Y, Wang W, et al. Efficient heterotrophic cultivation of Chlamydomonas reinhardtii [J]. Journal of Applied Phycology, 2019, 31: 1545-1554.).

[0008] However, due to the lack of cell walls, the requirements of cell wall-deficient Chlamydomonas reinhardtii for nutrients such as carbon, nitrogen, and phosphorus are quite different from those of cell wall-deficient Chlamydomonas reinhardtii, and it also has special requirements for the culture medium environment. The existing heterotrophic fed-batch culture method of Chlamydomonas reinhardtii is not effective when directly applied to cell wall-deficient Chlamydomonas reinhardtii, and it is difficult to achieve the same culture effect as Chlamydomonas reinhardtii, which needs to be improved urgently. Summary of the invention

[0009] In order to overcome the defects in the prior art, the present invention provides a new culture medium for culturing cell wall-defective Chlamydomonas reinhardtii, which is named "ZET culture medium" and is characterized by:

[0010] The ZET culture medium provides a carbon source through acetate, a nitrogen source through ammonium, and a phosphorus source through hydrogen phosphate, including an acetate concentration of 10-100 mM, an ammonium acetate concentration of 2-15 mM, and a potassium dihydrogen phosphate concentration of 0.5-10 mM. The pH of the ZET culture medium is controlled within the range of 6.0-8.0 through ammonia water; the ZET culture medium does not contain Tris (trishydroxymethylaminomethane), ammonium chloride, and potassium dihydrogen phosphate.

[0011] The cell wall components of Chlamydomonas reinhardtii are mainly glycoproteins. The wall-less Chlamydomonas reinhardtii lacks cell walls, and its demand for nitrogen sources is greatly reduced. Its demand for a suitable culture environment is also different from that of the walled Chlamydomonas reinhardtii. After sterilization in a fermenter, the culture medium containing ammonium chloride (such as TAP culture medium) will volatilize nearly half of the ammonia nitrogen, causing the pH of the culture medium to drop seriously, making it impossible to directly inoculate algae cells; if alkali is added to adjust the pH, the salinity of the culture medium will increase, affecting the growth of algae cells and increasing the risk of bacterial contamination in the fermenter, which makes the traditional TAP culture medium unsuitable for large-scale industrial production.

[0012] The ZET medium of the present invention removes the expensive Tris (trishydroxymethylaminomethane), ammonium chloride, and potassium dihydrogen phosphate in the TAP medium, and uses acetic acid and ammonium acetate (rather than ammonium chloride) as the main carbon source and nitrogen source, and the composition is simpler. The selected components are cheap and easy to obtain, and the culture effect is better. Therefore, this culture medium can be used as a culture medium for shake flask activation and as a tank culture medium, which can greatly reduce the cost of large-scale production. The ZET culture medium does not contain ammonium chloride components, and ammonia water is used to adjust the pH after sterilization, which can reduce the salinity of the culture medium and adjust the pH.

[0013] Preferably, the ZET culture medium further comprises magnesium chloride at a concentration of 0.01-5 mM; calcium chloride at a concentration of 0.02-3 mM; sodium molybdate at a concentration of 0.2-5 μM; and sodium selenite at a concentration of 0.1-10 μM.

[0014] The culture medium has some adjustments to the proportions of carbon, nitrogen, phosphorus, etc., so that the wall-less Chlamydomonas reinhardtii has a good adaptability to the culture medium when cultured on a large scale, which enables it to grow rapidly.

[0015] The present invention also provides a heterotrophic fed-batch culture method of cell wall-deficient Chlamydomonas reinhardtii, characterized in that:

[0016] Step 1: activating or culturing a cell wall-deficient Chlamydomonas reinhardtii strain in a shake flask using the ZET culture medium described in claim 1;

[0017] Step 2: sterilize the ZET medium described in claim 1 and add it into the fermentation tank as the upper tank medium, add ammonia water to adjust the pH to 7.0-8.0 after sterilization, and inoculate the algae species;

[0018] Step 3: The culture solution is subjected to fed-batch culture by adding acidic feed medium and ammonia feed for pH feedback control, so as to control the pH in the fermentation tank to be stable in the range of 7.0-8.0, until the Chlamydomonas reinhardtii ZY-22 cells grow to the stationary phase, and the culture is terminated;

[0019] Wherein, the carbon source and nitrogen source of the acidic feed medium in step 3 are from acetic acid and ammonium acetate, and the carbon-nitrogen ratio is greater than 15.

[0020] The traditional process is mainly aimed at the cultivation of walled Chlamydomonas reinhardtii, and the carbon-nitrogen ratio of the feed is generally around 10. However, since the wall-less Chlamydomonas reinhardtii lacks cell walls, the carbon-nitrogen ratio of the culture medium required for its growth is explored separately. The present invention finds that the carbon-nitrogen ratio of the feed medium of the wall-less Chlamydomonas reinhardtii process needs to be higher, at least greater than or equal to 15 to meet its rapid growth needs, and the effect is better when its carbon-nitrogen ratio is greater than 20.

[0021] Preferably, the feed medium also includes calcium chloride, magnesium sulfate, dipotassium hydrogen phosphate and other trace elements.

[0022] Preferably, the feed medium contains the following components: 1-20 M acetic acid, 0.5-10 M ammonium acetate, 20-30 mM calcium chloride, 20-40 mM magnesium sulfate, 30-70 mM potassium dihydrogen phosphate, and the remaining components are the same as 80 times the aforementioned ZET medium concentrate.

[0023] Preferably, the shake flask seed culture conditions in step 1 are: culture temperature is 20-35°C; light intensity is 20-240 μmol / m 2 / s; shaking table speed is 50-200rpm.

[0024] Preferably, the inoculation amount of the algae species in step 2 is 5%-30%.

[0025] Preferably, the specific method for feeding the feed medium in step 3 is: using pH feedback control for feeding, initially maintaining the pH at 7.9±0.1 for feeding, and then feeding at fixed intervals with a pH value range of 0.1-0.2, until the Chlamydomonas reinhardtii ZY-22 cells grow to a stationary phase and the culture is terminated; the pH of the culture process is always maintained in the range of 7.0-8.0.

[0026] Preferably, the specific method of feeding with ammonia water in step 3 is: when the acidic feed is too much and the pH drops beyond the control threshold, the addition of acidic feed medium is stopped, and after the pH is raised by adding ammonia water, the pH feedback set value of the original feeding program is restored.

[0027] Preferably, a defoaming agent is also added into the fermentation tank in step 3.

[0028] Beneficial Effects

[0029] The present invention designs a new culture medium (named as "ZET culture medium") for culturing cell wall defective Chlamydomonas reinhardtii ZY-22 and a heterotrophic fed-batch culture process matched therewith.

[0030] The ZET medium of the present invention removes the expensive Tris (trishydroxymethylaminomethane), ammonium chloride, and potassium dihydrogen phosphate in the traditional TAP medium, and uses acetic acid and ammonium acetate (rather than ammonium chloride) as the main carbon source and nitrogen source. While the ingredients are simplified, the culture effect is better. It can be used as a medium for shake flask activation and as a tank culture medium. The ZET medium does not contain ammonium chloride. After sterilization, ammonia water is used to adjust the pH, which can reduce the salinity of the culture medium while adjusting the pH. The selected ingredients of the ZET medium are cheap and easy to obtain, which reduces its cost by 97% compared with the traditional TAP medium, and can significantly reduce the cost of large-scale production of cell wall-deficient Chlamydomonas reinhardtii.

[0031] In the heterotrophic fed-batch culture process of pH feedback control involved in the present invention, the upper tank ZET culture medium is matched with feed culture medium and ammonia water after sterilization to realize feed control and pH range control. In this process, Chlamydomonas reinhardtii ZY-22 has good adaptability, fast growth rate, and excellent cell dry weight accumulation (in the case of 50L high-density culture, it reaches the highest cell dry weight of 30.2g / L in 215h, exceeding the wall-type Chlamydomonas reinhardtii CC1690, which reaches the highest cell dry weight of 25.44g / L in 237h). It is worth noting that the growth rate and the highest cell dry weight of Chlamydomonas reinhardtii ZY-22 in the technical solution of the present invention are greatly improved as the fermentation tank is enlarged and the oxygen supply is enhanced. Under the large-capacity culture environment of 500L and 10000L, its growth rate and cell dry weight are doubled, proving that the culture medium and culture scheme involved in the present invention are suitable for the production of large-scale cell wall defective Chlamydomonas reinhardtii.

[0032] In summary, the technical solution of the present invention realizes the high-density heterotrophic fed-batch culture of cell wall-deficient Chlamydomonas reinhardtii for the first time, and can obtain a higher cell density while controlling a relatively low cost, providing a new technical means for its commercial application in the field of food and genetically modified biological products. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1This is a process flow chart of the heterotrophic culture production process of the cell wall-deficient Chlamydomonas reinhardtii ZY-22 of the present invention;

[0034] Figure 2 : Comparison of heterotrophic growth of Chlamydomonas reinhardtii ZY-22 using TAP medium and ZET medium during activation in 500 mL shake flasks;

[0035] Figure 3 This is the growth curve of Chlamydomonas reinhardtii ZY-22 during heterotrophic fed-batch culture in a 5L tank;

[0036] Figure 4 The growth status of Chlamydomonas reinhardtii ZY-22 during heterotrophic fed-batch culture in a 50L tank;

[0037] Figure 5 This is the growth status of Chlamydomonas reinhardtii ZY-22 during the heterotrophic fed-batch culture process in a 500L tank.

[0038] Figure 6 This is the growth status of Chlamydomonas reinhardtii ZY-22 during the heterotrophic fed-batch culture process in a 10,000L tank. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with specific examples. Reagents and operations not mentioned in the examples are carried out according to conventional operations in the art:

[0040] The technical solution adopted by the present invention is: Chlamydomonas reinhardtii ZY-22 is a cell wall defective algae strain, provided and preserved by Yunnan Baoshan Zeyuan Algae Health Technology Co., Ltd. Yunnan Baoshan Zeyuan Algae Health Technology Co., Ltd. is a large-scale microalgae heterotrophic culture pilot base, which has 50L-50T heterotrophic bioreactors of various sizes and corresponding supporting equipment. At present, the base has achieved large-scale production of Chlorella and Haematococcus pluvialis, and has various conditions required for heterotrophic production of Chlamydomonas reinhardtii.

[0041] 1. ZET medium, feed medium and feed process:

[0042] (1) Configuration of ZET medium: Acetate and ammonium provide carbon and nitrogen sources respectively, with acetate concentration of 10-100 mM and ammonium acetate concentration of 2-15 mM; dimethyl phosphate is used as phosphorus source, with a concentration of 0.5-10 mM; magnesium chloride concentration of 0.01-5 mM; calcium chloride concentration of 0.02-3 mM; sodium molybdate concentration of 0.2-5 μM; sodium selenite concentration of 0.1-10 μM. The initial pH of the medium is adjusted to 6.0-8.0 by supplementing with ammonia water.

[0043] (2) Preparation of feed medium: The feed medium is an acidic medium containing the following ingredients: 1-20 M acetic acid, 0.5-10 M ammonium acetate, 20-30 mM calcium chloride, 20-40 mM magnesium sulfate, 30-70 mM potassium dihydrogen phosphate, and the remaining ingredients are the same as 80 times ZET medium concentrate. Feed medium is added to the fermenter during heterotrophic fed-batch culture to supplement nutrients and control the pH of the culture solution to 7.0-7.9.

[0044] Example 1: Shake flask culture activation

[0045] The examples in this section investigate the effects of traditional TAP medium and ZET medium on the growth of Chlamydomonas reinhardtii ZY-22 algae species required for tank cultivation under heterotrophic culture.

[0046] The above-mentioned Chlamydomonas reinhardtii ZY-22 is used as a seed, and the algae species required for tank loading are prepared by shaking flask culture, and the shaking flask culture is carried out in ZET culture medium. The specific method is as follows: the cell wall defective Chlamydomonas reinhardtii ZY-22 is inoculated into a 500mL shaking flask, and the formula of the ZET culture medium used in the shaking flask is: acetic acid concentration is 30mM, ammonium acetate concentration is 5mM, potassium hydrogen phosphate concentration is 2mM, magnesium chloride concentration is 2mM, calcium chloride concentration is 0.05mM, sodium molybdate concentration is 0.5μM, sodium selenite concentration is 0.8μM, and the initial pH of the culture medium is adjusted to 7.8 by adding ammonia water. The liquid volume of the 500mL shaking flask is 200mL, and the inoculation amount is 10%; the shaking table culture conditions are temperature 24°C, light 60μmol / m 2 / s, speed 150rpm.

[0047] The shake flask culture method for the control group was the same as above except that the culture medium used was TAP medium.

[0048] The comparison results of the two are as follows Figure 2 The experimental results showed that when Chlamydomonas reinhardtii ZY-22 was cultured heterotrophically using ZET medium, the cell dry weight reached a maximum value of 0.610 g / L at 60 h of culture, which was significantly higher than the 0.415 g / L achieved using TAP medium.

[0049] Example 2: 5L fermentation tank culture process and culture conditions

[0050] The examples in this section are about the establishment of a heterotrophic fed-batch culture process for cell wall-deficient Chlamydomonas reinhardtii ZY-22 in a 5L fermenter.

[0051] (1) Inoculation of algae in fermentation tank

[0052] Chlamydomonas reinhardtii ZY-22 was inoculated into a 5 L fermenter at an inoculation rate of 10%, and the ZET medium used in the upper tank culture medium was consistent with the formula described in Example 1. The initial pH of the culture medium was adjusted to 7.8 by supplementing ammonia water.

[0053] (2) Specific technology of fed-batch culture process

[0054] The composition of the feed medium used in the culture process after tank loading is as follows: 6.8M acetic acid, 0.6M ammonium acetate, 0.03M potassium hydrogen phosphate, 0.02M calcium chloride, 0.02M magnesium sulfate, and 80 times the mother solution of trace elements of the ZET medium. By adding the feed medium, the pH of the culture solution in the fermenter is controlled within the range of 7.0-7.9 by adopting the aforementioned feeding process. At the same time, the pressure of the fermenter is 0.06Mpa, the culture temperature is 24°C, the dissolved oxygen is 15%, the stirring speed is 150rpm, and the gas in the fermenter is air, which is maintained until the entire heterotrophic culture is completed.

[0055] The feeding process of the feed medium in the above-mentioned culture process is to feed the culture medium by pH feedback control. The specific steps are as follows: during the culture process, set the culture time to 0-48h, feed the culture medium according to the pH maintained at 7.9±0.1; during the culture time to 48-96h, feed the culture medium according to the pH maintained at 7.6±0.1; during the culture time to 96-144h, feed the culture medium according to the pH maintained at 7.3±0.1; after the culture time to 144h, feed the culture medium according to the pH maintained at 7.0±0.1 until the Chlamydomonas reinhardtii ZY-22 cells grow to the stationary phase, and the culture ends.

[0056] The fermentation tank is connected to a feed containing filtered and sterilized ammonia water. On the one hand, after the culture medium in the upper tank is sterilized, the ammonia water can regulate its initial pH value and provide a nitrogen source; on the other hand, during the culture process, the pH feedback control may be unstable. When too much feed medium is added, it will be too acidic, which can easily cause the death of algae. At this time, the ammonia water feed also plays a balancing role. While providing a nitrogen source, it regulates the pH in a dynamic balance. The addition method during the culture process is: stop the addition of acidic feed medium, increase the pH by adding ammonia water, and then restore the pH feedback setting value of the original feed.

[0057] A defoamer can be added during the culture process, and the defoamer is added by adding it to the feed medium or by feeding it separately. The defoamer is a modified polyether fatty acid ester emulsion, and the amount of the defoamer added to the feed medium is 20-50mL / L, and the amount of the defoamer added to the feed medium separately is 0.3-1% of the total volume of the culture medium each time.

[0058] (3) Termination of fermentation, separation and drying

[0059] When the fermentation tank is cultured to a dry weight of 20-100 g / L, the fermentation is terminated, and the resulting fermentation liquid is centrifuged through a bag centrifuge or a disc centrifuge, and then spray-dried to obtain algae powder. The obtained algae powder has a protein content of more than 30% and a crude polysaccharide content of more than 10%, which can meet the requirements of being a new food raw material.

[0060] The bag centrifuge uses a 1000-3000 mesh bag with a frequency of 10-40HZ to collect the solid product of Chlamydomonas reinhardtii through repeated centrifugation. The disc centrifuge has a slag discharge cycle of 120-300s, a slag discharge time of 0.1-0.4s, and a feed volume of 1-2m 3 The protein content of algae powder was determined by the Kjeldahl method, and the crude polysaccharide content was determined by the phenol-sulfuric acid method.

[0061] Cultivation status: The growth of Chlamydomonas reinhardtii ZY-22 in a 5L fermenter heterotrophic fed-batch culture process is shown in Figure 2. Figure 3 As shown. The experimental results show that Chlamydomonas reinhardtii ZY-22 reached a maximum cell dry weight of 22.1g / L and a growth rate of 94.6mg / L / h after 233h of culture in a 5L tank heterotrophic fed-batch culture. The algal cells obtained by the culture process were tested for nutrition, and the protein content was 39.0% and the crude polysaccharide content was 17.3%, which can meet the standards of Chlamydomonas reinhardtii as a new food raw material (standards for new food raw materials: protein content ≥30%, crude polysaccharide content ≥10%).

[0062] Example 3: 50L fermentation tank culture process and culture conditions

[0063] The example in this section is a heterotrophic fed-batch culture process of cell wall-deficient Chlamydomonas reinhardtii ZY-22 in a 50 L fermenter.

[0064] The Chlamydomonas reinhardtii ZY-22 algae species used in the 50L fermenter were 5L shake flask seeds cultured heterotrophically, with an inoculation amount of 10%. Increasing the initial algae density can shorten the lag period by about 18 hours (i.e., reaching the maximum density 18 hours earlier). The upper tank culture medium of the 50L fermenter is a ZET medium without ammonium chloride, which is consistent with the formula described in Example 1. After the upper tank culture medium is sterilized, the initial pH of the culture medium is adjusted to 8.0 by adding filtered and sterilized ammonia water.

[0065] The composition of the feed medium, the feeding process of the feed medium and ammonia water, the addition of the defoaming agent, and the separation and drying techniques are consistent with the techniques of the 5 L fermentation culture in Example 2.

[0066] Cultivation status: The growth of Chlamydomonas reinhardtii ZY-22 in a 50L fermenter heterotrophic fed-batch culture process is shown in Figure 2. Figure 4 The experimental results show that Chlamydomonas reinhardtii ZY-22 reaches a maximum cell dry weight of 30.2 g / L and a growth rate of 140 mg / L / h after 215 h of heterotrophic fed-batch culture in a 50L tank. Nutritional testing of the algae cells obtained by the culture process showed that the protein content was 34.7% and the crude polysaccharide content was 19.1%, which can meet the standards for new food raw materials.

[0067] Example 4: 500L fermentation tank culture process and culture conditions

[0068] This example is a heterotrophic fed-batch culture process of cell wall-deficient Chlamydomonas reinhardtii ZY-22 in a 500 L fermenter.

[0069] The algae species used in the 500L fermentation tank were the algae species used in the 50L tank culture for 160 hours, and the inoculation amount was 10%. The upper tank culture medium of the 500L fermentation tank was a ZET culture medium without ammonium chloride. After sterilization, the initial pH of the culture medium was adjusted to 8.0 by adding filtered and sterilized ammonia water. The ingredients of the feed medium, the feed process of the feed medium and ammonia water, the addition of the defoaming agent, and the separation and drying techniques were consistent with those of the 5L fermentation culture in Example 2.

[0070] Cultivation status: The growth of Chlamydomonas reinhardtii ZY-22 in a 500L fermenter heterotrophic fed-batch culture process is shown in Figure 2. Figure 5 As shown in the figure, after 144 hours of cultivation in a 500L tank, Chlamydomonas reinhardtii ZY-22 reached a maximum cell dry weight of 60.2 g / L and a growth rate of 403 mg / L / h. The obtained algal cells had a protein content of 33.7% and a crude polysaccharide content of 18.1%, which could meet the standards for new food raw materials.

[0071] Example 5: 10000L fermentation tank culture process and culture conditions

[0072] This example is a heterotrophic fed-batch culture process of cell wall-deficient Chlamydomonas reinhardtii ZY-22 in a 10,000 L fermenter.

[0073] The algae species used in the 10000L fermentation tank were the algae species used in the 500L tank culture for 120 hours, and the inoculation amount was 5%. The upper tank culture medium of the 10000L fermentation tank was a ZET culture medium without ammonium chloride. After sterilization, the initial pH of the culture medium was adjusted to 8.0 by adding filtered and sterilized ammonia water. The ingredients of the feed medium, the feed process of the feed medium and ammonia water, the addition of the defoaming agent, and the separation and drying techniques were consistent with those of the 5L fermentation culture in Example 2.

[0074] Cultivation status: The growth of Chlamydomonas reinhardtii ZY-22 in a 10000L fermenter heterotrophic fed-batch culture process is shown in Figure 2. Figure 6 As shown in the figure, after 168 hours of cultivation in a 10,000L tank, Chlamydomonas reinhardtii ZY-22 reached a maximum cell dry weight of 108.5 g / L and a growth rate of 634 mg / L / h. The obtained algal cells had a protein content of 35.7% and a crude polysaccharide content of 18.5%, which can meet the standards for new food raw materials.

[0075] The invention designs a ZET culture medium and a heterotrophic fed-batch culture process matched therewith for the culture of cell wall defective Chlamydomonas reinhardtii ZY-22.

[0076] Under this process, Chlamydomonas reinhardtii ZY-22 has good adaptability, fast growth rate, and excellent dry weight accumulation. Its 50L cell dry weight data (30.2g / L) has exceeded the highest cell dry weight (25.44g / L) obtained by high-density heterotrophic culture of Chlamydomonas reinhardtii CC1690 in 50L mentioned in the background technology, and the time to reach the highest dry weight is also shorter. In particular, in the technical solution of the present invention, the growth rate and the highest cell dry weight of Chlamydomonas reinhardtii ZY-22 increase significantly with the increase of the fermentation tank and the increase of oxygen supply, so that the growth rate and cell dry weight are doubled in a 10000L large-capacity culture environment (refer to the data comparison of each culture system in the table below), reflecting that the culture medium and culture scheme involved in the present invention are suitable for the production of large-scale cell wall-deficient Chlamydomonas reinhardtii.

[0077]

[0078]

[0079] At the same time, since the culture medium components used in the present invention are all low-priced inorganic salts except acetic acid, the cost of ZET culture medium is reduced by 97% compared with that of TAP culture medium, which can significantly reduce the cost of large-scale production of cell wall-deficient Chlamydomonas reinhardtii.

[0080] In summary, the technical solution of the present invention realizes the high-density heterotrophic fluid-batch culture of cell wall-defective Chlamydomonas reinhardtii for the first time, while controlling the low cost and obtaining a higher cell density, providing a new technical means for its commercial application in the field of food and genetically modified biological products.

[0081] Although the technical solutions of the present invention have been described and listed in detail, it should be understood that it is obvious to those skilled in the art to make modifications to the above embodiments or adopt equivalent alternatives. These modifications or improvements made without departing from the spirit of the present invention are within the scope of protection required by the present invention.

Claims

1. A ZET medium for culturing cell wall-deficient Chlamydomonas reinhardtii, characterized in that: The ZET culture medium provides a carbon source through acetate, a nitrogen source through ammonium, and a phosphorus source through hydrogen phosphate, including an acetate concentration of 10-100 mM, an ammonium acetate concentration of 2-15 mM, and a dipotassium hydrogen phosphate concentration of 0.5-10 mM. The pH of the ZET culture medium is controlled within the range of 6.0-8.0 through ammonia water; the ZET culture medium does not contain Tris (trishydroxymethylaminomethane) and ammonium chloride.

2. A ZET medium according to claim 1, characterized in that: The ZET culture medium also includes magnesium chloride at a concentration of 0.01-5 mM; calcium chloride at a concentration of 0.02-3 mM; sodium molybdate at a concentration of 0.2-5 μM; and sodium selenite at a concentration of 0.1-10 μM.

3. A heterotrophic fed-batch culture method for cell wall-deficient Chlamydomonas reinhardtii, characterized in that: Step 1: activating or culturing a cell wall-deficient Chlamydomonas reinhardtii strain in a shake flask using the ZET culture medium described in claim 1; Step 2: sterilize the ZET medium described in claim 1 and add it into the fermentation tank as the upper tank medium, add ammonia water to adjust the pH to 7.0-8.0 after sterilization, and inoculate the algae species; Step 3: The culture solution is subjected to fed-batch culture by adding acidic feed medium and ammonia feed for pH feedback control, so as to control the pH in the fermentation tank to be stable in the range of 7.0-8.0, until the Chlamydomonas reinhardtii ZY-22 cells grow to the stationary phase, and the culture is terminated; Wherein, the carbon source and nitrogen source of the acidic feed medium in step 3 are from acetic acid and ammonium acetate, and the carbon-nitrogen ratio is greater than 15.

4. The heterotrophic fed-batch culture method of a cell wall-deficient Chlamydomonas reinhardtii according to claim 3, characterized in that: The feed medium also includes calcium chloride, magnesium sulfate, dipotassium hydrogen phosphate and other trace elements.

5. The heterotrophic fed-batch culture method of a cell wall-deficient Chlamydomonas reinhardtii according to claim 4, characterized in that: The feed medium contains the following components: 1-20M acetic acid, 0.5-10M ammonium acetate, 20-30mM calcium chloride, 20-40mM magnesium sulfate, 30-70mM dipotassium hydrogen phosphate, and the remaining components are 80 times the concentrated ZET medium according to claim 2.

6. The heterotrophic fed-batch culture method of cell wall-deficient Chlamydomonas reinhardtii according to claim 3, characterized in that: The conditions for the shake flask seed culture in step 1 are: the culture temperature is 20-35°C; the light intensity is 20-240 μmol / m 2 / s; shaking table speed is 50-200rpm.

7. The heterotrophic fed-batch culture method for culturing cell wall-deficient Chlamydomonas reinhardtii according to claim 3, characterized in that: The inoculation amount of the algae species in step 2 is 5%-30%.

8. The heterotrophic fed-batch culture method of cell wall-deficient Chlamydomonas reinhardtii according to claim 3, characterized in that: The specific method for feeding the feed medium in step 3 is: using pH feedback control for feeding, initially maintaining the pH at 7.9±0.1 for feeding, and then feeding at a fixed time interval, adjusting the pH value range by 0.1-0.2 until the Chlamydomonas reinhardtii ZY-22 cells grow to the stationary phase and the culture is terminated; the pH of the culture process is always maintained in the range of 7.0-8.

0.

9. The heterotrophic fed-batch culture method of cell wall-deficient Chlamydomonas reinhardtii according to claim 3, characterized in that: The specific method of feeding with ammonia water in step 3 is: when the acidic feed is too much and the pH of the culture medium drops beyond the control threshold, the addition of acidic feed medium is stopped, and after the pH is raised by adding ammonia water, the pH feedback set value of the original feeding program is restored.

10. The heterotrophic fed-batch culture method for culturing cell wall-deficient Chlamydomonas reinhardtii according to claim 3, characterized in that: In step 3, a defoamer is also added into the fermentation tank.

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