Coupling fermentation method for synthesizing single-cell protein from methane and carbon dioxide
By domesticating aerobic methanogenic bacteria and microalgae through a coupled fermentation method, the problem of low gas-liquid mass transfer rate of methane and carbon dioxide was solved, and high-yield preparation of single-cell protein was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHAANXI DELIANGYUAN BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-02-02
- Publication Date
- 2026-05-08
AI Technical Summary
In existing technologies, the low gas-liquid mass transfer rates of methane and carbon dioxide limit the conversion of carbon sources by microorganisms and affect the production of single-cell proteins.
High-yield single-cell protein was prepared by mixing aerobic methanogenic bacteria and microalgae in a special culture medium, introducing a mixed gas for light fermentation, followed by centrifugation and vacuum drying.
It increases the yield of single-cell proteins, effectively utilizes greenhouse gases as a carbon source, and solves the problem of limited gas-liquid mass transfer rate.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of synthetic biology and fermentation engineering technology, specifically relating to a coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide. Background Technology
[0002] Greenhouse gases are gases that absorb and emit infrared radiation, primarily including carbon dioxide, methane, nitrous oxide, and water vapor. The main sources of greenhouse gases can be divided into natural and anthropogenic sources. Natural sources include volcanic eruptions and the decomposition of organic matter in soil and oceans; while anthropogenic sources mainly come from the combustion of fossil fuels, industrial production, agricultural activities, and land-use changes. The combustion of fossil fuels is the largest source of carbon dioxide, while agriculture is a significant source of methane and nitrous oxide. The increase in greenhouse gases has led to global climate change, resulting in more frequent extreme weather events such as heat waves, droughts, and floods, severely impacting human production and lives. Furthermore, global warming causes glacier melting and sea-level rise, threatening the safety of coastal areas. In addition, the imbalance of ecosystems puts many species at risk of extinction, severely threatening biodiversity. Therefore, reducing greenhouse gas emissions and promoting low-carbon development has become an important consensus. With breakthroughs in modern biotechnology and the promotion of energy conservation and emission reduction concepts, greenhouse gases have become an important carbon source that can be utilized by microorganisms.
[0003] Single-cell protein (SCP) is produced through fermentation based on microorganisms (such as bacteria, yeast, and fungi). Rich in protein, amino acids, vitamins, and minerals, SCP has high nutritional value and is widely used in the feed and food industries. With the increasing global demand for protein, SCP has gained attention as an emerging protein source. SCP production mainly involves three stages: cultivation, fermentation, and harvesting. First, suitable microbial strains are selected and cultivated to improve their growth rate and protein synthesis capacity. Second, fermentation technology is used, utilizing greenhouse gases as a carbon source, and through a series of biochemical reactions, microbial growth and protein accumulation are promoted. Finally, SCP is harvested through processes such as centrifugation and drying. Using greenhouse gases as a carbon source and fermenting with microorganisms not only effectively solves the pollution problem caused by greenhouse gas emissions but also creates high-value SCP, showing significant development potential. However, because CH4 and CO2 gases have lower solubility in water compared to glycosyl carbon sources, the gas-liquid mass transfer rate severely limits the conversion of carbon source gases by microorganisms, thus affecting SCP production.
[0004] Therefore, it is of great significance to discover how to use methane and carbon dioxide to ferment and produce high-yield single-cell proteins. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention solves the technical problems mentioned in the background by inoculating domesticated aerobic methanogenic bacteria and domesticated microalgae into a specially formulated culture medium for fermentation, followed by centrifugation and vacuum drying to obtain single-cell protein. Specifically, the technical solution of this invention includes the following steps: A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, the coupled fermentation method comprising the following steps: Domesticated aerobic methanogenic bacteria and domesticated microalgae were inoculated into a special culture medium and mixed. Then, a vacuum was drawn, and the first mixed gas was introduced and fermented under light to obtain fermentation broth. The fermentation broth was then centrifuged and vacuum dried to obtain single-cell protein.
[0006] Furthermore, the method for preparing the domesticated aerobic methanogenic bacteria includes the following steps: Aerobic methanotrophic bacteria and acclimatization culture medium were mixed at a weight ratio of 1:9~10, and then a second mixed gas was introduced and acclimatized in a temperature environment of 26℃~28℃ to obtain acclimatized aerobic methanotrophic bacteria. The method for preparing the acclimatization culture medium includes the following steps: The basic nutrient solution, iron-fortified nutrient solution, trace element mixture and nitrogen source solution were mixed in a volume ratio of 10~12:2~4:2~4:1 and deionized water was added to bring the volume to 1L. The mixture was then autoclaved at 121℃ for 20 minutes to obtain the acclimatization culture medium.
[0007] Furthermore, the concentration of the aerobic methanogenic bacteria is 3.5 × 10⁻⁶. 8 Methylomonas sp. (number of bacteria per mL)
[0008] Furthermore, the basic nutrient solution is obtained by mixing magnesium sulfate monohydrate and calcium chloride in a weight ratio of 14~15:2~3 and then bringing the volume to 1L.
[0009] Furthermore, the iron-fortified solution is an aqueous solution of sodium iron ethylenediaminetetraacetate with a mass concentration of 4 g / L to 4.5 g / L.
[0010] Furthermore, the trace element mixture is obtained by mixing boric acid, zinc sulfate monohydrate, copper sulfate pentahydrate and manganese chloride tetrahydrate in a weight ratio of 0.2~0.3:0.09~0.1:0.01~0.011:0.03~0.04 and then bringing the volume to 1L.
[0011] Furthermore, the nitrogen source solution is an aqueous solution of sodium nitrate with a mass concentration of 190 g / L to 195 g / L.
[0012] Furthermore, the second mixed gas is composed of methane and oxygen in a volume ratio of 3:7.
[0013] Furthermore, the method for preparing the domesticated microalgae includes the following steps: Microalgae and SE medium were mixed at a weight ratio of 1:15-16. Then, in an environment of 25°C, an acclimatization cycle was performed with 10 hours of light treatment and 14 hours of shading treatment. First, the microalgae were acclimatized for 1-2 days in a light intensity of 1000 Lux, then for 1-2 days in a light intensity of 1300 Lux, and finally for 1-2 days in a light intensity of 1500 Lux to obtain acclimatized microalgae.
[0014] Furthermore, the microalgae is Chlorella pyrenoidosa powder.
[0015] Furthermore, the method for preparing the special culture medium includes the following steps: 4g~4.6g ammonium chloride, 4.4g~5.0g dipotassium hydrogen phosphate, 2.1g~2.9g potassium dihydrogen phosphate, 9.4g~9.7g potassium nitrate, 2.6g~2.7g sodium chloride, 6.5g~6.6g disodium hydrogen phosphate, 0.2g~0.22g calcium chloride, 4.1g~4.3g magnesium sulfate heptahydrate, 0.021g~0.023g ferric chloride, 0.03g~0.031g ferric sulfate heptahydrate, and 0.08g~0.09g ethylenediaminetetraacetic acid were mixed with water and stirred until the volume was adjusted to 1L to obtain the first mixture. 0.1g~0.2g manganese sulfate monohydrate, 0.4g~0.41g zinc sulfate heptahydrate, 0.01g~0.02g copper sulfate pentahydrate and 0.08g~0.1g sodium molybdate dihydrate were added to water, mixed and stirred, and the volume was adjusted to 1L to obtain the second mixture. Take 80-90 mL of the first mixture and 10 mL of the second mixture, mix them, and bring the volume to 1 L. Then, autoclave at 121 °C for 20 min to obtain the special culture medium.
[0016] Furthermore, the weight ratio of the domesticated aerobic methanogenic bacteria: domesticated microalgae: specially prepared culture medium is 1:1:10~12.
[0017] Furthermore, the first mixed gas is composed of methane, oxygen, and carbon dioxide in a volume ratio of 2:1:2.
[0018] Furthermore, the conditions for the light-induced fermentation include a fermentation temperature of 30°C, a light intensity of 2000 Lux, and a fermentation time of 7 days.
[0019] Furthermore, the centrifugal separation conditions include a centrifugal speed of 7000 r / min to 8000 r / min, a centrifugation time of 10 min, and a centrifugation temperature of 4 °C.
[0020] Furthermore, the vacuum drying temperature is 40°C and the vacuum degree is 0.08 MPa.
[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a pre-acclimation method, pre-acclimating aerobic methanotrophic bacteria that utilize methane and microalgae that utilize carbon dioxide, respectively, to obtain acclimated aerobic methanotrophic bacteria and acclimated microalgae. Pre-acclimation helps improve the metabolic capacity of microorganisms, enabling them to better adapt to the environment of the new fermentation system when entering formal fermentation. The two are then mixed and inoculated into a special culture medium, and fermentation is carried out by introducing a mixed gas. Finally, the mixture is centrifuged and dried, which increases the yield of single-cell protein. Detailed Implementation
[0022] The technical solution of the present invention will be clearly and completely described below through embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0023] Unless otherwise stated, all raw materials and reagents used in this invention are commercially available or can be prepared by known methods.
[0024] Methylomonas sp. was purchased from the Hangzhou branch of Wuhan Gray Algae Biotechnology Co., Ltd. Both the Chlorella protein-nucleated powder and SE culture medium were purchased from Shanghai Guangyu Biotechnology Co., Ltd.
[0025] Preparation Example 1: The preparation of the basic nutrient solution includes the following processes: Weigh 14g of magnesium sulfate monohydrate and 2g of calcium chloride and add them to a beaker. Then, pour in 500mL of deionized water and stir until completely dissolved. Continue to add deionized water and bring the volume to 1L.
[0026] Preparation Example 2: The preparation of the basic nutrient solution includes the following processes: Weigh 15g of magnesium sulfate monohydrate and 3g of calcium chloride and add them to a beaker. Then, pour in 500mL of deionized water and stir until completely dissolved. Continue to add deionized water and bring the volume to 1L.
[0027] Preparation Example 3: The preparation of trace element mixtures specifically includes the following processes: Weigh out 0.2g boric acid, 0.09g zinc sulfate monohydrate, 0.01g copper sulfate pentahydrate and 0.03g manganese chloride tetrahydrate and add them to a beaker. Then pour in 500mL of deionized water and stir until completely dissolved. Continue to add deionized water and bring the volume to 1L.
[0028] Preparation Example 4: The preparation of trace element mixtures specifically includes the following processes: Weigh out 0.3g boric acid, 0.1g zinc sulfate monohydrate, 0.011g copper sulfate pentahydrate and 0.04g manganese chloride tetrahydrate and add them to a beaker. Then pour in 500mL of deionized water and stir until completely dissolved. Continue to add deionized water and bring the volume to 1L.
[0029] Preparation Example 5: The preparation method of the culture medium for acclimatization specifically includes the following steps: Weigh 10 mL of the basic nutrient solution obtained in Preparation Example 1, 2 mL of iron-fortified nutrient solution (4 g / L EDTA sodium iron aqueous solution), 2 mL of the trace element mixture obtained in Preparation Example 3, and 1 mL of nitrogen source solution (190 g / L sodium nitrate aqueous solution), mix and stir, then add deionized water to make up to 1 L, and then put it into an autoclave for autoclaving at 121 °C for 20 min (autoclaving pressure controlled at 0.1 MPa). After the sterilization process, let it cool naturally to room temperature to obtain the culture medium for acclimatization.
[0030] Preparation Example 6: The preparation method of the culture medium for acclimatization specifically includes the following steps: Weigh 11 mL of the basic nutrient solution obtained in Preparation Example 1, 3 mL of iron-fortified nutrient solution (4.3 g / L EDTA sodium iron aqueous solution), 3 mL of the trace element mixture obtained in Preparation Example 3, and 1 mL of nitrogen source solution (192 g / L sodium nitrate aqueous solution). Mix and stir, then add deionized water to make up to 1 L. Then place it in an autoclave and autoclave it at 121 °C for 20 min (autoclave pressure controlled at 0.1 MPa). After the sterilization process, allow it to cool naturally to room temperature to obtain the culture medium for acclimatization.
[0031] Preparation Example 7: The preparation method of the culture medium for acclimatization specifically includes the following steps: Weigh 12 mL of the basic nutrient solution obtained in Preparation Example 2, 4 mL of iron-fortified nutrient solution (4.5 g / L EDTA sodium iron aqueous solution), 4 mL of the trace element mixture obtained in Preparation Example 4, and 1 mL of nitrogen source solution (195 g / L sodium nitrate aqueous solution), mix and stir, then add deionized water to make up to 1 L, and then put it into an autoclave for autoclaving at 121 °C for 20 min (autoclaving pressure controlled at 0.1 MPa). After the sterilization process, let it cool naturally to room temperature to obtain the culture medium for acclimatization.
[0032] Preparation Example 8: The preparation method of the culture medium for acclimatization specifically includes the following steps: Weigh 16 mL of the basic nutrient solution obtained in Preparation Example 2, 8 mL of iron-fortified nutrient solution (4.5 g / L EDTA sodium iron aqueous solution), 8 mL of the trace element mixture obtained in Preparation Example 4, and 3 mL of nitrogen source solution (195 g / L sodium nitrate aqueous solution), mix and stir, then add deionized water to make up to 1 L, and then put it into an autoclave for autoclaving at 121 °C for 20 min (autoclaving pressure controlled at 0.1 MPa). After the sterilization process, let it cool naturally to room temperature to obtain the culture medium for acclimatization.
[0033] Preparation Example 9: The preparation method for domesticating aerobic methanogenic bacteria specifically includes the following steps: One part by weight of bacterial culture has a concentration of 3.5 × 10⁻⁶. 8 *Methylomonas* sp. (number of cells / mL) was inoculated into 9 portions of the acclimatization medium prepared in Example 5. The pH was then adjusted to 6.5, followed by the introduction of a mixed gas (methane and oxygen in a volume ratio of 3:7) for 2 minutes, and then the medium was sealed. After sealing, the medium was placed in an incubator at 26°C and 150 rpm for acclimatization culture with shaking for 2 days. During the culture period, the mixed gas was reintroduced for 2 minutes every 24 hours, followed by sealed culture, and finally, acclimatized aerobic methanogenic bacteria were obtained.
[0034] Preparation Example 10: The preparation method for domesticating aerobic methanogenic bacteria specifically includes the following steps: One part by weight of bacterial culture has a concentration of 3.5 × 10⁻⁶. 8*Methylomonas* sp. (number of cells / mL) was inoculated into 9.5 parts by weight of the culture medium prepared in Example 6. The pH was then adjusted to 6.5, followed by the introduction of a mixed gas (methane and oxygen in a volume ratio of 3:7) for 2 minutes, and then the medium was sealed. After sealing, the medium was placed in an incubator at 26°C and 150 rpm for 3 days of shaking acclimatization culture. During the culture period, the mixed gas was reintroduced for 2 minutes every 24 hours, followed by sealed culture, and finally, acclimatized aerobic methanogenic bacteria were obtained.
[0035] Preparation Example 11: The preparation method for domesticating aerobic methanogenic bacteria specifically includes the following steps: One part by weight of bacterial culture has a concentration of 3.5 × 10⁻⁶. 8 *Methylomonas* sp. (number of cells / mL) was inoculated into 10 portions of the acclimatization medium prepared in Example 7. The pH was then adjusted to 6.5, followed by the introduction of a mixed gas (methane and oxygen in a volume ratio of 3:7) for 2 minutes, and then the medium was sealed. After sealing, the medium was placed in an incubator at 28°C and 150 rpm for acclimatization culture with shaking for 4 days. During the culture period, the mixed gas was reintroduced for 2 minutes every 24 hours, followed by sealed culture, and finally, acclimatized aerobic methanogenic bacteria were obtained.
[0036] Preparation Example 12: The preparation method for domesticating aerobic methanogenic bacteria specifically includes the following steps: One part by weight of bacterial culture has a concentration of 3.5 × 10⁻⁶. 8 *Methylomonas* sp. (number of cells / mL) was inoculated into 10 portions of the acclimatization medium prepared in Example 8. The pH was then adjusted to 6.5, followed by the introduction of a mixed gas (methane and oxygen in a volume ratio of 3:7) for 2 minutes, and then the medium was sealed. After sealing, the medium was placed in an incubator at 28°C and 150 rpm for acclimatization culture with shaking for 4 days. During the culture period, the mixed gas was reintroduced for 2 minutes every 24 hours, followed by sealed culture, and finally, acclimatized aerobic methanogenic bacteria were obtained.
[0037] Preparation Example 13: The preparation method for domesticating aerobic methanogenic bacteria specifically includes the following steps: One part by weight of bacterial culture has a concentration of 3.5 × 10⁻⁶. 8*Methylomonas* sp. (number of cells / mL) was inoculated into 10 portions of the acclimatization medium prepared in Example 7. The pH was then adjusted to 6.5, followed by the introduction of a mixed gas (methane and oxygen in a volume ratio of 4:6) for 2 minutes, and then the medium was sealed. After sealing, the medium was placed in an incubator at 28°C and 150 rpm for 5 days of shaking acclimatization culture. During the culture period, the mixed gas was reintroduced for 2 minutes every 24 hours, followed by sealed culture, and finally, acclimatized aerobic methanogenic bacteria were obtained.
[0038] Preparation Example 14: The preparation method of domesticated microalgae specifically includes the following processes: Weigh 1 part by weight of Chlorella pyrenoidosa powder and add it to 15 parts by weight of SE medium. Then place it in a temperature environment of 25°C and treat it with a light intensity of 1000 Lux for 10 hours, followed by a 14-hour dark treatment. One acclimatization cycle is defined as 10 hours of light treatment followed by 14 hours of dark treatment. After acclimatization for 1 day, the light intensity is increased to 1300 Lux. The acclimatization cycle is again defined as 10 hours of light treatment followed by 14 hours of dark treatment. The acclimatization cycle is then defined as 1 day. Finally, the light intensity is increased to 1500 Lux. The acclimatization cycle is defined as 10 hours of light treatment followed by 14 hours of dark treatment. The acclimatized microalgae are obtained.
[0039] Preparation Example 15: The preparation method of domesticated microalgae specifically includes the following processes: Weigh 1 part by weight of Chlorella pyrenoidosa powder and add it to 16 parts by weight of SE medium. Then place it in a temperature environment of 25°C and treat it with light intensity of 1000 Lux for 10 hours, followed by 14 hours of occlusion. One acclimatization cycle is defined as 10 hours of light treatment followed by 14 hours of occlusion. After acclimatization for 2 consecutive days, the light intensity is increased to 1300 Lux. The acclimatization cycle is again defined as 10 hours of light treatment followed by 14 hours of occlusion. The acclimatization cycle is then defined as 10 hours of light treatment followed by 14 hours of occlusion. The acclimatization cycle is then defined as 1500 Lux. The acclimatization cycle is then defined as 10 hours of light treatment followed by 14 hours of occlusion. The acclimatized microalgae are obtained.
[0040] Preparation Example 16: The preparation method of domesticated microalgae specifically includes the following processes: Weigh 1 part by weight of Chlorella pyrenoidosa powder and add it to 16 parts by weight of SE medium. Then place it in a temperature environment of 25°C and treat it with a light intensity of 1000 Lux for 10 hours, followed by a 14-hour dark treatment. One acclimatization cycle is defined as 10 hours of light treatment followed by 14 hours of dark treatment. After acclimatization for 2 consecutive days, the light intensity is increased to 2000 Lux. The acclimatization cycle is again defined as 10 hours of light treatment followed by 14 hours of dark treatment. The acclimatization cycle is then defined as 3000 Lux. The acclimatization cycle is defined as 10 hours of light treatment followed by 14 hours of dark treatment. The acclimatized microalgae are obtained.
[0041] Preparation Example 17: The preparation method of the special culture medium specifically includes the following steps: Weigh out 4g of ammonium chloride, 4.4g of dipotassium hydrogen phosphate, 2.1g of potassium dihydrogen phosphate, 9.4g of potassium nitrate, 2.6g of sodium chloride, 6.5g of disodium hydrogen phosphate, 0.2g of calcium chloride, 4.1g of magnesium sulfate heptahydrate, 0.021g of ferric chloride, 0.03g of ferric sulfate heptahydrate, and 0.08g of ethylenediaminetetraacetic acid and add them to a flask. Then add 500mL of deionized water and stir until completely dissolved. Finally, add deionized water to make up to 1L to obtain the first mixture. Weigh out 0.1g manganese sulfate monohydrate, 0.4g zinc sulfate heptahydrate, 0.01g copper sulfate pentahydrate and 0.08g sodium molybdate dihydrate and add them to a flask. Then add 500mL of deionized water and stir until completely dissolved. Add more deionized water to make up to 1L to obtain the second mixture. Take 80 mL of the first mixture and 10 mL of the second mixture, mix and stir, then add deionized water to make up to 1 L, and then put it into an autoclave for autoclaving at 121°C for 20 min. After the autoclaving is completed, let it cool naturally to room temperature to obtain the special culture medium.
[0042] Preparation Example 18: The preparation method of the special culture medium specifically includes the following steps: Weigh out 4.3g of ammonium chloride, 4.8g of dipotassium hydrogen phosphate, 2.3g of potassium dihydrogen phosphate, 9.6g of potassium nitrate, 2.6g of sodium chloride, 6.6g of disodium hydrogen phosphate, 0.21g of calcium chloride, 4.2g of magnesium sulfate heptahydrate, 0.022g of ferric chloride, 0.03g of ferric sulfate heptahydrate, and 0.08g of ethylenediaminetetraacetic acid and add them to a flask. Then add 500mL of deionized water and stir until completely dissolved. Finally, add deionized water to make up to 1L to obtain the first mixture. Weigh out 0.15g of manganese sulfate monohydrate, 0.4g of zinc sulfate heptahydrate, 0.02g of copper sulfate pentahydrate and 0.09g of sodium molybdate dihydrate and add them to a flask. Then add 500mL of deionized water and stir until completely dissolved. Finally, add more deionized water to make up to 1L to obtain the second mixture. Take 85 mL of the first mixture and 10 mL of the second mixture, mix and stir, then add deionized water to make up to 1 L, and then put it into an autoclave for autoclaving at 121°C for 20 min. After the autoclaving is completed, let it cool naturally to room temperature to obtain the special culture medium.
[0043] Preparation Example 19: The preparation method of the special culture medium specifically includes the following steps: Weigh out 4.6g of ammonium chloride, 5.0g of dipotassium hydrogen phosphate, 2.9g of potassium dihydrogen phosphate, 9.7g of potassium nitrate, 2.7g of sodium chloride, 6.6g of disodium hydrogen phosphate, 0.22g of calcium chloride, 4.3g of magnesium sulfate heptahydrate, 0.023g of ferric chloride, 0.031g of ferric sulfate heptahydrate, and 0.09g of ethylenediaminetetraacetic acid and add them to a flask. Then add 500mL of deionized water and stir until completely dissolved. Finally, add more deionized water to bring the volume to 1L to obtain the first mixture. Weigh out 0.2g of manganese sulfate monohydrate, 0.41g of zinc sulfate heptahydrate, 0.02g of copper sulfate pentahydrate and 0.1g of sodium molybdate dihydrate and add them to a flask. Then add 500mL of deionized water and stir until completely dissolved. Add more deionized water to make up to 1L to obtain the second mixture. Take 90 mL of the first mixture and 10 mL of the second mixture, mix and stir, then add deionized water to make up to 1 L, and then put it into an autoclave for autoclaving at 121°C for 20 min. After the sterilization process, let it cool naturally to room temperature to obtain the special culture medium.
[0044] Preparation Example 20: The preparation method of the special culture medium specifically includes the following steps: The 90 mL first mixture and 10 mL second mixture in Preparation Example 19 were increased to 180 mL first mixture and 20 mL second mixture, and then deionized water was added to bring the volume to 1 L. The remaining preparation conditions were the same as in Preparation Example 19.
[0045] Example 1: A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: One part by weight of the domesticated aerobic methanogenic bacteria obtained in Preparation Example 9 and one part by weight of the domesticated microalgae obtained in Preparation Example 14 were inoculated together into 10 parts by weight of the specially prepared culture medium obtained in Preparation Example 17. All the air in the system was extracted by a vacuum pump, and then a first mixed gas (composed of methane, oxygen and carbon dioxide in a volume ratio of 2:1:2) was introduced for 2 minutes. Then the light intensity was adjusted to 2000 Lux and the temperature was 30°C. The timed photo-fermentation was started for 7 days (the first mixed gas was introduced again for 2 minutes every 24 hours during the fermentation period). The obtained fermentation broth was placed in a low-temperature centrifuge and the temperature was controlled at 4°C. Then it was centrifuged at 7000 r / min for 20 minutes. After centrifugation, the bottom precipitate was collected, washed with neutral phosphate buffer, and centrifuged again at 7000 r / min for 10 minutes. The bottom precipitate was collected and placed in a vacuum drying oven at 40°C. The vacuum degree was adjusted to 0.08 MPa and dried to remove water to obtain single-cell protein.
[0046] Example 2: A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: One part by weight of the domesticated aerobic methanogenic bacteria obtained in Preparation Example 10 and one part by weight of the domesticated microalgae obtained in Preparation Example 14 were inoculated together into 11 parts by weight of the specially prepared culture medium obtained in Preparation Example 18. All the air in the system was extracted by a vacuum pump, and then a first mixed gas (composed of methane, oxygen and carbon dioxide in a volume ratio of 2:1:2) was introduced for 2 minutes. Then the light intensity was adjusted to 2000 Lux and the temperature was 30°C. The timed photo-fermentation was started for 7 days (the first mixed gas was introduced again for 2 minutes every 24 hours during the fermentation period). The obtained fermentation broth was placed in a low-temperature centrifuge and the temperature was controlled at 4°C. Then it was centrifuged at 8000 r / min for 25 minutes. After centrifugation, the bottom precipitate was collected, washed with neutral phosphate buffer, and centrifuged again at 8000 r / min for 10 minutes. The bottom precipitate was collected and placed in a vacuum drying oven at 40°C. The vacuum degree was adjusted to 0.08 MPa and dried to remove water to obtain single-cell protein.
[0047] Example 3: A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: One part by weight of the domesticated aerobic methanogenic bacteria obtained in Preparation Example 11 and one part by weight of the domesticated microalgae obtained in Preparation Example 15 were inoculated together into 12 parts by weight of the specially prepared culture medium obtained in Preparation Example 19. The air in the system was completely extracted by a vacuum pump, and then a first mixed gas (composed of methane, oxygen and carbon dioxide in a volume ratio of 2:1:2) was introduced for 2 minutes. The light intensity was then adjusted to 2000 Lux and the temperature to 30°C. The timed photo-fermentation was started for 7 days (the first mixed gas was introduced again for 2 minutes every 24 hours during the fermentation period). The obtained fermentation broth was placed in a low-temperature centrifuge and the temperature was controlled at 4°C. Then, it was centrifuged at 8000 r / min for 30 minutes. After centrifugation, the bottom precipitate was collected, washed with neutral phosphate buffer, and centrifuged again at 8000 r / min for 10 minutes. The bottom precipitate was collected and placed in a vacuum drying oven at 40°C. The vacuum degree was adjusted to 0.08 MPa and dried to remove water to obtain single-cell protein.
[0048] Comparative Example 1: A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: The aerobic methanotrophic bacteria in Example 3 were replaced with the aerobic methanotrophic bacteria obtained in Preparation Example 12, and the rest of the preparation process was the same as in Example 3.
[0049] Comparative Example 2: A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: The aerobic methanotrophic bacteria in Example 3 were replaced with the aerobic methanotrophic bacteria obtained in Preparation Example 13, and the rest of the preparation process was the same as in Example 3.
[0050] Comparative Example 3: A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: The domesticated microalgae in Example 3 were replaced with the domesticated microalgae obtained in Preparation Example 16, and the rest of the preparation process remained the same as in Example 3.
[0051] Comparative Example 4: A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: The special culture medium in Example 3 was replaced with the special culture medium obtained in Preparation Example 20, and the rest of the preparation process was the same as in Example 3.
[0052] Comparative Example 5: A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: The first mixed gas in Example 3 (composed of methane, oxygen and carbon dioxide in a volume ratio of 2:1:2) was replaced with the first mixed gas (composed of methane, oxygen and carbon dioxide in a volume ratio of 1:2:3), and the rest of the preparation process was the same as in Example 3.
[0053] Comparative Example 6 A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: One part by weight of the domesticated aerobic methanogenic bacteria obtained in Preparation Example 11 and one part by weight of the domesticated microalgae obtained in Preparation Example 15 were inoculated together into 12 parts by weight of the specially prepared culture medium obtained in Preparation Example 19. All the air in the system was extracted by a vacuum pump, and then a first mixed gas (composed of methane, oxygen and carbon dioxide in a volume ratio of 2:1:2) was introduced for 2 minutes. Then the light intensity was adjusted to 1500 Lux and the temperature to 30°C, and the timed photo-fermentation was started for 7 days (during the fermentation, the first mixed gas was reintroduced for 2 minutes every 24 hours). The remaining steps were the same as in Example 3.
[0054] Comparative Example 7 A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: One part by weight of the domesticated aerobic methanogenic bacteria obtained in Preparation Example 11 and one part by weight of the domesticated microalgae obtained in Preparation Example 15 were inoculated together into 12 parts by weight of the specially prepared culture medium obtained in Preparation Example 19. All the air in the system was extracted by a vacuum pump, and then a first mixed gas (composed of methane, oxygen and carbon dioxide in a volume ratio of 2:1:2) was introduced for 2 minutes. Then the light intensity was adjusted to 2000 Lux and the temperature was 28°C. The timed photo-fermentation was started for 8 days (the first mixed gas was introduced again for 2 minutes every 24 hours during the fermentation period). The remaining steps were the same as in Example 3.
[0055] Comparative Example 8 A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, specifically comprising the following processes: One part by weight of the domesticated aerobic methanogenic bacteria obtained in Preparation Example 11 and one part by weight of the domesticated microalgae obtained in Preparation Example 15 were inoculated together into 12 parts by weight of the specially prepared culture medium obtained in Preparation Example 19. All the air in the system was extracted by a vacuum pump, and then a first mixed gas (composed of methane, oxygen and carbon dioxide in a volume ratio of 2:1:2) was introduced for 2 minutes. Then the light intensity was adjusted to 2000 Lux and the temperature to 35°C, and the timed photo-fermentation was started for 7 days (during the fermentation, the first mixed gas was reintroduced for 2 minutes every 24 hours). The remaining steps were the same as in Example 3.
[0056] Take 100 mL of the fermentation broth obtained in Examples 1-3 and Comparative Examples 1-8 respectively, place them in a low-temperature centrifuge, control the temperature at 4℃, and then centrifuge at 8000 r / min for 10 min. After centrifugation, collect the bottom precipitate, wash with neutral phosphate buffer, and centrifuge again at 8000 r / min for 10 min. Collect the bottom precipitate and place it in a vacuum drying oven at 40℃, adjust the vacuum degree to 0.08 MPa, dry and remove water to obtain single-cell protein, weigh it with an analytical balance, and calculate the yield of single-cell protein per 100 mL of fermentation broth according to the Kjeldahl nitrogen determination method. The results are shown in Table 1 below.
[0057] Table 1 Single-cell protein production
[0058] The following conclusions can be drawn from Table 1 above: (1) Through Examples 1-3, it can be found that the fermentation system constructed in this invention can make good use of methane and carbon dioxide to obtain a high yield of single-cell protein.
[0059] (2) Comparative Example 1 shows that the increase in basic nutrient solution, iron-enriched nutrient solution, trace element mixture and nitrogen source solution in the culture medium for domestication may cause the aerobic methanogenic bacteria to lose water through osmosis due to nutrient overload, inhibiting metabolic activity. As a result, the yield of the domesticated strain decreased when it was fermented to prepare single-cell protein.
[0060] (3) Comparative Example 2 shows that when the methane content in the mixed gas of the aerobic methanogenic bacteria is further increased and the acclimatization time is increased, although the high methane content helps to improve the metabolism of the strain and thus improve its own activity, it may be because in this system, excessive acclimatization may cause the strain to metabolize too fast and consume oxygen excessively, resulting in insufficient oxygen during subsequent acclimatization, which in turn inhibits the metabolic ability to ferment and prepare single-cell protein.
[0061] (4) Comparative Example 3 shows that when the light intensity of domesticated Chlorella proteoglycans is further increased, the yield of domesticated microalgae obtained by fermentation to prepare single-cell protein is low. This may be because in this system, although light intensity is beneficial to improve the metabolic capacity of Chlorella proteoglycans, excessive light intensity can easily lead to metabolic disorder of the domesticated microalgae obtained by over-domestication, reduce the light energy conversion efficiency, and thus weaken the fermentation yield of single-cell protein.
[0062] (5) Comparative Example 4 shows that when the nutrient content in the special culture medium is increased, the yield of single-cell protein prepared by fermentation is low. This may be because, in this system, on the one hand, excessive nutrient content can easily lead to an increase in the osmotic pressure of the culture medium, causing cells to lose water through osmosis and inhibiting metabolic activity; on the other hand, excessive nutrient content can easily cause the enzymes produced by metabolism to reach saturation too early, resulting in no further increase in the fermentation rate, which in turn weakens the yield of single-cell protein.
[0063] (6) Comparative Example 5 shows that the yield of single-cell protein prepared by fermentation is low. This may be because the composition ratio of the mixed gas has an important influence on the fermentation system constructed in this invention. Reducing methane and increasing oxygen and carbon dioxide may be detrimental to the fermentation of this system.
[0064] (7) Through comparative examples 6-8, it can be found that the method of preparing single-cell protein in this invention has specific requirements for temperature, light and fermentation time during the fermentation process. Insufficient light, excessive fermentation or insufficient temperature will lead to a decrease in the yield of single-cell protein.
[0065] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.
Claims
1. A coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide, characterized in that, The coupled fermentation method includes the following steps: Domesticated aerobic methanogenic bacteria and domesticated microalgae were co-inoculated into a specially prepared culture medium and mixed. Then, a vacuum was drawn, and the first mixed gas was introduced and fermented under light to obtain fermentation broth. The fermentation broth was then centrifuged and vacuum dried to obtain single-cell protein.
2. The coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide according to claim 1, characterized in that, The method for preparing the domesticated aerobic methanogenic bacteria includes the following steps: Aerobic methanotrophic bacteria and acclimatization culture medium were mixed at a weight ratio of 1:9~10, and then a second mixed gas was introduced and acclimatized in a temperature environment of 26℃~28℃ to obtain acclimatized aerobic methanotrophic bacteria. The method for preparing the acclimatization culture medium includes the following steps: The basic nutrient solution, iron-fortified nutrient solution, trace element mixture and nitrogen source solution were mixed in a volume ratio of 10~12:2~4:2~4:1 and deionized water was added to bring the volume to 1L. The mixture was then autoclaved at 121℃ for 20 minutes to obtain the acclimatization culture medium.
3. The coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide according to claim 2, characterized in that, The basic nutrient solution is obtained by mixing magnesium sulfate monohydrate and calcium chloride in a weight ratio of 14~15:2~3 and then bringing the volume to 1L.
4. The coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide according to claim 2, characterized in that, The iron-fortified solution is an aqueous solution of sodium iron ethylenediaminetetraacetate with a mass concentration of 4 g / L to 4.5 g / L.
5. The coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide according to claim 2, characterized in that, The trace element mixture is prepared by mixing boric acid, zinc sulfate monohydrate, copper sulfate pentahydrate and manganese chloride tetrahydrate in a weight ratio of 0.2~0.3:0.09~0.1:0.01~0.011:0.03~0.04 and then bringing the volume to 1L.
6. The coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide according to claim 2, characterized in that, The nitrogen source solution is an aqueous solution of sodium nitrate with a mass concentration of 190 g / L to 195 g / L.
7. The coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide according to claim 1, characterized in that, The method for preparing the domesticated microalgae includes the following steps: Microalgae and SE medium were mixed at a weight ratio of 1:15-16. Then, in an environment of 25°C, an acclimatization cycle was performed with 10 hours of light treatment and 14 hours of shading treatment. First, the microalgae were acclimatized for 1-2 days in a light intensity of 1000 Lux, then for 1-2 days in a light intensity of 1300 Lux, and finally for 1-2 days in a light intensity of 1500 Lux to obtain acclimatized microalgae.
8. The coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide according to claim 1, characterized in that, The preparation method of the special culture medium includes the following steps: 4g~4.6g ammonium chloride, 4.4g~5.0g dipotassium hydrogen phosphate, 2.1g~2.9g potassium dihydrogen phosphate, 9.4g~9.7g potassium nitrate, 2.6g~2.7g sodium chloride, 6.5g~6.6g disodium hydrogen phosphate, 0.2g~0.22g calcium chloride, 4.1g~4.3g magnesium sulfate heptahydrate, 0.021g~0.023g ferric chloride, 0.03g~0.031g ferric sulfate heptahydrate, and 0.08g~0.09g ethylenediaminetetraacetic acid were mixed with water and stirred until the volume was adjusted to 1L to obtain the first mixture. 0.1g~0.2g manganese sulfate monohydrate, 0.4g~0.41g zinc sulfate heptahydrate, 0.01g~0.02g copper sulfate pentahydrate and 0.08g~0.1g sodium molybdate dihydrate were added to water, mixed and stirred, and the volume was adjusted to 1L to obtain a second mixture. Take 80-90 mL of the first mixture and 10 mL of the second mixture, mix them, and bring the volume to 1 L. Then, autoclave at 121 °C for 20 min to obtain the special culture medium.
9. The coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide according to claim 1, characterized in that, The weight ratio of the domesticated aerobic methanogenic bacteria, domesticated microalgae, and specially prepared culture medium is 1:1:10~12.
10. The coupled fermentation method for synthesizing single-cell proteins from methane and carbon dioxide according to claim 1, characterized in that, The conditions for photo-fermentation include a fermentation temperature of 30°C, a light intensity of 2000 Lux, and a fermentation time of 7 days.