A method for simultaneously treating fermentation exhaust gas and producing oil by using azotobacter assisted microalgae

Abstract

The application discloses a method for producing oil by microalgae with the aid of nitrogen-fixing bacteria and treating fermentation waste gas, which comprises two stages of microalgae propagation and co-culture oil production. In the propagation stage, the microalgae take CO2 in the fermentation waste gas without any pretreatment as the carbon source, and realize high-density culture in the culture medium containing nitrogen. After the nitrogen source in the culture medium is consumed, multifunctional nitrogen-fixing bacteria are inoculated into the culture system to co-culture with the microalgae. The nitrogen in the fermentation waste gas is converted into the nitrogen source of the microalgae by the nitrogen-fixing bacteria, so that the low nitrogen source concentration is maintained to promote the oil production of the microalgae, and the secretions of the microalgae culture mainly in the form of extracellular polymers and volatile organic matters from the fermentation waste gas are decomposed. The application not only realizes the effective treatment of the fermentation waste gas and the high-density culture of the microalgae, but also improves the oil content of the microalgae, and through the reduction of the residual organic matters and total nitrogen content, the culture water can be repeatedly used for many times, so that the problem of large amount of wastewater in the microalgae culture is solved, and the application has good environmental benefits and application prospect.

Description

Technical Field

[0001] This invention relates to the fields of waste gas treatment, carbon dioxide emission reduction and utilization, and biological resource utilization, specifically to a method for using nitrogen-fixing bacteria to assist microalgae in oil production while simultaneously treating fermentation waste gas. Background Technology

[0002] Fermentation industry utilizes the metabolic activities of microorganisms to produce various products such as alcohol, antibiotics, amino acids, and enzyme preparations. Compared with chemical production, microbial fermentation is generally considered to have advantages such as mild production conditions, less environmental pollution, and lower energy consumption. However, the respiration of microorganisms during fermentation produces a large amount of CO2, accompanied by the generation of small amounts of volatile organic compounds (such as alcohols, esters, aldehydes, and volatile organic acids). How to effectively treat fermentation waste gas, especially reducing CO2 emissions, is a key concern for fermentation enterprises.

[0003] Microalgae can autotrophically grow by utilizing CO2 and releasing oxygen through photosynthesis; on average, obtaining 1 kg of dry weight of microalgae consumes 1.8 kg of CO2. In addition, microalgae cells contain various carbohydrates, proteins, trace elements, and other bioactive substances. In particular, their lipid content can reach over 30% of the cell's dry weight, making them suitable as raw materials for food, feed, energy, and the extraction of bioactive substances. Utilizing microalgae to treat waste gas from fermentation industries and reduce CO2 emissions has become an effective new approach, but the following problems still exist.

[0004] Volatile alcohols, esters, aldehydes, and organic acids contained in fermentation waste gas, when introduced into the microalgae culture system along with CO2, will inhibit microalgae growth and CO2 conversion efficiency if not effectively degraded in a timely manner and their concentration accumulates to a certain level. Patent CN113307377A addresses this issue by pre-treating and purifying the waste gas to remove volatile organic compounds before introducing the microalgae culture medium, but this increases the number of treatment steps and costs. Another major problem is how to treat the culture medium after microalgae cultivation and CO2 conversion. As mentioned earlier, microalgae cells have certain value, and the higher the oil content, the greater the utilization value. However, the biomass of microalgae in the culture medium is generally relatively low, and the large amount of culture medium remaining after removing the microalgae cells still needs to be treated as wastewater. Patent CN102443542B uses municipal wastewater with high inorganic nitrogen and phosphorus content that has undergone primary treatment as the microalgae culture medium. While this method can utilize microalgae to consume some of the nitrogen and phosphorus pollutants in municipal wastewater, it requires filtering out contaminating bacteria or algae from the wastewater before microalgae cultivation, and adding sea salt to increase osmotic pressure to inhibit the growth of other contaminating bacteria or algae during cultivation. Furthermore, the secretions from microalgae growth, especially extracellular polymers, also enter the municipal wastewater, increasing its organic matter and salt content. In addition, there are existing reports on the co-cultivation of microalgae with nitrogen-fixing microorganisms, where nitrogen-fixing bacteria convert N2 in the air or exhaust gas into a usable nitrogen source for microalgae. However, there are no successful cases of using co-cultivation of microalgae and nitrogen-fixing microorganisms to treat fermentation waste gas. This may be because co-cultivation also fails to solve the problems of volatile organic compounds in fermentation waste gas and the accumulation of microalgae secretions in the culture medium, and additional carbon sources are required for nitrogen-fixing bacteria during co-cultivation.

[0005] Therefore, developing a greener method for treating fermentation waste gas using microalgae, reducing CO2 emissions, and achieving high-density cultivation and high oil production of microalgae still has important research and application value. Summary of the Invention

[0006] To overcome the aforementioned shortcomings in the treatment of industrial fermentation waste gas, utilization, and conversion of CO2 by microalgae, and to achieve the goals of high-density microalgae cultivation and high oil production, this invention specifically aims to create a method that utilizes nitrogen-fixing bacteria in the co-cultivation of microalgae to assist in oil production and decompose and utilize microalgae secretions (mainly extracellular polymers) and volatile alcohols, esters, aldehydes, organic acids, etc. (mainly from fermentation waste gas), thereby mitigating or resolving the problems caused by the accumulation of these substances in the microalgae culture medium, which inhibits microalgae growth and the wastewater treatment issues that need to be addressed after cultivation. This invention provides a strain of Bacillus subtilis with nitrogen-fixing functions. subtleC1) and its method for assisting microalgae in oil production and degrading organic matter in the culture medium when co-cultured with microalgae. The bacterium was deposited at the China Center for Type Culture Collection on September 17, 2021, with accession number CCTCC NO: M20211184, deposited at Wuhan University, Wuhan, China.

[0007] This invention provides a use of nitrogen-fixing bacteria to assist microalgae in oil production, wherein the nitrogen-fixing bacteria is Bacillus subtilis. subtle C1 was deposited on September 17, 2021, at the China Center for Type Culture Collection (CCTCC) with accession number M20211184, located at Wuhan University, Wuhan, China.

[0008] The process of microalgae cultivation and oil production includes the microalgae reproduction stage and the oil production stage of co-culturing nitrogen-fixing bacteria and microalgae.

[0009] The microalgae mentioned include any one or more combinations of Cladophora, Chlorella, Crescentella, Oocystella, and Discella.

[0010] In some embodiments of the present invention, the microalgae may be selected from a variety of microalgae with autotrophic oil production function, that is, there is no specific limitation on the type of microalgae, as long as it has autotrophic oil production function. Chlorella sp., purchased from the National Aquatic Biological Germplasm Resource Bank, is preferred, with the algal species number: FACHB-5.

[0011] The process of microalgae cultivation and oil production also includes the co-culture mixed microbial cells of nitrogen-fixing bacteria and microalgae obtained in the oil production stage of co-culturing nitrogen-fixing bacteria and microalgae, and the mixed microbial cells obtained by co-culturing can be used for other purposes.

[0012] In some embodiments, another use of the present invention is the use of nitrogen-fixing bacteria to assist microalgae in oil production while simultaneously purifying industrial fermentation waste gas.

[0013] The industrial fermentation waste gas includes CO2, O2, N2, and a small amount of volatile organic compounds. The industrial fermentation waste gas originates from one of the following: fermentation tail gas generated during the production of dry yeast, fermentation tail gas generated during the production of neomycin sulfate, or fermentation tail gas generated during the production of active lactic acid bacteria for feed additives.

[0014] In the process of nitrogen-fixing bacteria-assisted microalgae oil production, the amount of nitrogen source required during the microalgae reproduction stage is reduced to a concentration of less than 600 mg / L.

[0015] To achieve the aforementioned reduction in nitrogen source usage, in the application of reducing the amount of nitrogen source required during the microalgae reproduction stage, nitrogen-fixing bacteria convert N2 in the waste gas and continuously provide nitrogen source for the microalgae.

[0016] A method for nitrogen-fixing bacteria-assisted microalgae oil production includes the following steps: (1) During the propagation stage, wet microalgae cells were inoculated into BG11 medium. Industrial fermentation waste gas was introduced into the inoculated microalgae culture system at a ventilation ratio of 0.05~0.15 vvm. Microalgae were cultured under light conditions to increase the cell concentration of microalgae in the culture medium. (2) During the oil production stage, Bacillus subtilis is used. subtle C1 wet cells were added to the culture system of step (1) and cultured under light and aeration. When the lipid content of the microalgae increased to a stable value, the mixed cells of nitrogen-fixing bacteria and microalgae obtained by co-culture were collected by centrifugation. (3) The mixed cells of nitrogen-fixing bacteria and microalgae obtained in step (2) can be used for other purposes. The supernatant obtained by centrifugation can be used as water for preparing BG11 culture medium. New microalgae can be re-inoculated and new nitrogen-fixing bacteria can be inoculated in the second stage. The reproduction stage of step (1) and the oil production stage of step (2) can be repeated to achieve the cultivation of microalgae and oil production.

[0017] Bacillus subtilis subtle The mass ratio of C1 wet bacterial cells to microalgal wet bacterial cells in step (1) is 1:1 to 1:100.

[0018] In some embodiments, the supernatant collected by centrifugation is used to prepare BG11 culture medium for recycling.

[0019] The industrial fermentation waste gas is derived from one of the following: fermentation tail gas generated during the production of dry yeast, fermentation tail gas generated during the production of neomycin sulfate, or fermentation tail gas generated during the production of active lactic acid bacteria for feed additives.

[0020] In some embodiments, the BG11 culture medium formulation is as follows: NaNO3 600 mg / L, K2HPO4 40 mg / L, Na2CO3 20 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, EDTA-Na 21 mg / L, and A5 mixture 1 mL / L, wherein the A5 mixture consists of H3BO3 2.86 g / L, MnCl2·4H2O 1.86 g / L, ZnSO4·7H2O 0.22 g / L, CuSO4·5H2O 0.08 g / L, Na2MoO4·2H2O 0.39 g / L, and Co(NO3)2·6H2O 0.05 g / L.

[0021] The BG11 medium has achieved a reduction in the concentration of nitrogen source (NaNO3) (originally 1500 mg / L).

[0022] The method for obtaining wet cells from microalgae seed culture and centrifugation is as follows: Chlorella sp. FACHB-5 is inoculated at a 10% inoculum into sterilized glucose + BG11 liquid medium (10 g / L glucose, 1500 mg / L NaNO3, and other contents are the same as BG11 medium). After incubation in shake flasks or fermenters at 28℃ and 250 rpm until the late logarithmic growth stage (about 4 days), the microalgae cells are collected by centrifugation at 1500g and 4℃ for 10 min. The cells are then resuspended in an appropriate amount of sterile water, centrifuged and washed for later use.

[0023] The method for obtaining wet bacterial cells by culturing and centrifuging the multifunctional nitrogen-fixing seed is as follows: Bacillus subtilis (Bacillus subtilis) is inoculated at a rate of 10%. subtle C1) Inoculate the cells into sterilized LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl), and culture in a shake flask or fermenter at 37°C and 250 rpm until the late logarithmic growth phase (approximately 24 h). Collect the cells by centrifugation at 1500 g and 4°C for 10 min, and resuspend in an appropriate amount of sterile water, centrifuge and wash before use.

[0024] The beneficial effects of this invention are as follows: (1) The method for treating fermentation waste gas simultaneously during microalgae cultivation and oil production provided by this invention eliminates the need for pretreatment to remove volatile alcohols, esters, aldehydes, organic acids, and other substances from the fermentation waste gas. The waste gas can be directly introduced into the microalgae culture medium, reducing the treatment steps and costs associated with fermentation waste gas. During the treatment process, the CO2 concentration in the fermentation waste gas can be reduced by up to 80%, which helps fermentation enterprises achieve CO2 emission reduction and green production, resulting in significant social benefits.

[0025] (2) The method for treating fermentation waste gas while simultaneously cultivating microalgae and producing oil provided by the present invention is divided into two stages: microalgae reproduction and oil production. Based on the characteristics of microalgae, which mainly grow in groups (cell reproduction and increase in number) when nitrogen source is abundant and mainly accumulate oil individually (microalgae number no longer increases significantly) when nitrogen source is limited, the method is divided into two stages: microalgae reproduction and oil production. The concentration of nitrogen source (NaNO3) in microalgae culture medium BG11 is reduced from 1500 mg / L to 600 mg / L. While ensuring the nitrogen source required for the microalgae reproduction stage, the amount of nitrogen source used is reduced, and high-density cultivation of microalgae is achieved (the final microalgae cell concentration can reach 6.2~6.8 g / L).

[0026] (3) Without changing other culture conditions, nitrogen-fixing bacteria and microalgae were co-cultured during the oil production stage, and the microalgae continued to use CO2 in the fermentation waste gas as a carbon source. Since the nitrogen source NaNO3 was consumed during the reproduction stage, the nitrogen-fixing bacteria converted N2 in the waste gas and continuously provided nitrogen source for the microalgae. Due to the balance between nitrogen fixation and nitrogen source utilization, the total nitrogen content in the culture medium during the oil production stage was maintained at a low level (1.0~1.2 mg / L), which was conducive to the accumulation of oil by the microalgae when the nitrogen source was limited (the oil content of the microalgae increased to more than 40%).

[0027] (4) The nitrogen-fixing bacteria provided by the present invention, with its unique functions of degrading organic matter and solubilizing phosphorus, decomposes and utilizes the microalgal secretions accumulated in the culture medium during the microalgal reproduction stage, especially the polysaccharides in the extracellular polymers and the volatile organic compounds from the fermentation waste gas, as a carbon source. It also decomposes the organic phosphorus in the extracellular polymers into soluble phosphorus sources that can be reused by microalgae and nitrogen-fixing bacteria. This is equivalent to purifying the culture medium during the oil production stage, so that the supernatant of the culture medium after collecting (removing) the co-cultured bacteria can be reused as culture water about 5 times (a total of 70 days), which greatly reduces the amount of wastewater.

[0028] (5) The method ultimately yields a co-culture mixture of nitrogen-fixing bacteria and microalgae, wherein the oil content of the microalgae is >40%, and the multifunctional nitrogen-fixing bacteria provided by the present invention is Bacillus subtilis, which is a feed microorganism permitted for use in my country. Therefore, the obtained mixture can be used as a feed additive. Attached Figure Description

[0029] Figure 1 Example 1 illustrates the changes in CO2 concentration in waste gas during the process of multifunctional nitrogen-fixing bacteria assisting microalgae in oil production while simultaneously treating waste gas from dry yeast fermentation.

[0030] Figure 2 Example 2 illustrates the changes in CO2 concentration in waste gas during the process of multifunctional nitrogen-fixing bacteria assisting microalgae in oil production while simultaneously treating neomycin fermentation waste gas.

[0031] Figure 3 Example 3 illustrates the changes in CO2 concentration in exhaust gas during the process of multifunctional nitrogen-fixing bacteria assisting microalgae in oil production while simultaneously treating exhaust gas from lactic acid bacteria fermentation.

[0032] Figure 4 As a comparative example, the CO2 concentration of exhaust gas during the process of microalgae cultivation (oil production) and simultaneous treatment of lactic acid bacteria fermentation exhaust gas was studied. Detailed Implementation

[0033] The present invention will be further explained below with reference to the embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0034] Bacillus subtilis subtle C1 was deposited at the China Center for Type Culture Collection on September 17, 2021, with accession number CCTCC NO: M20211184, at Wuhan University, Wuhan, China.

[0035] The microalgae were purchased from the National Aquatic Biological Germplasm Resource Bank as Chlorella sp., with species number FACHB-5.

[0036] The method for simultaneously treating fermentation waste gas during microalgae cultivation and oil production is divided into two stages: microalgae propagation and oil production. (1) Reproduction stage. The wet cells obtained by centrifuging the microalgae used as seed culture were added to BG11 medium with reduced nitrogen source concentration at an initial inoculum concentration of 0.1~0.4 g / L. The actual industrial fermentation waste gas without pretreatment and purification was introduced into the inoculated microalgae culture system at a constant ventilation ratio of 0.05~0.15 vvm. The culture was carried out at pH 6.5~8.5, 24~37℃, and light (light-dark cycle 12 h:12 h, light intensity 5000~10000 Lux) for 6~8 days to increase the cell concentration of microalgae in the culture medium to 5.5~6.0 g / L.

[0037] (2) Oil production stage. Bacillus subtilis, which has nitrogen-fixing, phosphorus-solubilizing, phosphorus-dissolving, and organic matter-degrading functions, will be cultured as seed culture. subtle C1) The wet bacterial cells obtained by centrifugation are added to the above culture system at a bacterial cell mass ratio of nitrogen-fixing bacteria to microalgae of 1:1 to 1:100. The culture is continued under the same conditions of light and aeration for 6 to 8 days. When the oil content of the microalgae increases from <10% at the end of the reproduction stage to more than 40%, the culture medium is centrifuged at 1500g. The mixed bacterial cells of nitrogen-fixing bacteria and microalgae obtained by co-culture are collected (removed). The supernatant produced by centrifugation is used as water for preparing BG11 medium. A new round of culture is started by inoculating microalgae seeds (nitrogen-fixing bacteria seeds are inoculated in the oil-producing stage). The above operation is repeated until the mass of the mixed bacterial cells of nitrogen-fixing bacteria and microalgae obtained by co-culture in the oil-producing stage drops to less than 80% of that in the first round of co-culture.

[0038] The BG11 medium with reduced nitrogen source (NaNO3) concentration is formulated as follows: NaNO3 600 mg / L, K2HPO4 40 mg / L, Na2CO3 20 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, EDTA-Na 21 mg / L, and A5 mixture 1 mL / L. The A5 mixture consists of H3BO3 2.86 g / L, MnCl2·4H2O 1.86 g / L, ZnSO4·7H2O 0.22 g / L, CuSO4·5H2O 0.08 g / L, Na2MoO4·2H2O 0.39 g / L, and Co(NO3)2·6H2O 0.05 g / L.

[0039] The method for obtaining wet cells from microalgae seed culture and centrifugation is as follows: Chlorella sp. FACHB-5 is inoculated at a 10% inoculum into sterilized glucose + BG11 liquid medium (10 g / L glucose, 1500 mg / L NaNO3, and other contents are the same as BG11 medium). After incubation at 28℃ and 250 rpm in shake flasks or fermenters until the late logarithmic growth stage (about 4 days), the microalgae cells are collected by centrifugation at 1500g and 4℃ for 10 min. The cells are then resuspended in an appropriate amount of sterile water, centrifuged, and washed for later use.

[0040] The method for obtaining wet bacterial cells by culturing and centrifuging the multifunctional nitrogen-fixing seed is as follows: Bacillus subtilis (Bacillus subtilis) is inoculated at a rate of 10%. subtle C1) Inoculate the cells into sterilized LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl), and culture in a shake flask or fermenter at 37°C and 250 rpm until the late logarithmic growth phase (approximately 24 h). Collect the cells by centrifugation at 1500 g and 4°C for 10 min, and resuspend them in an appropriate amount of sterile water, centrifuge and wash them for later use.

[0041] The untreated and unpurified industrial fermentation waste gas was collected directly from the outlet pipes of fermentation tanks at three companies: a certain active dry yeast production company, a certain neomycin sulfate production company, and a certain active lactic acid bacteria production company, using empty medical oxygen bags. Process gas mass spectrometry analysis revealed the following average compositions for the dry yeast fermentation waste gas: N 278.4%, O 24.5%, CO 215.7%, and volatile organic compounds (VOCs) 0.5% (the sum of volatile alcohols, esters, aldehydes, organic acids, etc., hereinafter the same); for the neomycin fermentation waste gas: N 278.8%, O 23.8%, CO 216.7%, and VOCs 0.2%; and for the lactic acid bacteria fermentation waste gas: N 277.4%, O 26.3%, CO 214.4%, and VOCs 0.2%.

[0042] Example 1 Multifunctional nitrogen-fixing bacteria assist microalgae in oil production while simultaneously treating waste gas from dry yeast fermentation. Wet cells obtained by centrifuging Chlorella sp. FACHB-5 seed culture were added to BG11 medium with reduced nitrogen source concentration at an initial inoculum concentration of 0.4 g / L. Untreated dry yeast fermentation exhaust gas (N2 78.4%, O2 4.5%, CO2 15.7%, volatile organic compounds 0.5%) was introduced into the inoculated microalgae culture system at a constant aeration rate of 0.09 vvm. After 7 days of cultivation at pH 7.5, 28℃, and a light / dark cycle of 12 h:12 h, light intensity 5000 Lux, the concentration of Chlorella FACHB-5 in the culture medium increased to 5.5 g / L. The multifunctional nitrogen-fixing bacterium Bacillus subtilis was also introduced. subtle The wet bacterial cells obtained by centrifuging the C1 seed culture medium were added to the above culture system at a bacterial cell mass ratio of nitrogen-fixing bacteria to microalgae of 1:10. After culturing under the same conditions of light and aeration for 7 days, the culture medium was centrifuged at 1500g, and the mixed bacterial cells of nitrogen-fixing bacteria and microalgae obtained by the culture were collected (removed). The supernatant produced by centrifugation was used as water for preparing BG11 culture medium (deionized water was added to the original culture medium volume, and the water added each time did not exceed 1 / 10 of the original volume). A new round of culture was started by inoculating the same amount of Chlorella seeds as the previous round (the oil-producing section was inoculated with the same amount of Bacillus subtilis C1 seeds as the previous round). The above operation was repeated for 6 consecutive rounds.

[0043] Depend on Figure 1Analysis shows that during the first five rounds of continuous cultivation using this method to treat dry yeast fermentation waste gas, the CO2 concentration changes in the waste gas treated by microalgae did not differ significantly between rounds. Days 1-7 of each round represent the microalgae reproduction stage, during which the number of microalgae increases exponentially, resulting in a significant decrease in the CO2 concentration at the outlet of the waste gas treated by microalgae, reaching a minimum of 2.8%, a reduction of 82.2% compared to the initial CO2 concentration of 15.7% in the introduced dry yeast fermentation waste gas. Days 8-14 of each round represent the oil production stage of the microalgae. During this stage, the CO2 concentration at the outlet of the waste gas treated by microalgae tends to stabilize, remaining at approximately 6.6%, a reduction of 58.0% compared to the initial concentration. This may be because the number of microalgae no longer increases significantly during the oil production stage, and the utilized CO2 is mainly used as a carbon source and energy source for oil accumulation. The CO2 concentration in the treated waste gas increased significantly in the sixth round compared to the previous five rounds, possibly due to the deterioration of the microalgae cultivation environment in the sixth round.

[0044] After each round of continuous culture, the wet weight of the mixed cells of nitrogen-fixing bacteria and microalgae obtained from the co-culture, the wet weight and lipid content of the microalgae, and the COD and total nitrogen (TN) content of the supernatant after centrifugation of the culture medium are shown in Table 1. After the first five rounds of culture, the wet weight of the mixed cells obtained by centrifugation was maintained at around 8.4 g / L, with microalgae content all >6.2 g / L and lipid content of microalgae >42%. The COD and TN content of the supernatant after centrifugation of the culture medium were less than 100 mg / L and 1.2 mg / L, respectively. However, after the sixth round of culture, the wet weight of the mixed cells obtained by centrifugation decreased to 5.9 g / L, the lipid content of microalgae dropped to below 24%, and the COD of the supernatant after centrifugation of the culture medium was 148.6 mg / L, indicating that the culture environment deteriorated and the growth of microalgae was restricted. However, this method allows the culture water to be reused continuously for five times (a total of 70 days), which has significantly reduced the amount of wastewater generated.

[0045] Table 1. Changes in key culture parameters after each round of six consecutive operations in Example 1.

[0046] Example 2 Multifunctional nitrogen-fixing bacteria assist microalgae in oil production while simultaneously treating neomycin fermentation waste gas Wet cells obtained by centrifuging Chlorella sp. FACHB-5 seed culture were added to BG11 medium with reduced nitrogen source concentration at an initial inoculum concentration of 0.2 g / L. Untreated neomycin fermentation waste gas (N2 78.8%, O2 3.8%, CO2 16.7%, volatile organic compounds 0.2%) was introduced into the inoculated microalgae culture system at a constant aeration rate of 0.08 vvm. After 7 days of cultivation at pH 7.5, 28℃, and a light / dark cycle of 12 h:12 h, light intensity 10000 Lux, the concentration of Chlorella FACHB-5 in the culture medium increased to 6.0 g / L. The multifunctional nitrogen-fixing bacterium Bacillus subtilis was also introduced. subtle C1) The wet bacterial cells obtained by centrifuging the seed culture medium were added to the above culture system at a bacterial cell mass ratio of nitrogen-fixing bacteria to microalgae of 1:10. After culturing under the same conditions of light and aeration for 7 days, the culture medium was centrifuged at 1500g to collect (remove) the mixed bacterial cells of nitrogen-fixing bacteria and microalgae obtained by co-culturing. All other operations were the same as in Example 1 and were repeated for 6 consecutive rounds.

[0047] Depend on Figure 2 Analysis showed that during the first five rounds of continuous cultivation using this method to treat neomycin fermentation waste gas, the CO2 concentration changes in the waste gas treated by microalgae were not significantly different in each round. The lowest CO2 concentration was 2.8% during the microalgae propagation stage, a decrease of 83.2% compared to the initial CO2 concentration of 16.7% in the neomycin fermentation waste gas. During the microalgae oil production stage, the CO2 concentration at the outlet of the waste gas treated by microalgae remained at approximately 6.8%, a decrease of 59.3% compared to the initial concentration. The CO2 concentration in the treated waste gas increased significantly in the sixth round compared to the previous five, possibly due to the deterioration of the microalgae cultivation environment in the sixth round.

[0048] As shown in Table 2, after the first five rounds of cultivation, the wet weight of the mixed bacterial cells obtained by centrifugation remained around 8.6 g / L, with microalgae cells all >6.4 g / L and microalgae lipid content >43%. The COD and TN contents of the supernatant after centrifugation were less than 100 mg / L and 1.2 mg / L, respectively. However, after the sixth round of cultivation, the wet weight of the mixed bacterial cells obtained by centrifugation decreased to 6.2 g / L, the microalgae lipid content dropped below 22%, and the COD of the supernatant after centrifugation was 161.3 mg / L, indicating a deterioration in the cultivation environment and limited microalgae growth. However, this method allows for the continuous reuse of the cultivation water five times (for a total of 70 days), significantly reducing the amount of wastewater generated.

[0049] Table 2. Changes in key culture parameters after each round of six consecutive operations in Example 2.

[0050] Example 3 Multifunctional nitrogen-fixing bacteria assist microalgae in oil production while simultaneously treating lactic acid bacteria fermentation waste gas. The wet cells obtained by centrifuging the seed culture of *Chlorella sp. FACHB-5* were added to BG11 medium with reduced nitrogen source concentration at an initial inoculum concentration of 0.4 g / L. Untreated lactic acid bacteria fermentation exhaust gas (N2 77.4%, O2 6.3%, CO2 14.4%, volatile organic compounds 0.2%) was introduced into the inoculated microalgae culture system at a constant aeration rate of 0.10 vvm. After 7 days of cultivation at pH 7.5, 28℃, and light (12 h:12 h light-dark cycle, 10000 Lux light intensity), the concentration of *Chlorella sp. FACHB-5* in the culture medium increased to 5.9 g / L. The multifunctional nitrogen-fixing bacterium *Bacillus subtilis* was also introduced into the culture. subtle C1) The wet bacterial cells obtained by centrifuging the seed culture medium were added to the above culture system at a bacterial cell mass ratio of nitrogen-fixing bacteria to microalgae of 1:10. After culturing under the same conditions of light and aeration for 7 days, the culture medium was centrifuged at 1500g to collect (remove) the mixed bacterial cells of nitrogen-fixing bacteria and microalgae obtained by co-culturing. All other operations were the same as in Example 1 and were repeated for 6 consecutive rounds.

[0051] Depend on Figure 3 Analysis showed that during the first five rounds of continuous cultivation using this method to treat lactic acid bacteria fermentation waste gas, the CO2 concentration changes in the waste gas treated by microalgae did not differ significantly between each round. The lowest CO2 concentration during the microalgae propagation stage was 2.5%, a decrease of 82.6% compared to the initial CO2 concentration of 14.4% in the introduced lactic acid bacteria fermentation waste gas. During the microalgae oil production stage, the CO2 concentration at the outlet of the waste gas treated by microalgae remained at approximately 6.3%, a decrease of 56.3% compared to the initial concentration. The CO2 concentration in the treated waste gas increased significantly in the sixth round compared to the previous five, possibly due to the deterioration of the microalgae cultivation environment in the sixth round.

[0052] As shown in Table 3, after the first five rounds of cultivation, the wet weight of the mixed bacterial cells obtained by centrifugation remained around 9.3 g / L, with microalgae cells all >6.8 g / L and microalgae lipid content >44%. The COD and TN contents of the supernatant after centrifugation were less than 100 mg / L and 1.2 mg / L, respectively. However, after the sixth round of cultivation, the wet weight of the mixed bacterial cells obtained by centrifugation decreased to 6.6 g / L, the microalgae lipid content dropped below 21%, and the COD of the supernatant after centrifugation was 133.3 mg / L, indicating a deterioration in the cultivation environment and limited microalgae growth. However, this method allows for the continuous reuse of the cultivation water five times (for a total of 70 days), significantly reducing the amount of wastewater generated.

[0053] Table 3. Changes in key culture parameters after each round of six consecutive operations in Example 3.

[0054] Comparative Example 1 Microalgae culture alone (for oil production) while simultaneously treating lactic acid bacteria fermentation waste gas The wet cells obtained by centrifuging the seed culture of Chlorella sp. FACHB-5 were added to BG11 medium with reduced nitrogen source concentration at an initial inoculum concentration of 0.4 g / L. Untreated lactic acid bacteria fermentation exhaust gas (N2 77.4%, O2 6.3%, CO2 14.4%, volatile organic compounds 0.2%) was introduced into the inoculated microalgae culture system at a constant aeration rate of 0.10 vvm. The pH was 7.5, the temperature was 28℃, and the light intensity was 10000 ppm (photo-dark cycle 12 h:12 h, light intensity 10000 ppm). After culturing for 14 days (Lux), the culture medium is centrifuged at 1500g to collect (remove) the microalgae cells. The supernatant produced by centrifugation is used as water for preparing BG11 culture medium (add deionized water to the original culture medium volume, the added water should not exceed 1 / 10 of the original volume). A new round of culture is started by inoculating the same amount of Chlorella seeds as the previous round (without inoculating Bacillus subtilis C1 seeds). The above operation is repeated for 2 consecutive rounds.

[0055] Depend on Figure 4 Analysis shows that when microalgae are cultured alone (for oil production) and used to treat lactic acid bacteria fermentation waste gas, the CO2 concentration at the outlet of the waste gas treated by microalgae from day 1 to day 7 is basically the same as that during the microalgae propagation stage in Example 3. This is because the nitrogen source concentration in the culture medium is relatively sufficient during this stage, and microalgae propagation is the main focus. However, in Comparative Example 1, the CO2 concentration at the outlet of the waste gas treated by microalgae from day 8 to day 14 of microalgae cultivation remained at approximately 11.3%, a decrease of only 21.5% compared to the initial concentration, which is significantly different from the CO2 concentration change during the oil production stage in Example 3. In Comparative Example 1, without the assistance of multifunctional nitrogen-fixing bacteria, after the first round of microalgae cultivation, the wet weight of the microalgae cells was only 4.2 g / L, and the oil content was only 9.5%. The COD and TN contents of the supernatant after centrifugation of the culture medium were 102.3 mg / L and 0.1 mg / L, respectively. The supernatant produced by centrifugation was used as water to prepare BG11 medium. After inoculating microalgae to start the second round of culture, the microalgae showed signs of death such as yellowing and precipitation after about 5 days of culture, and the second round of culture failed.

[0056] By comparing Comparative Example 1, especially with Example 3, it can be illustrated that the multifunctional nitrogen-fixing bacteria provided by the present invention, when co-cultured with microalgae, can decompose the secretions of microalgae, especially the polysaccharides in extracellular polymers and the volatile organic compounds from fermentation waste gas, and utilize them as a carbon source. Simultaneously, it can decompose the organic phosphorus in the extracellular polymers into soluble phosphorus sources that can be reused by the microalgae and nitrogen-fixing bacteria, effectively purifying the culture medium during the oil production stage. This allows the centrifuged supernatant after collecting (removing) the co-cultured bacteria to be reused as culture water up to approximately five times. Furthermore, after the nitrogen source in the culture medium is consumed, the nitrogen-fixing bacteria can convert N2 in the waste gas and continuously provide a nitrogen source for the microalgae. Due to the balance between nitrogen fixation and nitrogen source utilization, the total nitrogen content in the culture medium during the oil production stage is stably maintained at a low level (1.0~1.2 mg / L), which is beneficial for the accumulation of lipids by microalgae when nitrogen sources are limited (the lipid content of microalgae increases to over 40%).

Claims

1. A method for producing oil from microalgae assisted by nitrogen-fixing bacteria, characterized in that, The steps involved in nitrogen-fixing bacteria-assisted microalgae oil production include the following: (1) During the propagation stage, wet microalgae cells are inoculated into BG11 medium. Industrial fermentation waste gas is introduced into the inoculated microalgae culture system at a ventilation ratio of 0.05~0.15 vvm. Microalgae are cultured under light conditions to increase the cell concentration of microalgae in the culture medium. The industrial fermentation waste gas is derived from one or more of the following: fermentation tail gas generated during the production of dry yeast, fermentation tail gas generated during the production of neomycin sulfate, and fermentation tail gas generated during the production of active lactic acid bacteria for feed additives. (2) During the oil production stage, Bacillus subtilis is used. subtilis C1 wet cells were added to the culture system of step (1) and cultured under light and aeration. When the oil content of the microalgae increased to a stable value, the supernatant obtained by centrifugation was used to prepare water for BG11 culture medium. The propagation stage of step (1) and the oil production stage of step (2) were repeated to achieve the cultivation and oil production of the microalgae. The microalgae were Chlorella vulgaris and Bacillus subtilis. subtilis C1 was deposited on September 17, 2021, at the China Center for Type Culture Collection (CCTCC) with accession number M20211184, located at Wuhan University, Wuhan, China.

2. The method for nitrogen-fixing bacteria-assisted microalgae oil production according to claim 1, characterized in that, Bacillus subtilis subtilis The mass ratio of C1 wet cells to microalgal wet cells in step (1) is 1:1 to 1:

100.

3. The method for nitrogen-fixing bacteria-assisted microalgae oil production according to claim 1, characterized in that, Bacillus subtilis subtilis C1 assists microalgae in oil production and simultaneously purifies industrial fermentation waste gas.

4. The method for nitrogen-fixing bacteria-assisted microalgae oil production according to claim 1, characterized in that, The industrial fermentation waste gas includes CO2, O2, N2 and a small amount of volatile organic compounds.

5. The method for nitrogen-fixing bacteria-assisted microalgae oil production according to any one of claims 1-4, characterized in that, The nitrogen-fixing bacteria are used to reduce the amount of nitrogen source required during the microalgae's reproduction stage in the process of assisting microalgae in oil production, so that the required nitrogen source concentration is below 600 mg / L.

6. The method for nitrogen-fixing bacteria-assisted microalgae oil production according to claim 5, characterized in that, In the application of reducing the amount of nitrogen source required during the microalgae reproduction stage, nitrogen-fixing bacteria convert N2 in the exhaust gas and continuously provide nitrogen source for the microalgae.

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