Method for assisting microalgae oil production by using nitrogen-fixing bacteria and simultaneously treating fermentation waste gas

Abstract

The invention discloses a method for assisting microalgae oil production by nitrogen-fixing bacteria and treating fermentation waste gas at the same time. The method comprises two stages of microalgae breeding and co-culture oil production. In the breeding stage, the microalgae take CO2 in fermentation waste gas which is not pretreated as a carbon source, and high-density culture is realized in a nitrogen-containing culture medium. When the nitrogen source in the culture medium is consumed, inoculating multifunctional nitrogen-fixing bacteria into a culture system and co-culturing with the microalgae. Nitrogen in the fermentation waste gas is converted into a nitrogen source of the microalgae under the action of nitrogen-fixing bacteria, so that the concentration of the low nitrogen source is maintained to promote the microalgae to produce oil, and secretions mainly comprising extracellular polymeric substances and volatile organic compounds derived from the fermentation waste gas, which are generated by microalgae culture, are decomposed. Effective treatment of fermentation waste gas and high-density culture of microalgae are achieved, the oil content of the microalgae is increased, culture water can be repeatedly used by reducing the content of residual organic matter and total nitrogen, the problem that the amount of microalgae culture wastewater is large is solved, and good environmental benefits and application prospects are achieved.

Description

Technical Field

[0001] The invention relates to the technical fields of waste gas treatment, carbon dioxide emission reduction and utilization, and biological resource utilization, and in particular to a method for simultaneously treating fermentation waste gas by utilizing nitrogen-fixing bacteria to assist microalgae in oil production. Background Art

[0002] The fermentation industry uses the metabolic activities of microorganisms to produce a variety of products such as alcohol, antibiotics, amino acids, enzyme preparations, etc. Compared with chemical production, microbial fermentation is generally considered to have advantages such as mild production conditions, less environmental pollution, and low energy consumption. However, during the fermentation process, the respiration of microorganisms will produce a large amount of CO2, accompanied by the generation of a small amount of volatile organic compounds (such as alcohols, esters, aldehydes, volatile organic acids, etc.). How to effectively treat fermentation waste gas, especially to reduce CO2 emissions, is a key issue for fermentation companies.

[0003] Microalgae can grow autotrophically and release oxygen by using CO2 through photosynthesis. On average, 1 kg of dry weight of microalgae consumes 1.8 kg of CO2. In addition, microalgae also contain a variety of carbohydrates, proteins, trace elements and other active substances. In particular, its oil content can reach more than 30% of the cell dry weight, which can be used as food, feed, energy and raw materials for the extraction of biologically active substances. Using microalgae to treat fermentation industrial waste gas and reduce CO2 emissions has become an effective new way, but the following problems still exist.

[0004] After the volatile alcohols, esters, aldehydes, organic acids and other substances contained in the fermentation waste gas are introduced into the microalgae culture system with CO2, if they are not effectively degraded in time, when their concentration accumulates to a certain extent, they will inhibit the growth of microalgae and the conversion efficiency of CO2. The method of patent CN113307377A to solve this problem is to pre-treat and purify the waste gas, remove the volatile organic matter in the fermentation waste gas and then introduce the microalgae culture solution, but this also increases the processing steps and costs of the fermentation waste gas. Another major problem is how to deal with the culture solution after microalgae culture and conversion of CO2. As mentioned above, the microalgae body has a certain value, and the higher the oil content, the greater the utilization value, but the biomass of microalgae in the culture solution is generally relatively low, and the large amount of culture solution remaining after removing the microalgae body still needs to be treated as wastewater. Patent CN102443542B uses municipal sewage with high inorganic nitrogen and phosphorus content after primary treatment as microalgae culture solution. Although this method can use microalgae to consume some of the nitrogen, phosphorus and other pollutants in municipal sewage, it is necessary to filter and remove the bacteria or algae in the municipal sewage before culturing the microalgae, and add sea salt to increase the osmotic pressure to inhibit the growth of other bacteria or algae during the culture process. The secretions of the growing microalgae, especially the extracellular polymers, will also enter the municipal sewage, increasing the organic matter and salt content of the sewage. In addition, there are related literature reports on the co-cultivation of microalgae and nitrogen-fixing microorganisms, that is, the nitrogen-fixing bacteria convert the N2 in the air or exhaust gas into a nitrogen source that can be used by the microalgae, but there is no successful case of using microalgae and nitrogen-fixing microorganisms to co-cultivate fermentation waste gas. This may be because co-cultivation also fails to solve the problem of the accumulation of volatile organic compounds in the fermentation waste gas and the secretions of the microalgae culture in the culture medium, and it is also necessary to provide an additional carbon source for the nitrogen-fixing bacteria during co-cultivation.

[0005] Therefore, developing a greener method to use microalgae to treat fermentation waste gas, reduce CO2 emissions, and achieve high-density culture and high oil production of microalgae still has important research and application value. Summary of the invention

[0006] In order to overcome the above-mentioned deficiencies in microalgae treatment of industrial fermentation waste gas, utilization and conversion of CO2, and achieve the goal of high-density cultivation of microalgae and high oil production, in particular, to create a method for using nitrogen-fixing bacteria to co-cultivate microalgae to assist microalgae in producing oil and decompose and utilize microalgae secretions (mainly extracellular polymers) and volatile alcohols, esters, aldehydes, organic acids and other substances (mainly from fermentation waste gas), thereby alleviating or solving the problem of inhibiting microalgae growth caused by the accumulation of the two in the microalgae culture solution and the wastewater treatment problem that needs to be faced after the cultivation is completed. The present invention provides a strain of Bacillus subtilis with functions such as nitrogen fixation. subtleC1) and a method for assisting microalgae in oil production and degrading organic matter in culture medium when co-cultured with microalgae. The bacterium was deposited in the China Center for Type Culture Collection on September 17, 2021, with the deposit number CCTCC NO: M20211184, and the deposit address is: Wuhan University, Wuhan, China.

[0007] The present 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 in the China Center for Type Culture Collection on September 17, 2021, with the deposit number CCTCC NO: M20211184, and the deposit address is Wuhan University, Wuhan, China.

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

[0009] The microalgae include any one or more combinations of Cladophora, Chlorella, Closterium, Oocystis, and Pelagicola.

[0010] In some embodiments of the present invention, the microalgae can 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 the autotrophic oil production function. Preferably, Chlorella sp. purchased from the National Aquatic Biological Germplasm Resource Bank is purchased, and the algae species number is: FACHB-5.

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

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

[0013] The industrial fermentation waste gas includes CO2, O2, N2 and a small amount of volatile organic matter. The industrial fermentation waste gas comes from one of the fermentation tail gas generated in the production process of dry yeast, the fermentation tail gas generated in the production of neomycin sulfate, and the fermentation tail gas generated in the production of active lactic acid bacteria for feed additives.

[0014] In the process of nitrogen-fixing bacteria assisting microalgae in oil production, the amount of nitrogen source required in the microalgae reproduction stage is reduced so that the required nitrogen source concentration is below 600 mg / L.

[0015] In order to achieve the above-mentioned reduction in the amount of nitrogen source used, in the use of reducing the amount of nitrogen source required for the microalgae reproduction stage, the nitrogen-fixing bacteria convert the N2 in the exhaust gas and continue to provide a nitrogen source for the microalgae.

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

[0017] Bacillus subtilis subtle The mass ratio of the C1 wet bacteria to the microalgae wet bacteria 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 fermentation tail gas generated in the production process of dry yeast, the fermentation tail gas generated in the production of neomycin sulfate, and the fermentation tail gas generated in the production of active lactic acid bacteria for feed additives.

[0020] In some embodiments, the BG11 culture medium formula is: NaNO3600 mg / L, K2HPO440 mg / L, Na2CO320 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, citric acid 6 mg / L, ammonium ferric citrate 6 mg / L, EDTA-Na21 mg / L, A5 mixed solution 1mL / L, wherein the A5 mixed solution consists of H3BO32.86 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 the nitrogen source (NaNO3) (the original concentration was 1500 mg / L).

[0022] The method for culturing microalgae seeds and obtaining wet cells by centrifugation is as follows: Chlorella sp. FACHB-5 is inoculated into a sterilized glucose+BG11 liquid culture medium (glucose 10 g / L, NaNO3 1500 mg / L, and other substances are the same as those in the BG11 culture medium) at a 10% inoculation rate, and cultured in a shake flask or fermenter at 28°C and 250 rpm until the late logarithmic growth period (about 4 days), and then centrifuged at 1500g and 4°C for 10 min to collect the microalgae cells, and resuspended with an appropriate amount of sterile water, washed by centrifugation, and set aside.

[0023] The method of culturing the multifunctional nitrogen-fixing seeds and obtaining wet bacteria by centrifugation is as follows: inoculating Bacillus subtilis at a rate of 10% subtle C1) Inoculate into sterilized LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L), culture in a shake flask or fermenter at 37°C and 250 rpm until the late logarithmic growth period (about 24 h), then collect the cells by centrifugation at 1500 g and 4°C for 10 min, resuspend with an appropriate amount of sterile water, wash by centrifugation, and set aside.

[0024] The beneficial effects of the present invention are as follows: (1) The method provided by the present invention for treating fermentation waste gas while cultivating microalgae and producing oil, wherein the fermentation waste gas does not need to be pre-treated to remove volatile alcohols, esters, aldehydes, organic acids and other substances, and can be directly introduced into the microalgae culture solution, thereby reducing the processing steps and costs of the fermentation waste gas. During the treatment process, the CO2 concentration in the fermentation waste gas can be reduced by more than 80%, which helps fermentation enterprises to achieve CO2 emission reduction and green production, and has good social benefits.

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

[0026] (3) Without changing other culture conditions, nitrogen-fixing bacteria were introduced into the co-culture with microalgae 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 the N2 in the waste gas and continued to provide nitrogen source for the microalgae. In addition, 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 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, by virtue of their unique functions of degrading organic matter and solubilizing and dissolving phosphorus, decompose the microalgae secretions accumulated in the culture medium during the microalgae reproduction stage, especially the polysaccharides in the extracellular polymers and the volatile organic matter derived from the fermentation waste gas, and utilize them as carbon sources, and decompose the organic phosphorus in the extracellular polymers into soluble phosphorus sources for reuse by the microalgae and the nitrogen-fixing bacteria, which is equivalent to achieving purification of the culture medium in the oil production stage, so that the centrifugal supernatant of the culture medium after collecting (removing) the co-cultured bacteria can be reused as culture water for about 5 times (a total of 70 days), thereby greatly reducing the amount of wastewater.

[0028] (5) The method ultimately produces a co-cultured mixed bacterial cell of nitrogen-fixing bacteria and microalgae, wherein the oil content of the microalgae is >40%. 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 mixed bacterial cell can be used as a feed additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the change in waste gas CO2 concentration during the process of multifunctional nitrogen-fixing bacteria assisting microalgae in oil production and simultaneously treating dry yeast fermentation waste gas in Example 1.

[0030] Figure 2 This is Example 2, the change in waste gas CO2 concentration during the process of multifunctional nitrogen-fixing bacteria assisting microalgae in oil production and simultaneously treating neomycin fermentation waste gas.

[0031] Figure 3 This is Example 3, which shows the change in waste gas CO2 concentration during the process of multifunctional nitrogen-fixing bacteria assisting microalgae in oil production and simultaneously treating lactic acid bacteria fermentation waste gas.

[0032] Figure 4 This is the change in CO2 concentration in the waste gas during the process of treating the waste gas from lactic acid bacteria fermentation while cultivating microalgae alone (to produce oil) in Comparative Example 1. DETAILED DESCRIPTION

[0033] The present invention is further explained below in conjunction with the examples and drawings. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0034] Bacillus subtilis subtle C1) was deposited in the China Center for Type Culture Collection on September 17, 2021, with the deposit number CCTCC NO: M20211184, and the deposit address is: Wuhan University, Wuhan, China.

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

[0036] The method for treating fermentation waste gas while culturing and producing oil from microalgae is divided into two stages: microalgae propagation and oil production: (1) Reproduction stage. The wet cells obtained by centrifugation of the microalgae used as seed culture were added to the BG11 culture 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 rate of 0.05-0.15 vvm. The culture was carried out at pH 6.5-8.5, 24-37°C, and light intensity (light-dark cycle 12 h:12 h, light intensity 5000-10000 Lux) for 6-8 days to increase the cell concentration of the microalgae in the culture medium to 5.5-6.0 g / L.

[0037] (2) Oil production stage. Bacillus subtilis, which has the functions of nitrogen fixation, phosphorus solubilization, phosphate dissolution and organic matter degradation, is used as a seed culture. subtle C1) The wet cells obtained by centrifugation are added to the above culture system at a mass ratio of 1:1 to 1:100 between nitrogen-fixing bacteria and microalgae, and culture is continued under the same conditions of illumination and ventilation 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 solution is centrifuged at 1500g to collect (remove) the mixed cells of nitrogen-fixing bacteria and microalgae obtained by co-culture, and the supernatant produced by centrifugation is used as water for preparing BG11 culture medium, and microalgae seeds are newly inoculated (nitrogen-fixing bacteria seeds are inoculated in the oil-producing stage) to start a new round of culture, and the above operation is repeated until the mass of the mixed 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 culture 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, ammonium ferric citrate 6 mg / L, EDTA-Na2 1 mg / L, A5 mixed solution 1 mL / L, wherein the A5 mixed solution comprises 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 culturing microalgae seeds and obtaining wet cells by centrifugation is as follows: Chlorella sp. FACHB-5 is inoculated into a sterilized glucose+BG11 liquid culture medium (glucose 10 g / L, NaNO3 1500 mg / L, and other substances are the same as those in the BG11 culture medium) at a 10% inoculation rate, and cultured in a shake flask or fermenter at 28°C and 250 rpm until the late logarithmic growth period (about 4 days), and then centrifuged at 1500g and 4°C for 10 min to collect the microalgae cells, and resuspended with an appropriate amount of sterile water, washed by centrifugation, and set aside.

[0040] The method of culturing the multifunctional nitrogen-fixing seeds and obtaining wet bacteria by centrifugation is as follows: inoculating Bacillus subtilis at a rate of 10% subtle C1) Inoculate into sterilized LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L), culture in a shake flask or fermenter at 37°C and 250 rpm until the late logarithmic growth period (about 24 h), then collect the cells by centrifugation at 1500g and 4°C for 10 min, resuspend with an appropriate amount of sterile water, wash by centrifugation, and set aside.

[0041] The actual industrial fermentation waste gas without pretreatment and purification is: the fermentation waste gas directly collected at the outlet pipe of the fermentation tank of a certain active dry yeast production enterprise, a certain neomycin sulfate production enterprise, and a certain feed additive active lactic acid bacteria production enterprise using empty medical oxygen bags. Through the process gas mass spectrometer, the average composition of dry yeast fermentation waste gas is: N278.4%, O24.5%, CO215.7%, volatile organic matter 0.5% (the sum of volatile alcohols, esters, aldehydes, organic acids and other substances, the same below); the average composition of neomycin fermentation waste gas is: N278.8%, O23.8%, CO216.7%, volatile organic matter 0.2%; the average composition of lactic acid bacteria fermentation waste gas is: N277.4%, O26.3%, CO214.4%, volatile organic matter 0.2%.

[0042] Example 1 Multifunctional nitrogen-fixing bacteria assists microalgae in oil production and simultaneously treats waste gas from dry yeast fermentation The wet cells obtained by centrifugation of the seed culture of Chlorella sp. FACHB-5 were added to the BG11 medium with reduced nitrogen source concentration at an initial inoculum concentration of 0.4 g / L. The dry yeast fermentation waste gas (N278.4%, O24.5%, CO215.7%, volatile organic matter 0.5%) without pretreatment and purification was introduced into the inoculated microalgae culture system at a constant rate of 0.09 vvm. After 7 days of cultivation at pH 7.5, 28°C, and light (light-dark cycle 12 h:12 h, light intensity 5000 Lux), the cell concentration of Chlorella sp. FACHB-5 in the culture medium increased to 5.5 g / L. The multifunctional nitrogen-fixing bacteria Bacillus subtilis (Bacillus subtle C1) The wet cells obtained by centrifugation of the seed culture solution were added to the above culture system at a mass ratio of 1:10 between nitrogen-fixing bacteria and microalgae, and cultured under the same conditions of illumination and aeration for 7 days. The culture solution was centrifuged at 1500g to collect (remove) the mixed cells of nitrogen-fixing bacteria and microalgae obtained by culture. The supernatant produced by centrifugation was used as water for preparing BG11 culture medium (deionized water was added to the original culture volume, and the water added each time did not exceed 1 / 10 of the original volume), and the same amount of Chlorella seeds as in the previous round were newly inoculated (the same amount of Bacillus subtilis C1 seeds as in the previous round were inoculated in the oil-producing section) to start a new round of culture, and the above operation was repeated for 6 consecutive rounds.

[0043] Depend on Figure 1Analysis shows that during the first five rounds of continuous culture of dry yeast fermentation waste gas treated by this method, the CO2 concentration in the waste gas treated by microalgae in each round did not change significantly. The first to seventh days of each round were the reproduction stage of microalgae. In this stage, the CO2 concentration at the outlet of the waste gas treated by microalgae increased exponentially with the number of microalgae. The lowest CO2 concentration was 2.8%, which was 82.2% lower than the initial concentration of 15.7% of the dry yeast fermentation waste gas. The eighth to fourteenth day of each round was the oil production stage of microalgae. In this stage, the CO2 concentration at the outlet of the waste gas treated by microalgae tended to be stable, basically maintained at about 6.6%, which was 58.0% lower than the initial concentration. The reason may be that the number of microalgae no longer increased significantly in the oil production stage, and the CO2 used was mainly used as the carbon source and energy consumption required for the accumulation of oil. The CO2 concentration in the treated waste gas in the sixth round increased significantly compared with the previous five rounds, which may be due to the deterioration of the microalgae culture environment in the sixth round.

[0044] After each round of continuous culture, the wet weight of the mixed bacteria of nitrogen-fixing bacteria and microalgae obtained by co-culture, the wet weight and oil content of the microalgae, and the COD and total nitrogen (TN) content of the supernatant after centrifugation of the culture solution are shown in Table 1. The wet weight of the mixed bacteria obtained by centrifugation after the first five rounds of culture was maintained at about 8.4 g / L, of which the microalgae were all >6.2 g / L, and the oil content of the microalgae was >42%. The COD and TN contents of the supernatant after centrifugation of the culture solution 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 bacteria obtained by centrifugation decreased to 5.9 g / L, the oil content of the microalgae decreased to below 24%, and the COD of the supernatant after centrifugation of the culture solution was 148.6 mg / L, indicating that the culture environment deteriorated and the growth of microalgae was limited. However, this method can reuse the culture water for 5 consecutive times (a total of 70 days), which has greatly reduced the amount of wastewater generated.

[0045] Table 1 Changes in main culture parameters after each round of 6 consecutive operations in Example 1

[0046] Example 2 Multifunctional nitrogen-fixing bacteria assist microalgae in oil production and simultaneous treatment of neomycin fermentation waste gas The wet cells obtained by centrifugation of the seed culture of Chlorella sp. FACHB-5 were added to the BG11 medium with reduced nitrogen source concentration at an initial inoculum concentration of 0.2 g / L. The untreated and purified neomycin fermentation waste gas (N278.8%, O23.8%, CO216.7%, volatile organic matter 0.2%) was introduced into the inoculated microalgae culture system at a constant rate of 0.08 vvm. After 7 days of cultivation at pH 7.5, 28°C, and light intensity (light-dark cycle 12 h:12 h, light intensity 10000 Lux), the cell concentration of Chlorella sp. FACHB-5 in the culture medium increased to 6.0 g / L. The multifunctional nitrogen-fixing bacteria Bacillus subtilis (Bacillus subtle C1) The wet cells obtained by centrifugation of the seed culture solution were added to the above culture system at a mass ratio of 1:10 between the nitrogen-fixing bacteria and the microalgae, and cultured under the same conditions of illumination and aeration for 7 days. The culture solution was centrifuged at 1500 g to collect (remove) the mixed cells of the nitrogen-fixing bacteria and microalgae obtained by the co-culture. The other operations were the same as in Example 1, and repeated for 6 rounds.

[0047] Depend on Figure 2 Analysis shows that during the first five rounds of continuous culture of neomycin fermentation waste gas treated by this method, the CO2 concentration changes in the waste gas treated by microalgae in each round were not significant. The lowest CO2 concentration was 2.8% during the microalgae reproduction stage, which was 83.2% lower than the initial concentration of 16.7% of the neomycin fermentation waste gas CO2. The CO2 concentration at the outlet of the waste gas treated by microalgae during the microalgae oil production stage was basically maintained at around 6.8%, which was 59.3% lower than the initial concentration. In the sixth round, the CO2 concentration in the treated waste gas increased significantly compared with the first five rounds, which may be due to the deterioration of the microalgae culture environment in the sixth round.

[0048] As shown in Table 2, the wet weight of the mixed cells obtained by centrifugation after the first five rounds of culture was maintained at about 8.6 g / L, of which the microalgae cells were all >6.4 g / L, and the oil content of the microalgae was >43%. The COD and TN contents of the supernatant after centrifugation of the culture solution 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 6.2 g / L, the oil content of the microalgae decreased to less than 22%, and the COD of the supernatant after centrifugation of the culture solution was 161.3 mg / L, indicating that the culture environment deteriorated and the growth of microalgae was restricted. However, this method can reuse the culture water for 5 consecutive times (70 days in total), which has greatly reduced the amount of wastewater generated.

[0049] Table 2 Changes of main culture parameters after each round of 6 consecutive operations in Example 2

[0050] Example 3 Multifunctional nitrogen-fixing bacteria assists microalgae in oil production and simultaneously treats waste gas from lactic acid bacteria fermentation The wet cells obtained by centrifugation of the seed culture of Chlorella sp. FACHB-5 were added to the BG11 medium with reduced nitrogen source concentration at an initial inoculum concentration of 0.4 g / L. The untreated and purified lactic acid bacteria fermentation waste gas (N277.4%, O26.3%, CO214.4%, volatile organic matter 0.2%) was introduced into the inoculated microalgae culture system at a constant rate of 0.10 vvm at a pH of 7.5, 28°C, and light (light-dark cycle 12 h:12 h, light intensity 10000 Lux) for 7 days. The cell concentration of Chlorella sp. FACHB-5 in the culture medium increased to 5.9 g / L. The multifunctional nitrogen-fixing bacteria Bacillus subtilis (Bacillus subtle C1) The wet cells obtained by centrifugation of the seed culture solution were added to the above culture system at a mass ratio of 1:10 between the nitrogen-fixing bacteria and the microalgae, and cultured under the same conditions of illumination and aeration for 7 days. The culture solution was centrifuged at 1500 g to collect (remove) the mixed cells of the nitrogen-fixing bacteria and microalgae obtained by the co-culture. The other operations were the same as in Example 1, and repeated for 6 rounds.

[0051] Depend on Figure 3 Analysis shows that during the first five rounds of continuous culture of the waste gas from lactic acid bacteria fermentation using this method, the CO2 concentration in the waste gas treated with microalgae in each round was not significantly different. The lowest CO2 concentration was 2.5% during the microalgae reproduction stage, which was 82.6% lower than the initial CO2 concentration of 14.4% in the lactic acid bacteria fermentation waste gas. During the microalgae oil production stage, the CO2 concentration at the outlet of the waste gas treated with microalgae was basically maintained at around 6.3%, which was 56.3% lower than the initial concentration. In the sixth round, the CO2 concentration in the treated waste gas increased significantly compared with the first five rounds, which may be due to the deterioration of the microalgae culture environment in the sixth round.

[0052] As shown in Table 3, the wet weight of the mixed cells obtained by centrifugation after the first five rounds of culture was maintained at about 9.3 g / L, of which the microalgae cells were all >6.8 g / L, and the oil content of the microalgae was >44%. The COD and TN contents of the supernatant after centrifugation of the culture solution 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 6.6 g / L, the oil content of the microalgae decreased to less than 21%, and the COD of the supernatant after centrifugation of the culture solution was 133.3 mg / L, indicating that the culture environment deteriorated and the growth of microalgae was restricted. However, this method can reuse the culture water for 5 consecutive times (70 days in total), which has greatly reduced the amount of wastewater generated.

[0053] Table 3 Changes in main culture parameters after each round of 6 consecutive operations in Example 3

[0054] Comparative Example 1 Microalgae cultivation (oil production) and simultaneous treatment of lactic acid bacteria fermentation waste gas The wet cells obtained by centrifugation of the seed culture of Chlorella sp. FACHB-5 were added to the BG11 medium with reduced nitrogen source concentration at an initial inoculum concentration of 0.4 g / L. The waste gas of lactic acid bacteria fermentation (N277.4%, O26.3%, CO214.4%, volatile organic matter 0.2%) without pretreatment and purification was introduced into the inoculated microalgae culture system at a constant rate of 0.10 vvm. The pH was 7.5, 28°C, and the light intensity (light-dark cycle 12 h:12 h, light intensity 10000) was 100 μg / L. Lux) for 14 days, the culture solution was centrifuged at 1500g, the microalgae were collected (removed), and the supernatant produced by centrifugation was used as water for preparing BG11 culture medium (deionized water was added to the original culture volume, and the added water did not exceed 1 / 10 of the original volume), and a new round of cultivation was started by inoculating the same amount of Chlorella seeds as in the previous round (without inoculating Bacillus subtilis C1 seeds), and the above operation was repeated for 2 consecutive rounds.

[0055] Depend on Figure 4 Analysis shows that when microalgae are used to culture (oil production) and treat lactic acid bacteria fermentation waste gas, the CO2 concentration change at the outlet of the waste gas treated by microalgae on the 1st to 7th day is basically the same as that of the microalgae reproduction stage in Example 3. This is because the nitrogen source concentration in the culture solution at this stage is relatively sufficient, and the reproduction of microalgae is the main factor. However, the CO2 concentration at the outlet of the waste gas treated by microalgae on the 8th to 14th day of microalgae culture in Comparative Example 1 is basically maintained at about 11.3%, which is only 21.5% lower than the initial concentration, and is quite different from the CO2 concentration change in the oil production stage of Example 3. Comparative Example 1, under the condition of no multifunctional nitrogen-fixing bacteria, after the first round of microalgae culture, the wet weight of the microalgae was only 4.2 g / L, and the oil content of the microalgae was only 9.5%. The COD and TN contents of the supernatant after centrifugation of the culture solution were 102.3 mg / L and 0.1 mg / L, respectively. The supernatant produced by centrifugation was used as water for preparing BG11 culture medium and inoculated with microalgae to start the second round of culture. After about 5 days of culture, the microalgae showed signs of death such as yellowing and precipitation, and the second round of culture failed.

[0056] Through comparative example 1, especially the comparison with example 3, it is shown that the multifunctional nitrogen-fixing bacteria provided by the present invention can decompose the secretions of microalgae, especially the polysaccharides in the extracellular polymers and the volatile organic matter from the fermentation waste gas, when co-cultured with microalgae, and use them as carbon sources. At the same time, it can also decompose the organic phosphorus in the extracellular polymers into soluble phosphorus sources for reuse by microalgae and nitrogen-fixing bacteria, which is equivalent to achieving purification of the culture solution in the oil production stage, so that the centrifugal supernatant after collecting (removing) the co-cultured bacteria can be reused as culture water up to about 5 times. In addition, after the nitrogen source in the culture medium is consumed, the nitrogen-fixing bacteria can convert N2 in the waste gas and continue to provide nitrogen source for the microalgae, and due to the balance between nitrogen fixation and nitrogen source utilization, the total nitrogen content in the culture solution in the oil production stage is stably maintained at a low level (1.0~1.2mg / L), which is conducive to the accumulation of oil in microalgae when the nitrogen source is limited (the oil content of the microalgae increases to more than 40%).

Claims

1. A use of nitrogen-fixing bacteria to assist microalgae in oil production, characterized in that: The nitrogen-fixing bacteria is Bacillus subtilis subtilis C1 was deposited in the China Center for Type Culture Collection on September 17, 2021, with the deposit number CCTCC NO: M20211184, and the deposit address is Wuhan University, Wuhan, China.

2. The use according to claim 1, characterized in that The process of microalgae cultivation and oil production includes the microalgae reproduction stage and the nitrogen-fixing bacteria and microalgae co-cultivation oil production stage.

3. The use according to claim 2, characterized in that: The microalgae include any one or more combinations of Cladophora, Chlorella, Closterium, Oocystis, and Pelagicola.

4. The use according to claim 1, characterized in that The use of nitrogen-fixing bacteria to assist microalgae in oil production and simultaneously purify industrial fermentation waste gas.

5. The use according to claim 4, characterized in that The industrial fermentation waste gas includes CO2, O2, N2 and a small amount of volatile organic matter.

6. The use according to any one of claims 1 to 5, characterized in that: The nitrogen-fixing bacteria assists the microalgae in oil production by reducing the amount of nitrogen source required in the microalgae reproduction stage, so that the required nitrogen source concentration is below 600 mg / L.

7. The use according to claim 6, characterized in that In the use of reducing the amount of nitrogen source required for the reproduction stage of microalgae, nitrogen-fixing bacteria convert N2 in the exhaust gas and continuously provide a nitrogen source for the microalgae.

8. A method for nitrogen-fixing bacteria to assist microalgae in oil production, characterized in that: The steps of nitrogen-fixing bacteria-assisted microalgae oil production include the following: (1) In the propagation stage, the wet algae cells were inoculated into the BG11 culture medium, and the industrial fermentation waste gas was introduced into the inoculated microalgae culture system at a ventilation ratio of 0.05-0.15 vvm. The microalgae were cultured under light conditions to increase the concentration of the microalgae cells in the culture medium. (2) In the oil production stage, Bacillus subtilis subtilis The C1 wet bacteria are added to the culture system of step (1) and continue to be cultured under light conditions. When the oil content of the microalgae increases to a stable value, the supernatant obtained by centrifugation is used to prepare water for the BG11 culture medium. The propagation stage of step (1) and the oil production stage of step (2) are repeated to achieve the cultivation and oil production of the microalgae.

9. The method for producing oil by microalgae assisted by nitrogen-fixing bacteria according to claim 8, characterized in that: Bacillus subtilis subtilis The mass ratio of the C1 wet bacteria to the microalgae wet bacteria in step (1) is 1:1 to 1:

100.

10. The method for producing oil by microalgae assisted by nitrogen-fixing bacteria according to claim 8, characterized in that: The industrial fermentation waste gas comes from one or more combinations of fermentation tail gas generated in the production process of dry yeast, fermentation tail gas generated in the production of neomycin sulfate, and fermentation tail gas generated in the production of active lactic acid bacteria for feed additives.

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