Method for producing hydrogen and single-cell protein through bacteria-algae co-culture

Abstract

The invention provides a method for producing hydrogen and single-cell protein through bacteria-algae co-culture, which comprises the following steps: artificially constructing a bacteria-algae co-culture system through aerobic bacteria with an aerobic effect and hydrogen-producing microalgae with a hydrogen-producing and oxygen-producing effect, and consuming oxygen in the system through the aerobic bacteria so as to promote the hydrogen-producing microalgae to produce hydrogen, so that the hydrogen-producing microalgae can produce hydrogen; meanwhile, single-cell protein products are generated, co-production of hydrogen and protein is achieved, phycomycetes protein obtained through co-culture can also be used as animal feed and aquatic bait and even serve as a protein source of human food, the production cost is greatly reduced, and benefits are improved. The bacteria-algae co-culture system does not need to add an expensive and toxic interference agent which easily pollutes the environment, the operation is simple, and the workload and the technical difficulty are far lower than those of mutant strain screening and gene modification.

Description

Technical Field

[0001] The invention belongs to the technical field of bioenergy and relates to a method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae. Background Art

[0002] Hydrogen energy is a green, efficient and alternative energy source. The use of hydrogen energy is of great significance to building a clean and low-carbon energy system and achieving the "dual carbon" goal. Since hydrogen energy is a secondary energy source that needs to be produced by consuming primary energy sources such as coal, natural gas, and renewable energy, the cleanliness of hydrogen energy depends on how it is produced.

[0003] Compared with other existing hydrogen production methods, photobiological hydrogen production by algae has obvious advantages. Advantage 1: Algae itself has excellent light collection and energy conversion efficiency (Energy Conversion Efficiency, ECE). Biohydrogen (BioH2) is produced by algae through photosynthesis and is widely regarded as a promising and sustainable source of green hydrogen energy, playing a vital role in renewable energy generation and sustainable development. In theory, the ECE of BioH2 can reach 13.4%. Advantage 2: Photobiological hydrogen production by algae is a sustainable method because it relies on the natural process of photosynthesis. Algae can be cultivated using sunlight, water and carbon dioxide, making it a renewable and environmentally friendly source of hydrogen. Advantage 3: Algae are easy to cultivate on a large scale, and the large amount of hydrogen produced can be used by humans to meet the growing demand for clean energy. In traditional algae photosynthetic hydrogen production methods, it is usually necessary to frequently replace the culture medium or add expensive and environmentally polluting interfering agents, which poses a great challenge to the scale-up of hydrogen production technology.

[0004] Bacteria-algae co-cultivation has attracted much attention as a green hydrogen production method. Green algae directly utilize solar energy and water to produce hydrogen through photosynthesis system and hydrogen-producing enzymes. At present, the research on photosynthetic hydrogen production by algae-bacteria co-cultivation is mainly focused on freshwater green algae, especially Chlamydomonas reinhardtii, which has the characteristics of high hydrogenase activity, clear photosynthetic mechanism and easy cultivation. It is the main model strain of microalgae for hydrogen production. However, due to factors such as competition for nutrients or system instability, the synergy between different strains and algae is always not ideal, which affects the overall hydrogen production. In addition, the biomass produced by some existing bacteria-algae co-cultivation systems cannot be effectively utilized, which leads to increased technical costs. Summary of the invention

[0005] The purpose of the present invention is to provide a method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae, so as to solve the problems that the overall hydrogen production is low and the generated biomass cannot be effectively utilized in the existing bacteria and algae co-cultivation process.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In the present application, a bacteria-algae co-culture system is artificially constructed by using aerobic bacteria and hydrogen-producing microalgae that have strong complementarity and less demand for the same nutrients to reduce competition. At the same time, the symbiotic advantages are utilized to improve the efficiency of photosynthetic hydrogen production by green algae, while producing single-cell protein products, achieving the co-production of hydrogen and protein, and greatly improving efficiency.

[0007] In this application, hydrogen-producing microalgae that can use carbon dioxide as a carbon source are selected, and their oxygen production rate is controlled by light intensity; aerobic bacteria that can use glucose as a carbon source are selected, and the oxygen produced by the hydrogen-producing microalgae is used for respiration to maintain the anaerobic system and promote hydrogen production by the hydrogen-producing microalgae. At the same time, the concentration of glucose is used to control its respiration rate. On this basis, combination screening and testing are carried out to find the best bacteria and algae combination.

[0008] Specifically, the method for producing hydrogen and single-cell protein by co-culturing bacteria and algae provided in the present application includes: S01: After the aerobic bacteria are expanded and cultured, they are inoculated into the activated and expanded hydrogen-producing microalgae to obtain a microbial mixture.

[0009] After the hydrogen-producing microalgae were activated, they were inoculated into TAP medium. The hydrogen-producing microalgae were placed in a light incubator at a temperature of 26°C and a light intensity of 30-50 μmol•m -2 •s -1 The flask was shaken 3-5 times a day until the end of the logarithmic phase to form a seed algae solution and complete the expansion culture. The composition of the TAP medium used for hydrogen-producing microalgae is shown in Table 1.

[0010] Table 1: TAP medium components used for hydrogen-producing microalgae The hydrogen-producing microalgae in this application is selected from green algae. Green algae has high photosynthesis efficiency and hydrogen production enzyme activity, and can use light energy to effectively decompose water to produce hydrogen. At the same time, some green algae also have specialized hydrogen production mechanisms, which can stably produce hydrogen in anoxic environments. The green algae in this application are selected from Chlamydomonas reinhardtii, Chlorella vulgaris or Tetraselmis. Chlamydomonas reinhardtii, Chlorella vulgaris or Tetraselmis have all been proven to be safe for use and have been certified.

[0011] The aerobic bacteria were inoculated into the culture medium and cultured at 37°C until the end of the logarithmic phase, which was used as the seed culture to complete the expansion culture. After the culture was completed, the aerobic bacteria were inoculated into the hydrogen-producing microalgae to obtain a microbial mixture. The concentration of aerobic bacteria was OD600 = 0.138-0.6, and the final concentration of hydrogen-producing microalgae was 4.6*10 6 -2*10 7 cells / mL.

[0012] In the present application, the culture medium for aerobic bacteria is LB, TAP or GTAP culture medium, wherein the GTAP culture medium is TAP culture medium with 50 mM glucose added thereto.

[0013] In the present application, aerobic bacteria include other aerobic / facultative aerobic feed / edible protein strains such as Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus laterosporus, Bacillus lentus, Bacillus brevis, Aspergillus niger, Aspergillus oryzae, Rhodopseudomonas palustris, Saccharomyces cerevisiae, Candida utilis or Lactobacillus plantarum. Among them, Bacillus subtilis grows and reproduces fast, and its physiological functions have multiple benefits in agriculture, and it has no toxic effects and other side effects on the animal body. Adding Bacillus subtilis to livestock and poultry feed can regulate the intestines of livestock and poultry to more fully digest nutrients. At the same time, Bacillus subtilis as an intestinal probiotic can also inhibit the growth of harmful microbial flora. Bacillus subtilis will secrete secondary metabolites, including lipopeptides, antibiotics, surfactants and other molecules with antifungal effects, to inhibit the growth of some pathogens during growth. In addition, the biomass of Bacillus subtilis can also be used as single-cell protein, and has obtained domestic feed strain certification.

[0014] S02: The microbial mixture is heated at 26°C and the hydrogen production light intensity is 100-130 μmol•m -2 •s -1 The cells were cultured in a medium supplemented with glucose mother solution under the same conditions, and the produced hydrogen and single cell protein products were collected.

[0015] The microbial mixture was heated at 26°C and the hydrogen production light intensity was 100-130 μmol•m -2 •s -1 The cells were cultured in a medium supplemented with glucose mother solution under the same conditions and manually shaken 5-8 times a day during the culture process to collect the generated hydrogen and single-cell protein products.

[0016] The present invention has the following beneficial effects: (1) The present application artificially constructs a bacteria-algae co-culture system by using aerobic bacteria with aerobic effects and hydrogen-producing microalgae with hydrogen-producing and oxygen-producing effects. The oxygen in the system is consumed by aerobic bacteria, thereby promoting the production of hydrogen by the hydrogen-producing microalgae and producing single-cell protein products at the same time, thereby achieving the co-production of hydrogen and protein and greatly improving the efficiency.

[0017] (2) The bacteria-algae co-culture system in this application does not require the addition of expensive, toxic and environmentally polluting interfering agents. It is easy to operate, and the workload and technical difficulty are far less than mutant strain screening and genetic modification.

[0018] (3) The bacterial-algal co-culture system in this application can achieve the co-production of hydrogen and protein, which not only obtains clean energy but also recovers single-cell protein raw materials. At the same time, the algae protein obtained by co-culture can also be used as animal feed, aquatic bait, and even as a protein source for human food, which has the advantage of significantly reducing the overall production cost. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0020] Example 1 The present application provides a method for producing hydrogen and single-cell protein by co-culturing bacteria and algae, the method comprising: S101: After Chlamydomonas reinhardtii was activated, it was inoculated into TAP medium. The hydrogen-producing microalgae were placed in a light incubator at a temperature of 26°C and a light intensity of 30 μmol•m -2 •s -1 The flask was shaken 3 times a day for 24 hours under continuous light until the end of the logarithmic phase to form a seed algae solution and complete the expansion culture. Bacillus subtilis was inoculated into LB medium and cultured at 37°C until the end of the logarithmic phase to form a seed bacterial solution and complete the expansion culture.

[0021] 3 ml of seed bacterial solution was centrifuged and resuspended in 3 ml of sterilized TAP medium. At the same time, 40 ml of seed algae solution was centrifuged and resuspended in 40 ml of sterilized TAP medium. After the two TAP mediums were mixed, 6 ml of sterilized TAP medium and 1 ml of 250 mM glucose mother solution were added to form a 50 ml microbial mixture.

[0022] S102: Microbial mixture at 26°C and hydrogen production light intensity of 100 μmol•m -2 •s -1 The culture was carried out in a closed environment and manually shaken 8 times a day during the culture process to collect the generated hydrogen and single-cell protein products.

[0023] Example 2 The present application provides a method for producing hydrogen and single-cell protein by co-culturing bacteria and algae, the method comprising: S201: After Chlamydomonas reinhardtii was activated, it was inoculated into TAP medium. The hydrogen-producing microalgae were placed in a light incubator at a temperature of 26°C and a light intensity of 50 μmol•m -2 •s -1 The light was continuously illuminated for 24 hours, and the flask was shaken 5 times a day until the end of the logarithmic phase to form a seed algae solution and complete the expansion culture. Bacillus subtilis was inoculated into LB medium and cultured at 37°C until the end of the logarithmic phase to form a seed bacterial solution and complete the expansion culture.

[0024] 3 ml of seed bacterial solution was centrifuged and resuspended in 9 ml of sterilized TAP medium, and 40 ml of seed algae solution and 1 ml of 250 mM glucose mother solution were added to form 50 ml of microbial mixture.

[0025] S202: Microbial mixture at 26°C, hydrogen production light intensity of 130 μmol•m -2 •s -1 The culture was carried out in a closed environment and manually shaken 5 times a day during the culture process to collect the generated hydrogen and single-cell protein products.

[0026] Example 3 The present application provides a method for producing hydrogen and single-cell protein by co-culturing bacteria and algae, the method comprising: S301: After Chlamydomonas reinhardtii was activated, it was inoculated into TAP medium. The hydrogen-producing microalgae were placed in a light incubator at a temperature of 26°C and a light intensity of 40 μmol•m -2 •s -1 The light was continuously illuminated for 24 hours, and the flask was shaken 4 times a day until the end of the logarithmic phase to form a seed algae solution and complete the expansion culture. Bacillus subtilis was inoculated into LB medium and cultured at 37°C until the end of the logarithmic phase to form a seed bacterial solution and complete the expansion culture.

[0027] Mix 3 ml of seed bacterial solution, 40 ml of seed algae solution, 6 ml of sterilized TAP medium and 1 ml of 250 mM glucose mother solution to form a microbial mixture.

[0028] S302: Microbial mixture at 26°C, hydrogen production light intensity of 120 μmol•m -2 •s -1 The culture was carried out in a closed environment and manually shaken 7 times a day during the culture process to collect the generated hydrogen and single-cell protein products.

[0029] Example 4 The present application provides a method for producing hydrogen and single-cell protein by co-culturing bacteria and algae, the method comprising: S401: After Chlamydomonas reinhardtii was activated, it was inoculated into TAP medium. The hydrogen-producing microalgae were placed in a light incubator at a temperature of 26°C and a light intensity of 40 μmol•m -2 •s -1 The flask was shaken 4 times a day for 24 hours under continuous light until the end of the logarithmic phase to form a seed algae solution and complete the expansion culture. Bacillus subtilis was inoculated into GTAP medium and cultured at 37°C until the end of the logarithmic phase to form a seed bacterial solution and complete the expansion culture.

[0030] Mix 2.7 ml of seed bacterial solution, 40 ml of seed algae solution, 6.3 ml of sterilized TAP medium and 1 ml of 250 mM glucose mother solution to form a microbial mixture.

[0031] S402: Microbial mixture at 26°C, hydrogen production light intensity of 120 μmol•m -2 •s -1 The culture was carried out in a closed environment and manually shaken 7 times a day during the culture process to collect the generated hydrogen and single-cell protein products.

[0032] Example 5 The present application provides a method for producing hydrogen and single-cell protein by co-culturing bacteria and algae, the method comprising: S501: After Chlamydomonas reinhardtii was activated, it was inoculated into TAP medium. The hydrogen-producing microalgae were placed in a light incubator at a temperature of 26°C and a light intensity of 40 μmol•m -2 •s -1 The flask was shaken 4 times a day for 24 hours under continuous light until the end of the logarithmic phase to form a seed algae solution and complete the expansion culture. Bacillus subtilis was inoculated into TAP medium and cultured at 37°C until the end of the logarithmic phase to form a seed bacterial solution and complete the expansion culture.

[0033] Mix 6.8 ml of seed bacterial solution, 40 ml of seed algae solution, 2.2 ml of sterilized TAP medium and 1 ml of 250 mM glucose mother solution to form a microbial mixture.

[0034] S502: Microbial mixture at 26°C, hydrogen production light intensity of 120 μmol•m -2 •s -1 The culture was carried out in a closed environment and manually shaken 7 times a day during the culture process to collect the generated hydrogen and single-cell protein products.

[0035] Comparative Example 1 In the comparative example of the present application, Chlamydomonas reinhardtii was activated and then inoculated into TAP medium. The hydrogen-producing microalgae was placed in a light incubator at a temperature of 26°C and a light intensity of 40 μmol•m -2 •s -1 Continuous illumination for 24 hours, shake the flask 4 times a day until the end of the logarithmic phase, to form a seed algae solution, complete the expansion culture. Centrifuge 40 ml of the seed algae solution and resuspend it in 49 ml of sterilized TAP medium, add 1 ml of 250 mM glucose mother solution, and incubate at 26 ° C and a hydrogen production light intensity of 120 μmol•m -2 •s -1 The culture was carried out in a closed environment and manually shaken 7 times a day during the culture process to collect the generated hydrogen and single-cell protein products.

[0036] Comparative Example 2 In the comparative example of the present application, Bacillus subtilis was inoculated into LB medium and cultured at 37°C to the end of the logarithmic phase as a seed culture solution to complete the expansion culture. 3 ml of the seed culture solution was centrifuged and resuspended in 49 ml of sterilized TAP medium, 1 ml of 250 mM glucose mother solution was added, and the culture was continued at 26°C and the hydrogen production light intensity was 120 μmol•m -2 •s -1 The culture was carried out in a closed environment and manually shaken 7 times a day during the culture process to collect the generated hydrogen and single-cell protein products.

[0037] Comparative Example 3 In the comparative example of the present application, Chlamydomonas reinhardtii was activated and then inoculated into TAP medium. The hydrogen-producing microalgae was placed in a light incubator at a temperature of 26°C and a light intensity of 30 μmol•m -2 •s -1 The flask was shaken 3 times a day for 24 hours under continuous light until the end of the logarithmic phase to form a seed algae solution and complete the expansion culture. Bacillus subtilis was inoculated into LB medium and cultured at 37°C until the end of the logarithmic phase to form a seed bacterial solution and complete the expansion culture. 3 ml of seed bacterial solution and 40 ml of seed algae solution were used to collect single-cell protein products.

[0038] Comparative Example 4 In the comparative example of the present application, Chlamydomonas reinhardtii was activated and then inoculated into TAP medium. The hydrogen-producing microalgae was placed in a light incubator at a temperature of 26°C and a light intensity of 40 μmol•m -2 •s -1 The flask was shaken 4 times a day under 24-hour continuous light until the end of the logarithmic phase to form a seed algae solution. The algae were collected as single-cell protein products.

[0039] Comparative Example 5 In the comparative example of the present application, Bacillus subtilis was inoculated into LB medium and cultured at 37°C to the end of the logarithmic phase to serve as a seed bacterial liquid. The bacterial cells were collected as a single cell protein product.

[0040] In the examples of the present application, gas chromatography was used to periodically sample and measure the hydrogen content above the culture medium of Examples 1-5 and Comparative Examples 1-5, and Table 2 was obtained.

[0041] Table 2: Hydrogen and protein production data of Examples 1-5 and Comparative Examples 1 and 2 From Table 2, we can see that: (1) By comparing Comparative Examples 1 and 4, it can be seen that the protein content in Comparative Example 1 increased significantly from 40% to 43.37%, which indicates that the hydrogen production process can promote the increase of the protein content of the algae. It is speculated that this is because the hydrogen production process consumes the accumulated glycogen and may also promote the synthesis of a large amount of hydrogen production metabolic functional enzyme proteins.

[0042] (2) Comparison of Examples 1-5 and Comparative Example 3 shows that the biomass concentration, protein content and yield in Examples 1-5 are significantly improved compared with the initial system. This indicates that during the co-cultivation of bacteria and algae, hydrogen production is accompanied by the accumulation of biomass and protein.

[0043] (3) By comparing Example 1 with Comparative Examples 1 and 2, it can be seen that when only Bacillus subtilis was cultured in Comparative Example 2, no hydrogen and protein were produced. When only Chlamydomonas reinhardtii was cultured in Comparative Example 1, the unit hydrogen production was 7.15 ml / l, the protein content was 43.37%, and the protein yield was 0.76 g / l. This is because Chlamydomonas reinhardtii mainly uses carbohydrates as storage materials under aerobic conditions, and the protein content is only about 40%. After anaerobic hydrogen production induction, the partial degradation of glycogen produces hydrogen, which will cause the protein content of the algae itself to increase to 43.37%. When Chlamydomonas reinhardtii and Bacillus subtilis were co-cultured in Example 1, the unit hydrogen production was 9.91 ml / l, the protein content was 47.50%, and the protein yield was 0.79 g / l. This is because Bacillus subtilis has a high protein content, which can be as high as 68.16%. Although Bacillus subtilis cannot produce hydrogen and grow well alone under anaerobic conditions, it can grow in synergy with Chlamydomonas reinhardtii, consume oxygen produced by photosynthesis of Chlamydomonas reinhardtii in situ, maintain the anaerobic environment required for Chlamydomonas reinhardtii to produce hydrogen, and increase the protein content in the co-culture system to 47.5-59.13%. This shows that bacterial-algal co-culture can promote hydrogen production while increasing protein content and yield.

[0044] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae, characterized in that: include: After the aerobic bacteria are expanded and cultured, they are inoculated into the activated and expanded hydrogen-producing microalgae to obtain a microbial mixture; The microbial mixture is heated to 26°C and produces hydrogen at a light intensity of 100-130 μmol•m -2 •s -1 The cells were cultured in glucose mother solution under the following conditions, and the produced hydrogen and single cell protein products were collected.

2. The method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae according to claim 1, characterized in that: The aerobic bacteria include Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus laterosporus, Bacillus lentus, Bacillus pumilus, Aspergillus niger, Aspergillus oryzae, Rhodopseudomonas palustris, Saccharomyces cerevisiae, Candida utilis or Lactobacillus plantarum and Escherichia coli.

3. The method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae according to claim 1, characterized in that: The hydrogen-producing microalgae is green algae.

4. The method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae according to claim 3, characterized in that: The green algae include Chlamydomonas reinhardtii, Chlorella vulgaris or Tetraselmis.

5. The method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae according to claim 1, characterized in that: The concentration ratio of the aerobic bacteria to the hydrogen-producing microalgae is (OD600=0.138-0.6): (4.6*10 6 -2*10 7 cells / mL).

6. The method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae according to claim 1, characterized in that: In the microbial mixture, the concentration of the aerobic bacteria is OD600=0.138-0.

6.

7. The method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae according to claim 1, characterized in that: In the microbial mixture, the final concentration of the hydrogen-producing microalgae is 4.6*10 6 -2*10 7 cells / mL.

8. The method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae according to claim 1, characterized in that: The concentration of the glucose mother solution is 250 mM.

9. The method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae according to claim 1, characterized in that: The culture medium of the hydrogen-producing microalgae is TAP culture medium.

10. The method for producing hydrogen and single-cell protein by co-cultivation of bacteria and algae according to claim 1, characterized in that: The culture medium for the aerobic bacteria is LB, TAP or GTAP culture medium.

Images