Artificially-enhanced efficient anaerobic hydrogen production system and construction method thereof

Abstract

The invention belongs to the technical field of anaerobic biological fermentation hydrogen production, and particularly relates to an artificial intensified efficient anaerobic hydrogen production system and a construction method thereof. The artificially-enhanced efficient anaerobic hydrogen production system is constructed by adding artificially-synthesized flora from an external source. The artificially synthesized bacterial flora mainly comprises Klebsiella sp., Clostridium beijerinckii, Enterococcus faecalis, Ethanobacter haarabinensis and Clostridium pasteurianum, and the effective viable count of each strain in the artificially synthesized bacterial flora is more than 1 * 10 < 8 > CFU / mL, and the effective viable count of each strain in the artificially synthesized bacterial flora is more than 1 * 10 < 8 > CFU / mL, and the effective viable count of each strain in the artificially synthesized bacterial flora is more than 1 * 10 < 8 > CFU / mL, or more than 1 * 10 < 8 > CFU / mL, or more than 1 * 10 < 8 > CFU / mL or more than 1 * 10 < 8 > CFU / mL or more than 1 * 10 < 8 > CFU / mL. The artificially-enhanced efficient anaerobic hydrogen production system disclosed by the invention has rich efficient anaerobic hydrogen production bacteria and can realize efficient hydrogen production. The construction method of the artificially enhanced efficient anaerobic hydrogen production system has the advantages of simple process, easiness in industrial mass production and use and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of anaerobic biological fermentation hydrogen production, and particularly relates to an artificially enhanced high-efficiency anaerobic hydrogen production system and a construction method thereof. Background Art

[0002] Biohydrogen production technology is mainly divided into photosynthetic hydrogen production and anaerobic fermentation hydrogen production. Photosynthetic hydrogen production relies on light energy and is carried out by photosynthetic bacteria or algae under light conditions, while anaerobic fermentation hydrogen production uses the microbial fermentation process under anaerobic conditions to produce hydrogen. Compared with photosynthetic hydrogen production, anaerobic fermentation hydrogen production does not rely on light, has the advantages of simple operation, low energy consumption, and the use of a variety of organic wastes as substrates. The key to anaerobic fermentation hydrogen production lies in the selection and cultivation of hydrogen-producing microorganisms. However, in practical applications, the number of commonly used hydrogen-producing bacteria in the natural environment is limited, and the hydrogen production efficiency is not high, which is difficult to meet the needs of large-scale industrial production. In order to improve the hydrogen production efficiency of anaerobic fermentation, researchers have adopted a variety of methods, including optimizing fermentation conditions, screening and domesticating efficient hydrogen-producing strains, genetically engineering hydrogen-producing bacteria, and exogenously adding anaerobic hydrogen-producing bacteria. Among them, artificial bioaugmentation technology can significantly improve the hydrogen production efficiency in a relatively short period of time by exogenously adding efficient anaerobic hydrogen-producing bacteria. This method can not only overcome the limitation of the limited number of natural bacteria, but also achieve higher hydrogen production by regulating the fermentation environment. Therefore, the development of an artificially enhanced high-efficiency anaerobic hydrogen production system and its construction method has important theoretical significance and practical application value. Summary of the invention

[0003] Based on the above problems, the present invention provides an artificially enhanced high-efficiency anaerobic hydrogen production system, which can enrich the diversity and richness of anaerobic hydrogen-producing bacteria and achieve efficient and rapid hydrogen production.

[0004] Another object of the present invention is to provide a method for constructing the above-mentioned artificially enhanced high-efficiency anaerobic hydrogen production system.

[0005] In order to achieve the above invention object, the specific technical scheme of the present invention is as follows:

[0006] An artificially synthesized bacterial community mainly includes Klebsiella sp., Clostridum beijerinckii, Enterococcus lactis, Ethanoligenens harbinense, and Clostridium pasteurianum. The volume ratio of each bacterial species in the artificially synthesized bacterial community is 1-5:1-5:1-5:1-5:1-5, and the total effective viable bacteria count in the bacterial agent is 1×10 8 CFU / mL and above.

[0007] Preferably, the volume ratio of Klebsiella sp., Clostridum beijerinckii, Enterococcus lactis, Ethanoligenens harbinense, and Clostridium pasteurianum in the artificial synthetic bacterial flora is 1:1:1:1:1:1.

[0008] Preferably, the deposit number of Klebsiella sp. in the China Industrial Microorganism Culture Collection Center is CICC 24307, the deposit number of Clostridum beijerinckii in the China Industrial Microorganism Culture Collection Center is CICC 22954, the deposit number of Enterococcus lactis in the China Committee for the Collection of Microorganisms General Microorganism Center is GCMCC 1.15321, the deposit number of Ethanoligenens harbinense in the China Committee for the Collection of Microorganisms General Microorganism Center is GCMCC 1.5033, and the deposit number of Clostridium pasteurianum in the China Industrial Microorganism Culture Collection Center is CICC 10391, all of which are commercially available products.

[0009] A method for constructing an artificial synthetic bacterial community comprises activating and culturing five bacteria, namely Klebsiella sp., Clostridum beijerinckii, Enterococcus lactis, Ethanoligenens harbinense and Clostridium pasteurianum, under anaerobic conditions, and mixing and fermenting the bacteria according to a volume ratio to obtain an artificial synthetic bacterial community mother liquor after the bacteria grow to the end of a logarithmic phase.

[0010] Preferably, the components of the fermentation medium are: cellulose 10g / L, hemicellulose 5g / L, glucose 10g / L, xylose 5g / L, peptone 4g / L, yeast powder 1g / L, L-cysteine ​​0.5g / L, beef extract 5g / L, NaCl 4g / L, K2HPO41.5g / L, MgC12 0.1g / L, FeSO4 0.1g / L.

[0011] Preferably, the activation culture time is 3-6 days.

[0012] Preferably, the mixed fermentation conditions are as follows: pH 5.0-6.8, temperature 30-37° C., and fermentation culture time 2 days.

[0013] Preferably, the inoculation amount of the logarithmic phase bacterial solutions of the five bacteria mixed in a volume ratio is 1-10%.

[0014] A method for artificially enhancing efficient anaerobic hydrogen production, in which an artificially synthesized bacterial flora mother liquor is pumped into the reactor from the bottom of the reactor by a peristaltic pump in a running continuous flow anaerobic reactor at regular intervals every day, and the amount of the artificially synthesized bacterial flora mother liquor added each time is 1 / 4 to 1 / 6 of the effective volume of the reactor, until Klebsiella sp., Clostridum beijerinckii, Enterococcus lactis, Ethanoligenens harbinense, and Clostridium pasteurianum are successfully settled.

[0015] Preferably, the feed composition of the reactor is 10 g / L cellulose, 5 g / L hemicellulose, 1 g / L yeast powder, 0.5 g / L L-cysteine, 4 g / L NaCl, 1.5 g / L K2HPO4, 0.1 g / L MgC12, and 0.1 g / L FeSO4.

[0016] Preferably, the operating conditions of the reactor are: temperature of 30-37° C. and hydraulic retention time of 1 day.

[0017] The beneficial effects of the present invention are as follows: the artificial enhanced high-efficiency anaerobic hydrogen production system of the present invention has abundant high-efficiency anaerobic hydrogen-producing bacteria, which can achieve high-efficiency hydrogen production, and the hydrogen production rate of the tank volume can reach 8m 3 / m 3 The method for constructing the artificial enhanced high-efficiency anaerobic hydrogen production system of the present invention has the advantages of simple process and easy industrial mass production. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments, but this should not be understood as the scope of the above-mentioned subject matter of the present invention being limited to the following embodiments. Without departing from the above-mentioned technical ideas of the present invention, various substitutions and changes are made according to common technical knowledge and customary means in the art, which should all be included in the scope of the present invention.

[0019] Example 1: Screening of artificially synthesized bacterial strains

[0020] The inventors screened a variety of hydrogen-producing bacteria and selected five bacteria, including Klebsiella sp., Clostridum beijerinckii, Enterococcus lactis, Ethanoligenens harbinense, and Clostridium pasteurianum, which have fast utilization rates of cellulose, hemicellulose, glucose and xylose and high hydrogen yields for construction.

[0021] The five bacteria used in this embodiment are: Klebsiella sp., with a deposit number of CICC 24307 at the China Industrial Microbiological Culture Collection Center, Clostridum beijerinckii, with a deposit number of CICC 22954 at the China Industrial Microbiological Culture Collection Center, Enterococcus lactis, with a deposit number of GCMCC 1.15321 at the China Committee for the Collection of Microorganisms, General Microbiological Center, Ethanoligenens harbinense, with a deposit number of GCMCC 1.5033 at the China Committee for the Collection of Microorganisms, General Microbiological Center, and Clostridium pasteurianum, with a deposit number of CICC 10391 at the China Industrial Microbiological Culture Collection Center, all of which are commercially available products.

[0022] Example 2: Construction of artificial synthetic bacterial flora

[0023] (1) Klebsiella sp., Clostridum beijerinckii, Enterococcus lactis, Ethanoligenens harbinense, and Clostridium pasteurianum in Example 1 were activated and cultured under anaerobic conditions. When the bacteria grew to the end of the logarithmic phase, they were mixed and fermented in a volume ratio of 1:1:1:1:1 to obtain an artificial synthetic bacterial flora mother liquid.

[0024] (2) A 100 mL serum bottle was used as an anaerobic fermentation bottle, and 60 mL of culture medium was added. The bottle was stripped with nitrogen for more than 10 min to provide an oxygen-free environment, and then sterilized at 121°C for 20 min.

[0025] The components of the fermentation medium are: cellulose 10g / L, hemicellulose 5g / L, glucose 10g / L, xylose 5g / L, peptone 4g / L, yeast powder 1g / L, L-cysteine ​​0.5g / L, beef extract 5g / L, NaCl 4g / L, K2HPO4 1.5g / L, MgC120.1g / L, FeSO4 0.1g / L.

[0026] (3) The total inoculation volume is 10% of the culture medium, i.e. 6 mL. The mother solution of the artificially synthesized bacterial flora is added into the anaerobic fermentation bottle using a syringe.

[0027] (4) Anaerobic fermentation conditions: pH 5.0-6.8, temperature 30-37°C, fermentation culture time 2 days, daily use of syringes to record gas production, and gas chromatography to determine gas composition. Experiments show that the maximum cumulative hydrogen production of the artificial synthetic bacterial community is 190mL / 100mL, and the hydrogen content is 55.63%, which is significantly higher than the hydrogen production effect of a single bacterial species (the maximum cumulative hydrogen production of each single bacterial species is between 90 and 130mL / 100mL, and the hydrogen content is between 38 and 42%).

[0028] Example 3: An example of an artificially enhanced continuous flow anaerobic hydrogen production system

[0029] (1) The effective volume of the continuous flow anaerobic reactor is 4 L, the fermentation temperature is 37 °C, and the hydraulic retention time is 1 day.

[0030] The feed ingredients were 10 g / L cellulose, 5 g / L hemicellulose, 1 g / L yeast powder, 0.5 g / L L-cysteine, 4 g / L NaCl, 1.5 g / L K2HPO4, 0.1 g / L MgC12, and 0.1 g / L FeSO4.

[0031] (2) In the above-mentioned continuous flow anaerobic reactor, the artificial synthetic bacterial mother liquor constructed in Example 2 is pumped into the reactor from the bottom of the reactor through a peristaltic pump at regular intervals every day. The amount of artificial synthetic bacterial mother liquor added each time is one-fifth of the effective volume of the reactor, that is, 800 mL. The artificial synthetic bacterial mother liquor is added in a cycle of three weeks: one week of addition is followed by a two-week interval. After each cycle, the 16S rRNA gene high-throughput sequencing technology is used to monitor the microbial community in the reactor. If five bacteria, namely Klebsiella sp., Clostridum beijerinckii, Enterococcus lactis, Ethanoligenens harbinense, and Clostridium pasteurianum, are detected as dominant bacteria, the customization is considered successful. A wet gas flowmeter is used to record the gas production at regular intervals every day, and a gas chromatograph is used to determine the gas composition. After successful placement, the hydrogen content can reach more than 55%, and the hydrogen production rate per cell volume can reach 8m 3 / m 3 / d.

Claims

1. An artificially synthesized bacterial community, characterized in that: It includes Klebsiella sp., Clostridum beijerinckii, Enterococcus lactis, Ethanoligenens harbinense, and Clostridium pasteurianum, and the volume ratio of each species in the artificial synthetic bacterial community is 1~5:1~5:1~5:1~5:1~5:1~5.

2. The artificial bacterial flora according to claim 1, characterized in that: The volume ratio of Klebsiella, Clostridium beijerinckii, Enterococcus faecalis, Harbin ethanol Bacillus, and Clostridium pasteurianum in the artificially synthesized bacterial community is 1:1:1:1:1; Preferably, the deposit number of Klebsiella in the China Industrial Microorganism Culture Collection Center is CICC 24307, the deposit number of Clostridium beijerinckii in the China Industrial Microorganism Culture Collection Center is CICC 22954, the deposit number of Enterococcus faecalis in the China Committee for the Collection of Microorganisms General Microorganism Center is GCMCC 1.15321, the deposit number of Harbin Ethanol Bacillus in the China Committee for the Collection of Microorganisms General Microorganism Center is GCMCC 1.5033, and the deposit number of Clostridium pasteurianum in the China Industrial Microorganism Culture Collection Center is CICC 10391.

3. The artificial synthetic bacterial flora according to claim 1 or 2, characterized in that: The number of effective living bacteria in the artificially synthesized bacterial community is 1×10 8 CFU / mL and above.

4. The method for constructing an artificial synthetic bacterial community according to claim 1, 2 or 3, characterized in that: Five bacteria, namely Klebsiella sp., Clostridum beijerinckii, Enterococcus lactis, Ethanoligenens harbinense and Clostridium pasteurianum, were activated and cultured under anaerobic conditions, and after the bacteria grew to the end of the logarithmic phase, they were mixed and fermented according to a volume ratio to obtain an artificial synthetic bacterial flora mother liquid; The inoculation amount of the logarithmic phase bacterial solutions of the five bacteria mixed according to the volume ratio is 1-10%.

5. The method for constructing an artificial synthetic bacterial community according to claim 4, characterized in that: The components of the fermentation medium are: 10 g / L cellulose, 5 g / L hemicellulose, 10 g / L glucose, 5 g / L xylose, 4 g / L peptone, 1 g / L yeast powder, 0.5 g / L L-cysteine, 5 g / L beef extract, 4 g / L NaCl, 1.5 g / L K2HPO4, 0.1 g / L MgC12, and 0.1 g / L FeSO4.

6. The method for constructing an artificial synthetic bacterial community according to claim 4, characterized in that: The activation culture time is 3-6 days.

7. The method for constructing an artificial synthetic bacterial community according to claim 4, characterized in that: Anaerobic fermentation conditions: pH 5.0-6.8, temperature 30~37℃, fermentation culture time 2d.

8. The method for constructing an artificial synthetic bacterial community according to claim 4, characterized in that: The inoculation volume of the logarithmic phase bacterial solution of the three bacteria is 1-10%.

9. A method for artificially enhancing efficient anaerobic hydrogen production, characterized in that: In a running continuous flow anaerobic reactor, the artificial synthetic bacterial flora according to any one of claims 1 to 3 is pumped into the reactor from the bottom of the reactor by a peristaltic pump at regular intervals every day, and the amount of the artificial synthetic bacterial flora added each time is 1 / 4 to 1 / 6 of the effective volume of the reactor, until Klebsiella sp., Clostridum beijerinckii, Enterococcus lactis, Ethanoligenens harbinense, and Clostridium pasteurianum are successfully settled.

10. The method for artificially enhancing efficient anaerobic hydrogen production according to claim 9, characterized in that: The feed composition of the reactor was cellulose 10 g / L, hemicellulose 5 g / L, yeast powder 1 g / L, L-cysteine ​​0.5 g / L, NaCl 4 g / L, K2HPO4 1.5 g / L, MgC12 0.1 g / L, FeSO4 0.1 g / L; Preferably, the operating conditions of the reactor are: temperature of 30-37° C. and hydraulic retention time of 1 day.