CN107937327B

CN107937327B - Bacteria and algae composite preparation and application thereof in preparation of hydrogen

Info

Publication number: CN107937327B
Application number: CN201810080730.5A
Authority: CN
Inventors: 黄永根; 洪迪明; 胡三强; 白雪高; 白健明; 白永炜
Original assignee: Hangzhou Fuyang Youxin Technology Co ltd
Current assignee: HANGZHOU FUYANG YOUXIN TECHNOLOGY Co.,Ltd.
Priority date: 2018-01-28
Filing date: 2018-01-28
Publication date: 2021-05-04
Anticipated expiration: 2038-01-28
Also published as: CN107937327A

Abstract

The invention belongs to the technical field of biological new energy, and discloses a bacteria-algae composite preparation which comprises enterococcus faecium, rhodopseudomonas palustris and scenedesmus obliquus. The invention selects specific bacteria and algae for mixed culture, has good synergistic effect and can improve the yield of hydrogen and the biomass of algae.

Description

Bacteria and algae composite preparation and application thereof in preparation of hydrogen

Technical Field

The invention belongs to the technical field of new energy, and particularly relates to a bacteria-algae compound preparation and application thereof in preparation of hydrogen.

Background

With the development of industrialization and the improvement of the living standard of human beings, the utilization of fossil energy is increased year by year, on one hand, the utilization causes serious environmental pollution, and on the other hand, the fossil energy is non-renewable. Therefore, it is urgent to find a clean and efficient renewable new energy source. Compared with other energy substances, hydrogen has a series of advantages of rich resources, small density, light weight, convenience in transportation, various utilization forms, wide application, high combustion value, cleanness, renewability, water generation after combustion and the like, so that the hydrogen is considered to be the most potential environment-friendly renewable energy source in the future society. With the further development of fuel cell technology, the direct conversion of hydrogen energy into electric energy can be completely realized, and the research on hydrogen production technology has great development potential. Thus, many countries around the world have invested a great deal of research expenditures for the development of hydrogen energy.

At present, the preparation of hydrogen mainly comprises the steps of preparing hydrogen by using fossil raw materials, preparing hydrogen by electrolyzing water, preparing hydrogen by organisms and the like. However, at present, hydrogen generation mainly depends on two ways, namely thermochemistry and photoelectrochemistry, which are not only expensive in production cost, large in energy requirement and serious in environmental pollution, but also emit a large amount of greenhouse gases, so that the method is non-environment-friendly, and the former needs to consume a large amount of valuable non-renewable resources such as coal, petroleum, natural gas and the like: the latter is high in consumption

The amount of electrical energy comes at a cost. In order to enable the hydrogen to be widely applied in the future, the key point is to establish a method for efficiently, simply, quickly and sustainably preparing the hydrogen. Therefore, making hydrogen a clean, renewable energy source in future society faces great challenges. Therefore, the preparation of hydrogen by using low-cost and renewable biomass resources as raw materials is receiving much attention.

Bio-hydrogen production is a new technology with such characteristics. The biological hydrogen-producing group mainly comprises: photosynthetic organisms (anaerobic photosynthetic bacteria, cyanobacteria, and green algae), non-photosynthetic organisms (strictly photosynthetic bacteria, facultative anaerobic bacteria, and aerobic bacteria), and archaeal flora. Because the hydrogen production mechanisms are different, the potential application value of the method is also evaluated differently. Currently, the research of biohydrogen production mainly focuses on photosynthetic bacteria and microalgae. Although the photosynthetic bacteria do not emit oxygen when hydrogen is emitted, the solar energy cannot be directly utilized to produce hydrogen; meanwhile, the application range of the organic matter for hydrogen production is limited due to factors such as the production area and the quantity of the organic matter, and the like, so that the hydrogen source is difficult to provide for human beings on a large scale. The hydrogen production of microalgae (blue algae and green algae) is realized by decomposing water into hydrogen and oxygen by using solar energy through a photosynthesis system and a special hydrogen producing enzyme, so that the hydrogen production method has a good application prospect.

The blue algae and the green algae are different in enzyme for catalyzing hydrogen production, the blue algae is nitrogen fixation enzyme for catalyzing hydrogen production, and energy ATP is consumed in the hydrogen production process, so that the overall hydrogen production efficiency is low. The reversible hydrogen-producing enzyme for catalyzing hydrogen production in green algae takes solar energy as energy and water as raw materials, and the theoretical quantum efficiency of catalyzing hydrogen production is high, so that the research on hydrogen production by green algae is most likely to become the subject of hydrogen production research in the future. Because the hydrogen production mechanisms of green algae are different, more high-hydrogen-yield algae species are obtained, and the hydrogen discharge characteristics of the algae species are researched. The hydrogenase of green algae is extremely sensitive to oxygen and is easily inhibited by oxygen to lose activity, and the oxygen is a specific product of photosynthesis, so that the hydrogen production efficiency of the green algae is low, and the development of the hydrogen production of the green algae is limited to a great extent, so that the content of oxygen in cells of the green algae needs to be reduced when the hydrogen production of the green algae is improved. At present, the method for reducing the oxygen content mainly comprises the steps of removing sulfur elements in a culture medium, so that the oxygen release activity of a photosynthetic system is inhibited, and the oxygen content generated by water photolysis is reduced. Therefore, it is important to develop an algae preparation capable of producing hydrogen with high efficiency.

Disclosure of Invention

The invention aims to overcome the defects of low hydrogen production efficiency of algae in the prior art and the like, and provides a bacteria-algae compound preparation which can greatly improve the output of hydrogen through reasonable compatibility and mutual cooperation of bacteria and algae.

The invention is realized by the following technical scheme:

a bacteria-algae compound preparation comprises enterococcus faecium, Rhodopseudomonas palustris and Scenedesmus obliquus.

In particular, the amount of the solvent to be used,

the fungus and alga compound preparation is prepared according to the following process:

step 1) preparation of fermentation liquor: respectively culturing enterococcus faecium and Rhodopseudomonas palustris by conventional method to obtain 1 × 10⁸Mixing the seed solutions of CFU/ml according to a volume ratio of 1-2:2-3 to obtain a mixed seed solution, transferring the mixed seed solution into a fermentation culture medium according to an inoculation amount of 8-10%, and culturing at 30 ℃ for 12h to obtain a fermentation broth;

step 2) preparing an algae solution: inoculating Scenedesmus obliquus in logarithmic growth phase into TAP culture solution according to the inoculation amount of 5% for culturing to obtain an algae solution;

step 3) preparing hydrogen production culture solution: adding sodium glycerophosphate and linoleic acid glyceride into the TAP culture solution to obtain a hydrogen production culture solution;

step 4), preparing a fungus-algae composite preparation: and uniformly mixing the fermentation liquid, the algae liquid and the hydrogen production culture solution according to the volume ratio of 1-2:15-25:200 and 300 to obtain the hydrogen production culture solution.

Preferably, the first and second electrodes are formed of a metal,

the formula of the fermentation medium comprises the following components in percentage by mass: molasses 3%, corn steep liquor 2.5%, soybean meal 2%, K₂HPO₄0.1％、KH₂PO₄ 0.1％、CaCO₃0.01％、FeSO₄ 0.005％、MnSO₄0.005% and the balance water, pH 7.0.

Preferably, the first and second electrodes are formed of a metal,

the concentration of the sodium glycerophosphate is 0.5-0.8 mg/L.

Preferably, the first and second electrodes are formed of a metal,

the concentration of the linoleic acid glyceride is 0.8-1 mg/L.

Preferably, the first and second electrodes are formed of a metal,

in the step 2), the culture conditions are as follows: the illumination intensity is 100 mu mol.m^-2·s^-1Culturing for 2-3 days at water temperature of 25-28 deg.C and pH of 8 under light-dark ratio of 12h to 12 h.

The starting point and the beneficial effects of the invention mainly comprise but are not limited to the following aspects:

microalgae can usually release substances such as carbohydrates, amino acids, lipids and the like to the environment for bacteria to utilize during growth, meanwhile, the bacteria can provide growth promoting factors such as inorganic nitrogen, phosphorus, carbohydrate substances, vitamins and the like for the algae, and proper algae and bacteria are selected to possibly form a symbiotic relationship;

enterococcus faecium belongs to anaerobic bacteria and can be fermented to produce formate, acetate, organic acid and the like, and only hydrogen and carbon dioxide are produced in the fermentation process; the rhodopseudomonas palustris takes the substances as substrates, so that the hydrogen production effect is good;

the mixed bacteria liquid of the enterococcus faecium and the rhodopseudomonas palustris has good synergistic performance, can grow rapidly under the anaerobic illumination condition and can also grow under the aerobic condition, and the oxygen generated by the algae is effectively utilized, so that the oxygen content is reduced to the minimum, and the better hydrogen production capability is achieved; the carbon dioxide generated by the compound bacterial liquid can meet the requirement of the rapid growth of algae cells, and further the biomass of algae is improved.

The hydrogen yield of the invention is obviously superior to that of the method of singly adopting algae fermentation, the concentration of algae cells is also obviously improved, the oxygen concentration is kept at a reasonable lower level, and the invention is beneficial to keeping the activity of catalase and improving the hydrogen yield; the addition of a proper amount of chemical stimulant in the culture solution can improve the hydrogen production of algae cells and also has an obvious promotion effect on the biomass of the algae.

Drawings

FIG. 1: the influence of the compatibility of the strains on the yield of hydrogen and oxygen.

Detailed Description

Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.

Example 1

Specifically, the fungus-algae compound preparation is prepared according to the following process:

preparing a fermentation liquid: respectively culturing enterococcus faecium and Rhodopseudomonas palustris by conventional method to obtain 1 × 10⁸Mixing the CFU/ml seed solution according to the volume ratio of 2:3 to obtain a mixed seed solution, transferring the mixed seed solution into a fermentation culture medium according to the inoculation amount of 10%, and culturing for 12h at 30 ℃ to obtain a fermentation broth; the formula of the fermentation medium comprises the following components in percentage by mass: molasses 3%, corn steep liquor 2.5%, soybean meal 2%, K₂HPO₄ 0.1％、KH₂PO₄ 0.1％、CaCO₃0.01％、FeSO₄ 0.005％、MnSO₄0.005 percent, the balance being water, and the pH value being 7.0;

preparing an algae solution: collecting Scenedesmus obliquus (concentration of 5 × 10) in logarithmic growth phase⁵One seed/ml) of the cells were inoculated into TAP culture medium at an inoculum size of 5% and cultured with the light intensity controlled at 100. mu. mol. m^-2·s^-1Culturing for 3 days at a light-dark ratio of 12h to 12h, a water temperature of 25-28 deg.C and a pH of 8 to obtain an algae solution;

preparing hydrogen production culture solution: adding sodium glycerophosphate and linoleic acid glyceride into the TAP culture solution, and controlling the concentration of the sodium glycerophosphate to be 0.8mg/L and the concentration of the linoleic acid glyceride to be 1mg/L to obtain a hydrogen production culture solution;

preparing a fungus-algae compound preparation: and uniformly mixing the fermentation liquor, the algae liquid and the hydrogen production culture solution according to the volume ratio of 2:25:300 to obtain the product.

The conditions for preparing hydrogen by using the composite preparation are as follows: introducing N into the reactor₂Keeping an anaerobic environment, then placing the bacteria and algae compound preparation in a reactor, and culturing for more than 96 hours under the culture conditions: the illumination intensity is 100 mu mol.m^-2·s^-1The light-dark ratio is 12h:12 h.

Example 2

preparing a fermentation liquid: respectively culturing enterococcus faecium and Rhodopseudomonas palustris by conventional method to obtain 1 × 10⁸Mixing the CFU/ml seed solution according to the volume ratio of 1:2 to obtain a mixed seed solution, transferring the mixed seed solution into a fermentation culture medium according to the inoculation amount of 8%, and culturing for 12 hours at 30 ℃ to obtain a fermentation broth; the formula of the fermentation medium comprises the following components in percentage by mass: molasses 3%, corn steep liquor 2.5%, soybean meal 2%, K₂HPO₄ 0.1％、KH₂PO₄ 0.1％、CaCO₃0.01％、FeSO₄ 0.005％、MnSO₄0.005 percent, the balance being water, and the pH value being 7.0;

preparing an algae solution: collecting Scenedesmus obliquus in logarithmic growth phase(concentration 5X 10)⁵One seed/ml) of the cells were inoculated into TAP culture medium at an inoculum size of 5% and cultured with the light intensity controlled at 100. mu. mol. m^-2·s^-1Culturing for 2 days at a light-dark ratio of 12h to 12h, a water temperature of 25-28 deg.C and a pH of 8 to obtain an algae solution;

preparing hydrogen production culture solution: adding sodium glycerophosphate and linoleic acid glyceride into TAP culture solution, and controlling the concentration of the sodium glycerophosphate to be 0.5mg/L and the concentration of the linoleic acid glyceride to be 0.8mg/L to obtain hydrogen production culture solution;

preparing a fungus-algae compound preparation: and uniformly mixing the fermentation liquor, the algae liquid and the hydrogen production culture solution according to the volume ratio of 1:15:200 to obtain the product.

Example 3

The hydrogen production performance of the bacteria-algae composite preparation is tested as follows:

setting the liquid volume of the reactor to be 8L, setting the space above the liquid to be 2L, multiplying the gas composition by the volume of the gas to obtain the gas quantity generated in the reaction process, and measuring the gas phase product in the reactor by using a gas chromatography;

taking example 1 as an example, and setting a control group, wherein the ratio of the control group 1: the same procedure as in example 1 was repeated except that no fermentation broth was added; control group 2: the procedure of example 1 was repeated except that the hydrogen-producing culture solution was not supplemented with sodium glycerophosphate and linoleic acid glyceride.

The hydrogen production amount and the oxygen production amount of example 1 and the control groups 1 to 2 were respectively detected; the concentration of algal cells in the culture broth was also determined by cytometry. See table 1 specifically:

TABLE 1

Group of	Hydrogen (L)	Oxygen amount (L)	Algal cell concentration (. times.10)⁶cell/mL）
				Example 1	0.212	0.020	12.6
Control group 1	0.068	0.041	8.1
				Control group 2	0.159	0.026	10.7

And (4) conclusion: as shown in table 1, the hydrogen production of example 1 of the present invention is significantly better than that of control group 1 and control group 2, and is increased by more than two times compared with control group 1 and by more than 30% compared with control group 2; the concentration of the algae cells in the embodiment 1 is also better than that of the control group 1 and the control group 2, and is improved by about 50 percent compared with the control group 1 and about 18 percent compared with the control group 2; the oxygen concentration of example 1 is kept at a reasonably low level, and the hydrogen production is improved by keeping the activity of catalase, and Table 1 shows that the compound bacterial liquid and the chemical stimulant can improve the hydrogen production of algae cells, and also have obvious promotion effect on the biomass of algae.

Example 4

Influence of the composite bacterial liquid on hydrogen production of algae:

example 2 was used as the experimental group, and the detailed operation was performed according to example 3; a control group was also set, wherein control group 1: the procedure of example 2 was repeated except that enterococcus faecium was used; control group 2: the procedure of example 2 was followed except that Rhodopseudomonas palustris was used. As shown in fig. 1, the hydrogen production of the experimental group was significantly higher than that of the control group 1 and the control group 2 using a single strain, and the oxygen production of the experimental group was lower than that of the control group and the control group 2; probably because the enterococcus faecium belongs to anaerobic bacteria, organic matters such as formate, acetate and organic acid can be generated by fermentation, and only hydrogen and carbon dioxide can be generated in the fermentation process; the rhodopseudomonas palustris belongs to aerobic photosynthetic bacteria and can grow under the anaerobic condition, and the rhodopseudomonas palustris can take substances generated by the fermentation of enterococcus faecium as substrates, so that the hydrogen production effect is good; the mixed bacteria liquid can grow rapidly under the anaerobic illumination condition and can also grow under the aerobic condition, so that oxygen generated by algae is effectively utilized, the oxygen content is reduced to the minimum, and better hydrogen production capacity is achieved; the compound bacterial liquid produced by the synergistic fermentation of the two bacterial strains can effectively promote the hydrogen production of algae; carbon dioxide generated by the bacterial liquid can meet the requirement of rapid growth of algae cells, the biomass of algae is improved, and then positive effect is brought to the total hydrogen production amount.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. The bacteria-algae composite preparation is characterized by comprising enterococcus faecium (enterococcus faecium)Enterococcus faecium) Rhodopseudomonas palustris (a)Rhodopseudomonas palustris) And Scenedesmus obliquus (C. obliquus)Scenedesmus obliquus)；

step 1) preparation of fermentation liquor: respectively culturing enterococcus faecium and Rhodopseudomonas palustris by conventional method to obtain 1 × 10⁸CFU/ml seed solution, then 1: (1.5-2) mixing to obtain a mixed seed solution, transferring the mixed seed solution into a fermentation culture medium according to the inoculation amount of 8-10%, and culturing at 30 ℃ for 12h to obtain a fermentation liquid; the formula of the fermentation medium comprises the following components in percentage by mass: molasses 3%, corn steep liquor 2.5%, soybean meal 2%, K₂HPO₄ 0.1％、KH₂PO₄ 0.1％、CaCO₃0.01％、FeSO₄ 0.005％、MnSO₄0.005 percent, the balance being water, and the pH value being 7.0;

step 2) preparing an algae solution: taking the concentration of 5 × 10⁵Inoculating Scenedesmus obliquus per ml into TAP culture solution according to the inoculation amount of 5% for culturing to obtain an algae solution;

step 3) preparing hydrogen production culture solution: adding sodium glycerophosphate and linoleic acid glyceride into the TAP culture solution to obtain a hydrogen production culture solution; the concentration of the sodium glycerophosphate is 0.5-0.8 mg/L; the concentration of the linoleic acid glyceride is 0.8-1 mg/L;

step 4), preparing a fungus-algae composite preparation: and (3) mixing the fermentation liquor, the algae liquid and the hydrogen production culture solution according to the proportion of 1: (12.5-15): (150-200) and uniformly mixing to obtain the product.

2. The complex formulation as claimed in claim 1, wherein the culturing conditions in step 2) are as follows: the illumination intensity is 100 mu mol.m^-2·s^-1Culturing for 2-3 days at water temperature of 25-28 deg.C and pH of 8 under light-dark ratio of 12h to 12 h.