CN100355898C - Two-step method of hydrogen production by biologically photolyzing water using ocean green algae - Google Patents
Two-step method of hydrogen production by biologically photolyzing water using ocean green algae Download PDFInfo
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- CN100355898C CN100355898C CNB031109810A CN03110981A CN100355898C CN 100355898 C CN100355898 C CN 100355898C CN B031109810 A CNB031109810 A CN B031109810A CN 03110981 A CN03110981 A CN 03110981A CN 100355898 C CN100355898 C CN 100355898C
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Abstract
The present invention relates to a technology for hydrogen production by using microalgae, particularly to a method for hydrogen production by the biophotolysis of marine green algae with a two-step method. In the method, green algae is treated with photosynthesis to obtain biomass accumulation. The present invention is characterized in that the green algae is platymonas subcordiformis; the platymonas subcordiformis obtaining biomass accumulation is stored in an oxygen-free dark place for 20 to 45 hours for dark induction, and then is illuminated for hydrogen production. The present invention has the following advantages: 1, good marine microalage for hydrogen production can be obtained; 2, the route for hydrogen production is reasonable, and the operating process is simple; 3, the present invention develops sulfur-free marine water for the induction of the activity of a reversible hydrogen production enzyme system and hydrogen production by illumination; 4, by the single action of decoupling agents, or the combined action of decoupling agents and proton pump inhibiting agents, photosynthetic oxygen evolution can be effectively inhibited without inhibiting photosynthetic hydrogen production.
Description
Technical field
The present invention relates to little algae hydrogen producing technology, specifically a kind of marine green algae two-step approach biophotolysis water hydrogen production process.
Background technology
Less than 30 years, the algae kind that relates to mainly was blue-green algae and green alga to little algae hydrogen producing technology as the history of applied research; Blue-green algae utilizes intravital nitrogenase to produce hydrogen, and its biological mechanism has determined the capacity usage ratio of this technology can not reach 10% (referring to document 1:Carolyn C.Elam.IEA Agreement onthe production and utilization of hydrogen 1999:41-46; Document 2:Jun Miyake, Masato Miyake and Yasuo Asada (1999) Biotechnological hydrogen production:research for efficient light energy conversion, J.Biotechnology 70,89-111), therefore, international energy office (IEA) thinks that this technology does not have actual exploitation to be worth.Green alga (Platymonassubcordiformis) but the intravital nitrogenase of specific activity blue-green algae of intravital traitor's property hydrogen enzyme active approximately high 1000 times, and produce the hydrogen process and do not need ATP, optical energy utilization efficiency may reach 10% (referring to document 1:Carolyn C.Elam.IEA Agreement on the production and utilization ofhydrogen 1999:41-46; Document 3:John Benemann (1996) Hydrogen biotechnology:progress and prospects, Nature Biotechnology 14,1101-1103); But the direct photodissociation water of green alga traitor's property hydrogen enzyme hydrogen producing technology produces oxygen when producing hydrogen, when partial pressure of gaseous oxygen reaches 2%, but traitor's property hydrogen enzyme deactivation, product hydrogen process stops, and can not realize continuous large-scale hydrogen manufacturing.But the research that improves traitor's property hydrogen enzyme oxygen resistence is just begun at present, but the antioxygenic activity of traitor's property hydrogen enzyme does not also obviously improve (referring to document 4:Ghirardi et al.Process for selection ofoxygen tolerantalgal mutants that produce H
2Under aerobic conditions, USA Patent5,871,952 (2001)); Reagent such as adding weedicide can suppress oxygen and discharge, but have caused necrocytosis; Fall oxygen by adding chemical reagent consumption, or the feeding rare gas element is taken away hydrogen and the oxygen that is produced, produce hydrogen continuously though can obtain green alga, but these methods can not realize economy of large scale ground and make hydrogen (referring to document 5:Anastasios Melis et al., Hydrogen production usinghydrogenase-containing oxygenic photosynthetic organisms, United StatesPatent Application 20010053543 (2001)).
People such as the Melis of Univ California-Berkeley have proposed the indirect photodissociation water of two-step approach process for making hydrogen in 2000, wherein: the first step is that green alga carries out photosynthesis, and stabilizing carbon dioxide discharges oxygen, obtains the accumulation of biomass; But second step was to induce the high expression level of traitor's property hydrogen enzyme (to utilize no sulphur substratum in no sulphur, anaerobic environment, under illumination through 24～30 hours, the photosynthetic oxygen evolution speed of the chlamydomonas of sealing is reduced to is lower than the breathing keto consumpting speed, oxygen in the consumption systems, reach anaerobic state, but traitor's property hydrogen enzyme is induced), hydrogen photoproduction (beginning in 40 hours) is 6 * 10 with every liter of cell concn again
6The algae liquid of cells/mL, continuous 70 hours of the speed of per hour producing hydrogen 1.71mL produces 120 milliliters of hydrogen (referring to document 6:Melis, A.Et al., (2000) Sustainedphotobiological hydrogen gas production upon reversible inactivation ofoxygen evolution in the green alga Chlamydomanas reinhardtii, Plant Physiol.122,127-136).Calendar year 2001, USDOE estimates that this technology finally may realize hydrogen manufacturing cost 10$/GJH
2Target, make this technology finally reach the acceptable production cost in market (referring to document 7:Wade A.Amos, Margaret K.Mann, Pamela L.Spath, Janice M.Lane (2001) Process analysis work for the DOE hydrogen program-2000, Proceedings of the2001DOE hydrogen program review NREL/CP-570-30535).
Owing to contain element sulphur in the growth medium of little algae that Melis etc. are used, but and traitor's property hydrogen enzyme induce the substratum that all requires not have sulphur with the photosynthetic hydrogen production stage, so need carry out centrifugal collection, clean repeatedly repetitive operations such as cell to chlorella cell with fresh no sulphur substratum, both caused operational path long, complicated operation, also make the repeated use difficulty of micro algae biomass, the cost of this technology is increased; Because human available Freshwater resources are limited, if can utilize resourceful seawater to carry out the hydrogen manufacturing of little algae biophotolysis water, this technology will have more significant meaning again.Though but the indirect photolysis water hydrogen of green alga traitor's property hydrogen enzyme two-step approach has great development potentiality, also obtain certain progress, but make this technology really enter the practical stage, the technological difficulties that press for solution at present have: (1) obtains good ocean and produces the little algae of hydrogen; (2) but the raising of traitor's property hydrogen enzymic activity; (3) effectively suppress photosynthetic oxygen evolution and but do not suppress photosynthetic hydrogen production; (4) prolongation is produced the hydrogen time and is realized recycling of micro algae biomass etc.
Summary of the invention
The object of the present invention is to provide a kind of marine green algae two-step approach biophotolysis water hydrogen production process; It adopts good ocean to produce hydrogen algae kind, uses hydrogen production potential that rational hydrogen manufacturing route improves green alga, simplifies the operation course.
For achieving the above object, the technical scheme that the present invention takes is: operation in the usual way, and marine green alga carries out photosynthesis, obtains the accumulation of biomass, deposit in the anaerobic dark place and secretly induced in 20～45 hours obtaining marine green alga behind the biomass accumulation, again hydrogen photoproduction;
Described anaerobic is deposited and be can be simple sealing and deposit; Described secretly inducing preferably carried out in no sulphur seawater, obtained continuous 6～8 hours photosynthetic hydrogen of putting of green alga; If in natural sea-water, under illumination, discharge a large amount of oxygen immediately through the dark inductive algae of anaerobism liquid, thereby suppress the photosynthetic hydrogen production process rapidly, this product hydrogen process was only kept 1-3 hour;
Described no sulphur seawater (manually) better formula is:
| Component | NaCl | MgCl 2 | CaCl 2 | KCl | NaHCO 3 | H 3BO 3 | KBr | SrCl 2 | NaF |
| Weight content % | 27.23 | 5.079 | 1.123 | 0.667 | 0.196 | 0.098 | 0.098 | 0.024 | 0.003 |
The amount of micronutrient salt is 1.3mg FeCl in every liter of sea water medium
36H
2O, 0.36mg MnCl
24H
2O, 33.6mg H
3BO
3, 45.0mg EDTA, 20.0mg NaH
2PO
4H
2O, 100mg NaNO
3, 0.21mg ZnCl
2, 0.20mg CoCl
26H
2O, 0.09mg (NH
4)
4Mo
7O
244H
2O, 0.136mgCuCl
2, 0.1 μ g VB
12And 1.0 μ g VB
1
The cell density of described flat algae algae liquid is generally 1.0～6.0 * 10
6Cells/mL;
Described secretly inducing yet can or not have in the sulphur seawater and carry out at natural sea-water, adds micromole level uncoupling agents CCCP or FCCP (its Full Name in English CCCP:carbonyl cyanidem-chlorophenylhydrazone then in algae liquid; FCCP:carbonylcyanide-p-trifluoromethoxyphenyl); Making its concentration in algae liquid is 5～20 μ M; The use of uncoupling agents has suppressed photosynthetic oxygen evolution, has improved little algae photosynthetic hydrogen production speed and output again, and the concentration of adding can not made troubles to the operation of reactor capacity, hydrogen manufacturing whole process in micromole's level; The present invention uncoupling agents to the physiological regulating control effect of green alga be used to suppress green alga photosynthetic oxygen evolution, improve its photosynthetic hydrogen production, photosynthetic oxygen evolution is suppressed fully, different with mechanical means and chemical process; The use of uncoupling agents is suppressed photosynthetic oxygen evolution fully, and two of the gas-liquids of algae liquid all maintain anaerobic state, makes the photosynthetic hydrogen of putting be lasted till that every liter of cell concn was 1.0～1.2 * 10 6 hours
6The product hydrogen speed of the algae liquid of cells/mL reaches per hour 0.090～0.108mL.
After secretly inducing, also can in algae liquid, add proton pump inhibitor DCCD (its Full Name in English DCCD:N, N '-dicyclohexylcarbodiimide); Making its concentration in algae liquid is 1～5 μ M; Wherein uncoupling agents and proton pump inhibitor unite use, and do not add CCCP or FCCP relatively, make this algae product hydrogen time lengthening 1 times, lasted till that hydrogen output improved 79 times 6 hours, making every liter of cell concn is 1.0～1.2 * 10
6The product hydrogen speed of the algae liquid of cells/mL reaches per hour 0.234～0.281mL.
The present invention has following advantage:
1. obtain good ocean and produce the little algae of hydrogen.The present invention has found a kind of new product hydrogen ocean algae kind, takes the lead in having proposed a strain ocean and produces hydrogen green alga marine green alga; But it can utilize traitor's property hydrogen enzyme, makes it can photosynthetic hydrogen production through certain metabolic regulation means, makes the raw material of this technology expand seawater to from fresh water, has greatly enriched the source of its raw material.
2. the hydrogen manufacturing route is reasonable, and operating process is simple.The present invention proposes a kind of and marine green alga and produce the hydrogen operational path accordingly, this green alga at first carries out photosynthesis, discharges oxygen, stabilizing carbon dioxide, and the accumulation organism obtains to be fit to produce the biomass of hydrogen; Seal microalgae cell suspension then, but in the dark induce the activity of traitor's property hydrogen enzyme system, under illumination, utilize the luminous energy release hydrogen again:
3. but the present invention has developed activity and the hydrogen photoproduction that no sulphur seawater is used to induce traitor's property hydrogen enzyme system, compares with natural sea-water, and no sulphur seawater makes its hydrogen production potential improve 10 times.
4. can effectively suppress photosynthetic oxygen evolution and but not suppress photosynthetic hydrogen production.The present invention is after obtaining micro algae biomass, the algae liquid that only is sealed in the natural sea-water makes it just can secretly induce with isolated from atmosphere, add uncoupling agents inhibition photosynthetic oxygen evolution, the photosynthetic hydrogen of putting of promotion again after secretly inducing end, make algae liquid keep anaerobism and the continuous hydrogen that produces; Produce hydrogen end back green alga and can carry out photosynthesis, stabilizing carbon dioxide again, so present technique realizes the recycle of micro algae biomass easily; Utilize uncoupling agents, not only make photosynthetic oxygen evolution and photosyntheticly put the hydrogenolysis coupling, make little algae only produce the hydrogen non-oxygen-production, and broken the membranous sub-gradient of striding of thylakoid membrane, make proton stride the film transportation without what consume ATP, making every liter of cell concn is 1.0～1.2 * 10
6The product hydrogen speed of the algae liquid of cells/mL reaches per hour 0.090～0.108mL; Utilize the acting in conjunction of uncoupling agents and proton pump inhibitor, but proton is shifted to traitor's property hydrogen enzyme path, making every liter of cell concn is 1.0～1.2 * 10
6The product hydrogen speed of the algae liquid of cells/mL reaches per hour 0.234～0.281mL, and the result of the product hydrogen speed of same cell quantity and Melis etc. is close.The present invention utilizes same reactor to realize secretly inducing and two processes of hydrogen photoproduction.
Description of drawings
Fig. 1 is the present invention the utilizes product hydrogen graphic representation of no sulphur sea water medium under the dark induction time of difference.
Fig. 2 is the synoptic diagram of uncoupling agents of the present invention to the influence of product hydrogen.
Fig. 3 is the synoptic diagram that uncoupling agents of the present invention and proton pump inhibitor acting in conjunction influence product hydrogen.
Embodiment
Embodiment 1 (utilizing no sulphur sea water medium to produce the optimization of the dark induction time of hydrogen)
Collect the flat algae cell in logarithmic growth later stage,, adjust to 1.0～1.2 * 10 again with fresh no sulphur sea water medium centrifuge washing 3 times
6The cell concn of cells/mL; Trying to please long-pending is the plasma bottle of 300mL, and interior dress 295mL algae liquid seals bottleneck with soft rubber ball, closely is connected inflated with nitrogen 5 minutes with the plasma bottle upper space with the syringe needle of transfusion device; Under 25 ℃, be placed into the dark place and deposit the different time, utilize the dark respiration effect of frustule to consume the oxygen that is sealed in the bottle, make it to reach anaerobic state, and induce the activity of producing the hydrogen enzyme, again at 160 μ mol photo m
-2s
-1Illumination and 150rpm circle round and shake down illumination and put hydrogen.The transfusion device flexible pipe is put into inverted scale test tube, utilizes water seal to collect and measure gas volume.
The result is shown in Fig. 1 (dark induction time is to the influence of photosynthetic hydrogen production in the no sulphur seawater): as seen, the algae liquid of sealing through different dark induction times to the influence of photosynthetic hydrogen production amount greatly.Do not produce hydrogen without dark inductive algae liquid, the algae liquid of secretly inducing 40 hours produces 39000 and receives the hydrogen that rises, and the algae liquid of secretly inducing 48 hours does not produce hydrogen yet, so the optimal value of dark induction time is 40 hours.
Embodiment 2 (uncoupling agents is to producing the influence of hydrogen)
Flat algae cell suspending liquid with 295mL is made blank assay, its photosynthetic hydrogen production continuity 3 hours, and output is 6.3 μ L.Through the algae liquid of dark anaerobic induction in natural sea-water, begin to add conditioning agent in preceding 5 minutes in illumination, the results are shown in Figure 2 (DCMU, DCCD, CCCP, DBMIB are to producing the influence of hydrogen); The DCMU of 50 μ M makes the flat algae cell of same concentration and quantity produce hydrogen 6.7 μ L, than the hydrogen output increase by 6% of blank assay; It is 9.9 μ L that the DCCD of 2 μ M makes its hydrogen output, than the hydrogen output increase by 57% of blank test; The CCCP of 10 μ M makes it produce hydrogen process continuity 6 hours, and hydrogen output is 191 μ L, is 30 times of blank test; It is 1.7 μ L that the DBMIB of 5 μ M makes its hydrogen output, is 27% of blank test.Uncoupling agents has improved the photosynthetic hydrogen production amount greatly, and weedicide DCMU or other photosynthesis electron transfer inhibitors DBMIB suppress photosynthetic hydrogen production.
Embodiment 3 (uncoupling agents and proton pump inhibitor acting in conjunction are to producing the influence of hydrogen)
Through the algae liquid of dark anaerobic induction in natural sea-water, begin to add conditioning agent in preceding 5 minutes in illumination, the results are shown in Table 1 and Fig. 3 (DCMU, DBMIB, DCCD produce the influence of hydrogen to the flat algae that adds CCCP).With little algae photodissociation aquatic products hydrogen contrast of only using natural sea-water to carry out, uncoupling agents acts on separately and all makes the product hydrogen time extend to 6 hours from 3 hours with the proton pump inhibitor acting in conjunction.
Hydrogen output is 6.3 μ L in the blank test, and under the DCMU acting in conjunction of the CCCP of 10 μ M and 50 μ M, the flat algae cell of the equivalent under the same state produces hydrogen 2.4 μ L, is 0.38 of blank test; It is 10.6 μ L that the DBMIB of the CCCP of 10 μ M and 5 μ M makes its hydrogen output, is 1.68 times of blank test; The DCCD of the CCCP of 10 μ M and 2 μ M makes and produces hydrogen continuity 6 hours, and its hydrogen output is 498 μ L, is 79 times of blank test.
Table 1 uncoupling agents, proton pump inhibitor are handled hydrogen are produced in the influence of producing hydrogen
| Natural sea-water | Uncoupling agents+proton pump inhibitor | |
| Perdurability/hour | 3 | 6 |
| Hydrogen output/microlitre | 6.3 | 498.6 |
Claims (7)
1. marine green algae two-step approach biophotolysis water hydrogen production process, green alga carries out photosynthesis, obtain the accumulation of biomass, it is characterized in that: described green alga is a marine green alga, deposit in the anaerobic dark place and secretly induced in 20～45 hours obtaining marine green alga behind the biomass accumulation, again hydrogen photoproduction; Describedly secretly induce at natural sea-water or do not have in the sulphur seawater and carry out, in algae liquid, add micromole level uncoupling agents CCCP or FCCP then; Secretly inducing the back in algae liquid, to add proton pump inhibitor DCCD.
2. according to the described marine green algae two-step approach of claim 1 biophotolysis water hydrogen production process, it is characterized in that: deposit described anaerobic dark place is to deposit in sealed environment.
3. according to the described marine green algae two-step approach of claim 1 biophotolysis water hydrogen production process, it is characterized in that: described secretly inducing is at natural sea-water or do not have in the sulphur seawater and carry out.
4. according to the described marine green algae two-step approach of claim 3 biophotolysis water hydrogen production process, it is characterized in that: the amount of micronutrient salt is in described every liter of no sulphur sea water medium: 1.3mg FeCl
36H
2O, 0.36mg MnCl
24H
2O, 33.6mg H
3BO
3, 45.0mg EDTA, 20.0mg NaH
2PO
4H
2O, 100mg NaNO
3, 0.21mg ZnCl
2, 0.20mg CoCl
26H
2O, 0.09mg (NH
4)
4Mo
7O
244H
2O, 0.136mg CuCl
2, 0.1 μ g VB
12And 1.0 μ g VB
1
5. according to the described marine green algae two-step approach of claim 1 biophotolysis water hydrogen production process, it is characterized in that: the cell density of described marine green alga algae liquid is 1.0～6.0 * 10
6Cells/mL.
6. according to the described marine green algae two-step approach of claim 1 biophotolysis water hydrogen production process, it is characterized in that: behind the described adding uncoupling agents, making its concentration in algae liquid is 5～20 μ M.
7. according to the described marine green algae two-step approach of claim 1 biophotolysis water hydrogen production process, it is characterized in that: behind the described adding proton pump inhibitor, making its concentration in algae liquid is 1～5 μ M.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100392088C (en) * | 2005-07-27 | 2008-06-04 | 中国科学院大连化学物理研究所 | Method of promoting hydrogen production of sea green algae through direct illumination |
| CN1990872B (en) * | 2005-12-27 | 2010-04-14 | 中国科学院海洋研究所 | Method of preparing hydrogen gas by using castoff of mariculture organic matter |
| CN101275117B (en) * | 2007-03-30 | 2012-05-23 | 中国科学院大连化学物理研究所 | Method for fast algae proliferation directly used for bio-hydrogen production by using CO2 |
| CN102199577B (en) * | 2010-03-24 | 2013-01-16 | 中国科学院大连化学物理研究所 | Method for separating and purifying green alga hydrogenase in oxygen-free environment |
| CN103571876A (en) * | 2013-10-14 | 2014-02-12 | 清华大学 | Method for producing biogas with efficient utilization of solar energy by utilizing blue-green algae |
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Non-Patent Citations (1)
| Title |
|---|
| Sustained photobiological hydrogen gas production uponreversible inactivation of ovygen evolution in the green algachlamydomanas reinhardtii. Melis et al. Plant Physiol. 2000 * |
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