Abstract:
Hydrogen is biologically, effectively produced by an alga in an alternating light/dark cycle which comprises alternating a step for cultivating the alga in water under aerobic conditions in the presence of light to accumulate photosynthetic products in the alga and a step for cultivating the alga in water under microaerobic conditions in the dark to decompose accumulated material by photosynthesis to evolve hydrogen.

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates to a process for producing hydrogen by algae in an alternating light/dark cycle. 
     Hydrogen is one of the noteworthy clean energy sources which will be able to take the place of fossil fuel such as petroleum and coal. Hydrogen has various advantages as an energy source such that (1) it can be converted to electric energy effectively by means of a fuel cell, (2) its calorie per unit weight is 3 to 4 times of that of petroleum, and it burns to form only water so that it does not pollute the environment, and (3) water, which is one of the raw materials for producing hydrogen, is inexhaustible. 
     The processes for the production of hydrogen by means of solar energy are classified into two classes, one of which is a non-biological process using semiconductors and the other of which is a biological process using photosynthetic products. With respect to the latter biological process, there proposed some systems for biophotolyzing water to produce hydrogen by controlling the metabolism of higher plants and algae having the ability to decompose water or by combining said higher plants and algae with a microorganism having the ability to decompose water. However, in conventional systems, oxygen produced by photolysis of water deactivates or prohibits the activity of the hydrogen producing system (i.e. hydrogenase), which results in unstability in the production of hydrogen. Further, isolation and purification of hydrogen is difficult, and there is a possibility of explosion of a gaseous mixture of oxygen and hydrogen. Therefore, the conventional systems are not practical. 
     SUMMARY OF THE INVENTION 
     As a result of an extensive study to biologically produce hydrogen with good productivity and durability, it has now been found that in a certain system, hydrogen is effectively produced by temporary separation of oxygen and hydrogen. 
     According to the present invention, there is provided a process for producing hydrogen by an alga in an alternating light/dark cycle which comprises alternating a step for cultivating the alga in water under light aerobic conditions to accumulate photosynthetic products in the alga and a step for cultivating the alga in water under dark microaerobic conditions to decompose accumulated material by photosynthesis to evolve hydrogen. 
     The alga may be any alga which has ability to produce hydrogen. Preferably, a green alga having such ability (e.g. Chlamydomonas reinhardtii) and a blue-green alga having such ability (e.g. Synechococcus sp.) are used. The alga in any growth phase may be used. Preferably, the alga in a logarithmic growth phase, particularly in a midlogarithmic growth phase is used, since the alga in these growth phases produces hydrogen more effectively under the dark microaerobic conditions. 
     In the process of the invention, during the cultivation of the alga in the light aerobic conditions, organic materials (e.g. starch) are accumulated in the alga, and during the cultivation of the alga in the dark microaerobic conditions, the accumulated organic materials are decomposed to evolve hydrogen. 
     The cultivation in the light aerobic conditions is carried out in a medium containing adequate inorganic components in light with the passing of air through the medium. The cultivation temperature is usually from 15° to 70° C. Preferably, in case of the green alga, it is from 15° to 40° C., particularly from 25° to 35° C., and in case of the blue-green alga, from 15° to 60° C., particularly from 40° to 55° C. Addition of 2 to 5% by volume of carbon dioxide in air to be passed through preferably increases the amount of the accumulated organic materials. Preferred examples of the medium are modified Bristol medium (hereinafter referred to as &#34;MBM&#34;) for the green alga, and modified Detmer medium (hereinafter referred to as &#34;MDM&#34;) and BG-11 medium for the blue-green alga. 
     The compositions of these mediums are as follows: 
     
         ______________________________________Composition of MBMMgSO.sub.4.7H.sub.2 O    75     mg/lCaCl.sub.2.2H.sub.2 O    10     mg/lK.sub.2 HPO.sub.4        75     mg/lKH.sub.2 PO.sub.4        175    mg/lNaCl                     25     mg/lFeSO.sub.4.7H.sub.2 O    2.0    mg/lTrace-metal mixture A.sub.5 *.sup.1                    1.0    ml/lNa.sub.2 CO.sub.3        53     mg/lNH.sub.4 Cl              268    mg/lComposition of MDM (pH 8.0)KNO.sub.3                1.0    g/lCaCl.sub.2.2H.sub.2 O    0.1    g/lMgSO.sub.4.7H.sub.2 O    0.25   g/lNaCl                     0.1    g/lK.sub.2 HPO.sub.4        0.25   g/lFeSO.sub.4.7H.sub.2 O    0.02   g/lTrace-metal mixture A.sub.5 *.sup.1                    1.0    ml/lComposition of BG-11 medium (pH 7.0)NaNO.sub.3               1.5    g/lK.sub.2 HPO.sub.4        0.04   g/lMgSO.sub.4.7H.sub.2 O    0.075  g/lCaCl.sub.2.2H.sub.2 O    0.036  g/lCitric acid              0.006  g/lFerric ammonium citrate  0.006  g/lEDTA(disodium magnesium salt)                    0.006  g/lNa.sub.2 CO.sub.3        0.02   g/lTrace-metal mixture A.sub.5 *.sup.1                    1.0    g/l______________________________________ *.sup.1 The trace mixture A.sub.5 contains 2.86 g of H.sub.3 BO.sub.4, 1.81 g of MnCl.sub.2.4H.sub.2 O, 0.22 g of ZnSO.sub.4.7H.sub.2 O, 0.08 g of CuSO.sub.4.5H.sub.2 O, 0.021 g of Na.sub.2 MoO.sub.4, 1 drop of conc. H.sub.2 SO.sub.4 in 1 l of deionized water. 
    
     The cultivation under the dark microaerobic conditions is carried out in a light-shielded vessel containing the same medium as described above in an atmosphere of nitrogen containing a micro amount of oxygen. The amount of oxygen in nitrogen varies with other cultivation conditions. Since too much oxygen may inhibit the hydrogen producing system, usually not more than 0.10% by volume of oxygen is added in nitrogen. However, at the beginning of the cultivation under the dark microaerobic conditions, addition of not more than 0.30% by volume, preferably not more than 0.23% by volume of oxygen in nitrogen preferably increases the rate of hydrogen evolution. 
     The hydrogen evolution amount can be increased by agitating or shaking the medium. 
     The light/dark cycle may be an artificial cycle and preferably corresponds to a day/night cycle. 
     The process of the invention not only produces hydrogen very economically and effectively by utilizing the solar energy, but also produces useful organic materials, i.e. biomasses, by recovering the alga grown under the light conditions. Under the dark conditions, in addition to the production of hydrogen by the decomposition of the starch; ethanol, glycerol, formic acid, acetic acid, lactic acid, etc. are accumulated in the medium and recovered. Further, when these organic materials are decomposed to produce hydrogen by E. coli or photosynthetic bacteria which uses these materials as substrates, the productivity of hydrogen is greatly improved. 
     For example, E. coli decomposes formic acid with an enzyme system so called formic hydrogenlyase to produce hydrogen. The enzyme system is induced in the presence of glucose and casamino acid under anaerobic conditions. The induced amount of the enzyme system, however, greatly varies with the anaerobic degree and no enzyme is induced in aerobic conditions. As a result of an extensive study to induce the enzyme system in E. coli under aerobic conditions, it has been found that the addition of formate (e.g. sodium formate) as an inducer and sodium succinate as an electron donor for aerobic respiration induces E. coli to produce formic hydrogenlyase in amounts as high as induced anaerobically. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     FIG. 1 is a chart which plots the hydrogen evolution versus time for the experiment reported in Example 1; and 
     FIG. 2 is a chart which plots the hydrogen evolution versus time for the experiment reported in Example 2. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention will be hereinafter explained further in detail by the following examples. 
     EXAMPLE 1 
     Cultivation under light conditions 
     A green alga (Chlamydomonas reinhardtii) was added in a 1 l bottle containing 700 ml of MBM in the concentration of 3.5 μg.dry wt./ml and grown under air at a light intensity of 18 W/cm 2  at about 30° C. while air containing 5% by volume of carbon dioxide was passed through the medium at a rate of 0.5 l/min. 
     Dark hydrogen evolution 
     After growth of the alga, nitrogen was flushed in the bottle to make the interior microaerobic. Then, the bottle was shielded from light and the alga was cultivated at about 30° C. under stirring. The amount of evolved hydrogen was measured by gas chromatography. 
     The above described cultivation under light conditions and the dark hydrogen evolution were alternatively cycled for 12 hours. 
     The results are shown in FIG. 1 in which o denotes the amount (ml) of evolved hydrogen in the dark period and • denotes the algal mass represented by OD 660 . 
     From the results, it is understood that a nearly constant amount of hydrogen was evolved in every dark period but the amount does not increase with the increase of the algal mass. This may result from the aging of the alga and limited supply of light energy per unit area which results in the decrease of the amount of starch accumulated per unit weight of the alga. 
     EXAMPLE 2 
     In the same manner as in Example 1, but removing a part of the alga from the bottle after the dark hydrogen evolution and before the cultivation under the light conditions, the cyclic cultivation of the alga was carried out. The result are shown in FIG. 2. 
     EXAMPLE 3 AND COMPARATIVE EXAMPLES 1 AND 2 
     E. coli IFO 12713 (corresponding to 10 g of dry weight) was added to a substrate medium containing an inducer and shook at 35° C. for 2 hours under nitrogen or air atmosphere. 
     The substrate used in Example 3 comprised sodium formate (50M), sodium succinate (10 mM), casamino acid (0.2%) and potassium phosphate buffer (pH 7.0, 20 mM) and in Comparative Examples, glucose (50 mM), casamino acid (0.2%) and potassium phosphate buffer (pH 7.0, 20 mM). After 2 hours induction, the cells were collected and washed with phosphate buffer twice. The cells were added in a solution of 50 mM sodium formate, the gas phase was replaced with nitrogen and the mixture was shaken at 35° C. Formic hydrogenlyase activity was determined by measuring the amount of evolved hydrogen. The results are shown in Table 1. 
     
                       TABLE 1______________________________________                     Amount of formic                     hydrogenlyase  Inducer Condition  (unit*.sup.1 /mg. dry wt.)______________________________________Example 3    Sodium    Aerobic    12    formate +    sodium    succinateCompara- Glucose   Anaerobic  13tiveExample 1Compara- Glucose   Aerobic     0tiveExample 2______________________________________ Note *.sup.1 1 unit is an amount of the enzyme which produces 1 μmole of hydrogen per 1 hour by using formic acid as a substrate. 
    
     Chlamydomonas reinhardtii was cultivated under the dark conditions for 12 hours in the same manner as in Example 1 and thereafter the alga was removed by centrifugation. The supernatant was fed to E. coli in which formic hydrogenlyase had been aerobically induced as in Example 3. Hydrogen (0.63 μmole) was evolved from the supernatant containing formic acid (0.7 μmole). 
     EXAMPLE 4 
     Cultivation under light conditions 
     A single cell thermophilic blue-green alga (Synechococcus sp.) obtained from the Beppu hot spring (Ooita, Japan) was added in 1 l flask containing 500 ml of BG-11 medium and grown under a fluorescent lamp while the flask was reciprocally shaken at a rate of 100 rpm in a constant temperature room kept at 45° C. 
     Dark hydrogen evolution 
     When the algal concentration reached about 20 μg dry wt./ml, the alga was collected by centrifugation, washed with BG-11 medium twice and resuspended in the same medium (10 ml). The algal concentration was 0.17 mg/ml. The suspension was charged in a light-shielded 34 ml test tube and sealed with a rubber cap. The gas phase was replaced with nitrogen and a predetermined amount of oxygen was flushed by means of a syringe. The test tube was reciprocally shaken at a rate of 100 rpm in a constant temperature bath kept at 45° C. 
     After 2, 6.5, 12 and 20 hours of shaking, each 500 μl of a gas phase sample was collected and its composition was analyzed by gas chromatography. 
     The results are shown in Table 2. 
     
                       TABLE 2______________________________________        Amount of evolved hydrogenOxygen content        (μg/mg. dry wt.)in gas phase Shaking time (hr.)(% by vol.)  2       6.5      12    20______________________________________0.08         0       0         0     00.3          0       2        14    28______________________________________