Method for dissociating water using photosystem II (PSII)

Abstract

The invention relates to a method for dissociating an aqueous solution which includes electrochemical oxidation of the aqueous solution in the presence of pulsed light, said pulsed light being generated from a first source of light energy with a predetermined pulse frequency value, using an enzyme composition based on a first enzyme complex PSII, isolated from a second enzyme complex PSI, with production of oxygen, free electrons and free protons in the aqueous solution, characterised in that said light energy has a variable energy value over time, said method also including a step of modulating said predetermined pulse frequency value of said pulsed light.

Claims

1. A method for dissociating an aqueous solution, comprising: first electrochemical oxidating the aqueous solution in the presence of pulsed light, said pulsed light being generated from a first source of light energy at a predetermined pulse frequency value, and having a predetermined peak power, by an enzymatic composition based on a first enzymatic complex PSII, isolated from a second enzymatic complex PSI, with production of oxygen, free electrons and free protons in the aqueous solution, wherein said light energy from said first source of light energy has a variable energy value over time, said variable energy value over time having a light energy value per second determined over a plurality of pulses of said pulsed light; capturing said free electrons and of said free protons; capturing oxygen; and modulating said predetermined pulse frequency value of said pulsed light to a sufficient pulse frequency value in order to obtain a first oxygen production yield per unit of light energy which is greater by a factor (f) of 1.01 to 100.00 than a second oxygen production yield per unit of light energy obtained for a second electrochemical oxidation in the presence of continuous light, said first and second oxygen production yields (.sub.i) being each obtained from an oxygen production rate (K(O.sub.2).sub.i) per unit of light energy, i=1 or 2, such that: ${}_i=\frac{{K\left(O_2\right)}_i}{E_i},$ where E.sub.i refers to an amount of energy per second provided for obtaining said oxygen production rate (K(O.sub.2).sub.i), i=1 or 2, said factor (f) being equal to a ratio between said first oxygen production yield (.sub.1) and said second oxygen production yield (.sub.2) such that: $f=\frac{{}_1}{{}_2},$ said pulsed light and said continuous light having an equal predetermined wavelength and said continuous light have an equal power to said predetermined peak power of said pulsed light.

2. The method according to claim 1, comprising, before said electrochemical oxidation of said aqueous solution, extracting said enzymatic complex PSII from chloroplasts or tylakoids of chloroplasts.

3. The method according to claim 2, comprising, after extracting said PSII, purifying said enzymatic complex PSII in order to form an enzymatic composition substantially concentrated in PSII.

4. The method according to claim 2, wherein said chloroplasts or tylakoids of chloroplasts are chloroplasts or tylakoids of chloroplasts of plants from the family of Chenopodiaceae.

5. The method according to claim 1, comprising, before said electrochemical oxidation of said aqueous solution, synthetically manufacturing said enzymatic complex PSII.

6. The method according to claim 1, wherein said capture of free electrons and of free protons is carried out by an electron transport mediator selected from the group consisting of derivatives of quinone, 2,6-dimethylbenzoquinone, 2,6-dichloro-p-benzoquinone and 1,4-benzoquinone, and one of their mixtures.

7. The method according to claim 1, wherein said pulsed light is emitted at a pulse frequency comprised between 1 Hz and 100 MHz, preferably comprised between 1 Hz and 3,000 Hz, in a range of wavelengths comprised between 400 nm and 700 nm, at a peak power comprised between 1 mW and 800 mW.

8. The method according to claim 1, wherein said oxidation of water is carried out at a pH comprised between 4 and 8.

9. The method according to claim 1, wherein said energy value is determined based on a plurality of pulses over a time interval of 1 s.

Description

DESCRIPTION OF THE DRAWINGS

(1) The foregoing aspects and many of the attendant advantages of the claimed subject matter will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

(2) FIG. 1 illustrates the absorption spectra of PSII before and after purification according to a method for extracting the complex PSII from chloroplasts of spinach.

(3) FIG. 2 illustrates a curve of the time-dependent change in the maximum oxygen production rate obtained by the method in the presence of continuous coherent light of a wavelength of 673 nm, versus the light energy value per second.

(4) FIG. 3 illustrates a curve of the time-dependent change of the maximum oxygen production rate obtained by the method in the presence of pulsed coherent light at 800 Hz (pulses with a duration of 9.2 s) with a wavelength of 673 nm, versus the value of the light energy per second.

(5) FIG. 4 illustrates the curves of the time-dependent change of the maximum oxygen production rate obtained by the method in the presence of continuous coherent light, or of pulsed coherent light at 800 Hz (pulses with a duration of 1.5 s) with a wavelength of 673 nm, versus the value of the light energy per second.

(6) FIG. 5 illustrates the curves of the time-dependent change in the maximum energy yield of oxygen production rate obtained by the method in the presence of continuous coherent light, or pulsed coherent light at 800 Hz (pulses with a duration of 1.5 s) with a wavelength of 673 nm, versus the value of the light energy per second.

(7) FIG. 6 illustrates the curves of the time-dependent change in the maximum energy yield of oxygen production obtained with the method in the presence of continuous coherent light, or of pulsed coherent light at 800 Hz (pulses with a duration of 1.5 s), with a wavelength of 673 nm, depending on the value of the light energy per second (the energy being expressed in a logarithmic scale).

DETAILED DESCRIPTION

(8) The detailed description set forth below in connection with the appended drawings, where like numerals reference like elements, is intended as a description of various embodiments of the disclosed subject matter and is not intended to represent the only embodiments. Each embodiment described in this disclosure is provided merely as an example or illustration and should not be construed as preferred or advantageous over other embodiments. The illustrative examples provided herein are not intended to be exhaustive or to limit the claimed subject matter to the precise forms disclosed.

(9) The device used for applying the method according to the present disclosure, for which the performances are illustrated in the following examples, comprises:

(10) a source of light energy intended to produce a pulsed light for which the light energy has a variable energy value over time;

(11) a modulator of the pulse frequency of the pulsed light;

(12) a reactor for dissociating an aqueous solution comprising an aqueous phase, which reactor is laid out so that the solution may be irradiated with the pulsed light when the latter is generated;

(13) an enzymatic composition in solution in the aqueous phase, the enzymatic composition comprises a first complex PSII, isolated from a second enzymatic complex PSI, the enzymatic composition is laid out for, under the action of the pulsed light, electrochemically oxidizing the aqueous solution in order to produce oxygen, free electrons and free protons in the aqueous solution;

(14) a first means for capturing oxygen;

(15) a second means for capturing free electrons and free protons.

(16) During operation, the pulsed light is generated at a first predetermined pulse frequency and the pulsed light source is oriented so that the pulsed light irradiates the photo-enzymes in the aqueous solution.

(17) The irradiation of the enzymatic complex in suspension in the solution induces an oxidation reaction of the water contained in the reactor via the PSII enzymatic complex with production of oxygen which is captured by the first capture means which is for example an electrode of the Clark type immersed in the aqueous phase, the free electrons and the free protons are as for them captured by the second capture means which is a sensor of electrons, for example dimethylbenzoquinone (DMBQ) or 2,5-dichloro-p-benzoquinone (DCBQ).

(18) As the energy value of the light energy varies over time, the modulator of the pulse frequency of the pulsed light, which pulsed light is associated with this variable light energy, gives the possibility when it operates of modulating the predetermined pulse frequency value of this pulsed light at a sufficient pulse frequency value for obtaining a first oxygen production yield per unit of light energy which is greater by a factor comprised between 1.01 and 100.00 at a second oxygen production yield per unit of light energy obtained for a second electrochemical oxidation in the presence of continuous light, which pulsed light and continuous lights have an equal predetermined wavelength and an equal predetermined power.

(19) Preferably, the first oxygen production yield per unit of light energy which is greater by a factor comprised between 1.01 and 80.00 than a second oxygen production yield per unit of light energy obtained for a second electrochemical oxidation in the presence of continuous light, which pulsed lights and continuous lights have an equal predetermined wavelength and an equal predetermined power.

(20) By the terms of pulse frequency, should be understood in the present disclosure the frequency with which is modulated the energy value of the light energy emitted at a predetermined wavelength or in a predetermined range of wavelengths.

Example 1: Preparation of the Enzymatic Composition Based on PSII

(21) As mentioned earlier, the PSI complex which inhibits the activity of the PSII complex and which therefore limits the formation of oxygen should be removed.

(22) Chloroplasts from spinach (Spinacia oleracea) are extracted in accordance with the procedure developed by Barthelemy et al. (Journal of Photochemistry and Photobiology B: Biology, 1997, volume 39, pages 213-218), and then extracted from thylakoid membranes and dissociated from the PSI complex present in these membranes, the PSII complex according to the method of Berthold and al., see Febs Letters, 1981, volume 134, number 2, pages 231-234.

(23) As compared with the Barthelemy procedure, in the method according to the disclosure, mention is made of the following modifications:

(24) the absolute concentration of Triton X 100 is maintained constant in the sample of an aqueous solution by adjusting the chlorophyll concentration to 200 g/ml before adding 25 mg of Triton X 100 for 1 mg of chlorophyll to the sample, and

(25) the second treatment with Triton X 100 is excluded and replaced by rinsing of the PSII particles in an MES-NaOH 20 mM buffer (comprising 15 mM NaCl and 5 mM MgCl.sub.2) at pH=6.5 and a suspension of these particles in an MES-NaOH 20 mM buffer (comprising 15 mM NaCl, 5 mM MgCl.sub.2, 0.5 M sucrose) at pH=6.5.

(26) The PSII obtained by the modified Bertold and al. method, as described above is then kept at 80 C. after freezing in liquid nitrogen at a temperature of 196 C. (77K) for a period of 10 s.

(27) According to the modified extraction method described above, the PSII of the spinach is isolated from the PSI complex present in the chloroplast. Indeed, as shown by FIG. 1, the absorption peak appearing at 735 nm corresponds to that of the PSI complex while the absorption peak at 685 nm corresponds to the PSII complex, this peak decreasing the energy value by a factor 5 after purification according to the method of the present example, which corresponds to a decrease by a factor 5 of the PSI concentration.

Comparative Example 1: Measurement of the Maximum Oxygen Production Rate with the PSII Complex in Continuous Light and in Monochromatic Pulsed Light at 673 nm

(28) A photohydrolysis system consisting of a cell, with a volume of 2.5 ml, the walls of which are provided with glass windows, in which a PSII suspension is placed in an aqueous solution at a buffer pH of 6.5, containing dimethylbenzoquinone (DMBQ) or 2.5-dichloro-p-benzoquinone (DCBQ).

(29) The cell is illuminated with a laser light beam at 673 nm, in a continuous mode or in a pulsed mode. The characteristics of the laser are repeated in Tables 1a (DMBQ) and 1b (DCBQ).

(30) By the terms of pulsed light or pulsed mode is meant in the sense of the disclosure an intermittent light, i.e. for which the energy value alternately assumes a first zero value and a second non-zero predetermined value at a predetermined pulse frequency, for example 800 Hz, which means that the energy value passes 800 times per second from the zero value to the predetermined non-zero value by the power of the light.

(31) TABLE-US-00001 TABLE 1a Characteristics of the laser used for the PSII-DMBQ system Parameters Specifications Type of laser VCSEL Laser emission wavelengths 671 nm at 15 C. 676 nm at 35 C. Spectral width <2 nm FWHM Operating mode continuous or pulsed Average power 550 mW (continuous and pulsed mode) Peak power >700 mW (pulsed mode) Repetition rate from 1 Hz to 3,000 Hz (pulsed mode) Pulse duration 9.2 s (pulsed mode) Optical output collimated, linear, or focused

(32) TABLE-US-00002 TABLE 1b Characteristics of the laser used for the PSII-DCBQ system Parameters Specifications Type of laser VCSEL Laser emission wavelengths 671 nm at 15 C. 676 nm at 35 C. Spectral width <2 nm FWHM Operating mode continuous or pulsed Average power 642 mW (continuous and pulsed mode) Peak power 555 mW (pulsed mode) Repetition rate from 1 Hz to 5,000 Hz (pulsed mode) Pulse duration 1.5 s (pulsed mode) Optical output collimated, linear, or focused

(33) The rate of the increase in oxygen concentration under illumination was measured at 25 C. by using an electrode of the Clark type in an aqueous buffer solution with a concentration of 25 mM MES-NaOH (pH=6.5) and 1 mM of DMBQ.

(34) FIG. 2 illustrates a curve of the time-dependent change in the maximum oxygen production rate (in moles) for 25 g of chlorophyll (Chl), treated with the method according to example 1, per ml of aqueous solution and per minute (min). The curve is obtained in the presence of continuous coherent light with a wavelength of 673 nm, versus the value of the light energy per second (in mJ/sec).

(35) FIG. 3 illustrates a curve of the time-dependent change in the maximum oxygen production rate (in moles) for 25 g of chlorophyll (Chl), treated by the method according to example 1, per ml of aqueous solution and per minute (min). The curve is obtained in the presence of pulsed coherent light at 800 Hz with a wavelength of 673 nm, versus the value of the light energy per second (in mJ/sec).

(36) The analysis of FIGS. 2 and 3 gives the possibility of observing, for example, that having available a light power of 500 mW, the amount of energy per second in continuous light is equivalent to 500 mJ/s. To this amount of energy in continuous light corresponds an oxygen production rate K(O.sub.2) of 7 10.sup.2 moles/(25 g Chl ml min).

(37) In the presence of pulsed light, at a power of 500 mW, emitted at a pulse frequency of 100 Hz, i.e. an energy of 5 mJ/s, corresponds an oxygen production rate K(O.sub.2) (per energy) of 0.01535 moles/(25 g Chl ml min) per 500 mJ/s, i.e. an increase by a factor 21.9 of the energy yield, as compared with the rate measured in the presence of continuous light. The results of the experimental confirmation of this analysis are repeated in Table 2 below.

(38) TABLE-US-00003 TABLE 2 comparison of the oxygen production rate in the presence of pulsed or continuous light, with a power of 555 mW at 673 nm. Yield Energy/s K(O.sub.2) [K(O.sub.2)/energy Mode mJ/s mol/(25 g Chl ml min) unit]* Continuous 555 0.0846 1.52 10.sup.4 Pulsed 5.4 0.0174 32.22 10.sup.4 *mol/(25 g Chl ml min mJ)

(39) The analysis of FIGS. 4 to 6, and in particular of FIGS. 5 and 6, gives the possibility of demonstrating that the energy yield in pulsed mode is increased by a factor ranging from 1.03 to 61.75 according to the pulse frequency and therefore to the amount of energy per second, as compared with the application of the method in the presence of continuous light at an equal power.

(40) The results of FIG. 5 are copied in the following Table 3:

(41) TABLE-US-00004 TABLE 3 Factor of Energy yield K (O.sub.2) increase in the Energy (mJ/s) moles/(25 g Chl ml min)/mJ yield ** 1.10 0.32727 61.75 3.21 0.22804 43.03 5.50 0.16364 30.87 6.42 0.15576 29.39 32.10 0.04984 9.40 64.20 0.02741 5.17 256.80 0.01137 2.15 385.20 0.00779 1.47 513.60 0.00631 1.19 577.80 0.00547 1.03 642 0.00530 1.00 ** as compared with continuous light at an energy of 642 mJ/s

(42) It should be understood that the present disclosure is by no means limited to the embodiments described above and that many modifications may be brought thereto within the scope of the appended claims.

(43) The present application may also reference quantities and numbers. Unless specifically stated, such quantities and numbers are not to be considered restrictive, but exemplary of the possible quantities or numbers associated with the present application. Also in this regard, the present application may use the term plurality to reference a quantity or number. In this regard, the term plurality is meant to be any number that is more than one, for example, two, three, four, five, etc. The terms about, approximately, near, etc., mean plus or minus 5% of the stated value. For the purposes of the present disclosure, the phrase at least one of A, B, and C, for example, means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C), including all further possible permutations when greater than three elements are listed.

(44) The principles, representative embodiments, and modes of operation of the present disclosure have been described in the foregoing description. However, aspects of the present disclosure which are intended to be protected are not to be construed as limited to the particular embodiments disclosed. Further, the embodiments described herein are to be regarded as illustrative rather than restrictive. It will be appreciated that variations and changes may be made by others, and equivalents employed, without departing from the spirit of the present disclosure. Accordingly, it is expressly intended that all such variations, changes, and equivalents fall within the spirit and scope of the present disclosure, as claimed.