METHOD FOR THE ELECTROCHEMICAL CONTROL OF THE PHOTOSYNTHETIC METABOLISM OF PURPLE NON-SULFUR BACTERIA AND REDOX MEDIATORS THEREOF

Abstract

The present invention relates to a method for the electrochemical control of the level of gene expression in purple non-sulfur bacteria, in particular Rhodobacter, and relative applications for the treatment of wastewater. The invention also relates to the use of fat-soluble redox mediators capable of permeabilizing the bacterial membrane and altering the oxidation state of the disulfide bond present in thioredoxins.

Claims

1. A method for the electrochemical control of the photosynthetic metabolism of purple non-sulfur bacteria using an electrochemical cell, said electrochemical cell comprising a working electrode, a counter electrode and, optionally, a reference electrode in the presence of an irradiation source, said method comprising adding a redox mediator having an equilibrium potential more negative than cytoplasmic thioredoxins in said electrochemical cell, wherein said working electrode has an equilibrium potential more negative than the redox mediator.

2. The method for the electrochemical control of the photosynthetic metabolism of purple non-sulfur bacteria according to claim 1, wherein said purple non-sulfur bacteria are selected from the group consisting of Rhodobacter, Rhodopseudomonas and Rhodospirillum.

3. The method for the electrochemical control of the photosynthetic metabolism of purple non-sulfur bacteria according to claim 2, wherein said purple non-sulfur bacteria belong to the Rhodobacter sphaeroides or Rhodobacter capsulatus species.

4. The method for the electrochemical control of the photosynthetic metabolism of purple non-sulfur bacteria according to claim 1, wherein the working electrode of the electrochemical cell is selected from the group consisting of gold, platinum and stainless steel; the counter electrode is selected from graphite and stainless steel, and the reference electrode, if present, is selected from the group consisting of hydrogen electrode (SHE), SCE and Ag/AgCl.

5. The method for the electrochemical control of the photosynthetic metabolism of purple non-sulfur bacteria according to claim 1, wherein the redox mediator is a safranin.

6. The method for the electrochemical control of the photosynthetic metabolism of purple non-sulfur bacteria according to claim 1, wherein the redox mediator is added in the electrochemical cell at a concentration ranging from 25 nM to 250 nM.

7. The method for the electrochemical control of the photosynthetic metabolism of purple non-sulfur bacteria according to claim 1, wherein the working electrode has an equilibrium potential of −660 mV.

8. An electrochemical cell comprising a culture of purple non-sulfur bacteria, a redox mediator with an equilibrium potential of less than −460 mV, a working electrode, a counter electrode and, optionally, a reference electrode; wherein said working electrode has an equilibrium potential more negative than the redox mediator.

9. The electrochemical cell according to claim 8, wherein the redox mediator is a safranin.

10. The electrochemical cell according to claim 8, wherein the working electrode is selected from the group consisting of gold, platinum and stainless steel; the counter electrode is selected from the group consisting of graphite and stainless steel, and the reference electrode, if present, is selected from the group consisting of hydrogen electrode (SHE), SCE and Ag/AgCl.

11-12. (canceled)

13. A method for the chemical oxidation of a substrate comprising one or more carbon compounds, comprising: (a) cultivating purple non-sulfur bacteria in an electrochemical cell of a reactor in the presence of an irradiation source and a redox mediator having an equilibrium potential more negative than the cytoplasmic thioredoxins of purple non-sulfur bacteria; wherein said electrochemical cell is characterized by the presence of a working electrode, a counter electrode and, optionally, a reference electrode; wherein said working electrode has an equilibrium potential more negative than the redox mediator; and (b) putting purple non-sulfur bacteria with an activated photosynthetic metabolism in contact with a substrate comprising one or more carbon compounds.

14. The method for the chemical oxidation of a substrate according to claim 13, wherein said purple non-sulfur bacteria is selected from the group consisting of Rhodobacter, Rhodopseudomonas and Rhodospirillum, preferably the Rhodobacter sphaeroides and Rhodobacter capsulatus species.

15. The method for the chemical oxidation of a substrate according to claim 13, wherein said substrate is a liquid.

16. The method for the chemical oxidation of a substrate according to claim 13, wherein the redox mediator is a safranin.

17. The method for the chemical oxidation of a substrate according to claim 13, wherein the redox mediator is added at a concentration ranging from 25 nM to 250 nM.

18. The method for the chemical oxidation of a substrate according to claim 5, wherein the safranin is safranin T having the following formula: ##STR00003## and an equilibrium potential of −540 mV.

19. The method for the electrochemical control of the photosynthetic metabolism of purple non-sulfur bacteria according to claim 6, wherein the redox mediator is added in the electrochemical cell at a concentration of 50 nM.

20. The electrochemical cell according to claim 9, wherein the safranin is safranin T having the following formula: ##STR00004## and an equilibrium potential of −540 mV.

21. The method for the chemical oxidation of a substrate according to claim 15, wherein the liquid is a wastewater of the food industry.

22. The method for the chemical oxidation of a substrate according to claim 16, wherein the safranin is safranin T having the following formula: ##STR00005## and an equilibrium potential of −540 mV.

23. The method for the chemical oxidation of a substrate according to claim 17, wherein the redox mediator is added at a concentration of 50 nM.

Description

[0067] The present invention will now be described for illustrative, but non-limiting, purposes according to a preferred embodiment with particular reference to the attached figures, wherein:

[0068] FIG. 1 shows the action scheme of thioredoxins, on the part of gyrase B, in relation to the oxygen tension in Rhodobacter.

[0069] FIG. 2 illustrates the aerobic growth curve of R. sphaeroides in M550 wherein the ordinates show the absorbance at 660 nm at time t-at time 0, and in the abscissas the hours that have elapsed since inoculation.

[0070] FIG. 3 illustrates the safranin formula and a diagram with the equilibrium potentials of safranin and thioredoxin.

[0071] FIG. 4 shows the optical absorption spectra of bacteriochlorophyll extracted from two different preparations of R. sphaeroides.

[0072] FIG. 5 exemplifies an experimental setup of the reactor used for the abatement of c.o.d. (chemical oxygen demand) in wastewater using the method according to the invention.

[0073] FIG. 6 shows the trend of the c.o.d. (chemical oxygen demand) in relation to time and for different initial dosages of c.o.d. (sugar or addition of a known volume and c.o.d. effluents).

[0074] The following non-limiting examples are now provided for a better illustration of the invention wherein the authors have implemented an electrochemical control method of the level of gene expression in Rhodobacter.

EXAMPLE 1: PREPARATION METHOD FOR PROMOTING THE EXPRESSION OF PHOTOSYNTHETIC GENES IN RHODOBACTER TO THE DETRIMENT OF THOSE OF THE RESPIRATORY CHAIN

[0075] A strain of Rhodobacter sphaeroides (ATCC® 55304 ™) was used whose growth curve in optimized medium M550 is shown in FIG. 2.

[0076] A homemade potentiostat was used as an external source of free energy, capable of providing a current of up to 100 mA and keeping the potential constant within the range of −2 V÷+2 V).

[0077] Safranin T was used for reducing the disulfide bonds of the thioredoxins (which has an equilibrium potential of about −540 mV (vs SCE, reference electrode), i.e. more negative than the corresponding potential of the thioredoxins which typically is around −460 mV (vs SCE), as shown in FIG. 3.

[0078] Using these molecules inside a reactor in which a working electrode, a counter electrode and a possible reference electrode are also inserted and keeping the working electrode at a more negative potential than the equilibrium potential of the mediator, the latter will remain reduced and will thus be capable of reducing the disulfide bonds of the cytoplasmic thioredoxins, regardless of the presence or absence of atmospheric oxygen in the fermenter.

[0079] Thanks to the diffusion, the redox mediator, after conditioning the oxidative state of the bacterial cytoplasm, will be able to come back into contact with the working electrode and will therefore be reduced again by the working electrode kept at a negative potential by the potentiostat, being recharged with electrons and being able to repeat the cycle described.

[0080] The materials of the electrodes used are: [0081] Working electrode: stainless steel grid with 1 mm mesh spacing. [0082] Counter electrode: graphite bars (0.8 cm in diameter, 30 cm long) [0083] Reference electrode: (SCE)

[0084] Safranin T, molecular mass equal to 350.85. (Fluka) at a concentration equal to 50 nM, was used as redox mediator in the cell.

[0085] The solution inside the fermenter/reactor was kept under mild stirring to facilitate the recirculation of the redox mediator molecules.

[0086] The overall ionic strength of the solution must be >10 mM to allow the potentiostat to control the potential of the working electrode [4].

[0087] The periodic dosage of vitamins such as biotin (0.06-0.6 mg/1), nicotinamide (1-10 mg/1), nicotinic acid (1-10 mg/1) and thiamine (2-20 mg/i) is also essential for supporting cell duplication during the increase in the biomass and the functioning of the system.

[0088] A comparison between the quantity of bacteriochlorophyll, an essential component of photosynthetic pigments (reaction centers, antenna pigments LHI and LHII) synthesized by the population of Rhodobacter sphaeroides subjected to the electrochemical control conditions with the above parameters, and that produced by a population of the same bacteria but maintained in aerobic growth, is indicated in the data shown in the graph of FIG. 4. From the absorption spectra it can be observed that the absorbance of the solution obtained from the processing of the bacterial suspension with acetone/methanol, normalized to that at 660 nm (proportional to the bacterial number for R. sphaeroides) shows the absorption bands of bacteriochlorophyll. The intensity of the bands of the population subjected to electrochemical control in the presence of 50 nM safranin appears almost tripled compared to the corresponding aerobic population.

[0089] This result indicates that the use of these conditions actually simulates a shortage of oxygen that induces the overexpression of photosynthetic genes. In other words, these results indicate the capacity of the technology proposed of electrochemically modulating the expression level of Rhodobacter genes (photosynthesis vs respiration).

EXAMPLE 2: USE OF THE ELECTROCHEMICAL MODULATION TECHNOLOGY OF THE EXPRESSION LEVEL OF RHODOBACTER GENES FOR WASTEWATER TREATMENT

[0090] The technology illustrated in Example 1 was used for reducing the c.o.d. (chemical oxygen demand) value in solutions of a known composition or following the addition of the volume of wastewater of an industrial food origin as an example of the effectiveness of the method found.

[0091] After growing the biomass of R. sphaeroides (ATCC® 55304™) up to desired values within the range of values of sst-total suspended solids—equal to 1-5 g/l, degradation kinetic measurements of the c.o.d. were carried out in relation to time. After reaching a threshold value of 200-300 mg/l of c.o.d., the reactor was enriched again by dosing sugar or adding known volumes of wastewater with a known c.o.d value.

[0092] The reactor used (FIG. 5) was configured with the following parameters: [0093] Reactor volume: 40 l [0094] T=28° C. [0095] pH=7 [0096] Redox potential reactor solution=−300÷−400 mV [0097] SST=1÷0.3 g/l [0098] SSV=0.9÷0.28 g/l [0099] NH.sub.4.sup.+=8÷10 mg/l [0100] NO.sub.2=0 [0101] NO.sub.3=0 [0102] PO.sub.4.sup.+>100 mg/l [0103] V.sub.WE=−660 mV (vs SCE) [0104] Safranin concentration=50 nM [0105] Working electrode: stainless steel grid [0106] Counter electrode: graphite bar [0107] Reference electrode: SCE

[0108] The reactor is continuously irradiated during the whole operating period by 3 LED lamps (15 W, T=6400K, Aigostar) kept at a distance of 30 cm from the upper surface.

[0109] FIG. 6 shows a graph of the trend of the c.o.d. in relation to time for a reactor configured as described above. It can be noted that the abatement of c.o.d. follows a “sawtooth” trend with discontinuity in correspondence with the various additions of oxidizable carbon (sugar or wastewater). The abatement kinetics are naturally faster in correspondence with high values of c.o.d. (corresponding to abundant nourishment available for the bacteria), but it can be observed how the c.o.d. abatement rate increases with the same c.o.d. values in the various subsequent cycles, indicating an adaptation of the bacterial population to environmental conditions. Whereas, in fact, in the initial (linear) part of the first cycle the abatement rate was 294 mg/l per day, in the cycle relating to the second dosage of c.o.d. this rate becomes equal to 482 mg/l per day with a substantially equal biomass present (approximately 1 g/l).

[0110] These preliminary results suggest that R. sphaeroides, when subjected to electrochemical control in the presence of light irradiation and an appropriate concentration of fat-soluble redox mediator and with a more negative equilibrium potential than that of cytoplasmic thioredoxins, behaves as an excellent c.o.d. reducer.

[0111] Furthermore, under the conditions used, no excess biomass is produced and no forced aeration is required by the microorganism, unlike the activated sludge composed of cocktails of aerobic microorganisms commonly used in the purifiers of the state of the art.

BIBLIOGRAPHY

[0112] [1] Kuanyu Li et al., Nucleic Acids Research, 2004, 32, 4563-4575. [0113] [2] E. Katz, A. N. Shipway and I. Willner Biochemical fuel cells in Handbook of Fuel Cells—Fundamentals, Technology and Applications, Eds. Wolf Vielstich. [0114] [3] Hubert A. Gasteiger. Arnold Lamm. Vol. 1: Fundamentals and Survey of Systems. 2003 John Wiley & Sons, Ltd. [0115] [4] A. Bard & L. Faulkner Electrochemical Applications, second Edition, Wiley, 2001.