Life Sciences Division

Biohydrogen; hydrogen photoproduction; photofermentation; biodegradation; environment; biodiversity; meatbolism.

Agricultural waste products and by-products represent a major source of organic material that is potentially convertible to hydrogen by fermentation. Classical or “dark” fermentation has a maximal theoretical yield of 4 mol H₂ and 2 mol acetate per mol glucose. On the other hand, “light” fermentation or photofermentation, which is carried out by photosynthetic, purple non-sulphur bacteria, is capable of completely converting organic substrates into H₂ et CO₂, giving a theoretical maximal yield of 12 mol H₂/mol de glucose. In practice, the maximum experimental yield of H₂ from dark fermentation is around 2 mol/mol glucose for mesophilic bacteria, with organic acids such as lactate, acetate and butyrate also being formed, and around 3 mol/mol glucose (i.e. 75%) for hyperthermophilic bacteria. In the case of photofermentation, the experimental H₂ yield is fairly low for sugars (around 30% for glucose) but is much higher for organic acids that are produced by dark fermentation. We are therefore investigating the possibility of coupling a stage of dark fermentation of sugars at high temperature with a stage of light fermentation of acetate to H₂, with an expected efficiency for the overall process of 75%.

The photosynthetic bacterium we are studying is Rhodobacter capsulatus strain B10. This strain has been extensively characterized in terms of its physiology, biochemistry and genetics, and its genome has been sequenced. The genetics and regulation of nitrogen fixation, catalysed by nitrogenase, has been particularly studied. Nitrogenase is a metalloenzyme, containing Fe and Mo at the active site, and the reducing power and ATP necessary for its activity are supplied by photosynthesis. Unlike plants and microalgae, photosynthetic bacteria do not produce oxygen, and electrons entering the photosystem are supplied by the oxidation of organic compounds or reduced inorganic compounds such as sulphur, rather than water. Electron transfer to nitrogenase involves a 4Fe-4S ferredoxin and a membrane-bound “Rnf” complex comprised of peripheral Fe-S and transmembrane proteins [Jouanneau et al., 1998].

During its catalytic cycle, the nitrogenase produces one mol H₂ per mol N₂ reduced, and in the absence of N₂, the nitrogenase continues to reduce protons to H₂. R. capsulatus contains an uptake hydrogenase that is able to reoxidize H₂ to H⁺, but no reversible hydrogenase, so nitrogenase is the sole enzyme responsible for H₂ production in this organism. The input of light energy enables the complete oxidation of substrates to H₂ and CO₂, as shown in the following equation for lactate:

C₃H₆O₃ + 3H₂O -----> 6H₂ + 3CO₂

Most of the CO₂ produced during the oxidation of organic acids is trapped in the growth medium as bicarbonate. The biogas evolved is therefore very pure (>95% H₂) and can be used directly by fuel cells to produce electricity. The direct coupling of photosynthetic cultures with a polyelectrolyte membrane fuel cell (PEM-FC) [He et al., 2005] and a solid oxide fuel cell (SOFC) (Picture 01) was demonstrated in collaboration with the LEPMI laboratory (Grenoble-INP) as part of the SOBBRE project financed by the Rhône-Alpes region from 2003-2006.



Picture 1:
A - 1 L photosynthetic culture of Rhodobacter capsulatus IR3 grown on lactate and producing hydrogen at a rate of 2 ml/min.
B - 1 W capacity PEMFC-type fuel cell connected to a voltmeter and operating a small electric-powered propellor.

The experiment with coupling to a PEM fuel cell has been demonstrated at the Palais de la Découverte in Paris (Picture #2).



Picture 2:
A - 20 L photosynthetic culture of Rhodobacter capsulatus B10 producing hydrogen, with a carrier gas flow (argon) of 2 L/min.
B - 800°C oven containing a home-made solid oxide fuel cell (SOFC) connected to a recording apparatus.

Several types of mutant strain have been isolated that show an increased capacity for hydrogen production. One mutant in particular, strain IR3, produced hydrogen at a greater rate, and for a longer time, than other strains tested under the same conditions [He et al., 2005] and we are attempting to identify the mutation or mutations responsible for its phenotype. This mutant was first identified on the basis of its inability to grow under autotrophic conditions, i.e., with H₂ + CO₂ as source of carbon and energy [Willison et al., 1984]. However, the activities of the enzymes specifically required for autotrophic growth (Rubisco, phosphoribulokinase, hydrogenase) are unaffected or only slightly reduced, and it is possible that indirect metabolic or regulatory effects are responsible for the increased photoproduction of hydrogen.

More generally, we wish to understand why the conversion efficiency of organic substrates into hydrogen is always < 100%, varying between 30% for sugars and 70-80% for organic acids under optimal conditions. We have identified a number of additional photofermentation products in H₂-producing cultures, but they are formed in insufficient quantities to account fir the difference between the theoretical and experimental yields of H₂. Quantitative analysis of both intracellular and extracellular components, particularly metabolites and polysaccharides, should enable us to determine the precise fate of the growth substrate during conditions of H₂-photoevolution. We especially wish to apply the technique of metabolic flux analysis (MFA) in order to model cellular metabolism under H₂-producing conditions and hence optimize the electron flow from carbon sources to nitrogenase by metabolic engineering [Hadike et al., 2011; Tao et al., 2012].

This work will be carried out as part of the HYCOFOL_BV project (Production of hydrogen by coupling of dark and light fermentation processes using plant biomass), which is financed by the Bioenergy programme of the French National Association for Research (ANR). This project, which runs from 2010-2013, is coordinated by John Willison at the CEA-Grenoble, in collaboration with 4 institutional partners (CNRS, Grenoble; IRD, Marseille; BRGM, Orléans; ARD, Pomacle (Reims). Its objective is to propose a process for the biological production of hydrogen from a plant biomass (wheat straw) by associating a stage of fermentation at high temperature, using hyperthermophilic bacteria of the genus Thermotoga, with a stage of photofermentation using a photosynthetic bacterium of the genus Rhodobacter. Each stage will be optimised with respect to the other, using microbiological and biochemical approaches combined with process engineering (design of experiments, studies of bioreactor configuration, etc.).

Polycyclic aromatic hydrocarbons (PAH) are recalcitrant pollutants, on account of their chemical stability and their poor solubility in water. The use of a water-immiscible organic phase to solubilise PAH increases their bioavailability and facilitates their biodegradation. We have developed a biphasic culture medium, in which PAH are dissolved in silicone oil, to isolate novel bacterial strains that are able to grow on high-molecular-weight (HMW) PAH, such as pyrene Krivobok et al., 2003] and chrysene [Willison, 2004]. In this medium, sufficient biomass is formed to enable biochemical analysis of the enzymes involved in biodegradation and the identification of metabolic intermediates in the biodegradation pathways. Some of these metabolites may be useful as environmental indicators of PAH degradation [Jouanneau et al., 2005].

Physiological studies of Mycobacterium sp. 6PY1, which degrades pyrene, and Sphingomonas sp. CHY-1, which degrades chrysene, have revealed the existence of adaptive mechanisms to growth in biphasic media, involving hydrophobic interactions with the oil phase [Abdelhay et al., 2008, 2009] (Picture #3).



Picture 3 A:
Micelle of approx. 100 µm diameter containing cells of Mycobacterium sp. 6PY1 growing on pyrene in a biphasic culture medium.



Picture 3 B:
View of an oil emulsion drop of approx. 200 µm diameter showing bacteria adhering to the surface (Mycobacterium sp. 6PY1, growing on biphasic culture medium with pyrene).

These factors affect the rate of degradation of PAH in the organic phase and lead to significant changes in the profile of metabolites that accumulate during biodegradation. The understanding of these mechanisms will be important from the point of view of using biphasic culture media for bioremediation. In this regard, the design of experiments (DOE) method has been used to optimise PAH biodegradation in industrial sludge from a top furnace that is also contaminated with heavy metals. Under optimised conditions, 95% degradation of PAH was achieved in 10 days. Inoculation of the sludge with Sphingomonas sp. CHY-1, which degrades a wide range of PAH, including alkylated PAH, had no effect on biodegradation, most likely because of the high levels of heavy metals, particularly Zn. Several bacterial strains resistant to heavy metals were isolated from the sludge [Cheik et al., 2010] and Zn-resistance was successfully transferred from one of these strains, Methylobacterium sp. BHF005, to Sphingomonas sp. CHY-1, by conjugation.

Pierre Caumette / Robert Duran, Université de Pau et des Pays de l'Adour, IPREM-EEM, UMR CNRS 5254, Équipe Environnement et Microbiologie, IBEAS, UFR Sciences et Technologies, BP 1155, 64013 Pau cedex

Jean-Pierre Magnin, Grenoble-INP, LEPMI, UMR 5631 (CNRS-INPG-UJF), Laboratoire d'Electrochimie et de Physico-chimie des Matériaux et des Interfaces, BP 75, 38402 St. Martin d'Hères cedex.

Muriel Raveton, Université Joseph Fourier, LECA-P3E, UMR CNRS 5553, Laboratoire d'Ecologie Alpine, BP 53, 38041 Grenoble Cedex 09.

Eric Latrille/ Eric Trably, Laboratoire de Biotechnologie de l'Environnement, INRA-LBE, Avenue des Etangs, 11100 Narbonne

Arwa A, Baup S, Gondrexon N, Magnin JP and Willison J
Enhancement of mass transfer characteristics and phenanthrene degradation in a two-phase partitioning bioreactor equipped with internal static mixers.
Biotechnology and Bioprocess Engineering, 2011, 16(2): 413-418
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Jouanneau Y, Martin F, Krivobok S and Willison J
Ring-hydroxylating dioxygenases involved in PAH biodegradation: Structure, function and biodiversity.
In A-I Koukkou ed., Microbial bioremediation of non-metals: Current research, 2011, pp. 149-175, Caister Academic Press, Norfolk, UK

Al Bsoul A, Magnin JP, Commenges-Bernole N, Gondrexon N, Willison J and Petrier C
Effectiveness of ultrasound for the destruction of Mycobacterium sp strain (6PY1).
Ultrasonics Sonochemistry, 2010, 17(1): 106-110
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Cheikh M, Magnin JP, Gondrexon N, Willison J and Hassen A
Zinc and lead leaching from contaminated industrial waste sludges using coupled processes.
Environmental Technology, 2010, 31(14): 1577-1585
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Obeid J, Flaus J-M, Adrot O, Magnin J-P and Willison JC
State estimation of a batch hydrogen production process using the photosynthetic bacteria Rhodobacter capsulatus.
International Journal of Hydrogen Energy, 2010, 35(19): 10719-10724
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Sérandour J, Willison J, Thuiller W, Ravanel P, Lempérière G and Raveton M
Environmental drivers for Coquillettidia mosquito habitat selection: A method to highlight key field factors.
Hydrobiologia, 2010, 652(1): 377-388
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Abdelhay A, Magnin JP, Gondrexon N, Baup S and Willison J
Adaptation of a Mycobacterium strain to phenanthrene degradation in a biphasic culture system: Influence on interfacial area and droplet size.
Biotechnology Letters, 2009, 31(1): 57-63
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Obeid J, Magnin JP, Flaus JM, Adrot O, Willison JC and Zlatev R
Modelling of hydrogen production in batch cultures of the photosynthetic bacterium Rhodobacter capsulatus.
International Journal of Hydrogen Energy, 2009, 34(1): 180-185
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Abdelhay A, Magnin JP, Gondrexon N, Baup S and Willison J
Optimization and modeling of phenanthrene degradation by Mycobacterium sp. 6PY1 in a biphasic medium using response-surface methodology.
Applied Microbiology and Biotechnology, 2008, 78(5): 881-888
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Sérandour J, Reynaud S, Willison J, Patouraux J, Gaude T, Ravanel P, Lempérière G and Raveton M
Ubiquitous water-soluble molecules in aquatic plant exudates determine specific insect attraction.
PLoS One, 2008, 3: e3350
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Raveton M, Aajoud A, Willison J, Cherifi M, Tissut M and Ravanel P
Soil distribution of fipronil and its metabolites originating from a seed-coated formulation.
Chemosphere, 2007, 69(7): 1124-1129
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He D, Bultel Y, Magnin JP and Willison J
Kinetic analysis of photosynthetic growth and photohydrogen production of two strains of Rhodobacter capsulatus.
Enzyme and Microbial Technology, 2006, 38(1-2): 253-259
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Raveton M, Aajoud A, Willison JC, Aouadi H, Tissut M and Ravanel P
Phototransformation of the insecticide fipronil: Identification of novel photoproducts and evidence for an alternative pathway of photodegradation.
Environmental Science and Technology, 2006, 40(13): 4151-4157
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Vignais PM, Magnin JP and Willison JC
Increasing biohydrogen production by metabolic engineering.
International Journal of Hydrogen Energy, 2006, 31(11): 1478-1483
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He D, Bultel Y, Magnin JP, Roux C and Willison JC
Hydrogen photosynthesis by Rhodobacter capsulatus and its coupling to a PEM fuel cell.
Journal of Power Sources, 2005, 141(1): 19-23
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Jouanneau Y, Willison JC, Meyer C, Krivobok S, Chevron N, Besombes JL and Blake G
Stimulation of pyrene mineralization in freshwater sediments by bacterial and plant bioaugmentation.
Environmental Science and Technology, 2005, 39: 5729-5735
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Willison JC
Isolation and characterization of a novel sphingomonad capable of growth with chrysene as sole carbon and energy source.
FEMS Microbiology Letters, 2004, 241: 143-150

Krivobok S, Kuony S, Meyer C, Louwagie M, Willison JC and Jouanneau Y
Identification of pyrene-induced proteins in Mycobacterium sp. 6PY1: Evidence for two ring-hydroxylating dioxygenases.
Journal of Bacteriology, 2003, 185: 3828-3841

Jouanneau Y, Jeong HS, Hugo N, Meyer C and Willison JC
Overexpression in Escherichia coli of the rnf genes from Rhodobacter capsulatus--characterization of two membrane-bound iron-sulfur proteins.
European Journal of Biochemistry, 1998, 251(1-2): 54-64

Willison JC, Madern D and Vignais PM
Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata.
Biochemical Journal, 1984, 219(2): 593-600

Jouanneau Y, Martin F, Krivobok S and Willison J
Ring-hydroxylating dioxygenases involved in PAH biodegradation: Structure, function and biodiversity.
In A-I Koukkou ed., Microbial bioremediation of non-metals: Current research, 2011, pp. 149-175, Caister Academic Press, Norfolk, UK