Etude de la production d'hydrogène en bioréacteur par une bactérie photosynthétique Rhodobacter capsulatus 1. Photobioréacteur et conditions optimales de production d'hydrogène Hydrogen production in bioreactor by a photosynthetic bacterium Rhodobacter capsulatus 1. Photobioreactor and optima conditions of hydrogen production

Résumé

Un réacteur de 10 litres, automatisé pour la culture continue en anaérobiose de bactéries photosynthétiques, a été réalisé et mis au point. Ce réacteur parfaitement agité a été utilisé dans différentes conditions de fonctionnement en système fermé (batch), en système semi-ouvert (atouts de substrats concentrés en discontinu), en système ouvert (chémostat) avec et sans recyclage de la biomasse, afin d'étudier la consommation, par les bactéries, d'un substrat carboné, le lactate. La production d'hydrogène par la bactérie photosynthétique Rhodobacter capsulatus, souche B10, résultant de la dégradation du lactate, est optimale pour des cultures en continu diluées, limitées en source azotée. Ainsi, à un taux de dilution de 0,04 h^-1, avec 5 mM glutamate dans le milieu nutritif, la densité bactérienne étant de 2,1 è 660 nm, on a observé une production continue moyenne de 65 ml • h^-1• l^-1 pendant une période de 200 heures. Pour des concentrations bactériennes élevées, la limitation d'énergie lumineuse entrain une perte d'activité nitrogénase et, de ce fait, une chute de la production d'hydrogène.

Mots-clés:

• Rhodobacter capsulatus,
• dégradation du lactate,
• photobioréacteur,
• production d'hydrogène

Abstract

A photobioreactor was set up to cultivate a photosynthetic bacterium in continuous cultures. The bioreactor was designed so as to 1) allow the capture of light energy by bacteria through a spiral transparent flexible tube placed under the light, in a water bath maintaining the growth temperature at 30 °C; 2) male the suspension of bacteria circulate continuously in the reactor with a volumetric pump to maintain the medium homogeneous; 3) allow degassing of the suspension in a degassing chamber; 4) feed the culture with nutritive media, add neutralizing solution (pH 7) and withdrax aliquots white maintaining constant the volume of the culture; 5) recycle the bacteria by filtration when the bioreactor was used as e closed system (batch).

The photosynthetic bacterium was Rhodobacter capsulatus strain B10 is known to lie a good H₂ producer [Hillmer and Gest (1977) J. Bacteriol. 129, 724-731]. The bioreactor was run using 10 l of a synthetic medium containing lactate as carbon source and glutamate as nitrogen source. It was studied for its capacity to degrade lactate. Glutamate was the growth-liliting substrate allowing a maximum derepression of nitrogenase, the enzyme catalysing the reduction of protons to H₂. The bacterial suspension was continuously circulated in the photoreactor, conceived as a plane light captor of 1 m², to avoid bacterial self-shading and allow regeneration of ATP by photophosphorylation at high rates. The circuit was tightly closed to avoid air entry, which would prevent H₂ production due to respiration of the bacteria.

To run it under automated conditions, the bioreactor was equipped with two temperature sensors, two pH electrodes, a water level detector, a manometer and a computer-controlled electric valve. The bioreactor, of the well-mixed type, was used under various working conditions, namely as a closed (batch) system, as a fed-batch system (discontinuous additions of concentrated substrates), and as an open system (chemostat) with or without biomass recycling.

Control of key parameters (pH, temperature, dilution rates) allowed us to define the culture conditions producing maximal amounts of molecular hydrogen. The production of H₂ accompanying lactate degradation was maximal in diluted nitrogen-limited continuous cultures. It was observed at a dilution rate of 0.04 h^-1 with 5 mM glutamate in the influent medium, the optical density of the culture being 2.1 at 660 nm. Under these conditions an average production H₂ of 85 ml • h^-1 • l^-1 was observed over a 200 h period. At higher bacterial concentrations, the limitation of light energy resulted in a decrease in nitrogenase activity and therefore in a drop in the production of hydrogen.

The interdependence of various parameters (pH, dilution rates, N and C sources light intensity) renders the system complex and not easily controlled by computer. Indeed, we observed that during recycling of the bacteria by the use of an ultrafiltration cell, the bacteria became a fermentative-type of metabolism accompanied by a decrease in nitrogenase activity and therefore in a drop in the production of hydrogen.

Keywords:

• Rhodobacter capsulatus,
• lactate degradation,
• photobioreactor,
• hydrogen production