Hoekema, S., Janssen, M., Tramper, J. and Wijffels, R.H. Introduction Hydrogen is a good alternative energy source in comparison to fossil fuels. Sustainable production technologies need to be developed to provide the hydrogen that will be needed in the near future. At the Food and Bioprocess Engineering Group the photoheterotrophic conversion of acetate to hydrogen is studied and optimised. Acetate to hydrogen conversion takes place according to the following reaction: 2 CH 3 COO H 2 O + “light energy”  4 HCO H + +8 H 2 [1] We use a purple non-sulphur bacterium, grown photoheterotrophically, for this conversion. The conversion has a positive  G 0 and is therefore energetically unfavourable. Light energy has to be utilised to overcome this energy barrier in a process called anoxygenic photosynthesis. The produced gas consists for about 90% of H 2, the remainder is CO 2. Lab-scale photo bioreactor The feasibility of the process depends on the efficiency of hydrogen production from acetate and light. Equation 1 indicates that the theoretical maximal yield of hydrogen on acetate is 4 mol mol -1. Growth of biomass will consume part of the acetate and decrease this efficiency. Nitrogen starvation seems a good way of decreasing growth. Ammonium is used as nitrogen source, and since ammonium inhibits hydrogen production at higher concentrations, a continuous fermentation system is needed. A new and fully autoclavable flat panel reactor system for continuous fermentation, depicted on the right (Figure 1), is developed. In this system, the light regime is simple when compared to other systems (one dimensional). The light path is only three centimetres and this, combined with turbulent mixing, could make light utilisation more efficient *. As important, the simple light regime allows accurate measurement of light absorption by the cells. The mixing is facilitated by a gas recirculation system also shown in Figure 1. Planned experiments To gain more insight in the behaviour of our bacterium in a continuous system, a so called A-stat experiment will be performed in the flat panel reactor. Upon steady state at low growth rate (about 20 % of the maximal growth rate,  max ) the dilution rate will be increased smoothly until the growth rate does not increase any further. This process is schematically depicted in Figure 2. While the dilution rate increases, the biomass concentration decreases. At a certain point the growth rate cannot keep up with the dilution rate, indicating that the maximal growth rate has been reached. Biomass productivity can be calculated by multiplying C x and . This biomass productivity clearly indicates an optimum. A similar curve can be made for the volumetric hydrogen production. This experiment will provide information on the efficiency of the bacterium with respect to light and acetate used for growth and hydrogen production under varying environmental conditions. * Efficiency of light utilisation is defined as the energy stored in hydrogen as a fraction of the light energy absorbed by the cells. Food and Bioprocess Engineering Group Department of Agrotechnology and Food Sciences, Wageningen University P.O. Box 8129, 6700 EV, Wageningen, The Netherlands  Optimisation of continuous biological hydrogen production using purple non-sulphur bacteria Figure 1 Lab-scale flat panel photo bioreactor On the right a picture of the developed bioreactor is presented, on the left a schematic drawing explaining the gas recirculation system. A pump [1] recirculates the gas in the system while produced gas can be collected [2]. Pressures are held constant using pressure vessels [3] and overpressure valves [4]. The flow through the system is controlled by a mass flow controller [5] and a condenser [6] prevents water vapour from entering the gas recirculation. Figure 2 Typical A-stat curves The curves of the dilution rate (D), growth rate (µ), biomass concentration (C x ) and biomass productivity (P x ) as a function of time during a typical A-stat experiment.         90% H 2 10% CO 2 D µ PxPx CxCx