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OBJECTIVES
The main objective of the EU Biohydrogen project is the production of hydrogen from energy crops and wastes employing (hyper)thermophilic and photoheterotrophic microorganisms to supply the fuel cell industry with clean hydrogen gas.
The project involves several disciplines which are aimed at the production of biomass, the processing, the conversion and the application of the final product hydrogen. The first objective is the selection and pretreatment of the biomass from energy crops and waste streams to match the requirements of a suitable feedstock for (hyper)thermophilic fermentation. The modified biomass will subsequently feed the ‘hydrogen’ factory which is composed of two fermentations (Fig. 1). The first stage involves the (hyper)thermophilic conversion of the feedstock to hydrogen, carbon dioxide and organic acids, at the theoretical efficiency of 4 moles hydrogen per mole glucose consumed. The second fermentation addresses the photoheterotrophic conversion of organic acids to hydrogen and carbon dioxide, at an efficiency approaching the theoretical maximum of 8 moles hydrogen per 2 moles acetate (derived from 1 mole glucose). There are several objectives in this part of the project. Firstly, existing and newly isolated microorganisms will be employed for the optimalisation of the fermentations. When applicable, genetic engineering will be used for strain improvement to achieve the theoretically maximum production of 12 moles of hydrogen per mole consumed glucose in the combined bioprocess. The ‘hydrogen’ factory is ready when the production of hydrogen from both fermentations is coupled to an efficient hydrogen recovery process in which hydrogen is collected and carbon dioxide is removed.

PROJECT WORKPLAN
The raw materials utilised in the project for the production of fermentation feedstock are derived from two European biomass streams: the first from an energy crop yielding mostly sugars and lignocellulose and the other one from wastes streams yielding mainly cellulose. The production of these biomass streams will be optimised with respect to yield and supply but also qualitatively in view of processing to suitable feedstock for anaerobic, (hyper)thermophilic fermentations. Therefore, chemical, physical, and biotechnological protocols for processing of the raw materials will be developed. Dependent on the outcome from physiological, molecular biological and processtechnological studies in this project, hydrolytic enzymes potentially excreted by the hydrogen producers themselves, may be employed in the pretreatment of the raw materials. Finally, in view of whole crop utilisation, attention will be given to apply one of the main byproducts, lignin, as a secondary biofuel in the bioprocess and other applications will be sought for secondary byproducts.
The following step in the project is the development of the bioprocess for the conversion of the fermentable feedstock, in 2 stages, to hydrogen. In the first stage, the biomass is heterotrophically fermented to mainly acetate, carbon dioxide and hydrogen. Thereafter, a second stage for phototrophic conversion of acetate to carbon dioxide and hydrogen will be applied. Available microorganisms will be screened and new strains will be isolated for optimal performance in the conversion of the biomass to hydrogen according to:

Heterotrophic fermentation in stage 1:
C6H12O6 + 4 H2O ----> 2 CH3COO- + 4 H2 + 2 HCO3- + 4 H+

Photoheterotrophic fermentation in stage 2:
CH3COOH + 2 H2O + photons ---> 4 H2 + 2 CO2

Besides physiological studies aimed at the optimisation of hydrogen production by existing or newly isolated strains, genetic engineering is included in the bioconversion as hydrogen production efficiency may be further increased by strain improvement at the level of the hydrogenase or nitrogenase activities at the one hand, or by increasing the rate of substrate utilisation at the level of the hydrolytic activities on the other.
The work associated with the bioconversion step will also deal with the technological aspects related to working with biomass-derived substrates (slurries), linkage of two different fermentations and optimisation of the bioprocess, starting with using the commercially available model organisms.
Finally, a safe and cost-efficient system for the recovery, handling and storage of hydrogen will be developed. The end-use performance of the produced hydrogen will be assessed and this will be done in close collaboration with the partners involved in the production of biohydrogen. In this way, the specific constraints encountered in industrial requirements and in the production processes of biomass and hydrogen will be taken into consideration during the whole course of the project, enabling the development of an integrated, well-balanced biohydrogen production process.

Overall methodologies

Partner 1 (CO: ATO-DLO) will start assessing the technological adaptations which are needed to enable the smooth operation of a fermentation running on the expected feedstock. The latter will not be a clear liquid but, potentially, a viscous liquid or slurry. The required modifications will be implemented and scale-up of hydrogen producing fermentations using already available microorganisms will be initiated. At the same time, continuous fermentations will be run on a smaller scale for the selection of the best performing organisms. Finally, processtechnological methodologies will be employed to combine the heterotrophic and photoheterotrophic fermentations in one integrated bioprocess. The development of a cost efficient system for the recovery of hydrogen will be done in close contact with partner 6 (CR 6: AL).

Partner 2 (CR 2: NTUA) will characterise the energy crop selected for this project with respect to the requirements related to quality and security of supply, dealing with methodologies for harvesting, mechanical conversion, storage and quality control in order to meet subsequent processing requirements. The emphasis will be on storage systems that preserve the carbohydrate fraction of the raw material and pretreatment systems that increase the recovery of fermentable feedstock upstream to the bioreactors. This includes the development of local processing technologies which add value, reduce transport costs, and improve whole crop utilisation, by application of non-fermentable fractions ¨¢nd fermentable fractions. Overall, this partner will assess the applicability of sweet sorghum for the purposes of the project, taking into account the regional conditions, as well as the agricultural production and handling logistics.

Partner 3 (CR 3: BUTE) will investigate and characterise the potential of plant biomass derived from waste streams from industries processing agricultural produce. There will be a special focus on the potential utilisation of paper sludge as a waste stream containing mainly cellulose. The general methodology will be similar as described for partner 2 (CR 2: NTUA) albeit that the chemical difference of the biomass, i.e. cellulose instead of, to a large extent, sugars, will confer its own, specific, approaches. Furthermore, this partner will investigate the production of (hemi)cellulolytic enzymes by the (hyper)thermophilic organisms of this project in order to determine their suitability for increased hydrolysis.

Partner 4 (CR 4: WAU) will work on the development of a biological conversion of biomass to hydrogen employing specialist microorganisms or isolating new strains of hydrogen-producing microorganisms, aimed at a complete conversion of carbohydrates to hydrogen and carbon dioxide, in a heterotrophic and subsequent photoheterotrophic fermentation. The research will concern optimisation of growth and production methodologies on the basis of acquired physiological knowledge (from literature and experiments). Partner 4 will also study the requirements of a pilot scale reactor for the photoheterotrophic fermentation. The methodologies involved in this part of the project are related to the exploration of scale-up processes for the design of a reactor in which efficient light penetration together with adequate mixing of feedstock and microorganisms are warranted.

Partner 5 (CR 5: JATE/BRC) will also play a role in the optimisation of the biological conversion but then through the application of genetic engineering. For strain improvement with respect to the efficiency of hydrogen production, research will be focussed on the overexpression of desired and silencing of undesired genes coding for hydrogenase and nitrogenase activities. When necessary, an attempt will be made to increase the substrate range of the hydrogen producing microorganisms by introducing genes coding for hydrolytic enzymes.

Partner 6 (CR 6: AL) will develop a system for the safe and cost-efficient recovery and handling of hydrogen from the designed bioprocess and assist other partners in hydrogen technology related aspects, especially safety, of the fermentation studies. This partner will also perform the quality testing of the delivered product according to the specific end-use (fuel cells) and determine the specifications of the biohydrogen in order to make it a promising product with good socio-economic prospectives.