Production of hydrogen from plant biomass using a combination of light and dark fermentation processes – HYCOFOL_BV

The most efficient strain for high-temperature was selected by screening a collection of hyperthermophilic bacterial strains, maintained at the IRD, for their ability to produce hydrogen from pre-treated wheat straw, supplied by the industrial partner, ARD. Scale-up for optimisation was carried out in a 5L bioreactor integrated into a high-temperature fermentation platform.
The best photosynthetic bacterial strain was selected from species obtained from the DSMZ culture collection and from a collection of mutants isolated by the CEA. The optimisation of H2 photoproduction from acetate was carried out at the CNRS, in purpose-built installations for phototrophic cultures, using the design of experiments (DOE) methodolo-gy.
Microbiological control of the fermentation steps was carried out by the BRGM, either by 16S rRNA gene diversity fingerprinting or by fluorescent labelling of the bacterial cells with 16S rRNA-specific probes. This involved the use of image analysis software, experimental validation of the oligonucleotide probes, and optimisation of the protocol for the extraction of bacteria from wheat straw, as well as the subsequent hybridization steps.

Sugars derived from the hydrolysis of wheat straw were converted into H2 with a yield of 75% of the maximal theoretical amount, by means of a sequential process associating treat-ment by the hyperthermophilic bacterium Thermotoga maritima with treatment by photosynthetic bacteria of the species Rhodobacter capsulatus and R. sphaeroides. No microbial contamination of the process due to the hydrolysed or micronized wheat straw is expected, except in the case of wheat straw after enzymatic hydrolysis, which necessitated sterilization prior to high-temperature fermentation.

The results obtained during the HYCOFOL_BV project are sufficiently promising to justify the development of a pre-industrial pilot-scale reactor for biohydrogen production, combining a high-temperature fermentation stage and a photofermentation stage. The coupled process will have an expected sugar-to-H2 conversion yield of at least 75%, and will allow the valorisation of a renewable agricultural waste product, the utilisation of solar energy and the production of a biogas of high purity. However, additional research on strain improvement and bioreactor configuration will be needed to achieve this goal.

The project partners have published 2 scientific articles describing results obtained during the project, and 4 additional articles are in preparation. They have also presented 10 communications at international scientific conferences (5 invited oral presentations and 5 poster presentations), notably at the Bioenergy DSV/CEA Conference in Paris in February 2012, at the 19th World Hydrogen Energy Conference (WHEC2012) in Toronto in June 2012, and at the IAHE BioH2 conference in Montreal in August 2013.

The fundamental research project HYCOFOL_BV (Production of hydrogen from plant biomass by a combination of light and dark fermentation processes) involves 4 public research laboratories (CEA, CNRS, IRD and BRGM) and 1 industrial (ARD). Its aim is to propose a bioprocess for the production of hydrogen from wheat straw, an agricultural by-product. The treatment of this biomass releases an effluent rich in pentose and hexose sugars that can be converted biologically into H2 by fermentation. The first stage of the bioprocess involves fermentation at high temperature (70-80°C) by hyperthermophilic bacteria of the order Thermotogales, which can ferment pentose and hexose sugars into acetate, CO2 and H2. The second stage involves photofermentation of acetate in the light, using mesophilic, photoheterotrophic bacteria of the genus Rhodobacter. The coupling of these two processes can theoretically lead to a complete conversion of sugars into hydrogen, with a maximum theoetical yield of 12 mol H2/mol glucose.

At first, the industrial substrate (wheat straw), after pretreatment by the industrial partner according to a procedure developed during the project SPPECABBE (ANR PNR-B 2005-2008), will be characterized physicochemically and microbiologically. A laboratory collection of hyperthermophilic bacteria will be screened for their ability to degrade cellulose and hemicellulose, with or without pretreatment, and to tranform the free sugars into H2. The fermentation at high temperature will then be studied, using the best strain or strains selected, using first a synthetic medium based on the composition of the industrial substrate and then the industrial substrate itself. The fermentation and culture parameters will be determined in terms of the conversion efficiency of the different sugars into H2, but also taking into account the compatibility of the end-products with the photofermentation stage and the microbiological stability of the process. In fact, the fate of the hyperthermpohilic bacteria, as well as that of the microbial poulation present intially in the industrial substrate, will be followed throughout the process using the tehcniques of molecular microbial ecology.

Photosynthetic bacteria are theoretically able to convert organic acids, such as acetate, completely into H2 and CO2. However, in practice, the yields are much lower and depend on the strain and the operating conditions. In the present project we propose to use molecular gentic, biochemicaland process engineering approaches to optimize the photoproduction of H2 from acetate. H2-overproducing strains of Rhodobacter capsulatus, isolated in the laboratory, will be studied for their ability to degrade acetate into H2 and the limiting steps wil be identified. The efficiency of photfemprenation will then be determined using, first, the effluent from high temperature fermentation of the synthetic medium, and second, the effluent from high temperature fermentation of the industrial substrate. The microbial population will also be followed during the photofermentation stage in order to evaluate the microbiological stabiltiy of the process. The effect of the photobioreactor configuration on the efficiency of H2 production will also be studied.

The pluridisciplinarity of the different partners involved in the project, and their competence in the areas of high-temperature fermenation, photofermentation and molecular microbial ecology, will alow them to solve eventual problems, such as inhibition of the photofermentation step by the effluent from the high temperature reactor or interference by contaminating microorganisms. The results obtained will be used to evaluate the feasability of a coupled process involving high-temperature fermenation and photofermentation for the transfomation of a plant biomass into H2.