The production rates and yields were investigated at 30 °C in a 2.3 l bioreactor operated in 19 batch and sequenced-batch mode using glucose and starch as substrates. In order to study the 20 precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, 21 cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 22 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 23 mol H 2 mol -1 glucose) was measured when the pH was maintained at 5.

12This paper investigated the enhancement effect of nanometre-sized metallic (Pd, Ag and Cu)

In this paper, a simple and rapid method was developed in order to assess in comparative tests the production of binary biogas mixtures containing CO 2 and another gaseous compound such as hydrogen or methane. This method was validated and experimented for the characterisation of the biochemical hydrogen potential of different pure strains and mixed cultures of hydrogen-producing bacteria (HPB) growing on glucose.The experimental results compared the hydrogen production yield of 19 different pure strains and sludges : facultative and strict anaerobic HPB strains along with anaerobic digester sludges thermally pre-treated or not. Significant yields variations were recorded even between different strains of the same species by i.e. about 20% for three Clostridium butyricum strains. The pure Clostridium butyricum and pasteurianum strains achieved the highest yields i.e. up to 1,36 mol H 2 /mol glucose compared to the yields achieved by the sludges and the tested Escherichia and Citrobacter strains.

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Fermentative hydrogen production has often been described as inhibited by its own gas production. In this work, hydrogen production by Clostridium butyricum was investigated in batch Biochemical Hydrogen Potential (BHP) tests and in a 2.5 L Anaerobic Sequenced Batch Reactor (AnSBR) under different operating conditions regarding liquid-to-gas mass transfer.Through the addition of both stirring up to 400 RPM and nitrogen sparging, the yields were enhanced from 1.6 to 3.1 mol H2 ·mol glucose -1 and the maximum hydrogen production rates from 140 to 278 mL·h -1 . These original results were achieved with a pure Clostridium strain. They showed that hydrogen production was improved by a higher liquid-to-gas hydrogen transfer resulting in a lower dissolved hydrogen concentration in the culture medium and therefore in a lower bacterial inhibition. In addition, biohydrogen partitioning between the gas and the liquid phase did not conform to Henry's Law due to critical supersaturation phenomena up to seven-fold higher than the equilibrium conditions. Therefore dissolved hydrogen concentration should be systematically measured instead of the headspace hydrogen partial pressure. A model was proposed to correlate H 2 production yield and rate by the pure C.butyricum strain CWBI1009 with mass transfer coefficient K L a.

14This study investigates the mesophilic biohydrogen production from glucose using a strictly 15 anaerobic strain, Clostridium butyricum CWBI1009, immobilized in a trickling bed sequenced 16 batch reactor (TBSBR) packed with a Lantec HD Q-PAC ® packing material (132 ft²/ft³ specific 17 surface). The reactor was operated for 62 days. The main parameters measured here were 18 hydrogen composition, hydrogen production rate and soluble metabolic products. pH, 19 temperature, recirculation flow rate and inlet glucose concentration at 10 g/l were the controlled 20 parameters. The maximum specific hydrogen production rate and the hydrogen yield found from 21 this study were 146 mmol H 2 /L.d and 1.67 mol H 2 /mol glucose. The maximum hydrogen 22 composition was 83%. Following a thermal treatment, the culture was active without adding 23 fresh inoculum in the subsequent feeding and both the hydrogen yield and the hydrogen 24 1 production rate were improved. For all sequences, the soluble metabolites were dominated by the 25 presence of butyric and acetic acids compared to other volatile fatty acids. The results from the 26 standard biohydrogen production (BHP) test which was conducted using samples from TBSBR 27 as inoculum confirmed that the culture generated more biogas and hydrogen compared to the 28 pure strain of Clostridium butyricum CWBI1009. The effect of biofilm activity was studied by 29 completely removing (100%) the mixed liquid and by adding fresh medium with glucose. For 30 three subsequent sequences, similar results were recorded as in the previous sequences with 40% 31 removal of spent medium. The TBSBR biofilm density varied from top to bottom in the packing 32 bed and the highest biofilm density was found at the bottom plates. Moreover, no clogging was 33 evidenced in this packing material, which is characterized by a relatively high specific surface 34 area. Following a PCA test, contaminants of the Bacillus genus were isolated and a standard 35 BHP test was conducted, resulting in no hydrogen production. 36

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A horizontal tubular fixed bed bioreactor (HFBR) and an anaerobic biodisc-like reactor (AnBDR) were designed to both fix Clostridium biomass and enable rapid transfer of the hydrogen produced to gas phase in order to decrease the strong effect of H 2 partial pressure and H 2 supersaturation on the performances of Clostridium strains. The highest H 2 production rate (703 mL H 2 /L.h) and yield (302 mL/g glucose consumed i.e. 2.4 mol/mol) with the pure culture were recorded in the AnBDR with 300 mL culture medium (total volume 2.3 L) at pH 5.2 and a glucose loading rate of 2.87 g/L.h. These results are about 2.3 and 1.3-fold higher than those achieved in the same bioreactor with 500 mL liquid medium and with the same glucose consumption rate. Therefore, our experimentations and a short review of the literature reported in this paper emphasize the relevance of performing bioreactors with high L/G transfer.Keywords: Clostridium; hydrogen production; biofilm; bioreactor; pure strain CWBI -ULg 2/35 IntroductionThe fermentative production of hydrogen has drawn increased attention in recent years. This biological process called "dark fermentation" (DF) offers new opportunities to produce "green" energy from various renewable resources and organic wastes [1][2][3]. While significant improvements have been made in development of such alternative H 2 production systems, more technical progress and cost reduction needs to occur for them to compete with current large scale technologies e.g. methane-reforming process. By contrast, for local and smaller scale DF and some other opportunities, biohydrogen production processes would be cost competitive since the feedstocks are available almost anywhere and crucial interest is paid for both energy independence and efficient utilization [4,5]. However, optimization is still needed for DF regarding the bioreactor design, rapid removal and purification of gases, use of cheaper feedstock, genetic and molecular engineering to redirect metabolic pathway [6][7][8][9][10].Moreover, DF is only likely to be viable as an industrial process if integrated with a process that maximizes energy recovery from the fermentation end-products. The traditional methaneproducing anaerobic digestion process is the most promising since about 10 to 30 % more energy could be generated in the two-stage integrated system comparing to a single stage methanogenic process [11]. Besides, very prospective processes to convert acetate from DF spent medium exist such as further biohydrogen production (towards the maximum theoretical yield of 12 mol/mol glucose) by photosynthetic non-sulfur bacteria or direct electricity production in microbial fuel cells [6,11].In the past decades, most studies on biohydrogen production processes dealt with suspended culture systems such as the conventional (dis-)continuous stirred tank reactors (CSTR) since they are relatively simple and easy to operate. These investigations, several times reviewed [5,10,12,13], enabled to optimize number of operating parameters such as the ...