Novel FISH and quantitative PCR protocols to monitor artificial consortia composed of different hydrogen-producing Clostridium spp.

in International Journal of Hydrogen Energy (2011), 36

The use of an artificial consortium composed of selected hydrogen-producing species, instead of a natural anaerobic sludge, has been proposed for biohydrogen production. In order to monitor such a ... [more ▼]

The use of an artificial consortium composed of selected hydrogen-producing species, instead of a natural anaerobic sludge, has been proposed for biohydrogen production. In order to monitor such a consortium composed of different Clostridium spp., new protocols were tested for two different assays, FISH and qPCR. New species-specific FISH probes and qPCR primer sets were developed and optimised for three strains: Clostridium butyricum, Clostridium felsineum and Clostridium pasteurianum, that were used in a consortium. Application of a fast two-step FISH protocol, with pre-treatment step at 90 C for 5 min and a subsequent hybridisation step at higher temperature (55 C) for 20 min resulted in a much shorter analytical time compared to the standard FISH procedure (46 C for 2e3 h) and gave a high hybridisation performance. Moreover, to accurately quantify each microorganism by qPCR assay, two innovations were tested: the direct use of cell lysates (omitting the DNA extraction step) and the use of two alternative molecular markers, recA and gyrA. These markers are present in single copies in the genome, whereas there are multiple copies of the ribosomal operons. This resulted in the development of accurate, reliable and fast FISH and qPCR assays for routine monitoring of the dynamics of artificial hydrogen-producing microbial consortia. Moreover, both techniques can be easily adapted to new Clostridium strains. [less ▲]

Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009

in International Journal of Hydrogen Energy (2010), 35(8), 3371-3378

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production ... [more ▼]

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 l bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H2 mol-1 glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol-1 glucose and 0.6 mol butyrate mol-1 glucose. For starch a maximum yield of 2.0 mol H2 mol-1 hexose, and a maximum production rate of 15 mol H2 mol-1 hexose h-1 were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol-1 hexose and 0.67 mol butyrate mol-1 hexose. [less ▲]