A culture apparatus for maintaining H2 at sub-nanomolar concentrations

Abstract

We devised a microbial culture apparatus capable of maintaining sub-nanomolar H concentrations. This apparatus 2 provides a method for study of interspecies hydrogen transfer by externally fulfilling the thermodynamic requirement for low H concentrations, thereby obviating the need for use of cocultures to study some forms of metabolism. The culture vessel is 2 constructed of glass and operates by sparging a liquid culture with purified gases, thereby removing H as it is produced. We 2 used the culture apparatus to decouple a syntrophic association in an ethanol-consuming, methanogenic enrichment culture, allowing ethanol oxidation to dominate methane production. We also used the culture apparatus to grow pure cultures of the ethanol-oxidizing, proton-reducing Pelobacter acetylenicus (WoAcy 1), and to study the bioenergetics of growth.

Pure-Culture Growth of Fermentative Bacteria, Facilitated by H 2 Removal: Bioenergetics and H 2 Production

“…As seen in the initial study using the H 2 -purging culture vessel (29) and as also observed in other studies with true syntrophic organisms (21), these experiments showed that thermodynamics are an important control on hydrogen production in cultures of H 2 -producing syntrophic organisms. When faced with changes in environmental conditions that would alter the Gibbs free energy yield, hydrogen production always shifted in such a way as to minimize the change in ⌬G.…”

“…Cultures were grown in the H 2 -purging culture vessel described by Valentine et al (29). The reactor continuously purges a liquid culture with H 2 -and O 2 -free gas (80% N 2 , 20% CO 2 ), replacing the H 2 -consuming syntrophic partner.…”

“…Alanine was quantified by an enzymatic assay using alanine aminotransferase and lactate dehydrogenase (32). The concentration of H 2 in the gas flowing out of the reaction vessel was measured using a gas chromatograph equipped with a reducing gas analyzer (Trace Analytical, Menlo Park, CA), as described by Valentine et al (29). Bacterial cell counts were determined by DAPI (4Ј,6Ј-diamidino-2-phenylindole) staining (18).…”

“…Valentine et al (29) designed a culture vessel that purges H 2 as it is produced and then used this apparatus to grow an ethanol-oxidizing syntrophic organism in the absence of an H 2 -consuming partner. By decoupling the syntrophic association, it is possible to manipulate the single organism's growth environment, to quantify the concentrations of catabolic substrates and products, and to calculate the Gibbs free energy (⌬G) available to the fermentative organisms under these conditions.…”

Effect of Temperature on Anaerobic Ethanol Oxidation and Methanogenesis in Acidic Peat from a Northern Wetland

“…In a pilot study we became aware that ethanol was a major intermediate in the samples, and the concentrations were up to 10.5 mM. Methanogens are virtually unable to use primary alcohols (62), but they may take advantage of the H 2 released during anaerobic oxidation of ethanol (32,51,59). To determine the most probable pathways involved in anaerobic ethanol oxidation, we combined inhibitor experiments with mass balance and thermodynamic calculations.…”