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

Abstract

We used an H 2 -purging culture vessel to replace an H 2 -consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (⌬G) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H 2 production changed such that ⌬G remained nearly constant for each organism (؊11.1 ؎ 1.4 kJ mol butyrate ؊1 for S. lipocalidus and ؊58.2 ؎ 1.0 kJ mol alanine ؊1 for A. colombiense). The cellular maintenance energy, determined from the ⌬G value and the hydrogen production rate at the point where the cell number was constant, was 4.6 ؋ 10 ؊13 kJ cell ؊1 day ؊1 for S. lipocalidus at 55°C and 6.2 ؋ 10 ؊13 kJ cell ؊1 day ؊1 for A. colombiense at 37°C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.In anoxic environments, syntrophic organisms play an important role in the degradation of organic material (20). In syntrophic degradation, a single substrate is consumed by the concerted action of two or more microorganisms which are incapable of consuming the substrate individually. This process is mediated by interspecies electron transfer, and H 2 is a key intermediate. H 2 is both a product of fermentation reactions and a substrate for terminal electron-accepting processes such as methanogenesis and sulfate reduction. In the absence of an H 2 -consuming organism, the H 2 partial pressure (P H 2 ) rapidly reaches a level that thermodynamically inhibits further fermentation. This has led to great difficulty in growing the H 2 -producing organisms that perform these processes in pure cultures, although many of them are capable of pure culture growth on alternate substrates (7,17,25).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. The ⌬G of a reaction is dependent on both the temperature and the concentrations of the reactants and products, such that, for the reactions shown in Table 1, ⌬G is defined by the following equations:where ⌬G°Ј (T ) is the Gibbs free energy yield for the reaction under standard conditions, corrected for the entropy change caused by variation of the temperature (ϪT⌬S); T is the temperature (kelvin); and R is the universal gas constant (0.008314 kJ K Ϫ1 mol Ϫ1 ). A more negative value indicates a more energetically favorable reaction.Previous studies (5,10,11,12) have suggested that...

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