CARBON ISOTOPE EFFECTS ASSOCIATED WITH MIXED-ACID FERMENTATION OF SACCHARIDES BY CLOSTRIDIUM PAPYROSOLVENS

Abstract

In anoxic environments, microbial fermentation is the first metabolic process in the path of organic matter degradation. Since little is known about carbon isotope fractionation during microbial fermentation, we studied mixed-acid fermentation of different saccharides (glucose, cellobiose, and cellulose) in Clostridium papyrosolvens. The bacterium was grown anaerobically in batch under different growth conditions, both in pure culture and in co-culture with Methanobacterium bryantii utilizing H2/CO2 or Methanospirillum hungatei utilizing both H2/CO2 and formate. Fermentation products were acetate, lactate, ethanol, formate, H2, and CO2 (and CH4 in methanogenic co-culture), with acetate becoming dominant at low H2 partial pressures. After complete conversion of the saccharides, acetate was 13C-enriched (αsacc/ac = 0.991-0.997), whereas lactate (αsacc/lac = 1.001-1.006), ethanol (αsacc/etoh = 1.007-1.013), and formate (αsacc/form = 1.007-1.011) were 13C-depleted. The total inorganic carbon produced was only slightly enriched in 13C, but was more enriched, when formate was produced in large amounts, as 12CO2 was preferentially converted with H2 to formate. During biomass formation, 12C was slightly preferred (αsacc/biom ≈ 1.002). The observations in batch culture were confirmed in glucose-limited chemostat culture at growth rates of 0.02-0.15 h-1 at both low and high hydrogen partial pressures. Our experiments showed that the carbon flow at metabolic branch points in the fermentation path governed carbon isotope fractionation to the accumulated products. During production of pyruvate, C isotopes were not fractionated when using cellulose, but were fractionated to different extents depending on growth conditions when using cellobiose or glucose. At the first catabolic branch point (pyruvate), the produced lactate was depleted in 13C, whereas the alternative product acetyl-CoA was 13C enriched. At the second branch point (acetyl-CoA), the ethanol formed was 15.6-18.6‰ depleted in 13C compared to the alternative product acetate. At low hydrogen partial pressures, as normally observed under environmental conditions, fermentation of saccharides should mainly result in the production of acetate that is only slightly enriched in 13C (<3‰). © 2006 Elsevier Inc. All rights reserved.