Experimental studies on the stable carbon isotope biogeochemistry of acetate in lake sediments

Abstract

Acetate is an important intermediate in the anaerobic degradation of organic matter and highly relevant for the cycling of carbon in nature. The water soluble C₂ compound is produced by fermentation of organic matter as well as by reduction of CO₂ with H₂ via the acetyl-CoA pathway (acetogenesis) and serves as an important substrate for a variety of microorganisms including sulfate reducing bacteria and methanogenic archaea. The relative importance of the different metabolic processes in acetate production and consumption is thought to be reflected in the stable carbon isotopic composition of pore water acetate. δ¹³C values of acetate have been shown to be highly variable in marine sediments, ranging from –85‰ to –3‰, and distinct isotopic fractionations have previously been reported to be associated with fermentation, acetogenesis and acetoclastic methanogenesis in pure microbial cultures. Aiming to better understand the stable carbon isotope biogeochemistry of sedimentary acetate, this study investigates if process related carbon isotopic signals of acetate are also expressed in the pore water pool of complex freshwater environments. We report findings from incubation experiments with lake sediments, in which we manipulated the relative importance of single processes by addition of specific substrates and inhibitors. In particular, we found: (a) δ¹³C values of acetate closely resembled δ¹³C values of total organic carbon in the sediment’s solid phase (TOC) where fermentation was the dominant process; (b) a distinct ¹³C enrichment of the acetate pool relative to δ¹³C_TOC where acetate was consumed by acetoclastic methanogenesis; and (c) a distinct ¹³C depletion of acetate relative to both δ¹³C_TOC and δ¹³C values of dissolved inorganic carbon (DIC) where high levels of H₂ stimulated acetogenesis. Our study provides further evidence for the diagnostic value of the stable carbon isotope chemistry of acetate for the detection of biogeochemical processes in natural systems.

Introduction

The decomposition of organic matter plays an important role in global and local carbon budgets in that it recycles complex organic molecules into small molecules such as CO₂ and CH₄. Under anaerobic conditions, organic matter is hydrolyzed and fermented, and the fermentation products are converted to H₂, acetate and other short chain organics (Sansone and Martens, 1982). In sediments, where inorganic electron acceptors other than CO₂ are not available (e.g., nitrate, ferric iron and sulfate), subsequent consumption of H₂ is mediated by methanogenic archaea and acetogenic bacteria (Conrad, 1999, Drake et al., 2006). Acetate in turn, which is produced either by fermentation of organic matter or by reduction of CO₂ with H₂ via the acetyl-CoA pathway (acetogenesis) (Drake et al., 2006), serves as an important substrate for a variety of microorganisms including acetoclastic methanogens (Sansone and Martens, 1981). Because of its central role in carbon cycling (Fig. 1), acetate deserves particular attention and has previously been studied in a variety of sedimentary environments including rice field soils (Krüger et al., 2001, Conrad et al., 2002, Penning and Conrad, 2007), peat bogs (Shannon and White, 1996, Duddleston et al., 2002, Beer and Blodau, 2007), lake sediments (Lovley and Klug, 1982, Schulz and Conrad, 1995), marine surface sediments (Barcelona, 1980, Wellsbury and Parkes, 1995, Wu et al., 1997), the deep marine sub-seafloor (Wellsbury et al., 1997, Wellsbury et al., 2002, Egeberg and Barth, 1998, Heuer et al., 2009) and the deep terrestrial subsurface (Chapelle and Bradley, 1996, Krumholz et al., 1997).

In sediments, where the supply of H₂ is low, microbial populations will consume H₂ to the threshold of bioenergetic limitation (Hoehler et al., 2002). In such systems, the competition of acetogenic and methanogenic microbial groups for H₂ is of particular ecological importance (Kotsyurbenko et al., 2001). Since methanogenic CO₂ reduction is thermodynamically more favorable than acetogenic CO₂ reduction, methanogens are expected to outcompete acetogens for H₂ (Hoehler et al., 2002). However, in natural sediments processes are not driven by thermodynamics alone. Other factors like temperature have important effects on the biochemical capabilities of microorganisms and community structure, as well. At the cold temperatures typical for surface sediments of deep stratified lakes (5–10 °C), acetogens have been found to outcompete methanogens for H₂ (Schulz and Conrad, 1996, Schulz et al., 1997, Kotsyurbenko et al., 2001, Glissmann et al., 2004, Nozhevnikova et al., 2007). Under these conditions, H₂/CO₂ was converted into CH₄ by a two step process. First acetate was formed by acetogenic CO₂ reduction, followed by acetoclastic methanogenesis. However, the exact quantitative role of hydrogenotrophic and acetotrophic methanogenesis in cold freshwater sediments is not yet fully understood (Nozhevnikova et al., 2007).

The relative importance of the different metabolic processes in acetate production and consumption is thought to be reflected in the stable carbon isotopic composition of pore water acetate (Blair et al., 1987, Gelwicks et al., 1989, Blair and Carter, 1992). In principle, the stable carbon isotopic composition of acetate should be controlled by (1) the isotopic composition of its precursors, (2) the isotopic fractionations associated with its formation and consumption, and (3) the relative rates of all acetate producing and consuming processes which influence its pool size. Distinct isotopic fractionations have previously been observed during acetate production and consumption in pure microbial cultures (Meinschein et al., 1974, Rinaldi et al., 1974, Blair et al., 1985, Krzycki et al., 1987, Gelwicks et al., 1989, Gelwicks et al., 1994, Preuss et al., 1989, Londry and Marais, 2003, Valentine et al., 2004, Penning et al., 2006, Penning and Conrad, 2006, Goevert and Conrad, 2008), but so far merely a few studies have employed the stable carbon isotopic composition of pore water acetate as an indicator of biogeochemical processes that occur in natural sediments in situ. Nevertheless, δ¹³C values of acetate have been shown to be highly variable in natural sediments, ranging from −85‰ up to −3‰ (Blair et al., 1987, Blair and Carter, 1992, Heuer et al., 2006, Heuer et al., 2009). In addition, δ¹³C values of acetate have been found to change systematically with depth in deeply buried sediments thus indicating the presence of biogeochemical zones with different modes of carbon turnover (Heuer et al., 2009). These findings underline the large potential of the stable carbon isotopic composition of acetate as a sensitive indicator of metabolic processes in sediments.

The available data on the stable carbon isotope biogeochemistry of acetate can be summarized in three working hypotheses: (1) Fermentation is accompanied by little carbon isotopic fractionation and the resulting pore water acetate has similar δ¹³C values as total organic carbon (TOC) in the sediment’s solid phase. Experimental evidence comes from pure cultures of Clostridium papyrosolvens, a bacterium typically found in estuarine sediments, freshwater swamps and rice fields soils in which mixed acid fermentation of saccharides resulted in the production of acetate that was only slightly enriched in ¹³C (<3‰) (Penning and Conrad, 2006). (2) Acetogenesis produces pore water acetate that is distinctly depleted in ¹³C relative to TOC. This hypothesis is supported by the observation of fractionation factors of up to −60‰ (ε_ace/DIC) during acetogenic CO₂ reduction in pure cultures of Acetobacterium woodii (Gelwicks et al., 1989). (3) The preferential consumption of acetate during acetoclastic methanogenesis results in the enrichment of ¹³C in the residual pore water acetate pool. The latter hypothesis is supported by the observation of ¹³C depleted CH₄. Krzycki et al. (1987) and Gelwicks et al. (1994) reported fractionation factors of −21.2‰ for CH₄ produced from acetate by Methanosarcina barkeri, whereas two members of the family Methanosaetaceae showed distinctly smaller fractionations, i.e., −10‰ in Methanosaeta concilii (Penning et al., 2006) and −7‰ in Methanosaeta thermophila (Valentine et al., 2004).

With this study we aimed to provide further information for the interpretation and use of the carbon isotopic composition of acetate as a tool to monitor the cycling of carbon in natural environments. We investigated the expression of isotope effects in two lake sediments that were incubated under controlled conditions in the laboratory. In particular, we wanted to test if fermentation, acetogenesis and acetoclastic methanogenesis, processes which have been reported to be associated with distinct isotopic fractionations in pure cultures, create characteristic carbon isotopic signals in the acetate pools of complex natural sediments. We present carbon isotopic evidence for acetogenesis that will help unraveling the competition between CO₂ reducing acetogenic bacteria and methanogenic archaea for H₂, based on carbon isotope analysis of pore water acetate.