Species related to Clostridium or Clostridiales were abundant in the 16S rRNA gene clone libraries of the formate- and lactate-fed KB-1 subcultures (Table 1). In addition, RFLP demonstrated the presence of Clostridium in both KB-1 subcultures (Fig. 2), as well as in the upper sand layer part of the lactate-fed diffusion-cell and the diffusion-cell BIO1 (Figs 3b and 4). Clostridium and related species are known for their fermenting diversity (Cato et al., 1986). In the KB-1 subcultures, these species potentially played an important role in converting the amended electron donors. Formate and lactate cannot directly support dechlorination by G. lovleyi SZ, which requires H2, acetate or pyruvate as electron donor (Sung et al., 2006). In the KB-1 culture, however, acetate does not support dechlorination (Duhamel et al., 2002; Haest et al., 2011). Therefore, dechlorination in the examined KB-1 subcultures requires the conversion of formate or lactate likely to hydrogen. Lactate is a common growth substrate for fermentative Clostridia (Cato et al., 1986) and in the lactate-fed KB-1 subculture and diffusion-cells, the fermentation of lactate to H2, acetate and bicarbonate (McCarty et al., 2007) likely provided Geobacter with hydrogen. In addition, formate is easily converted to H2 and bicarbonate, but this dehydrogenation reaction rarely supports microbial growth (Ferry, 1990; Kim et al., 2010). As such, this conversion cannot explain the recorded dominance of Clostridium in the formate-fed KB-1 subculture and in some of the formate-fed diffusion-cells. Therefore, we hypothesize that the Clostridium species grew on the fermentation of yeast extract and cysteine, two constituents of the anaerobic medium which were found to serve as electron donors (J. Philips, unpublished results). This hypothesis is supported by the finding that RFLP profiles corresponding with Clostridium had a low intensity, if formate-fed diffusion-cells used anaerobic medium with strongly reduced yeast extract and cysteine concentrations (J. Philips, unpublished results). During the cultivation of the KB-1 subcultures, however, no additional doses of yeast extract and cysteine were applied. As such, the initial growth of Clostridium on yeast extract and cysteine was likely sufficient to support long-term conversion of formate. Moreover, Clostridium cells have on average nine 16S rRNA gene copies per cell (Klappenbach et al., 2001), and, as such, the lack of sustained growth does not explicitly disagree with the high number of Clostridium 16S rRNA gene clones obtained from the formate-fed KB-1 subculture (Fig. 2). Alternatively, the presence of Clostridium species in the KB-1 subcultures could be explained by acetogenesis, in which Clostridium produce acetate from hydrogen resulting from the formate dehydrogenation (Drake et al., 2008). Clostridium would then compete with Geobacter for H2. However, none of the Clostridium OTUs were phylogenetically related to known acetogenic Clostridia (Table 1) (Drake et al., 2008). In addition, little to no acetate was produced in other formate-fed KB-1 subcultures, in which high H2 concentrations were measured (L. Paul, unpublished results). The role of Clostridium in the examined KB-1 subcultures, however, remains uncertain, because neither acetate, nor formate, lactate or H2 were monitored in the current study. As such, extended experiments are required to clarify the recorded dominance of Clostridium.
Several other studies previously identified Clostridium as dominant fermentative species in dechlorinating enrichment cultures (Fletcher et al., 2008; Ise et al., 2011). In contrast, Duhamel & Edwards (2006) did not identify any Clostridium species in their KB-1 subcultures, maybe because they used a different electron donor, that is, methanol, than in the current study. Alternatively, the presence of Clostridium species in the examined KB-1 subcultures, in contrast to in those of Duhamel & Edwards (2006), could be due to the high TCE concentration amended in the current study. Bowman et al. (2009) demonstrated that many Clostridium species are tolerant to chlorinated solvents and are able to produce hydrogen even in the presence of PCE concentrations close to saturation. In addition, two studies on the microbial community in DNAPL source zones, where elevated chlorinated solvent concentrations are expected, showed the dominance of Clostridium (Macbeth et al., 2004) or of members of the order Clostridiales (Bowman et al., 2006).
In the diffusion-cells, differences in the apparent fermentative population were observed (Figs 3 and 4), which could not be related to the amended electron donor and did not affect the dechlorination rate. Freeborn et al. (2005) also found a constant dechlorination rate for subcultures differing in fermentative population, which for their study was related to different electron donors used. In addition, several other studies described fermentative communities, which were functionally stable despite their highly dynamical community composition (Fernandez et al., 1999, 2000; Carballa et al., 2011; Pycke et al., 2011).