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3.4. Die Rolle von Mitgliedern der Familie Geobacteraceae bei der anaeroben
Martina Metje und Peter Frenzel Zielsetzung
Bei vorangegangenen Experimenten mit Proben aus dem estnischen Hochmoor wurde die syntrophe Oxidation von Acetat nachgewiesen (Ergebnisse 3.3.). Fe(III) spielte als alternativer Elektronenakzeptor keine Rolle. Mit Hilfe von speziellen 16S rRNA-Primern konnten allerdings Mitglieder der Familie Geobacteraceae detektiert werden, die bisher nur aus Fe(III)-reichen Habitaten isoliert wurden und die Fähigkeit zur Eisenreduktion besitzen. Die hier detektierten Geobacteraceae müssen sich also an andere Elektronenakzeptoren wie zum Beispiel Huminsäuren angepasst haben. In den im Folgenden beschriebenen Experimenten wurde die Rolle dieser Organismen bei der anaeroben Acetatoxidation untersucht. Um das Potential der mikrobiellen Gemeinschaft zur Fe(III)-Reduktion und die Auswirkungen auf die Mineralisation zu testen, wurden die Torfsuspensionen entweder mit Ferrihydrit oder mit FeCl3 inkubiert.
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The role of Geobacteraceae in Anaerobic Acetate Oxidation in an Acidic Peat Bog
(in preparation)
Martina Metje and Peter Frenzel
Max-Planck-Institute for Terrestrial Microbiology, D-35043 Marburg, Germany Abstract
We investigated the effect of the amorphous Fe(III) oxyhydroxide ferrihydrite and of FeCl3
on anaerobic acetate oxidation (AAcO) and methanogenesis. Mineralization rates were not stimulated by ferrihydrite compared to control samples. In control samples CH4 and CO2
were produced at a rate of 0.46 µmol · gDW-1 ± 0.03 and 1.6 µmol · gDW-1 ± 0.1, respectively. However, methanogenesis together with Fe(III) was transiently inhibited in samples amended with ferrihydrite. In addition, in samples amended with ferrihydrite BES (bromoethanesulfonate) led to the inhibition of [2-14C]-acetate oxidation. However, only 4.8% ± 0.7 of the available ferrihydrite were reduced to Fe(II). Amendment with FeCl3
resulted in a complete inhibition of methanogenesis. In addition, mineralization rates, measured as CO2, were starkly increased immediately. Similar to samples amended with ferrihydrite, only 6.3% ± 0.4 of the FeCl3 were reduced to Fe(II). Further experiments with autoclaved and non-autoclaved peat revealed that Fe(III) reduction in samples with FeCl3
was completely abiotic. Phylogenetic analysis of the 16S rRNA gene amplified with primers specifically designed for the detection of Geobacteraceae (Cummings et al., 2003) revealed that all sequences were closely related to Geobacter chapelleii. Members of the family Geobacteraceae until today have been found mainly in Fe(III)-rich soils or sediments. They have never been shown to play an essential role in Fe(III)-depleted peat bogs. Our results indicate that Geobacteraceae are not necessarily involved in Fe(III) reduction, which strongly depends on the availability of labile organic substrates. Instead they seem to be adapted to other electron acceptors like humic substances or even H+.
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Introduction
Wetlands emit up to 550 Tg of the greenhouse gas CH4 per year and, hence, are the most important source of naturally emitted CH4 (Fung et al., 1991; IPCC, 1996; Matthews and Fung, 1987), representing 10-30% of all CH4 emitting sources (Mitra et al., 2005).
Wetlands of the Northern Hemisphere play a prominent role: ~50% of all wetlands are situated between 50 and 70°N (Mitra et al., 2005). These peat-rich regions contribute 35-60% of CH4 emitted from wetlands (Bartlett and Harriss, 1993; Matthews and Fung, 1987).
Despite low rates of primary production, northern peatlands have accumulated 200-455 Pg carbon since the last glaciation, corresponding to 20-30% of the global soil organic carbon pool (Frolking et al., 2001; Gorham, 1991). Hence, they have been and may still be substantial sinks for atmospheric CO2. However, a global warming, which is predicted by global circulating models, may increase mineralization rates, leading to higher emissions of CH4 and CO2.
In the absence of alternative electron acceptors like Fe(III), NO3-, or SO42-, methanogenesis is the terminal step in anaerobic mineralization. The main precursors of CH4 are H2/CO2
and acetate, whereas theoretically 2/3 of CH4 originate from acetate, when glucose is the primary substrate. However, (i) in nature the portion of acetoclastic methanogenesis can diverge considerably from this ratio and (ii) an alternative terminal acetate degrading process is the syntrophic oxidation of acetate.
In the peat investigated here, recently anaerobic acetate oxidation (AAcO) has been observed (Metje and Frenzel, submitted). Based on the absence of alternative electron acceptors like Fe(III), SO42-, and NO3- and based on further results, we proposed that AAcO was based on a syntrophic relationship. The syntrophic acetate oxidizers isolated today are homoacetogens, oxidizing acetate via the acetyl-CoA-pathway (Hattori et al., 2000; Hattori et al., 2005; Lee and Zinder, 1988; Schnürer et al., 1996; Schnürer et al., 1999; Zinder and Koch, 1984).
However, not only homoacetogens are capable of syntrophic acetate oxidation: This process has also been evidenced for Geobacter sulfurreducens, oxidizing acetate syntrophically in cooperation with Wolinella succinogenes (Cord-Ruwisch et al., 1998).
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Geobacteraceae are the most important Fe(III) reducers known today, and have been isolated from Fe(III)-rich habitats like aquifers, sediments, and flooded mineral soils (Lovley, 1997). Geobacter sulfurreducens, a well-studied representative out of the family Geobacteraceae, is known to completely oxidize acetate to CO2 via the citric acid cycle (Cord-Ruwisch et al., 1998; Galushko and Schink, 2000). Since Fe(III) reduction did not take place, Geobacteraceae were not expected to be present in the investigated peat bog or must have been adapted to other electron acceptors. However, in addition to Fe(III) reduction Geobacteraceae are capable of humic acid reduction. Humic substances are chemically heterogeneous organic compounds and are widespread also in peatlands.
Hence, these substances may have been operated as electron acceptors during anaerobic acetate oxidation in the investigated peat bog.
Nevertheless, in search for potentially important microbiota we phylogenetically analysed the 16S rRNA gene with primers directed towards conserved regions within the family Geobacteraceae (Cummings et al., 2003). An extensive clone library of 91 sequences revealed that the next cultivated relatives were Geobacter chapelleii (U 41561), which has been isolated from an aquifer (Coates et al., 1996; Coates et al., 2001; Lonergan et al., 1996) and Pelobacter propionicus (Evers et al., 1993; Schink, 1984). The presence of Geobacteraceae in this habitat was totally unexpected, because Geobacter spp. have never been described for peat before. However, all 16S rRNA sequences retrieved could unambiguously be affiliated to the family Geobacteraceae.
Here we study the ability of this family to oxidise acetate anaerobically even in the absence of Fe(III). In order to determine the Fe(III)-reducing capacity of Geobacteraceae and to shift the metabolism to higher mineralization rates we amended the acidic peat bog with (i) the amorphous Fe(III) oxyhydroxide ferrihydrite, an amorphous iron oxyhydroxides are presumably the predominant source for Fe(III) reduction (Lovley, 1991), and (ii) FeCl3, which is very soluble in water and is mainly used as flocculating and precipitating agent in the treatment of both drinking water and waste water. Acetate turnover and
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Material and Methods
Peat was sampled from a raised peat bog (Männikjärve Bog) at the Endla Nature Reserve in central Estonia (58°52’27.9’’N; 26°15’6.8’’E) in August 2004. The mean annual temperature in this region is around 4.8°C, mean annual precipitation is 589 mm (). The average temperature during summer time is 17°C (Edgar Karofeld, personal communication). The samples were taken from a Sphagnum-cuspidatum-hollow located in the central ridge-hollow-complex. The only other macroscopic plant at the sampling plot was Rhynchospora alba. Samples were taken below the water table (20-40 cm) and filled bubble-free into sterile polyethylene terephthalate bottles (Nalgene®). Samples were stored at 4°C until further analysis. Loss on ignition was 98%. The pH was 3.6.
Incubation
Peat samples were homogenized as described previously (Metje and Frenzel, 2005), and incubated at 25°C in pressure tubes (V = 16 mL, Ochs, Göttingen, Germany) containing 6 ml slurry or in serume flasks (V = 150 mL) 60 mL slurry. An oxygen-free solution of sodium 2-bromoethanesulfonate (BES, final concentration 40mM) was used to completely inhibit methanogenesis (Metje and Frenzel, 2005) (controls, n=4). Headspace concentrations of CH4, CO2 and H2 were measured by gas chromatography after equilibrating gas and liquid phases. Samples were incubated for 63 days. CH4, CO2 and H2
were measured regularly during incubation. Pore water samples were taken at day 1, 15, 22, 43 and 63 and were analyzed for fatty acids. Similarly, slurry samples were taken at day 15, 20, 30, 43 and 63 to measure Fe(II) concentrations.
In order to determine acetate turnover, we followed the turnover of [2-14C]-acetate sodium salt (57.0 mCi/mmol, Amersham Life Sciences, UK) or 14C-Bicarbonate (53.0 mCi/mmol, Moravek Biochemicals, Brea, CA, USA). The total radioactivity per tube was 165,000 Bq for [2-14C]-acetate and 55,000 Bq for 14C-bicarbonate (n=4).The substrates were added at day 14.
In order to follow the effect of different Fe(III) species, on day 14 we added ferrihydrite and FeCl3 to the slurry incubations. Ferrihydrite was prepared as described by Jäckel and Schnell (Jäckel and Schnell, 2000). The ferrihydrite stock solution contained 0.1 g
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ferrihydrite /mL. 0.005 g (0.75 mL of the stock) and 0.05 g ferrihydrite (4.7 mL of the stock) were added to the 16 mL pressure tubes and the 150 mL serume flasks, respectively.
This corresponds to an end concentration of ~ 1.7 mM. FeCl3 was dissolved in O2-free sterile Aqua bidest. and added to the slurry samples to an end concentration of 10 mM.
Summed up, the following incubations were run: BES, ferrihydrite, ferrihydrite + BES, FeCl3, FeCl3 + BES (V = 16 mL; n = 8). Thereof, four parallels of each treatment were amended with [2-14C]-acetate and four parallels with 14C-bicarbonate, respectively. In addition: Control, BES, ferrihydrite, ferrihydrite + BES, FeCl3, FeCl3 + BES (V = 150mL;
n = 5), which were not amended with labeled substrates.
Analytical Techniques
Radioactive and non-radioactive CH4 and CO2 were analyzed with a gas chromatograph equipped with a methanizer, flame ionization detector and RAGA radioactivity gas proportional counter (Raytest, Straubenhardt, Germany) (Conrad, 1989). H2 was analyzed by GC-TCD (Shimadzu, Kyoto, Japan). Liquid samples were filtered through 0.2 µm membrane filters (FP 30/0.2 CA-S, non-pyrogenic, Schleicher & Schuell, Dassel, Germany) and stored at –20°C until analysis. 14C-labeled and unlabeled organic acids (lactate, formate, acetate, propionate, butyrate and caproate) were measured by high-performance liquid chromatography (HPLC) with an Aminex HPX – 87H Ion Exclusion Column (300 mm x 7.8 mm, BIO-RAD, Hercules, California, USA), a refraction index detector (RI2000, Sykam, Gilching, Germany) and a UV-detector (UV/VIS S3200, Sykam, Gilching, Germany). The detection limits were 5-50 µM (Krumböck and Conrad, 1991).
14C-labeled fatty acids and ethanol were detected via a Radioactivity Monitoring Analyzer (Raytest, Straubenhardt, Germany).
Radioactivity of added substrates ([2-14C]-acetate and 14C-bicarbonate) was determined by a Multi Purpose Scintillation Counter LS6500 (Beckman Instruments, Inc., Fullerton, CA, USA) using Quicksafe A as scintillation cocktail (Zinsser, Frankfurt, Germany). The total
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Fe(II) was extracted with 0.5 M HCl and measured as described by Lovley and Phillips (Lovley and Phillips, 1987) and modified by Ratering and Schnell (Ratering and Schnell, 2000). Samples for Fe(II) analysis were taken from peat slurries amended with ferrihydrite or FeCl3 and with or without BES, respectively at day 15, 20, 30, 42, and 63, respectively.
Concentrations of Fe(II) are given in µmol · gDW-1. DNA-Extraction and PCR amplification
Slurries were sampled at the beginning of the experiment and after incubation for 28 days at 25°C. The samples were homogenized by pestle and mortar to break up macroscopic peat structures. DNA was extracted with the FastDNA SPIN Kit for soil according to the manufacturer’s instructions (Qbiogene, Carlsbad, USA). In order to clean the extract from PCR-inhibiting compounds, two further purification steps with guanidine-thiocyanate (5.5 mM; Sigma) were necessary. Primers Geo564F (5’-AAG CGT TGT TCG GAW TTA T-3’) (Coates and Achenbach, 2002) and Geo840R (5’-GGC ACT GCA GGG GTC AAT A-3’) (Cummings et al., 2003), corresponding to approximate positions 564 and 840 of the 16S rRNA gene, were used to target the 16S rRNA genes of Geobacteraceae spp.. PCR was carried out as described earlier (Cummings et al., 2003). PCR-products were purified with the QIAquick PCR purification kit (Quiagen, Hilden, Germany).
Cloning, sequencing, and phylogenetic analysis
A Gene library for 16S rRNA sequences was constructed using DNA extracts from the original peat sample and after incubation for 28 days at 25°C. PCR products were ligated into pGEM-T vector plasmids (Promega, Mannheim, Germany) and transformed into Escherichia coli JM109 competent cells (Promega, Mannheim, Germany), according to the manufacturer’s instructions. 16S rRNA genes were directly amplified with the Geobacteraceae specific primers Geo564F and Geo840R (Cummings et al., 2003).
Plasmid DNA was sequenced with an automated ABI Prism BigDye terminator cycle Ready Reaction kit with AmpliTaq polymerase FS (Applied Biosystems) according to the manufacturer’s instructions using primers M13 rev-29 (5`-CAGGAAACAGCTATGACC-3`) and T7 (5`-TAATACGACTCACTATAGGG-(5`-CAGGAAACAGCTATGACC-3`). 16S rRNA gene sequences were assembled with SeqMan II (DNASTAR) and compared with the sequences available in the
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phylogenetic affiliations. Chimeric sequences of 16S rRNA genes were identified by Chimera Check of Ribosomal Database Project II (release 8.1) (Cole et al., 2005).
Alignment and phylogenetic analysis of 16S rRNA gene sequences were done with ARB (Ludwig et al., 2004). Additional sequences that were potentially related to the retrieved clones were added to the existing tree using the ARB parsimony tool. 16S rRNA gene sequences (≥800 nt) were selected to construct an archaeal base frequency filter (50 to 100% similarity), which was subsequently used to generate an initial neighbor-joining tree, as implemented in ARB. Escherichia coli was used as the outgroup. 16S rRNA gene sequences generated in this study were then added to the existing tree by quick add parsimony as implemented in ARB.
Results
Previous studies have been shown that the acidic peat bog investigated here lacks alternative electron acceptors like Fe(III), NO3- or SO42- (Metje and Frenzel, submitted).
Hence, observed anaerobic acetate oxidation (AAcO) was not due to Fe(III) reduction.
Further experiments have suggested that (i) an homoacetogen may have been involved in syntrophic acetate oxidation and (ii) the citric acid may have been involved in AAcO (Metje and Frenzel, submitted).
Analysis of the 16S rRNA genes (91 clones) amplified with primers directed towards conserved regions within the family Geobacteraceae showed that the next cultivated relatives were Geobacter chapelleii (U 41561) (Coates et al., 1996; Coates et al., 2001;
Lonergan et al., 1996), isolated from an aquifer and Pelobacter propionicus (Evers et al., 1993; Schink, 1984) (Figure 6). Hence, a Geobacter sp. may have been responsible for anaerobic acetate oxidation in the absence of Fe(III) and other electron acceptors. The clone sequences fell into the newly defined groups I to VI (Figure 6). Most clone sequences grouped with group II (32%) and IV (20%), respectively. The lowest number of clone sequences grouped with group III (3.4%) and V (4.5%), respectively.
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± 0.03. CH4 production together with Fe(III) reduction, which was measured as Fe(II) accumulation (Figure 1 and 3), transiently stopped in samples amended with ferrihydrite.
CH4 production and Fe(III)-reduction started again at day 30, with CH4 being produced at a rate of 0.34 µmol · gDW-1 ± 0.07 (Figures 1 and 3).
Considering CO2 accumulation (Figure 1), mineralization rates were not enhanced by ferrihydrite, but instead were decreased by ~33%: In control samples CO2 was produced at a rate of 2.4 µmol · gDW-1 ± 0.09, in samples with ferrihydrite CO2 was produced at a rate of 1.6 µmol · gDW-1 ± 0.1. Mass balances between CH4, CO2 (Figure 1) and fatty acids (Figure 2) were calculated for day 28. Mass balance calculations showed that carbon atoms, which went into CH4 and CO2 in control samples, were nearly completely recovered as fatty acid carbon when CH4 and CO2 accumulation were decreased by ferrihydrite (Table 1). Hence, ferrihydrite seems to change the carbon flow of terminal mineralization to fatty acids.
Measurements of fatty acids showed that acetate, propionate and butyrate occurred as intermediates or methanogenic precursors (Figure 2). Acetate was the main end product with a start concentration of 42 µM. End concentrations in the differently treated slurry samples are listed in Table 2. Acetate accumulation was decreased by BES in samples with and without ferrihydrite or FeCl3, respectively. The same was true for butyrate. In contrast the accumulation of propionate was not decreased by BES.
In samples with ferrihydrite oxidation of [2-14C]-acetate, measured as 14C-bicarbonate accumulation, was lower with BES than without BES (Figure 4). Hence, acetate oxidation seemed to be coupled to an actively CH4 producing population, indicating that methanogens themselves were involved in Fe(III) reduction. However, in total only 4.8% ± 0.7 of the available Fe(III) were reduced to Fe(II), indicating that Fe(III) did not play an important role in the acidic peat bog and that Fe(III) could not be forced to play a more important role by addition of ferrihydrite.
With FeCl3 CH4 production was completely inhibited (data not shown). However, mineralization rates, measured as CO2 accumulation, were increased dramatically with and
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without BES (Figure 5), indicating that Fe(III) was reduced abiotically. Further experiments with autoclaved and non-autoclaved peat samples confirmed this assumption (Figure 5): In autoclaved samples without FeCl3 CO2 accumulation was largely inhibited.
CO2-accumulation in non-autoclaved samples with FeCl3 was similar to control samples.
Only between day 0 and 10 mineralization was higher in samples with FeCl3, which was consistent with measurements in Figure 1 and 5A. Interestingly, autoclaved and non-autoclaved samples with FeCl3 showed nearly no difference in CO2 accumulation, indicating that Fe(III) reduction was completely abiotic in the presence of FeCl3.
Discussion
Despite the lack of Fe(III) in the investigated peat bog, all 16S rRNA gene sequences could be affiliated to the family Geobacteraceae. In addition, the amplification of Geobacteraceae 16S rRNA genes was specific, which is in contrast to Cummings and colleagues, who in addition detected members of other δ-Proteobacteria (Syntrophic, Desulfomonile, and Anaeromyxobacter) (Cummings et al., 2003). Here, exclusively Geobacteraceae were detected, indicating that (i) PCR touch down conditions were stringent enough or (ii) representatives of the δ-Proteobacteria mentioned above were not present in the peat.
Until today Geobacteraceae sequences have been mainly retrieved from contaminated aquifers (Snoeyenbos-West et al., 2000), flooded mineral soils, landfill leachate and aquatic sediments with high Fe(III) content (Coates et al., 1996; Cummings et al., 2003;
Holmes et al., 2002). In this context Geobacteraceae are the most important representatives of Fe(III) reducers. However, they have never been shown to play an essential role in Fe(III)-free peat.
An amendment of ferrihydrite to other environmental samples or to pure cultures often resulted in a stimulation of anaerobic mineralization and in the inhibition of
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methanogens and Fe(III)-reducing bacteria for the common substrates H2 and acetate.
However, since Fe(III) reduction was low, the methanogenic community after a lag phase may have been adapted to the amendment with ferrihydrite (Figure 1).
In contrast to samples amended with ferrihydrite, mineralization rates in samples amended with FeCl3 were increased dramatically by abiotic Fe(III) reduction (Figures 5A and 5B).
Pracht and colleagues have demonstrated the abiotic mineralization of phenolic compounds by soluble Fe(III) (Pracht et al., 2001). Since polyphenoles are part of Sphagnum mosses this process may have been contributed to abiotic Fe(III) reduction in the peat bog investigated here as well.
However, the contribution of Fe(III) reduction to anaerobic carbon metabolism most importantly depends on the availability of labile organic matter and Fe(III) (Roden and Wetzel, 2002). The acidic peat bog investigated here, due to the refractive character of the Sphagnum-cuspidatum-peat bog, is characterized by low availability of labile organic carbon substrates (Metje and Frenzel, submitted). The concentration of acetate was around 40 µM, which is substantially below the threshold for growth of hydrogenotrophic and acetoclastic methanogens (Jetten et al., 1990) and also below the Ks for acetate during growth for Geobacter sulfurreducens (Esteve-Nunez et al., 2005). In addition, the bioavailability of acetate can strongly differ from the measured pore water concentration (Parkes et al., 1984; Wellsbury and Parkes, 1995). Hence, the low ratios of Fe(III) reduction in samples amended with ferrihydrite or FeCl3, respectively, are consistent with the observation that anaerobic respiration is kinetically limited by the availability of electron donors (Lovley and Goodwin, 1988; Lovley and Phillips, 1987).
In addition, the surface area and thermodynamic properties of the Fe minerals may influence Fe(III) reduction rates. At pH 7 ferrihydrite is available as Fe oxyhydroxide.
Most investigations focusing on the effects of ferrihydrite amendments were made with circumneutral soils (Lueders and Friedrich, 2002; Weiss et al., 2004). Since the peat bog investigated here was acidic (pH 4.0), Fe(III) was primarily available as soluble ions.
However, several Fe(III) reducers, e.g. Acidiphilium cryptum and Acidithiobacillus ferrooxidans, can grow at low pH (Küsel and Drake, 1999; Ohmura et al., 2002).
Geobacter sulfurreducens as well is able to reduce soluble Fe(III) (Khare et al., 2006).
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Further explanations for the low Fe(III) reduction capacity may have been low densities of Fe(III) reducing bacteria and low amounts of electron-shuttling humic substances. A lot of Fe(III)-reducing bacteria are capable of electron transfer to humic acids as well as to the humic acid analogue AQDS (anthraquinone-2, 6-disulfonate) (Lovley et al., 1996; Lovley et al., 1998). The fermenting bacterium Propionibacterium freudenreichii, e.g. is only capable of transferring electrons towards Fe(III) from anaerobic respiration, when humic acids are present (Benz et al., 1998). However, we cannot completely exclude the presence of oxidized humines and humic acids, the most abundant form of organic matter in many soils and sediments, which may work as appropriate electron acceptors for anaerobic respiration (Coates et al., 1998; Lovley et al., 1996).
It has been observed that the capability of Shewanella putrefaciens to reduce Fe(III) oxides was limited by nutrients (Fredrickson et al., 1998; Kukkadapu et al., 2001). This was not true for Geobacter metallireducens (Weber et al., 2006), but may be true for other representatives of the family Geobacteraceae. Since the stability of Fe(II) increases with decreasing pH, we can exclude the recycling of Fe(III) via anaerobic Fe(II) oxidation, which may mimic low Fe(III) reduction rates.
However, Geobacter sulfurreducens has been shown to be capable of both H2 consumption and H2 production (Caccavo et al., 1994; Cord-Ruwisch et al., 1998) and to posses several hydrogenases(Coppi, 2005), which in part are proposed to convert excess NADH-reducing equivalents to H2. Hence, Geobacteraceae may be capable of anaerobic acetate oxidation via H+ reduction, becoming completely independent of Fe(III) reduction.
Conclusions
Considering the ecology of the investigated site, the absence of Fe(III) reduction seems to be reasonable. However, our results reveal that Geobacteraceae are distributed more widely than it was assumed before. Usually they are present in Fe(III) rich microniches of
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adapt to alternative electron acceptors like humic acids or even H+. Our results let arise the question about the general function of Geobacteraceae in different ecosystems. Are they main actors in anaerobic degradation also in Fe(III)-poor or -free environments?
Outlook
In order to solve their relevance in peat bogs, Geobacteraceae have to be quantified in relation to the total bacterial community. For enumeration of Geobacter spp. We will perform the most probable number method. In order to resolve the high diversity of Geobacteraceae, DGGE will be performed. The next step will be the enrichment of Geobacteraceae on graphite electrodes. Several organisms are capable of energy conservation to support growth by oxidising organic compounds to CO2 with a quantitative electron transfer to electrodes (Lovley, 2006). If this approach will work for the acidic peat bog, it may be possible to isolate the corresponding Geobacter sp. directly from the electrode. First attempts to amplify Geobacteraceae from electrode DNA extracts yielded a PCR product, which seems to be promising for future experiments.
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Legends
Figure 1: Time dependent accumulation of CH4 (A) and CO2 (B) [µmol · gDW-1] and H2
mixing ratio [ppmv] (C) in samples incubated +/- ferrihydrite and +/- BES. At day 15 ferrihydrite was added, as indicated by the dashed line. Means ± SE; n=4.
Figure 2: Time dependent net accumulation of acetate, propionate and butyrate [µmol · gDW-1] in control samples and in samples with ferrihydrite ± BES. Means ± SE; n=4.
Figure 3: Time dependent accumulation of Fe(II) [µmol · gDW-1] in samples with ferrihydrite with and without BES. Means ± SE; n=4.
Figure 4: Time dependent accumulation of 14CO2 from [2-14C]-acetate [Bq · gDW-1] in samples with ferrihydrite +/- BES. Means ± SE; n=4.
Figure 5: Time dependent accumulation of CO2 in samples amended with FeCl3 ± BES [µmol · gDW-1] (A; Means ± SE; n=4) and in autoclaved and non-autoclaved samples ± FeCl3 [ppmv] (B; Means ± SE; n=3).
Figure 6: Neighbour Joining tree of the 16S rRNA gene specific for Geobacterceae (Cummings et al., 2003). As outgroup Escherichia coli was used. Sequences were retrieved from the original peat sampled in 2003 and 2004, respectively, and were merged into the newly defined groups I-VI.
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Tables
Table 1: Carbon mass balance at day 28, considering control samples and samples amended with ferrihydrite. Measured concentrations on day 0, 22, 43, and 63 were interpolated for concentrations on day 28. *sum of carbon atoms in [µmol · gDW-1] originating from CH4, CO2 and fatty acids. Means ± SE; n=4.
Sample CH4
[µmol · gDW-1]
CO2
[µmol · gDW-1]
Fatty acids carbon [µmol · gDW-1]
Sum*
Control 13.5 68.4 109.5 191.4
Ferrihydrite 4.3 46.5 154.2 205.0
Table 2: End concentrations [µM] of acetate, propionate and butyrate, respectively, in samples amended with FeCl3 or ferrihydrite with and without BES and in control samples after 63 days of incubation at 25°C. Means ± SE; n=8.
Sample Acetate [µM] Propionate [[µM] Butyrate [µM]
BES 0.26 ± 0.02 0.06 ± 0.004 0.06 ± 0.01 FeCl3 0.41 ± 0.02 0.01 ± 0.002 0.03 ± 0.002 FeCl3 BES 0.16 ± 0.01 0.06 ± 0.01 0.07 ± 0.01 Ferrihydrite 0.77 ± 0.06 0.03 ± 0.01 0.07 ± 0.01 Ferrihydrite BES 0.31 ± 0.01 0.11 ± 0.01 0.16 ± 0.02
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Figures
0 20 40 60
0 400 800 1200
0 20 40 60
0 5 10 15 20 25 30 35
0 20 40 60
0 20 40 60 80 100 120
H2 [ppmv]
Time [day]
CH4 [µmol · gDW-1 ]
Time [day]
ferrihydrite ferrihydrite + BES
BES Control
C B
A
CO2 [µmol · gDW-1 ]
Time [day]
Figure 1: Time dependent accumulation of CH4 (A) and CO2 (B) [µmol · gDW-1] and H2
mixing ratio [ppmv] (C) in samples incubated +/- ferrihydrite and +/- BES. At day 15 ferrihydrite was added, as indicated by the dashed line. Means ± SE; n=4.
20 40 60
0 25 50 75 100 125
20 40 60
0 3 6 9 12 15
20 40 60
0 2 4 6 8 10
Time [day]
BES ferrihydrite ferrihydrite + BES control
acetate [µmol · gDW-1 ]
Time [day]
propionate [µmol · gDW-1 ]
Time [day]
butyrate [µmol · gDW-1 ]
Figure 2: Time dependent net accumulation of acetate, propionate and butyrate [µmol · gDW-1] in control samples and in samples with ferrihydrite ± BES. Means ± SE; n=4.
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20 40 60
20 30 40
50 ferrihydrite ferrihydrite + BES
Fe(II) [µmol · gDW-1 ]
Time [day]
Figure 3: Time dependent accumulation of Fe(II) [µmol · gDW-1] in samples with ferrihydrite with and without BES. Means ± SE; n=4.
20 40 60
0 4 8 12
16 ferrihydrite ferrihydrite + BES
14 CO 2 · 104 [Bq · gDW-1 ]
Time [day]
Figure 4: Time dependent accumulation of 14CO2 from [2-14C]-acetate [Bq · gDW-1] in samples with ferrihydrite +/- BES. Means ± SE; n=4.
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0 20 40 60
0 40 80 120 160
0 10 20 30
0 1500 3000 4500 6000 7500
CO 2 [µmol · gDW-1 ]
Time [day]
FeCl3 FeCl
3 + BES
B A
CO 2 [ppm v]
Time [day]
control autoclaved FeCl3 FeCl
3 autoklaved
Figure 5: Time dependent accumulation of CO2 in samples amended with FeCl3 ± BES [µmol · gDW-1] (A; Means ± SE; n=4) and in autoclaved and non-autoclaved samples ± FeCl3 [ppmv] (B; Means ± SE; n=3).
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Figure 6: Neighbour Joining tree of the 16S rRNA gene specific for Geobacterceae (Cummings et al., 2003). As outgroup Escherichia coli was used. Sequences were retrieved from the original peat sampled in 2003 and 2004, respectively, and were merged into the newly defined groups I-VI.