Inorganic carbon uptake in the presence of external inorganic

2.2.3 Inorganic carbon uptake in the presence of external inorganic electron

can be made, which are discussed below. Possible external substrate limitation has been discussed above and seems unlikely.

Electron donors provided internally. The assumption that some electron donors might be provided internally is supported by sulfate reduction rates and flux calculations (Dubilier et al. 2001). Internal sulfide production by the sulfate-reducing symbionts of O. algarvensis was estimated to exceed sulfide influx from the surrounding sediment, were sulfide concentrations are low (Dubilier et al. 2001). Thus, internal sulfide production might be – for a certain time – sufficient for the sulfur-oxidizing symbionts, making sulfide uptake from the environment almost unnecessary.

This internal sulfur loop would be particularly advantageous in the sulfide-poor habitat of O. algarvensis and might have occurred in the incubation experiments presented.

High sulfate concentrations in the anoxic medium used might have even stimulated high sulfate-reduction rates by the sulfate-reducing symbionts resulting in high sulfide production (or other reduced sulfur compounds) provided that an energy source for sulfate reduction was available. This sulfide (or other intermediates) could have been used as internal electron donor by the sulfur-oxidizing symbionts.

Figure 2.9: ¹⁴C-inorganic carbon fixation rates by the O. algarvensis symbiosis under anoxic incubation conditions in the presence of combinations of electron donors and electron acceptors. See Figure 2.7 for further explanations.

Thiosulfate would be another possible internal electron donor. Many symbiotic and non-symbiotic marine invertebrates produce thio-sulfate as a means of sulfide detoxification (Grieshaber and Volkel 1998, O'Brien and Vetter 1990, Powell and Somero 1986).

This was also proposed for a gut-bearing, ectosymbiotic marine oligo-chaete (Thiermann et al. 1996). Thiosulfate can be accumulated in symbiotic hosts after being exposed to sulfide or thiosulfate, and much beyond the usually low porewater concentrations (Anderson et al. 1987, Hentschel et al. 1999). It is unknown how gutless oligochaetes detoxify sulfide and if thiosulfate is accumulated or generated

substrates

control white (5/11) control pale (2/4) sulfide, 0.05 mmol/l and nitrate 0.1 mmol/l (2/4) sulfide, 0.1 mmol/l and nitrate 0.5 mmol/l (1/3) sulfide, 0.1 mmol/l and nitrate 0.1 mmol/l (1/3) thiosulfate, 0.2 mmol/l and oxygen (1/3) thiosulfate,0.5 mmol/l and fumarate, 0.5 mmol/l (2/7)

carbon uptake rate [%]

0 200 400 600 1200 1400

at sufficient rates considering the low in situ sulfide concentrations (C. Lott, unpublished data, Dubilier et al. 2001, Perner 2003).

Stored sulfur is present in white oligochaetes and might serve as internal electron donor for the sulfur-oxidizing symbionts. However, field observations, incubation experiments with oxygen, and similar inorganic carbon uptake rates for pale and white worms under anoxic conditions suggest that sulfur is used as electron donor under oxic conditions. Under anoxic conditions, sulfur stores are rather recharged than consumed.

Substrates used as electron donor supporting autotrophy. Most worms incubated with sulfide or thiosulfate did not display an increase in inorganic carbon uptake rates compared to the unsupplemented control. It was suggested that further stimulation is not possible by external reduced sulfur compounds when internal sulfur stores were present in the symbiosis (Hentschel et al. 1999). Individual worms though had increased inorganic carbon uptake rates under anoxic conditions in the presence of sulfide and thiosulfate. This suggests that stimulation was possible and that the oxidation of these reduced sulfur compounds provided energy for inorganic carbon uptake, most likely carried out by the chemoautotrophic sulfur-oxidizing symbionts of O. algarvensis. Even higher stimulation of uptake rates were detected when sulfide and thiosulfate were combined with the electron acceptors nitrate and oxygen, indicating that both electron acceptors and electron donors might have been limiting in unsupplemented incubations and that stimulation of carbon fixation with external substrates was possible in at least some worms. Suggestions why these large differences between individual worm uptake rates might have occurred are presented in Chapter 2.2.4.

Hydrogen was investigated as potential electron donor that can support autotrophy in the sulfate-reducing symbionts. Even this substrate might have been provided by the symbiosis (Chapter 1.3.4, Woyke et al. 2006) and could explain why the addition of hydrogen did not affected carbon fixation in the O. algarvensis symbiosis. Another explanation might be that the sulfate-reducing symbionts of O. algarvensis do not live autotrophically. Especially under anoxic conditions they could benefit from the waste products the worm excretes when switching to an anaerobic metabolism. Metagenomic analysis and preliminary proteome data have identified a wide range of genes coding for enzymes involved in transporting carbon compounds across the membrane (M. Kleiner, C. Wentrup, N. Dubilier, unpublished data, Woyke et al. 2006). This suggests that a heterotrophic metabolism for the sulfate-reducing symbionts is not only energetically more advantageous, but also more likely. Only under starving conditions the sulfate-reducing symbionts might switch to autotrophy.

Finally, acetate was added as a heterotrophic carbon fixation control. However, inorganic carbon uptake did not increase in the presence of an organic carbon compound. Thus, acetate did not stimulate anapleurotic nor autotrophic carbon fixation under the conditions used.

Electron acceptor lacking or limiting. Lack of electron acceptor might explain the lack of carbon fixation stimulation. This is indicated by the results of the mixed incubations with sulfide and nitrate, and thiosulfate and oxygen, which showed a strong increase in inorganic carbon uptake rates in individual worms when electron donor and electron acceptor were added. However, only very few worms were analyzed under these conditions, thus more data is clearly needed to confirm this result.

Preliiminiary data from another experiment, which was meant as control experiment for oxygen consumption studies, supports the assumption that electron acceptors and donors were limiting in the anoxic incubations (Figure 2.10). Based on oxygen consumption rates of O. algarvensis (Häusler 2008) these incubations were depleted in oxygen already after a few hours. The almost constant carbon uptake rate of the control worms independent of their sulfur content indicates that oxygen as electron acceptor for the oxidation of stored sulfur was used up. Thus oxidation of sulfur stopped and with that, autotrophic carbon fixation (Figure 2.10). However, when external substrates were added, in particular thiosulfate and nitrate, carbon fixation rates increased above the rates of the control worms even when sulfur contents were low.

Figure 2.10: Elemental sulfur content and

14C-inorganic carbon fixation rates of the O. algarvensis symbiosis in the presence of different electron donors, electron acceptors and organic carbon compounds under oxygen limiting conditions. Oxygen was used up within the first half of the 6 h incubation based on oxygen consumption rates (Häusler 2008). Worms in this experiment were collected in the field a few days prior to the incubation experiment and were not maintained in the laboratory as the worms used in most other experiments. All substrates were added to a final concentration of 1 mM.

To summarize, the addition of external electron acceptors had little to no effect on inorganic carbon uptake for most worms studied. Sulfide and thiosulfate though could increase uptake rates in individual worms. These substrates were most likely used by the sulfur-oxidizing symbionts. When both external electron donors and electron acceptors were added, sulfide, thiosulfate, nitrate and oxygen can stimulate carbon uptake, indicating that electron donor and electron acceptor were limiting in most anoxic incubations.

0 2 4 6 8 10

0 20 40 60 80 100

elemental s ulfur [%]

nmol C mm-3 h-1

control nitrate fumarate TMAO thiosulfate acetate lactate succinate