Methanogenic symbionts of anaerobic ciliates and their contribution to methanogenesis in an anoxic rice field soil

Abstract

Methanogenesis in rice field soils starts soon after flooding while potentially competing processes like reduction of sulphate and iron take place. Early methanogenesis is mainly driven by hydrogen, while later in the season acetate tends to become more important. Anaerobic ciliates are abundant during this period, and their endosymbionts use hydrogen produced by the ciliates to reduce carbon dioxide to methane. These endosymbiotic methanogens are protected from the competition for substrates with other bacteria that may control methanogenesis outside the protozoan cells. Thus, we focussed on early methanogenesis and on the potential contribution from ciliates and their endosymbionts. Only ciliates of the genus Metopus were found to harbour methanogens, as identified by the F(420)-fluorescence of the endosymbionts. We followed the population dynamics of the ciliates with time, and calculated the ratio of symbiotic methane production to overall methanogenesis. Symbiotic methane production was calculated from the species-specific numbers of methanogenic endosymbionts times the cell-specific methane production of the symbionts. According to this calculation, the symbionts' contribution to overall methane production was only 6.4% at the beginning and decreased with time. In a second experiment, colchicine and cycloheximide were used to inhibit all eukaryotes, comparing the remaining methane production rate to a control without inhibitors. In the inhibition experiment, the contribution from symbionts decreased from 40% to 6% during the first days after flooding, and dropped to near zero within 2 weeks. However, nearly all methane produced from H(2)/CO(2) could be attributed to the ciliates' symbionts between days 5 and 10 after flooding. Both experiments showed that the contribution of methanogenic symbionts to overall methane production is a transient phenomenon, restricted to the first 2 weeks.

Impact of Protists on the Activity and Structure of the Bacterial Community in a Rice Field Soil

“…For the bacteria, we could rely on a clone library generated previously from the same soil (51). However, in previous work on protists from the soil we focused on ciliates and relied on morphology-based identification (57,58). To cover the full diversity of protists, we had to use a molecular approach.…”

“…However, most work published so far has ignored the role of microbial mortality. From recent work on ciliates it became evident that grazing may have an effect on soil bacteria in rice fields (57,58). However, ciliates were outnumbered by flagellates, which might have a much greater effect on microbes (58).…”

Possible sources of methane-derived carbon for chironomid larvae

“…Consequently, either methanogens and MOB have been degraded rapidly after consumption, thus escaping molecular analysis, or the larvae do not feed directly on these microorganisms. Instead, it might be possible that either methylo-or autotrophic microorganisms feed on carbon metabolites released by MOB (like CO 2 or methanol) or that members of the meiofauna (such as ciliates) feed on MOB or methanogen biomass (Schwarz & Frenzel 2005). These microorganisms or protists are then, in turn, taken up by the grazing chironomid larvae, indirectly transferring the methane δ 13 C signature into the larvae.…”

“…Feeding of protists on (e.g.) methanogens has recently been reported for rice field soil (Schwarz & Frenzel 2005) and could occur in lake sediments too. Because microbial cells would already be digested within the protists, MOB and methanogens would not be detectable in the larval gut by our methods, but the δ 13 C signature of the strongly 13 C-depleted carbon source would be 'transported' to the larvae also.…”

“…Symbiotic relationships between bacteria and animals or protists have been reported for a variety of phyla and systems (Hackstein & Stumm 1994, Dubilier et al 1995, Schwarz & Frenzel 2005, Yang et al 2005, with methanotrophic symbionts playing an important role in deep-sea environments (Lee & Childress 1994, Stewart et al 2005 and references therein). Assuming a similar mechanism as for example in bivalves, symbiotic MOB in chironomid larvae could provide the host with an otherwise unavailable carbon source.…”