Hydrogen Concentration Profiles at the Oxic-Anoxic Interface: a Microsensor Study of the Hindgut of the Wood-Feeding Lower Termite Reticulitermes flavipes (Kollar)

Abstract

Molecular hydrogen is a key intermediate in lignocellulose degradation by the microbial community of termite hindguts. With polarographic, Clark-type H 2 microelectrodes, we determined H 2 concentrations at microscale resolution in the gut of the wood-feeding lower termite Reticulitermes flavipes (Kollar). Axial H 2 concentration profiles obtained from isolated intestinal tracts embedded in agarose Ringer solution clearly identified the voluminous hindgut paunch as the site of H 2 production. The latter was strictly coupled with both a low redox potential (E h ‫؍‬ ؊200 mV) and the absence of oxygen, in agreement with the growth requirements of the cellulolytic, H 2-producing flagellates located in the hindgut paunch. Luminal H 2 partial pressures were much higher than expected (ca. 5 kPa) and increased more than threefold when the guts were incubated under a N 2 headspace. Radial H 2 concentration gradients showed a steep decrease from the gut center towards the periphery, indicating the presence of H 2-consuming activities both within the lumen and at the gut epithelium. Measurements under controlled gas headspace showed that the gut wall was also a sink for externally supplied H 2 , both under oxic and anoxic conditions. With O 2 microelectrodes, we confirmed that the H 2 sink below the gut epithelium is located within the microoxic gut periphery, but the H 2-consuming activity itself, at least a substantial part of it, was clearly due to an anaerobic process. These results are in accordance with the recently reported presence of methanogens attached in large numbers to the luminal side of the hindgut epithelium of R. flavipes. If the oxygen partial pressure was increased, O 2 penetrated deeper and H 2 production was suppressed; it ceased completely as soon as the gut was fully oxic. In experiments with living termites, externally supplied H 2 (20 kPa) stimulated methane formation five-to sixfold to 0.93 mol (g of termite) ؊1 h ؊1 , indicating that the methanogenic activity in R. flavipes hindguts is not saturated for hydrogen under in situ conditions. This rate was in good agreement with the H 2 uptake rates exhibited by isolated hindguts, which would account for more than half of the CH 4 formed by living termites under comparable conditions.

Simultaneous methanogenesis and oxygen reduction by Methanobrevibacter cuticularis at low oxygen fluxes

“…This observation agreed with the findings that at least part of the H 2 -oxidizing activities encountered in the gut periphery of R. flavipes must be attributed to anaerobic microorganisms and that hindgut methanogenesis in Reticulitermes species is strongly stimulated by exogenous H 2 (Ebert & Brune, 1997;. However, this also placed methanogens furthest downstream in the outwardly diffusing H 2 gradient and directly within a site exhibiting the highest O 2 concentrations within the gut (Ebert & Brune, 1997).…”

“…The present study was prompted by the observation that methanogenic Archaea in the wood-feeding lower termite Reticulitermes flavipes are located in the microoxic periphery of the hindgut, either associated with the gut epithelium or adhering to other prokaryotes colonizing the gut wall (Leadbetter & Breznak, 1996;Leadbetter et al, 1998). This observation agreed with the findings that at least part of the H 2 -oxidizing activities encountered in the gut periphery of R. flavipes must be attributed to anaerobic microorganisms and that hindgut methanogenesis in Reticulitermes species is strongly stimulated by exogenous H 2 (Ebert & Brune, 1997;. However, this also placed methanogens furthest downstream in the outwardly diffusing H 2 gradient and directly within a site exhibiting the highest O 2 concentrations within the gut (Ebert & Brune, 1997).…”

“…Interestingly, there is a considerable discrepancy between the amount of CH 4 emitted by R. flavipes and the potential CH 4 production rate of the methanogenic community (Leadbetter & Breznak, 1996), which cannot be explained by CH 4 oxidation . Rather, the severe H 2 -limitation of methanogenesis in Reticulitermes species (Ebert & Brune 1997;) and the capacity of H 2 -dependent O 2 reduction of Methanobrevibacter cuticularis suggest that the low rates of methanogenesis in the hindgut are caused by a redirection of electron flow from methanogenesis towards O 2 reduction.…”

“…It remains enigmatic how Methanobrevibacter cuticularis can thrive directly below the gut wall, a region where oxygen is present in appreciable concentrations at least in agaroseembedded guts (Brune et al, 1995a;Ebert & Brune, 1997), while it remains metabolically active in vitro only if O 2 is below the detection limit. Possibly, Methanobrevibacter cuticularis is capable of performing methanogenesis in situ also at low O 2 partial pressures.…”

“…Influence of increasing O 2 partial pressure in the gas headspace on the depth position relative to the meniscus of the growth bands of Methanobrevibacter cuticularis formed in O 2 -gradient agar tubes, the resulting O 2 fluxes, and the maximal H 2 fluxes possible at this depth (nmol min À1 cm À2 ) H 2 flux à (nmol min À1 cm À2 ) Areal fluxes were calculated assuming zero concentration at the top of the growth band, using Fick's first law of diffusion, Henry's law, and the diffusion coefficients and solubility values for O 2 and H 2 in pure water (details as inEbert & Brune, 1997).…”