Denitrification, Dissimilatory Nitrate Reduction, and Methanogenesis in the Gut of Earthworms (Oligochaeta): Assessment of Greenhouse Gases and Genetic Markers

The earthworm gut is an anoxic microzone in aerated soils, shows a high water content and high amounts of nitrite and organic carbon. These conditions are in marked contrast to those in the pre-ingested material (i.e., substrate) and ideal for microorganisms as those capable of fermentations, for denitrifiers, and dissimilatory nitrate reducers (DNR). Thus, substrate-derived denitrifiers in the earthworm gut (Lumbricidae) emit the greenhouse gas N2O as well as N2. One large species (Megascolecidae) emitted no N2O, leading to the hypothesis that large earthworms cannot emit nitrogenous gases, i.e., N2O and N2. There was no emission of the greenhouse gas CH4 reported for earthworm species. The current study analyzed ten earthworm species of different families, sizes, and feeding guilds (i.e., burrow and feeding habits) from Brazil for the emission of nitrogenous gases and CH4. Taxa affiliated with these emissions, i.e., denitrifiers, DNR, and methanogens were analyzed with cloning and pyrosequencing of marker genes, from gut contents and substrates of earthworm species from Brazil (Amynthas gracilis, Glossoscolex paulistus, Eudrilus eugeniae), Germany (Aporrectodea caliginosa, Lumbricus terrestris, Lumbricus rubellus), and New Zealand (Octochaetus multiporus) on gene and partly on transcript levels. Potential denitrifiers and methanogens were isolated and enriched from earthworm gut contents, respectively. Sequences of narG (encoding for a nitrate reductase; targets denitrifiers and DNR), nirK, nirS (both encoding for a nitrite reductase; target denitrifiers), nosZ (encoding for a N2O reductase; targets denitrifiers), and mcrA/mrtA (encoding for the methyl-CoM reductase and its isoenzyme; target methanogenic Archaea) were analyzed. For nirK and nirS, cutoff values were calculated to define species-level affiliations according to their sequence similarities. Perionyx excavatus, A. gracilis (both Megascolecidae), Pontoscolex corethrurus, Rhinodrilus alatus (both Glossoscolecidae), Dichogaster annae, Dichogaster sp. (both Acanthodrilidae), and E. eugeniae (Eudrilidae) emitted nitrogenous gases; G. paulistus, Glossoscolex sp. (both Glossoscolecidae), and Eisenia andrei (Lumbricidae) did not. Earthworm substrates emitted smaller amounts of nitrogenous gases, predominantly N2. When provided with nitrite, G. paulistus emitted nitrogenous gases but total emissions and the ratio of N2O to N2 were higher for A. gracilis. It was shown that earthworms of all families, sizes, and feeding guilds can emit nitrogenous gases, and that the earthworm substrate, size, and feeding guild were influencing but not determinative factors taken alone. In earthworms gut contents, denitrifiers were predominantly affiliated with Bradyrhizobiaceae (Rhizobiales), indicating that here, these taxa might be responsible for the emission of nitrogenous gases. Active DNR were predominantly affiliated with Mycobacterium (Actinomycetales), and it is anticipated that these Bacteria compete with denitrifiers for nitrate. Gene analyses and isolation approaches demonstrated that (i) both denitrifiers and DNR in the earthworm gut were derived from ingested material and (ii) diversity in the gut was influenced by the earthworm feeding guild. Analyses of genes and transcripts from earthworms from Germany demonstrated that there was a selective activation of substrate-derived denitrifiers and DNR in the gut. E. eugeniae emitted the highest amounts of CH4, P. corethrurus and R. alatus emitted less, all other tested species no CH4. One substrate emitted minor amounts of CH4, all others did not emit CH4. Certain substrates appeared to influence the emission of CH4 by earthworms but the substrate taken alone was not a determinative factor. The capacity to emit CH4 by E. eugeniae was not significantly affected by supplemental H2/CO2 and was at least partly retained when maintained on diverse alternative substrates. Analysis of mcrA/mrtA revealed that selectively activated hydrogenotrophic and acetoclastic methanogens of the Methanosarcinaceae and Methanobacteriacea were the source of the CH4 emitted by E. eugeniae. These methanogens were assumed to be substrate-derived although a symbiotic affiliation with the earthworm cannot be excluded. Certain earthworms emitted both CH4 and nitrogenous gases, suggesting that methanogenesis and denitrification can be concomitant processes in the earthworm gut. This study demonstrated that (i) earthworms from all families, sizes, and feeding guilds can emit N2O and N2, (ii) substrate-derived and selectively activated denitrifiers within the Rhizobiales are the main source of N2O and N2 whereas Actinomycetales are the main active DNR, (iii) the earthworm feeding guild affects the selective activation of ingested denitrifiers and DNR, (iv) certain earthworms emit CH4, and Methanosarcinaceae and Methanobacteriaceae appear to be the main source of this CH4, and (v) certain earthworms can concomitantly emit N2O, N2, and CH4.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bayreuth

Year

2013

Author dc:creator

• Depkat-Jakob, Peter

Contributors dc:contributor

• Drake, Harold

Repository record source_url

https://epub.uni-bayreuth.de/id/eprint/93/

OAI identifier oai:identifier

oai:epub.uni-bayreuth.de:93