Cultivation and growth experiments

2.3.2.1. Isolation of denitrifiers

The two experimental approaches to isolate denitrifiers from earthworm gut contents used nitrite and N₂O separately as main electron donors. These nitrogenous compounds are typical substrates for denitrifiers (1.2.1.1). Avoiding nitrate as electron acceptor aimed to predominantly isolate denitrifiers instead of dissimilatory nitrate reducers (1.2.2).

Dilution steps ranging from 10^-2 to 10^-4 of gut contents prepared from L. rubellus, L. terrestris, A. caliginosa, and O. lacteum (2.2.4.2) were conducted with anoxic phosphate buffer in anoxic tubes. In the oxygen-free chamber (100 % N2), approximately 100 µl of the highest dilution step (10^-4) of the gut contents of each earthworm species was plated out on plates of the DE/NO2 and DE/N2O-medium.

The DE/NO2 medium was used to isolate denitrifiers with nitrite as electron donor, and therefore only contained nitrite (3 mM) as electron donor. Plates of this isolation approach were placed into anoxic jars (approximately 5 l; University of Bayreuth, Germany), flushed with 100 % argon, and incubated at 15 °C in the dark.

The DE/N2O-medium was used to isolate denitrifiers with N2O as main electron donor, and contained minor amounts of nitrite (0.05 mM) only. Agar plates of this isolation approach were placed into anoxic jars and flushed with 100 % argon. N2O (100 %) was added to the gas phase of the anoxic jar to a final concentration of approximately 10 mM. In addition, small amounts of NO (100 %) were added (approximately 100 nM). Nitrite and NO were needed in the approach with N₂O as main electron acceptor because nitrite and NO are

important signal molecules to activate the transcription and expression of denitrification genes, enzymes, and other signal proteins (1.2.1.2).

For both isolation approaches, agar plates were checked for growth of colonies in the oxygen-free chamber every 6 to 10 weeks. There were no visible differences of the prokaryotic colonies between the agar plates of the four earthworm species, or the two isolation approaches. Approximately 200 colonies were picked randomly, plated out on new agar plates containing the medium they were isolated from, and again incubated in anoxic jars as described above. This procedure was repeated three times. During the last plating out, a subsample of each of the remaining 159 colonies was dissolved in a small volume (20 µl) of anoxic phosphate buffer in 2 ml Eppendorf tubes. Polymerase chain reactions (PCRs; 2.5.7) amplifying the bacterial 16S rRNA gene were conducted (2.5.7.3). Isolate 201 and Isolate 208 were checked for the appearance of genes indicative of denitrification, i.e., narG, napA, nirK, nirS, and nosZ (2.5.7.2.2).

2.3.2.2. Growth experiments under oxic and anoxic condition with Isolate 201 and Isolate 208

For Isolate 201 and Isolate 208, basic physiological features were determined under oxic and anoxic conditions. Therefore, colonies of Isolate 201 and Isolate 208 isolated under anoxic conditions (2.3.2.1) were transferred to oxic DE-plates (2.3.1.2.3) containing the same substances as the plates used during their isolation (2.3.2.1) but no nitrite. Isolates were incubated under oxic conditions at 20 °C in the dark. Both isolates grew up to colonies with approximately 2 mm diameter within approximately 3 days. Colonies were used as inoculums for growth experiments under oxic (2.3.2.2.1) and anoxic conditions (2.3.2.2.2).

2.3.2.2.1. Growth under oxic conditions

Growth under oxic conditions was analyzed with medium DE/ISO in flasks that had an additional protuberance (2.3.1.2.4) that enables the non-invasive measurement of the OD during growth (2.3.2.4). The 24 ml-tubes used for the anoxic experiments (2.3.2.2.2) could not be used as the amount of oxygen in the tube was assumed to be insufficient to enable oxic growth for a sufficient period of time. 0.1 ml of a suspension of colonies (2.3.2.2) was injected and flasks were incubated for 48 hours to gain an active pre-culture. 0.1 ml of this pre-culture was used as inoculum for growth experiments, i.e., the measurement of the OD every hour over a period of 8 hours. All incubations were at 28 °C in the dark with a HT Infors Shaker (Infors, Bottmingen, Switzerland) at 150 rotations per minute.

2.3.2.2.2. Growth under anoxic conditions

Growth under anoxic conditions was analyzed in anoxic tubes that enable the direct measurement of the OD during growth (2.3.2.4). 0.1 ml of a suspension of colonies (2.3.2.2) was injected into anoxic tubes with DE/ISO/NO3-medium, i.e., a medium containing nitrate (2.3.1.2.5). Tubes were incubated for 48 hours to gain an active pre-culture. 0.1 ml of this pre-culture in DE/ISO/NO3-medium was used as inoculum for growth experiments, i.e., the measurement of the optical density OD over a period of 24 hours. These growth experiments under anoxic conditions, were conducted either with DE/ISO-medium to analyze anoxic growth without nitrate, or with DE/ISO/NO3-medium to analyze anoxic growth with nitrate. All incubations were at 28 °C in the dark with a HT Infors Shaker (Infors, Bottmingen, Switzerland) at 150 rotations per minute.

2.3.2.3. Enrichment and isolation of methanogens

Methanogens were enriched from a mixture of gut contents, coelom fluid and gut sections from the anterior part of the digestive system of E. eugeniae raised and maintained on Substrate 1. The enrichment was aimed to finally get methanogenic, archaeal isolates.

Approximately 1 ml of the aqueous phase (2.2.4.1) was transferred into an anoxic tube with RUP-medium without earthworm extract but with the 20-fold amount of yeast and tryptone (2.3.1.2.6) with syringes that were flushed with sterile argon (100 %) before. After 4 weeks, CH₄ and H₂ were measured via GC (2.4.1) yielding approximately 5 % CH₄ and no detectable amounts of H2 in the headspace (data not shown). Aliquots (1 ml) of this enrichment step (10^-1 to 10^-5; dilution with RUP-medium) were used as inoculums for further enrichment steps with RUP-medium. After two additional transfers to new medium after 8 to 12 weeks each, GC measurements (2.4.1) were conducted after 50 days of incubation of the last enrichment step. Ratios between utilized H2 and produced CH4 were calculated in which a possible production of H₂ from fermentations during the incubation was disregarded.

An aliquot of the 10^-5 dilution was used after 50 days for T-RFLP analysis with amplified mcrA/mrtA gene fragments (2.5.8) to check purity and phylogeny of enriched methanogens.

Therefore, the aliquot (2 ml) was centrifuged (10,000 × g, 10 min, 4 °C), resuspended in PCR-H₂O, and subsequently used for PCR to amplify the fluorescence-labeled mcrA/mrtA fragments (2.5.8.1) for the T-RFLP analysis.

2.3.2.4. Optical density

The optical density (OD) was measured at 660 nm (OD₆₆₀) for growth experiments with Isolate 201 and Isolate 208 (2.3.2.2) in a photometer (Spectroquant Multy, Merck, Darmstadt,

Germany). A wavelength of more than 600 nm ensured to measure only the cell mass but no compounds as cytochromes and FeS clusters (Green & Sambrook 2012). The OD was normalized with a non-inoculated sample of the medium that was used in the corresponding experiment.