= 1 − × 100
, coverage (%); , number of OTUs that occur once; , total number of sequences.
Rarefaction analyses enable to compare the observed richness in samples that have not been sampled equally. Rarefaction curves and 95%-convidence intervals were calculated according to the Hurlbert-rarefaction concept [157, 179] using the aRarefact-Win software available at http://strata.uga.edu/software/. Flat and plateauing curves indicate that sampling was sufficient and most of the expected diversity was covered by the sampling effort. Steeply increasing curves indicate that sampling was not sufficient and a more thorough sampling would be sufficient to cover most of the expected diversity in the sample.
2.6.4. Calculation of phylogenetic trees
Phylogenetic trees were calculated using the maximum parsimony, neighbor joining, or maximum likelihood method in the ARB software [264]. Branch lengths are based on the maximum parsimony tree unless otherwise stated. Nodes congruent with all three treeing methods are indicated with filled circles. Nodes congruent with two treeing methods are indicated with open circles. Bootstrap values are averages from the maximum parsimony tree (1000 resamplings), the neighbor joining tree (1000), and the maximum likelihood tree (100).
Bootstrap values were shown for nodes that were congruent with all three treeing methods.
2.6.5. Nucleotide sequence accession numbers
Sequences obtained during this study were submitted to the European Nucleotide Archive with the following accession numbers: FR715716-FR715893, FR717338-FR717357, FR717732-FR717817, HG324304-HG325724, LK024545-LK026322, LN555754-LN556287.
2.7. Calculations and statistical analyses
2.7.1. Carbon and electron balances
2.7.1.1. Recoveries of carbon and reductant in cellulose-supplemented peat soil microcosms (3.2.1)
Concentrations of a compound after the preincubation in cellulose supplemented and unsupplemented treatments (2.1.2.1) were subtracted from concentrations of this compound at the end of the incubation to calculate net amounts. Net amounts from controls were subtracted from that of cellulose treatments to calculate recoveries. Approximately 37 mM carbon and 148 mM reductant were supplemented by the addition of cellulose. The following numbers of electrons/carbon atoms per molecule were used to calculate net amounts of reductant/carbon: CH4, 8/1; CO2, 0/1; Acetate, 8/2; Butyrate, 20/4; Propionate, 14/3.
2.7.1.2. Recoveries of carbon and reductant in soil-free root and root-free soil microcosms (3.2.2)
Concentrations of a compound at the start of the incubation were subtracted from concentrations of this compound at the end of the incubation to calculate net amounts. Net amounts from controls were subtracted from that of formate treatments to calculate recoveries.
A total of 5.6 and 5.9 mM formate were supplemented in formate treatments of soil-free root and root-free soil microcosms, respectively. The amount of carbon and reductant derived from formate was set to 100%. The following numbers of electrons/carbon atoms per molecule were used to calculate net recoveries of reductant/carbon: CH4, 8/1; CO2, 0/1; acetate, 8/2;
butyrate, 20/4; propionate, 14/3; formate, 2/1; H2, 2/0.
2.7.1.3. Recoveries of carbon and reductant in peat soil microcosms supplemented with ethanol, butyrate, or propionate (3.2.3)
Cumulative amounts of CH4 and CO2 were calculated from amounts of gases formed in the timeframe between two flushing events (i.e., when gasphases were exchanged with sterile
N2 as indicated in Figure 31). Cumulative amounts of CO2 were corrected as follows: When gas phases were flushed, CO2 could not be removed completely since some CO2 remained dissolved in the liquid phase. Those leftover concentrations of CO2 could not be detected during the gas chromatographic measurments directly after flushing as CO2 in gas and liquid phases were not yet in equilibrium. This resulted in very low CO2 concentrations (close to the detection limit) measured directly after flushing. At the second measurement after each flushing event, gaseous and dissolved CO2 were back in equilibrium, and relatively high concentrations of CO2 were detected. As a result of the re-emergeing equilibrium, the increase in CO2 per time interval was much higher between the first two CO2 measurements after each flushing event compared to the increase in CO2 in the same time interval before flushing and after the second measurement following a flushing event. To correct the CO2 concentrations, linear CO2 production was assumed after flushing of the gas phase with N2. The correction of cumulative CO2 concentrations influences carbon recoveries and overall stoichiometries but not the recoveries of reductant (Table 23). This correction was not necessary for CH4 since the solubility of CH4 is rather low compared to CO2 (Table 8). Cumulative amounts of CH4 and CO2 in unsupplemented controls were subtracted from cumulative amounts of CH4 and CO2
formed in butyrate, ethanol, and propionate treatments between the end of the preincubation and the end of the main incubation (2.1.2.2). This resulted in net-amounts of CH4 and CO2. Finally, amounts of carbon atoms and electrons from net-amounts of CH4 and CO2 were divided by the amounts of carbon atoms and electrons supplemented as either butyrate, ethanol, or propionate. The following numbers of electrons/carbon atoms per molecule were used for the calculations: CH4, 8/1; CO2, 0/1; Ethanol, 12/2; Butyrate, 20/4; Propionate, 14/3.
2.7.1.4. Recoveries of carbon and reductant in earthworm gut content microcosms supplemented with S. cerevisiae cell lysate
An elemental formula of CH1.613O0.557N0.158 and a molar weight of 26.09 g·C-mol-1 for S. cerevisiae biomass [462] was used to calculate carbon recoveries for anoxic earthworm gut content microcosms supplemented with lysed S. cerevisiae cells (Table 27). The total carbon recovery was calculated according to Equation 9. Approximately 2400 µmol S. cerevisiae-derived carbon per gram of earthworm gut content fresh weight (gFW) was supplemented to treatments with cell lysate. The average redox state of carbon was calculated to be 0 based on the elemental formula above. Thus, each C-mol may donate 4 mols of electrons if it is completely oxidized to the redox state of carbon in CO2 (+4). This factor of 4 mols of electrons per mol of carbon was used to calculate the electron recovery according to Equation 10.
Electrons derieved from the oxidation of organic nitrogen were not included into electron recoveries since nitrogen-containing compounds (e.g., N2, N2O or NH4+) were not measured.