Supplementary Materials s9

Supplementary Materials

Materials and Methods

Analysis of terminal restriction fragment length polymorphism data

The abundance of individual terminal restriction fragments (TRFs) was calculated as previously outlined (Clement et al., 1998; Enright et al., 2007) except that TRFs representing <5% of the total area of respective restriction patterns were excluded from further analyses. Terminal restriction fragment length polymorphism (TRFLP) analysis could subsequently be successfully applied to monitor the fate of specific clones throughout the trial, taking into consideration that discrepancies of ± 2 to 3 base-pairs (bp) have to be taken into account when comparing predicted and measured

TRFs (Liu et al., 1997; Kitts, 2001; Lueders and Friedrich, 2003). The identity of

TRFs was assigned through (i) performing in silico enzyme restrictions of clone library sequences (ii) the TAP-TRFLP tool of the Ribosomal Database Project database (Marsh et al., 2000) and (iii) an in-house database of simulated archaeal and bacterial in silico restrictions (Collins et al., 2003; McHugh et al., 2003; McHugh et al., 2004; Enright et al., 2007, 2009). TRFLP data analysis was conducted either qualitatively, by creating binary matrices whereby the presence (‘1’) or absence (‘0’) of individual TRFs was scored, or semi-quantitatively, by calculating the relative abundance of TRFs normalised by the total area of the respective TRF patterns.

Cluster analysis was performed using those matrices and a Multivariant Statistical

Package (MVSP; Kovach, 1999). Dendrograms were constructed using unweighted pair-group methods using arithmetic averages (UPGMA) analysis and Jaccard distance measure. Ordination by non-metric multidimensional scaling (NMS; Kruskal,

1964) was also performed based on the presence/absence matrices to track the

1 temporal changes in community structure. NMS condenses a data set to a point on the resulting plot without loss of information so that the communities with similar TRFLP profiles will be plotted close together (McCune and Grace, 2002). In this study, the

NMS ordination was carried out based on Sorensen (Bray-Curtis) distance measure with PC-ORD software (version 5.0; Grandin, 2006).

Quantitative polymerase chain reaction analysis

Quantitative polymerase chain reaction amplifications were performed in a two-step thermal cycling: pre-denaturation for 10 min at 94○C, followed by 40 cycles of 10 s at

94○C and 30 s at 60○C. All templates were analysed in triplicate. The quantification standard curves were constructed as previously described (Yu et al., 2006) using representative strains of the target groups: Methanospirillum hungatei JF1 (DSM 864) and Methanomicrobium mobile BP (DSM 1539) for MMB set and Methanosaeta concilli GP6 (DSM 3671) for Mst-set. Three custom recombinant plasmids containing the full-length 16S rRNA gene sequences of the representative strains were used as the templates for constructing standard curves. For MMB-set, the equimolar mixture of its two different standard plasmids was used. The mass concentrations of plasmids were measured in duplicate using a Qubit system (Invitrogen, U.S.A.) and converted to their copy concentrations (Yu et al., 2006). A 10-fold dilution series of 101 to 109 copies l-1 was generated for each standard solution and analysed in triplicate by qPCR. The threshold cycle values (CT) determined were plotted against the logarithm of their template copy concentrations. The 16S rRNA gene copy concentrations of target groups were determined against the corresponding standard curves within the linear range (r2 >0.995).

2 Table SM1. Archaeal clone library analysis; In silico TRFLP restrictions of clonal sequences. Coverage Sequence Homology TRFLP in silico restriction length3 Library Accession No. Lineage1 (%) (%)2 HhaI-forward HhaI-reverse Seed DQ679927 13 Uncultured Crenarchaeota 99 n.d. 231 DQ679928 3 Uncultured Crenarchaeota 99 n.d. 231 DQ679929 3 Methanosaeta 99 n.d. 577 DQ679930 10 Methanosaeta 99 n.d. 582 DQ679931 7 Methanomethylovorans 97 175 n.d. DQ679932 13 Methanosaeta 98 193 n.d. DQ679933 51 Methanosaeta 99 193 n.d.

Day 1,228 FJ347527 43 Methanosaeta 99 195 581 FJ347528 19 Methanocorpusculum 97 326 122 FJ347529 30 Methanocorpusculum 99 327 122 FJ347530 4 Methanocorpusculum 99 326 122 FJ347531 3 Methanosaeta 94 193 582 FJ347532 1 Methanocorpusculum 98 327 364 1 Based on Ribosomal Database Project classification. 2 Based on National Centre for Biomedical Information classification. 3 In base-pairs. n.d. - not determined.

3 Figure SM1. Non metric multidimensional scaling (NMS) plot for the TRFLP spectra derived from (a) bacterial and (b) archaeal HhaI-reverse primer profiles. Percentage of variance for each axis is given in parenthesis.

4 Figure SM2. Cluster analysis (UPGMA) of bacterial terminal restriction fragments resolved using (a) HhaI-reverse and (b) HhaI-forward primer profiles.

5 Figure SM3. Cluster analysis (UPGMA) of archaeal terminal restriction fragments resolved using (a) HhaI-reverse and (b) HhaI-forward primer profiles.

6 References

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