SUPPLEMENTAL MATERIALS
Heterogeneity in 13C and 15N assimilation in sediment incubations exhibiting low
anaerobic oxidation of methane activity
While most sediment incubations from the Eel River Basin seeps were stimulated
by the addition of CH4, the PC-59 sediment incubation showed very few signs of active
AOM or ANME/DSS growth throughout the 89-day experiment. Unlike PC-76, sulfide
only reached a final concentration of 500 µM, and displayed little to no change in the
percentage of ANME/DSS recorded by FISH (Supplemental Table S1). The 15N
aggregate enrichment in PC-59 after ~3 month incubation was low and highly variable
relative to other incubation experiments, with 9 of 17 shell aggregates containing 15N
values equivalent to the enrichment observed in just 5 days by shell aggregates in PC-76
and PC-55 (Figure 4; Supplemental Figure S2). All signs indicated methanotrophic
ANME/DSS aggregates from the PC-59 incubation were inactive relative to other 15N
incubated samples, however there was some evidence of metabolic activity in monospecific ANME aggregates, with 3 out of 7 ANME-2 clusters containing modest 15N
values similar to values recorded in the PC-59 shell aggregates (15N ~1-3 atom %;
Supplemental Figure S2). FISH documentation of a 10% increase in the relative
proportion of hybridized ANME-2 cell clusters without a bacterial partner provided
additional evidence of potential metabolic activity and growth by mono-specific ANME2 in PC-59 during the incubation (Supplemental Table S1).
The documentation of mono-specific ANME-2 aggregates with enriched 15Nbiomass
values is interesting and implies that some members the ANME-2 lineage are capable of
active metabolism without a physically attached bacterial partner (Supplemental Figure
S2). The observation of heavy 13Cbiomass in these 15N enriched mono-specific ANME-2
aggregates (13C ranging from -20 to -38‰) may also be significant, suggesting the
possibility for methanogenic growth which frequently results in 13C enriched biomass
compared with organisms growing methanotrophically (Supplemental Figure S2; (Londry
et al., 2008). Taken together, these results illustrate the potential metabolic plasticity of
the ANME-2 clade, where subpopulations may be capable of methanotrophy and/or
methanogenesis depending on physicochemical conditions and neighboring microbial
assemblages.
Table S1: FISH quantification of percent change in ANME and bacteria over time
PC-76 (clam)
PC-59 (mat)
incubation days
0
33
0
85
15N
amendment
NH4
AA
NH4
AA All N-1* All N-2 All N-1 All N-2
% hybridized
aggregatesb
ANME-1
8a
8
6
11
0
0
0
0
ANME-2
26
28
27
21
18
29
27
37
Bacteria
9
9
7
3
6
4
9
10
Mixed aggregate
3
14
7
14
28
27
30
27
Shell aggregate
54
42
54
51
45
38
36
26
Number of total
76
95
104
103
97
102
88
101
hybridized cells
a
Values represent percentage of total hybridized aggregates analyzed for each time point
*All N-1 and All N-2 are replicate incubation vials from PC-59 amended with both
amino acids (AA) and ammonium (NH4).
bMicrobial cells and cell aggregates were hybridized with FISH probes EelMSMX_932
and DSS_658 (ANME only and ANME/Desulfosarcina consortia) or with the general
bacterial probe Eub_338 (bacteria).
Table S2: Temporal variation in ratio of ANME-2/DSS shell aggregate size in PC-76
Incubation Time (days)
0
33
112
a
Treatment
AA
NH4
AA
NH4
AA
NH4
shell diameter (µm)
Mean
5.0
4.4
4.6
4.6
5.3
4.5
Std Dev
1.8
1.5
1.4
1.4
1.9
2.0
Range
8.4
7.7
5.8
5.6
8.8
11.4
Aggregate total
Mean
93.5
61.5
66.4
66.4
114.2
82.1
biovolume (µm3)
Std Dev 126.7
75.2
56.8
63.3
146.5
159.6
Range
629.7 434.7 260.4 252.8 692.7 1195.2
ANME-2 total
biovolume (µm3)
DSS total
biovolume (µm3)
Ratio biovolume
ANME/DSS
ratio ANME/DSS cells
per aggregate
Mean
41.3
19.9
25.9
24.6
57.0
33.1
Std Dev
Range
Mean
74.2
444.4
52.1
21.3
93.9
41.6
31.1
152.0
40.6
29.9
161.1
41.8
84.9
443.6
57.2
69.2
493.5
49.0
Std Dev
Range
Mean
61.6
305.2
57.9
341.6
32.3
123.8
38.9
172.7
70.2
383.1
92.3
704.1
0.71
0.66
0.68
0.65
1.10
0.96
Std Dev
0.45
0.54
0.56
0.58
1.09
1.35
Range
Mean
2.23
2.88
2.39
3.33
5.38
7.87
0.36
0.34
0.34
0.33
0.57
0.51
Std Dev
Range
N
0.24
1.10
0.28
1.50
0.29
1.20
0.30
1.70
0.56
2.80
0.69
4.00
39
41
57
53
66
Total number
51
hybridized aggregates
a
Amino Acid (AA) ammonium (NH4)
Fig. S1: Relationship between 13C (‰) and 15N (atom %) for shell ANME-2/DSS after a
5-day incubation with 15N labeled ammonium or amino acids from (a) PC-55 (diamonds;
n=6) and PC-76 (squares; n=10). ‘z’ symbol denotes paired 13C /15N values for a mixed
ANME-2/DSS aggregate. (b) Paired 13C /15N values for PC-59 cell aggregates after 4day incubation (open symbols) and 85-day incubation (closed symbols) with ammonium
and amino acids. Shell aggregates are represented by a square symbol, mixed aggregates
(triangle) and mono-specific ANME-2 clusters are represented by a circle. Plus signs
denote ANME/DSS shell aggregates from control incubation without exogeneous 15N
labeled nitrogen. In both panels, plotted values include heaviest 13C (red color) and
lightest 13C (black) data points for each ANME-2 or ANME/DSS aggregate measured
during the FISH-SIMS analysis.
Fig. S2: Comparison of the relationship between aggregate size and enrichment in
15
Nbiomass (atom %) for shell consortia. a) Maximum 15N enrichment after 5-day
incubation for shell aggregates in PC-76 and PC-55. b) Box plot showing the average 15N
value, range, and 95% confidence intervals for shell aggregates with diameters between
2-7µm (mean 15N atom %= 1.8, n=9) and aggregates ranging between 7µm and 20µm
(mean 15N atom %=1.4, n=6). c) Maximum 15N enrichment after 112-day incubation for
shell aggregates in PC-76. Although a general trend of greater 15N assimilation by smaller
shell aggregates was present, the statistical significance of aggregate size and 15N
enrichment was not observed (P=0.44, Wilcoxon test).