3. R ESULTS
3.2. Effect of microbial cell lysate, protein, and RNA on the fermentative microbiota of
3.2.9. Effect of ribose and transient intermediates on gut fermentative bacterial families
A total of 606,090 bacterial 16S rRNA gene and 16S rRNA sequences, associated to 25 phyla (including candidate phyla), were obtained from the saccharide and transient intermediate treatments, and the rarefaction analyses indicated that the most abundant taxa were targeted (Figure 53 and Figure 54). The analysis of the relative sequence abundances demonstrated that Aeromonadaceae were stimulated by ribose and glucose (Figure 55, Figure 56, Figure 57, and Table 47). Fusobactariaceae-affiliated sequences displayed a net increase of affiliated sequences in succinate treatments, whereas formate mostly stimulated Peptrostreptococcaceae (Figure 55). The associated relative sequence abundances of these families were significantly greater in the respective supplemented treatment than in the control at the end of incubation (Table 47). Consistent with the strong fermentative response of Aeromonadaceae to glucose (Figure 51 and Figure 55), the number of detected phylotypes, the number of expected phylotypes (Chao1), and Shannon indices of the glucose treatment were lower than those of unsupplemented control (Figure 54 B and Table 48). These findings indicated that shifts in the community occurred during the incubation, and NMDS analysis (Section 2.6.2.2) of all detected phylotypes confirmed that the microbial communities changed during the incubation (Figure 58).
Figure 53. Rarefaction analyses of bacterial 16S rRNA gene (DNA) and 16S rRNA (RNA) sequences obtained from anoxic L. terrestris gut content microcosms supplemented with ribose. Phylotypes were based on a 97% sequence similarity cutoff. Samples of the three replicates of the 16S rRNA gene control treatment at 0 h and 30 h, 16S rRNA control treatment at 0 h, and all 16S rRNA treatments at 30 h were analyzed separately. Samples of the three replicates were pooled for each of the other treatments at 0 h or 30 h. Abbreviations: 0 h and 30 h indicate the time of sampling in hours; C, unsupplemented control; R, ribose. Numbers assigned to a treatment (e.g., C1) indicate the respective replicate. Figure modified and used with permission from Zeibich et al., 2019b.
Figure 54. Rarefaction analyses of bacterial 16S rRNA gene (A) and 16S rRNA (B) sequences obtained from anoxic L. terrestris gut content microcosms supplemented with glucose and transient intermediates.
Phylotypes were based on a 97% sequence similarity cutoff. Samples of the three replicates of the 16S rRNA control treatment at 0 h, and all 16S rRNA treatments at 30 h were analyzed separately. Samples of the three replicates were pooled for each of the other treatments at 0 h, 10 h, 22 h or 30 h.
Abbreviations: C, unsupplemented control; S, succinate; F, formate; G, glucose. Identification numbers (e.g., C1) indicate the respective replicates. Figure modified and used with permission from Zeibich et al., 2019b.
RESULTS 131
Figure 55. Net increases in 16S rRNA gene (DNA) and 16S rRNA (RNA) relative abundances of bacterial families stimulated by supplemental saccharides, succinate, and formate in L. terrestris gut content microcosms. The graph is limited to families that displayed a net increase in relative abundance of ≥ 4%
in at least one treatment and the families are color-coded to the respective phyla (see Figure 56 and Figure 57 for the complete 16S rRNA and 16S rRNA gene analyses). Net increases of relative abundances were calculated as follows: (a) the calculation is based either on mean relative abundances when samples from the three replicates were analyzed separately (i.e., all RNA and DNA samples of control treatments and RNA samples at 30 h of supplemented treatments) or on single relative abundances when samples of the three replicates were pooled for sequence analyses (i.e., DNA samples at 0 h and 30 h and RNA samples at 0 h of supplemented treatments); (b) mean or single relative abundances at the beginning of incubation were subtracted from those at the end of incubation for control and supplemented treatments; (c) the resulting time-corrected relative abundances of control treatments were subtracted from those of supplemented treatments (negative time-corrected relative abundances of control treatments were ignored). Figure modified and used with permission from Zeibich et al., 2019b.
Table 47. Statistical analyses of stimulated families in ribose, succinate, formate, and glucose treatments.a
Family Treatment Mean Standard
Deviation Median
Enterobacteriaceae ControlA 2.0 0.1 1.9
Ribose 4.4 0.5 4.1 4.6(2)
Family Treatment Mean Standard
Peptostreptococcaceae ControlB 3.3 0.2 3.2
Formate 8.3 1.0 7.9 5.0(1)
Glucose 4.0 0.4 4.2 4.6(3)
aFamilies reaching a LDA score ≥ 4.0 were considered. LEfSe analysis, mean value, standard deviation, and median are based on the relative abundance of 16S rRNA sequences of the three replicates per treatment at 30 h of incubation. Table modified and used with permission from Zeibich et al., 2019b.
bLDA scores were calculated using LEfSe. Numbers in parentheses display the rank in the LDA analysis (i.e., higher ranking families exhibited a stronger response to supplement compared to lower ranking ones).
Figure 56. 16S rRNA gene (DNA) and 16S rRNA (RNA) analyses of control and ribose treatments. The most abundant families (i.e., families with ≥ 4% relative abundance in at least one sampling period) are displayed in the color of the respective phylum. Process data are shown Figure 51 and Table 44, and information on all detected taxa is provided in Table A7. Abbreviations: C, unsupplemented control; R, ribose treatment. Single bars indicate that 16S rRNA or 16S rRNA gene samples of the three replicates were pooled for the sequence analysis and grouped bars indicate that the sequence analysis was performed individually for the three replicates. Figure modified and used with permission from Zeibich et al., 2019b.
RESULTS 133
Figure 57. 16S rRNA (RNA) and 16S rRNA gene (DNA) analyses of control, succinate, formate, and glucose treatments. The most abundant families (i.e., families with ≥ 4% relative abundance in at least one sampling period) are displayed in the color of the respective phylum. Process data are shown in Figure 52, and information on all detected taxa is provided in Table A8. Abbreviations: C, unsupplemented control;
S, succinate; F, formate; G, glucose. Panel A: Single bars without asterisk indicate that DNA or RNA samples of the three replicates were pooled for the sequence analysis. Asterisk indicates analysis was performed individually for the three replicates (see grouped bars Panel B). Figure modified and used with permission from Zeibich et al., 2019b.
Table 48. Alpha diversity of the microbial community in control, ribose, glucose and transient intermediate treatments.a
Sample (Sampling Time)
Treatment Number of sequences
Observed phylotypesb (normalized)c
Chao1 (normalized)c
Shannon (normalized)c DNA (0 h) ControlA 1 15345 776 (198) 1046 (241) 4.0 (3.5)
ControlA 2 10377 681 (199) 984 (234) 4.1 (3.6)
ControlA 3 11331 734 (203) 1019 (236) 4.3 (3.7)
Ribose 10234 650 (188) 974 (227) 3.8 (3.4)
ControlB 15359 965 (235) 1315 (253) 4.6 (3.9)
Succinate 17131 896 (221) 1311 (239) 3.9 (3.3)
Formate 20373 1060 (232) 1380 (245) 4.5 (3.8)
Glucose 16530 1022 (232) 1337 (247) 4.6 (3.8)
DNA (10 h) Control 14156 742 (216) 1109 (243) 3.5 (3.0)
Glucose 12798 416 (139) 720 (185) 1.7 (1.5)
DNA (22 h) Control 18327 789 (216) 1193 (244) 3.4 (3.0)
Glucose 17591 583 (175) 912 (223) 2.3 (2.1)
DNA (30 h) ControlA 1 8937 571 (179) 317 (223) 3.5 (3.1)
ControlA 2 9669 624 (192) 995 (237) 3.7 (3.3)
ControlA 3 9364 597 (190) 958 (232) 3.7 (3.3)
Ribose 7845 364 (136) 611 (196) 2.6 (2.4)
Control 18096 787 (223) 1105 (255) 3.7 (3.3)
Succinate 18342 733 (209) 1071 (237) 3.3 (2.9)
Formate 17229 772 (220) 1105 (249) 3.9 (3.3)
Glucose 15892 669 (204) 998 (236) 3.1 (2.4)
RNA (0 h) ControlA 1 3684 339 (144) 634 (175) 3.1 (2.7)
ControlA 2 2958 307 (152) 581 (183) 3.1 (2.9)
ControlA 3 4255 556 (213) 866 (242) 4.0 (3.4)
Ribose 9677 643 (188) 10110 (234) 3.5 (3.1)
ControlB 1 10695 768 (216) 1178 (237) 3.5 (3.0)
ControlB 2 10734 802 (225) 1185 (240) 3.8 (3.2)
ControlB 3 10158 816 (227) 1242 (246) 3.9 (3.3)
Succinate 15874 883 (218) 1325 (240) 3.5 (2.9)
Formate 15238 925 (232) 1332 (248) 3.8 (3.2)
Glucose 9185 772 (224) 1163 (240) 3.8 (3.2)
RNA (10 h) Control 13621 668 (197) 1074 (229) 3.0 (2.7)
Glucose 20734 556 (142) 1019 (199) 1.9 (1.7)
RNA (22 h) Control 9473 517 (202) 891 (228) 3.2 (2.9)
Glucose 15076 480 (157) 865 (211) 2.2 (2.0)
RESULTS 135 Sample
(Sampling Time)
Treatment Number of sequences
aFor the ribose experiment: Samples of the three replicates of the 16S rRNA gene (DNA) control treatment at 0 h and 30 h, 16S rRNA (RNA) control treatment at 0 h, and all 16S rRNA treatments at 30 h were analyzed separately. Samples of the three replicates were pooled for each of the other treatments at 0 h or 30 h. For the transient intermediate experiment: Samples of the three replicates of the 16S rRNA control treatment at 0 h, and all 16S rRNA treatments at 30 h were analyzed separately. Samples of the three replicates were pooled for each of the other treatments at 0 h, 10 h, 22h, or 30 h. Identification numbers (e.g., ControlA1) indicate the respective replicates. Table modified and used with permission from Zeibich et al., 2019b.
bPhylotypes were clustered based on a sequence similarity cut-off of 97%.
cFor comparison of amplicon libraries of different sizes, the transient data sets were normalized to 5,000 and the ribose data set were normalized to 2,500 sequences.
Control Ribose
Figure 58. NMDS plot of the microbial community composition in ribose (A and B), and succinate, formate, and glucose (C and D) treatments. Distance matrices (Bray-Curtis) are based on the relative abundances of all detected phylotypes in the different treatments (Table A8). Proximity of symbols represent the degree of similarity between the different treatments. Panel A and B: Samples of the three replicates of the 16S rRNA gene (DNA) control treatment at 0 h and 30 h, 16S rRNA (RNA) control treatment at 0 h, and all 16S rRNA treatments at 30 h were analyzed separately. Samples of the three replicates were pooled for each of the other treatments at 0 h or 30 h. Panel C and D: Samples of the three replicates of the 16S rRNA control treatment at 0 h, and all 16S rRNA treatments at 30 h were analyzed separately. Samples of the three replicates were pooled for each of the other treatments at 0 h or 30 h. Figure modified and used with permission from Zeibich et al., 2019b.