Effect of lysed S. cerevisiae cells on the composition and activity of the bacterial

16S rRNA gene and transcript analyses were conducted to target both activity and community composition of the Bacteria present in anoxic gut content microcosms [122, 144, 473]. A total of 1715804 bacterial 16S rRNA gene and transcript sequences were obtained from DNA and RNA samples. The sequences were clustered in 19763 OTUs (99% sequence similarity threshold) and assigned to 26 different phyla (inclusive candidate phyla). Rarefaction analyses indicated that the sampling effort was sufficient to cover most of the diversity present in the different samples (Figure 41). However, more exhaustive sampling would most likely result in the detection of additional rare OTUs. Interestingly, the number of detected OTUs at a distinct number of sequences (e.g., 70000 sequences) was lower for RNA and DNA samples from microcosms supplemented with lysed cells compared to samples from unsupplemented controls or samples collected prior to the incubation (Figure 41). This suggests a lower bacterial diversity in treatments with lysed cells compared to unsupplemented controls and thus a stimulation of only a subset of the microbial community by the supplemented lysed cells.

Figure 41 Rarefaction analyses of of bacterial 16S rRNA transcript and gene sequences obtained from RNA and DNA samples of anoxic earthworm gut content microcosms.

OTUs were calculated based on 99% sequence similarity cutoff. Legend: D / R, DNA / RNA; +C / -C, microcosms supplemented with lysed S. cerevisiae cells / unsupplemented controls (2.2.2); 0 / 6 / 12 / 20 /30, incubation time in hours; A / B / C, identifies the triplicate. See also legend of Figure 42 for details on how samples were pooled for molecular analyses.

16S rRNA gene analyses revealed that Actinobacteria and Proteobacteria (of the alpha and gamma subgroup) dominanted the bacterial community in earthworm gut contents prior to the incubation in microcosms (Figure 42). Planctomycetes, Verrucomicrobia, and Tenericutes of the family Mycoplasmataceae were also abundant. Similar taxa were detected on RNA level

No. of sequences

0 20000 40000 60000 80000 100000 120000 140000

No. of OTUs

0 2000 4000 6000

8000 ^D_0 _{D_-C_30}

D_+C_30 R_0 R_-C_6 R_-C_12 R_-C_20 R_-C_30_A R_-C_30_B R_-C_30_C R_+C_6 R_+C_12 R_+C_20 R_+C_30_A R_+C_30_B R_+C_30_C

but 16S rRNA transcripts of the Mycoplasmataceae were more abundant and those of the Actinobacteria were less abundant compared to 16S rRNA genes, suggesting that the Mycoplasmataceae were more active than the Actinobacteria in the freshly collected gut content material. The Mycoplasmataceae were represented by a single species level OTU that shared 99% similarity to Lumbricincola sp. LR-B2. Representatives of the candidatus genus Lumbricincola were detected in tissues, gut contents, and casts of earthworms and a symbiotic relationship between these microbes and their hosts was suggested [311]. However, no cultured representatives of this genus are available so far and their physiological role is not resolved yet. In unsupplemented gut content microcosms, the microbial community changed marginally over time, indicating that the conditions in the microcosms resembled the in situ conditions. Solely the Peptostreptococcaceae increased on RNA and DNA level.

Distinct changes in the relative abundancies of 16S rRNA transcripts between the fresh gut content material and the samples taken after 6 hours of anoxic incubation in microcosms supplemented with lysed cells suggested that the bacterial community immediately responded to the substrate addition (Figure 42). Especially, Peptostreptococcaceae and Aeromonadaceae rapidly increased while the Actinobacteria and Mycoplasmataceae decreased in relative abundance. The rapid increase of the Peptostreptococcaceae and Aeromonadaceae could be largely attributed to single species level OTUs that were closely related to Clostridium bifermentans (99% identity) and several Aeromonas sp. (e.g., 99%

identity to A. media and A. hydrophila), respectively (Figure 43). Both, the obligate fermenter Clostridium bifermentans and the facultative aerobic species of Aeromonas, can ferment proteinaceous substrates as well as sugars [2, 46] and therefore, these taxa may profit from various organic compounds that will be released when cells get disrupted by the grinding activity of the gizzard. Subsequently, Clostridiaceae, Enterobacteriaceae, and Lachno-spiraceae partially replaced the initially responding taxa at later timepoints. The majority of the Clostridiaceae were represented by specialiced proteolytic species (e.g., C. peptidovorans, C.

sulfidigenes, and C. frigidicarnis [34, 285, 363]). In contrast, the single most abundant OTU of the Enterobacteriaceae was 100% similar to different species (e.g., Enterobacter aerogenes, Buttiauxella agrestis, and Klyvera intermedia) that are not proteolytic (i.e., do not liquefy gelatin and therefore do not excrete proteases) but are saccharolytic [33]. The most abundant OTU of the Lachnospiraceae was not closely related to any cultured organism (94% identiy to Epulopiscium fishelsoni) and may therefore represent a novel genus. However, the physiology of this taxa is unknown.

OTUs Y5 and Y18 were closely related to C. glycolicum and C. magnum, two species that comprise acetogenic strains [28, 175, 222]. Both OTUs were abundant predominantly in treatments with cell lysate and at later time points. This is in accordance with the assumption that formate- and (at the end of the incubation) H2-dependent acetogenesis were ongoing

processes. Some strains of C. glycolicum could grow by succinate decarboxylation to propionate [45], and this process may have been a source for propionate in the treatments with cell lysate. Thus, C. glycolicum may have consumed various fermentation products derived from cell lysates.

In summary, fermenters that can degrade sugars and proteins were rapidly stimulated by cell lysates and these taxa were partially replaced by more specialized fermenters with time.

Acetogens likely profit from the accumulation of acetogenic substrates (formate and H2) and some may also utilize succinate.

Figure 42 Relative abundace of bacterial phyla and dominant families in earthworm gut content microcosms over time based on 16S rRNA transcript and gene sequence analyses.

+C / -C, microcosms supplemented with lysed S. cerevisiae cells / unsupplemented controls (2.2.2). 0 h, samples from triplicates of the lysed cell treatments and unsupplemented controls were pooled prior to the incubation; 6 h / 12 h / 20 h, samples from triplicates of the lysed cell treatments or unsupplemented controls were pooled at given time points; 30 h, samples from each triplicate of the lysed cell treatments and unsupplemented controls at the end of the incubation were prepared separately and each bar represents one replicate (the high similarity between different replicates indicates that the results of the microbial community analyses were reproducible).

Figure 43 16S rRNA phylogenetic tree of abundant species level OTUs from earthworm gut content microcosms (bold) and reference sequences.

The phylogenetic tree was calculated using the neighbor-joining, maximum parsimony and maximum likelihood method. Empty and solid circles at nodes indicate congruent nodes in two and three trees, respectively. Branch length and bootstrap values (1,000 resamplings) are from the maximum parsimony tree. The bar indicates 0.1 change per nucleotide. Thermotoga maritima (AE000512) was used as outgroup. Relative abundancies (in %) of the species level OTUs are shown for the different samplings after 0, 6, 12, 20, and 30 h of incubation. +/- indicates microcosms +/- supplemental yeast cell lysate.

Clostridium carnis, M59091