1. ABSTRACT
Three years ago, we began anaerobic soil-seeded microcosms in minimal media with switchgrass as
the carbon source. Our intent was ultimately to maintain a stable microbial community in vitro,
while selecting for active members. These microcosms have been serially transferred in five parallel
experiments. Recent Illumina sequencing data shows that the soil inoculum was a very diverse sam-
ple, containing approximately 30 different phyla, though composed mainly of Acidobacteria (29% to
41%) Proteobacteria (22% to 35%), and Verrucomicrobia (17% to 24%). However, by only the second
transfer, the number of phyla represented had decreased to an average of 8.6 (SD=1.67),with Proteo-
bacteria (~5%) and Firmicutes (~95%) dominating. By transfer 30 the microcosms consisted almost
entirely of Firmicutes (>99%), though they comprised only 0.26% to 0.67% of the initial inoculum.
From transfer 2 through transfer 62, the average number of phyla had decreased to 7.35 (SD=0.22).
Despite the phylum-level diversity decrease, rare OTUs (less than 1% abundance) within the Firmic-
utes continue to account for most of the diversity we see (>97% of OTUs) at transfer 62. Through se-
rial transfer, we have developed a stable and presumably specialized microbial community, while
maintaining high species diversity, even among closely related organisms. This community structure
conjures questions about the existence of the rare biosphere, the concept of functional redundancy,
and community interactions. We have isolated and characterized several abundant species from the
microcosms, to investigate these questions further.
A special thank you to the Gordon Research Conference for the support in
presenting my work here.
Community Structural Changes Occur Early then Stabilize
Sequencing was done on a MiSeq platform, resulting in average read lengths of 254
basepairs. Community analysis was done using Qiime (Quantitative Insights into
Microbial Ecology).
Methods
Enriched Community is Comprised of Rare Members
Family Level Changes in Replicate 2 Over Time
Phylum Level Changes in Replicate 2 Over Time
Is this Diversity Real?
Rare Members Make Up Almost 100% of OTUs, but
only a Small Fraction of Total Sequence Reads
Isolate
ID OTU ID # Abundance Taxonomy
Percent
Abundance
#14 83876 147720 p__Firmicutes; c__Clostridia; o__Clostridiales; f__Clostridiaceae; g__Clostridium 44.11%
#6 91612 55757 p__Firmicutes; c__Clostridia; o__Clostridiales; f__Lachnospiraceae 16.65%
#12 102238 25023 p__Firmicutes; c__Clostridia; o__Clostridiales; f__Lachnospiraceae 7.47%
#16 102244 23820 p__Firmicutes; c__Clostridia; o__Clostridiales; f__Ruminococcaceae; g__Ruminococcus 7.11%
E faecalis 82296 22838 p__Firmicutes; c__Bacilli; o__Lactobacillales; f__Enterococcaceae; g__Enterococcus 6.82%
#5 52849 21746 p__Firmicutes; c__Clostridia; o__Clostridiales; f__Lachnospiraceae 6.49%
#8 51275 8859 p__Firmicutes; c__Clostridia; o__Clostridiales; f__Lachnospiraceae 2.65%
#9 57493 5027 p__Firmicutes; c__Clostridia; o__Clostridiales; f__Lachnospiraceae 1.50%
NA 83560 990 p__Firmicutes; c__Clostridia; o__Clostridiales; f__Clostridiaceae; g__Clostridium 0.30%
NA 117553 827 p__Firmicutes; c__Bacilli; o__Lactobacillales; f__Enterococcaceae 0.25%
NA 84367 826 p__Firmicutes; c__Clostridia; o__Clostridiales; f__Clostridiaceae; g__Clostridium 0.25%
Table 1. The Shift Between Real and Artifactual OTUs
S1T62 S2T62 S3T62 S4T62 S5T62
# OTUs at >1% Abundance 7 8 6 6 7
Total OTUs 6298 4631 6961 4655 7438
Dominant OTUs Abundance 85.17% 82.02% 82.85% 86.83% 78.75%
Discussion and Future Work
We were able to develop a stable, replicable, and less diverse microbial communi-
ty in vitro. Changes occur early and persist, though resilience and resistance to dis-
turbance have not been tested. This technique may used to create simplified consor-
tia that can more easily be studied and manipulated than they can be in situ. It can
also be used to select for rare members prior to isolation.
Due to chimera formation during the sequencing process, we cannot say just how
much less diverse the community is after evolving. The incredible number of aber-
rant OTUs formed between PCR and data analysis urges caution in statements about
diversity and the rare biosphere. Optimization of PCR protocols, along with full-
length reads and refined data analysis may be able to someday eliminate these se-
quencing artifacts.
This work continues with the characterization of novel isolates and with the opti-
mization of the community by recombining isolates.
T0_1 T0_2 T2 T10 T20 T30 T40 T49 T62
Transfer
RelativeAbundance
RelativeAbundance T0_1 T0_2 T2 T10 T20 T30 T40 T49 T62
Transfer