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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%


#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%




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

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T0_1 T0_2 T2 T10 T20 T30 T40 T49 T62

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