ABSTRACT There are an estimated 325,000 species of bacteria on planet earth, with only approximately 4,500 genomes

published to date. These estimates are based on 16S ribosomal DNA community surveys across a broad range

of habitats. Bacterial communities typically exhibit a log normal distribution, with a few members comprising

most of the reads, and hundreds or thousands of low-abundance members accounting for most of the diversity.

As new technologies have developed it has been possible to identify bacterial species at ever lower abundanc-

es. These low abundance species, most of which have previously evaded detection by sequencing or culture

methods were termed the “rare biosphere” by Sogin et al. 2006. This seminal paper raised the questions: 1)

How much diversity are we still missing? 2) Do these members serve an ecological purpose? 3) Are these

members rare by nature?

Continued and expanded sequencing efforts have cued us in to how much diversity we may not have yet

discovered, and many temporal studies have shown that rare members may act as microbial seed banks, be-

coming abundant when favorable conditions arise. Yet, without removing rare members from natural systems,

it is difficult, if not impossible, to speak to the contribution of rare members to ecosystem function. We have a

collection of abundant community members isolated from an evolved switchgrass-degrading microcosm. By

recombining only the abundant members and comparing their function with the original community, we can

assay the impact of the rare biosphere.

For over three years, we maintained soil-seeded anaerobic switchgrass-degrading microcosms, from which

we isolated at least six novel species and genera. These strains, along with Enterococcus faecalis, make up the

most abundant species in the microcosms, comprising 88% to 89% of all 16S rDNA amplicon reads. Over 300

OTUs account for the remaining ~11% of reads. We believe that many of these rare members are artifacts due

to chimera formation and sequencing error, as 16S rDNA amplicon sequencing on pooled isolate DNA resulted

in 113 erroneous OTUs. And yet, we know some of these rare taxa are real, as we were able to isolate strains

making up as few as 0.04% of reads.

Rare Members Make Up Almost 100% of OTUs, but only a

Small Fraction of Total Sequence Reads

Discussion and Future Work

The communities vary significantly in their acetate production, with the rebuilt

community outperforming the whole community in MQM media but the whole com-

munity outperforming in Mic media. More ethanol was produced and more

switchgrass was degraded in MQM media for both communities compared to those

in Mic media. This reflects isolate studies in both media types where the only

switchgrass/cellulose-degrading strains from the community are able to degrade

switchgrass much faster in MQM than in Mic. These isolates are also ethanol produc-

ers.

Quantifying proteins and total DNA would help explain if these differences are

based on extracellular enzymes or total cells in the community. We did not measure

gas, but significant carbon could be “lost” as carbon dioxide in this system, so in or-

der to track the total flux, that must be taken into account.

DNA will be sequenced to better understand the role of the abundant and rare

members, specifically how the rare members have persisted in these systems.

The rare members affected community function in both degradation rate and in

products, but were not necessary for either. They must be taken into account when

assaying community function and developing models of carbon/nutrient cycling.

Switchgrass degradation is fastest with the whole

community (S2) in MQM, but does not crash without rare members

Probing the Significance of the Rare Biosphere: Reconstructing a Complex Anaerobic Plant-Degrading Community without the Rare Members

Haas KN1,3, Kulkarni M2, and Blanchard JL1,2,3

1Graduate Program in Microbiology, 2Department of Biochemistry, 3Department of Biology University of Massachusetts

The products are the same but the rebuilt community produces

more acetate than the whole community in MQM media

The whole community produces more acetate than the rebuilt

community in Mic media

We isolated the most abundant taxa from the microcosm,

making up ~85% of total Illumina 16S rDNA amplicon reads

Experimental Design:

“Removing” rare members by rebuilding the communities without

them and comparing differences in whole and rebuilt communities

switchgrass degradation and fermentation products

DNA pellet

for sequencing

Switchgrass degradation HPLC