Historically, the difficulty of obtaining pure cultures of abundant marine
microbial plankton has an obstacle to reconstructing the underlying
mechanisms of biogeochemistry in the ocean. While a number of dominant
marine species from the ocean surface have been cultured, the dominant
microbial plankton of the dark ocean proved far more difficult to tame.
Genomic analyses of single cells emerged as a powerful means to expand
knowledge of the diverse biochemical potential of these communities.
Chapter 1 reviews the timeline of events in this field and summarizes current
research with single-cell genomics and metagenomics within the framework
of marine microbial ecology.
The defining step in single-cell genomics approaches to environmental
studies is the physical isolation of wild-type cells from heterogeneous
microbial populations. In Chapter two I detail the construction and application
of new instrumentation for optical trapping in conjunction with microfluidic
devices (optofluidics) that allows for the selection of individual cells for
genome amplification and sequencing. This approach has unique advantages
for analyses of rare community members, cells with irregular morphologies,
small quantity samples, and studies that employ advanced optical microscopy
approaches to cell visualization.Fluorescence-activated cell sorting (FACS) approaches to single-cell genomics have reached full development and have been applied effectively to
explore microbial diversity in the deep. In Chapter 3 I explore single amplified
genomes obtained with FACS approaches, from several single-amplified
genomes (SAGs) of the SAR202 clade, which has been shown to be
ubiquitously abundant in the meso- and bathypelagic waters of the open
ocean. Prior to this study the metabolism and geochemical role of the
SAR202 clade was unknown, but their high abundance suggested they
played an important role in nutrient cycling in the dark ocean. Due to their
distinctive vertical profile, early accounts of the SAR202 clade speculated that
they might be major mediators of recalcitrant organic carbon sequestration
and turnover in the deep ocean, contributing to the "microbial carbon pump"
through the conversion of labile carbon forms to more heterogeneous and
refractory forms that could remain in the deep sea for thousands of years. I
discovered that SAR202 encodes several families of oxidative enzymes and
hypothesize that they are involved in the cycling of a major class of refractory
deep-water marine dissolved organic matter (DOM), known as carboxyl-rich
alicyclic matter, or CRAM.
In Chapter 4 I revisit the optofluidic approach and describe its use to
isolate single-amplified genomes (SAGS) from the marine environment.
Several of these SAGs were shown to be representatives of groups of
microbial plankton that are abundant in the ocean but not represented by
genome sequences. In this chapter we evaluate the performance of this
technique for single-cell genomics and outline the encoded metabolic features
of three relatively-unstudied groups of marine microbes isolated using this
In Chapter 5, I outline potential areas of improvement for the optofluidic
technology described in this thesis and discuss where the future of single-cell
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