The history of sea ice microbial ecology

Doctoral dissertations typically include an introduction, a Chapter 1 that summarizes the work and the motivation for undertaking it.  Last week I submitted my dissertation to the UW and the introduction, which includes citations from some long-ago work on psychrophiles and sea ice microbial ecology, might be of interest to some.  I’ve posted the introduction here in its entirety.  If you would like a pdf of any of the hard-to-find early works cited, please let me know and I’d be happy to provide.

Chapter 1: An introduction to sea ice microbial ecology

Our universe is a cold place, with a background temperature of only 2.73 degrees above absolute zero (de Bernardis et al., 2000). Yet scattered throughout this universe are pinpoints of warmth, the result of nuclear fusion, as in the case of our sun, and gravitational interactions, as in the case of the moon Europa). It is much too hot for life to exist near the energetic centers of these pinpoints, but around each is a thin veneer of conditions that enable liquid water, chemical interactions, and reaction rates familiar to those that permit life on Earth. If we consider each of these pinpoints as a sphere, we find that the habitable space around each increases exponentially with a linear decrease in the temperature minima of life (Fig. 1). Thus these spheres succinctly demonstrate that the lower the temperature that life can tolerate, the more space there is for habitation. With this in mind, the function of life at low temperature becomes a central question to the ecology of life in our universe and on Earth.

In seven chapters this dissertation explores the adaptation of microbial life on Earth to low temperature with a particular emphasis on sea ice environments. Chapter 1 reviews the current state of our understanding of sea ice microbial ecology. Chapter 2 compares the microbial community structure of Arctic multiyear sea ice to that of surface seawater — two proximate but ecologically disparate low temperature environments. Chapter 3 explores the microbial community in frost flowers, highly saline structures on the surface of newly formed sea ice, and the underlying newly formed or young sea ice. Chapter 4 uses metagenomics to probe the functional diversity within this same environment. Chapter 5 evaluates the role that horizontal gene transfer might play in microbial adaptation to low temperature environments through quantification of genomic plasticity in psychrophile genomes and taxonomically related mesophile genomes. This chapter also explores the rate of horizontal gene transfer among psychrophiles and mesophiles throughout the Phanerozoic Eon. Chapter 6 explores the adaptation of putatively cold active enzymes to low temperature and describes a simple model, the Protein Evolution Parameter Calculator (PEPC), to evaluate the difficulty of producing enzymes optimized to multiple environmental challenges. Chapter 7 explores the distribution of genes coding for alkane hydroxylase enzymes in psychrophile genomes and the degree to which these enzymes might be optimized to low temperatures. Three appendices accompany this dissertation as well. Appendix 1 describes the Cryosphere Frost Flower Reactor for Organic Geochemistry (CRYO-FFROG), an experimental apparatus for exploring photochemical reactions in the surface ice environment. Appendix 2 reports a strong correlation between bacterial abundance and salt in frost flowers and other components of the young sea ice environment, and it discusses the potential implications for microbial transport and chemical reactions at the sea ice surface. Appendix 3 explores the potential microbial exchange between two geographically separate ice environments linked by aerial transport: the supraglacial environment and the young sea ice environment.

1.1 A brief history of research on sea ice microbial communities

Cold-active microbes first appeared in the scientific literature as early as 1887, when Forster isolated bioluminescent bacteria capable of growth at 0 °C from cold-stored flounder (Forster, 1887). Likewise, the scientific exploration of sea ice microbial communities dates back to at least that same year, when Nansen described diatoms that adhered to the bottom of Arctic sea ice (see reference in Nansen, 1906). McLlan extended these observations during the Australian Antarctic Expedition between 1911 and 1914, describing “practically the whole of low life which exists” in Antarctic sea ice— including protists, rotifers, and bacteria — and in fact, was able to culture bacteria from preparations of sea ice algae (McLlan, 1918). Despite these tantalizing early observations, the study of life in low temperature environments proceeded slowly until after World War II, when increased exploration of the Arctic and Antarctic provided new opportunities to study cold microbial habitats. Prior to 1960, this work was motivated primarily by the role bacteria played in food spoilage and focused on a relatively narrow range of isolates (e.g. Pseudomonas spp.). Despite these limitations, important advances in our overall understanding of psychrophiles were made during this period; these included adoption of the reaction rate terminologies Q₁₀ and µ to describe the temperature dependence of enzymes (Ingraham & Bailey, 1959) and an overall appreciation that psychrophilic microbes are ecologically distinct from mesophiles (Ingraham, 1958).

The International Geophysical Year (IGY) of 1957-1958 heralded an era of increased scientific activity in the Arctic and Antarctic. While we cannot link any microbiological studies directly to IGY, it serves as a useful boundary between studies that preceded it, which were primarily limited to laboratory work, and those that followed it, which would be augmented by environmental observations. The first focused study of sea ice bacteria proceeded from the fifth Japanese Research Expedition of 1961. Researchers obtained several isolates from surface seawater and from melted sea ice samples stored at -5 °C for several months (Iizuka, Tanabe, & Meguro, 1966). Isolation of these samples, however, took place at 25 °C, a temperature that is deleterious to most sea ice bacteria, thus the dominant members of the community were missed. The authors however, did note a co-occurrence of bacteria and ice algae in the sampled “plankton ice” and speculated on possible ecological interactions, a re-occurring theme in sea ice microbial ecology (e.g. Sullivan & Palmisano, 1984, Feng et al., 2014). While the isolation and characterization of marine psychrophiles from seawater continued throughout the 1960’s (e.g. Colwell et al., 1964), studies of sea ice microbial ecology focused almost exclusively on ice algae. The most visible component of the sea ice microbial ecosystem, ice algae can reach densities in excess of 670 µg chlorophyll a L^-1 in spring and summer (H Meguro, 1962). Descriptive work of this element of the ecosystem (Bunt, 1963b; Iizuka et al., 1966) rapidly gave way to more quantitative analyses (Apollonio, 1965; Bunt, 1963a; Burkholder & Mandelli, 1965; Horner & Alexander, 1972; Hiroshi Meguro, Ito, & Fukushima, 1967) regarding chlorophyll concentrations, primary production, and the specific challenges of life in ice. These studies clarified that ice algae are wide-spread, important to the polar carbon cycle, and uniquely adapted to the sea ice environment.

During the 1970’s and early 1980’s, the works of Pomeroy (1974), Azam et al. (1983), and others brought forward the role that heterotrophic bacteria play in the marine carbon cycle. The “microbial loop,” wherein dissolved organic carbon (DOC) is recycled via bacterial assimilation and predation by protozoa, became recognized as an important component of the marine food web. Sullivan and a host of co-authors transferred this concept to the sea ice ecosystem in a pivotal series of papers in the 1980s (Grossi, Kottmeier, & Sullivan, 1984; Kottmeier, Grossi, & Sullivan, 1987; Kottmeier & Sullivan, 1988; Sullivan & Palmisano, 1984). Their work confirmed that sea ice bacteria are not only abundant and active within sea ice but also closely coupled to the occurrence of ice algae. These observations, all on mature, land-fast ice within McMurdo Sound, Antarctica, were extended to more variable ice types by Helmke and Weyland (1994) and Grossmann and Diekmann (1994). Working on newly formed pelagic sea ice, Grossmann and Diekmann (1994) observed significant bacterial growth rates even in relatively oligotrophic sea ice as well as bacterial production rates far in excess of those observed in seawater. Helmke and Weyland (1994) extended these observations to pelagic winter sea ice via the observation of high rates of activity and bacterial biomass relative to the underlying water column; at times, the ATP concentration, a measure of metabolic activity, in a single meter of sea ice exceeded the 100 m depth-integrated value for the underlying water column. These high levels of activity, however, were limited to the bottom-most, warmest zone of sea ice.

By the mid-1990s, it was clear that sea ice bacterial communities were composed of physiologically unique, psychrophilic bacteria capable of survival under conditions of severe environmental stress and had developed a fast response to new inputs of carbon. The taxonomic and functional diversity of this community, however, was almost entirely unknown, with the exception of the phenotype and morphology-based classifications of a few isolates for the former (Iizuka et al., 1966) and the limited observations of extracellular enzyme activity for the latter (Helmke & Weyland, 1994). Concurrent with the growing appreciation of sea ice bacteria as a unique and potentially important component of the polar marine ecosystem came major advances in understanding taxonomic diversity within microbial communities. In a groundbreaking 1977 paper, Woese and Fox used 16S and 18S rRNA gene sequences to classify life into three broad domains (Woese & Fox, 1977). Improvements in sequence technology nearly a decade later (Smith et al., 1986) opened the door for more wide-spread sequencing of 16S rRNA genes from environmental samples and a rapid shift in the existing paradigms of microbial diversity (e.g. Giovannoni, et al., 1990, Ward, et al., 1990). These methods were further utilized to identify isolates from sea ice (John P Bowman et al., 1998; J. P. Bowman, McCammon, Brown, Nichols, & McMeekin, 1997) and, in a novel application of the technique, to identify sequences from an environmental clone library (Brown & Bowman, 2001). These and later studies established that while most genera observed in sea ice have members common to other environments, there are specific strains associated with sea ice.

Studies during this time also introduced astrobiology as a new motivation for studying sea ice microbial communities (Deming & Huston, 2000) (Fig. 2).  Broadly defined as the study of life in a universal context, astrobiology could be considered the purest form of ecology.  A common approach is to study Earth environments that are analogous to potential extraterrestrial habitats.  Since, as discussed previously, the universe is a cold place, the study of sea ice, glacial ice, and permafrost are central, but certainly not exclusive of, this approach.

Reports of the first extra-solar planet orbiting a main sequence star in 1995 (Mayor & Queloz, 1995) and the thousands of candidate and confirmed extrasolar planets since have catalyzed the research on Earth analogues, as has the now hotly disputed  putative microbial fossils in the Martian meteor ALH84001 in 1996 (McKay et al., 1996) and the now widely accepted liquid-water ocean beneath Europa’s icy exterior in 1998 (Carr et al., 1998).

Although the studies of the 1990s began to elucidate the composition of the sea ice microbial community, they did not address its functional role.  In some cases, function could be inferred from specific experiments; Gerdes et al. (Gerdes, Brinkmeyer, Dieckmann, & Helmke, 2005) and Brakstad et al. (Brakstad, Nonstad, Faksness, & Brandvik, 2008) used diesel and crude oil perturbation experiments to explore the ability of the sea ice microbial community to respond to these carbon sources.  Analyses of specific functional genes within sea ice, however, have been surprisingly limited even though they are the most high-throughput measure of community metabolic capability.  Koh et al. identified proteorhodopsin genes (Koh et al., 2010) and genes for anoxygenic photosynthesis (Koh, Phua, & Ryan, 2011) within Antarctic sea ice, which suggests that bacterial energy acquisition in sea ice is not limited to the oxidation of ice algal photosynthates, and Møller et al. (2011) found mercury resistance genes in Arctic sea ice brines.  These insights into sea ice microbial community function have been extended by a small but growing number of sequenced genomes from sea ice isolates.  Although inferences of function from genomes are restricted to the geography and ecology of the original isolate, commonalities between isolates can provide a broader picture of adaptation to the sea ice environment.

Colwellia psychrerythraea 34H was the first sequenced sea ice bacterium (Methe et al., 2005). Although this strain was isolated from sediment, 16S rRNA gene sequences associated with the genus Colwellia have also been observed in sea ice (John P Bowman et al., 1998; J. P. Bowman et al., 1997; K. Junge, Imhoff, Staley, & Deming, 2002), although it is not ubiquitous in the sea ice environment. Other sequenced genomes from sea ice bacteria include Glaciecola pyschrophila 170 (Yin et al., 2013), Octadecabacter arcticus 238 and Octadecabacter antarcticus 307 (Vollmers et al., 2013), and Pyschroflexus torquis ATCC 700755 (Feng, Powell, Wilson, & Bowman, 2014). Considering that in July of 2014 there were nearly 12,000 completed and draft genomes in Genbank, it is clear that the genetic diversity of sea ice has been under sampled compared to some other environments. The tiny glimpse into the function of the sea ice microbial community afforded by these genome sequences suggest that the community may be not only exceptionally genetically and physiologically plastic, but also highly adapted to the environmental constraints imposed by sea ice.

1.2 The present-day understanding of sea ice microbial ecology

Like many microbial habitats, ice is a porous media with a solid phase composed of ice crystals and a liquid phase composed of water and solutes excluded from the crystals during growth. Aside from temperature alone, water ice is distinct from other porous media due to its dynamic nature over a temperature range that is relevant to microbial life. While tap water begins to freeze at 0 °C, seawater, with a salinity of roughly 35 ppt, begins to freeze at -1.8 °C and does not complete the process until -36 °C (G. Marion, Farren, & Komrowski, 1999). The higher the salinity of the starting solution, the lower the temperature required to initiate freezing. Once freezing is initiated for a solution with the brine composition of seawater, however, the salinity of the interstitial brines is almost entirely a function of temperature, although organic matter content and other factors do have some affect. At -5 °C the brine salinity of sea ice is approximately 87 ppt; at -20 °C it will have reached 209 ppt.

The concentration of solutes by freezing is most evident with salt but applies to other components of seawater as well. The lower the salinity and the colder the temperature of the starting solution, the more concentrated the solutes are in the brine phase relative to their starting concentrations. This extends even to bacteria and virus particles as demonstrated by Junge et al. (Karen Junge, Krembs, Deming, Stierle, & Eicken, 2001) and Wells and Deming (Wells & Deming, 2006). For any solute, the degree to which it is concentrated in ice relative to its concentration in the source material is a function of temperature and the bulk salinity of the ice, the total quantity of salt contained in a volume of melted ice (Cox & Weeks, 1983). Thus the degree of concentration varies widely between ice types (Fig. 3).

Because primary production is most concentrated at the sea ice-seawater interface and because DOC, as with other solutes, is concentrated in sea ice brines (Giannelli et al., 2001), bacterial sea ice specialists are optimized to high concentrations of organic carbon. This may aid growth at the coldest temperatures and allow enzymes to maintain uptake rates sufficient to support growth at temperature well below their optimum if substrate concentrations are high (Nedwell, 1999; Pomeroy & Wiebe, 2001). Likewise, this discovery has aided the laboratory study of sea ice bacteria, as sea ice specialists tend to also be specialists for common organic-rich culture conditions (K. Junge et al., 2002).

Observations of sea ice microbial community structure during the winter suggest that, while metabolic activity is present (Karen Junge, Eicken, & Deming, 2004), bacterial growth is extremely limited. Observing Arctic sea ice throughout the winter, Collins et al. (2010) reported little change to microbial community structure, while that of the underlying seawater changed considerably in the same time period. The authors hypothesized that photoplankton-produced exopolymers (EPS) and bacteria known to act as cryoprotectants (Krembs, Eicken, Junge, & Deming, 2002) may enable the survival of even non-ice associated genera within sea ice. The over-wintering community observed by Collins et al. is similar to a typical seawater community and distinct from the community observed in late-spring and summer sea ice. To date, the transition from winter to summer has not been observed with molecular methods, a surprising deficiency given the relevance of this transition to the polar carbon cycle. By tracking bacterial abundance and chlorophyll a concentrations, the sea ice microbial community can be seen rapidly responding to the initiation of the spring algal bloom, though the response may be slower than that observed for seawater (Fig. 4). This response is presumed to reflect the rapid growth of the psychrophilic sea ice microbial community as soon as DOC concentrations are sufficient to overcome the temperature inhibition of enzymes.

1.3 The future of sea ice microbial ecology

The over-arching research objectives in microbial ecology follow the classic set of questions: who, what, where, when, why, and how. When these questions are known in sufficient detail, it is possible to make predictions about the ecosystem in question. A goal of sea ice microbial ecology, for example, is to predict how the microbial ecosystem will respond to environmental perturbations, including slow perturbations, like changing climate, or fast perturbations, such as a release of crude oil. The latter issue has particular significance in the Arctic, where exploration and extraction continues on significant marine petroleum reserves (Gautier et al., 2009). The Macondo Well disaster in the Gulf of Mexico demonstrated that indigenous deep sea bacteria can degrade a considerable quantity of crude oil despite high pressure and low temperatures (Redmond & Valentine, 2012). Interestingly, one of the bacteria observed responding to this input of crude oil was Colwellia sp., a genus known to sea ice as discussed above. Colwellia psychrerythraea 34H is one of the few sequenced sea ice associated bacteria and is commonly used in laboratory studies of cold adaptation. Neither C. psychrerythraea 34H nor a close relative sequenced from water in the vicinity of the Macondo wellhead (Mason, Han, Woyke, & Jansson, 2014), however, have recognizable genes for catabolizing the low molecular weight alkanes that the latter is implicated in. One explanation is that this bacterium is using a gene without close homology to known alkane degradation genes, an exciting and realistic possibility, or that it is responding to secondary metabolites produced by Oceanospiralles, the primary responder.

Either of these scenarios has important implications for the bioremediation of oil released in the proximity of sea ice. Because sea ice bacteria are optimized to high carbon concentrations, they may be resistant to crude oil toxicity and capable of more rapid bioremediation. This idea is supported by the work of Gerdes et al. (Gerdes et al., 2005) and Brakstad et al. (Brakstad et al., 2008), who observed that the indigenous sea ice microbial community is capable of crude oil degradation. The potential rate of crude oil catabolism under the environmental conditions imposed by sea ice, however, remains an unknown. At low temperatures, many crude oil components have reduced bioavailability (Colwell, Walker, & Cooney, 1977), and in sea ice, the rate of bacterial production is generally below the rate of primary production (Pomeroy & Wiebe, 2001). This indicates the limit to which increased substrate concentration can make up for reduced substrate affinity. While sea ice bacteria produce enzymes optimized for low temperatures (Adrienne L. Huston, Krieger-Brockett, & Deming, 2000; Adrienne L Huston, Methe, & Deming, 2004; Methe et al., 2005), this optimization may be insufficient to keep pace with either carbon fixation or a rapid input of crude oil. Predicting the fate of crude oil or any other perturbation to the sea ice ecosystem, will require a much more complete understanding of microbial functional diversity and physiology and their impact on biogeochemistry.

One pathway to develop a predictive biogeochemical model is the metabolic flux model, an idea that was explored for crude oil degradation in a review by Rӧling and Bodegom (Röling & van Bodegom, 2014). This model makes use of community metabolic potential and gene expression data, information derived from environmental and isolate sequencing experiments, to predict the flow of energy and material between the biotic and abiotic components of the biosphere as well as between members of the microbial community. Coupled to a traditional biogeochemical model, the metabolic model becomes predictive at the ecosystem level; as the flow of energy, carbon, or nutrients into the system change, or as members of the community change in presence or abundance, the impact on biogeochemistry can be quantified. This idea is conceptualized in the Taxonomy-Metabolic potential-Metabolism-Biogeochemistry (TMMB) model framework (Fig. 5). As in any model, however, the predictive value of the TMMB framework depends on the level of detail built into the model itself.

Despite decades of research on sea ice microbial ecology, our grasp of the details of sea ice ecosystem function trails far behind our grasp for the marine microbial ecosystem in general. As outlined in the section: A brief history of research on sea ice microbial communities, our understanding of sea ice microbial ecology has generally lagged the broader field of marine microbial ecology by a decade or more. The first measurement of primary production by the ¹⁴C method, for example, came in 1952 for seawater (Steeman-Nielsen, 1952) and 1965 for sea ice (Burkholder & Mandelli, 1965). The first study of in situ gene expression in seawater came in 1990, while gene transcripts were not studied in sea ice until 2010 (Koh et al., 2010). Reasons for this delay may include the novelty of the environment, which reduces the need for new or sophisticated techniques to warrant funding or publication, technical challenges regarding the application of these techniques to sea ice, the limited number of researchers in the field, and the significant logistical challenge of accessing the sea ice environment and conducting research there. While these are valid reasons, an alternate paradigm for the future may be to view the challenges of sea ice microbial ecology as a strong motivator for greater innovation in research. The field of sea ice microbial ecology is well placed to lead the development of a new framework for understanding microbial ecology.

This preferential placement is because, compared to many other microbial ecosystems, the sea ice microbial ecosystem is relatively simple. A large number of its dominant members can be cultured (K. Junge et al., 2002) and thus sequenced and subjected to detailed physiological evaluation. Due to the static nature of sea ice, the flux of nutrients and materials into the system is easier to constrain than for many other marine environments. A strong seasonality not only defines the sea ice environment, but also provides a predictable annual cycle of perturbation and community succession that makes it ideal to test hypothesis regarding the biogeochemical impact of community structure. Sea ice represents an optimal environment to develop and test integrated models connecting community structure, metabolic potential, biological activity, biogeochemistry, and the resulting feedback loop on community structure. Such an undertaking, however, will require a coordinated and long-term research effort involving both modelers and observationalists, with both groups including specialists in physiology, genetics, and biogeochemistry.

While this dissertation does not solve the problem of implementing a TMMB model for the sea ice microbial ecosystem, it instead seeks to clarify further details of the sea ice microbial ecology through a better understanding of microbial community structure within several under-explored ice types, the genomic plasticity and metabolic function of psychrophiles, and the evolution of cold-adapted communities. In time, a deeper appreciation of these aspects of the microbial community will become part of the foundation for a more complete understanding of the sea ice ecosystem as a whole.

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