Polar Microbiology: Life in a Deep Freeze

This chapter talks about the development of culture-independent, molecular methods that have revolutionized the field and the understanding of molecular ecology. Through the use of these techniques, it is now apparent that the earlier culture-based studies were not a representative reflection of the dominant microorganisms in many psychrophilic habitats. Cyanobacteria present in Dry Valleys mineral soils are considered to be the major primary producers and contribute significantly to microbial diversity. Lithic communities are classified by the specific environmental niche they reside in, and hypoliths, chasmoliths, and cryptoendoliths are further discussed in this chapter. The majority of bacteria isolated from permafrost are aerobic and include a number of coryneforms, endospore formers, sulfate reducers, nitrifying and denitrifying bacteria, and cellulose degraders. The microbial mat bacterial diversity of 10 Dry Valleys lakes was assessed by culturing techniques (heterotrophic growth conditions and fatty acid analysis). Microbial mats from Markham and Ward Hunt Ice Shelves showed species homogeneity in the vertical profile, which has not been seen previously in Antarctic mats, possibly due to differences in mat thickness. The stratified Antarctic mats from the McMurdo Ice Shelf were up to 8 cm thick in places, while the Arctic mats in this study were approximately 2 cm. Using metagenomic methods researchers can assess the diversity of culturable and uncultured organisms, including rare taxa.

Modern molecular PCR-based methods, typically targeting the 16S rRNA gene, have now revolutionized the field of environmental microbiology and have allowed culture-independent surveys of natural in situ microbial communities. These new approaches have unearthed a wide diversity and ubiquitous presence of Archaea in nonextreme environments such as soils, sediments, and oceans. The archaeal domain is split into two major phyla, the Crenarchaeota and Euryarchaeota. In spite of the extreme environmental conditions in the polar regions, through the application of culture-independent 16S rRNA gene-based surveys, Archaea have been found to inhabit a wide range of polar environments. This chapter reviews the current literature describing archaeal presence and diversity in polar and subpolar habitats. Marine and terrestrial ecosystems are discussed individually for Antarctic and Arctic ecosystems, with final sections discussing comparative studies of archaeal communities between polar regions, the potential response and contribution of Archaea to future climate change models, highlights of recent findings, and future research needs. The first wide-ranging PCR-based survey of archaeal 16S rRNA genes in terrestrial Antarctic sites has recently been reported. 16S rRNA gene clone library-based methods were used to analyze archaeal communities from Kirkpatrickia varialosa, Latrunculia apicalis, Mycale acerata, Homaxinella balfourensis, and Sphaerotylus antarcticus. FISH-based studies have shown that Archaea are typically minor components of pelagic microbial communities in Arctic water bodies. The majority of microbes in any given environment are typically recalcitrant to laboratory cultivation, and as such only a handful of Archaea have been isolated from polar environments.

This chapter explores the data collected on the importance of bacteriophages to the ecology of the earth's polar regions. It examines the numbers of phage-like particles that have been observed in the ecosystems and their importance in regulating bacterial numbers and the food chain of the extreme oligotrophic environments. To understand the potential impact of bacteriophages on polar ecosystems, it is first necessary to understand the different life choices of phages and how environmental factors are known to affect them. Perhaps not surprisingly, these data suggest that bacteriophages are an important shunt in carbon cycling in the Antarctic regions as well as in the Arctic. Unlike many other studies, the authors conclude that neither bacteriovores nor bacteriophages are important regulators of bacterial numbers but that numbers are regulated by algae blooms and other factors that affect bacterial growth. Pseudolysogeny was first defined by Baess in 1971. In his review, pseudolysogeny was identified as a phage-host interaction in which the phage, following infection of the host, elicits an unstable, nonproductive response. Thus any study that enumerates total virus-like particles (VLPs) must be tempered with the understanding that not all will be infective. The data obtained in this study support the hypothesis that bacteriophages are of quantifiable significance in the carbon-flow cycle of Antarctic oligotrophic lakes. Few researchers have investigated bacteriophages in both the Arctic and Antarctic in the same study. Phylogenetic analysis of their data revealed five previously uncharacterized subgroups of T4-like bacteriophages in many environments, including the Arctic samples.

Although some fungi that are endemic to the extreme polar regions show psychrophilic behavior, the majority are instead psychrotolerant and globally distributed, ranging from the Arctic to Antarctica. It is important to note that although their natural ecological niches are in the polar environments, such fungi can also grow in human proximity; they can inhabit freezers and cold-storage rooms, and refrigerated and even frozen food. The methods used for fungal detection have been time appropriate, from the classical early microscope visualization to the more recent sophisticated DNA-based techniques, which have been complemented lately by metagenomic studies, although these have generally not been focused on fungi. Permafrost in polar regions covers more than 25% of the land surface and significant parts of the coastal sea shelves. Fungal diversity in the Arctic and Antarctic permafrost has been studied intensively over the last decade. The dominant species in Arctic glaciers environments was Penicillium crustosum, which represented on average half of all of the isolated strains from the glaciers studied. Penicillia were the most frequently occurring filamentous fungi in all of our samples, including seawater, sea ice, snow/coastal ice in tidal zones, puddles on snow, subglacial ice, and glacial meltwater.

Although interest in microbes inhabiting low-temperature environments has increased in recent years, significant gaps remain in understanding what makes certain microorganisms cold adapted. Interest in the structural, biochemical, and physiological properties of psychrophilic microorganisms has motivated investigations to characterize the adaptations that maintain enzymatic reaction rates, macromolecular stability, and homeostasis at cold temperature. This chapter provides an overview on the state of knowledge about adaptations that allow certain bacteria and archaea to persist in the coldest regions of the biosphere. In general, the proteins of psychrophilic microorganisms must maintain flexibility to perform catalysis at low temperatures, whereas thermophilic proteins are rigid to protect them from thermal denaturation. Importantly, adaptations that enhance protein flexibility reduce the activation energy needed for the formation of the enzyme-substrate complex, resulting in enhanced catalytic activity at low temperature. The chapter discusses many of the most common and generally understood biochemical and physiological adaptations that appear unique to the psychrophilic lifestyle. Psychrophilic microorganisms use a range of strategies to persist at low temperatures, including possessing catalytically efficient enzymes, synthesizing specialized lipids that increase membrane flexibility, and producing proteins that affect freezing and ice structure. Coupled with technological advances in high-throughput DNA sequencing and proteomics, one can expect that information on cold-adapted bacteria and archaea will increase in the future.

Examination of the transcriptome and proteome enables the investigation of the underlying gene and protein expression, respectively, that results in cold adaptation and ultimately permits the successful colonization of cold environments by cold-adapted microorganisms. Genomics can be used to investigate cold adaptation at the level of whole genes by examining gene content, gene expression, protein expression, and other unique features, while at the molecular level, genomic analyses may identify trends in amino acid composition, codon usage, and nucleotide content that result from cold adaptation. This chapter discusses (i) use of ecological information to discern cold-adapted microorganisms, (ii) unique gene- and protein-expression adaptations for coping with cold environment stresses, (iii) sequence adaptations that facilitate protein function at low temperature, and (iv) a case study comparing cold-adapted and warm-adapted species of the genus Exiguobacterium. The genera Exiguobacterium and Psychrobacter represent gram-positive and gram-negative bacteria, respectively. Strains of these two genera were among the psychrophile genomes sequenced and used, along with other examples, to illustrate various aspects of cold adaptation. Five prominent eurypsychrophiles including the permafrost firmicute E. sibiricum 255-15 have been subjected to functional genomics experimentation at low temperature. Findings from studies with these organisms with reference to other psychrophilic and mesophilic microbes where appropriate, are presented in the chapter. The results suggested that E. sibiricum requires active transport of nutrients at lower temperature to increase substrate uptake.

Polar environments can be among the simplest environments on Earth, making them excellent models to understand ecosystem processes and the responses of microorganisms to environmental perturbations. This vulnerability and the rate of the ongoing change make polar environments a priority in climate change and bioremediation studies. This chapter covers studies based on large-scale 16S rRNA gene libraries, environmental microarrays, large-insert clone libraries, and shotgun genome sequencing that are applying metagenomic techniques to characterize and understand polar ecosystems. A recent study at sites ranging from the Falkland Islands all the way to the base of the Antarctic Peninsula used 454 GS FLX Titanium sequencing of the 16S rRNA gene to compare the bacterial community structure and diversity in warmed versus control plots. This study revealed that soil warming induced significant shifts in the major soil bacterial groups like Acidobacteria and α-Proteobacteria, which led to changes in soil respiration. A recent shotgun metagenomic study in the Canadian High Arctic sequenced a permafrost sample and its overlying active-layer soil and focused particularly on genes that might be important for greenhouse gas emissions following permafrost thaw. The only metagenomic study of polar freshwater ecosystems published to date looked at the viral communities of an Antarctic lake. This study revealed that a large proportion of the viral sequences retrieved from the lake were from eukaryotic viruses and not from bacteriophages. Emerging technologies could also be interesting to apply to polar environments. For instance, bioremediation studies could benefit from metabolomics, proteomics, and newly developed reactome arrays.

The various organisms thriving in extreme environments on Earth, psychrophiles (cold-loving organisms) are the most abundant in terms of biomass, diversity, and distribution. The chapter presents an overview of the biotechnological uses of psychrophiles and of their biomolecules using selected examples. Site-specific mutants of psychrophiles are a useful tool to study cold adaptation and expression of cold-active enzymes at low temperature. At the industrial level, the best-known representative of polar microorganisms is certainly the yeast Candida antarctica, as its species name unambiguously refers to the sampling origin. Antarcticine-NF3 is a glycoprotein with antifreeze properties produced by the bacterium Pseudoalteromonas antarctica, which has been patented by Spanish researchers. It was found that Antarcticine is effective for scar treatment and re-epithelialization of wounds. Most studies on hydrocarbon bioremediation in polar regions have focused on the treatment of petroleum hydrocarbons, since increased petroleum exploration increases the risk of accidental oil release. Polar plants and animals have also found diverse applications and are worth citing in the context of the present survey. Recent developments based on cold-adapted organisms and their biomolecules have clearly demonstrated the huge potential of psychrophiles.

Terrestrial and submarine permafrost is identified as one of the most vulnerable carbon pools on Earth. In some areas, permafrost comprises upwards of 80% ice in the form of large features, such as massive ice sheets many kilometers in length; or on smaller scales, such as ice wedges and ice lenses, and as ice that fills soil pore space. Residual pockets of seawater, from the subsidence of the polar ocean, exist as saturated, salt-rich permafrost environments known as salt lenses or cryopegs. All of these permafrost features sustain microbial communities that contribute to carbon cycling in polar regions. The way in which gas is released from permafrost, i.e., the rate and pathway, determines the ratio of methane and carbon dioxide emitted to the atmosphere. This chapter describes the different carbon pools, carbon fluxes, and freeze-thaw stresses related to microbial activities. It then examines methane-cycling communities in Arctic active-layer and permafrost environments. The fast recovery of the microbial activity during spring suggests that carbon mineralization in thawing Arctic sediment may rapidly respond to warming, resulting in substantial changes in microbial carbon cycling and growth of microbial populations. Environmental sequences from the Laptev Sea coast consist of four specific permafrost clusters. It was hypothesized that these clusters comprise methanogenic Archaea with a specific physiological potential to survive under harsh environmental conditions. A first study on submarine permafrost of the Laptev Sea shelf demonstrated that intact DNA was extractable from late Pleistocene permafrost deposits with an age of up to 111,000 years.

At the highest taxonomic levels, microbial communities in the polar oceans are similar to those in temperate oceans, and contain diverse representatives from the three domains of life: Eukarya (protists), Bacteria, and Archaea. This chapter presents a brief overview of pelagic microbes and their diversity, vertical distribution, and influences on biogeochemistry and upper food webs. The focus is on recent advances following the application of molecular biological techniques to polar marine systems. A revolution has occurred with the application of molecular biological techniques, especially small-subunit rRNA gene surveys, and identification of Bacteria, Archaea, and picoeukaryotes is now possible. The two most abundant archaeal phyla in the ocean belong to the Euryarchaeota and Thaumarchaeota Marine Groups (MG) I, which were originally classified as part of another phylum, the Crenarchaeota. A microbial loop begins with organic matter in the ocean being taken up by bacteria; the bacteria are eaten by small flagellates that excrete organic matter, which is used by bacteria that are eaten by small flagellates. All higher trophic levels, including whales, seals, and birds at both poles and polar bears in the Arctic, ultimately depend on microbes to convert inorganic carbon and solar energy into organic carbon, maintain it in a biologically available form, and recycle nutrients.

This chapter focuses on glaciers and ice sheets, sea ice, and ice shelves of the polar regions, i.e., those latitudes above the Arctic and Antarctic Circles where glaciers and ice sheets cover a significant proportion of the land mass and where large expanses of the surface waters of the Arctic and Southern Oceans undergo an annual cycle of freezing and melting. This chapter further introduces sea ice as a microbial habitat and summarizes from some of the aforementioned reviews what is known to date about the abundance, activity, diversity, and ecology of prokaryotic sea-ice microorganisms. It provides a brief outline of the role of microorganisms in biogeochemical cycling of elements in sea ice. The majority of bacteria isolated from sea ice are pigmented and highly cold adapted, with some able to form gas vesicles. Possible cold-adaptation strategies revealed by whole-genome sequence analysis also include the production of cryoprotective osmolytes and exopolymers. Polar ice shelves are thick masses of ice floating on the ocean. They are formed through glacial ice and ice sheets pushing onto the sea or long-term accumulations of sea ice. Analysis of ice-shelf heterotrophic bacteria and microbial eukaryotes suggests phylogenetic affiliation with taxa from diverse environments and climatic zones ranging from Antarctica and other cryosphere habitats to temperate ecozones. Microbial investigations on polar glaciers, ice sheets, and ice shelves are still largely in their infancy, with sea-ice research being somewhat more established.

Depending on wind speed and direction, microbes are swept up from diverse terrestrial and oceanic environments and blown onto glacial ice. The activation energy for survival metabolism turns out to be ~110 kJ, but with an ~106-fold smaller preexponential factor than for unlimited growth. The arrival rates of bacteria and nonmicrobial dust blown from African desert sources to an air collector on Barbados showed similar patterns of seasonal and daily. In seeking to interpret the rapid decrease in fluorescence intensities of tryptophan (Trp) in the top 120 m of ice and the flattening of intensity values at greater depth, researchers carried out ground-truth measurements of cell concentrations in ice from several sites in Antarctica and Greenland. The main conclusion is that the depth dependence of cell concentration seen with epifluorescence microscopy is far weaker than the ~20-fold decrease with depth of the chlorophyll (Chl) and Trp fluorescence shown. The weak decrease in microbial concentration with depth suggests that both psychrophiles and nonpsychrophiles are equally able to adapt to the lower temperatures, lower nutrient availability, and immobility in ice than in oceans and soil. By using new techniques of single-cell genomics, it should be possible to track changes in their genome as a function of depth in the ice and thus to infer their mutation rates in the ocean before they reached the ice.

In light of increasing interest in the functioning of extremophilic environments and growing concerns over global warming, this chapter attempts to address some of the factors that influence life in cold polar habitats. The cryobiosphere is especially sensitive to changes in climate and itself plays an important role in gas fluxes and environmental shifts at the poles and, by extension, on the earth as a whole. This sensitivity and its consequent global impact make an understanding of polar bioclimatic interactions critical to predicting future climate change trends. During the 2001 season Warner and Miller collected data on RecA antigen concentrations in marine bacterioplankton near Palmer Station. Although the data were scattered, a direct correlation between an increased ratio of midday-to-evening induction and the extent of the stratospheric ozone depletion was observed. This chapter addresses the mechanisms by which microbes repair UV damage in their most fundamental molecule, the DNA that encodes the proteins and molecules that mediate all their life functions. Many of the hypotheses regarding the effect of global warming at the poles have focused on the high concentration of carbon trapped in the permafrost and the concept that its release will result in massive increases in microbial activity that will produce positive feedback, further exaggerating climate change. Microbial nitrous oxide production appears to be enhanced at higher water-activity concentrations and atmospheric release of nitrogen species with a biological origin from snow has been shown to occur even under conditions of low or absent light without ice-/snowmelt.

All life on Earth represents a common genetic and biochemical system descendent from a common ancestor. The other worlds in our solar system that are the most promising targets in the search for life are Mars, Europa (a moon of Jupiter), and Enceladus (a moon of Saturn). Perchlorates are also metabolically active. It is known that microorganisms on Earth are capable of using perchlorates as electron acceptors, allowing anaerobic microbial respiration to occur where perchlorate replaces oxygen as the terminal electron acceptor. Studies of the microbes in the ground ice below dry permafrost in University Valley show that there is an adapted microbial community, and RNA data show that there is microbial activity. The availability of liquid water within the Martian subsurface (permafrost or regolith) would be concentrated into eutectic brines. As such, the microorganisms that could survive and potentially remain viable under such growth conditions would most likely be halophilic cryophiles. While the northern plains represent the most likely site of recent life due to the melting of near-surface ice, the southern highlands represent the best location to find long-frozen remains of ancient life on Mars. In the outer solar system there are two worlds that potentially have liquid water under layers of ice: Europa and Enceladus. In addition to Mars, Europa, and Enceladus, there are other worlds of interest to astrobiology-and they are also icy worlds.