Antarctic ecosystems (such as the ponds by Bratina Island, Antarctica) provide a great opportunity to examine organisms that can live in one of the most extreme and geochemically varied environments in the world. These ponds are of such interest as each one can vary greatly in size, depth, and age as well as profiles of dissolved oxygen, metal concentrations, pH and salinity. Even within ponds geochemically distinct stratified layers can form which can greatly influence their microbial communities. This study aims to increase our knowledge of microbial biodiversity and the environmental factors which structure them paying special attention to the stratification transition zones within ponds. A thorough set of biological samples will be taken from four selected ponds during mid-summer in the 09-10 season to complement those taken during the winter freeze-up in the 07-08 extended season by Hawes et al. Oxygen concentration, pH, salinity and temperature of the ponds will be measured in the field then split water samples will be taken back to the University of Waikato for further analysis. This research will use a suite of modern genetic approaches matched with geochemistry to identify and characterise the resident and functional members in the microbial community and how their community is structured in relation to their environmental conditions. This information should lead to a better understanding of diversity and spatial variability of microbial populations in relation to the extreme physicochemical characteristics of their environment. It will also help to formulate and develop a model of how the microbial communities of the ponds are structured in response to their environment and what drives the community composition and structure.
Chapter 1 : Literature Review
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One of the fundamental challenges in biology today is to understand how organisms respond and evolve in relation to changing environments (Kussell et al., 2005). The ponds on Bratina Island (78° 01' S latitude, 165° 32' E longitude on the McMurdo ice shelf (MIS) in the Ross Sea area of Antarctica) provide an extraordinary and tractable opportunity to examine metabolic functioning and the adaptations that allow microbial communities to thrive under these extreme conditions. Yet, after decades of research we still know little about the microbial biodiversity and processes that occur in Antarctic ecosystems and the environmental factors that structure them.
1.1. The Physical Environment of Antarctica
The Antarctic continent is one of the harshest and most demanding environments in the world due to its broad range of extreme conditions (Cowan, Ah Tow, 2004). It is Antarctica's combination of extremes that make it unlike any other place on Earth: extreme isolation; past loss of habitat from ice formation; extreme selection pressure particularly from temperature and water stress (Bergstrom et al, 2006b); along with solar UV radiation (Hughes et al , 2006); long periods of complete seasonal darkness (Cowan, Ah Tow, 2004); and strong, dry katabatic winds, which ensure that any water that falls on the rocky valley floors is quickly lost to the atmosphere by the process of sublimation; (NIWA Science, 2007b). As a result the continent has a highly reduced biodiversity (Bergstrom et al, 2006b); in fact in 1903 when Captain Scott's party first discovered the dry valleys they considered them to be a barren land absent of any kind of life. Today we now know that this harsh landscape harbors a variety of diverse and productive ecosystems, each with its characteristic community of organisms, although little is known about what specifically is in the majority of Antarctic ecosystems (NIWA Science, 2007d).
1.1.1. Bratina Island Ponds
Bratina Island Ponds experience a unique, extreme and fluctuating climate which makes it difficult for pond systems to establish themselves and survive (Cowan and Tow, 2004). Their existence is driven by the short period of the year when liquid water is available to collect in the depressions of the undulating landscape of the McMurdo Ice Shelf (MIS) near to Bratina Island (Victoria Land, Antarctica) during the peak of summer (Howard-Williams and Hawes, 2005). The balance between water supply and evaporation means that each pond will have a broad range of physiochemistry's that can rapidly change (Laybourn-Parry et al., 2002). Ponds within metres of each other can vary greatly in size, depth, and age (Gibson et al., 2006) as well as chemical evolution and profiles of dissolved oxygen (Koob and Leister, 1972), metal concentrations, pH and salinity (Matsumoto et al., 1992; Schmidt et al., 1991). These ponds also experience extreme fluctuations in temperature and light regimes throughout the year (Hughes et al., 2006). However the most interesting feature of these ponds is how some have stratified layers within the water column with characteristic geochemistry. Detailed sampling at the interfaces of these stratified layers should show rapid changes in the microbial community profile showing how they are structured by the environmental factors around them. Put together this project provides a great opportunity to examine organisms that can live in one of the most extreme and geochemically varied environments in the world (Bergstrom et al., 2006).
1.1.2. Antarctic Ponds as a microbial environment
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Marked to Standard
Even though Antarctic ponds are constantly exposed to some of the harshest conditions known in the world they can still contain and maintain a diverse microbial population (Howard-Williams, Hawes, 2006). One particular stress put on organisms in ponds in Antarctica is that most ponds completely freeze each winter and thaw each summer. To survive freezing they must have some sort of mechanism which allows them to survive. Some have the ability to produce a resting state while the water around them is frozen (Heywood, 1984), and then become active again when the ice melts (Wynn-Williams, 1996). Another stress not found in more temperate regions is that Antarctica has highly seasonal light meaning that organisms can only photosynthesize for a limited period of the year, in ponds light is further reduced by the presence of ice which blocks the sunlight. This means that only organisms which are adapted to low and seasonal light will be able to maintain themselves; they can do this by being highly efficient at harvesting what light is present when it is present and retain the fixed carbon gained from this time, or be able to switch nutritional modes between autotrophy and heterotrophy so that they can live off others (Heywood, 1984). The combination of low light, low temperature and slow supply of nutrients leads to very slow growth (Hawes, Howard-Williams, 2007). The combination of the permanently cold temperature which limits metabolic processes, hypersalinity and low light levels which inhibits growth (Lyons et al, 2006) makes the life that has persisted in this environment very interesting.
Although there is a substantial planktonic population in most lakes (Hawes, Howard-Williams, 2007), the majority of past microbial research carried out in Antarctic limnetic systems has focused on the dense microbial mats on pond and lake floors (Fernandez-Valiente et al., 2001; Hawes et al., 2001; Hitzfeld et al., 2000; Moorhead et al., 1997; Taton et al., 2006; Taton et al., 2003) and the sediments underneath (Bowman et al., 2000; Cowan and Tow, 2004; Wasell and Hakansson, 1992); however little is known about the microbes that reside within the ponds water column. There was an extensive study made by Hawes et al, 1999 between January 1997 and January 1998 on three shallow ponds on the McMurdo Ice Shelf which followed the environmental conditions in and around them. They found that the microbial mats remained photosynthetically active as long as there was still light and liquid water. It was found that even in such harsh conditions these organisms manage to successfully grow and maintain a large and diverse population (Hawes et al., 1999). From this it is assumed that the planktonic populations should show an even greater response and require greater resistance to the conditions as their survival is dictated by their own ability and not the collective metagenome of a microbial mat.
1.1.3. Pond physical-chemical Characteristics
To date it seems that the majority of research done on Antarctic aquatic systems has focused on lake physical, chemical and biological processes (Priscu et al, 1997; Tyler et al, 1998; James et al, 1994; Labrenz, 1998; Aiken, Miller, 1996; Pearce, 2002; Priscu et al, 2005), not on ponds. An important note is that when defining a polar pond it is a body of water that freezes solid right to the bottom during winter, and some can completely thaw in summer. Lakes in these areas on the other hand, don't necessarily freeze to the bottom (Hawes et al, 1999), they can maintain a pool of liquid water under a protective ice layer and frequently do not completely thaw in summer (Howard-Williams, Hawes, 2006). This fact markedly changes what the environments in lakes and ponds are like throughout the year (Hawes et al, 1999). When ponds do freeze in the winter it is from the top down which means that there is a time when there is a protective ice-cover with a liquid water core (Hawes et al, 1999). This greatly affects the physical and biological processes occurring in lakes (Parker, Simmons, 1985). The ice-covered lakes can have oxygen levels in excess of two times the expected level due to oxygen production exceeding consumption coupled with the ice cap stopping atmospheric release and equilibration (Lyons et al, 2006). The ice cover will filter out natural light decreasing the total amount available to be used by the bacteria, this will cause a depletion of oxygen production, furthermore the ice freezes pure water first leaving the dissolved salted in solution making it hypersaline as it does. The benefit of this ice cover is that it shields the inside of the pond from the rapid air temperature changes, however this is temporary eventually everything gets to the extremely cold temperature (Hawes et al, 1999).
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Antarctic ponds have a broad variety of trace metals in varying concentrations depending on depth, size of pool, age, experimenter and proximity to the major sources of metals which are atmospheric salt fallout, the weathering of rocks and soils (input via meltwater), seawater from pools which have been separated from the sea some time ago and hydrothermal activity (Matsumoto et al, 1992). There is a constant input of inorganic material and micro-organisms from surface sediment which makes it way into the ponds during the summer, the extent and rate of this depends on the temperature and duration of the summer (Brambilla et al, 2001). All of these factors make ponds physical and chemical characteristics highly varied between ponds and time.
1.1.4. Pond environment during freezing
Little is known about how polar ponds are affected by the extremes of an Antarctic winter (Hawes et al, 1999). “Generally ice grows downward throughout February and concentrates dissolved material below it through a freezing out process” (Goldman et al, 1972). It was found by Koob and Leister (1972) that there was high Dissolved oxygen present from middle of November to the middle of January which was thought to occur due to the high levels of photosynthesis during this time resulting in high oxygen production, with the highest levels in early December. There was an extensive study done by Hawes et al, 1999 between January 1997 and January 1998, it followed the environmental conditions in and around three shallow ponds on the McMurdo Ice Shelf. The ponds melted during summer during the short period when the air temperatures rose above 0 °C (the water temperature was warmer than that). Between late February and early March the shallow “shores” began to froze with the bottom of the lake remaining unfrozen until early June. The microbial mats remained photosynthetically active as long as there was still light and liquid water, so deeper ponds have more shielded microbial mats from the elements so remain active for longer (Hawes et al, 1999).
1.2. Microbial Communities in Antarctic Ponds
Microorganisms are fundamental to the functioning of Antarctic (and in fact all) ecosystems (Franzmann et al, 1997). However unlike organisms in more temperate regions there are more restrictions put on growth in Antarctica and this reduces the number species that can be found there (Wynn-Williams, 1996). To survive the microbes must use a variety of survival mechanisms such as modifications to its physiology, biochemistry and behavior (Hughes et al, 2006). For instance to survive over winter some microbes can produce large amounts of starch bodies within their cells to be used as energy storage or encysted themselves (Bell et al, 1999).Even though these organisms are put under a range of environmental challenges they still manage to accumulate a large volume of biomass and there is an extremely high biodiversity present in these ponds (Hawes et al, 1999). There have been some intensive studies done on some of these organisms such as Psychromonas antarcticus a psychrophilic, halophilic, aerotolerant anaerobic bacterium isolated from the anaerobic sediments of a high salinity pool by Mountfort et al, (1997) and Flavobacterium gondwanense, a halophilic orange- and yellow-pigmented, gram negative, nonmotile bacteria which is readily cultured from Organic lake, a hypersaline meromictic lake in the Vestfold Hills region (Dobson et al, 1993), however the majority of planktonic life remains undescribed.
A psychrophile is “an organism with an optimal growth temperature of 15 °C or lower, a maximum growth temperature below 20 °C and a minimum growth temperature at 0 °C or lower” (Madigan, Martinko, 2006, Bowman et al, 1997). They are able to succeed in cold environments due to a range of unique mechanisms they have evolved over thousands of years (Deming, 2002). To grow Psychrophiles require liquid water, saline water freezes at colder temperatures than pure water and even in solid ice there are microscopic pockets of water that contain liquid water between ice crystals (Madigan, Martinko, 2006) known as the eutectic zone which allows microbial growth (Deming, 2002). Some bacteria can also produce an anti-freeze protein which inhibits the formation of ice crystals (Gilbert et al, 2003). Generally the most successful organisms have the ability to remain active over the winter period (Laynourn-Parry, 2002) however most bacteria are believed to go into inactive states when the ice is frozen such as the viable by non culturable state (VBNC) to protect themselves during extreme cold (Chattopadhyay, 1999). It is believed that the planet Earth has had several global periods of extreme cold (ice ages) where the entire planet would have resembled Antarctica. By this logic it is thought that cold tolerance was an essential skill to have in an organism's repertoire so the ability to live psychrophilically is not a new phenomenon but a legacy to early life on Earth (Howard-Williams, Hawes, 2006).
Antarctica is unique in the fact that it has been relatively isolated from the rest of the world since its separation from Gondwanaland more than 10 million years ago (Vincent, 2000). It has also had the majority of its indigenous life removed from it during periods of climatic cooling and major glacial advances which occurred between 32-5 mya. This means that most life on Antarctica is the result of relatively recent (Ellis-Evans, Walton, 1990) and continuous colonization by invading organisms (Wynn-Williams, 1990) the most common invaders being prokaryotes (Franzmann et al, 1997). Microbial colonisation in Antarctica is generally carried out by air or borne on birds or mammals (especially humans) (Wynn-Williams, 1990). Colonisation can also occur due to movements of the ground which isolates marine communities from the sea and over time these lakes and ponds significantly change both chemically and biologically (Laybourn-Parry et al, 2002). Wynn-Williams (1990) says that “colonization occurs in two phases: Firstly, the immigration, survival and establishment of the microorganisms, and secondly the stabilization of the environment for subsequent higher organisms”.
The abundance of an organism is based on two factors: Firstly how well they are adapted to dispersal and secondly how well they can survive in the environment they land in. An organisms may be a good disperser but not be able to survive well so won't be very common, conversely if an organism is well adapted to a habitat but is a poor disperser it may not reach this habitat so again will not be very common in that environment (Bergstrom et al, 2006b; Ellis-Evans & Walton, 1990). It is thought by some that most microbes only utilize certain parts of their genome (the parts that are needed for survival in the environment they are in) and that some microbes in temperate regions actually harbor the ability to live in the extreme Antarctic climate which is how microbes are able to constantly invade and colonise Antarctica (Hughes et al , 2006).
Based on a molecular clock made by a 16s rRNA analysis it is believed that Anatarctic strains diverged from the rest of the world approximately 75 million years ago, however Antarctica has only been like it is today for the past 3 million years so the organisms present are a product of fairly recent evolution (Wynn-Williams, 1996). Even though Antarctica may seem like the perfect place for truly unique species to sustain themselves the above facts indicate that even Antarctica would not be host to truly endemic species (Stevens, Hogg, 2006; Vincent, James, 1995).
The question of how microbes are distributed in time and space has been argued for decades (whether it is the current climate or historical events which have the greatest impact on the structure of a population (Martiny et al., 2006)). In 1934 Baas Becking summarised previous thoughts about biogeography in the famous microbiological tenet “Everything is everywhere, but the environment selects” meaning that organisms will be found wherever their ecological requirements are met. This means that the communities in different environments will be structured based on the geochemical conditions in which they are in, which is the assumption this project is working under (Baas-Becking, 1934). There are a number of studies which support this idea (Gray et al., 2007) where populations are structured based on the environment they are in however there are also those which prove that contemporary environmental conditions can be overridden by a long historical legacy (Papke et al., 2003; Whitaker et al., 2003) meaning that populations of the same chemistry will be different over distance (Martiny et al., 2006). Due to the extreme environment and mostly young ecosystems Bratina Pond populations will most likely be structured based on the environment in which they reside.
1.2.3. Community Structure
Almost all Antarctic ponds have a dense microbial mat at the bottom of it (Howard-Williams, Hawes, 2006) which consists largely of cyanobacteria. Cyanobacteria are an essential part of the ecosystem as they make up a substantial portion of the benthic mats, planktonic populations and are the major primary producer (Gibson et al 2006). Because of Antarctic ponds wide range of environmental variables it is not surprising that it has been found that each one generally has its own specific composition of planktonic composition. For instance the plankton composition in Algal Lake, Ross Island, is largely flagellated and coloured (Goldman et al, 1972) whereas in Ace Lake, in the Vestfold Hills, there have been two plankton populations found due to the highly depth related oxygen levels down the water column (Bell et al, 1999).
Bacterial community structure in Antarctic lakes (there is no ready information on ponds) is highly related to the time of year as this affects light levels and availability of liquid water. During summer there is light for photosynthesis and liquid water from melted ice and this results in an explosion of life from photosynthetic organisms such as dinoflagellates and Phytoflagellates making them the dominant life in the ponds during this time however these species are outcompeted throughout winter, autum and spring (Bell et al, 1999) by populations that can sustain temporary or permanent heterotrophy (Laybourn-Parry, 2002). These studies indicate that the populations found in Antarctic ponds will be highly diverse and variable due to the unique environment they are in.
With advancements in culture independent molecular ecology, the knowledge base of extreme organisms and more detailed examinations of previously uninvestigated habitats there has been a major change in the perception that Antarctic microbial biodiversity levels are low (Hughes et al , 2006). Cowan and Sjoling 2003 investigated the microbial diversity of pond sediments from Bratina Island using 16S rDNA-dependent molecular phylogeny. They showed that there was a highly diverse bacterial population and a relatively low Archaeal diversity in the sediments. They found that “the sequenced clones fell into seven major lineages of the domain Bacteria; the α, γ, and δ subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides, the Spirochaetaceae, and the Actinobacteria. All of the archaeal clones sequenced belonged to the group Crenarchaeota” (Sjoling, D, Cowan, 03). However Kemp et al 2003 found that biodiversity in sediments can be highly varied so this may not be a constant thing in all Antarctic pond sediments.
1.2.5 Superoxide Dismutase
Superoxide Dismutase is an essential enzyme in aerobic environments (McCord et al, 1971) which is produced by aerobic bacteria (Mikell et al, 1986) to protect them against the free radicals produced as a byproduct of oxygen metabolism (Hassett et al, 1999). Although oxygen gives a higher energy yield (Scandalious, 1993) from its metabolism the free radicals produced are highly reactive and can destroy cellular machinery essential to life (Fridovich, 1995; Touati, 2000). This is a relevant enzyme to many planktonic pond dwelling microbes in Antarctica because of the supersaturated oxygen levels which can be found in the ponds at certain times in the year (Lyons et al, 2006). Oxygen becomes supersaturated due to a range of things firstly it is supplied by meltwater inflow, produced by photosynthesizing organisms in the pond and once it begins to get cold again around autumn an ice cap will form over the pond which will effectively cap its release into the atmosphere (Craig et al, 1991). This causes a buildup in oxygen which can reach such high levels that when experimenters breach the ice cap with a drill a torrent of gas and water can spurt out like in December 1962 (Koob and Leister, 1972).
1.3 Antarctic Molecular Ecology
After decades of research on Antarctica lakes and major advances in molecular techniques there still has not been very much molecular work on the bacteria in them (Laybourn-Parry et al, 2002). When determining an environment's biodiversity you must first be able to isolate the organisms present and identify them either physiologically or genetically (Madigan, Martinko, 2006). The method used for the organisms isolation and identification depends on what you want to know, how much of each sample and how many samples you have, the time to analyse them, (Casamayor et al, 2002) and the funding available to. The method chosen also needs to be simple as each step in the molecular analysis of an environment will be a source of error (Osborn et al, 2000).
In a diversity study you generally want to know how many species are there and the relative ratio of each species (Lui et al, 1997). The introduction of culture-independent molecular techniques in the late 1970's gave a huge leap forward in identifying previously unknown organisms (Bowman et al, 1997) which may not have been found in culturing studies alone (Brambilla et al, 2001). It is hard to evaluate the approaches used today as a best or worst as which one used depends on the environment and desired data, 16S rDNA sequences identify more organisms that culture dependant studies (Brambilla et al, 2001), however culture dependant studies will generally reveal a different set of organisms that independent studies alone. When studies using both techniques have been done there is generally very few genera identified using both sets of techniques (Pearce et al 2003).
1.3.1 Culture dependant
The first man to grow bacteria using culture dependant techniques called Robert Koch who used potato slices as his media. His work was instrumental in the development of microbiology as we know it today (Madigan, Martinko, 2006). However, culture dependant techniques are limited as any particular culture will not grow all of the microbes from the environmental samples optimally (Sjoling, Cowan, 2003) (its believed that at present less than 5% of bacteria in nature can be cultured (Pearce et al 2003)) so the entire diversity will be difficult to determine using these techniques alone (Sjoling, Cowan, 2003). They are good for determining the physiological properties of organisms in the environment but are not as effective as other culture independent techniques on determining the community composition of an organisms significance in situ (Pearce et al 2003). Culture based approaches also have the limitation that any change in the culture conditions from the environment will put new selective pressures on the sample changing its community structure (Lui et al, 1997). This is why many investigators do not entirely rely on culture dependant methods for characterizing microbial community composition (Pearce et al 2003).
1.3.2 Terminal Restriction Fragment Length Polymorphism (TRFLP)
TRFLP is a commonly used and highly effective culture independent technique used to rapidly compare bacterial species (Dunbar et al, 2000). It uses the same principle as the polymerase chain reaction where specific primers are used to amplify a section of DNA from each individual in a sample, the only difference is that one primer is labeled (Egert et al, 2003). The DNA is then digested with a restriction enzyme making each organisms DNA a separate length (Dunbar et al, 2000), this variation in size can then be identified through electrophoresis where the larger pieces move slower through the gel or capillary, and then the labeled fragments are detected as the pass through a laser. When analyzing the profiles generated from different environmental samples they will vary in the number (the more there are the more different species in the samples) and size (representing the proportion of that species in a sample) of peaks present in the profile (Osborn et al, 2000).
TRFLP is a highly useful method of rapidly processing a broad range of environmental samples (Dunbar et al, 2000) which has its own internal standard increasing its reproducibility (Osborn et al, 2000). There are however some limitations: To give the best profile which allows a clear and accurate determination of the species composition ideally the organisms must be genetically very different and the DNA must be in high concentration and of good quality (Dunbar et al, 2000); Since TRFLP works off the same principles as PCR it is liable to the same biases such as the use of universal primers, incomplete cell lysis and amplification of contamination (Casamayor et al, 2002); if the protocol used does not allow the enzyme to completely digest all of the product there will be partially digested products in the sample which will produce more peaks in the profile which if not recognized will be interpreted as more diversity (Osborn et al, 2000); conversely if there are two DNA fragments with the same length but from different organisms the diversity will be underestimated (Casamayor et al, 2002); diversity may also be underestimated if there are organisms in such low numbers that it is not picked up with the analysis (Lui et al, 1997). That being said it has been found that when multiple enzymes are used the bands from the profile are sufficient to estimate the total biodiversity present(Martinez-Murcia et al, 1995) and that the hypervariable partial sequence used will give similar results as when larger sequences are used (Kemp, Aller, 2003).
For this study only genetic based techniques will be used due to the shortcomings of culture dependent techniques (it is believed that at present less than 5% of bacteria in nature can be cultured (Pearce, 2003)). The development on culture-independent molecular techniques over the past few decades have allowed many unculturable organisms to be identified and is often found to be a superior alternative to conventional culture dependent methods (Bowman et al., 1997; Davidov et al., 2006; Hugenholtz et al., 1998; Pearce, 2003). This study will primarily use a number of DNA community level fingerprinting techniques which have already been successfully applied to Antarctic microorganisms such as Automated Ribosomal Intergenic Spacer Analysis (Wood et al., 2008) and terminal restriction fragment length polymorphisms (Dunbar et al., 2001) coupled to the creation of 16S clone libraries and sequence comparison.
(Dunbar et al., 2001). Both ARISA and tRFLP utilise fluorescently labelled PCR primers to amplify variable DNA sequences within the community's respective genomes. When run through a DNA sequencer a ‘fingerprint' is formed which can be used to tentatively assign microbial biodiversity and species abundance. From the detailed fingerprinting analysis representative sites will be selected for clone library construction so that the 16S genes of the communities can be sequenced and their individuals identified using basic local alignment search tool (BLAST). Additionally a survey of the 16s rRNA will provide an assessment of the metabolically active component of the community.
The microbial diversity data will be used for between pool comparisons and within pool comparisons, which will then be linked to the geochemical data and specific environmental drives for each of these processes will be determined. All of the methods necessary for the proposed work are currently available and routinely used in the Cary Lab. Sequenced clones will be aligned using the Clustal W analysis (Thompson et al., 1994) in DNASTAR's MegAlign sequence manipulation program (Lasergene Inc, Madison, WI).
Statistical analysis will be conducted on the ARISA fingerprints using a Multivariate analysis and analysis of similarities (ANOSIM). Linkage between the microbial abundance and geochemical parameters will be subjected to extensive statistical analyses (BEST analysis using the PRIMER 6 software package (PRIMER-E, Ltd., UK)) to resolve which key environmental parameters control community composition and structure and to elucidate which members are adapted to survive this amazing physicochemical shift in their environment. Finally correlation analysis will be performed using Statistica (StatSoft, Tulsa, OK).
1.4 Why there is interest in Antarctic Ponds
There is such interest in Antarctic organisms because they are useful in a multitude of studies which will: give a better understanding of ancient life and evolution (Hawes, Howard-Williams, 2007); help us postulate what life may be like on other planets (Deming, 2002); find previously undiscovered and useful biological products (Nichols et al, 1999); Furthermore many Antarctic microbial communities are particularly sensitive to external impacts such as global warming (Cowan, Ah Tow, 2004) allowing investigators to use them as indicators for global change; further our understanding of the mysteries of life; give a great insight into controlling pathogenic organisms which are responsible for food spoiling even at low temperatures; and allow us to create organisms which can break down man made wastes even at extremely cold temperatures (Chattopadhyay, 2006).
1.4.1 Environmental Impact
The primary goal for environmental impact assessments in most environments is to ensure human welfare is maintained which means that the environment can be damaged as long as it will not impact the humans living in the area. However in Antarctica “the aims should be to maintain the unspoiled environment and fragile ecosystems of the land, inland waters and ice-covered areas of the continent". It is so important to keep a close eye on the Antarctic environment so that we can distinguish what we humans are doing locally to an area there as opposed to the changes occurring globally. (Benninghoff, Bonner, 1985). From this careful separation of facts it has been found that some organisms in Antarctica will be dramatically affected by temperature increases (Hennion et al, 2006) such as is seen by the spreading of mosses onto previously barren areas (Walther et al, 2002) which seems good however these kinds of effects may allow “weeds” to outcompete potentially useful organisms. By monitoring these environmental changes we can increase our understanding of humans environmental impact this environment (NIWA Science, 2007c) before irreversible damage is done (Vincent, 2000).
1.4.2 Uniqueness of the Environment
The Antarctic environment offers one of the most extreme and unique habitats in the world, which is perfect for a range of scientific studies on all aspects of its environment. One very important attribute some of its organisms have is their sensitivity to a range of environmental pressures which means that they can be used to monitor how the ecosystems function and how the climate changes over time (Wynn-Williams, 1996). There are ecosystems which have been sealed from the outside world for thousands of years which have a special evolutionary value as they have been removed from outside influences experienced by the rest of the world such as environmental fluctuations, competition by other organisms and gene flow (Vincent, 2000). Another unique evolutionary feature Antarctica holds is in its cyanobacterial mats which are highly similar to the oldest recognisable Precambrian fossils. It may seem strange to see prehistoric organism analogues in a cold environment however the general conception of early earth now is that it went through extreme ice ages where the entire world was similar to how Antarctica is today (Hawes, Howard-Williams, 2007).
1.4.3 Possible Industrial Applications
One of the main reasons so much research has been done in Antarctica is so microbiologists can search for biotechnologically exploitable organisms (Brambilla et al, 2001). The organisms there and the products they produce can be used in a range of applications making them highly valuable, in fact the use of autofluorescing cyanobacterial primary produces as indicators of climate change has already been evaluated and advocated by Wynn-Williams, 1996. Research has also revealed the existence of poly unsaturated fatty acids (PUFA) producing strains from Antarctica, the traditional sources of PUFA's are fish oils for use in aquaculture feeds and human health supplements (Nichols et al, 1999). Antarctic psychrophiles can produce enzymes that function optimally in the cold (Feller et al, 1996) which can be used commercially for a range of uses (Madigan, Martinko, 2006). These include: cleaning agents, leather processing, degradation of xenobiotic compounds in cold climes, food processing (fermentation, cheese manufacture, bakery, confectionary and meat tenderization), and in molecular biology. There has been some oppourtunistic research done in Antarctica on the degradation of hydrocarbons by Antarctic microorganisms due to several accidental fuel spills left by human activity (Nichols et al, 1999; Delille et al, 1997; Delille et al, 1996) which could assist in the breakdown of oil after other spills throughout the world.
1.5 Aims, approach and outline of the thesis
This study aims to identify the microbial communities present in the water column throughout Antarctic ponds on the edge of Bratina Island while the ponds are stratified during mid-summer and to correlate this data to their geochemical surroundings. This is a unique project which is a continuation of the National Institute of Water & Atmospheric Research's (NIWA) ‘Life in the Cold and Dark' project. The scientists involved in the previous project stayed later in the season than ever before to study how Antarctic organisms tolerate the freeze-up period in the ponds leading into the dark winters. The main goals of this project are to formulate and develop a model of how the microbial communities of the ponds are structured in response to their environment, and how they function within it. Another interesting feature of these ponds is the lack of complex food webs. This means that the dynamics (community structure and physiology) of the microbial populations in ponds of the McMurdo Ice Shelf throughout the summer months should mainly be directly correlated to the extreme chemical and physical structure of the environment. So the question will be what is driving the community composition and structure? Is there a single keystone parameter or are there multiple parameters that structure the communities in such a way that each pond is unique? I have already received funding through Antarctica New Zealand to pay for the required sampling rig, the trip down to Antarctica and other research related costs for this project.
Preliminary data shows that within the ponds on Bratina Island there are highly stratified layers with steep chemical gradients which should drastically effect the microbial composition of the pond populations. This project will focus on the transitions between stratified layers using a detailed study of microbiology coupled with extensive geochemistry in the field and back at the university to see if there is a correlation between population structure and one or a range of geochemistry's. As such the aims of this research are to:
1. Integrate modern molecular genetic approaches (such as ARISA and TRFLP) with geochemistry to study the diversity and ecology of microbial communities in the Bratina melt-water ponds during mid summer and the winter freeze.
2. Examine potential correlations between the geochemistry and the microbiology in the ponds so that the microbial community structure and function can be linked to the intense physicochemical gradients of the ponds.
3. Develop a molecular genetic study of rDNA coupled and compared with rRNA to investigate the metabolically active members of the populations in the meltwater ponds, as dictated by the geochemistry.
This investigation should lead to a better understanding of how planktonic communities are structured by the physiochemical characteristics of the environment. It may also help to create a basic ecosystem model which can be applied to more complex ecosystems and increase the understanding of Antarctic biological processes such as freezing, or desiccation resistance.