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In many natural environments, organisms get exposed to low temperature and/or to strong temperature shifts. Also, standard preservation protocols for live cells or tissues involve ultradeep freezing in or above liquid nitrogen (-196°C or -150°C, respectively). To which extent these conditions cause cold- or cryostress has rarely been investigated systematically. Using ATP content as an indicator of the physiological state of cells, we found that representatives of bacteria, fungi, algae, plant tissue, as well as plant and human cell lines exhibited similar responses during freezing and thawing. Compared to optimum growth conditions, the cellular ATP content of most model organisms decreased significantly upon treatment with cryoprotectant and cooling to up to -196°C. After thawing and a longer period of regeneration, the initial ATP content was restored or even exceeded the initial ATP levels. To assess the implications of cellular ATP concentration for the physiology of cryostress, cell viability was determined in parallel using independent approaches. A significantly positive correlation of ATP content and viability was detected only in the cryosensitive algae Chlamydomonas reinhardtii SAG 11-32b and Chlorella variabilis NC64A, and in plant cell lines of Solanum tuberosum. When comparing mesophilic with psychrophilic bacteria of the same genera, and cryosensitive with cryotolerant algae, ATP levels of actively growing cells were generally higher in the psychrophilic and cryotolerant representatives. During exposure to ultralow temperatures, however, psychrophilic and cryotolerant species showed a decline in ATP content similar to their mesophilic or cryosensitive counterparts. Nevertheless, psychrophilic and cryotolerant species attained better culturability after freezing. Cellular ATP concentrations and viability measurements thus monitor different features of live cells during their exposure to ultralow temperatures and cryostress.

Antarctic glacier forfields are extreme environments and pioneer sites for ecological succession. The Antarctic continent shows microbial community development as a natural laboratory because of its special environment, geographic isolation and little anthropogenic influence. Increasing temperatures due to global warming lead to enhanced deglaciation processes in cold-affected habitats and new terrain is becoming exposed to soil formation and accessible for microbial colonisation. This study aims to understand the structure and development of glacier forefield bacterial communities, especially how soil parameters impact the microorganisms and how those are adapted to the extreme conditions of the habitat. To this effect, a combination of cultivation experiments, molecular, geophysical and geochemical analysis was applied to examine two glacier forfields of the Larsemann Hills, East Antarctica. Culture-independent molecular tools such as terminal restriction length polymorphism (T-RFLP), clone libraries and quantitative real-time PCR (qPCR) were used to determine bacterial diversity and distribution. Cultivation of yet unknown species was carried out to get insights in the physiology and adaptation of the microorganisms. Adaptation strategies of the microorganisms were studied by determining changes of the cell membrane phospholipid fatty acid (PLFA) inventory of an isolated bacterium in response to temperature and pH fluctuations and by measuring enzyme activity at low temperature in environmental soil samples. The two studied glacier forefields are extreme habitats characterised by low temperatures, low water availability and small oligotrophic nutrient pools and represent sites of different bacterial succession in relation to soil parameters. The investigated sites showed microbial succession at an early step of soil formation near the ice tongue in comparison to closely located but rather older and more developed soil from the forefield. At the early step the succession is influenced by a deglaciation-dependent areal shift of soil parameters followed by a variable and prevalently depth-related distribution of the soil parameters that is driven by the extreme Antarctic conditions. The dominant taxa in the glacier forefields are Actinobacteria, Acidobacteria, Proteobacteria, Bacteroidetes, Cyanobacteria and Chloroflexi. The connection of soil characteristics with bacterial community structure showed that soil parameter and soil formation along the glacier forefield influence the distribution of certain phyla. In the early step of succession the relative undifferentiated bacterial diversity reflects the undifferentiated soil development and has a high potential to shift according to past and present environmental conditions. With progressing development environmental constraints such as water or carbon limitation have a greater influence. Adapting the culturing conditions to the cold and oligotrophic environment, the number of culturable heterotrophic bacteria reached up to 108 colony forming units per gram soil and 148 isolates were obtained. Two new psychrotolerant bacteria, Herbaspirillum psychrotolerans PB1T and Chryseobacterium frigidisoli PB4T, were characterised in detail and described as novel species in the family of Oxalobacteraceae and Flavobacteriaceae, respectively. The isolates are able to grow at low temperatures tolerating temperature fluctuations and they are not specialised to a certain substrate, therefore they are well-adapted to the cold and oligotrophic environment. The adaptation strategies of the microorganisms were analysed in environmental samples and cultures focussing on extracellular enzyme activity at low temperature and PLFA analyses. Extracellular phosphatases (pH 11 and pH 6.5), β-glucosidase, invertase and urease activity were detected in the glacier forefield soils at low temperature (14°C) catalysing the conversion of various compounds providing necessary substrates and may further play a role in the soil formation and total carbon turnover of the habitat. The PLFA analysis of the newly isolated species C. frigidisoli showed that the cold-adapted strain develops different strategies to maintain the cell membrane function under changing environmental conditions by altering the PLFA inventory at different temperatures and pH values. A newly discovered fatty acid, which was not found in any other microorganism so far, significantly increased at decreasing temperature and low pH and thus plays an important role in the adaption of C. frigidisoli. This work gives insights into the diversity, distribution and adaptation mechanisms of microbial communities in oligotrophic cold-affected soils and shows that Antarctic glacier forefields are suitable model systems to study bacterial colonisation in connection to soil formation. Gletschervorfelder der Antarktis stellen extreme Habitate dar und sind Pionierstandorte biologischer Sukzession. Insbesondere unter Berücksichtigung zuletzt beobachteter und vorausgesagter Erwärmungstrends in der Antarktis und der Relevanz der Mikroorganismen für das Antarktische Ökosystem, ist es essentiell mehr Informationen über die Entwicklung frisch exponierter Gletschervorfelder zu erlangen. Ziel dieser Studie ist es, die Struktur und Entwicklung bakterieller Gletschervorfeldgemeinschaften zu verstehen, insbesondere wie die Mikroorganismen von den Bodenparametern beeinflusst werden und wie diese sich an die extremen Bedingungen des Habitats anpassen. Für die Untersuchung der Proben von zwei Gletschervorfeldern aus den Larsemann Bergen der Ostantarktis diente eine Kombination aus Kultivierungsexperimenten und molekularen, geophysikalischen und geochemischen Analysen. Die untersuchten Gletschervorfelder sind durch extrem niedrige Temperaturen, einer geringen biologischen Wasserverfügbarkeit und oligotrophe Nährstoffgehalte charakterisiert und zeigen unterschiedliche Entwicklungsstufen in Verbindung zu den Bodenparametern. In einem frühen Schritt der Bodenbildung in der Nähe der Gletscherzunge sind die Gemeinschaften undifferenziert, doch mit fortschreitender Entwicklung nimmt de Einfluss von Wasser- und Nährstofflimitationen zu. Nachdem die Kultivierungsbedingungen den kalten und nährstoffarmen Bedingungen des Habitats angepasst wurden, konnten 108 koloniebildende Einheiten heterotropher Bakterien pro Gramm Boden angereichert und daraus 148 Isolate gewonnen werden. Zwei neue psychrotolerante Bakterien, Herbaspirillum psychrotolerans PB1T und Chryseobacterium frigidisoli PB4T, wurden detailiert charakterisiert und als jeweils neue Spezies beschrieben. Die Anpassungsstrategien der Mikroorganismen an die extremen antarktischen Bedingungen zeigten sich in der Aktivität extrazellulärer Enzyme bei niedriger Temperatur, die mit derer temperierter Habitate vergleichbar ist, und in der Fähigkeit der Mikroorganismen, die Fettsäurezusammensetzung der Zellmembran zu ändern. Eine neue Fettsäure, die bisher in keinen anderen Mikroorganismus gefunden wurde, spielt eine entscheidende Rolle in der Anpassung des neu-beschriebenen Bakteriums C. frigidisoli an niedrige Temperaturen und saure pH-Werte. Diese Arbeit gibt einen Einblick in die Vielfalt, Verteilung und Anpassung mikrobieller Gemeinschaften in nährstoffarmen und Kälte-beeinflussten Habitaten und zeigt, dass Antarktische Gletschervorfelder geeignete Modellsysteme, um bakterielle Besiedelung in Verbindung zu Bodenbildung zu untersuchen.