This PhD research project was designed to provide further information about the biological properties of red cells found in the rains that fell for three months from July 2001 during the monsoon season in Kerala, Southern India. The studies involved using a variety of staining methodologies as well as spectroscopic and microscopic analyses. The red rain cells display an exceptionally thick, multilayered cell wall and contain high concentrations of UV absorbing components. The cells often live in clumps forming a bio- film rich in silicon, most of which is readily precipitated in the outer layers of their thick cell wall. These properties may suggest that the cells can survive in multiple extreme environments. DAPI staining method demonstrating the presence of DNA in these cells contradicted earlier work by Louis and Kumar (2006) that the red cells were devoid of DNA. The positive detection of DNA was only possible if the red cells were pre-treated with DMSO prior to DAPI staining. The DMSO treated cells showed no structural damage, but instead released the red compounds. This solvent thus seemed to affect the binding of the red compounds to the outer layers, but not the structural integrity of the cells. Additional data indicated that the red rain cells are possible hyperthermophiles. The data provided in the current study tentatively suggests that the red cells are unusual prokaryotes of hyperthermophilic nature.

To date, the fundamental question of how life originated remains unanswered. The main mystery that has to be solved is how prebiotic chemistry has evolved into primitive biotic chemistry. The primitive record of biological evolution is no longer preserved on Earth due to destructive tectonic processes. However, examples of the organic inventory of the early solar system are available in carbonaceous chondrites. The varied organic content of these meteorites could have provided an exogenous source of organic molecules for the onset of life on the early Earth. My research examines the amino acid contents of carbonaceous and ordinary chondrites analysed with gas chromatography–mass spectrometry (GC-MS). The enantiomeric (D/L) ratios of amino acids were obtained to distinguish materials produced biotically or abiotically as life on Earth is dominated by L-asymmetry. This study reveals that the ordinary chondrite Chainpur contains abundant amino acids. Despite the D/L ratios indicating a certain level of terrestrial contamination, the presence of non-protein amino acid suggests the presence of indigenous materials in the meteorites. Once arisen, life was undoubtedly successful on Earth. But it may have also existed on Mars. This hypothesis will be tested by forthcoming life-detection missions that will carry out in situ analyses on the Red Planet. Fluorescence spectroscopy is a technique that targets biologically significant organic molecules such as amino acids. These molecules are important building blocks of life. The focus of this research is to analyse the fluorescence patterns of a range of standards representing terrestrial and extraterrestrial amino acids and their mixtures, alongside a Martian soil analogue, Salten Skov. This study identifies the optimal excitation and emission wavelengths for amino acids and evaluates the problems associated with photochemical quenching. Spectrofluorometry is a quick and simple analytical technique that offers high sensitivity and immediate identification. Overall, this study aids in understanding the organic reservoir that may have contributed to the origin of life and provides imperative information for successful life-detection missions.

The aim of the thesis is to improve the strategy for exploration of life on Mars and potentially other planetary bodies. This was achieved through the analysis of terrestrial analogues, particularly for targets on the surface of Mars. The search for evidence of life requires optimising several aspects including choice of samples, sample preparation and sample analysis. Analogues selected include snow/ice, evaporites and weathered volcanics. The determination of optimal parameters for extraction of biomarkers was performed on carbon-rich and carbon-poor samples using powdered limestone-shale pairs and on weathered basalt. For all samples, extraction efficiency increased (i) up to about 35°C then did not increase further with temperature; (ii) with smaller grain size; (iii) up to 24 hours extraction time then did not increase further. These data indicate that extraction protocols could be optimised in advance. Dry core drilling experiments showed that the resultant powder obscured visual observation, the range of particle sizes did not allow for the most efficient extraction of organics, and the core powder contained less biomarkers than the core as a result of heating. Extracts of melted Cairngorm snow/ice contained a range of n-alkanols up C18 and extracts of particulate matter contained n-alkanols up to C29. High molecular weight biomarkers are poorly soluble in water, emphasising the importance of optimising the extraction protocol. Samples of snow containing snow algae were successfully analysed for biological pigments using Surface Enhanced Raman Spectroscopy. The development of a bioluminescence assay for the detection of ATP was undertaken in the Cairngorm mountains during a summer and winter season. The detection of ATP was optimised by filtration. An assessment of the potential for site selection, on basalt, from visual parameters, proved that fracture density had the clearest correlation to ATP levels determined by bio-assay. Areas of high fracture density can be detected from Mars orbit, therefore such data could be used to highlight areas most likely to harbour microbes on Mars. The research has shown that terrestrial analogues can yield valuable information on how to optimise different stages of the analysis of a range of rock types that may be expected on Mars.

The aim of the research documented in this thesis was to explore issues associated with the development of instrumentation for life detection and characterisation in a planetary exploration context. Within this aim, the following objectives had to be achieved: 1. To consider current and near-future single molecule detection (ultra-low lower limit of detection) analytical techniques that would be compatible with development into a Space qualifiable in situ analytical instrument for the detection of biomarkers in a planetary exploration context. 2. To practically consider the consequences of Planetary Protection and Contamination Control on the development of a sample return instrumentation in a planetary exploration context. 3. To consider the implications of flying an in situ instrument on-board a stratospheric balloon platform in order to apply them into a specific planetary exploration mission: In order to achieve the objectives described above, the following work was pursued:  A desk-based European Space Agency (ESA) study was carried out which entailed producing a literature review on single molecule detection technologies that had to be validated by the expert community. This was done by organising an International Workshop on Single Molecule Detection Technologies for Space Applications in March 2009 at Cranfield University, UK. The approved technologies then had to be analysed with standard analytical techniques (i.e., tradeoffs) in order to propose a specific technology for development and present its breadboard implementation and test plans at the end of the study.  A sample return experiment implementing PP&CC constraints and protocols was designed, built, tested and flown on-board the ESA, Swedish Space Corporation (SSC), Swedish National Space Board (SNSB) and German Space Agency (DLR) BEXUS stratospheric balloon platform. The biological and engineering results obtained from the sample return flight were then analysed and lessons learnt obtained for future flights.  Another desk-based study was performed to research future stratospheric balloon platforms for the exploration of Venus’ cloud layer. The in situ instrument previously proposed for the detection of biomarkers for planetary exploration missions was then put forward as a possible payload for a Venusian stratospheric balloon platform and approved by experts during the Venus Exploration Analysis Group (VEXAG) conference held in August 2011 in Washington D.C, USA. The first part of the research involved studying ultra-low lower limit of detection technologies as these have the potential to impact significantly on the technological and scientific requirements of future Space missions. Two systems were proposed: one based on Tandem Mass Spectrometry (with Cylindrical Ion Trap analysers) followed by Surface Enhanced Raman Scattering spectroscopy to create an MS/MS-SERS instrument for the detection of astrobiology biomarkers in Martian regolith, Europan ice and samples from Titan’s hydrocarbon lakes; and a second one as a Stand-Alone SERS system for the detection of biomarkers in Enceladean plumes, Venusian clouds and cometary coma. The second part of the research practically explored the design of instrumentation for stratospheric balloon platforms. CASS•E, the Cranfield Astrobiological Stratospheric Sampling Experiment, was a life detection experiment that aimed to be capable of detecting stratospheric microorganisms. The experiment consisted of a pump which drew air from the Stratosphere through a 0.2 μm collection filter which retained any microorganisms and >0.2 μm particulates present in the pumped air. Due to the expected rarity of microbes in the Stratosphere compared to the known levels of contamination at ground level, Planetary Protection and Contamination Control (PP&CC)constraints were introduced. Therefore PP&CC protocols were followed to implement Space qualified cleaning and sterilisation techniques; biobarrier technology was implemented to prevent re-contamination of the instrument after sterilisation; and cleanliness and contamination was monitored throughout assembly, integration and testing. The third part of the research demonstrated how an instrument from the first part of the study could be proposed as a payload on-board a stratospheric balloon platform with a focused mission context, i.e., a life detection mission for Venus. Therefore, the research concluded with the proposal of a payload for a Venus mission based on SERS technology on-board a stratospheric balloon platform to search for life above or in the mid Venusian cloud cover.

The Life Marker Chip (LMC) instrument was developed with the aim to detect evidence of life on Mars. The detection was based on an inhibition immunoassay. In this work aptamers were evaluated as potential alternative to antibodies for the LMC. Aptamers were synthetic oligonucleotides able to bind specifically with high affinity to a wide range of target molecules, and have been also integrated as bioreceptors in several detection instruments. The generation of new aptamers against two small molecules using the FluMag-SELEX method was tested and was verified the adaptability of pre-existing aptamers against small targets to the LMC assay type. Based on the fact that the LMC was going to be integrated into the space programme ExoMars, it was also implemented into a small scale experiment the Planetary Protection and Contamination Control requirements found on a life-search mission. In addition to that aptamers compatibility with a sterilisation procedure used in life-search missions was also tested. Furthermore because of the nature of the small molecules studied, multiple analytical chemistry techniques were assessed to verify covalent chemistry surface immobilisation. Within the project timeline it was not possible to achieve a full aptamer generation process but it was possible to understand the methodology behind the procedure and give input for future work. It was found that the direct implementation of existing aptamers against small molecules into the LMC assay was not successful. It was also seen that in the case of aptamer integration onto the LMC some assay changes would probably have to be made. This information was very useful to understand if aptamers could be an alternative to antibodies and be implemented directly into the LMC. It was found that aptamers survived the preliminary sterilisation method applied, which might open the possibility of making aptamers convenient space bioreceptors, reducing time and costs of instrument Planetary Protection implementation. In conclusion aptamers were not straightforward alternatives to antibodies for the LMC because aptamers interacted differently with their targets in comparison to antibodies, particularly with small molecules. Also the biochemical simplicity of the small molecule targets introduced difficulties in aptamers generation that more complex targets would have not. Although aptamers shown incompatibility with the LMC assay format against small targets, they presented resilience to a sterilisation procedure implemented on space missions which could lead to the development of more robust bioreceptors for space missions. This information was helpful in understanding which assay formats were better for detection of small molecules using aptamers and that might contribute for future assay choices applied in detection instruments. It was also possible to make recommendations for the LMC regarding design and validation methods used in life-search missions based on the lessons learn from the developed of a small scale experiment. The developed work was presented at conferences and mentioned in an article journal, and in that way contributed to the knowledge of the space community in general.