David D. Wynn-Williams

Ecological Aspects of Antarctic Microbiology

If the science of microbiology is approaching maturity, then Antarctic microbiology is only just emerging from its infancy. The early expeditions of the 20th century used classical medical methodology to isolate and identify bacteria, yeasts, and fungi from sea water, soil, snow, air, and animals (Ekelof, 1908; Tsiklinsky, 1908; Gazert, 1912; McLean, 1918, 1919). The initial emphasis was on survey and taxonomy, although Gazert (1912) noted the influence of marine bacteria on nutrient cycling during the German Antarctic Expedition of 1901–03. However, it is Ekelof of the Swedish National Antarctic Expedition 1901–03 who may be regarded as the father of Antarctic microbial ecology. Between February 1902 and November 1903, he made a seasonal study of the soil and air microbiota at Snow Hill Island (64° 30′S) off the east coast of the Antarctic Peninsula (Fig. 1). Using rich medical media, he monitored viable bacteria, yeasts, and other microfungi but made no mention of the organisms resembling cyanobacteria and microalgae which are frequently the dominant primary producers in terrestrial Antarctic ecosystems (Ekelof, 1908).

Ambient air levels and atmospheric long-range transport of persistent organochlorines to Signy Island, Antarctica

Levels of persistent organic pollutants (POPs), such as polychlorinated biphenyls and pesticides have been determined in ambient air at Signy Island, Antarctica, over a period of 17 weeks. Mean concentrations for single polychlorinated biphenyls (0.02–17 pg/m3), for chlordanes (0.04–0.9 pg/m3), DDT compounds (0.07–0.40 pg/m3) and γ-hexachlorocyclohexane (HCH, 22 pg/m3) were comparable to those in Arctic air. However, α-HCH levels were approximately one order of magnitude lower. Compared to the Arctic, differences were also observed in the concentration ratios of α-/γ-HCH and chlordane compounds. Two possible atmospheric long-range transport episodes from South America were found by comparing 10-day back trajectories with observed concentration changes. The lower limits of determination (LOD) were mainly governed by the field blanks. They were satisfactory for the most volatile PCBs. However, many concentrations for DDT and chlordane compounds were below the LODs (range 0.1–1 pg/m3) or even the instrumental detection limit (0.01–0.03 pg/m3).

Pigmentation as a survival strategy for ancient and modern photosynthetic microbes under high ultraviolet stress on planetary surfaces

Solar radiation is the primary energy source for surface planetary life, so that pigments are fundamental components of any surface-dwelling organism. They may therefore have evolved in some form on Mars as they did on Earth. Photosynthetic microbes are major primary producers on Earth, but are concurrently vulnerable to ultraviolet (UV) damage. Using non-intrusive laser Raman spectroscopy to recognize the component parts of biomolecules, we have shown not only the abundance of microbial photosynthetic and photoprotective pigments in situ, but also their spatial distribution within their microhabitat. This essential aspect of their screening or avoidance survival strategies is lost on extraction with solvents. This precise approach is eminently suited to analysis of epilithic (surface) and endolithic (within rocks) communities in Antarctic desert habitats, which are putative analogues of early Mars. Raman spectra for key biomolecules (e.g. the UV screen parietin and the antioxidant b-carotene in epilithic lichens) enable not only the detection of organics in light-stratified habitats, but also the characterization of unknown pigments. Typical biomarkers of astrobiological relevance in our Raman spectral database include scytonemin (a UV screen), chlorophyll (primary photosynthetic pigment), phycocyanin (accessory pigment for shade adaptation) and a hopanoid extracted from 2-5 Gya microbial stromatolite from Australia. This compound dates from the same time period when a wetter Mars could have had a potentially flourishing surface microbial community of its own. Analyses with a laboratory Raman instrument have been extended to a novel miniature Raman spectrometer, operating at the same optimal excitation wavelength (1064 nm) via an In-Ga-As detector. After evaluation in Antarctica, this instrument will be space-qualified for a proposed Mars rover mission to detect biomolecules in the near- surface sediment profile of palaeolakes, using experience with Antarctic biomarkers to interpret alien spectra of fundamental components, without the need for prior knowledge of the identity of the target compounds.

A novel miniature confocal microscope/Raman spectrometer system for biomolecular analysis on future Mars missions after Antarctic trials

Biomolecules, such as the productive and protective pigments of photosynthetic organisms, are good biomarkers in extreme Antarctic deserts as analogues of early Mars. Laser Raman technology at long wavelengths which minimize fluorescence is ideal for remote analysis of biomolecules in situ. We report Raman spectra obtained with a prototype miniature laser-Raman spectrometer/confocal microscope (specification < 1 kg) under development for a Mars lander and evaluation in Antarctic deserts. We compare the efficiency of its 852 nm laser/CCD detector system with an optimal bench-top 1064 nm FT Raman spectrometer which excels with biomolecules. Using a yellow Antarctic lichen, Acarospora chlorophana, we show good correlation between both instruments restricted to the 460–1350 cm−1 wavenumber range.

Why Raman spectroscopy on Mars?--a case of the right tool for the right job.

We provide a scientific rationale for the astrobiological investigation of Mars. We suggest that, given practical constraints, the most promising locations for the search for former life on Mars are palaeolake craters and the evaporite deposits that may reside within them. We suggest that Raman spectroscopy offers a promising tool for the detection of evidence of former (or extant) biota on Mars. In particular, we highlight the detection of hopanoids as long-lived bacterial cell wall products and photosynthetic pigments as the most promising targets. We further suggest that Raman spectroscopy as a fibre optic-based instrument lends itself to flexible planetary deployment.

Functional biomolecules of Antarctic stromatolitic and endolithic cyanobacterial communities

For activity and survival in extreme terrestrial Antarctic habitats, lithobiontic cyanobacteria depend on key biomolecules for protection against environmental stress and for optimization of growth conditions. Their ability to synthesize such molecules is central to their pioneering characteristics and major role as primary producers in Antarctic desert habitats. Pigmentation is especially important in protecting them against enhanced UVB damage during stratospheric ozone depletion (the Ozone Hole) during the Antarctic spring and subsequent photoinhibition in the intense insolation of the summer. To be effective, especially for the screening of highly shade-adapted photosystems of cyanobacteria, protective pigments need to be located strategically. Antarctic lithic cyanobacterial communities are therefore stratified, as in soil biofilms of Alexander Island, the benthic stromatolitic mats of ice-covered hypersaline lakes in the McMurdo Dry Valleys, and the endolithic communities within translucent Beacon s...

FT-Raman spectroscopic analysis of endolithic microbial communities from Beacon sandstone in Victoria Land, Antarctica

Laser-based Fourier-Transform Raman spectroscopy (FTRS) has been used to identify in situ compounds of ecophysiological significance in diverse field-fresh Antarctic cryptoendolithic microbial communities. FTRS does not disrupt the community and permits characterization of visible and invisible compounds in their natural configuration within cells and their current or former microhabitat. The small “footprint” of the microscopic laser beam permits accurate analysis of discrete zones of compounds produced by extant or degraded micro-organisms with minimum destruction of the biota. This spatial chemical analysis is applicable to any translucent or exposed habitat or biotic assemblage. Two hydrated forms of biodegradative calcium oxalate were differentiated in black-pigmented and hyaline lichen zones of endolithic communities. The oxalate was restricted to zones containing fungi. Communities dominated by cyanobacteria at Battleship Promontory (77°S) and a newly discovered site at Timber Peak (74°S) contrasted chemically with those dominated by eukaryotic algae at East Beacon (78°S). FTRS also showed the zonation of pigments including chlorophyll and UV-protective carotenoids in situ. At extreme sites on the polar plateau, it revealed the presence of “fossil” endolithics where detrimental climatic changes had made the microbes non-viable or amorphous, being represented solely by their residual bio-molecules. The technique has potential for past or present life-detection anywhere in the world without destruction of the microniche

Vibrational Raman spectroscopic study of scytonemin, the UV-protective cyanobacterial pigment

The Raman spectrum of the photoprotective pigment scytonemin found in cyanobacterial sheaths has been obtained for the first time. Its skeletal structure is extensively conjugated and unique in nature. Detailed molecular vibrational assignments are proposed and a distinctive group of four corroborative vibrational bands have been identified as unique indicators for the compound. These bands, especially a prominent feature at wavenumber 1590 cm(-1), are sufficiently conspicuous to be detectable in the mixed biomolecular pools of undisturbed natural microbial communities. This has been confirmed by demonstrating the Raman spectral bands for scytonemin in a sample of an intact intertidal cyanobacterial mat.

Simulation of seasonal changes in microbial activity of maritime antarctic peat

Abstract Seasonal variations in temperature and moisture in moss peat were monitored in the field at Signy Island, Antarctica. When simulated in intact peat cores in vitro after frozen storage, these variations caused changes on O 2 -uptake which closely reproduced the results for fresh samples. Respiration rate was used as a measure of aerobic decomposer activity. Supplements of sugars indicated the predominance of microbial respiration and its dependence on the availability of dissolved organic C (DOC). Low temperatures of 0° to 1°C were not rate-limiting for respiration in vivo or in vitro , and O 2 -uptake was detected at −1°C. Repeated peaks of O 2 -uptake under wet conditions resulting from simulated spring freeze-thaw cycles, and a solitary peak during an autumn simulation, suggested release of DOC substrates from frost-damaged cells. Desiccation, microfaunal predation and microaerophily were thought to contribute to respiratory declines. O 2 -uptake and CO 2 -evolution were equivalent in peat beneath Polytrichum sampled in autumn. Peat respiration was not generally proportional to microbial biomass, but saccharolytic yeasts were dominant during the respiratory maximum in spring and correlated with O 2 -uptake in a mixed culture of indigenous microflora. Yeasts grew exponentially in freezethaw cycle simulations but percolated into the peat profile in the field. The basal O 2 -uptake, which may be attributable to the decomposition of redalcitrant molecules such as cellulose, was lower in simulations of spring than autumn. Although bacterial biomass increased and diversified during summer, the ratio of fungal-to-bacterial contributions to O 2 -uptake in an incubated homogenate of peat sampled in autumn was 4:1.

Molecular structural studies of lichen substances II: atranorin, gyrophoric acid, fumarprotocetraric acid, rhizocarpic acid, calycin, pulvinic dilactone and usnic acid

The FT-Raman and infrared vibrational spectra of some important lichen compounds from two metabolic pathways are characterised. Key biomolecular marker bands have been suggested for the spectroscopic identification of atranorin, gyrophoric acid, fumarprotocetraric acid rhizocarpic acid, calycin, pulvinic dilactone and usnic acid. A spectroscopic protocol has been defined for the detection of these molecules in organisms subjected to environmental stresses such as UV-radiation exposure, desiccation and low temperatures. Use of the protocol will be made for the assessment of survival strategies used by stress-tolerant lichens in Antarctic cold deserts.