Andrew Steele

Questioning the evidence for Earth's oldest fossils.

Structures resembling remarkably preserved bacterial and cyanobacterial microfossils from ∼3,465-million-year-old Apex cherts of the Warrawoona Group in Western Australia currently provide the oldest morphological evidence for life on Earth and have been taken to support an early beginning for oxygen-producing photosynthesis. Eleven species of filamentous prokaryote, distinguished by shape and geometry, have been put forward as meeting the criteria required of authentic Archaean microfossils, and contrast with other microfossils dismissed as either unreliable or unreproducible. These structures are nearly a billion years older than putative cyanobacterial biomarkers, genomic arguments for cyanobacteria, an oxygenic atmosphere and any comparably diverse suite of microfossils. Here we report new research on the type and re-collected material, involving mapping, optical and electron microscopy, digital image analysis, micro-Raman spectroscopy and other geochemical techniques. We reinterpret the purported microfossil-like structure as secondary artefacts formed from amorphous graphite within multiple generations of metalliferous hydrothermal vein chert and volcanic glass. Although there is no support for primary biological morphology, a Fischer–Tropsch-type synthesis of carbon compounds and carbon isotopic fractionation is inferred for one of the oldest known hydrothermal systems on Earth.

The use of atomic force microscopy for studying interactions of bacterial biofilms with surfaces

Biofilms formed in different environments under either field or laboratory conditions on naturally occurring and man-made surfaces have been extensively studied in various stages of their development using a wide range of microscopy techniques. The majority of these methods are, however, qualitative and, with the exception of scanning electron microscopy (SEM), do not provide information on the effect that the biofilm exerts on the underlying substratum. In contrast, atomic force microscopy (AFM) has proven to be a potent tool for characterising, both qualitatively and quantitatively, aspects of biofilm/substratum interactions. This communication provides an overview of the application of AFM for the investigation of bacterial biofilms focusing on specific studies related to metallic surfaces such as stainless steel and copper alloys in freshwater and marine environments.

Polymeric substances and biofilms as biomarkers in terrestrial materials: Implications for extraterrestrial samples

Organic polymeric substances are a fundamental component of microbial biofilms. Microorganisms, especially bacteria, secrete extracellular polymeric substances (EPS) to form slime layers in which they reproduce. In the sedimentary environment, biofilms commonly contain the products of degraded bacteria as well as allochthonous and autochthonous mineral components. They are complex structures which serve as protection for the colonies of microorganisms living in them and also act as nutrient traps. Biofilms are almost ubiquitous wherever there is an interface and moisture (liquid/liquid, liquid/solid, liquid/gas, solid/gas). In sedimentary rocks they are commonly recognized as stromatolites. We also discuss the distinction between bacterial biofilms and prebiotic films. The EPS and cell components of the microbial biofilms contain many cation chelation sites which are implicated in the mineralization of the films. EPS, biofilms, and their related components thus have strong preservation potential in the rock record. Fossilized microbial polymeric substances (FPS) and biofilms appear to retain the same morphological characteristics as the unfossilized material and have been recognized in rock formations dating back to the Early Archaean (3.5 b.y.). We describe FPS and biofilms from hot spring, deep-sea, volcanic lake, and shallow marine/littoral environments ranging up to 3.5 b.y. in age. FPS and biofilms are more commonly observed than fossil bacteria themselves, especially in the older part of the terrestrial record. The widespread distribution of microbial biofilms and their great survival potential makes their fossilized remains a useful biomarker as a proxy for life with obvious application to the search for life in extraterrestrial materials.

Morphologic and spectral investigation of exceptionally well-preserved bacterial biofilms from the Oligocene Enspel formation, Germany

Abstract The fossilised soft tissues of a tadpole and an associated coprolitic structure from the organic-rich volcanoclastic lacustrine Upper Oligocene Enspel sediments (Germany) were investigated using high-resolution imaging techniques and nondestructive in situ surface analysis. Total organic carbon analysis of the coprolite and the sediment revealed values of 28.9 and 8.9% respectively. The soft tissues from the tadpole and the coprolite were found to be composed of 0.5 to 1 μm-sized spheres and rod shapes. These features are interpreted as the fossil remains of bacterial biofilms consisting probably of heterotrophic bacteria and fossilised extracellular polymeric substances. They became fossilised while in the process of degrading the organic matter of the organism and the coprolite. Comparison with a modern marine biofilm revealed morphologic details identical to those observed in the fossil bacterial biofilms. Although the fossil biofilms on both macrofossils exhibited identical microtextures, their mode of preservation was inhomogeneous and varied between calcium phosphate and an as yet unidentified mineral phase consisting mainly of Si, Ca, Ti, P, and S, but also showing the presence of Mg, Al, and Fe. The coprolite consists purely of fossilised bacterial cells in a densely packed arrangement and associated fossilised extracellular polymeric substances. In addition to preliminary imaging and energy-dispersive X-ray analysis, both the fossil biofilms and the sediment were investigated by nondestructive in situ analysis using time of flight-secondary ion mass spectroscopy (ToF-SIMS). The mass spectra obtained on the coprolite in mass-resolved chemical mapping mode allowed the tentative identification of a number of organic secondary ion species. Some spectra appear to indicate the presence of bacterial hopanoids, but further work using standard techniques such as gas chromatography mass spectroscopy is needed to conclusively verify the presence of these substances. Nevertheless, ToF-SIMS chemical maps were successfully correlated with electron microscopy images, allowing the correlation of molecular spectra, the spatial distribution of individual organic species, and specific morphologic features to demonstrate the potential of this approach in the analysis of microfossils.

Life on Mars: evaluation of the evidence within Martian meteorites ALH84001, Nakhla, and Shergotty

Abstract Analyses both support and are in opposition to the hypothesis that the Martian meteorite ALH84001 contains evidence for possible biogenic activity on Mars. New observations in two additional Martian meteorites, Nakhla (1.3 Ga old) and Shergotty (300–165 Ma old) indicate possible biogenic features. Features in the three Martian meteorites compare favorably with the accepted criteria for terrestrial microfossils and evidence for early life on the Earth. There is strong evidence for the presence of indigenous reduced carbon, biogenic magnetite, and the low-temperature formation of carbonate globules. The morphological similarities between terrestrial microfossils, biofilms, and the features found in the three Martian meteorites are intriguing but have not been conclusively proven. Every investigation must recognize the possibility of terrestrial contamination of the meteorites, whether or not the meteorites are Martian. The search for evidence of ancient life in Martian meteorites has emphasized the difficulties confronting the scientific community with the respect to the positive identification of evidence of past biogenic activity.

An atomic force microscopy study of the biodeterioration of stainless steel in the presence of bacterial biofilms

Atomic Force Microscopy (AFM), a technique requiring little or no preparation of biological samples prior to viewing and allowing observation of bacteria and bacterial expolymers in their hydrated forms, has been used to elucidate the phenomena of stainless steel corrosion due to the development of bacterial biofilms. Biofilms formed by pure and mixed cultures of Pseudomonas aeruginosa, the sulphate-reducing bacterium Desulfovibrio gigas and a consortium isolated from a corroding, cast iron pipe carrying potable water were grown for 7 and 14 days in batch cultures at 25°C on surfaces of stainless steel (316) coupons, polished to obtain a 1 μm finish. Surfaces with biofilms present and removed were examined by AFM and by scanning electron microscopy (SEM). The study revealed that the greatest deterioration of steel, in a form of pitting corrosion, occurred in the presence of an isolated pipe consortium. The degree of corrosion observed in mixed cultures of P. aeruginosa and D. gigas was higher than that recorded in pure cultures of these bacteria. The advantage of using AFM as a qualitative method of biocorrosion assessment and its potential for quantitative analysis of microbially influenced corrosion are emphasised.

Time of flight secondary ion mass spectrometry (ToFSIMS) of a number of hopanoids

Abstract Time of flight secondary ion mass spectrometry (ToFSIMS) has been applied to a number of bacterial hopanoids in an attempt to characterise these geologically important molecules in situ by a surface sensitive technique. Our results show that these molecules can be detected using this instrumentation to a high degree of mass accuracy. We believe that ToFSIMS can, therefore, be used to identify these molecules in environmental samples where sample size may be an issue and contraindicate the use of more traditional techniques such as GC–MS.

Speciation of l-DOPA on Nanorutile as a Function of pH and Surface Coverage Using Surface-Enhanced Raman Spectroscopy (SERS)

The adsorption configuration of organic molecules on mineral surfaces is of great interest because it can provide fundamental information for both engineered and natural systems. Here we have conducted surface-enhanced Raman spectroscopy (SERS) measurements to probe the attachment configurations of DOPA on nanorutile particles under different pH and surface coverage conditions. The Raman signal enhancement arises when a charge transfer (CT) complex forms between the nanoparticles and adsorbed DOPA. This Raman signal is exclusively from the surface-bound complexes with great sensitivity to the binding and orientation of the DOPA attached to the TiO(2) surface. Our SERS spectra show peaks that progressively change with pH and surface coverage, indicating changing surface speciation. At low pH and surface coverage, DOPA adsorbs on the surface lying down, with probably three points of attachment, whereas at higher pH and surface coverage DOPA stands up on the surface as a species involving two attachment points via the two phenolic oxygens. Our results demonstrate experimentally the varying proportions of the two surface species as a function of environmental conditions consistent with published surface complexation modeling. This observation opens up the possibility to manipulate organic molecule attachment in engineered systems such as biodetection devices. Furthermore, it provides a perspective on the possible role of mineral surfaces in the chemical evolution of biomolecules on the early Earth. Adsorbed biomolecules on mineral surface in certain configurations may have had an advantage for subsequent condensation reactions, facilitating the formation of peptides.

Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001

A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy‐dispersive X‐ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al. (Search for past life on Mars — possible relic biogenic activity in martian meteorite ALH84001. Science, 1996, pp. 924–930) of NASA who used SEM imaging of gold‐coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.

Effects of sterilizing doses of gamma radiation on Mars analog rocks and minerals

Rock and soil samples from the planet Mars are due to be returned to Earth within a decade. Martian samples initially will be tested for evidence of life and biological hazard under strict biological containment. Wider distribution of samples for organic and inorganic analysis may occur only if neither evidence of life nor hazard is detected, or if the samples are first sterilized. We subjected a range of Mars analog rocks and minerals to high doses of gamma radiation in order to determine the effects of gamma sterilization on the samples isotopic, chemical, and physical properties. Gamma photons from 60 Co (1.17 and 1.33 MeV) in doses as high as 3 x 10 7 rads did not induce radioactivity in the samples and produced no measurable changes in their isotopic and chemical compositions. This level of irradiation also produced no measurable changes in the crystallographic structure of any sample, the surface areas of soil analogs, or the fluid inclusion homogenization temperature of quartz. The only detectable effects of irradiation were dose-dependent changes in the visible and near-infrared spectral region (e.g., discoloration and darkening of quartz and halite and an increase in albedo of carbonates) and increases in the thermoluminescence of quartz and plagioclase. If samples returned from Mars require biological sterilization, gamma irradiation provides a feasible option.