Eric A. Marchand

Development and Application of Small-Subunit rRNA Probes for Assessment of Selected Thiobacillus Species and Members of the Genus Acidiphilium

ABSTRACT Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus andAcidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria:Thiobacillus ferrooxidans and T. thiooxidans(probe Thio820) and members of the genus Acidiphilium(probe Acdp821). Using 32P radiolabels, probe specificity was characterized by hybridization dissociation temperature (Td) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determinedTds. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.

The Role of Enhanced Heterotrophic Bacterial Growth on Iron Oxidation by Acidithiobacillus ferrooxidans

Addition of organic carbon substrate (glucose) profoundly affected the growth of cultures of acidophilic bacteria typical of acid mine drainage (AMD) sites: the iron-oxidizing autotrophic bacteria, Acidithiobacillus ferrooxidans , and a common heterotrophic strain, Acidiphilium acidophilum . Growth of A. ferrooxidans on soluble ferrous iron media was significantly inhibited in the presence of 1,000 mg/L glucose, regardless of the initial cell density, and in spite of favorable pH and aeration conditions. Interestingly, inhibition of A. ferrooxidans was reduced with addition of the heterotroph, apparently due to the consumption of glucose because the onset of iron oxidation coincided with reduction in glucose concentration in the medium. Another mechanism, local production of CO 2 by A. acidophilum provided inorganic carbon required by A. ferrooxidans cells, was investigated. Although no direct proof of interspecies CO 2 exchange was identified, iron oxidation was enhanced and glucose inhibition reduced with incubation of A. ferrooxidans cultures with 5% CO 2 in air. When oxygen was limited and glucose was added to the acidic coculture, the ultimate amount of iron oxidized was significantly lower and the ferric iron produced was subsequently reduced by the heterotrophs as conditions became anoxic. Attribution of ferric iron reduction to A. acidophilum was confirmed in pure culture experiments where a zero-order iron-reduction rate of 458 - 26.9 w mol Fe/L-day (25.6 - 1.5 mg Fe/L-day) was observed. Bacterial iron reduction also led to an increase in pH from 2.5 to 4.0. Thus, the addition of glucose or some other organic electron donor could provide an in situ or ex situ bioremediation strategy to raise pH at AMD sites resulting in a lower amount of metal leaching into drainage water by promoting reducing conditions favorable to iron reduction.

Modelling salinity inhibition effects during biodegradation of perchlorate

Aims:  To determine the mathematical kinetic rates and mechanisms of acclimated perchlorate (ClO)‐reducing microbial cultures by incorporating a term to relate the inhibitory effect of high salinity during biological reduction of concentrated perchlorate solutions.

Physical Simulation for Low-Energy Astrobiology Environmental Scenarios

Speculations about the extent of life of independent origin and the potential for sustaining Earth-based life in subsurface environments on both Europa and Mars are of current and relevant interest. Theoretical modeling based on chemical energetics has demonstrated potential options for viable biochemical metabolism (metabolic pathways) in these types of environments. Also, similar environments on Earth show microbial activity. However, actual physical simulation testing of specific environments is required to confidently determine the interplay of various physical and chemical parameters on the viability of relevant metabolic pathways. This testing is required to determine the potential to sustain life in these environments on a specific scenario by scenario basis. This study examines the justification, design, and fabrication of, as well as the culture selection and screening for, a psychrophilic/halophilic/anaerobic digester. This digester is specifically designed to conform to physical testing needs of research relating to potential extent physical environments on Europa and other planetary bodies in the Solar System. The study is a long-term effort and is currently in an early phase, with only screening-level data at this time. Full study results will likely take an additional 2 years. However, researchers in electromagnetic biosignature and in situ instrument development should be aware of the study at this time, as they are invited to participate in planning for future applications of the digester facility.

Probing microplatform for the study of biological adhesion forces

A tool for the study of biological adhesion forces with the atomic force microscope (AFM) is introduced. The tool, a “microplatform,” can be functionalized with variety of specimens such as bacterial cells and used to study adhesion between the specimen and a surface. This tool is easily created using commercially available silicon AFM tips and an AFM, and can be customized in size to fit specific applications. Two custom fabricated microplatforms, ∼1 and ∼2.5 μm were tested. The method of microplatform fabrication, as well as adhesion force data between E. coli bacteria and a nanofiltration membrane is presented.

Engineering Methods for Europan Relevant Biosignature Development

One of the primary driving forces for space exploration in the foreseeable future is astrobiology, and specifically the search for a plausible sign of life beyond Earth. Because of the size of the potential saltwater ocean involved, Europa is potentially the most interesting, and possibly the only, currently viable (for life) environment in the solar system. It also presents the possibility of remote sensing evaluation for presence or absence of biotic and/or pre-biotic organic material. The material of interest is the non-ice (referring to water ice) surface material near features that have the potential of being in recent communication with the postulated ocean below. An analysis of this material using a full range of inorganic, pre-biotic organic, and metabolically relevant biologic materials as spectrum calibrating target materials, examined under Europan surface conditions, is a daunting proposition. A comprehensive attempt is still pending. This study involves the collection and growth of psychrophilic (low temperature), halophilic (high salt), anaerobic cultures in high sulfate environments. These cultures are intended as reflected spectrum target materials, based on relevant biomass, for comparison to Europan non-ice surface materials. The selection, collection, development (growth) and examination of this biotic material (blomass) requires the use of stringent, and in some cases extreme, environmental controls. It also requires the extrapolation of standard environmental engineering sampling and laboratory analysis procedures for use in comparison with, and interpretation of, data from a profoundly extreme and unfamiliar environment. The function of this study is to demonstrate the use of environmental engineering techniques and processes (test methodology) necessary to develope a reasonable biosignature related to the Europan target environment.