University of Houston • University of Houston-Clear Lake • ISSO Annual Report Y2005 • 116-118,126
Low-Frequency Dielectric Spectroscopy of Martian Soil Samples
Abstract--Investigation concentrates on Martian soil simulants and live cell suspensions using low-frequency dielectric spectroscopy and related techniques, such as nonlinear harmonic response. Such methods hold tremendous potential in the development of sensors that could test for subsurface microbial life on Mars with numerous additional applications. In previous work, UH and NASA-JSC researchers measured the low-frequency dielectric properties of soil samples known to be Mars analogues, as well as known live cell suspensions. In this initial study, they tested common soil and JSC Mars-1, a sample of volcanic ash from Hawaii developed for use as a Mars regolith simulant. Biologically active, JSC Mars-1 contains microorganisms and biomolecules equivalent to 106 -107 cells/gram, less than common soils. More recently, researchers have focused on nonlinear harmonic response as a method of revealing potential signatures of live organisms. Recent investigations include studies of whole cells as well as extracted organelles, such as mitochondria and chloroplasts.
The possible existence of life on Mars, either in the past or at present,1 has profound scientific implications for the evolution of life on Earth and the distribution of life in the cosmos. The Viking Program, which made the first serious attempt to detect the presence of living or fossilized organisms in Martian soil, yielded ambiguous results.2 More recent studies3 of Martian meteorite Allan Hills 84001 (ALH84001) suggest that microbial life existed on Mars about four billion years ago. Compelling evidence includes the existence of magnetite (Fe3O4) crystals found within carbonate globules and their associated rims in the meteorite.4 About one fourth of these tens-of-nanometer-sized magnetites are nearly identical to those produced by magnetotactic bacteria on Earth and are not known or expected to be produced by abiotic means. It has, therefore, been argued that these Martian magnetite crystals are, in fact, magnetofossils, which, if true, would constitute evidence of the oldest life forms known.5
Further evidence suggests that subsurface Martian life could potentially survive even today.6 There is abundant geological evidence that ice was once deposited in the regolith, where it should still be present above mid-latitudes.7 This ice, which probably extends several kilometers below the surface, could be a source of liquid water near magmatic intrusions. On Earth, the biomass of subterranean organisms may equal or exceed that at the surface.9 These organisms can live in highly saline conditions at temperatures from 115°C to -20°C.10,11 Such conditions might prevail beneath the surface in an aquifer or hydrothermal system. For these and other reasons, there is considerable interest in developing new techniques for detecting subsurface life on Mars. Moreover, the likelihood that oceans of liquid water exist below the icy surfaces of Europa and other moons make these exciting candidates for the existence of extraterrestrial life in our solar system. Therefore, there is a need to develop technologies that could lead to portable devices useful in robotic missions or for use by astronauts for the detection of extant life forms.
Goals of the Project
The goal of this project is to study possible techniques for the detection and
characterization of live organisms, including dielectric spectroscopy12,13 and
related methods, such as nonlinear harmonic response.14 One objective is to
detect possible signatures unique to live organisms that could be used to search for life
elsewhere in the solar system. A challenge for astrobiological investigation of Mars and
other extraterrestrial bodies is to develop in situ instruments capable of
distinguishing environmental samples or extracts containing life forms from those that do
not. At the same time, the life-detection technology must not be geocentric; that is, it
must not be targeted to characteristics that, although specific to life, may be limited to
those life forms native to Earth. We are thus investigating dielectric spectroscopy (DS)
as a life detection tool because life throughout the Cosmos, regardless of its
biochemistry and the nature of its genetic material, must utilize a variety of complex,
charged macromolecules.
Results
Our Y2004 report discussed measurements of complex dielectric constant, which represents
the linear response to an applied ac electric field. In those experiments, we employed a a
liquid capacitor cell coupled to a Solartron Analytical Model 1260 Impedance Analyzer.
This setup enabled us to obtain the complex dielectric response, with both the real and
imaginary parts, where the imaginary part of the dielectric response is proportional to
the conductivity divided by the frequency. We tested common soil and JSC Mars-1, volcanic
ash from Hawaii, developed for use as a Mars regolith simulant.15 Biologically
active, JSC Mars-1 contains microorganisms and biomolecules equivalent to 106-107
cells/gram, less than common soils (which can contain quantities of up to 109
cells/gram). Portions of each environmental sample were left untreated, while other
portions were sterilized: autoclaved for 60 minutes at 121°C, at 2 atm, then heated in an
oven at 220° for 3 hours followed by exposure to ultraviolet light for 16 hours. Water
extractions were then performed on sterilized and untreated soil and JSC Mars-1 samples.
Extracts of untreated soil and JSC Mars-1 yielded multiple microbial strains when
incubated on Luria-Bertani (LB) agar for 24 hours at three temperatures: 23°C, 30°C, and
37°C. Extracts of sterilized soil and JSC Mars-1 showed no growth at any of these
temperatures, indicating that the sterilization protocol had indeed destroyed all living
forms within the environmental samples.
Dielectric spectroscopy conducted on extracts showed that the dielectric constant and conductivity were higher for sterilized samples compared with untreated samples. We hypothesized that the sterilization protocol results in increased dielectric constant and conductivity due to lysis of cells and consequent release of charged molecules. Samples containing living cells may thus be distinguishable from those containing only macromolecules by performing dielectric spectroscopy at variable temperatures. We therefore performed measurements at two temperatures, 4°C and 37°C, where we tested a suspension of the bacterium, E. coli (2.5 × 109 cells/ml), as well as three examples of large, charged biomolecules: deoxyribonucleic acid (DNA), hemoglobin (Hb), and bovine serum albumin (BSA). At 10 Hz, dielectric constant for E. coli suspension increased by 70% at 37° as compared to 4°, while dielectric constants for DNA, Hb, and BSA increased by 28%, 17%, and 49% respectively. However, the DNA, Hb, and BSA used in this preliminary study were then found to be contaminated with microorganisms, and as in the case of E. coli, the effects of temperature on life may be the reason the dielectric differences measured for these compounds. Therefore, we used fetal bovine serum (FBS) (containing proteins, cell wall lipids, and other compounds), specially treated to eliminate all known or suspected life forms, including the controversial entities known as nanobacteria.17,18 Results showed that at 10 Hz the dielectric constant for sterile FBS increases by only approximately 6.5% for the FBS at 37°C vs. 4°C.
More recently, we have been investigating nonlinear harmonic response to probe signals produced by active enzyme complexes unique to live organisms. Experimental setups employ a Stanford Research SR 780 Vector Signal Analyzer. A superconducting quantum interference device (SQUID) is used to directly probe the magnetic fields produced by the induced currents at frequencies below 100 Hz, while a four-electrode setup is used at kilohertz frequencies. (See p. 33.) For example, we have discovered resonant-like peaks in the frequency-dependent harmonic responses of live yeast cell suspensions, as shown in Fig. 1.
Figure 1. Induced 3rd harmonic amplitude vs. applied fundamental frequency (5-V applied amplitude) for three different concentrations of S. cerevisiae, (budding yeast). Note that two peaks, centered around 5 kHz and 12 kHz, appear to "grow" out of the background as the cell concentration is increased. These results on whole cells have led us to investigate extracted organelles, including mitochondria and chloroplasts. (See pp. 76-79.)
Preliminary evidence suggested that this behavior may result from active molecular motor complexes unique to live organisms, thus providing another tool for detecting life forms and for fundamental research in biophysics. Figure 1 shows the magnitudes of the induced harmonics (across the inner two electrodes) vs. applied fundamental frequency, for an applied voltage amplitude of 5 V across the outer two electrodes. Note that two peaks, centered around 5 kHz and 12 kHz, appear to "grow" out of the background as the cell concentration is increased. In addition, we find that potassium cyanide suppresses the observed peaks. Potassium cyanide (KCN) is a known respiratory inhibitor that binds to the cytochrome c oxidase complex. This enzyme is the fourth complex of the electron transport chain, which pumps protons (H+ ions) across the mitochondrial inner membrane against the concentration gradient. As it does so, it oxidizes the electron carrier cytochrome c, and is responsible for 90 percent of the oxygen consumption by all living organisms on the planet. Importantly, the yeast cells are not necessarily killed by cyanide, and they remain capable of fermentation.
These results led us to embark on a study of extracted organelles, particularly mitochondria and chloroplasts. Several highlights of this study are discussed on pp. 33-38.
In addition, using SQUIDs for low-frequency measurements, we continued studies of whole cells in which we investigated harmonic response signals that appear to be generated by active membrane pumps in the plasma membrane. For example, we found that the addition of glucose dramatically affects the observed induced harmonics at low frequencies, as shown in Fig. 2, which provides an additional tool for studying behavior unique to live organisms.
Figure 2. Time dependent 2nd (top) and 3rd (bottom) harmonic responses of a yeast cell suspension (108 cells/ml) after adding 0.1 M of D-glucose. The glucose was added to the resting cell suspension a few minutes before the measurements (Time = 0 minutes). H+-ATPase and other transporters apparently increase activity in the presence of glucose, as reflected by an increase in the 2nd harmonic response, and decreased (by about 60%) in the 3rd harmonic response. After about 25-30 minutes, the 2nd harmonic drops significantly while the 3rd harmonic increases back to its original value as the glucose concentration becomes depleted. The frequency and amplitude of the applied signal was set to 45 Hz and 3 V/cm, respectively. The horizontal lines show the original 2nd and 3rd harmonic responses without any added glucose.
In conclusion, our results indicate that variable-temperature dielectric spectroscopy and nonlinear harmonic response measurements may be useful for astrobiology studies and for numerous terrestrial applications as well.
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Presentations
Miller, J., D. Nawarathna, D. Warmflash, F. Pereira, and W. Brownell. "Dielectric
Properties of Live Yeast Cells Expressed with the Motor Protein Prestin," Bull.
Am. Phys. Soc. 50 (2005): 1340; March Meeting of the American Physical Society, Los
Angeles, CA, March 21-25, 2005.
Institute for Space Systems Operations - Y2005 Annual Report
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