5

Microbial Detection and Identification

A key aspect of planetary protection is the determination of the bioload on the spacecraft prior to launch. From the Viking era to the present day, the bioload has been monitored by a routine and ongoing procedure that continues until shortly before launch. The monitoring entails swabbing accessible external and internal surfaces of the spacecraft with cotton and then determining the number of culturable bacteria from a known surface area. The standard procedure is to transfer the cells from a swabbed surface to a liquid medium, which is then heat shocked at 80 °C for 8 minutes. The surviving cells are then cultured to determine the number of colony-forming units (cfu). A cfu is defined as a colony on a culture plate (using a standard growth medium) that develops from a cell that survived heat shock.

Although the techniques employed in NASA’s planetary protection protocols have remained virtually unchanged for the last 25 years, the methods now available for bioload characterization have changed dramatically thanks to advances in biotechnology. It is now possible to survey with good confidence the microbial diversity on a spacecraft or within its assembly area using explicit molecular criteria. A molecular approach circumvents many of the problems associated with culture-based characterization (e.g., delays caused by the time needed to grow the microbial colonies or by the inability to successfully culture most microbes).

The most established methods are based on selective recovery and sequencing of genes encoding the ribosomal ribonucleic acids (RNA). 1 In addition to new technologies such as high-density deoxyribonucleic acid (DNA) hybridization arrays,2 refinements of existing molecular methods (e.g., the polymerase chain reaction (PCR)) and analytical methods (e.g., mass spectrometry and immunochemistry) provide improved detection capability using a variety of diagnostic cellular biopolymers, including proteins, lipids, and carbohydrates. A variety of advanced detection methods are now in common use.3,4 Improvements in the sensitivity and specificity of microbial detection should be incorporated into new assessment standards.

It is important to bear in mind that most DNA-based techniques do not necessarily distinguish between living and dead materials. The preservation of DNA in some noncellular contexts is well known, and DNA bound to surfaces such as clay is resistant to degradation.5Nevertheless, other biopolymers, such as RNA and phospholipids, are much less stable and generally degrade rapidly following cell death. 6For example, reverse transcriptase PCR (RT-PCR) has been used to confirm that microbial populations detected at the DNA level are metabolically active.7Also, viable microbes have an intact membrane that contains phospholipids. Cellular enzymes hydrolyze the phosphate group from phospholipids within minutes to hours of cell death.8Therefore, determination of the total amount of phospholipid ester-linked fatty acids (PLFA) in a particular sample provides a quantitative measure of the viable or potentially viable biomass.9Research is needed to develop other techniques able to discriminate between living and dead material.

Acceptable bioburden standards for spacecraft should consider both total abundance and the presence of specific physiological groups. Standards should be defined that take into account the likelihood of survival during transit from Earth and dispersal following landing or impact. Of concern is the possible transport of viable cells to environments supportive of growth of particular classes of terrestrial organisms.

Among the terrestrial microorganisms of most concern are those deriving all of their carbon and energy requirements from inorganic compounds (the chemolithoautotrophs), since they would be most likely to proliferate in a europan ocean. Given current understanding of Europa, it is not unreasonable to suggest that reduced chemical species (e.g., H2, HS-, and Fe+2) might be produced by geothermal processes within an ocean and that oxidized species (e.g., O2, CO2, SO3-, and SO4-) might be transported by geologic activity from the surface to an ocean. A variety of recognized chemoautotrophs are capable of growth using these chemical species as substrates for energy generation and growth.

Represented by Archaea and Bacteria, chemoautotrophs are phylogenetically diverse. Although some lineages are composed solely of chemoautotrophic representatives (e.g., methanogens), others (such as the homoacetogens) are intertwined with heterotrophic lineages and could not be easily recognized by phylogenetic affiliation alone. The identification of these lineages may therefore need to consider the presence of key enzymes or genes required for chemoautotrophic growth. For example, genes encoding enzymes for CO2 fixation (e.g., ribulose bisphosphate carboxylase or carbon monoxide dehydrogenase) are possible diagnostic targets.



The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement



Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.

OCR for page 20
Preventing the Forward Contamination of Europa 5 Microbial Detection and Identification A key aspect of planetary protection is the determination of the bioload on the spacecraft prior to launch. From the Viking era to the present day, the bioload has been monitored by a routine and ongoing procedure that continues until shortly before launch. The monitoring entails swabbing accessible external and internal surfaces of the spacecraft with cotton and then determining the number of culturable bacteria from a known surface area. The standard procedure is to transfer the cells from a swabbed surface to a liquid medium, which is then heat shocked at 80 °C for 8 minutes. The surviving cells are then cultured to determine the number of colony-forming units (cfu). A cfu is defined as a colony on a culture plate (using a standard growth medium) that develops from a cell that survived heat shock. Although the techniques employed in NASA’s planetary protection protocols have remained virtually unchanged for the last 25 years, the methods now available for bioload characterization have changed dramatically thanks to advances in biotechnology. It is now possible to survey with good confidence the microbial diversity on a spacecraft or within its assembly area using explicit molecular criteria. A molecular approach circumvents many of the problems associated with culture-based characterization (e.g., delays caused by the time needed to grow the microbial colonies or by the inability to successfully culture most microbes). The most established methods are based on selective recovery and sequencing of genes encoding the ribosomal ribonucleic acids (RNA). 1 In addition to new technologies such as high-density deoxyribonucleic acid (DNA) hybridization arrays,2 refinements of existing molecular methods (e.g., the polymerase chain reaction (PCR)) and analytical methods (e.g., mass spectrometry and immunochemistry) provide improved detection capability using a variety of diagnostic cellular biopolymers, including proteins, lipids, and carbohydrates. A variety of advanced detection methods are now in common use.3,4 Improvements in the sensitivity and specificity of microbial detection should be incorporated into new assessment standards. It is important to bear in mind that most DNA-based techniques do not necessarily distinguish between living and dead materials. The preservation of DNA in some noncellular contexts is well known, and DNA bound to surfaces such as clay is resistant to degradation.5Nevertheless, other biopolymers, such as RNA and phospholipids, are much less stable and generally degrade rapidly following cell death. 6For example, reverse transcriptase PCR (RT-PCR) has been used to confirm that microbial populations detected at the DNA level are metabolically active.7Also, viable microbes have an intact membrane that contains phospholipids. Cellular enzymes hydrolyze the phosphate group from phospholipids within minutes to hours of cell death.8Therefore, determination of the total amount of phospholipid ester-linked fatty acids (PLFA) in a particular sample provides a quantitative measure of the viable or potentially viable biomass.9Research is needed to develop other techniques able to discriminate between living and dead material. Acceptable bioburden standards for spacecraft should consider both total abundance and the presence of specific physiological groups. Standards should be defined that take into account the likelihood of survival during transit from Earth and dispersal following landing or impact. Of concern is the possible transport of viable cells to environments supportive of growth of particular classes of terrestrial organisms. Among the terrestrial microorganisms of most concern are those deriving all of their carbon and energy requirements from inorganic compounds (the chemolithoautotrophs), since they would be most likely to proliferate in a europan ocean. Given current understanding of Europa, it is not unreasonable to suggest that reduced chemical species (e.g., H2, HS-, and Fe+2) might be produced by geothermal processes within an ocean and that oxidized species (e.g., O2, CO2, SO3-, and SO4-) might be transported by geologic activity from the surface to an ocean. A variety of recognized chemoautotrophs are capable of growth using these chemical species as substrates for energy generation and growth. Represented by Archaea and Bacteria, chemoautotrophs are phylogenetically diverse. Although some lineages are composed solely of chemoautotrophic representatives (e.g., methanogens), others (such as the homoacetogens) are intertwined with heterotrophic lineages and could not be easily recognized by phylogenetic affiliation alone. The identification of these lineages may therefore need to consider the presence of key enzymes or genes required for chemoautotrophic growth. For example, genes encoding enzymes for CO2 fixation (e.g., ribulose bisphosphate carboxylase or carbon monoxide dehydrogenase) are possible diagnostic targets.

OCR for page 20
Preventing the Forward Contamination of Europa REFERENCES 1 D.A. Stahl, “Molecular Approaches for the Measurement of Density, Diversity, and Phylogeny,” C.J. Hurst, G.R. Knudsen, M.J. McInerney, M.V. Walters, and L.D. Stetzenbach (eds.), Manual of Environmental Microbiology, ASM Press, Washington, D.C., 1996, page 102. 2 M. Schena et al., “Quantitative Monitoring of Gene-Expression Patterns with a Complementary-DNA Microarray,” Science 270: 467, 1995. 3 C.J. Hurst, G.R. Knudsen, M.J. McInerney, M.V. Walters, and L.D. Stetzenbach (eds.), Manual of Environmental Microbiology,ASM Press, Washington, D.C., 1996. 4 C. Edwards (ed.), Environmental Monitoring of Bacteria: Methods in Biotechnology, Humana Press, Totowa, N.J., 1999. 5 A.J. Alvarez et al., “Amplification of DNA Bound on Clay Minerals,”Molecular Ecology 7: 775, 1998. 6 D.C. White et al., “Determination of the Sedimentary Microbial Biomass by Extractable Lipid Phosphate,” Oecologia 40: 51, 1979. 7 M.M. Moeseneder et al., “Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis,” Applied Environmental Microbiology 65: 3518, 1999. 8 D.C. White et al., “Determination of the Sedimentary Microbial Biomass by Extractable Lipid Phosphate,” Oecologia 40: 51, 1979. 9 D.L. Balkwill et al., “Equivalence of Microbial Biomass Measures Based on Membrane Lipid and Cell Wall Components, Adenosine Triphospate, and Direct Counts in Subsurface Sediments,” Microbial Ecology 16: 73, 1988.