Cave biosignature suites: microbes, minerals, and Mars.

Earth's subsurface offers one of the best possible sites to search for microbial life and the characteristic lithologies that life leaves behind. The subterrain may be equally valuable for astrobiology. Where surface conditions are particularly hostile, like on Mars, the subsurface may offer the only habitat for extant lifeforms and access to recognizable biosignatures. We have identified numerous unequivocally biogenic macroscopic, microscopic, and chemical/geochemical cave biosignatures. However, to be especially useful for astrobiology, we are looking for suites of characteristics. Ideally, "biosignature suites" should be both macroscopically and microscopically detectable, independently verifiable by nonmorphological means, and as independent as possible of specific details of life chemistries--demanding (and sometimes conflicting) criteria. Working in fragile, legally protected environments, we developed noninvasive and minimal impact techniques for life and biosignature detection/characterization analogous to Planetary Protection Protocols. Our difficult field conditions have shared limitations common to extraterrestrial robotic and human missions. Thus, the cave/subsurface astrobiology model addresses the most important goals from both scientific and operational points of view. We present details of cave biosignature suites involving manganese and iron oxides, calcite, and sulfur minerals. Suites include morphological fossils, mineral-coated filaments, living microbial mats and preserved biofabrics, 13C and 34S values consistent with microbial metabolism, genetic data, unusual elemental abundances and ratios, and crystallographic mineral forms.

Density, activity, and diversity of bacteria indigenous to a karstic aquifer.

The microbial ecology of karstic ground water is largely unknown. The density, activity, and diversity of bacteria indigenous to subsurface karstic material in Mammoth Cave National Park, Mammoth Cave, Kentucky were studied using minimally disruptive, on-site procedures. Two sites, located 100 m below the surface and consisting of saturated fine to coarse sand in pooled water, were examined. Samples were taken aseptically using modified, sterile 60-cc syringes. Total cell and total respiring cell densities were determined using an acridine orange/p-iodonitrotetrazolium violet (AO/INT) staining procedure. Cells in selected cores were stained with INT and incubated in the cave for 4 h prior to fixing with glutaraldehyde and subsequent transport to the laboratory. Cells were stained with AO in the laboratory. Low- and high-nutrient media were used to determine viable cell counts. Plates were incubated in the cave for 1 day at ambient temperature prior to transportation to the laboratory in an insulated cooler. Viable cell counts ranged from 1.0 × 106 to 8.1 × 106 cells wet g(-1) of sediment. Total direct counts were 3.9 × 106 and 1.4 × 107 cells wet g(-1) for the Olivia's Dome and the Catherine's Dome sites, respectively. Viable cell counts were highly similar to respiring cell counts at both sites. At the Olivia's Dome site, viable cell counts represented 26-31% of the direct cell counts, while 58% of the total cell count were actively respiring. At the Catherine's Dome site, viable cell counts represented 11-58% of the direct counts, while 53% of the cells were actively respiring. A total of 237 strains recovered from low- and high-nutrient media at both Olivia's and Catherine's Domes, and 10 reference strains were examined for 117 morphological, biochemical, and physiological characteristics. Results were coded in a binary fashion and analyzed using numerical taxonomic techniques. Similarity values were calculated using a simple matching coefficient. Fifty-two clusters, ranging in size from 2 to 13 members, were defined at the 80-85% similarity level with the weighted pair-group mathematical average algorithm (WPGMA). The matrix was examined using the Jaccard coefficient and WPGMA clustering to control for distortion due to negative matches and varying group size. Presumptively identified genera include, Arthrobacter, Brevibacterium, Bacillus, Cornyebacterium, Actinomyces, Aureobacterium, Chromobacterium, and Mycobacterium. Pseudomonas spp. were not recovered. Fifty percent of the clustered operational taxonomic units (OTUs) were not identified. Thirty percent of the clustered OTUs were irregular, asporogenous, Gram-positive rods. The bacterial communities varied between sites, and isolation medium had a strong influence on the strains recovered. The bacterial community in the karstic sediments sampled exhibits a high degree of diversity having no dominant strain or strains.

The community structure of sessile heterotrophic bacteria stressed by acid mine drainage.

Microbial communities that developed on glass slides suspended in acid-polluted (pH=2.9) and nonpolluted (pH=6.5) but otherwise similar waters showed evidence of stress when suspended at the opposite station. Glucose incorporation was inhibited in both translocated communities, but the inhibition was not as severe and recovery of activity was faster for the acid-developed community as compared to the circumneutral community. The communities contained a substantially different set of members with little overlap. The range of pH values at which the members of the acid-developed community could function suggested that the members of that community were generalists, as opposed to narrowly constrained members of the community from the circumneutral station. Based on the proportion of test characters that received positive responses, the organisms from the acidic site were more general in their abilities (47.6% positive) as compared with the neutral counterparts (18.7% positive). The results support the concept that communities developed in extreme environments tend to be generalists, whereas those from mesic environments, due to the higher levels of competition present, tend to be specialists. Furthermore, the study of microbial communities in dynamic systems such as streams and reservoir inflows is facilitated by the use of solid surfaces which allow an assemblage of nontransient microbes to develop.

Reasons for possible failure of inoculation to enhance biodegradation.

Pseudomonas strains capable of mineralizing 2,4-dichlorophenol (DCP) and p-nitrophenol (PNP) in culture media were isolated from soil. One DCP-metabolizing strain mineralized 1.0 and 10 micrograms of DCP but not 2.0 to 300 ng/ml in culture. When added to lake water containing 10 micrograms of DCP per ml, the bacterium did not mineralize the compound, and only after 6 days did it cause the degradation of 1.0 microgram of DCP per ml. The organism did not grow or metabolize DCP when inoculated into sterile lake water, but it multiplied in sterile lake water amended with glucose or with DCP and supplemental nutrients. Its population density declined and DCP was not mineralized when the pseudomonad was added to nonsterile sewage, but the bacterium grew in sterile DCP-amended sewage, although not causing appreciable mineralization of the test compound. Addition of the bacterium to nonsterile soil did not result in the mineralization of 10 micrograms of DCP per g, although mineralization was evident if the inoculum was added to sterile soil. A second DCP-utilizing pseudomonad failed to mineralize DCP when added to the surface of sterile soil, although activity was evident if the inoculum was mixed with the soil. A pseudomonad able to mineralize 5.0 micrograms of PNP per ml in culture did not mineralize the compound in sterile or nonsterile lake water. The bacterium destroyed PNP in sterile sewage and enhanced PNP mineralization in nonsterile sewage. When added to the surface of sterile soil, the bacterium mineralized little of the PNP present at 5.0 micrograms/g, but it was active if mixed well with the sterile soil.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of FAME (fatty acid methyl ester) analysis in the numerical taxonomic determination of bacterial guild structure.

Comparative numerical taxonomic analyses, using fatty acid methyl ester (FAME) profiles and phenetic characteristics, were conducted to examine bacterial guild structure in freshwater sediments. Both approaches were used to examine a subset of 60 OTUs obtained from a previously well characterized microbial community in sediments of a shallow fast flowing stream. For both classifications, greater than 80% of the OTUs were recovered in 11 and 12 major groups for FAME and phenetics approaches. However, there was not complete correspondence for the groupings of the 2 classifications, with most FAME groups being distributed among the phenetic groups and 2 phenetic groups not providing usable characteristics for FAME analysis. The results did demonstrate significant taxonomic variation in bacteria capable of occupying the same or a similar fundamental niche.

Alternative prey: a mechanism for elimination of bacterial species by protozoa.

Antibiotic-resistant strains of Salmonella typhimurium and Klebsiella pneumoniae died readily after their addition to raw sewage, but they grew in sterilized sewage. The decline was not a result of abiotic stresses, and because the bacteria were able to survive in large numbers for at least 15 days in solutions containing no organic nutrients, it was not a result of competition. Toxin production, bacteriophages, and Bdellovibrio sp. did not cause the disappearance of the two bacterial species. A decline was also evident if the sewage was first passed through a 3-micron (pore size) filter or treated with cycloheximide or cycloheximide plus nystatin, but protozoa developed under these conditions. Little or no decline occurred if the sewage was filtered and treated with the eucaryotic inhibitors before the addition of S. typhimurium or K. pneumoniae, and protozoa were not detected. S. typhimurium increased in abundance if cycloheximide, streptomycin, and erythromycin or large amounts of glucose were added to sewage. Tetrahymena thermophilus did not significantly reduce the population of S. typhimurium in buffer when the density of the bacterium was about 10(4)/ml. However, when more than 10(8) Enterobacter agglomerans cells per ml were added to the buffer, T. thermophilus reduced the abundance of E. agglomerans and S. typhimurium to 10(6) and 10/ml, respectively. The density of S. typhimurium was further decreased by a second increment of E. agglomerans cells. The disappearance of S. typhimurium and K. pneumoniae from sewage thus is the result of predation by protozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterotrophic bacterial guild structure: Relationship to biodegradative populations.

Numerical taxonomic analysis of a freshwater bacterial guild demonstrated that the bacteria capable of growth on phenanthrene and polychlorinated biphenyl media were representative of the taxa obtained from low nutrient oligotrophic media. The diversity of heterotrophic bacteria and members of new taxa recovered from the guild followed a poisson distribution relative to the number of isolation media used. Moderately high nutrient, yeast extract peptone and glucose agar was found to be the most selective isolation medium relative to the total number of taxa recovered whereas low nutrient, lake water agar was the least selective medium used. Carbon source utilization patterns of the isolated taxa indicated that taxa within the guild had broad niche ranges and could potentially occupy many niches within a dynamic environment. The structure of the bacterial guild was dominated by mesophilic oligotrophs.The results of this investigation demonstrate that potential biodegradative populations are representative of the diverse taxa found in uncontaminated freshwater environments.

Impact of coal-coking effluent on sediment microbial communities: a multivariate approach.

The functional response to and recovery from coal-coking waste effluent was evaluated for sediment microbial communities. Twenty estimates of microbial population density, biomass, and activity were measured five times during a 15-month period. Significant effects on microbial communities were observed in response to both wastewater contamination and diversion of the wastewater. Multivariate analysis of variance and discriminant analysis indicated that accurate differentiation between uncontaminated and contaminated sediments required a minimum of nine estimates of community response. Total viable population density, ATP, alkaline phosphatase, naphthalene, and phenanthrene mineralization rates were found to be highly weighted variables in site discrimination. Lipid and glucose mineralization, nitrogen fixation, and sediment protein also contributed significantly to explaining variation among sites. Estimates of anaerobic population densities and rates of methane production contributed little to discrimination among sites in the environment examined. In general, total viable population density, ATP, and alkaline phosphatase activity were significantly depressed in contaminated sediments. However, after removal of this contamination, the previously affected sites demonstrated greater temporal variability but a closer approximation of the mean response at the control site. Naphthalene and phenanthrene mineralization did not follow the general trend and were elevated at the contaminated sites throughout the investigation. Results of the investigation supported the hypothesis that multiple functional measures of microbial community response are required to evaluate the effect of and recovery from environmental contamination. In addition, when long-term effects are evaluated, select physiological traits, i.e., polyaromatic hydrocarbon mineralization, may not reflect population and biomass estimates of community response.

Fate in model ecosystems of microbial species of potential use in genetic engineering.

The changes in populations of Staphylococcus aureus, Bacillus subtilis, Salmonella typhimurium, Klebsiella pneumoniae, Agrobacterium tumefaciens, Rhizobium meliloti, and Saccharomyces cerevisiae were measured after their introduction into samples of sewage, lake water, and soil. Enumeration of small populations was possible because the strains used were resistant to antibiotics in concentrations and combinations such that few species native to these ecosystems were able to grow on agar containing the inhibitors. Fewer than 2 cells per ml of sewage or lake water and 25 cells per g of soil could be detected. A. tumefaciens and R. meliloti persisted in significant numbers with little decline, but S. aureus, K. pneumoniae, S. typhimurium, S. cerevisiae, and vegetative cells of B. subtilis failed to survive in samples of sewage and lake water. In sterile sewage, however, K. pneumoniae, B. subtilis, S. typhimurium, A. tumefaciens, and R. meliloti grew; S. cerevisiae populations were maintained at the levels used for inoculation; and S. aureus died rapidly. In sterile lake water, the population of S. aureus and K. pneumoniae and the number of vegetative cells of B. subtilis declined rapidly, R. meliloti grew, and the other species maintained significant numbers with little or a slow decline. The populations of S. aureus, K. pneumoniae, A. tumefaciens, B. subtilis, and S. typhimurium declined in soil, but the first four species grew in sterile soil. It is suggested that some species persist in environments in which they are not indigenous because they tolerate abiotic stresses, do not lose viability readily when starved, and coexist with antagonists. The species that fails to survive need only be affected by one of these factors.

Numerical taxonomy and ecology of oligotrophic bacteria isolated from the estuarine environment.

Slow-growing bacteria, isolated on nutrient-rich and nutrient-limited media, from Chesapeake Bay water and sediment samples, were examined for 119 biochemical, cultural, morphological, nutritional, and physiological characters. Those bacteria which grow on low nutrient media, termed oligotrophs, a total of 162 strains, were subjected to taxonomic analysis, as a preliminary step in determining their ecological significance. The data for all strains included in the study were examined by computer and the simple matching (S SM) and Jaccard (SJ) coefficients calculated. Clustering was achieved by the unweighted average-linkage method. From sorted similarity matrices and dendrograms, 148 strains, 90% of the total, were recovered in 24 phenetic groups defined at the 80 to 85% similarity level. Only 12 phena could be presumptively identified and these included representatives of Alcaligenes, Corynebacterium, Hyphomicrobium, Hyphomonas polymorpha, Listeria, Nocardia marina, Pedomicrobium, Planococcus citreus, Sphaerotilus, Streptothrix, and Streptomyces. Of the remaining organisms, 10% were unidentified sheathed bacteria. It is concluded that slow-growing bacteria are distributed throughout the estuarine environment and can account for a large proportion of the colonies observed on media after prolonged periods of incubation. The oligotrophic bacteria appear to predominate in areas where the concentration of available nutrients is low and are more characteristic of non-eutrophic aquatic systems.