David B. Ringelberg

Quantitative comparisons ofin situ microbial biodiversity by signature biomarker analysis

Microscopic examinations have convinced microbial ecologists that the culturable microbes recovered from environmental samples represent a tiny proportion of the extant microbiota. Methods for recovery and enzymatic amplification of nucleic acids from environmental samples have shown that a huge diversity existsin situ, far exceeding any expectations which were based on direct microscopy. It is now theoretically possible to extract, amplify and sequence all the nucleic acids from a community and thereby gain a comprehensive measure of the diversity as well as some insights into the phylogeny of the various elements within this community. Unfortunately, this analysis becomes economically prohibitive if applied to the multitude of niches in a single biome let alone to a diverse set of environments. It is also difficult to utilize PCR amplification on nucleic acids from some biomes because of coextracting enzymatic inhibitors. Signature biomarker analysis which potentially combines gene probe and lipid analysis on the same sample, can serve as a complement to massive environmental genome analysis in providing quantitative comparisons between microniches in the biome under study. This analysis can also give indications of the magnitude of differences in biodiversity in the blome as well as provide insight into the phenotypic activities of each community in a rapid and cost-effective manner. Applications of signature lipid biomarker analysis to define quantitatively the microbial viable biomass of portions of an Eastern USA deciduous forest, are presented.

Pore‐size constraints on the activity and survival of subsurface bacteria in a late cretaceous shale‐sandstone sequence, northwestern New Mexico

To investigate the distribution of microbial biomass and activities to gain insights into the physical controls on microbial activity and potential long‐term survival in the subsurface, 24 shale and sandstone cores were collected from a site in northwestern New Mexico. Bacterial biomass in the core samples ranged from below detection to 31.9 pmol total phospholipid fatty acid (PLFA) g‐1 of rock with no apparent relationship between lithology and PLFA abundance. No metabolic activities, as determined by anaerobic mineralization of [14C]acetate and [14C]glucose and 35SO4 2‐ reduction, were detected in core samples with pore throats <0.2 fan in diameter, smaller than the size of known bacteria. However, enrichments revealed the presence of sulfate‐re‐ducing bacteria, and 35SO4 2‐ reduction was detected upon extended (14 days) incubation in some small‐pore‐throat samples. In contrast, relatively rapid rates of metabolic activity were more common in core samples containing a significant fraction of pore throat...

Soil Microbial Communities Beneath Populus Grandidentata Grown Under Elevated Atmospheric CO2

In most terrestrial ecosystems, the amount of substrate entering the soil from plant litter production is only sufficient to meet the maintenance requirements of soil microorganisms, allowing for no net annual growth. However, the rising atmospheric CO2 concentration has the potential to alter such a balance by increasing plant litter production, and hence the amount of substrate available for heterotrophic metabolism in soil. In a recent experiment, we observed that greater belowground plant litter production at elevated at- mospheric CO2 significantly increased the biomass of soil microorganisms in both rhizo- sphere and non-rhizosphere soil. Because soil microorganisms differ in their ability to convert substrate into biomass, we hypothesized that greater plant litter production at elevated CO2 should shift community composition as fungal populations increase in re- sponse to greater substrate availability. We used a molecular technique, phospholipid fatty acid (PLFA) analysis, to gain insight into the composition of soil microbial communities beneath Populus grandidentata growing at ambient and twice-ambient atmospheric CO2. PLFAs extracted from rhizosphere and non-rhizosphere soil were derivatized and identified using gas chromatography and mass spectrometry. After one growing season the proportions of bacterial, actinomycetal, and fungal PLFAs were not significantly influenced by elevated atmospheric CO2 in either rhizosphere or non-rhizosphere soil. However, clear differences were present between microbial communities in rhizosphere and non-rhizosphere soil. Al- though enhanced belowground plant litter production under elevated atmospheric CO2 in- creased the biomass of soil microorganisms, we have no evidence to suggest that such an increase occurred through a shift in community composition, at least in the short term.

Taxonomic Study of Aromatic-Degrading Bacteria from Deep- Terrestrial-Subsurface Sediments and Description of Sphingomonas aromaticivorans sp. nov., Sphingomonas subterranea sp. nov., and Sphingomonas stygia sp. nov.

Phylogenetic analyses of 16S rRNA gene sequences by distance matrix and parsimony methods indicated that six strains of bacteria isolated from deep saturated Atlantic coastal plain sediments were closely related to the genus Sphingomonas. Five of the strains clustered with, but were distinct from, Sphingomonas capsulata, whereas the sixth strain was most closely related to Blastobacter natatorius. The five strains that clustered with S. capsulata, all of which could degrade aromatic compounds, were gram-negative, non-spore-forming, non-motile, rod-shaped organisms that produced small, yellow colonies on complex media. Their G + C contents ranged from 60.0 to 65.4 mol%, and the predominant isoprenoid quinone was ubiquinone Q-10. All of the strains were aerobic and catalase positive. Indole, urease, and arginine dihydrolase were not produced. Gelatin was not liquified, and glucose was not fermented. Sphingolipids were present in all strains; 2OH14:0 was the major hydroxy fatty acid, and 18:1 was a major constituent of cellular lipids. Acid was produced oxidatively from pentoses, hexoses, and disaccharides, but not from polyalcohols and indole. All of these characteristics indicate that the five aromatic-degrading strains should be placed in the genus Sphingomonas as currently defined. Phylogenetic analysis of 16S rRNA gene sequences, DNA-DNA reassociation values, BOX-PCR genomic fingerprinting, differences in cellular lipid composition, and differences in physiological traits all indicated that the five strains represent three previously undescribed Sphingomonas species. Therefore, we propose the following new species: Sphingomonas aromaticivorans (type strain, SMCC F199), Sphingomonas subterranea (type strain, SMCC B0478), and Sphingomonas stygia (type strain, SMCC B0712).

Membrane fatty acids as phenotypic markers in the polyphasic taxonomy of methylotrophs within the proteobacteria

A polyphasic approach to bacterial taxonomy attempts to integrate phylogenetic relationships with phenotypic marker analysis. This study describes the application of membrane fatty acids as a phenotypic marker for methylotrophs. Detailed phospholipid, ester-linked fatty acid (PLFA) profiles are reported for 17 methylotrophic eubacterial strains. These profiles included verification of double bond positions and geometries, both critical features for this analysis. Multivariate cluster analysis was used to indicate groupings of these strains along with literature values of both methylotrophs and non-methylotrophs based on the PLFA phenotype. Like many phenotypic characteristics, PLFA profiles were influenced by environmental conditions. The instabilities displayed, however, were predictable from physiological studies including increased trans/cis and cyclopropyl/cis ratios. Cluster analysis of PLFA profiles generated by separate investigators with different culture conditions indicated reproducibility by strain and species. The PLFA phenotype relationships compare favourably with phylogenetic associations based on 16S rRNA data for methylotrophs and will continue to be a valuable phenotypic marker for Proteobacteria taxonomy.

Combined microbial community-level analyses for quality assurance of terrestrial subsurface cores☆

Bacterial communities from surface soils, groundwater, drilling muds and deep subsurface cores were profiled by sole carbon source utilization and by phospholipid ester-linked fatty acid analysis. The combination of these functional and structural methods successfully distinguished communities from disparate origins. Multivariate analysis of the data showed good agreement between the results of the two methods. Subsurface communities tended to respire amino acids over carbohydrates and demonstrated preferential use of individual compounds such as acetate and Tween as sole carbon sources. PLFA profiles indicated that the groundwaters predominately contained gram negative aerobic heterotrophic populations, the drilling muds and cuttings were populated by gram negative anaerobes and the core communities were composed of anaerobic gram negative bacteria and gram positive bacteria. The utility of this approach as a component of quality assurance of core samples obtained for microbiological analysis during mud rotary coring was demonstrated. Monitoring of controlled bioprocesses, environmental remediation and detection of environmental disturbance are some of the numerous potential applications for these community-level characterization methods. Since combined analyses such as these can simultaneously provide specific information about individual community members and about community-level function, it is hoped that these methods will prove useful in answering fundamental questions in microbial ecology, such as the relationship between in situ community structure and its measurable function.

Succession of Phenotypic, Genotypic, and Metabolic Community Characteristics during In Vitro Bioslurry Treatment of Polycyclic Aromatic Hydrocarbon-Contaminated Sediments

ABSTRACT Dredged harbor sediment contaminated with polycyclic aromatic hydrocarbons (PAHs) was removed from the Milwaukee Confined Disposal Facility and examined for in situ biodegradative capacity. Molecular techniques were used to determine the successional characteristics of the indigenous microbiota during a 4-month bioslurry evaluation. Ester-linked phospholipid fatty acids (PLFA), multiplex PCR of targeted genes, and radiorespirometry techniques were used to define in situ microbial phenotypic, genotypic, and metabolic responses, respectively. Soxhlet extractions revealed a loss in total PAH concentrations of 52%. Individual PAHs showed reductions as great as 75% (i.e., acenapthene and fluorene). Rates of 14C-PAH mineralization (percent/day) were greatest for phenanthrene, followed by pyrene and then chrysene. There was no mineralization capacity for benzo[a]pyrene. Ester-linked phospholipid fatty acid analysis revealed a threefold increase in total microbial biomass and a dynamic microbial community composition that showed a strong correlation with observed changes in the PAH chemistry (canonicalr2 of 0.999). Nucleic acid analyses showed copies of genes encoding PAH-degrading enzymes (extradiol dioxygenases, hydroxylases, and meta-cleavage enzymes) to increase by as much as 4 orders of magnitude. Shifts in gene copy numbers showed strong correlations with shifts in specific subsets of the extant microbial community. Specifically, declines in the concentrations of three-ring PAH moieties (i.e., phenanthrene) correlated with PLFA indicative of certain gram-negative bacteria (i.e., Rhodococcus spp. and/or actinomycetes) and genes encoding for naphthalene-, biphenyl-, and catechol-2,3-dioxygenase degradative enzymes. The results of this study suggest that the intrinsic biodegradative potential of an environmental site can be derived from the polyphasic characterization of the in situ microbial community.

Observations pertaining to the origin and ecology of microorganisms recovered from the deep subsurface of Taylorsville Basin, Virginia

To understand the conditions under which microorganisms exist in deep hydrocarbon reservoirs, sidewall cores were collected from a natural gas‐bearing formation, 2800 m below the surface in Taylorsville Basin, Virginia. Data from chemical and microbial tracers and controls indicate that the interiors of some sidewall cores contained microorganisms indigenous to the rock formation. The cultured microorganisms were composed primarily of saline‐tolerant, thermophilic fermenting, Fe(III)‐reducing, and sulfate‐reducing bacteria (1 to 104 cells/g). The physiological capabilities of the cultured microorganisms are compatible with the temperature (76°C), pressure (32 MPa), and salinity (≈0.8 wt.% NaCl equivalent) in the sampled interval. The petrological data indicated that the strata contain intercrystalline pores of micrometer size, that occur between late diagenetic cement in siltstone and within cross‐cutting, mineralized fractures in shale. These pores made up only 0.04% of the rock volume, were mostly gas‐f...

Microbial Communities in High and Low Recharge Environments: Implications for Microbial Transport in the Vadose Zone

A bstractMicrobial communities along vertical transects in the unsaturated zone were evaluated at five sites in the Pasco Basin, in southeastern Washington State. Sites with contrasting recharge rates were chosen to maximize or minimize the potential for microbial transport. Pore water ages along the vertical transects were established using natural chloride tracers, and ranged from modern to either ∼15,000 yBP (years before present) or ∼30,000 yBP at the two low-recharge sites. Unsaturated flow processes were short-circuited by preferential flow at two of the three high-recharge sites, resulting in rapid movement of water through the vertical transects. Microbial numbers and biomass, based on plate counts, and phospholipid fatty acid (PLFA) concentrations decreased with depth at all sites. The majority (55–90%) of the culturable chemoheterotrophs recovered from most samples were streptomycete bacteria. 16S rRNA gene sequence and MIDI analyses indicated that 75% of the remaining isolates were Gram-positive bacteria (most likely species of Arthrobacter and Bacillus) 25% were Gram-negative bacteria (probably members of several genera in the alpha- and gamma-Proteobacteria). Comparison of microbial communities at low-recharge sites vs. high-recharge sites, where preferential flow occurs, revealed several differences that might be attributed to vertical transport of microbial cells at the high-recharge sites. Plate counts and PLFA analyses indicated that the proportion of streptomycetes, which were abundant at the surface but present in the subsurface as spores, decreased, or remained constant, with depth at the low-recharge sites, but increased with depth at the high-recharge sites. PLFA analyses also indicated that Gram-negative bacteria displayed increased nutrient stress with depth at the high-recharge sites characterized by preferential flow, but not at the low recharge site. This may be a result of advective transport of microbes to depths where it was difficult for them to compete effectively with the established community. Moreover, PLFA community structure profiles fluctuated considerably with depth at the low-recharge sites, but not at the high-recharge sites. This might be expected if transport were distributing the microbial community along the vertical profile at the high-recharge sites. In contrast to the high-recharge sites at which preferential flow occurs, filtration likely prevented vertical transport of microorganisms at the high-recharge site that was characterized by unsaturated flow.

Description of Two New Thermophilic Desulfotomaculum spp., Desulfotomaculum putei sp. nov., from a Deep Terrestrial Subsurface, and Desulfotomaculum luciae sp. nov., from a Hot Spring

Six strains of thermophilic, endospore-forming, sulfate-reducing bacteria were enriched and isolated from 2.7 km below the earths surface in the Taylorsville Triassic Basin in Virginia. The cells of these strains were motile rods that were 1 to 1.1 μ in diameter and 2 to 5 μ long. The cells grew by oxidizing H2, formate, methanol (weakly), lactate (incompletely, to acetate and CO2), or pyruvate (incompletely) while reducing sulfate to sulfide; acetate did not serve as a catabolic substrate. Thiosulfate or sulfite could replace sulfate as an electron acceptor. The results of a phylogenetic analysis of the 16S rRNA gene indicated that these strains belong to the genus Desulfotomaculum, but are distinct from previously described Desulfotomaculum species. Thus, we propose a new species, Desulfotomaculum putei, for them, with strain TH-11 (= SMCC W459) as the type strain. The results of our phylogenetic analysis also indicated that strain SLTT, which was isolated from a hot spring and has been described previously (T. M. Karnauchow, S. F. Koval, and K. F. Jarrell, Syst. Appl. Microbiol. 15:296-310, 1992), is also a member of the genus Desulfotomaculum and is distinct from other species in this genus. We therefore propose the new species Desulfotomaculum luciae for this organism; strain SLT (= SMCC W644) is the type strain of D. luciae.