Anne M. Spain

Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere

ABSTRACT Soil bacterial communities typically exhibit a distribution pattern in which most bacterial species are present in low abundance. Due to the relatively small size of most culture-independent sequencing surveys, a detailed phylogenetic analysis of rare members of the community is lacking. To gain access to the rarely sampled soil biosphere, we analyzed a data set of 13,001 near-full-length 16S rRNA gene clones derived from an undisturbed tall grass prairie soil in central Oklahoma. Rare members of the soil bacterial community (empirically defined at two different abundance cutoffs) represented 18.1 to 37.1% of the total number of clones in the data set and were, on average, less similar to their closest relatives in public databases when compared to more abundant members of the community. Detailed phylogenetic analyses indicated that members of the soil rare biosphere either belonged to novel bacterial lineages (members of five novel bacterial phyla identified in the data set, as well as members of multiple novel lineages within previously described phyla or candidate phyla), to lineages that are prevalent in other environments but rarely encountered in soil, or were close relatives to more abundant taxa in the data set. While a fraction of the rare community was closely related to more abundant taxonomic groups in the data set, a significant portion of the rare biosphere represented evolutionarily distinct lineages at various taxonomic cutoffs. We reason that these novelty and uniqueness patterns provide clues regarding the origins and potential ecological roles of members of the soils rare biosphere.

Abundance, composition, diversity and novelty of soil Proteobacteria.

Small subunit (16S) rRNA gene surveys generating near full-length 16S rRNA clones offer a unique opportunity for in-depth phylogenetic analysis to highlight the breadth of diversity within various major bacterial phyla encountered in soil. This study offers a detailed phylogenetic analysis of the Proteobacteria-affiliated clones identified from 13 001 nearly full-length 16S rRNA gene clones derived from Oklahoma tall-grass prairie soil. Proteobacteria was the most abundant phylum in the community, and comprised 25% of the total clones. The most abundant and diverse class within the Proteobacteria was Alphaproteobacteria, followed by the Delta-, Beta- and Gammaproteobacteria. Members of the Epsilon- and Zetaproteobacteria were not detected in the dataset. Our analysis identified 15 novel order-level and 48 novel family-level Proteobacteria lineages. In addition, we show that the majority of Proteobacteria clones in the dataset belong to orders and families containing no described cultivated representatives (50% and 65%, respectively). An examination of the ecological distribution of the six most abundant Proteobacteria lineages in this dataset with no characterized pure culture representatives provided important information regarding their global distribution and environmental preferences. This level of novel phylogenetic diversity indicates that our understanding of the functions of soil microorganisms, even those belonging to phyla with numerous and diverse well-characterized cultured representatives such as the Proteobacteria, remains far from adequate.

Identification and Isolation of a Castellaniella Species Important during Biostimulation of an Acidic Nitrate- and Uranium-Contaminated Aquifer

ABSTRACT Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.

Changes in Microbial Community Composition and Geochemistry during Uranium and Technetium Bioimmobilization

ABSTRACT In a previous column study, we investigated the long-term impact of ethanol additions on U and Tc mobility in groundwater (M. M. Michalsen et al., Environ. Sci. Technol. 40:7048-7053, 2006). Ethanol additions stimulated iron- and sulfate-reducing conditions and significantly enhanced U and Tc removal from groundwater compared to an identical column that received no ethanol additions (control). Here we present the results of a combined signature lipid and nucleic acid-based microbial community characterization in sediments collected from along the ethanol-stimulated and control column flow paths. Phospholipid fatty acid analysis showed both an increase in microbial biomass (∼2 orders of magnitude) and decreased ratios of cyclopropane to monoenoic precursor fatty acids in the stimulated column compared to the control, which is consistent with electron donor limitation in the control. Spatial shifts in microbial community composition were identified by PCR-denaturing gradient gel electrophoresis analysis as well as by quantitative PCR, which showed that Geobacteraceae increased significantly near the stimulated-column outlet, where soluble electron acceptors were largely depleted. Clone libraries of 16S rRNA genes from selected flow path locations in the stimulated column showed that Proteobacteria were dominant near the inlet (46 to 52%), while members of candidate division OP11 were dominant near the outlet (67%). Redundancy analysis revealed a highly significant difference (P = 0.0003) between microbial community compositions within stimulated and control sediments, with geochemical variables explaining 68% of the variance in community composition on the first two canonical axes.

Nitrate-Reducing Bacteria at the Nitrate and Radionuclide Contaminated Oak Ridge Integrated Field Research Challenge Site: A Review

Mining and enrichment of uranium (U) for nuclear weapons and energy has left this radionuclide an important groundwater contaminant in the United States and worldwide. The use of nitric acid in these processes has also resulted in low pH and high nitrate concentrations in U-contaminated subsurface sediments. This presents problems for bioremediation strategies to control the migration of U in groundwater, usually achieved through microbial reduction of soluble U(VI) to insoluble U(IV) upon electron donor addition to the subsurface. Nitrate, which serves as a competitive electron acceptor in the subsurface, can inhibit or retard U(VI) reduction efforts; as well, intermediates of nitrate respiration (or denitrification), such as nitrite, can lead to U(IV) oxidation and remobilization. The Integrated Field Research Challenge site in Oak Ridge, Tennessee provides an ideal location to address the challenges nitrate poses to uranium bioreduction as the site encompasses several different geochemical conditions: two acidic U- high-nitrate U-contaminated sites (both Areas 1 and 3), a low-nitrate U-contaminated site (Area 2), and a pristine (uncontaminated) background site. In this paper, we review 24 studies examining the microbial communities from these sites within the OR-IFRC as well as denitrifying fluidized bed reactors (FBRs) treating high-nitrate groundwater in an effort to describe the overall potential denitrifying community composition at these sites. Pseudomonas was the most widely detected genus among all sites, but was not detected in either of two studies describing metabolically active populations from community total RNA extracts. Collectively, 16S rRNA gene surveys indicate the following genera may be of potential importance in nitrate reduction and denitrification at the OR-IFRC: Ralstonia and Dechloromonas in the low nitrate neutral pH Area 2, Castellaniella and Burkholderia in Area 1, Thiobacillus and Ferribacterium in Area 3, and Acidovorax in FBRs. This work begins to help us understand how geochemical conditions can determine the composition of nitrate-reducing microbial communities at uranium contaminated sites as well as how population structure and physiologies of the microorganisms present affect in situ rates of denitrification and radionuclide immobilization.

Cooperation of Three Denitrifying Bacteria in Nitrate Removal of Acidic Nitrate- and Uranium-Contaminated Groundwater

In uranium-contaminated aquifers co-contaminated with nitrate, denitrifiers play a critical role in bioremediation. Six strains of denitrifying bacteria belonging to Rhizobium, Pseudomonas, and Castellaniella were isolated from the Oak Ridge Integrated Field Research Challenge Site (OR-IFRC), where biostimulation of acidic (pH 3.5–6.5), nitrate-contaminated (up to 140 mM) groundwater occurred. Three isolates were characterized in regards to nitrite tolerance, denitrification kinetic parameters, and growth on different denitrification intermediates. Kinetic and growth experiments showed that Pseudomonas str. GN33#1 reduced NO− 3 most rapidly (Vmax = 15.8 μmol e−·min−1·mg protein−1) and had the fastest generation time (gt) on NO− 3 (2.6 h). Castellaniella str. 4.5A2 was the most low pH and NO− 2 tolerant and grew rapidly on NO− 2 (gt = 4.0 h). Rhizobium str. GN32#2 was also tolerant of low pH values and reduced NO− 2 rapidly (Vmax = 10.6 μmol e−·min−1·mg protein−1) but was far less NO− 2 tolerant than Castellaniella str. 4.5A2. Growth of and denitrification by these three strains incubated together and individually were measured in OR-IFRC groundwater at pHs 5 and 7 to determine whether they cooperate or compete during denitrification. Mixed assemblages reduced NO− 3 more rapidly and more completely than any individual isolate over the course of the experiment. The results described in this article demonstrate 1) that this synthetic assemblage comprised of three physiologically distinct denitrifying bacterial isolates cooperate to achieve more complete levels of denitrification and 2) the importance of pH- and nitrite-tolerant bacteria such as Castellaniella str. 4.5A2 in minimizing NO− 2 accumulation in high-NO− 3 groundwater during bioremediation. Supplemental materials are available for this article. Go to the publishers online edition of Geomicrobiology Journal to view the free supplemental files.

Metatranscriptomic analysis of a high-sulfide aquatic spring reveals insights into sulfur cycling and unexpected aerobic metabolism

Zodletone spring is a sulfide-rich spring in southwestern Oklahoma characterized by shallow, microoxic, light-exposed spring water overlaying anoxic sediments. Previously, culture-independent 16S rRNA gene based diversity surveys have revealed that Zodletone spring source sediments harbor a highly diverse microbial community, with multiple lineages putatively involved in various sulfur-cycling processes. Here, we conducted a metatranscriptomic survey of microbial populations in Zodletone spring source sediments to characterize the relative prevalence and importance of putative phototrophic, chemolithotrophic, and heterotrophic microorganisms in the sulfur cycle, the identity of lineages actively involved in various sulfur cycling processes, and the interaction between sulfur cycling and other geochemical processes at the spring source. Sediment samples at the spring’s source were taken at three different times within a 24-h period for geochemical analyses and RNA sequencing. In depth mining of datasets for sulfur cycling transcripts revealed major sulfur cycling pathways and taxa involved, including an unexpected potential role of Actinobacteria in sulfide oxidation and thiosulfate transformation. Surprisingly, transcripts coding for the cyanobacterial Photosystem II D1 protein, methane monooxygenase, and terminal cytochrome oxidases were encountered, indicating that genes for oxygen production and aerobic modes of metabolism are actively being transcribed, despite below-detectable levels (<1 µM) of oxygen in source sediment. Results highlight transcripts involved in sulfur, methane, and oxygen cycles, propose that oxygenic photosynthesis could support aerobic methane and sulfide oxidation in anoxic sediments exposed to sunlight, and provide a viewpoint of microbial metabolic lifestyles under conditions similar to those seen during late Archaean and Proterozoic eons.

Phylum XVIII. Fibrobacteres Garrity and Holt 2001

The phylum Fibrobacteres currently consists of three classes circumscribed on the basis of phylogenetic analysis of 16S rRNA gene sequences, including one cultivated class, Fibrobacteria class. nov. Fibrobacterales is the type order, and contains a single family and genus.

Effects of Microbial Community Structure, Terminal Electron Accepting Conditions, and Molybdate on the Extent of U(VI) Reduction in Landfill Aquifer Sediments

Addition of an electron donor, such as ethanol, glucose, or acetate, to the subsurface in order to stimulate biological reduction of soluble U(VI) to insoluble U(IV) is an important strategy for uranium immobilization in contaminated aquifers. Electron donor addition typically results in anaerobic conditions and the respiratory process (sulfate-reducing, iron-reducing, and/or methanogenic) will depend on site geochemistry. Although previous studies have found that U(VI) reduction can occur under any of these conditions, the goal of this study was to examine the relative extent of U(VI) reduction under different geochemical conditions and the influence of the different microbial populations on the reduction process. Sulfate-reducing (SR), iron-reducing (FeR), and methanogenic (Meth) conditions were stimulated by electron donor addition in sediment batch microcosms, and 100 μM U(VI) was added upon depletion of alternate electron acceptors. Within seven days, 89, 96, and 66% of soluble U(VI) was immobilized by sorption and/or precipitation from sulfate-reducing, iron-reducing, and methanogenic bottles, respectively. After 26 days, however, bicarbonate and nitric acid extractions of solid-associated U(VI) and total U showed that (i) there was no significant difference in the amount of U(VI) reduced between the different terminal electron accepting conditions stimulated in pre-incubations (0.204 ± 0.052, 0.263 ± 0.023, and 0.247 μmol total reduced U(IV) per g dry sediment in SR, FeR, and Meth bottles, respectively); (ii) geochemical conditions formed in FeR bottles contributed most to abiotic U(VI) reduction, with 0.0123 ± 0.0105, 0.0737 ± 0.0111, and 0.0464 ± 0.0002 μmol total reduced U(IV) per g dry sediment in formaldehyde-killed SR, FeR, and Meth bottles, respectively; and, (iii) molybdate inhibited biotic U(VI) reduction to some degree under each terminal electron accepting condition stimulated, with an average of 33% less U(IV) per g dry sediment in live bottles containing molybdate compared to controls. PLFA analysis of sediments showed that different major PLFAs groups were associated with the amount of U(IV) per g dry sediment with each terminal electron accepting condition stimulated during pre-incubations, with positive correlations between monounsaturates and U(IV) in SR bottles (r = 0.926), branched monounsaturates and U(IV) in FeR bottles (r = 0.886), and terminal-branched saturates and U(IV) in Meth bottles (r = 0.999). Collectively, these data suggesting that different populations may have been involved in U(VI) reduction under each condition and that the extent of U(VI) reduction does not differ whether sediments were sulfate-reducing, iron-reducing, or methanogenic prior to U(VI) addition.