R. Michael Lehman

Attached and unattached microbial communities in a simulated basalt aquifer under fracture- and porous-flow conditions.

ABSTRACT Bench scale column studies were used to examine the partitioning of microorganisms between groundwater and a geologic medium and to examine the effect of hydrogeology (i.e., porous- versus fracture-flow) on organism partitioning. Replicated columns were constructed with intact basalt core segments that contained natural fractures and with the same basalt crushed into particles. The columns were perfused with groundwater, and upon reaching a steady state, the columns were sacrificed and the attached and unattached communities were analyzed by multiple approaches. The analyses included the total number of cells, the phylogenetic affiliation of the cells (i.e., the α, β, and γ subclasses of the class Proteobacteria and gram positives with high G+C DNA content) by fluorescent in situ hybridization (FISH), number and taxonomic affiliation by fatty acid methyl ester profiles of culturable heterotrophs, most-probable-number estimates of methanotrophs and phenol oxidizers, and whole-community sole carbon source utilization patterns from Biolog GN microplates. In the packed columns, about 99% of the total biomass (per cubic centimeter of porous medium) was attached to the geologic medium. Lack of equitable units precluded a comparison of attached and unattached biomasses in the fractured columns where the attached biomass was expressed per unit of surface area. Compositional differences in the attached and unattached communities were evidenced by (i) the recovery ofPseudomonas stutzeri, an Enterococcus sp., andBacillus psychrophilus from the groundwater and not from the basalt, (ii) differences between community carbon source utilization patterns, and (iii) the relative abundances of different phylogenetic groups estimated by FISH in both column types. In the packed columns, attached communities were depleted of members of the α- and β-Proteobacteria subclasses in comparison to those in the corresponding groundwater. In the fractured columns, attached communities were enriched in gram-positive Bacteriaand γ-Proteobacteria and depleted of β-Proteobacteria, in comparison to those in the corresponding groundwater. Segregation of populations and their activities, possibly modified by attachment to geologic media, may influence contaminant fate and transport in the subsurface and impact other in situ applications.

Attached and Unattached Bacterial Communities in a 120-Meter Corehole in an Acidic, Crystalline Rock Aquifer

ABSTRACT The bacteria colonizing geologic core sections (attached) were contrasted with those found suspended in the groundwater (unattached) by examining the microbiology of 16 depth-paired core and groundwater samples using a suite of culture-independent and culture-dependent analyses. One hundred twenty-two meters was continuously cored from a buried chalcopyrite ore hosted in a biotite-quartz-monzonite porphyry at the Mineral Park Mine near Kingman, Ariz. Every fourth 1.5-m core was acquired using microbiologically defensible methods, and these core sections were aseptically processed for characterization of the attached bacteria. Groundwater samples containing unattached bacteria were collected from the uncased corehole at depth intervals corresponding to the individual cores using an inflatable straddle packer sampler. The groundwater was acidic (pH 2.8 to 5.0), with low levels of dissolved oxygen and high concentrations of sulfate and metals, including ferrous iron. Total numbers of attached cells were less than 105 cells g of core material−1 while unattached cells numbered about 105 cells ml of groundwater−1. Attached and unattached acidophilic heterotrophs were observed throughout the depth profile. In contrast, acidophilic chemolithotrophs were not found attached to the rock but were commonly observed in the groundwater. Attached communities were composed of low numbers (<40 CFU g−1) of neutrophilic heterotrophs that exhibited a high degree of morphologic diversity, while unattached communities contained higher numbers (ca. 103 CFU ml−1) of neutrophilic heterotrophs of limited diversity. Sulfate-reducing bacteria were restricted to the deepest samples of both core and groundwater. 16S ribosomal DNA sequence analysis of attached, acidophilic isolates indicated that organisms closely related to heterotrophic, acidophilic mesophiles such as Acidiphilium organovorum and, surprisingly, to the moderately thermophilic Alicyclobacillus acidocaldariuswere present. The results indicate that viable (but possibly inactive) microorganisms were present in the buried ore and that there was substantial distinction in biomass and physiological capabilities between attached and unattached populations.

Comparison of extracellular enzyme activities and community composition of attached and free-living bacteria in porous medium columns

ABSTRACT Free-living and surface-associated microbial communities in sand-packed columns perfused with groundwater were compared by examination of compositional and functional characteristics. The composition of the microbial communities was assessed by bulk DNA extraction, PCR amplification of 16S ribosomal DNA fragments, separation of these fragments by denaturing gradient gel electrophoresis, and sequence analysis. Community function was assessed by measurement of β-glucosidase and aminopeptidase extracellular enzyme activities. Free-living populations in the aqueous phase exhibited a greater diversity of phylotypes than populations associated with the solid phase. The attached bacterial community displayed significantly greater β-glucosidase and aminopeptidase enzyme activities per volume of porous medium than those of the free-living community. On a per-cell basis, the attached community had a significantly higher cell-specific aminopeptidase enzyme activity (1.07 × 10−7 nmol cell−1 h−1) than the free-living community (5.02 × 10−8 nmol cell−1 h−1). Conversely, the free-living community had a significantly higher cell-specific β-glucosidase activity (1.92 × 10−6 nmol cell−1 h−1) than the surface-associated community (6.08 × 10−7 nmol cell−1 h−1). The compositional and functional differences observed between these two communities may reflect different roles for these distinct but interacting communities in the decomposition of natural organic matter or biodegradation of xenobiotics in aquifers.

Bacterial Communities Associated with the Digestive Tract of the Predatory Ground Beetle, Poecilus chalcites, and Their Modification by Laboratory Rearing and Antibiotic Treatment

Ground beetles such as Poecilus chalcites (Coleoptera: Carabidae) are beneficial insects in agricultural systems where they contribute to the control of insect and weed pests. We assessed the complexity of bacterial communities occurring in the digestive tracts of field-collected P. chalcites using terminal restriction fragment length polymorphism analyses of polymerase chain reaction-amplified 16S rRNA genes. Bacterial identification was performed by the construction of 16S rRNA gene clone libraries and sequence analysis. Intestinal bacteria in field-collected beetles were then compared to those from groups of beetles that were reared in the lab on an artificial diet with and without antibiotics. Direct cell counts estimated 1.5 × 108 bacteria per milliliter of gut. The digestive tract of field-collected P. chalcites produced an average of 4.8 terminal restriction fragments (tRF) for each beetle. The most abundant clones were affiliated with the genus Lactobacillus, followed by the taxa Enterobacteriaceae, Clostridia, and Bacteriodetes. The majority of the sequences recovered were closely related to those reported from other insect gastrointestinal tracts. Lab-reared beetles produced fewer tRF, an average of 3.1 per beetle, and a reduced number of taxa with a higher number of clones from the family Enterobacteriaceae compared to the field-collected beetles. Antibiotic treatment significantly (p < 0.05) reduced the number of tRF per beetle and selected for a less diverse set of bacterial taxa. We conclude that the digestive tract of P. chalcites is colonized by a simple community of bacteria that possess autochthonous characteristics. Laboratory-reared beetles harbored the most common bacteria found in field-collected beetles, and these bacterial communities may be manipulated in the laboratory with the addition of antibiotics to the diet to allow study of functional roles.

Microbiological Comparison of Core and Groundwater Samples Collected from a Fractured Basalt Aquifer with that of Dialysis Chambers Incubated In Situ

Microorganisms associated with basalt core were compared to those suspended in groundwater pumped from the same well in the eastern Snake River Plain Aquifer (Idaho, USA). Two wells located at different distances from the source of a mixed-waste plume in the fractured basalt aquifer were examined. In the well more distal from the plume source, an array of dialysis chambers filled with either deionized water or crushed basalt was equilibrated to compare the microorganisms collected in this fashion with those from core and groundwater samples collected in a traditional manner from the same well. The samples were characterized to determine the total amount of biomass, presence of specific populations or physiological groups, and potential community functions. Microorganisms and their activities were nearly undetectable in core and groundwater collected from the well farthest from the plume source and substantially enriched in both core and groundwater from the well closest to the plume source. In both wells, differences (statistically significant for some measures) were found between bacteria associated with the cores and those suspended in the groundwater. Significantly higher populations were found in the basalt- and water-filled dialysis chambers incubated in the open well compared with core and groundwater samples, respectively. For a given parameter, the variation among dialysis chambers incubated at different depths was much less than the high variation observed among core samples. Analyses on selected basalt- and water-filled dialysis chamber samples suggested that these two communities were compositionally similar but exhibited different potential functions. Documented knowledge of cell physiological changes associated with attachment and potential differences between attached and unattached communities in aquifers indicate that careful consideration should be given to the type of sample media (i.e., core, groundwater, substrata incubated in a well) used to represent a subsurface environment.

Understanding of Aquifer Microbiology is Tightly Linked to Sampling Approaches

Primary samples of groundwater or core are collected and analyzed to characterize the microbiology of aquifers and to predict biogeochemical transformations. Alternative sampling devices have been developed that are incubated for some length of time in the aquifer to accrue biomass for analysis. Considering data generated from different types of aquifer samples, it appears that the type of sample collected and analyzed may strongly influence the resulting view of aquifer microbiology. Borehole artifacts need to be rigorously considered when incubated substrata are used. The indigenous attached populations in deeper, fractured rock aquifers remain understudied and await new sampling approaches.

Bacterial Communities within Digestive Tracts of Ground Beetles (Coleoptera: Carabidae)

Abstract We identified the bacterial communities within the alimentary tracts of two granivorous ground beetles as a first step in the exploration of bacteria–ground beetle symbioses. Terminal-restriction fragment length polymorphism analyses of bacterial rRNA extracted from the guts of field-collected individuals of Harpalus pensylvanicus (DeGeer) and Anisodactylus sanctaecrucis (F.) (Coleoptera: Carabidae) revealed that gut-associated bacterial communities were of low diversity. Individuals from the same beetle species possessed similar bacterial community profiles, but the two species exhibited unique profiles. Bacterial 16S rRNA clone libraries constructed for the two beetle species showed that H. pensylvanicus had a more diverse community (six operational taxonomic units [OTUs]) compared with A. sanctaecrucis (three OTUs). Only one OTU, closely related to Hafnia alvei, was common between the two beetle species. Cloned partial 16S rRNA sequences for each OTU were most closely matched to the following cultivated bacteria: Serratia sp., Burkholderia fungorum, and H. alvei and Phenylbacterium sp., Caedibacter sp., Spiroplasma sp., Enterobacter strain B-14, and Weissella viridescens, representing the divisions Alpha-, Beta- and Gammaproteobacteria, Mollicutes, and Bacilli. Some, but not all of these organisms have been previously associated with insects. The identification of bacteria uniquely and consistently associated with these ground beetles provides the basis for further investigation of species-specific functional roles.

Detection of Euryarchaeota and Crenarchaeota in an oxic basalt aquifer

Groundwater from an oxic, fractured basalt aquifer was examined for the presence of Archaea. DNA was extracted from cells concentrated from groundwater collected from five wells penetrating the eastern Snake River Plain Aquifer (Idaho, USA). Polymerase chain reaction (PCR) amplification of 16S rDNA was performed with Archaea-specific primers using both nested (ca. 200-bp product) and direct (ca. 600-bp product) PCR approaches. Estimates of the archaeal diversity were made by separating PCR products from all five wells by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis of partial 16S rDNA sequences from two wells was performed following cloning procedures. Archaea were detected in all wells and the number of DGGE bands per well ranged from two to nine and varied according to PCR approach. There were 30 unique clonal 16S rDNA partial sequences (ca. 600 bp) within a total of 100 clones that were screened from two wells. Twenty-two of the 16S rDNA fragments recovered from the aquifer were related to the Crenarchaeota and Euryarchaeota kingdoms (one large clade of clones in the former and six smaller clades in the latter), with sequences ranging from 23.7 to 95.4% similar to those found in other investigations. The presence of potentially thermophilic or methanogenic Archaea in this fully oxic aquifer may be related to deep thermal sources or elevated dissolved methane concentrations. Many sequences were similar to those that represent non-thermophilic Crenarchaeota of which there are no known cultured members and therefore no putative function.

Soil biology for resilient, healthy soil

What is a resilient, healthy soil? A resilient soil is capable of recovering from or adapting to stress, and the health of the living/biological component of the soil is crucial for soil resiliency. Soil health is tightly coupled with the concept of soil quality (table 1), and the terms are frequently used interchangeably. The living component of soil or soil biota represents a small fraction (<0.05% dry weight), but it is essential to many soil functions and overall soil quality. Some of these key functions or services for production agriculture are (1) nutrient provision and cycling, (2) pest and pathogen protection, (3) production of growth factors, (4) water availability, and (5) formation of stable aggregates to reduce the risks of soil erosion and increase water infiltration (table 2). Soil resources and their inherent biological communities are the foundation for agricultural production systems that sustain the human population. The rapidly increasing human population is expanding the demand for food, fiber, feed, and fuel, which is stretching the capacity of the soil resource and contributing to soil degradation. Soil degradation decreases a soils production capacity to directly supply human demands and decreases a soils functional capacity to perform numerous critical services, which…

Detection of fatty acids from intact microorganisms by molecular beam static secondary ion mass spectrometry.

We report the use of a surface analysis approach, static secondary ion mass spectrometry (SIMS) equipped with a molecular (ReO(4)(-)) ion primary beam, to analyze the surface of intact microbial cells. SIMS spectra of 28 microorganisms were compared to fatty acid profiles determined by gas chromatographic analysis of transesterfied fatty acids extracted from the same organisms. The results indicate that surface bombardment using the molecular primary beam cleaved the ester linkage characteristic of bacteria at the glycerophosphate backbone of the phospholipid components of the cell membrane. This cleavage enables direct detection of the fatty acid conjugate base of intact microorganisms by static SIMS. The limit of detection for this approach is approximately 10(7) bacterial cells/cm(2). Multivariate statistical methods were applied in a graded approach to the SIMS microbial data. The results showed that the full data set could initially be statistically grouped based upon major differences in biochemical composition of the cell wall. The gram-positive bacteria were further statistically analyzed, followed by final analysis of a specific bacterial genus that was successfully grouped by species. Additionally, the use of SIMS to detect microbes on mineral surfaces is demonstrated by an analysis of Shewanella oneidensis on crushed hematite. The results of this study provide evidence for the potential of static SIMS to rapidly detect bacterial species based on ion fragments originating from cell membrane lipids directly from sample surfaces.