Aaron L. Mills

Multi-scale variation in spatial heterogeneity for microbial community structure in an eastern Virginia agricultural field

To better understand the distribution of soil microbial communities at multiple spatial scales, a survey was conducted to examine the spatial organization of community structure in a wheat field in eastern Virginia (USA). Nearly 200 soil samples were collected at a variety of separation distances ranging from 2.5 cm to 11 m. Whole-community DNA was extracted from each sample, and community structure was compared using amplified fragment length polymorphism (AFLP) DNA fingerprinting. Relative similarity was calculated between each pair of samples and compared using geostatistical variogram analysis to study autocorrelation as a function of separation distance. Spatial autocorrelation was found at scales ranging from 30 cm to more than 6 m, depending on the sampling extent considered. In some locations, up to four different correlation length scales were detected. The presence of nested scales of variability suggests that the environmental factors regulating the development of the communities in this soil may operate at different scales. Kriging was used to generate maps of the spatial organization of communities across the plot, and the results demonstrated that bacterial distributions can be highly structured, even within a habitat that appears relatively homogeneous at the plot and field scale. Different subsets of the microbial community were distributed differently across the plot, and this is thought to be due to the variable response of individual populations to spatial heterogeneity associated with soil properties.

Bacterial transport in porous media: Evaluation of a model using laboratory observations

The factors that control the transport of bacteria through porous media are not well understood. The relative importance of the processes of dispersion, of immobilization of bacterial cells by various mechanisms (deposition), and of subsequent release of these trapped cells (entrainment) in describing transport has not been elucidated experimentally. Moreover, the variability of the phenomenological coefficients used to model these processes, given changes in such primary factors as grain size, organism, and ionic strength of the water, is unknown. We report results of fitting solutions of an advection-dispersion equation, modified to account for deposition and entrainment, to breakthrough curves from packed sand columns using two sizes of sand, two ionic strengths of the carrier solution, and two organisms with different sizes. A solution to the advection-dispersion equation including three processes, that is, dispersion, deposition, and entrainment, provides a match to the data that is superior to that achieved by solutions ignoring one of the processes. Fitted values of the coefficient describing deposition vary in a consistent manner with the control variables (organism, grain size, and ionic strength) and are generally within one order of magnitude of those predicted on the basis of theory.

The influence of mineralogy and solution chemistry on the attachment of bacteria to representative aquifer materials.

Abstract The rate and extent of bacterial attachment to mineral surfaces (chips of quartz, muscovite, limestone, and Fe-hydroxide-coated quartz and muscovite) was investigated by counting the numbers of bacterial cells (Lula-D, an indigenous groundwater organism) associated with each surface over time. The degree of attachment of cells to mineral surfaces was correlated with the sign of the surface charge as estimated from literature values for the isoelectric point; attachment of the negatively charged bacteria was much greater to the positively charged surfaces of limeston, Fe-hydroxide-coated quartz, and Fe-hydroxide-coated muscovite than to the negatively charged surfaces of clean quartz and clean muscovite. Batch experiments determined that the numbers of bacteria attached to the clean muscovite increased with increasing ionic strength of the solution, and the numbers attached to clean quartz were greater at pH 5 than at pH 7. In columns of clean quartz sand under saturated flow conditions, bacteria initially broke through at 1 pore volume but continued to elute for at least 7 pore volumes. Columns of Fe-hydroxide-coated sand retained more of the bacteria added to the columns (99.9% vs 97.4%), and the elution of cells ceased after the primary breakthrough. The results indicate that surface interactions between the mineral grains in an aquifer and the bacterial cells must play an essential role in determining the movement of bacteria through saturated porous media.

Spatial distribution of deposited bacteria following Miscible Displacement Experiments in intact cores

Miscible displacement experiments were performed on intact sand columns ranging from 15 to 60 cm in length to determine whether bacterial deposition varies at the centimeter scale within aquifer sediments. A 1-pore-volume pulse of radiolabeled cell suspension was introduced into the columns followed by a 2-pore-volume flush of artificial groundwater. The columns were then drained and dissected along the axis of flow. At ∼1-cm intervals, nine samples were removed for the enumeration of sediment-associated bacteria. Concentrations of sediment-associated (deposited) bacteria varied by up to 2 orders of magnitude in the direction perpendicular to flow demonstrating that bacterial deposition cannot be described mechanistically by a single rate coefficient. Incorporation of a distribution of sediment size and porosity values into Monte Carlo simulations indicates that physical heterogeneities are only partially responsible for the observed variability in deposited bacteria. A simple first-order model (classic filtration theory) adequately described the average spatial distribution of bacteria with depth within the 15-cm column. For the longer columns, however, the average concentration of deposited bacteria did not decrease exponentially with depth. A second-order model, modified to include an influent suspension of bacteria consisting of two subpopulations with separate sticking efficiencies (dual-alpha population), was required to describe the observed decreases of deposited bacteria with depth. A sensitivity analysis was performed with a first-order dual-alpha model to understand the effects of an influent suspension with two subpopulations of bacteria on the decrease of deposited bacteria with flow path length. Numerical simulations show that even for small fractions (0.01) of nonsticky bacteria, the decrease in deposited bacteria may deviate substantially from the exponential decrease expected from colloid-filtration theory. Results from experimental as well as numerical studies demonstrate the importance of column dissections for understanding bacterial deposition in saturated porous media.

Effect of surface coatings, grain size, and ionic strength on the maximum attainable coverage of bacteria on sand surfaces.

The injection of bacteria in the subsurface has been identified as a potential method for in situ cleanup of contaminated aquifers. For high bacterial loadings, the presence of previously deposited bacteria can result in decreased deposition rates--a phenomenon known as blocking. Miscible displacement experiments were performed on short sand columns (approximately 5 cm) to determine how bacterial deposition on positively charged metal-oxyhydroxide-coated sands is affected by the presence of previously deposited bacteria. Approximately 8 pore volumes of a radiolabeled bacterial suspension at a concentration of approximately 1 x 10(9) cells ml-1 were introduced into the columns followed by a 2-pore-volume flush of cell-free buffer. It was found that the presence of Al- and Fe-coated sand increased both deposition rates and maximum fractional surface coverage of bacteria on the sediment surfaces. The effect of grain size on maximum bacterial retention capacity, however, was not significant. Decreasing ionic strength from 10(-1) to 10(-2) M KCl resulted in noticeable decreases in sticking efficiency (alpha) and maximum surface coverage (thetamax) for clean silica sand--results consistent with DLVO theory. In columns containing positively charged Al- and Fe-coated sands, however, changes in alpha and thetamax due to decreasing ionic strength were minimal. These findings demonstrate the importance of geochemical controls on the maximum bacterial retention capacity of sands.

Effect of bacterial cell shape on transport of bacteria in porous media

Parameters used to describe the transport of colloids (including bacteria) through porous media either implicitly or explicitly account for colloidal particle size but assume that the particles are spheres of uniform size. Bacteria found in soils and in aquifers exhibit a variety of shapes as well as sizes. We sought to determine if there exists a systematic effect of cell shape on the transport of bacteria in columns packed with clean quartz sand. A pulse of resting cells (14 strains of bacteria isolated from aquifers) suspended in an artificial groundwater was passed through a short column. Properties of the bacteria in the influent pulse were compared with those in the eluent from the columns. Cell shape, as quantified by the ratio of cell width to cell length, affects the transport of bacterial cells through porous media. In addition, the distributions of size and shape of cells in the effluent differed from those in the influent suspension with cells in the effluent being smaller and rounder. Short rods with low water contact angles (a measure of cell-surface hydrophobicity) showed the greatest decrease in cell length during passage through short columns. 21 refs., 3 figs.

Relative effectiveness of kinetic analysis vs single point readings for classifying environmental samples based on community-level physiological profiles (CLPP)

The relative effectiveness of average-well-color-development-normalized single-point absorbance readings (AWCD) vs the kinetic parameters mu(m), lambda, A, and integral (AREA) of the modified Gompertz equation fit to the color development curve resulting from reduction of a redox sensitive dye from microbial respiration of 95 separate sole carbon sources in microplate wells was compared for a dilution series of rhizosphere samples from hydroponically grown wheat and potato ranging in inoculum densities of 1 x 10(4)-4 x 10(6) cells ml-1. Patterns generated with each parameter were analyzed using principal component analysis (PCA) and discriminant function analysis (DFA) to test relative resolving power. Samples of equivalent cell density (undiluted samples) were correctly classified by rhizosphere type for all parameters based on DFA analysis of the first five PC scores. Analysis of undiluted and 1:4 diluted samples resulted in misclassification of at least two of the wheat samples for all parameters except the AWCD normalized (0.50 abs. units) data, and analysis of undiluted, 1:4, and 1:16 diluted samples resulted in misclassification for all parameter types. Ordination of samples along the first principal component (PC) was correlated to inoculum density in analyses performed on all of the kinetic parameters, but no such influence was seen for AWCD-derived results. The carbon sources responsible for classification differed among the variable types with the exception of AREA and A, which were strongly correlated. These results indicate that the use of kinetic parameters for pattern analysis in CLPP may provide some additional information, but only if the influence of inoculum density is carefully considered.

Impact of dilution on microbial community structure and functional potential: comparison of numerical simulations and batch culture experiments

ABSTRACT A series of microcosm experiments was performed using serial dilutions of a sewage microbial community to inoculate a set of batch cultures in sterile sewage. After inoculation, the dilution-defined communities were allowed to regrow for several days and a number of community attributes were measured in the regrown assemblages. Based upon a set of numerical simulations, community structure was expected to differ along the dilution gradient; the greatest differences in structure were anticipated between the undiluted–low-dilution communities and the communities regrown from the very dilute (more than 10−4) inocula. Furthermore, some differences were expected among the lower-dilution treatments (e.g., between undiluted and 10−1) depending upon the evenness of the original community. In general, each of the procedures used to examine the experimental community structures separated the communities into at least two, often three, distinct groups. The groupings were consistent with the simulated dilution of a mixture of organisms with a very uneven distribution. Significant differences in community structure were detected with genetic (amplified fragment length polymorphism and terminal restriction fragment length polymorphism), physiological (community level physiological profiling), and culture-based (colony morphology on R2A agar) measurements. Along with differences in community structure, differences in community size (acridine orange direct counting), composition (ratio of sewage medium counts to R2A counts, monitoring of each colony morphology across the treatments), and metabolic redundancy (i.e., generalist versus specialist) were also observed, suggesting that the differences in structure and diversity of communities maintained in the same environment can be manifested as differences in community organization and function.

Bacterial utilization of formic and acetic acid in rainwater

Abstract Rain samples were collected aseptically, during 1983 and 1984, in Charlottesville, Virginia to determine the ability of bacteria in precipitation to utilize formate and acetate. The total number of bacteria, as counted by Acridine Orange Direct Counts, was one to two orders of magnitude greater from April to September (105 cells ml−1) than during the rest of the year (103−104 cells ml−1). Formate and acetate concentrations ranged between 6–23 and 3–9 μM, respectively and were higher from June to September. Heterotrophic uptake on the day of collection was not different from the controls, but after incubation at room temperature for a minimum of three days, the turnover rate constants were 0.14 and 0.17 h−1 for formate and acetate, respectively. Total bacterial counts increased an order of magnitude during that interval. These turnover rate constants were used to calculate losses of 44 and 24 μmoll−1day−1 of formic and acetic acid, respectively. Turnover times were 1.5 and 34 days for formate and acetate, respectively. This study demonstrated that there are viable microorganisms in the atmosphere capable of utilizing formate and acetate for growth.

Characterization of microbial communities using randomly amplified polymorphic DNA (RAPD).

Similarity among a number of aquatic microbial communities was examined using randomly amplified polymorphic DNA (RAPD), a common polymerase chain reaction (PCR)-based DNA fingerprinting technique. After amplification of whole-community DNA extracts, the PCR products were resolved by agarose gel electrophoresis and the band patterns compared to determine percent similarity. Twelve different primers were used to amplify approximately 100 fragments (total) from each DNA sample; the bands were scored as present or absent and the similarity between each sample was determined using Jaccards coefficient. From this information. dendrograms were constructed and a bootstrapping procedure was used to assess how well supported the tree topologies were. Principal component analyses were also conducted as a means of visualizing the relationships among samples. Results obtained for two different experimental systems (a pair of tidal creeks and several wells in a shallow groundwater aquifer) correlated well with the temporal and spatial variations in environmental regime at the sites confirming that arbitrarily primed PCR-based DNA fingerprinting techniques such as RAPD are useful means of discriminating among microbial communities and estimating community relatedness. Moreover, this approach has several advantages over other DNA-based procedures for whole-community analysis; it is less laborious and uses smaller quantities of DNA, making it amenable to sample-intensive monitoring, and it does not depend on culturing or the use of selective PCR primers.