Microbial astronauts: assembling microbial communities for advanced life support systems.

Extension of human habitation into space requires that humans carry with them many of the microorganisms with which they coexist on Earth. The ubiquity of microorganisms in close association with all living things and biogeochemical processes on Earth predicates that they must also play a critical role in maintaining the viability of human life in space. Even though bacterial populations exist as locally adapted ecotypes, the abundance of individuals in microbial species is so large that dispersal is unlikely to be limited by geographical barriers on Earth (i.e., for most environments "everything is everywhere" given enough time). This will not be true for microbial communities in space where local species richness will be relatively low because of sterilization protocols prior to launch and physical barriers between Earth and spacecraft after launch. Although community diversity will be sufficient to sustain ecosystem function at the onset, richness and evenness may decline over time such that biological systems either lose functional potential (e.g., bioreactors may fail to reduce BOD or nitrogen load) or become susceptible to invasion by human-associated microorganisms (pathogens) over time. Research at the John F. Kennedy Space Center has evaluated fundamental properties of microbial diversity and community assembly in prototype bioregenerative systems for NASA Advanced Life Support. Successional trends related to increased niche specialization, including an apparent increase in the proportion of nonculturable types of organisms, have been consistently observed. In addition, the stability of the microbial communities, as defined by their resistance to invasion by human-associated microorganisms, has been correlated to their diversity. Overall, these results reflect the significant challenges ahead for the assembly of stable, functional communities using gnotobiotic approaches, and the need to better define the basic biological principles that define ecosystem processes in the space environment.

Distribution of microbial communities associated with the dominant high marsh plants and sediments of the United States East Coast.

Microbial communities in the sediment and associated with the dominant type of standing dead plant were collected from the high marsh zones of 10 sites along the eastern coast of the United States from Maine to Florida. Microbial community composition was examined using T-RFLP, and bacterial and fungal abundance was determined microscopically. Within the sediment, community composition was strongly correlated with latitude, indicating that biogeographical factors are important determinants of sediment community composition, whereas abundance was positively and strongly correlated with sediment organic matter content. A strong biogeographical effect was observed for both bacterial and fungal abundance on standing dead plants, but there was no clear relationship between community composition and latitude. Microbial community composition was more similar among plants of the same type (i.e., related plant species) suggesting that plant type (i.e., substrate quality) is primarily responsible for the determining community composition on standing dead plants.

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.

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

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.

Transport of Escherichia coli in sand columns with constant and changing water contents.

Understanding how changes in volumetric water content (theta) affect bacterial adsorption could help reduce transport of pathogenic and indicator bacteria that may be present in infiltrating wastewater. Three flow regimes that simulated infiltration from a household septic system were evaluated: saturated, unsaturated with a constant volumetric water content theta (constant unsaturated flow), and unsaturated with cyclic changes in theta (variable unsaturated flow). Escherichia coli was suspended in artificial sewage (AS) and applied as step inputs to sand columns, with regular interruptions in input for variable unsaturated flow. A transport model was fit to the saturated and constant unsaturated flow breakthrough curves to determine retardation (R), the first-order filtration coefficient (mu), and the maximum outflow relative concentration (Cmax). The total cells transported as a fraction of input (tau) in all three flow regimes was calculated. Constant unsaturated flow resulted in a significantly lower Cmax (0.633) in comparison with saturated flow (0.803, P < or = 0.05), although unsaturated mu (0.0693 h(-1)) was not significantly different from saturated mu (0.0259 h(-1)). Constant unsaturated flow also resulted in a significantly smaller tau (0.617) than saturated (0.806) or variable unsaturated flow (0.734). In variable unsaturated flow, cell concentrations were out of phase with theta--as the column drained, cell concentrations in the outflow increased; and when a pulse of suspension was applied, cell concentrations decreased. Constant unsaturated flow is probably the best for removal of pathogenic bacteria because this regime resulted in lower maximum concentrations of E. coli and greater cell removal, in comparison with saturated and variable unsaturated flow.

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.

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 Jaccard's 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.

Bacterial enrichment at the gas-water interface of a laboratory apparatus.

The gas-water interface (GWI) is likely to have important effects on bacterial adsorption and transport in unsaturated porous media. A glass apparatus that isolated GWIs in ports above a flowthrough suspension of a groundwater bacterial isolate was used to represent unsaturated porous media. The surface microlayer was collected by placing a polycarbonate filter on the GWI. The filter was stained, and the bacteria were enumerated by direct count. The significance of five independent variables on the surface density of cells (s, in cells per square millimeter) was determined by nonlinear multiple regression. Three of the variables were shown to be significant: surfactant concentration (d), time (t), and bulk bacterial concentration (B). The surface density decreased with increasing d and increased with increasing t and B. When surfactant was absent, the GWI became highly enriched in bacteria. For example, when d = 0, 48 h < t < 72 h, and 5,000 cells mm(sup-3) < B < 10,000 cells mm(sup-3), s averaged 3.0 x 10(sup4) cells mm(sup-2). This surface density occupied about 6.0% of the GWI, and the surface microlayer concentration of cells was 190 times the bulk concentration. The other two variables: pH (p) and ionic strength (I) were shown to be insignificant. The strong effect of d and the lack of effect of I and p support the hypothesis that hydrophobic interaction dominates bacterial adsorption to the GWI.

Effect of solution ionic strength and iron coatings on mineral grains on the sorption of bacterial cells to quartz sand.

Understanding the interaction between bacterial cells and solid surfaces is essential to our attempts to quantify and predict the transport of microbes in groundwater aquifers, whether from the point of view of contamination or from that of bioremediation. The sorption of bacterial cells suspended in groundwater to porous medium grains was examined in batch studies. Bacterial sorption to clean quartz sand yielded equilibrium, linear, adsorption isotherms that varied with the bacterial strain used and the ionic strength of the aqueous solution. Values of K(d) (the slope of the linear sorption isotherm) ranged from 0.55 to 6.11 ml g, with the greatest sorption observed for the highest groundwater ionic strength. These findings are consistent with the interpretation that an increasingly compressed electrical double layer results in stronger adsorption between the like-charged mineral surface and the bacterial cells. When iron-oxyhydroxide-coated sand was used, however, all of the added bacteria were adsorbed up to a threshold of 6.93 x 10 cells g of coated sand, beyond which no further adsorption occurred. The irreversible, threshold adsorption is the result of a strong electrostatic attraction between the sesquioxide coating and the bacterial cells. Experimental results of adsorption in mixtures of quartz and Fe(III)-coated sand were successfully predicted by a simple additive model for sorption by the two substrate phases. Even small amounts of Fe(III)-coated sand in a mixture influenced the extent of adsorption of bacterial cells. A quantitative description of adsorption in the mixtures can be realized by using a linear isotherm for reversible adsorption to the quartz grains with a y intercept that represents the number of cells irreversibly adsorbed to the Fe(III)-coated sand.

Physical and chemical factors influencing transport of microorganisms through porous media.

Resting-cell suspensions of bacteria isolated from groundwater were added as a pulse to the tops of columns of clean quartz sand. An artificial groundwater solution (AGW) was pumped through the columns, and bacterial breakthrough curves were established and compared to test the effects of ionic strength of the AGW, cell size (by using strains of similar cell surface hydrophobicity but different size), mineral grain size, and presence of heterogeneities within the porous media on transport of the bacteria. The proportion of cells recovered in the effluent ranged from nearly 90% for AGW of a higher ionic strength (I = 0.0089 versus 0.00089 m), small cells (0.75-micron-diameter spheres versus 0.75 by 1.8-micron rods), and coarse-grained sand (1.0 versus 0.33 mm) to less than 1% for AGW of lower ionic strength, large cells, and fine-grained sand. Differences in the widths of peaks (an indicator of dispersion) were significant only for the cell size treatment. For treatments containing heterogeneities (a vein of coarse sand in the center of a bed of fine sand), doubly peaked breakthrough curves were obtained. The first peak represents movement of bacteria through the transmissive coarse-grained vein. The second peak is thought to be dominated by cells which have moved (due to dispersion) from the fine-grained matrix to the coarse-grained vein near the top of the column and thus had been retarded, but not retained, by the column. Strength of effects tests indicated that grain size was the most important factor controlling transport of bacteria over the range of values tested for all of the factors examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization.

The BLOLOG redox technology based on tetrazolium dye reduction as an indicator of sole-carbon-source utilization was evaluated as a rapid, community-level method to characterize and classify heterotrophic microbial communities. Direct incubation of whole environmental samples (aquatic, soil, and rhizosphere) in BIOLOG plates containing 95 separate carbon sources produced community-dependent patterns of sole-carbon-source utilization. Principal-component analysis of color responses quantified from digitized images of plates revealed distinctive patterns among microbial habitats and spatial gradients within soil and estuarine sites. Correlation of the original carbon source variables to the principal components gives a functional basis to distinctions among communities. Intensive spatial and temporal analysis of microbial communities with this technique can produce ecologically relevant classifications of heterotrophic microbial communities.

Impact of water column acidification on protozoan bacterivory at the lake sediment-water interface.

Although the impact of acidification on planktonic grazer food webs has been extensively studied, little is known about microbial food webs either in the water column or in the sediments. Protozoon-bacterium interactions were investigated in a chronically acidified (acid mine drainage) portion of a lake in Virginia. We determined the distribution, abundance, apparent specific grazing rate, and growth rate of protozoa over a pH range of 3.6 to 6.5. Protozoan abundance was lower at the most acidified site, while abundance, in general, was high compared with other systems. Specific grazing rates were uncorrelated with pH and ranged between 0.02 and 0.23 h, values similar to those in unacidified systems. The protozoan community from an acidified station was not better adapted (P = 0.95) to low-pH conditions than a community from an unacidified site (multivariate analysis of variance on growth rates for each community incubated at pHs 4, 5, and 6). Both communities had significantly lower (P < 0.05) growth rates at pHs 4 and 5 than at pH 6. Reduced protozoan growth rates coupled with high grazing rates and relatively higher bacterial yields (ratio of bacterial-protozoan standing stock) at low pH indicate reduced net protozoan growth efficiency and a metabolic cost of acidification to the protozoan community. However, the presence of an abundant, neutrophilic protozoan community and high bacterial grazing rates indicates that acidification of Lake Anna has not inhibited the bacterium-protozoon link of the sediment microbial food web.

Ion-chromatographic analysis of mixtures of ferrous and ferric iron.

Determinations of the aqueous iron species Fe(II) and Fe(III) are essential for a fully-informed understanding of redox processes involving iron. Most previous methods for speciation of iron have been based on the calorimetric determination of Fe(II) followed by reduction of Fe(III) and analysis for total iron. The indirect determination of Fe(III) and the consumption of relatively large sample volumes have limited the accuracy and utility of such methods. A method based on ion-chromatography has been developed for simultaneous direct determination of Fe(II) and Fe(III). Sample pretreatment involves only conventional filtration and acidification. No interferences with the iron(II) determination were found; in determination of iron(III) the only interference observed was an artifact peak (of unknown origin) that occurred only when iron(II) was present, and had an area that was a function of the iron(II) concentration and could hence be corrected for. Solutions of iron(II) free from iron(III) can be prepared by treatment with a mixture of hydrogen and nitrogen in the presence of palladium black as catalyst, to reduce the iron(III). Photoreduction of iron(III) in acidified samples increases the Fe(II)/Fe(III) ratio; no means of circumventing this effect is known, other than storing the samples in the dark and analysing them as soon as possible.

Tests of the critical assumptions of the dilution method for estimating bacterivory by microeucaryotes.

The critical assumptions of the dilution method for estimating grazing rates of microzooplankton were tested by using a community from the sediment-water interface of Lake Anna, Va. Determination of the appropriate computational model was achieved by regression analysis; the exponential model was appropriate for bacterial growth at Lake Anna. The assumption that the change in grazing pressure is linearly proportional to the dilution factor was tested by analysis of variance with a lack-of-fit test. There was a significant (P < 0.0001) linear (P > 0.05) relationship between the dilution factor and time-dependent change in ln bacterial abundance. The assumption that bacterial growth is not altered by possible substrate enrichment in the dilution treatment was tested by amending diluted water with various amounts of dissolved organic carbon (either yeast extract or extracted carbon from lake sediments). Additions of carbon did not significantly alter bacterial growth rates during the incubation period (24 h). On the basis of these results, the assumptions of the dilution method proved to be valid for the system examined.

Biogeochemical Conditions Favoring Magnetite Formation during Anaerobic Iron Reduction.

Several anaerobic bacteria isolated from the sediments of Contrary Creek, an iron-rich environment, produced magnetite when cultured in combinations but not when cultured alone in synthetic iron oxyhydroxide medium. When glucose was added as a carbon source, the pH of the medium decreased (to 5.5) and no magnetite was formed. When the same growth medium without glucose was used, the pH increased (to 8.5) and magnetite was formed. In both cases, Fe was released into the growth medium. Geochemical equilibrium equations with E(h) and pH as master variables were solved for the concentrations of iron and inorganic carbon that were observed in the system. Magnetite was predicted to be the dominant iron oxide formed at high pHs, while free Fe or siderite were the dominant forms of iron expected at low pHs. Thus, magnetite formation occurs because of microbial alteration of the local E(h) and pH conditions, along with concurrent reduction of ferric iron (direct biological reduction or abiological oxidation-reduction reactions).

Inadequacy of the eucaryote inhibitor cycloheximide in studies of protozoan grazing on bacteria at the freshwater-sediment interface.

Four guilds from a lake sediment-water interface microbial community were isolated and tested for sensitivity to cycloheximide (0.1 to 200 mg liter). Field experiments were conducted to compare the inhibition, dilution, and filtration methods for determining grazing rates. Cycloheximide inhibited anaerobic bacteria at 50 mg liter, and inhibition of bacterial growth was observed in the grazing experiments. The results show that the assumption of selective inhibition of heterotrophic eucaryotes was violated and preclude the use of cycloheximide in grazing experiments.

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.

Sulfate reduction in freshwater sediments receiving Acid mine drainage.

One arm of Lake Anna, Va., receives acid mine drainage (AMD) from Contrary Creek (SO(4) concentration = 2 to 20 mM, pH = 2.5 to 3.5). Acid-volatile sulfide concentrations, SO(4) reduction rates, and interstitial SO(4) concentrations were measured at various depths in the sediment at four stations in four seasons to assess the effects of the AMD-added SO(4) on bacterial SO(4) reduction. Acid-volatile sulfide concentrations were always an order of magnitude higher at the stations receiving AMD than at a control station in another arm of the lake that received no AMD. Summer SO(4) reduction rates were also an order of magnitude higher at stations that received AMD than at the control station (226 versus 13.5 mmol m day), but winter values were inconclusive, probably due to low sediment temperature (6 degrees C). Profiles of interstitial SO(4) concentrations at the AMD stations showed a rapid decrease with depth (from 1,270 to 6 muM in the top 6 cm) due to rapid SO(4) reduction. Bottom-water SO(4) concentrations in the AMD-receiving arm were highest in winter and lowest in summer. These data support the conclusion that there is a significant enhancement of SO(4) reduction in sediments receiving high SO(4) inputs from AMD.

The behavior of toxic compounds in the environment.

The generation of toxic and hazardous chemicals in the United States can be considered as one of the nation's healthier growth industries. The vast quantities of compounds generated provide opportunities for human exposure from a variety of both occupational and environmental sources. The solubility, adsorption, evaporation, and movement in the environment of these substances are discussed.