Biomagnification of cadmium selenide quantum dots in a simple experimental microbial food chain.

Previous studies have shown that engineered nanomaterials can be transferred from prey to predator, but the ecological impacts of this are mostly unknown. In particular, it is not known if these materials can be biomagnified-a process in which higher concentrations of materials accumulate in organisms higher up in the food chain. Here, we show that bare CdSe quantum dots that have accumulated in Pseudomonas aeruginosa bacteria can be transferred to and biomagnified in the Tetrahymena thermophila protozoa that prey on the bacteria. Cadmium concentrations in the protozoa predator were approximately five times higher than their bacterial prey. Quantum-dot-treated bacteria were differentially toxic to the protozoa, in that they inhibited their own digestion in the protozoan food vacuoles. Because the protozoa did not lyse, largely intact quantum dots remain available to higher trophic levels. The observed biomagnification from bacterial prey is significant because bacteria are at the base of environmental food webs. Our findings illustrate the potential for biomagnification as an ecological impact of nanomaterials.

Relationships between sediment microbial communities and pollutants in two California salt marshes.

Salt marshes are important ecosystems whose plant and microbial communities can alter terrestrially derived pollutants prior to coastal water discharge. However, knowledge regarding relationships between anthropogenic pollutant levels and salt marsh microbial communities is limited, and salt marshes on the West Coast of the United States are rarely examined. In this study, we investigated the relationships between microbial community composition and 24 pollutants (20 metals and 4 organics) in two California salt marshes. Multivariate ordination techniques were used to assess how bacterial community composition, as determined by terminal restriction fragment length polymorphism and phospholipid fatty acid analyses, was related to pollution. Sea urchin embryo toxicity measurements and plant tissue metabolite profiles were considered two other biometrics of pollution. Spatial effects were strongly manifested across marshes and across channel elevations within marshes. Utilizing partial canonical correspondence analysis, an ordination technique new to microbial ecology, we found that several metals were strongly associated with microbial community composition after accounting for spatial effects. The major patterns in plant metabolite profiles were consistent with patterns across microbial community profiles, but sea urchin embryo assays, which are commonly used to evaluate ecological toxicity, had no identifiable relationships with pollution. Whereas salt marshes are generally dynamic and complex habitats, microbial communities in these marshes appear to be relatively sensitive indicators of toxic pollutants.

Extracellular DNA in single- and multiple-species unsaturated biofilms.

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.

A mechanistic model of runoff-associated fecal coliform fate and transport through a coastal lagoon.

Fecal coliform (FC) contamination in coastal waters is an ongoing public health problem worldwide. Coastal wetlands and lagoons are typically expected to protect coastal waters by attenuating watershed pollutants including FC bacteria. However, new evidence suggests that coastal lagoons or marshes can also be a source of high indicator organism concentrations in coastal waters. We asked for a Mediterranean-type climate, what is the fate of runoff-associated FC through a coastal lagoon? To address this question, we developed a mass balance-based, mechanistic model of FC concentration through a coastal lagoon and simulated, for summer and winter conditions, FC within the lagoon water column, lagoon sediments, and in the ocean water just downstream of the lagoon mouth. Our model accounts for advective flow and dispersion, decay and sedimentation and resuspension of FC-laden sediments during high flow, erosional conditions. Under low flow conditions that occur in the summer, net FC decay and FC storage in lagoon sediments are predicted. Under high flow conditions that occur in the winter, FC-laden sediments are predicted to erode, resuspend and flow out of the lagoon where they elevate FC concentrations in the coastal ocean. For both seasonal conditions, the predicted water column FC concentrations were within an order of magnitude of field measurements for a reference site in southern California. Our results suggest that there are seasonally varying roles for coastal lagoons in mediating FC contamination to coastal waters.

Influence of drying-rewetting frequency on soil bacterial community structure.

Soil drying and rewetting represents a common physiological stress for the microbial communities residing in surface soils. A drying-rewetting cycle may induce lysis in a significant proportion of the microbial biomass and, for a number of reasons, may directly or indirectly influence microbial community composition. Few studies have explicitly examined the role of drying-rewetting frequency in shaping soil microbial community structure. In this experiment, we manipulated soil water stress in the laboratory by exposing two different soil types to 0, 1, 2, 4, 6, 9, or 15 drying-rewetting cycles over a 2-month period. The two soils used for the experiment were both collected from the Sedgwick Ranch Natural Reserve in Santa Ynez, CA, one from an annual grassland, the other from underneath an oak canopy. The average soil moisture content over the course of the incubation was the same for all samples, compensating for the number of drying-rewetting cycles. At the end of the 2-month incubation we extracted DNA from soil samples and characterized the soil bacterial communities using the terminal restriction fragment length polymorphism (T-RFLP) method. We found that drying-rewetting regimes can influence bacterial community composition in oak but not in grass soils. The two soils have inherently different bacterial communities; only the bacteria residing in the oak soil, which are less frequently exposed to moisture stress in their natural environment, were significantly affected by drying-rewetting cycles. The community indices of taxonomic diversity and richness were relatively insensitive to drying-rewetting frequency. We hypothesize that drying-rewetting induced shifts in bacterial community composition may partly explain the changes in C mineralization rates that are commonly observed following exposure to numerous drying-rewetting cycles. Microbial community composition may influence soil processes, particularly in soils exposed to a significant level of environmental stress.

Comparison of subsurface and surface soil bacterial communities in California grassland as assessed by terminal restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes.

The integrated biomass beneath the surface horizon in unsaturated soils is large and potentially important in nutrient and carbon cycling. Compared to surface soils, the ecology of these subsurface soils is weakly understood, particularly in terms of the composition of bacterial communities. We compared soil bacterial communities along two vertical transects by terminal restriction fragment length polymorphisms (TRFLPs) of PCR-amplified 16S rRNA genes to determine how surface and deep bacterial communities differ. DNA yield from soils collected from two Mediterranean grassland transects decreased exponentially from the surface to 4 m deep. Richness, as assessed by the number of peaks obtained after restriction with HhaI, MspI, RsaI, or HaeIII, and diversity, as assessed by the Shannon diversity indices, were lowest in the deepest sample. Lower diversity at depth is consistent with species-energy theory, which would predict relatively low diversity in the low organic matter horizons. Principal components analysis suggested that, in terms of HhaI and HaeIII generated TRFLPs, bacterial communities differed between depths. The most abundant amplicons cloned from the deepest sample contained sequences with restriction sites consistent with the largest peaks observed in TRFLPs generated from deep samples. These more abundant operational taxonomic units (OTUs) appeared related to Pseudomonas and Variovorax. Several OTUs were more related to each other than any previously described ribotypes. These OTUs showed similarity to bacteria from the divisions Actinobacteria and Firmicutes.

Comparison of free-living and particle-associated bacterial communities in a coastal lagoon.

We analyzed, by terminal restriction fragment length polymorphisms (T-RFLPs) of PCR-amplified 16S rDNA, microbial diversity in water collected during the dry and wet seasons in a human-impacted coastal lagoon. Water samples were fractionated by prefiltration to differentiate particle-associated and free-living microbes. From a sample collected during the dry season, prefiltration removed 23 to 44% of bacteria, as assessed by direct counts and MPN, and 99% of phytoplankton, as assessed by chlorophyll a. Restriction with RsaI yielded fewer peaks than restriction with HhaI. Diversity indices calculated from T-RFLPs were higher in the lagoon than adjoining coastal waters and higher in the particle-associated than the free-living fraction. In the dry season, peaks found only in bulk and particle-associated T-RFLPs were consistent with plastid and cyanobacterial ribotypes. These peaks matched those observed in the sequence of a clone generated from the bulk fraction with plastid and cyanobacterial specific primers. This clone appeared related to plastids found in the diatom genus Skeletonema. Principal component analysis of T-RFLPs suggested that the difference between the free-living and particle-associated fractions in the dry season was less than temporal variability in this lagoon and that these fractions varied significantly only in the wet season. This fractionation of microbial populations into particle-associated and free-living guilds during the wet season, when water residence time in the lagoon is relatively low, suggests an external source of particle-associated bacteria such as erosion of upland soils by runoff.

Elongation correlates with nutrient deprivation in Pseudomonas aeruginosa-unsaturates biofilms.

Bacteria in nature frequently grow as biofilms, yet little is known regarding how biofilm bacteria morphologically adapt to low nutrient availability, which is common in unsaturated environments such as the terrestrial subsurface or on plant leaves. For unsaturated biofilms, in which the substratum may provide all nutrients, what are the relationships between nutrition and cell size and shape-the simplest metrics of cellular morphology? To address this question, we cultured Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium that is environmentally and medically important, on membranes overlaying solid media, and then measured cellular dimensions using atomic force microscopy (AFM). Nutrition was controlled chemically by media composition and physically by stacking membranes to increase the path length for nutrient diffusion. Under conditions of carbon-nitrogen imbalance, low carbon bioavailability, or increased nutrient diffusional path length, cells elongated while maintaining constant width. A mathematical relationship suggests that, by elongating, biofilm bacteria strategically enlarge their nutrient collection surface without substantially changing the ratio of surface area to volume (SA/V). We conclude that P. aeruginosa growing as unsaturated biofilm with a planar nutrient source morphologically adapt to starvation by elongating. This adaptation, if generalizable, differs from a better-understood starvation response (i.e., cell size decreases; thus SA/V in-creases) for planktonic bacteria in well-mixed environments.

Assessing the role of Pseudomonas aeruginosa surface-active gene expression in hexadecane biodegradation in sand.

Low pollutant substrate bioavailability limits hydrocarbon biodegradation in soils. Bacterially produced surface-active compounds, such as rhamnolipid biosurfactant and the PA bioemulsifying protein produced by Pseudomonas aeruginosa, can improve bioavailability and biodegradation in liquid culture, but their production and roles in soils are unknown. In this study, we asked if the genes for surface-active compounds are expressed in unsaturated porous media contaminated with hexadecane. Furthermore, if expression does occur, is biodegradation enhanced? To detect expression of genes for surface-active compounds, we fused the gfp reporter gene either to the promoter region of pra, which encodes for the emulsifying PA protein, or to the promoter of the transcriptional activator rhlR. We assessed green fluorescent protein (GFP) production conferred by these gene fusions in P. aeruginosa PG201. GFP was produced in sand culture, indicating that the rhlR and pra genes are both transcribed in unsaturated porous media. Confocal laser scanning microscopy of liquid drops revealed that gfp expression was localized at the hexadecane-water interface. Wild-type PG201 and its mutants that are deficient in either PA protein, rhamnolipid synthesis, or both were studied to determine if the genetic potential to make surface-active compounds confers an advantage to P. aeruginosa biodegrading hexadecane in sand. Hexadecane depletion rates and carbon utilization efficiency in sand culture were the same for wild-type and mutant strains, i.e., whether PG201 was proficient or deficient in surfactant or emulsifier production. Environmental scanning electron microscopy revealed that colonization of sand grains was sparse, with cells in small monolayer clusters instead of multilayered biofilms. Our findings suggest that P. aeruginosa likely produces surface-active compounds in sand culture. However, the ability to produce surface-active compounds did not enhance biodegradation in sand culture because well-distributed cells and well-distributed hexadecane favored direct contact to hexadecane for most cells. In contrast, surface-active compounds enable bacteria in liquid culture to adhere to the hexadecane-water interface when they otherwise would not, and thus production of surface-active compounds is an advantage for hexadecane biodegradation in well-dispersed liquid systems.

Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis.

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.

Water Content Mediated Microaerophilic Toluene Biodegradation in Arid Vadose Zone Materials.

We investigated the conditions promoting toluene biodegradation for gasoline-contaminated near-surface (0.6 m depth) and subsurface (4.7 to 5.0 m depth) vadose zone soils sampled from an arid environment. At both depths, water addition was required for toluene biodegradation to occur. In near-surface samples, no inorganic nutrient addition was necessary and (i) biodegradation was fastest at 0.0 MPa, (ii) biodegradation rates decreased with decreasing water potential down to ?1.0 MPa, and (iii) biodegradation was undetectable at ?1.5 MPa. For subsurface material, toluene depletion was stimulated either by slurrying with a nutrient solution or by adjusting the moisture content to 20% (0.0 MPa) with nutrient solution and lowering the oxygen concentration (to effectively 1 mg L-1 in the aqueous phase). Thus, in the subsurface material, toluene depletion was microaerobic and nutrient-limited, occurring only under low oxygen and with inorganic nutrient addition. Our studies implicate microaerophily as an important characteristic of the toluene-degrading communities in these dry soils, with soil water as a primary controller of oxygen availability.

Probing biopolymers with the atomic force microscope: a review.

This short review presents an overview of atomic force microscopy (AFM) of biopolymers and specific examples of some of the biopolymers that have been analyzed by AFM. These specific examples include extracellular polymeric substances on the surfaces of bacterial biofilms, condensed DNA, DNA constructs, and DNA-protein interactions. In addition, two examples are presented for AFM analyses of proteins: laminin flexing its arms in solution and neurofilaments entropically brushing away the space around themselves.

Physical morphology and surface properties of unsaturated Pseudomonas putida biofilms.

Unsaturated biofilms of Pseudomonas putida, i.e., biofilms grown in humid air, were analyzed by atomic force microscopy to determine surface morphology, roughness, and adhesion forces in the outer and basal cell layers of fresh and desiccated biofilms. Desiccated biofilms were equilibrated with a 75.5% relative humidity atmosphere, which is far below the relative humidity of 98 to 99% at which these biofilms were cultured. In sharp contrast to the effects of drying on biofilms grown in fluid, we observed that drying caused little change in morphology, roughness, or adhesion forces in these unsaturated biofilms. Surface roughness for moist and dry biofilms increased approximately linearly with increasing scan sizes. This indicated that the divides between bacteria contributed more to overall roughness than did extracellular polymeric substances (EPS) on individual bacteria. The EPS formed higher-order structures we termed mesostructures. These mesostructures are much larger than the discrete polymers of glycolipids and proteins that have been previously characterized on the outer surface of these gram-negative bacteria.

Water stress effects on toluene biodegradation by Pseudomonas putida.

We quantified the effects of matric and solute water potential on toluene biodegradation by Pseudomonas putida mt-2, a bacterial strain originally isolated from soil. Across the matric potential range of 0 to -1.5 MPa, growth rates were maximal for P. putida at -0.25 MPa and further reductions in the matric potential resulted in concomitant reductions in growth rates. Growth rates were constant over the solute potential range 0 to -1.0 MPa and lower at -1.5 MPa. First order toluene depletion rate coefficients were highest at 0.0 MPa as compared to other matric water potentials down to -1.5 MPa. Solute potentials down to -1.5 MPa did not affect first order toluene depletion rate coefficients. Total yield (protein) and carbon utilization efficiency were not affected by water potential, indicating that water potentials common to temperate soils were not sufficiently stressful to change cellular energy requirements. We conclude that for P. putida: (1) slightly negative matric potentials facilitate faster growth rates on toluene but more negative water potentials result in slower growth, (2) toluene utilization rate per cell mass is highest without matric water stress and is unaffected by solute potential, (3) growth efficiency did not differ across the range of matric water potentials 0.0 to -1.5 MPa.

Toluene diffusion and reaction in unsaturated Pseudomonas putida biofilms.

Biofilms are frequently studied in the context of submerged or aquatic systems. However, much less is known about biofilms in unsaturated systems, despite their importance to such processes as food spoilage, terrestrial nutrient cycling, and biodegradation of environmental pollutants in soils. Using modeling and experimentation, we have described the biodegradation of toluene in unsaturated media by bacterial biofilms as a function of matric water potential, a dominant variable in unsaturated systems. We experimentally determined diffusion and kinetic parameters for Pseudomonas putida biofilms, then predicted biodegradation rates over a range of matric water potentials. For validation, we measured the rate of toluene depletion by intact biofilms and found the results to reasonably follow the model predictions. The diffusion coefficient for toluene through unsaturated P. putida biofilm averaged 1.3 x 10(7) cm(2)/s, which is approximately two orders of magnitude lower than toluene diffusivity in water. Our studies show that, at the scale of the microbial biofilm, the diffusion of toluene to biodegrading bacteria can limit the overall rate of biological toluene depletion in unsaturated systems. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 656-670, 1997.

Coupling transport and biodegradation of VOCs in surface and subsurface soils.

Volatile organic chemicals present at Superfund sites preferentially partition into the soil gas and may be available for microbial degradation. A simple mass transfer model for biodegradation for volatile substrates has been developed for the aerobic decomposition of aromatic and aliphatic hydrocarbons. The mass transfer analysis calculates diffusive fluxes from soil gas through water and membrane films and into the cell. This model predicts an extreme sensitivity of potential biodegradation rates to the air-water partition coefficients of the compounds. Aromatic hydrocarbons are removed rapidly while the aliphatic hydrocarbons are much slower by orders of magnitude. Furthermore, oxygen transfer is likely to limit aromatic hydrocarbon degradation rates. The model presents results that cast doubt on the practicality of using methane or propane for the co-metabolic destruction of trichloroethylene in a gas phase bioreactor. Toluene as a primary substrate has better mass transfer characteristics to achieve more efficient trichloroethylene degradation. Hence, in sites where these contaminants coexist, bioremediation could be improved.