Importance of the Colmation Layer in the Transport and Removal of Cyanobacteria, Viruses, and Dissolved Organic Carbon during Natural Lake-Bank Filtration.

This study focused on the importance of the colmation layer in the removal of cyanobacteria, viruses, and dissolved organic carbon (DOC) during natural bank filtration. Injection-and-recovery studies were performed at two shallow (0.5 m deep), sandy, near-shore sites at the southern end of Ashumet Pond, a waste-impacted, kettle pond on Cape Cod, MA, that is subject to periodic blooms of cyanobacteria and continuously recharges a sole-source drinking-water aquifer. The experiment involved assessing the transport behaviors of bromide (conservative tracer), sp. IU625 (cyanobacterium, 2.6 ± 0.2 µm), AS-1 (tailed cyanophage, 110 nm long), MS2 (coliphage, 26 nm diameter), and carboxylate-modified microspheres (1.7 µm diameter) introduced to the colmation layer using a bag-and-barrel (Lee-type) seepage meter. The injectate constituents were tracked as they were advected across the pond water-groundwater interface and through the underlying aquifer sediments under natural-gradient conditions past push-point samplers placed at ∼30-cm intervals along a 1.2-m-long, diagonally downward flow path. More than 99% of the microspheres, IU625, MS2, AS-1, and ∼44% of the pond DOC were removed in the colmation layer (upper 25 cm of poorly sorted bottom sediments) at two test locations characterized by dissimilar seepage rates (1.7 vs. 0.26 m d). Retention profiles in recovered core material indicated that >82% of the attached IU625 were in the top 3 cm of bottom sediments. The colmation layer was also responsible for rapid changes in the character of the DOC and was more effective (by three orders of magnitude) at removing microspheres than was the underlying 20-cm-thick segment of sediment.

Mineralogical variables that control the antibacterial effectiveness of a natural clay deposit.

As antibiotic-resistant bacterial strains emerge and pose increased global health risks, new antibacterial agents are needed as alternatives to conventional antimicrobials. Naturally occurring antibacterial clays have been identified which are effective in killing antibiotic-resistant bacteria. This study examines a hydrothermally formed antibacterial clay deposit near Crater Lake, OR (USA). Our hypothesis is that antibacterial clays buffer pH and Eh conditions to dissolve unstable mineral phases containing transition metals (primarily Fe(2+)), while smectite interlayers serve as reservoirs for time release of bactericidal components. Model pathogens (Escherichia coli ATCC 25922 and Staphylococcus epidermidis ATCC 14990) were incubated with clays from different alteration zones of the hydrothermal deposit. In vitro antibacterial susceptibility testing showed that reduced mineral zones were bactericidal, while more oxidized zones had variable antibacterial effect. TEM images showed no indication of cell lysis. Cytoplasmic condensation and cell wall accumulations of <100 nm particles were seen within both bacterial populations. Electron energy loss analysis indicates precipitation of intracellular Fe(3+)-oxide nanoparticles (<10 nm) in E. coli after 24 h. Clay minerals and pyrite buffer aqueous solutions to pH 2.5-3.1, Eh > 630 mV and contain elevated level (mM) of soluble Fe (Fe(2+) and Fe(3+)) and Al(3+). Our interpretation is that rapid uptake of Fe(2+) impairs bacterial metabolism by flooding the cell with excess Fe(2+) and overwhelming iron storage proteins. As the intracellular Fe(2+) oxidizes, it produces reactive oxygen species that damage biomolecules and precipitates Fe-oxides. The ability of antibacterial clays to buffer pH and Eh in chronic non-healing wounds to conditions of healthy skin appears key to their healing potential and viability as an alternative to conventional antibiotics.

Effect of dissolved organic carbon on the transport and attachment behaviors of Cryptosporidium parvum oocysts and carboxylate-modified microspheres advected through temperate humic and tropical volcanic agricultural soil.

Transport of Cryptosporidium parvum oocysts and microspheres in two disparate (a clay- and Fe-rich, volcanic and a temperate, humic) agricultural soils were studied in the presence and absence of 100 mg L(-1) of sodium dodecyl benzene sulfonate (SDBS), and Suwannee River Humic Acid (SRHA) at pH 5.0-6.0. Transport of carboxylate-modified, 1.8 µm microspheres in soil columns was highly sensitive to the nature of the dissolved organic carbon (DOC), whereas oocysts transport was more affected by soil mineralogy. SDBS increased transport of microspheres from 48% to 87% through the tropical soil and from 43% to 93% in temperate soil. In contrast, SRHA reduced transport of microspheres from 48% to 28% in tropical soil and from 43% to 16% in temperate soil. SDBS also increased oocysts transport through the temperate soil 5-fold, whereas no oocyst transport was detected in tropical soil. SRHA had only a nominal effect in increasing oocysts transport in tropical soil, but caused a 6-fold increase in transport through the temperate soil. Amendments of only 4 mg L(-1) SRHA and SDBS decreased oocyst hydrophobicity from 66% to 20% and from 66% to 5%, respectively. However, SDBS increased microsphere hydrophobicity from 16% to 33%. Soil fines, which includes clays, and SRHA, both caused the oocysts zeta potential (ζ) to become more negative, but caused the highly hydrophilic microspheres to become less negatively charged. The disparate behaviors of the two colloids in the presence of an ionic surfactant and natural organic matter suggest that microspheres may not be suitable surrogates for oocysts in certain types of soils. These results indicate that whether or not DOC inhibits or promotes transport of oocysts and microspheres in agricultural soils and by how much, depends not only on the surface characteristics of the colloid, but the nature of the DOC and the soil mineralogy.

Effects on groundwater microbial communities of an engineered 30-day in situ exposure to the antibiotic sulfamethoxazole.

Effects upon microbial communities from environmental exposure to concentrations of antibiotics in the µg L(-1) range remain poorly understood. Microbial communities from an oligotrophic aquifer (estimated doubling rates of only once per week) that were previously acclimated (AC) or unacclimated (UAC) to historical sulfamethoxazole (SMX) contamination, and a laboratory-grown Pseudomonas stutzeri strain, were exposed to 240-520 µg L(-1) SMX for 30 days in situ using filter chambers allowing exposure to ambient groundwater, but not to ambient microorganisms. SMX-exposed UAC bacterial communities displayed the greatest mortality and impairment (viable stain assays), the greatest change in sensitivity to SMX (dose-response assays), and the greatest change in community composition (Terminal Restriction Fragment Length Polymorphism; T-RFLP). The sul1 gene, encoding resistance to SMX at clinically relevant levels, and an element of Class I integrons, was not detected in any community. Changes in microbial community structure and SMX resistance over a short experimental period in previously nonexposed, slow-growing aquifer communities suggest concentrations of antibiotics 2-3 orders of magnitude less than those used in clinical applications may influence ecological function through changes in community composition, and could promote antibiotic resistance through selection of naturally resistant bacteria.

Differential effects of dissolved organic carbon upon re-entrainment and surface properties of groundwater bacteria and bacteria-sized microspheres during transport through a contaminated, sandy aquifer.

Injection-and-recovery studies involving a contaminated, sandy aquifer (Cape Cod, Massachusetts) were conducted to assess the relative susceptibility for in situ re-entrainment of attached groundwater bacteria (Pseudomonas stuzeri ML2, and uncultured, native bacteria) and carboxylate-modified microspheres (0.2 and 1.0 µm diameters). Different patterns of re-entrainment were evident for the two colloids in response to subsequent injections of groundwater (hydrodynamic perturbation), deionized water (ionic strength alteration), 77 µM linear alkylbenzene sulfonates (LAS, anionic surfactant), and 76 µM Tween 80 (polyoxyethylene sorbitan monooleate, a very hydrophobic nonionic surfactant). An injection of deionized water was more effective in causing detachment of micrsopheres than were either of the surfactants, consistent with the more electrostatic nature of microsphere's attachment, their extreme hydrophilicity (hydrophilicity index, HI, of 0.99), and negative charge (zeta potentials, ζ, of -44 to -49 mv). In contrast, Tween 80 was considerably more effective in re-entraining the more-hydrophobic native bacteria. Both the hydrophilicities and zeta potentials of the native bacteria were highly sensitive to and linearly correlated with levels of groundwater dissolved organic carbon (DOC), which varied modestly from 0.6 to 1.3 mg L(-1). The most hydrophilic (0.52 HI) and negatively charged (ζ -38.1 mv) indigenous bacteria were associated with the lowest DOC. FTIR spectra indicated the latter community had the highest average density of surface carboxyl groups. In contrast, differences in groundwater (DOC) had no measurable effect on hydrophilicity of the bacteria-sized microspheres and only a minor effect on their ζ. These findings suggest that microspheres may not be very good surrogates for bacteria in field-scale transport studies and that adaptive (biological) changes in bacterial surface characteristics may need to be considered where there is longer-term exposure to contaminant DOC.

Effects of sediment-associated extractable metals, degree of sediment grain sorting, and dissolved organic carbon upon Cryptosporidium parvum removal and transport within riverbank filtration sediments, Sonoma County, California.

Oocysts of the protozoan pathogen Cryptosporidium parvum are of particular concern for riverbank filtration (RBF) operations because of their persistence, ubiquity, and resistance to chlorine disinfection. At the Russian River RBF site (Sonoma County, CA), transport of C. parvum oocysts and oocyst-sized (3 µm) carboxylate-modified microspheres through poorly sorted (sorting indices, σ(1), up to 3.0) and geochemically heterogeneous sediments collected between 2 and 25 m below land surface (bls) were assessed. Removal was highly sensitive to variations in both the quantity of extractable metals (mainly Fe and Al) and degree of grain sorting. In flow-through columns, there was a log-linear relationship (r(2) = 0.82 at p < 0.002) between collision efficiency (α, the probability that colloidal collisions with grain surfaces would result in attachment) and extractable metals, and a linear relationship (r(2) = 0.99 at p < 0.002) between α and σ(1). Collectively, variability in extractable metals and grain sorting accounted for ∼83% of the variability in α (at p < 0.0002) along the depth profiles. Amendments of 2.2 mg L(-1) of Russian River dissolved organic carbon (DOC) reduced α for oocysts by 4-5 fold. The highly reactive hydrophobic organic acid (HPOA) fraction was particularly effective in re-entraining sediment-attached microspheres. However, the transport-enhancing effects of the riverine DOC did not appear to penetrate very deeply into the underlying sediments, judging from high α values (∼1.0) observed for oocysts being advected through unamended sediments collected at ∼2 m bls. This study suggests that in evaluating the efficacy of RBF operations to remove oocysts, it may be necessary to consider not only the geochemical nature and size distribution of the sediment grains, but also the degrees of sediment sorting and the concentration, reactivity, and penetration of the source water DOC.

What makes a natural clay antibacterial?

Natural clays have been used in ancient and modern medicine, but the mechanism(s) that make certain clays lethal against bacterial pathogens has not been identified. We have compared the depositional environments, mineralogies, and chemistries of clays that exhibit antibacterial effects on a broad spectrum of human pathogens including antibiotic resistant strains. Natural antibacterial clays contain nanoscale (<200 nm), illite-smectite and reduced iron phases. The role of clay minerals in the bactericidal process is to buffer the aqueous pH and oxidation state to conditions that promote Fe(2+) solubility. Chemical analyses of E. coli killed by aqueous leachates of an antibacterial clay show that intracellular concentrations of Fe and P are elevated relative to controls. Phosphorus uptake by the cells supports a regulatory role of polyphosphate or phospholipids in controlling Fe(2+). Fenton reaction products can degrade critical cell components, but we deduce that extracellular processes do not cause cell death. Rather, Fe(2+) overwhelms outer membrane regulatory proteins and is oxidized when it enters the cell, precipitating Fe(3+) and producing lethal hydroxyl radicals.

Effects of the antimicrobial sulfamethoxazole on groundwater bacterial enrichment.

The effects of "trace" (environmentally relevant) concentrations of the antimicrobial agent sulfamethoxazole (SMX) on the growth, nitrate reduction activity, and bacterial composition of an enrichment culture prepared with groundwater from a pristine zone of a sandy drinking-water aquifer on Cape Cod, MA, were assessed by laboratory incubations. When the enrichments were grown under heterotrophic denitrifying conditions and exposed to SMX, noticeable differences from the control (no SMX) were observed. Exposure to SMX in concentrations as low as 0.005 µM delayed the initiation of cell growth by up to 1 day and decreased nitrate reduction potential (total amount of nitrate reduced after 19 days) by 47% (p=0.02). Exposure to 1 µM SMX, a concentration below those prescribed for clinical applications but higher than concentrations typically detected in aqueous environments, resulted in additional inhibitions: reduced growth rates (p=5×10(-6)), lower nitrate reduction rate potentials (p=0.01), and decreased overall representation of 16S rRNA gene sequences belonging to the genus Pseudomonas. The reduced abundance of Pseudomonas sequences in the libraries was replaced by sequences representing the genus Variovorax. Results of these growth and nitrate reduction experiments collectively suggest that subtherapeutic concentrations of SMX altered the composition of the enriched nitrate-reducing microcosms and inhibited nitrate reduction capabilities.

Effect of cell physicochemical characteristics and motility on bacterial transport in groundwater.

The influence of physicochemical characteristics and motility on bacterial transport in groundwater were examined in flow-through columns. Four strains of bacteria isolated from a crystalline rock groundwater system were investigated, with carboxylate-modified and amidine-modified latex microspheres and bromide as reference tracers. The bacterial isolates included a gram-positive rod (ML1), a gram-negative motile rod (ML2), a nonmotile mutant of ML2 (ML2m), and a gram-positive coccoid (ML3). Experiments were repeated at two flow velocities, in a glass column packed with glass beads, and in another packed with iron-oxyhydroxide coated glass beads. Bacteria breakthrough curves were interpreted using a transport equation that incorporates a sorption model from microscopic observation of bacterial deposition in flow-cell experiments. The model predicts that bacterial desorption rate will decrease exponentially with the amount of time the cell is attached to the solid surface. Desorption kinetics appeared to influence transport at the lower flow rate, but were not discernable at the higher flow rate. Iron-oxyhydroxide coatings had a lower-than-expected effect on bacterial breakthrough and no effect on the microsphere recovery in the column experiments. Cell wall type and shape also had minor effects on breakthrough. Motility tended to increase the adsorption rate, and decrease the desorption rate. The transport model predicts that at field scale, desorption rate kinetics may be important to the prediction of bacteria transport rates.

Bacterial transport experiments in fractured crystalline bedrock.

The efficiency of contaminant biodegradation in ground water depends, in part, on the transport properties of the degrading bacteria. Few data exist concerning the transport of bacteria in saturated bedrock, particularly at the field scale. Bacteria and microsphere tracer experiments were conducted in a fractured crystalline bedrock under forced-gradient conditions over a distance of 36 m. Bacteria isolated from the local ground water were chosen on the basis of physicochemical and physiological differences (shape, cell-wall type, motility), and were differentially stained so that their transport behavior could be compared. No two bacterial strains transported in an identical manner, and microspheres produced distinctly different breakthrough curves than bacteria. Although there was insufficient control in this field experiment to completely separate the effects of bacteria shape, reaction to Gram staining, cell size, and motility on transport efficiency, it was observed that (1) the nonmotile, mutant strain exhibited better fractional recovery than the motile parent strain; (2) Gram-negative rod-shaped bacteria exhibited higher fractional recovery relative to the Gram-positive rod-shaped strain of similar size; and (3) coccoidal (spherical-shaped) bacteria transported better than all but one strain of the rod-shaped bacteria. The field experiment must be interpreted in the context of the specific bacterial strains and ground water environment in which they were conducted, but experimental results suggest that minor differences in the physical properties of bacteria can lead to major differences in transport behavior at the field scale.

Effects of altered groundwater chemistry upon the pH-dependency and magnitude of bacterial attachment during transport within an organically contaminated sandy aquifer.

The effects of a dilute (ionic strength=5x10(-3)M) plume of treated sewage, with elevated levels (3.9 mg/L) of dissolved organic carbon (DOC), upon the pH-dependency and magnitude of bacterial transport through an iron-laden, quartz sand aquifer (Cape Cod, MA) were evaluated using sets of replicate, static minicolumns. Compared with uncontaminated groundwater, the plume chemistry diminished bacterial attachment under mildly acidic (pH 5.0-6.5) in-situ conditions, in spite of the 5-fold increase in ionic strength and substantively enhanced attachment under more alkaline conditions. The effects of the hydrophobic neutral and total fractions of the plume DOC; modest concentrations of fulvic and humic acids (1.5 mg/L); linear alkyl benzene sulfonate (LAS) (25 mg/L); Imbentin (200 microg/L), a model nonionic surfactant; sulfate (28 mg/L); and calcium (20 mg/L) varied sharply in response to relatively small changes in pH, although the plume constituents collectively decreased the pH-dependency of bacterial attachment. LAS and other hydrophobic neutrals (collectively representing only approximately 3% of the plume DOC) had a disproportionately large effect upon bacterial attachment, as did the elevated concentrations of sulfate within the plume. The findings further suggest that the roles of organic plume constituents in transport or bacteria through acidic aquifer sediments can be very different than would be predicted from column studies performed at circumneutral pH and that the inorganic constituents within the plume cannot be ignored.

Comparison of transport and attachment behaviors of Cryptosporidium parvum oocysts and oocyst-sized microspheres being advected through three minerologically different granular porous media.

In order to gain more information about the fate of Cryptosporidium parvum oocysts in tropical volcanic soils, the transport and attachment behaviors of oocysts and oocyst-sized polystyrene microspheres were studied in the presence of two soils. These soils were chosen because of their differing chemical and physical properties, i.e., an organic-rich (43-46% by mass) volcanic ash-derived soil from the island of Hawaii, and a red, iron (22-29% by mass), aluminum (29-45% by mass), and clay-rich (68-76% by mass) volcanic soil from the island of Oahu. A third agricultural soil, an organic- (13% by mass) and quartz-rich (40% by mass) soil from Illinois, was included for reference. In 10-cm long flow-through columns, oocysts and microspheres advecting through the red volcanic soil were almost completely (98% and 99%) immobilized. The modest breakthrough resulted from preferential flow-path structure inadvertently created by soil-particle aggregation during the re-wetting process. Although a high (99%) removal of oocysts and microsphere within the volcanic ash soil occurred initially, further examination revealed that transport was merely retarded because of highly reversible interactions with grain surfaces. Judging from the slope of the substantive and protracted tail of the breakthrough curve for the 1.8-µm microspheres, almost all (>99%) predictably would be recovered within ∼4000 pore volumes. This suggests that once contaminated, the volcanic ash soil could serve as a reservoir for subsequent contamination of groundwater, at least for pathogens of similar size or smaller. Because of the highly reversible nature of organic colloid immobilization in this soil type, C. parvum could contaminate surface water should overland flow during heavy precipitation events pick up near-surface grains to which they are attached. Surprisingly, oocyst and microsphere attachment to the reference soil from Illinois appeared to be at least as sensitive to changes in pH as was observed for the red, metal-oxide rich soil from Oahu. In contrast, colloidal attachment in the organic-rich, volcanic ash soil was relatively insensitive to changes in pH in spite of the high iron content. Given the fundamental differences in transport behavior of oocyst-sized colloids within the two volcanic soils of similar origin, agricultural practices modified to lessen C. parvum contamination of ground or surface water would necessitate taking the individual soil properties into account.

Surface complexation of carboxylate adheres Cryptosporidium parvum oocysts to the hematite-water interface.

The interaction of viable Cryptosporidium parvum oocysts at the hematite (alpha-Fe2O3)-water interface was examined over a wide range in solution chemistry using in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Spectra for hematite-sorbed oocysts showed distinct changes in carboxylate group vibrations relative to spectra obtained in the absence of hematite, indicative of direct chemical bonding between carboxylate groups and Fe metal centers of the hematite surface. The data also indicate that complexation modes vary with solution chemistry. InNaCl solution, oocysts are bound to hematite via monodentate and binuclear bidentate complexes. The former predominates at low pH, whereas the latter becomes increasingly prevalent with increasing pH. In a CaCl2 solution, only binuclear bidentate complexes are observed. When solution pH is above the point of zero net proton charge (PZNPC) of hematite, oocyst surface carboxylate groups are bound to the mineral surface via outer-sphere complexes in both electrolyte solutions.

Field evidence for a protistan role in an organically-contaminated aquifer.

The association between protists, bacteria, and dissolved organic carbon (DOC) in an oxygen-depleted, 6 km-long wastewater contaminant plume within a sandy aquifer (Cape Cod, MA) was investigated by comparing abundance patterns along longitudinal and vertical transects and at a control site. Strong linear correlations were observed between unattached bacterial abundance and DOC for much of the upgradient-half of the plume (0.1-2.5 km downgradient from the source) that is characterized by quasi-steady state chemistry. However, a logarithmic decrease was observed between the number of protists supported per mg of DOC and the estimated age of the DOC within the plume. The relatively labile dissolved organic contaminants that characterize the groundwater sampled from the plume < or = 0.1 km downgradient from the contaminant source appeared to indirectly support 3-4 times as many protists (per mg of DOC) as the older, more recalcitrant DOC in the alkylbenzene sulfonate (ABS)-contaminated zone at 3 km downgradient (approximately 30 years travel time). Substantive numbers of protists (>10(4)/cm3) were recovered from suboxic zones of the plume. The higher than expected ratios of protists to unattached bacteria (10 to 100:1) observed in much of the plume suggest that protists may be grazing upon both surface-associated and unattached bacterial communities to meet their nutritional requirements. In closed bottle incubation experiments, the presence of protists caused an increase in bacterial growth rate, which became more apparent at higher amendments of labile DOC (3-20 mgC/L). The presence of protists resulted in an increase in the apparent substrate saturation level for the unattached bacterial community, suggesting an important role for protists in the fate of more-labile aquifer organic contaminants.

Effect of growth conditions and staining procedure upon the subsurface transport and attachment behaviors of a groundwater protist.

The transport and attachment behaviors of Spumella guttula (Kent), a nanoflagellate (protist) found in contaminated and uncontaminated aquifer sediments in Cape Cod, Mass., were assessed in flowthrough and static columns and in a field injection-and-recovery transport experiment involving an array of multilevel samplers. Transport of S. guttula harvested from low-nutrient (10 mg of dissolved organic carbon per liter), slightly acidic, granular (porous) growth media was compared to earlier observations involving nanoflagellates grown in a traditional high-nutrient liquid broth. In contrast to the highly retarded (retardation factor of approximately 3) subsurface transport previously reported for S. guttula, the peak concentration of porous-medium-grown S. guttula traveled concomitantly with that of a conservative (bromide) tracer. About one-third of the porous-medium-grown nanoflagellates added to the aquifer were transported at least 2.8 m downgradient, compared to only approximately 2% of the broth-grown nanoflagellates. Flowthrough column studies revealed that a vital (hydroethidine [HE]) staining procedure resulted in considerably less attachment (more transport) of S. guttula in aquifer sediments than did a staining-and-fixation procedure involving 4',6'-diamidino-2-phenylindole (DAPI) and glutaraldehyde. The calculated collision efficiency (approximately 10(-2) for porous-medium-grown, DAPI-stained nanoflagellates) was comparable to that observed earlier for the indigenous community of unattached groundwater bacteria that serve as prey. The attachment of HE-labeled S. guttula onto aquifer sediment grains was independent of pH (over the range from pH 3 to 9) suggesting a primary attachment mechanism that may be fundamentally different from that of their prey bacteria, which exhibit sharp decreases in fractional attachment with increasing pH. The high degree of mobility of S. guttula in the aquifer sediments has important ecological implications for the protistan community within the temporally changing plume of organic contaminants in the Cape Cod aquifer.