Transport and Retention of Fecal Indicator Bacteria in Unsaturated Porous Media: Effect of Transient Water Flow.

For production of clean drinking water, the processes governing bacterial remobilization in the unsaturated zone at transient water flow are critical. Although managed aquifer recharge is an effective way to dispose of pathogens, there are concerns about recontamination after heavy precipitation. To better understand how bacteria that were initially retained in porous media can be released to groundwater due to transient water content, transport experiments and modeling for Escherichia coli and Enterococcus moraviensis were conducted at the soil column scale. After inoculating dune sand columns with a bacteria suspension for 4 h, three rainfall events were performed at 24-h intervals. The effluent from sand columns was collected to analyze bacteria breakthrough curves (BTCs). After the rainfall experiments, the bacteria distribution in the sand column was determined. The collected BTCs and profile retentions were modeled with HYDRUS-1D, using different model concepts, including one-site kinetic attachment/detachment (M1), Langmuirian (M2), Langmuirian and blocking (M3), and two-site attachment/detachment (M4). After inoculation, almost 99% of the bacteria remained in the soil. The M1 and M2 bacteria models had a high agreement between observed and modeled concentrations, and attachment and detachment were two significant mechanisms for regulating bacteria movement in a porous medium with fluctuations in water flow. At the end of the experiment, the majority of bacteria were still found within the depth range of 5 cm to 15 cm. Our experiments show that E. coli is more mobile in sandy soils than E. moraviensis. The results of this study also suggest that the unsaturated zone is an important barrier between microbial contamination at the soil surface and groundwater. Follow-up studies are needed to completely understand the variables that regulate bacteria remobilization in the unsaturated zone of dune sands. IMPORTANCE At managed artificial recharge sites in the Netherlands, recontamination of infiltrated water with fecal indicator bacteria has been observed. The results of this study suggest that the unsaturated zone is an important barrier between microbial contamination at the soil surface and groundwater. Bacteria that accumulate in the unsaturated zone, on the other hand, can multiply to such an extent that they can be released into the saturated zone when saturation increases due to major rain events or a rise in groundwater level.

Freeze-thaw treatment of bacteria significantly affects bacterial transport behaviors in both saturated and unsaturated porous media: Deep investigation the roles of flagella and extracellular polymeric substances.

The effects of freeze-thaw (FT) treatment and mechanisms on bacteria transport/retention in porous media with different moisture contents remain unclear. The transport/retention behaviors of bacteria with different FT treatment cycles (0, 1, and 3) in sand columns with different moisture contents (100 %, 90 %, 60 %, and 30 %) in NaCl solutions (10 and 100 mM) thus were investigated. Regardless of moisture content and solution chemistry, FT treatment increased bacteria deposition in sand columns, consistent with the results of QCM-D and visible parallel plate flow chamber (PPFC) systems. Via deep investigation of the contribution of flagella through using genetic-modified bacteria strain without flagella and that of extracellular polymeric substances (EPS) through analyzing its overall quantity, composition as well as the secondary structure of its two major components (proteins and polysaccharides), the mechanisms of FT treatment controlling bacterial transport/deposition were revealed. Although FT treatment induced flagella loss, it was not the major contributor to driving to the enhanced FT-treated cell deposition. Instead, FT treatment stimulated EPS secretion and increased its hydrophobicity (via increasing hydrophobicity of both proteins and polysaccharides), mainly contributing to the enhanced bacterial deposition. Even with copresent humic acid, FT treatment still enhanced bacterial deposition in sand columns with different moisture contents.

Chemorepellent-Loaded Nanocarriers Promote Localized Interference of Escherichia coli Transport to Inhibit Biofilm Formation.

To mitigate antimicrobial resistance, we developed polymeric nanocarrier delivery of the chemorepellent signaling agent, nickel, to interfere with Escherichia coli transport to a surface, an incipient biofilm formation stage. The dynamics of nickel nanocarrier (Ni NC) chemorepellent release and induced chemorepellent response required to effectively modulate bacterial transport for biofilm prevention were characterized in this work. Ni NCs were fabricated with the established Flash NanoPrecipitation method. NC size was characterized with dynamic light scattering. Measured with a zincon monosodium salt colorimetric assay, NC nickel release was pH-dependent, with 62.5% of total encapsulated nickel released at pH 7 within 0-15 min, competitive with rapid E. coli transport to the surface. Confocal laser scanning microscopy of E. coli (GFP-expressing) biofilm growth dynamics on fluorescently labeled Ni NC coated glass coupled with a theoretical dynamical criterion probed the biofilm prevention outcomes of NC design. The Ni NC coating significantly reduced E. coli attachment compared to a soluble nickel coating and reduced E. coli biomass area by 61% compared to uncoated glass. A chemical-in-plug assay revealed Ni NCs induced a chemorepellent response in E. coli. A characteristic E. coli chemorepellent response was observed away from the Ni NC coated glass over 10 µm length scales effective to prevent incipient biofilm surface attachment. The dynamical criterion provided semiquantitative analysis of NC mechanisms to control biofilm and informed optimal chemorepellent release profiles to improve NC biofilm inhibition. This work is fundamental for dynamical informed design of biofilm-inhibiting chemorepellent-loaded NCs promising to mitigate the development of resistance and interfere with the transport of specific pathogens.

Bacteria transport and deposition in an unsaturated aggregated porous medium with dual porosity.

Bacterial transport and deposition play an important role in the assessment and prediction of subsurface pollution risks. Bacteria transport experiments were performed under unsaturated flow conditions in an aggregated porous medium at the laboratory column scale, to investigate how the inter- and intra-aggregated pore space of this medium could affect transport and deposition under unsaturated flow conditions, where inter- and intra-pore spaces are not fully activated. The results obtained through experimental observations and numerical simulations showed that some intra- and inter-pore space of this medium was excluded from bacteria transport and retention, as confirmed by the non-uniform transport of bacteria pathways in the aggregated porous media under unsaturated flow conditions. Capillary energy was higher the than other forces acting at bacteria air-water-solid interfaces. If this energy should contribute in increasing bacteria deposition under unsaturated conditions, similar to what has been reported for sandy media, similar overall retention of E. coli and R. rhodochrous was obtained under unsaturated flow conditions, suggesting that capillary energy was not the driving force for bacteria deposition.

Transport, retention, and release of Escherichia coli and Rhodococcus erythropolis through dry natural soils as affected by water repellency.

Microbial transport in soil affects pathogen retention, colonization, and innoculant delivery in bioremediating agricultural soils. Various bacteria strains residing in the fluid phases of soils are potential contaminants affecting human health. We measured the transport of hydrophilic Escherichia coli (E. coli) and hydrophobic Rhodococcus erythropolis (R. erythropolis) bacteria through initially air-dried wettable or water-repellent soil columns to understand the effect of water repellency and the hydrophobicity of the organism on its retention, release, and transport properties. Bacteria suspensions infiltrated the top of the columns under saturated (0 cm) and unsaturated (-5 cm) flows in the air-dried (pulse 1) and rewetting (pulse 2) conditions. Cells were recovered from the leachates and the soil extracts by the viable counts. Wettable soil efficiently retained both hydrophobic and hydrophilic bacteria (>80%) in initial air-dried conditions (pulse 1). Even after rewetting, and the formation and expansion of water films and corresponding reduction of the air-water interfacial area (pulse 2), few bacteria were released (maximum 31.5% and 10.1% for saturated and unsaturated flows, respectively), whereas more cells were released from the water-repellent counterpart (more that 72%). The smaller size of hydrophobic R. erythropolis made cell transport possible within the thinner water films of both soils compared to hydrophilic E. coli through pulses 1 and 2. The shape of each strain's retention profiles was uniform and exponential as influenced by soil, strain, and water flow conditions. The results suggest that hydrophobic bacteria will disperse readily when leached into initially dry soil, while hydrophilic bacteria are more susceptible to leaching, posing a risk of pathogen contamination. Clearly the wettability of soil and organisms affects fate and transport.

The influence of different charged poly (amido amine) dendrimer on the transport and deposition of bacteria in porous media.

The influence of dendrimer on the bacterial transport and deposition behaviors in saturated porous media (quartz sand) was investigated in both NaCl (10 and 25 mM) and CaCl2 solutions (1.2 and 5 mM). 3.5G and 4G poly (amido amine) (PAMAM) dendrimer was employed as negatively and positively charged dendrimer, respectively. Three dendrimer concentrations (10 µg/L, 1 and 10 mg/L) were considered in present study. We found that regardless of the solution chemistry (ionic strength and ion types) and dendrimer concentrations, the presence of negatively charged PAMAM 3.5G in suspensions enhanced bacterial transport and inhibited their deposition in quartz sand; while the presence of positive charged PAMAM 4G yet induced the opposite effects (decreased bacterial transport and increased their deposition in quartz sand). The increased repulsive force between cell and quartz sand due to the adsorption of PAMAM 3.5G onto both cell and sand surfaces, the competition deposition sites as well as the steric repulsion via the suspended PAMAM 3.5G drove to the increased bacterial transport with PAMAM 3.5G copresent in suspensions in quartz sand. While the reduced repulsive force between cell and quartz sand induced by the chemical heterogeneity on both cell and sand surfaces (due to the adsorption of positive charged PAMAM 4G) increased bacterial retention in quartz sand with copresence of PAMAM 4G (10 µg/L and 1 mg/L) in suspensions. Steric repulsion due to the presence of great amount of suspended PAMAM 4G yet lead to the enhanced bacterial transport with furthering increasing PAMAM 4G to 10 mg/L relative to the lower PAMAM 4G concentration.

Modeling Escherichia coli and Rhodococcus erythropolis transport through wettable and water repellent porous media.

Water protection and bioremediation strategies in the vadose zone require understanding the factors controlling bacterial transport for different hydraulic conditions. Breakthrough experiments were made in two different flow conditions: i) an initial bacteria pulse under ponded infiltration into dry sand (-15,000 cm); ii) a second bacteria pulse into the same columns during subsequent infiltration in constant water content and steady-state flow. Escherichia coli (E. coli) and Rhodococcus erythropolis (R. erythropolis) were used to represent hydrophilic and hydrophobic bacteria, respectively. Equilibrium and attachment/detachment models were tested to fit bromide (Br-) and bacteria transport data using HYDRUS-1D. Derjaguin-Landau-Verwey-Overbeek (DLVO) and extended DVLO (XDLVO) interaction energy profiles were calculated to predict bacteria sorption at particles. Adsorption of bacteria at air-water interfaces was estimated by a hydrophobic force approach. Results suggested greater retention of bacteria in water repellent sand compared with wettable sand. Inverse parameter optimization suggested that physico-chemical attachment of both E. coli and R. erythropolis was thousands of times lower in wettable than repellant sand and straining was 10-fold lower in E. coli for wettable vs repellant sand compared to the exact opposite by orders of magnitude with R. erythropolis. HYDRUS did not provide a clear priority of importance of solid-water or air-water interfaces in bacteria retention. Optimized model parameters did not show a clear relation to the (X)DLVO adsorption energies. This illustrated the ambivalence of (X)DLVO to predict bacterial attachment at solid soil particles of different wetting properties. Simultaneous analysis of mass recovery, numerical modeling, and interaction energy profiles thus suggested irreversible straining due to bacteria sizing as dominant compared to attachment to liquid-solid or liquid-air interfaces. Further studies are needed to distinguish straining mechanisms (i.e. pore structure or film straining) in different hydraulic conditions.

Different electrically charged proteins result in diverse bacterial transport behaviors in porous media.

The influence of proteins on bacterial transport and deposition behaviors in quartz sand was examined in both NaCl (10 and 25 mM) and CaCl2 solutions (1.2 and 5 mM). Bovine Serum Albumin (BSA) and bovine trypsin were used to represent negatively and positively charged proteins in natural aquatic systems, respectively. The presence of negatively charged BSA in suspensions increased the transport and decreased bacterial deposition in quartz sand, regardless of the ionic strength and ion types. Whereas, positively charged trypsin inhibited the transport and enhanced bacterial deposition under all experimental conditions. The potential mechanisms controlling the changes of bacterial transport behaviors varied for different charged proteins. The steric repulsion resulting from BSA adsorption onto both bacteria and quartz sand was found to play a dominant role in the transport and deposition of bacteria in porous media with BSA copresent in suspension. BSA adsorption onto bacterial surfaces and competition for deposition sites onto sand surfaces (adsorption of quartz sand surfaces) contributed to the increased cell transport with BSA in suspension. In contrast, the attractive patch-charged interaction induced by the adsorption of trypsin onto both bacteria and quartz sand had great contribution to the decreased bacterial transport in porous media with trypsin copresent in suspension. The increase in bacteria size, and the adsorption of trypsin onto cell surfaces (resulting in less negative cell surface charge) and quartz sand surfaces (providing extra deposition sites) were found to be the main contributors to the decreased transport and increased deposition of bacteria in quartz sand with trypsin in suspension.

Natural freeze-thaw cycles may increase the risk associated with Salmonella contamination in surface and groundwater environments.

Groundwater contamination by bacteria poses a serious threat to our drinking water supplies. In cold climate regions, microorganisms introduced to upper soil layers by spreading of animal manure are subject to low temperatures and multiple cycles of freezing and thawing at the beginning of winter and during spring melt. We investigated the influence of temperature fluctuations around the freezing point, known as freeze-thaw (FT), on the inactivation rates, growth, and biofilm formation of a manure-isolated strain of Salmonella typhimurium. Moreover, the effects of FT on the transport characteristics of S. typhimurium in quartz sand were monitored in model porewater solutions of two different ionic strengths (IS: 10 and 100 mM KCl) and two different humic acid (HA) concentrations (1 and 5 mg/L). Increasing numbers of FT cycles were found to decrease the deposition of S. typhimurium onto quartz sand and increase the percentage of detached cells in sand-packed column experiments. Based on the calculated bacterial attachment efficiencies, the predicted minimum setback distances between the location of water supply wells and manure spreading activities are higher when the effects of FT are taken into consideration. While FT treatment significantly affected cell viability (in the presence of HA), most cells were in a viable but non-culturable (VBNC) state with compromised ability to form biofilm. This investigation demonstrates the effects of spring temperature variations in upper soil layers on S. typhimurium properties and the potential increased risk of bacterial contamination in representative aquifer environments in cold climate regions.

Influence of Bisphenol A on the transport and deposition behaviors of bacteria in quartz sand.

The influence of Bisphenol A (BPA) on the transport and deposition behaviors of bacteria in quartz sand was examined in both NaCl (10 and 25 mM) and CaCl2 solutions (1.2 and 5 mM) by comparing the breakthrough curves and retained profiles of cell with BPA in suspensions versus those without BPA. Gram-negative Escherichia coli and Gram-positive Bacillus subtilis were employed as model cells in the present study. The extended Derjaguin-Landau-Verwey-Overbeek interaction energy calculation revealed that the presence of BPA in cell suspensions led to a lower repulsive interaction between the cells and the quartz sand. This suggests that, theoretically, increased cell deposition on quartz sand would be expected in the presence of BPA. However, under all examined solution conditions, the presence of BPA in cell suspensions increased transport and decreased deposition of bacteria in porous media regardless of cell type, ionic strength, ion valence, the presence or absence of extracellular polymeric substances. We found that competition by BPA through hydrophobicity for deposition sites on the quartz sand surfaces was the sole contributor to the enhanced transport and decreased deposition of bacteria in the presence of BPA.

Quantifying bacterial attachment and detachment using leaching solutions of various ionic strengths after bacterial pulse.

In this study, we quantified the attachment and detachment of bacteria during transport in order to elucidate the contributions of reversible attachment on bacterial breakthrough curves. The first set of breakthrough experiment was performed for a laboratory sand column using leaching solutions of deionized water and mineral salt medium (MSM) of 200 mM with reference to KCl solution by employing Pseudomonas putida as a model bacterium. In the second set of experiment, the ionic strengths of leaching solutions immediately after bacterial pulse were lowered to tenfold and 100-fold diluted system (2 and 20 mM MSM) to focus on the influence of physicochemical factor. Results have shown that bacterial retention occurred in the sand column due to the physical deposition and physicochemical attachment. The physicochemical attachment was attributed to the high ionic strength (200 mM MSM) of leaching solution and the formation of primary energy minimum. Replacing the 200 mM leaching solution with the lower ionic strengths after pulse resulted in the increased tailing of breakthrough curve due to the detachment from the attached bacteria. The detachment could be well explained by DLVO theory, which showed the formation of energy barrier and disappearance of the secondary minimum as the ionic strength gradually decreased. Analysis of mass recovery revealed that 12-20% of the attachment was due to physical and physicochemical attachment, respectively, where the latter consisted of 25-75% of irreversible and reversible attachment respectively.

Modeling fecal bacteria transport and retention in agricultural and urban soils under saturated and unsaturated flow conditions.

Pathogenic bacteria, that enter surface water bodies and groundwater systems through unmanaged wastewater land application, pose a great risk to human health. In this study, six soil column experiments were conducted to simulate the vulnerability of agricultural and urban field soils for fecal bacteria transport and retention under saturated and unsaturated flow conditions. HYDRUS-1D kinetic attachment and kinetic attachment-detachment models were used to simulate the breakthrough curves of the experimental data by fitting model parameters. Results indicated significant differences in the retention and drainage of bacteria between saturated and unsaturated flow condition in the two studied soils. Flow under unsaturated condition retained more bacteria than the saturated flow case. The high bacteria retention in the urban soil compared to agricultural soil is ascribed not only to the dynamic attachment and sorption mechanisms but also to the greater surface area of fine particles and low flow rate. All models simulated experimental data satisfactorily under saturated flow conditions; however, under variably saturated flow, the peak concentrations were overestimated by the attachment-detachment model and underestimated by the attachment model with blocking. The good match between observed data and simulated concentrations by the attachment model which was supported by the Akaike information criterion (AIC) for model selection indicates that the first-order attachment coefficient was sufficient to represent the quantitative and temporal distribution of bacteria in the soil column. On the other hand, the total mass balance of the drained and retained bacteria in all transport experiments was in the range of values commonly found in the literature. Regardless of flow conditions and soil texture, most of the bacteria were retained in the top 12 cm of the soil column. The approaches and the models used in this study have proven to be a good tool for simulating fecal bacteria transport under a variety of initial and boundary flow conditions, hence providing a better understanding of the transport mechanism of bacteria as well as soil removal efficiency.

Bacteria cell properties and grain size impact on bacteria transport and deposition in porous media.

The simultaneous role of bacteria cell properties and porous media grain size on bacteria transport and deposition behavior was investigated in this study. Transport column experiments and numerical HYDRUS-1D simulations of three bacteria with different cell properties (Escherichia coli, Klebsiella oxytoca, and Rhodococcus rhodochrous) were carried out on two sandy media with different grain sizes, under saturated steady state flow conditions. Each bacterium was characterized by cell size and shape, cell motility, electrophoretic mobility, zeta potential, hydrophobicity and potential of interaction with the sand surface. Cell characteristics affected bacteria transport behavior in the fine sand, but similar bacteria breakthroughs and retardation factors observed in the coarse sand, indicated that bacteria transport was more depended on grain size than on bacteria cell properties. Retention decreased with increasing hydrophobicity and increased with increasing electrophoretic mobility of bacteria for both sand. The increasing sand grain size resulted in a decrease of bacteria retention, except for the motile E. coli, indicating that retention of this strain was more dependent on cell motility than on the sand grain size. Bacteria deposition coefficients obtained from numerical simulations of the retention profiles indicated that straining was an important mechanism affecting bacteria deposition of E. coli and Klebsiella sp., in the fine sand, but the attachment had the same importance as straining for R. rhodochrous. The results obtained in the coarse sand did not permit to discriminate the predominant mechanism of bacteria deposition and the relative implication of bacteria cell properties of this process.

Transport and viability of Escherichia coli cells in clean and iron oxide coated sand following coating with silver nanoparticles.

A mechanistic understanding of processes controlling the transport and viability of bacteria in porous media is critical for designing in situ bioremediation and microbiological water decontamination programs. We investigated the combined influence of coating sand with iron oxide and silver nanoparticles on the transport and viability of Escherichia coli cells under saturated conditions. Results showed that iron oxide coatings increase cell deposition which was generally reversed by silver nanoparticle coatings in the early stages of injection. These observations are consistent with short-term, particle surface charge controls on bacteria transport, where a negatively charged surface induced by silver nanoparticles reverses the positive charge due to iron oxide coatings, but columns eventually recovered irreversible cell deposition. Silver nanoparticle coatings significantly increased cell inactivation during transit through the columns. However, when viability data is normalised to volume throughput, only a small improvement in cell inactivation is observed for silver nanoparticle coated sands relative to iron oxide coating alone. This counterintuitive result underscores the importance of net surface charge in controlling cell transport and inactivation and implies that the extra cost for implementing silver nanoparticle coatings on porous beds coated with iron oxides may not be justified in designing point of use water filters in low income countries.