ARTICLE pubs.acs.org/est
Transport and Bacterial Interactions of Three Bacterial Strains in Saturated Column Experiments Christine Stumpp,*,†,‡ John R. Lawrence,§ M. Jim Hendry,† and Piotr Maloszewski‡ †
Department of Geological Sciences, University of Saskatchewan, Saskatoon, SK S7N 5E2, Canada Helmholtz Zentrum M€unchen, Institute of Groundwater Ecology, Ingolstaedter Landstrasse 1, D-85764 Neuherberg, Germany § Environment Canada, 11 Innovation Blvd., Saskatoon, SK S7N 3H5, Canada ‡
bS Supporting Information ABSTRACT: The impact of bacteria-solid and bacteria-bacteria interactions on the transport of Klebsiella oxytoca, Burkholderia cepacia G4PR1, and Pseudomonas sp. #5 was investigated in saturated sand column experiments (L = 114 mm; ø = 33 mm) under constant water velocities (∼5 cm 3 h-1). Bacterial strains were injected into the columns as pulses either individually, simultaneously, or successively. A one-dimensional mathematical model for advective-dispersive transport and for irreversible and reversible bacterial kinetic sorption was used to analyze the bacterial breakthrough curves. Different sorption parameters were obtained for each strain in each of the three experimental setups. In the presence of other bacteria, sorption parameters for B. cepacia G4PR1 remained similar to results from individual experiments, indicating the presence of other bacteria generally had a lesser influence on its migration than for the other bacteria. K. oxytoca is more competitive for the sorption sites when simultaneously injected with the other bacteria. Ps. sp. #5 generally yielded the greatest detachment rates and the least affinity to attach to the sand, indicative of its mobility in groundwater systems. The results of this study clearly indicate both bacteria-solid and bacteria-bacteria interactions influence the migration of bacteria. A more complete understanding of such interactions is necessary to determine potential migration in groundwater systems.
’ INTRODUCTION Investigations of bacterial migration in porous media are required to improve understanding of the factors controlling fate and transport of microbes.1,2 Pathogenic microorganisms, such as some viruses, protozoa, or bacteria, can pollute drinking water resources and are introduced to the subsurface during riverbank infiltration 3 or by land application of wastewater effluents or animal manure;4-6 the practice of sewage injection also raises concerns.7,8 Similar to other colloids, bacteria can enhance facilitated transport and therefore the mobility of inorganic and organic toxic pollutants.9 Accurate prediction of bacterial behavior is also valuable when employing in situ bioremediation techniques (e.g., bioaugmentation) or natural attenuation to control the degradation of groundwater contamination.10 Both the efficiency of remediation strategies and the protection of groundwater resources can be improved through greater understanding of the complexity of bacterial transport. Although microbial transport in the saturated subsurface is an important subject, some influential factors have been investigated. These include biological parameters, such as cell size and morphology, hydrophobicity or charge of the cell surface, growth cycles, and motility (e.g., refs 11-13); chemical factors, such as solution composition, the presence of particle coatings, and concentration gradients stimulating chemotaxis (e.g., refs 13-17); and physical factors, such as sediment properties or flow velocities (e.g., refs 18-20). In addition to advection, diffusion, and dispersion as transport mechanisms, sorption, both reversible and irreversible, has a major influence on bacterial migration.21,22 Reversible sorption of bacteria has been described r 2011 American Chemical Society
with first-order kinetic reactions.7,21,23 Irreversible sorption occurs (e.g., when cells produce adhesive substances to anchor themselves to surfaces), and is defined as a time-dependent process in which bacteria can no longer be easily removed from solid surfaces.24 In addition, migration of bacteria to the primary or secondary energy minima can result in irreversible sorption to a surface.25 Bacterial transport mechanisms, sorption rates and the influential factors mentioned above, have to date typically only been studied for individual species. To the best of the our knowledge, only one study26 has investigated the simultaneous transport of two bacterial strains in soil columns, wherein bacteria appeared before the tracer, and sorption rates were smaller for both strains in the simultaneous compared to the individual experiments. These results demonstrated bacterial interactions can indeed impact transport processes; however, no further conclusions were drawn regarding differences between individual and simultaneous transport. Developing an understanding of bacteriabacteria and bacteria-sediment interactions and their impact on transport is necessary to understand the complex processes in groundwater ecosystems.27,28 The objective of the current study was to determine the impact of bacterial interactions on transport and sorption processes and thus improve on our limited understanding of bacterial migration Received: February 11, 2010 Accepted: January 19, 2011 Revised: January 5, 2011 Published: February 14, 2011 2116
dx.doi.org/10.1021/es103569u | Environ. Sci. Technol. 2011, 45, 2116–2123
Environmental Science & Technology as it pertains to bacteria-bacteria and bacteria-sediment interactions. This objective was attained by conducting a series of column experiments (in quadruplicate) in saturated medium to coarse grained silica sand columns under constant pore water velocities (5.2 ( 0.5 cm h-1), wherein three gram-negative, rodshaped bacteria were added individually, simultaneously, and successively. The resulting bacterial breakthrough curves (BTC) in the column effluent were modeled as a function of time in conjunction with an associated conservative tracer (chloride) to determine sorption parameters (i.e., irreversible and reversible attachment) and their variability resulting from interaction with other bacteria.
’ METHODOLOGY Characterization and Preparation of Bacteria. Interactions among species as well as between species and the geologic media during bacterial transport experiments were investigated using three gram-negative, rod shaped bacteria: Burkholderia cepacia G4PR1, Klebsiella oxytoca, and Pseudomonas sp. #5. These bacteria were selected because they are environmentally relevant in groundwater ecosystems. G4PR1 is a Tn5 insertion mutant that constitutively expresses the toluene orthomonooxygenase responsible for TCE degradation.29 As such, this nonmotile organism is a candidate for use in remediation of contaminated aquifers; determination of its transport characteristics is a prerequisite for its application in the subsurface. K. oxytoca is a nonmotile bacterium producing exopolysaccharides (polysaccharides, proteins, and lipids) when stimulated with nutrients. The formation of such exopolysaccharides has the potential to create microbial barriers in porous media, thus isolating groundwater contaminants from the natural flow system.30 Ps. sp. #5 is a motile plant pathogen isolated from surficial brown, fractured, and oxidized glacial until at a study site located 140 km south of Saskatoon, Canada.31 This strain was selected as being representative of motile, pathogenic bacteria. Cell size and morphology were evaluated using confocal scanning laser microscopy in conjunction with SYTO 9, a fluorescent DNA stain (Invitrogen, Molecular Probes, Eugene OR), and negative staining.32 The relative hydrophobicity of the three bacteria was determined using the bacterial adherence to hydrocarbons (BATH) technique.33 The nature of cell surfaces was also examined by quantifying the attachment of fluorescent 0.1 μm hydrophobic and hydrophilic beads (Invitrogen, Molecular Probes, Eugene, OR).34 These observations were repeated on the input solutions of bacteria over the time course of the column breakthrough experiments. Input bacterial solutions were also subjected to a Live-Dead (Invitrogen, Molecular Probes, Eugene, OR) staining procedure 32 before and after centrifugation and washing procedures to assess the impact of these treatments on the status of input bacteria. The potential for microbial interactions was determined through standard crossstreaking on agar media and through coaggregation tests.35 Cell density (g 3 cm-3) was determined using NaCl Percoll gradients with internal standards (Sigma Chemical, St Louis, MI). Zeta potentials were measured with a PALS Zeta potential analyzer (Brookhaven Instruments). For preparation, the cells were grown overnight in a shaking water bath (250 rpm) in tryptic soy broth at room temperature. The cell pellets were obtained by centrifugation (7000 rpm) for five minutes and washed with MilliQ water. This procedure was repeated three times. To facilitate recovery and enumeration of the bacteria, a K. oxytoca colony with an antibiotic resistance marker (kanamycin)
ARTICLE
was prepared by transposon 5 mutagenesis.36 K. oxytoca was routinely cultured in half strength brain heart infusion (BHI) media at pH 7.4. Flasks containing 500 mL of 50% BHI were inoculated from an isolated colony of the kanamycin resistant K. oxytoca growing on BHI agar plates. The flasks were incubated for 48 h on an orbital shaker at 23 ( 2 C. Cultures were harvested by centrifugation (t = 10 min at 25 000G), resuspended, and washed three times with artificial groundwater, which contained the conservative tracer Cl- (AGWt), to remove all available carbon. Centrifugation may influence the cell surface chemistry and bacterial behavior. The implication of these effects for extrapolation of these data to other studies, such as field studies, is unknown. G4PR1 and Ps. sp. #5 were prepared in the same manner as K. oxytoca but in 10% strength tryptic soy broth or agar as appropriate growth media. Cells were added to the AGWt to produce input solutions with similar bacterial concentrations. The concentration of viable cells in the input solutions was monitored for 200 h by plating dilutions on selective media. All solutions were used immediately after preparation. All stock cultures were stored at -80 C in half strength BHI and 15% glycerol.37 Porous Medium and Aqueous Solutions. A medium to coarse grained commercially available silica sand was used as porous medium. From sieve analysis, 77% of the sand was in the size range of 500-1000 μm diameter (coarse sand), and the remainder of the sand was in the size range of 250-500 μm diameter (medium sand). Total inorganic carbon content of the sand was 0.11% (w 3 w-1); total organic carbon was below detection ( tpulse
ð5Þ
where Cout(t) is the concentration of the tracer at the outlet, C0 is the concentration of the injected pulse, tpulse is the duration of the pulse, and g(τ) is the solution of eqs 2 and 3 for the Dirac input function, δ(t) 8 " #9 ð1 - τ=t0 Þ2 > > > > > > > = pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi gðτÞ ¼ > > τ 4πPD τ=t0 > > > > > > ; : (rffiffiffiffiffiffiffiffiffiffiffi Z kf kr t0 þ exp½-ðkirr þ kf Þτ 4πPD
"
ð1 - ητ=t0 Þ2 4PD ητ=t0 0 ) pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi dη - ðkf - kr ÞητI1 ½2τ kr kf ð1 - ηÞη pffiffiffiffiffiffiffiffiffiffiffi ð6Þ η 1-η 1
exp
where PD is the dispersion parameter (PD = RL/x), I1 is the Bessel function of the first kind and of the first order, and τ is an integration variable. The solution for eq 5 with 6 consists of five unknown parameters (v, RL, kirr, kf, and kr), neglecting bacterial death or growth, which were minimized for the experimental setup and controlled in the AGWt.19,21 By using values of v and RL determined from the BTC for the conservative tracer (kirr = kf = kr = 0), the number of unknowns is reduced to three (kirr, kf, and kr) and the equations can be solved using a trial and error approach optimizing the model calibration efficiency.19
’ RESULTS AND DISCUSSION Characterization of Bacteria. Assessments of bacterial interactions including all possible combinations indicated no detectable coaggregation. Similarly, cross streaking analyses revealed no negative or positive growth interactions. Live-Dead analyses confirmed that the cell membranes remained intact and there was no change in viability. Microscopic examination confirmed no changes in morphology of the bacteria. The impact of growth, death, aggregation, antagonism, or predators was therefore neglected in the experiments. The hydrophobicity tests were 2118
dx.doi.org/10.1021/es103569u |Environ. Sci. Technol. 2011, 45, 2116–2123
Environmental Science & Technology
ARTICLE
Table 1. Characteristics of the Bacterial Strains; The Shape Factor Is the Coefficient of Width and Length
hydrophobicity ranking -3
buoyant density (g 3 cm )
a
G4PR1
K. oxytoca
Ps. sp. #5
(27%)a slightly hydrophobic
(
