Chemotaxis Increases the Retention of Bacteria in Porous Media with

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Chemotaxis increases the retention of bacteria in porous media with residual NAPL entrapment Joanna S.T. Adadevoh, C. Andrew Ramsburg, and Roseanne Marie Ford Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01172 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Environmental Science & Technology

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Chemotaxis increases the retention of bacteria in

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porous media with residual NAPL entrapment

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Joanna S. T. Adadevoh,† C. Andrew Ramsburg,‡ and Roseanne M. Ford*,†

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†Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904

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‡Department of Civil and Environmental Engineering, Tufts University, Medford, Massachusetts

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02155

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Keywords: chemotaxis, bioremediation, porous media, NAPL, transport phenomena,

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Pseudomonas putida

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ABSTRACT

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Chemotaxis has the potential to decrease the persistence of non-aqueous phase liquid (NAPL)

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contaminants in aquifers by allowing pollutant-degrading bacteria to move toward sources of

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contamination and thus influence dissolution. This experimental study investigated the migratory

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response of chemotactic bacteria to a distribution of residual NAPL ganglia entrapped within a

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laboratory-scale sand column under continuous-flow at a superficial velocity of 0.05 cm/min.

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Naphthalene dissolved in a model NAPL 2,2,4,4,6,8,8-heptamethylnonane partitioned into the

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aqueous phase to create localized chemoattractant gradients throughout the column. A pulse

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mixture of equal concentrations of Pseudomonas putida G7, a strain chemotactic to naphthalene,

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and Pseudomonas putida G7 Y1, a non-chemotactic mutant, was introduced to the column and

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effluent bacterial concentrations were measured with time. Breakthrough curves (BTCs) for the

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two strains were noticeably different upon visual inspection. Differences in BTCs (compared to

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non-chemotactic controls) were quantified in terms of percent recovery and were statistically

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significant (p< 0.01). Chemotaxis reduced percent recovery in the effluent by 45% thereby

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increasing the population of bacteria that were retained within the column in the vicinity of

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residual NAPL contaminants. An increase in flow rate to a superficial velocity of 0.25 cm/min

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did not diminish cell retention associated with the chemotactic effect.

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INTRODUCTION

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Non-aqueous phase liquids (NAPLs) typically comprise persistent organic pollutants that pose

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long-term threats to groundwater quality.1–5 In fact, complete dissolution of entrapped NAPL

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contaminants may take many decades.2,6,7 Chemotaxis-aided bioremediation has been recently

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highlighted as a promising technology for more rapid aquifer restoration.8–10 Chemotaxis is a

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phenomenon in which motile cells, such as bacteria, can detect the concentration gradient of a

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chemical and move preferentially towards a region of higher chemical/chemoattractant

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concentration if it is advantageous to do so.11–13 Chemotaxis has the potential to improve

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bioremediation strategies by augmenting the mass transfer of pollutant-degrading bacteria to the

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source of NAPL contamination.

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Bacterial chemotaxis in porous media is well studied. In microfluidic devices, and laboratory-

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scale sand columns and microcosms, researchers documented enhanced migration of chemotactic

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bacteria towards attractant sources in directions transverse to flow in both physically

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homogeneous and heterogeneous porous media.14–16 Controlled field studies showed that the

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coupled effects of cell chemotaxis and proliferation greatly enhanced bacterial transport towards


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a chemical source.17 In all these studies, bacteria exhibited chemotaxis towards macroscopic

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contaminant plumes. NAPLs entrapped in porous media typically form discrete ganglia, each

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serving as a source for contaminant dissolution.2–4 In essence, what results is a distribution of

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multiple, localized, concentration gradients within a NAPL source zone. A few studies have

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investigated chemotactic bacterial response to residual NAPL entrapment at the micro-scale.5,18

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However, this phenomenon is yet to be explored in the meso- and macro-scales. This work with

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NAPLs builds on a recent study in which localized concentration gradients in a sand column

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were created by randomly distributing solid naphthalene crystals within the packed bed.19 While

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similar in design, solid contaminants present different physical interfaces and chemical

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characteristics than are present in porous media containing entrapped NAPL.

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Hence, the purpose of this study was to investigate the migratory response of chemotactic

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bacteria to a distribution of residual NAPL ganglia entrapped within a lab-scale sand-packed

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column. This work is important for assessing the role of bacterial chemotaxis on the

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bioremediation and biorestoration of NAPL source zones.

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MATERIALS AND METHODS

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Bacteria and Culture Conditions. Two motile bacterial strains were used in this study:

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Pseudomonas putida G7 (PpG7), which is chemotactic to naphthalene, and Pseudomonas putida

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G7 Y1 (PpG7 Y1), a nahY::Km non-chemotactic mutant strain.20 A detailed description of the

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bacterial culture conditions is provided in a previous article by Adadevoh and co-workers.19

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Before proceeding with transport experiments, cell motility was verified under oil immersion at

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100× with a Zeiss microscope (F100/1.25 oil). According to a previous study no difference in

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bacteria swimming speed was found between the two strains. 19


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Column Assembly. Sand column experiments were performed in duplicates for each type of

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column set-up (i.e., NAPL experiments, and No NAPL experiments); for each replicate, the sand

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column was re-assembled as follows. A glass chromatography column (diameter 4.8 cm, length

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15.5 cm) was dry packed with 30-40 mesh quartz sand (VWR item BDH9274) in 1-2 cm

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increments under mixing and vibration. The average gravimetrically estimated porosity for

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replicate sand columns was 0.38. To aid subsequent buffer saturation, air was displaced from the

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sand-packed column by flushing the column with several pore volumes of CO2 as CO2 is more

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soluble in the aqueous phase than air. The sand column was then saturated with 10% (v/v)

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random motility buffer (RMB; 11.2 g/L K2HPO4 (Fisher, 7758-11-4), 4.8 g/L KH2PO4

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(Amresco, 7778-77-0), 0.029 g/L EDTA (Sigma-Aldrich, 60-00-4)) at a superficial flow rate of 5

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mL/min, against gravity, using an ÄKTAexplorer (Amersham Pharmacia Biotech, 18-1300-00).

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After fully saturating the sand column with 10% RMB, a conservative, non-reactive, solute tracer

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test (10 mL pulse of 0.05 M NaNO3 (Sigma-Aldrich, 221341) at a superficial rate of 0.905

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mL/min) was conducted to quantify the dispersivity within the column. To capture tracer

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breakthrough, 5 mL effluent samples were collected continuously via a fraction collector

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(Pharmacia Biotech, Frac-900) for 1.6 pore volumes (i.e., 190 minutes). Nitrate concentrations in

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the effluent samples were quantified via absorbance at 300 nm using a Beckman Coulter

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spectrophotometer (DU® 640).

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2,2,4,4,6,8,8-Heptamethylnonane (HMN; Acros Organics, 4390-04-9) was selected as a model

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NAPL based on its prior use when examining the influence of chemotaxis on desorption and

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degradation of naphthalene.8,22 HMN is not known to elicit a chemotactic response in PpG7 and

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was not toxic to the bacteria at the concentrations used in this study. The HMN NAPL contained

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33 g/L naphthalene (Fisher, 91-20-3) for chemotaxis experiments and no naphthalene for HMN


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control experiments. The aqueous-phase concentration of naphthalene in equilibrium with the

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HMN NAPL containing 33 g/L naphthalene was 15.4 mg/L (see the Supporting Information).

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For experiments containing NAPL, a residual NAPL saturation was produced following the

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tracer test using well established protocols.21 In brief, NAPL was introduced into the column at a

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constant flow rate of 1 mL/min using the ÄKTAexplorer. Upon reaching the maximum HMN

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NAPL saturation of 75%, aqueous flow (10% RMB buffer) was re-established and continued

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until reaching a residual, entrapped NAPL saturation of 19%. The NAPL saturation was

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determined gravimetrically. After establishing the residual NAPL saturation, a second tracer test

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was conducted, in a manner similar to that of the first tracer test, to determine the resulting

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aqueous-phase dispersivity.3 All tracer tests were conducted at a superficial flow rate of 0.905

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mL/min.

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Bacterial Transport Experiment and Sample Analysis. Bacterial transport experiments

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were performed in duplicate and for each run, the column packing and NAPL entrapment

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process was repeated, as previously described. A 10 mL mixture (~10% of the pore volume) of

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equal concentrations of PpG7 and PpG7 Y1 (~2 × 108 cells/mL in 10% RMB) was introduced

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into the sand column containing entrapped globules of naphthalene dissolved in HMN at a

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superficial velocity of 0.905 mL/min against gravity. Prior to bacterial transport experiments,

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PpG7 and PpG7 Y1 were stained with red (FM4-64; Molecular Probes, T3166) and green

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(calcein AM; Molecular Probes, C1430) fluorescent dyes, respectively, to aid cell differentiation.

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FM4-64 and calcein AM have excitation/emission wavelengths of ~515/640 nm and ~495/516

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nm, respectively. A previous study showed that these stains had no effect on the cell size, swim

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speed, or zeta potential of PpG7 and PpG7 Y1.19 The same study also showed that the transport

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of either of these strains is not influenced by the presence of the other. Bacterial effluent samples


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(i.e., 5 mL) were collected continuously from the column via a fraction collector (Pharmacia

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Biotech, Frac-900) for 1.7 pore volumes (i.e., 200 min). Once 1.7 pore volumes was reached, the

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flow rate was increased by a factor of 5, while 5 mL effluent samples were still being collected,

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to observe the effect of increased flow rate on bacterial transport within the column. Bacterial

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concentrations in effluent samples were enumerated via a flow cytometer (BD Accuri™ C6).

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The flow cytometry procedure utilized in this study is the same as that previously provided by

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Adadevoh and co-workers.19 Concentrations as low as 105 cells/mL were detectable using this

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method. After each transport experiment, cell motility, and hence viability, was verified via

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visual inspection under oil immersion at 100× with a Zeiss microscope (F100/1.25 oil).

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Naphthalene concentration in the column effluent was also measured through the course of the

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experiment once residual NAPL entrapment was achieved. Samples collected during the

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bacterial experiments were first filtered using 0.22 µm PTFE syringe filters (Celltreat Scientific

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Products, 229757) to remove bacterial cells. Naphthalene was quantified via absorbance at 220

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nm using a Shimadzu Prominence UFLC equipped with a UV detector. Isocratic (85%

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acetonitrile and 15% DI H2O) separation was accomplished on a C-18 column at a flow rate of

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0.6 mL/min.22 To serve as control experiments, bacterial transport was also observed in columns

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containing only sand (i.e., no NAPL) and in columns containing entrapped HMN with no

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dissolved naphthalene. Interstitial velocities for columns containing entrapped NAPL and for

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columns containing no NAPL were 2.33 ± 0.04 m/d and 1.88 ± 0.02 m/d, respectively (note that

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± represents 1 standard deviation about a mean for replicate experiments, unless noted

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otherwise). For columns containing no NAPL, bacterial experiments were conducted for 2 pore

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volumes (i.e., 240 min) before an increase in flow rate. Cell proliferation on naphthalene over the

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course of these experiments was negligible.19,23


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Quantitative Assessment of Breakthrough Curves. Previous studies have used a one-

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dimensional advection-dispersion equation to quantify differences in experimentally acquired

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bacterial breakthrough curves (BTCs).15,19,24,25 One such study employed fitted parameters of the

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1D equation as apparent values used to empirically assess differences in transport between

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chemotactic and non-chemotactic bacteria.19 We have adopted a similar approach in this study.

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The following equation was used to quantify differences in shape and magnitude of BTCs that

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correspond to certain features (e.g. spread in the peak, position of the peak, and area under the

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peak) for chemotactic and non-chemotactic bacterial populations = − − (1)

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where b is the dimensionless species concentration in the aqueous phase [-], t is time [T], z is the

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longitudinal position [L], R is used to quantify the reversible retention of bacterial mass [-], Dbz

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is the longitudinal hydrodynamic dispersion coefficient [L2T-1], vf is the interstitial pore water

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velocity [LT-1], and km represents irreversible, first-order retention of bacteria within the medium

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[T-1]. The nonlinear least-squares parameter optimization method in CXTFIT was employed to

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fit the 1D equation to the experimental BTC data.26 Specifically, R, Dbz, and km parameter values

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were fit. Longitudinal dispersivity, αz [L], was calculated via = + where Deff

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[L2T-1] is the effective motility coefficient for bacteria in porous media.9 For P. putida strains in

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30-40 mesh quartz sand, Deff was reported to be 1.3 × 10-5 cm2/s.11,15,17,27

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Note that the parameters in Equation 1 do not explicitly represent chemotaxis – the 1D

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advection-dispersion equation was employed only to show that there are quantitative differences

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in BTCs for chemotactic bacteria by comparing apparent or effective parameters obtained by

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fitting Equation 1. Thus, the reader is cautioned against attributing these parameter values to the

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description of any particular physical process. Also note that Equation 1 does not include kinetic


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sorption/desorption terms, so it cannot capture the tailing phenomenon that is evident in the

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experimental BTCs. Our goal in this study was not to develop a mathematical model for bacterial

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transport, rather we used Equation 1 to quantify differences between various characteristics of

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BTCs that were evident upon visual inspection.

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Cell normalized mean travel time (τ) and percent recovery from the sand column were also

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calculated via the first and zeroth moment of the bacterial BTCs, respectively.28 Additionally, the

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1D advection-dispersion equation was fit to tracer BTCs in a manner similar to that described for

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bacterial BTCs with the exception that only Dbz was fit while R and km were prescribed at 1.0 and

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0, respectively.

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RESULTS AND DISCUSSION

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Sand Column Characterization. Three types of sand column experiments were conducted in

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this study: (1) water-saturated sand columns with no HMN NAPL and no naphthalene (i.e., No

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NAPL); (2) sand columns with entrapped HMN NAPL, but no naphthalene (i.e., HMN NAPL);

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and (3) sand columns with entrapped HMN NAPL containing 33 g/L naphthalene (i.e., HMN-

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NAP NAPL). Table 1 lists characteristic parameters for two replicates of each type of sand

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column experiment. Prior to NAPL entrapment, the average pore water velocity and standard

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deviation within the sand column was determined to be 1.88 ± 0.02 m/d for six experimental runs

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(i.e., duplicate experiments for HMN-NAP NAPL, HMN NAPL, and No NAPL). After

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entrapment, residual NAPL saturation within the column was gravimetrically estimated to be 19

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± 2% and the resulting interstitial velocity was 2.33 ± 0.04 m/d. For HMN-NAP NAPL

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experiments, effluent naphthalene concentrations were observed to remain at a steady-state value

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of 10.2 ± 0.1 mg/L. Longitudinal dispersivity assessed by the nitrate tracer tests was 0.39 ± 0.12


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mm. Values for the dispersivity parameter assessed from bacterial BTCs are reported in Table 2

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with an average of 1.8 ± 0.75 mm.

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Influence of NAPL on Bacterial Transport. Chemotactic (PpG7) and non-chemotactic

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(PpG7 Y1) bacterial transport was observed in duplicate experiments in each of the previously

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described types of sand columns: (1) water-saturated sand columns with no HMN NAPL and no

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naphthalene; (2) sand columns with entrapped HMN NAPL, but no naphthalene; and (3) sand

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columns with entrapped HMN NAPL containing 33 g/L naphthalene. BTCs in Figure 1 c-d were

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from columns that contained HMN, but no naphthalene. Because there was no chemoattractant,

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the BTCs were expected to look the same for both chemotactic and non-chemotactic species and

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this was the case. The open circles in panels c and d were consistent with what we expected to

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observe. Bacteria did not exhibit a chemotactic response in the absence of naphthalene. The open

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diamond symbols also showed no difference between chemotactic and nonchemotactic species.

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Although we see differences in the qualitative aspects of the BTCs (comparing diamonds to

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circles), the differences between the percent recoveries – one of the main quantitative descriptors

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– is similar to that observed between the two BTCs in Figure 1b. There is the possibility that the

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column packing yielded differences in the qualitative trend of the BTCs, such as the peak values.

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This, however, should not detract from the main message that we obtain from the BTCs –

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bacteria did not exhibit a chemotactic response in the absence of naphthalene.

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To focus on the effect of residual NAPL entrapment on bacterial transport, we direct our

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attention to bacterial migration in columns containing only HMN and in those with no NAPL

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(Figure 1c-f). The presence of NAPL within the column led to an earlier breakthrough of cells

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and resulted in an average of 17% decrease in normalized mean travel time, τ, (Table 2),

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compared to results obtained in the absence of NAPL for both PpG7 and PpG7 Y1. The earlier


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cell breakthrough is a result of the greater interstitial velocity arising in those experiments

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occurring in the presence of NAPL (i.e., 19% lower water saturation with a similar superficial

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velocity). For experiments with no NAPL, bacterial normalized mean travel time was greater

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than 1 due to reversible retention of cells in the sandy medium; values of τ are comparable to

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those previously reported for these bacterial strains in 30-40 mesh quartz sand (e.g., τ = 1.26).19

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Bacterial cell recovery was found to be approximately 50% lower in the presence of entrapped

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NAPL (38-46% in the absence of NAPL versus 18-25% in the presence of the HMN NAPL).

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Correspondingly, the parameter associated with irreversible retention (km) was found to be

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approximately double, while reversible retention (R) remained relatively similar across all

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experiments (Table 2). The decrease in cell recovery was expected as a previous study reported

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that due to bacterial motility alone, cells may encounter the NAPL-aqueous interface more

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frequently and subsequently be retained at/near the interface.22 In that study Law and Aitken22

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observed greater accumulation of motile bacteria on the surface of an HMN drop by direct visual

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inspection with microscopy. Another study documented that bacterial cells had a stronger

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affinity for an oil-water interface compared to a glass-water interface and suggested that the

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increased affinity was due to attractive energies and capillary forces.29

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Differences in colloidal attachment and retention at immiscible liquid interfaces and solid-fluid

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interfaces have been explained using interaction energy profiles.30 Thus, energy profiles of PpG7

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interaction with HMN NAPL, and PpG7 interaction with sand were calculated based on the

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Derjaguin-Landau-Verwey-Overbeek (DLVO) theory (see Figure S1 in the Supporting

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Information). Repulsive energy barriers were compared given that this energy has to be

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overcome for irreversible attachment of cells to occur.31 The repulsive energy barrier between

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the cells and HMN NAPL was found to be 715 kT less than the repulsive energy barrier between


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the cells and the sand. The reduced repulsive energy barrier for bacterial cell interaction with

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NAPL implies that the cells have a greater tendency to be irreversibly retained near the NAPL

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surface as compared to the sand surface. A greater attachment of cells to the NAPL surface

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results in the decreased cell percent recovery observed in this study for NAPL experiments, even

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when chemotaxis is not occurring.

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Influence of Chemotaxis on Bacterial Transport in Sand Columns with Residual NAPL.

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Chemotaxis-directed bacterial transport resulted in a distinct decrease in the recovery of PpG7

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(10-13%, Figure 1a) as compared to both the NAPL control (18-25%, Figure 1c) conducted with

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PpG7 and the chemotaxis control conducted with PpG7 Y1 (18-24%, Figure 1b). A one-tailed T-

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test revealed a statistically significant decrease (p

Chemotaxis Increases the Retention of Bacteria in Porous Media with

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