Chemotaxis increases the retention of bacteria in porous media with

Subscriber access provided by University of Winnipeg Library

Environmental Processes

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23

Environmental Science & Technology

1

Chemotaxis increases the retention of bacteria in

2

porous media with residual NAPL entrapment

3

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

4

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

5

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

6

02155

7

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

8

Pseudomonas putida

9 10

ABSTRACT

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

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

ACS Paragon Plus Environment

1


Page 2 of 23

20

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

21

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

22

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

23

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

24

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

25

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

26

did not diminish cell retention associated with the chemotactic effect.

27 28

INTRODUCTION

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

source of NAPL contamination.

38

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

39

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

40

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

41

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

42

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


2

Page 3 of 23


43

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

44

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

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

characteristics than are present in porous media containing entrapped NAPL.

53

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

54

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

55

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

56

bioremediation and biorestoration of NAPL source zones.

57

MATERIALS AND METHODS

58

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

59

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

60

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

61

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

62

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

63

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

64

bacteria swimming speed was found between the two strains. 19


3


Page 4 of 23

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

spectrophotometer (DU® 640).

83

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

84

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

85

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

86

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

87

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


4

Page 5 of 23


88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

mL/min.

99

Bacterial Transport Experiment and Sample Analysis. Bacterial transport experiments

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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


5


Page 6 of 23

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

course of these experiments was negligible.19,23


6

Page 7 of 23


134

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

135

dimensional advection-dispersion equation to quantify differences in experimentally acquired

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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


7


Page 8 of 23

156

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

157

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

158

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

159

BTCs that were evident upon visual inspection.

160

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

161

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

162

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

163

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

164

0, respectively.

165

RESULTS AND DISCUSSION

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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


8

Page 9 of 23


178

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

179

with an average of 1.8 ± 0.75 mm.

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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


9


Page 10 of 23

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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


10

Page 11 of 23


224

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

225

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

226

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

227

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

228

when chemotaxis is not occurring.

229

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

230

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

231

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

232

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

233

test revealed a statistically significant decrease (p

Chemotaxis increases the retention of bacteria in porous media with

Recommend Documents