pH Dependency in Anode Biofilms of Thermincola ferriacetica

Subscriber access provided by UNIV OF SCIENCES PHILADELPHIA

pH Dependency in Anode Biofilms of Thermincola ferriacetica Suggests a Proton-Dependent Electrochemical Response Bradley G. Lusk, Isaias Peraza, Gaurav Albal, Andrew K. Marcus, Sudeep C Popat, and César I. Torres J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 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 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

82x44mm (300 x 300 DPI)

ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

pH Dependency in Anode Biofilms of Thermincola ferriacetica Suggests a ProtonDependent Electrochemical Response

3 4 5

Bradley G. Lusk†,¤, Isaias Peraza†, Gaurav Albal†, Andrew K. Marcus†, Sudeep C. Popat*, Cesar I. Torres†,§

6 7 8 9 10 11 12 13 14 15

†

Swette Center for Environmental Biotechnology, The Biodesign Institute at Arizona State University, P.O. Box 875701, Tempe, Arizona 85287−5701, United States of America ¤

ScienceTheEarth, Mesa, AZ, 85201

*Department of Environmental Engineering and Earth Sciences, Clemson University, 342 Computer Ct, Anderson, SC 29625, United States of America §

School for Engineering of Matter, Transport and Energy, Arizona State University, 501 E Tyler Mall, Tempe, Arizona 85287, United States of America

16

1 ACS Paragon Plus Environment

Page 2 of 31

Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

17


ABSTRACT

18

Monitoring the electrochemical response of anode respiring bacteria (ARB) helps

19

elucidate the fundamental processes of anode respiration and their rate limitations.

20

Understanding these limitations provides insights on how ARB create the complex interfacing of

21

biochemical metabolic processes with insoluble electron acceptors and electronics. In this study,

22

anode biofilms of the thermophilic (60 oC) Gram-positive ARB Thermincola ferriacetica were

23

studied to determine the presence of a proton-dependent electron transfer response. The effects

24

of pH, the presence of an electron donor (acetate), and biofilm growth were varied to determine

25

their influence on the electrochemical midpoint potential (EKA) and formal redox potential (Eo`)

26

under non-turnover conditions. The EKA and Eo` are associated to an enzymatic process within

27

ARB’s metabolism that controls the rate and energetic state of their respiration. Results for all

28

conditions indicate that pH was the major contributor to altering the energetics of T. ferriacetica

29

anode biofilms. Electrochemical responses measured in the absence of an electron donor and

30

with a minimal proton gradient within the anode biofilms resulted in a 48 ± 7 mV/ pH unit shift

31

in the Eo`, suggesting a proton-dependent rate-limiting process.

32

available for anode respiration (< 200 mV when using acetate as electron donor), our results

33

provide a new perspective in understanding proton-transport limitations in ARB biofilms, one in

34

which ARB are thermodynamically limited by pH gradients. Since the anode biofilms of all

35

ARB which perform direct extracellular electron transfer (EET) investigated thus far exhibit an n

36

= 1 Nernstian behavior, and because this behavior is affected by changes in pH, we hypothesize

37

that the Nernstian response is associated to membrane proteins responsible for proton

38

translocation. Finally, this study shows that the EKA and Eo` are a function of pH within the

39

physiological range of ARB and thus, given the significant effect pH has on this parameter, we

40

recommend reporting the EKA and Eo` of ARB biofilms at a specific bulk pH. 2 ACS Paragon Plus Environment

Given the limited energy


41

42

Key words: Microbial Electrochemical Cell, Nernst-Monod Kinetics, Midpoint Potential, pH,

43

Biofilm

44


Page 4 of 31

Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

45 46


INTRODUCTION For over a century, researchers have attempted to interface biology and electrochemistry

47

by connecting cells, enzymes, or cell components to electrodes [1, 2]. These efforts have led to

48

research developments on enzymatic and microbial-based technologies centered on bio-

49

electricity including microbial electrochemical cells (MxCs) for wastewater treatment [3]

50

biosensors for detecting environmental contaminants [4], and self-powered human-implanted

51

devices [5]. While most existing technologies rely on synthetic molecules to interface biology

52

and electrodes, a unique opportunity arises through the discovery of anode respiring bacteria

53

(ARB): a natural, self-assembled, bioelectrochemical interface.

54

Understanding how ARB form an electrochemical network will elucidate how to

55

efficiently interface biology and electronics, and thus provide insights for technologies based on

56

bio-electricity. The study of MxCs, for example, has been an active research area for more than a

57

decade [3, 6, 7]; nevertheless, implementation of commercial MxCs is still limited. The

58

development of this emerging technology is impacted by the fundamental knowledge of how the

59

ARB connect to the MxC anode. Yet, many details of this process are unknown, from the

60

complex set of proteins that allows ARB to transport electrons to their outer membrane, to the

61

extracellular electron transfer (EET) that leads electrons to the anode surface [8, 9, 10].

62

Despite the complexity of microbial metabolism involving anode respiration, many ARB

63

including Geobacter sulfurreducens [11], Thermincola ferriacetica [12, 13],

64

Thermoanaerobacter pseudethanolicus [14], Geoalkalibacter ferrihydriticus [15], and

65

Geoalkalibacter subterraneus [15] exhibit a simple Nernstian response to changes in anode

66

potential, which can be modeled with an n = 1 irreversible Nernst-Monod equation [16]. This

67

thermodynamic/kinetic response of ARB suggests that a rate-limiting enzymatic reaction 4 ACS Paragon Plus Environment


Page 6 of 31

68

controls the overall rate of respiration, and thus current generation in Gram-negative and Gram-

69

positive ARB [17, 18]. Through the study of the electrochemical response of ARB, it has been

70

assumed that the n =1 reaction was that of a simple electron transfer [16, 19, 20, 21], like that

71

carried out by most cytochromes as shown in Equation 1: Mredn−1 → Moxn + e−

72

(1)

73

Given the importance of cytochromes in the electron transport chain, their involvement in

74

transport of electrons to the outer membrane in Gram-negative bacteria, and most likely in EET,

75

the assumption that the electrochemical response of ARB follows Equation 1 has been widely

76

accepted [16, 19, 20, 21].

77

Changes in pH also affect the energetics of ARB metabolism. Due to the nature of their

78

respiration, ARB have a net metabolic reaction in which one proton is released per electron, as

79

shown by Equation 2:

80

CH3COO- + 3H2O CO2 + HCO3- + 8H+ + 8e-

(2)

81

Since the overall oxidation of the electron donor (e.g., acetate) leads to the production of one

82

proton per electron, the Nernst equilibrium potential of the electron donor shifts 59.1 mV/ pH

83

unit at 30 oC (66.1 mV/ pH unit at 60 oC) [22]. In G. sulfurreducens, the midpoint potentials

84

(EKA) (−0.16 V and -0.10 vs SHE) [23] compared to the standard potential of acetate (−0.29 V vs

85

SHE) provides less than 200 mV for growth. Thus, a single pH unit increase or decrease

86

significantly changes the potential available for ARB growth. However, if the electrochemical

87

response of ARB is that of a single electron transfer as in Equation 1, the reaction is proton-

88

independent and cyclic voltammograms (CVs) should not change as a function of pH.


Page 7 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


89

However, [24] reported a shift in the electrochemical response of G. sulfurreducens non-

90

turnover (electron-donor depleted) CVs as a function of pH that followed a ~49 mV/ pH unit

91

shift. This is the only report that has quantified the shift in EKA in G. sulfurreducens biofilms as

92

a function of pH and it is unknown if the catalytic curve also follows the same dependency.

93

Nonetheless, the shift in electrochemical response as a function of pH in G. sulfurreducens

94

suggests that the electrochemical response is linked to a proton-dependent electron transfer

95

reaction [24].

96

simultaneous release of a proton and an electron such as the case of quinones as shown in

97

Equation 3:

98

This proton linkage can be related to the net reaction being linked to the

MnHred → Mox + nH+ + ne−

(3)

99

Alternatively, some proteins, mostly those involved in proton translocation at the

100

electron-transport chain, will have a pH dependency, known as the redox-Bohr effect, in which

101

the protonation of a site within the protein causes a shift in its thermodynamic equilibrium [21,

102

25, 26]. Cytochromes within the periplasmic space of G. sulfurreducens, including PpCA and

103

PccH, have been shown to have a redox-Bohr effect and have been linked to proton translocation

104

for ATP synthesis [27, 28, 29, 30].

105

The pH dependency of ARB’s electrochemical response greatly affects the interpretation

106

of electron transfer kinetics and the modeling efforts performed so far [16, 19, 20, 31, 32]. It also

107

limits the possible biochemical reactions that could be responsible for this electrochemical

108

signal, providing new insights on how ARB perform this unique bioelectrochemical interface.

109

The redox-pH dependency of individual proteins has been widely studied [25, 27, 28, 29, 30] but

110

studying this phenomenon on a complex ARB biofilm network is a major challenge [24].



111

We are interested in determining whether the Nernstian response of ARB is proton-

112

dependent. For this, we conducted the first systematic study to observe the effects of pH on the

113

energetics of anode respiration by ARB under catalytic and non-catalytic conditions. pH

114

gradients in ARB biofilms have been widely documented [13, 31, 33] and are known to limit

115

current production. Since G. sulfurreducens has a narrow pH range for growth (5.8-8.0) [31,

116

34], it would be difficult to document significant pH changes without affecting the physiological

117

and metabolic conditions of this ARB. Thus, in this study, we use T. ferriacetica, a thermophilic

118

(60 oC) Gram-positive ARB that has shown a decreased response to pH gradients in the anode

119

biofilm [13].

120

In addition, T. ferriacetica grows in a wider pH range (5.2-8.3) than most known ARB

121

[13] and contains 35 multi-heme c-type cytochromes [35]. To assess the pH-dependent response

122

of anode biofilms of T. ferriacetica, we used low-scan cyclic voltammetry (LSCV) to determine

123

the EKA under catalytic and formal redox potential (Eo) under non-turnover conditions as a

124

function of pH. In addition, LSCV was used to monitor the EKA for the anode biofilm as a

125

function of biofilm growth - showing that pH-dependency is the major factor influencing shifts

126

in the EKA and Eo of T. ferriacetica anode biofilms. We conclude the manuscript with an in depth

127

discussion of our understanding of this phenomenon and its implications in the field of

128

bioelectrochemistry.

129

130

MATERIALS AND METHODS

131

Growth Medium and Culture Conditions. A modified DSMZ Medium 962:

132

Thermovenabulum medium was used to grow T. ferriacetica strain 14005 (DSMZ, Braunshweig,

133

Germany). The modified medium contained per liter: 3.4 g NaCH3COO•3H2O, 0.33 g NH4Cl , 7 ACS Paragon Plus Environment

Page 8 of 31

Page 9 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


134

0.33 g K2HPO4, 0.33 g MgCl2•H2O, 0.1 g CaCl2•2H2O, 0.33 g KCl, 0.05 g yeast extract, 1 mL

135

selenite−tungstate stock solution (prepared by dissolving 3 mg Na2SeO3•5H2O, 4 mg

136

Na2WO4•2H2O, and 0.5g NaOH in 1 L distilled water), Wolfe’s vitamin solution (10 ml), and

137

trace elements solution (10 ml). The trace elements solution was composed of the following

138

ingredients (DSMZ 141 Methanogenium Medium) in 1 L deionized water: 1.5 g nitrilotriacetic

139

acid, 3.0 g MgSO4•7H2O, 0.5 g MnSO4•H2O, 1.0 g NaCl, 0.1g FeSO4•7H2O, 0.18 g

140

CoSO4•7H2O, 0.1 g CaCl2•2H2O, 0.18 g ZnSO4•7H2O, 0.01 g CuSO4•5H2O, 0.02 g

141

KAl(SO4)2•12H2O, 0.01 g H3BO3, 0.01 g Na2MoO4•2H2O, 0.03 g NiCl2•6H2O, and 0.3 mg

142

Na2SeO3•H2O. Medium was prepared in a condenser apparatus under N2:CO2 (80:20) gas

143

conditions. Medium was brought to boil and allowed to boil for 15 minutes per liter. Medium

144

was stored in 100 ml serum bottles and autoclaved for 15 minutes at 121 °C. ATCC Vitamin

145

Solution and NaHCO3 were added after autoclaving; NaHCO3 concentrations were 50 mM for

146

each experimental condition. Initial T. ferriacetica stock cultures were grown in 100 ml serum

147

bottles containing 10 mM Fe(OH)3 in an Excella E24 Incubator Shaker (New Brunswick

148

Scientific) at 60 ºC and 150 RPM. Subsequent cultures were inoculated using culture grown in

149

the laboratory.

150

H-Type Microbial Electrolysis Cell Construction for All Experiments with T.

151

ferriacetica. H-type microbial electrolysis cells (MECs) were constructed and operated in batch.

152

Each MEC consisted of two 350 mL compartments separated by an anion exchange membrane

153

(AMI 7001, Membranes International, Glen Rock, NJ). For all MECs, operating temperature

154

was 60 °C. All MECs contained two cylindrical graphite anodes with varying surface areas and

155

an Ag/AgCl reference electrode (BASi MF-2052). Reference potential conversion to a standard

156

hydrogen electrode (SHE) was conducted by constructing a two-chambered cell with one



Page 10 of 31

157

chamber containing modified DSMZ Medium 962 and the other containing 1M KCl [12, 36].

158

Anode potential was poised at -0.06 V vs SHE and current was continuously monitored every

159

two minutes using a potentiostat (Princeton Applied Research, Model VMP3, Oak Ridge, TN).

160

For all MECs, the anode chambers were mixed via agitation from a magnetic stir bar. The

161

cathode consisted of a single cylindrical graphite rod (0.3 cm diameter and a total area of 6.67

162

cm2). Cathode pH was adjusted to 12 via addition of NaOH. Gas collection bags were placed on

163

the anode compartments to collect volatile products and on the cathode to collect hydrogen.

164

Sterile conditions were maintained as reported in [13].

165

Effect of pH on Electrochemical Response at Non-turnover Conditions.

T.

166

ferriacetica stock cultures (10 ml) from 100 ml serum bottles were used to inoculate MECs.

167

Mature biofilms were established on anodes with media containing 50 mM bicarbonate buffer

168

and 25 mM acetate. Then, biomass from the biofilms was transferred to subsequent MECs to

169

eliminate background response from Fe(OH)3. Anode surface areas were 3.02 and 3.64 cm2.

170

After a mature biofilm was established in each reactor, the medium was replaced by flowing 1 L

171

(~3 reactor volumes) of 50 mM bicarbonate buffer with no acetate to achieve a current density (j)

172

= ~0.05 A m-2. pH was altered by either the addition of HCl or NaOH and at each pH condition,

173

after achieving a steady j, LSCV was performed at 1.0 mV s-1 (~45 minutes). Graphs were

174

created by performing a baseline subtraction for all conditions and then normalizing to j/jmax. All

175

measurements were performed in triplicate. For all conditions, a one-way ANOVA with post-hoc

176

Tukey Honest Significant Difference (HSD) test was performed as reported in [13]. Cell viability

177

was confirmed in Supporting Information Fig S8 by showing steady current (~0.05 A m-2) for

178

~30 minutes after each scan. Positive (unresponsive biofilms) and negative (bare electrode)

179

controls are shown in Supporting Information Fig S9. 9 ACS Paragon Plus Environment

Page 11 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

180 181


For all non-turnover experiments, Eo was determined by using the mean of the cathodic (Epc) and anodic peaks (Epa) as shown in Equation 4: ° =

182

(4)

183

Where Eo is the formal redox potential, Epc is the cathodic peak potential, and Epa is the anodic

184

peak potential.

185

Effect of pH on Electrochemical Response at Catalytic Conditions. MECs were

186

inoculated and mature biofilms were established in an identical matter to the non-turnover

187

experiments. To determine the effect of pH on EKA of T. ferriacetica, MECs were operated using

188

50 mM bicarbonate buffer and 25 mM acetate as the electron donor. Each MEC had an anode

189

surface area of 3.89 cm2. At each pH condition, after achieving a steady j, pH was altered by

190

either the addition of HCl or NaOH and LSCV was performed at 1.0 mV s-1 (~45 minutes).

191

Graphs were plotted as normalized current density, j/jmax. All measurements were performed in

192

triplicate. For all conditions, a one-way ANOVA with post-hoc Tukey Honest Significant

193

Difference (HSD) test was performed as reported in [13]. Cell viability was confirmed in

194

Supporting Information Fig S7 by showing steady current for ~30 minutes after each scan.

195

Positive (unresponsive biofilms) and negative (bare electrode) controls are shown in Supporting

196

Information Fig S10.

197

Effect of Biofilm Growth Stage on T. ferriacetica’s Electrochemical Response. To

198

test whether growth was a determining factor in the midpoint potential response, MECs were

199

operated using 25 mM acetate, 50 mM bicarbonate. All MECs were inoculated following the

200

protocols from the non-turnover and catalytic experiments. MEC anodes had surface area of

201

3.89 cm2 and were monitored over 36 hours as j increased from 0 A m-2 to 12.8 A m-2. LSCV 10 ACS Paragon Plus Environment


Page 12 of 31

202

was conducted at a scan rate of 1.0 mV s-1 (~45 minutes) at each gain in j of ~1 A m-2. Samples

203

were taken for pH at the beginning (j = 1 A m-2) and at the end (j = 12.8 A m-2) of the

204

experiment. The impact of the change in current density in young biofilms on EKA was assessed

205

by subtracting the impact caused by change in pH (48 ± 7 mV/ pH unit) determined from

206

observing the non-catalytic response to change in pH. All measurements were performed in

207

triplicate. For all conditions, a one-way ANOVA with post-hoc Tukey Honest Significant

208

Difference (HSD) test was performed as reported in [13].

209

Determining pH Gradients Inside Thermophilic T. ferriacetica Biofilms. To estimate

210

the pH gradients inside the ARB biofilm, we used a modified version of the Nernst–Monod

211

equation developed by [16], shown in Equation (5). This kinetic expression uses an empirical

212

pH-inhibition function based on the changes of the pH inside the ARB-biofilm [37].

213

= (

214

Where qmaxXf is the maximum substrate consumption rate by the T. ferriacetica (mol Ace cm-3

215

day-1), S is substrate donor (acetate) concentration (mol m-3), KS,app is the half-saturation constant

216

for T. ferriacetica (mol m-3), E4node is the anode potential (V), E◦KA is the potential at which

217

current (j) is ½ jmax at pH 6.9 (V), F is the Faraday constant (96485 C mol-1), R is the ideal gas

218

constant (8.314 J mol-1 K-1), T is temperature of the system (K), pHbulk is pH in the bulk solution,

219

pH is the pH inside the T. ferriacetica biofilm, and pHopt is the optimum pH at which T.

220

ferriacetica grows [13, 31]. To simulate the forward scan cyclic voltammetry, the Eanode was

221

made a time-dependent variable with the initial value of -0.6 V vs SHE and scan rate of 1.0 mV

222

s-1.

,

)(

(

( ° $.$&&('('()*+, ) -./0 ) ! "#


)(

1 2

) (5)

( ('('(.3 )

Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


223

To compute the gradients of acetate, buffer, and pH inside the biofilm, we used Equation

224

5 coupled to a set of two time-dependent partial differential equations with diffusion and reaction

225

terms for acetate (Equation 6) and bicarbonate buffer in its protonated form (Equation 7):

226

9[;

9>

9

= 9? @ABC 9

9[;

pH Dependency in Anode Biofilms of Thermincola ferriacetica

Recommend Documents