- Email: [email protected]
The yield and decay coefficients of exoelectrogenic bacteria in bioelectrochemical systems
Accepted Manuscript The yield and decay coefficients of exoelectrogenic bacteria in bioelectrochemical systems Erica L. Wilson, Younggy Kim PII:
S0043-1354(16)30113-0
DOI:
10.1016/j.watres.2016.02.054
Reference:
WR 11870
To appear in:
Water Research
Received Date: 1 December 2015 Revised Date:
25 February 2016
Accepted Date: 26 February 2016
Please cite this article as: Wilson, E.L., Kim, Y., The yield and decay coefficients of exoelectrogenic bacteria in bioelectrochemical systems, Water Research (2016), doi: 10.1016/j.watres.2016.02.054. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
pH = 9 & anode = −0.1 V vs. SHE
Bioelectrochemical systems
Cell yield
Organic substrate
Exoelectrogen
ACCEPTED MANUSCRIPT
SC TE D
M AN U
Cathode
EP
Bioanode
AC C
CO2
RI PT
pH = 5 & anode = −0.3 V vs. SHE Cell yield
ACCEPTED MANUSCRIPT
1 2
Submitted to: Water Research Date of submission: Feb. 25, 2016 (Original submission: Nov. 28, 2015)
5
Erica L. Wilson & Younggy Kim*
6 7 8 9
Department of Civil Engineering, McMaster University 1280 Main St. W., JHE 301, Hamilton, Ontario, L8S 4L7, Canada *corresponding author: [email protected]; 1-905-525-9140 ext.24802
RI PT
4
The yield and decay coefficients of exoelectrogenic bacteria in bioelectrochemical systems
3
Abstract
11
In conventional wastewater treatment, waste sludge management and disposal contribute the
12
major cost for wastewater treatment. Bioelectrochemical systems, as a potential alternative for
13
future wastewater treatment and resources recovery, are expected to produce small amounts of
14
waste sludge because exoelectrogenic bacteria grow on anaerobic respiration and form highly
15
populated biofilms on bioanode surfaces. While waste sludge production is governed by the yield
16
and decay coefficient, none of previous studies have quantified these kinetic constants for
17
exoelectrogenic bacteria. For yield coefficient estimation, we modified McCarty’s free energy-
18
based model by using the bioanode potential for the free energy of the electron acceptor reaction.
19
The estimated true yield coefficient ranged 0.1 to 0.3 g-VSS (volatile suspended solids) g-COD-1
20
(chemical oxygen demand), which is similar to that of most anaerobic microorganisms. The yield
21
coefficient was sensitively affected by the bioanode potential and pH while the substrate and
22
bicarbonate concentrations had relatively minor effects on the yield coefficient. In lab-scale
23
experiments using microbial electrolysis cells, the observed yield coefficient (including the effect
24
of cell decay) was found to be 0.020 ± 0.008 g-VSS g-COD-1, which is an order of magnitude
25
smaller than the theoretical estimation. Based on the difference between the theoretical and
26
experimental results, the decay coefficient was approximated to be 0.013 ± 0.002 d-1. These
AC C
EP
TE D
M AN U
SC
10
1
ACCEPTED MANUSCRIPT
findings indicate that bioelectrochemical systems have potential for future wastewater treatment
28
with reduced waste sludge as well as for resources recovery. Also, the found kinetic information
29
will allow accurate estimation of wastewater treatment performance in bioelectrochemical
30
systems.
RI PT
27
31
Keywords
33
Wastewater sludge; microbial electrolysis cells; microbial fuel cells; microbial kinetics and
34
energetics; yield coefficient; cell decay coefficient
M AN U
SC
32
35
Nomenclature
37
Abbreviations
38
COD
chemical oxygen demand
39
FID-GC
flame ionization detector-gas chromatography
40
MDC
microbial desalination cell
41
MEC
microbial electrolysis cell
42
MFC
43
RPM
44
SHE
standard hydrogen electrode
45
TSS
total suspended solids
AC C
EP
TE D
36
microbial fuel cell rotations per min
2
ACCEPTED MANUSCRIPT
46
VSS
volatile suspended solids
47
Symbols
48
A
RI PT
number of moles of electrons transferred to terminal electron acceptor out of A+1 moles (-)
49
b
cell decay coefficient (d-1)
51
c
sodium acetate concentration (mg L-1)
52
CE
Coulombic efficiency (-)
53
Ean
bioanode potential (V vs. SHE)
54
F
Faraday constant (96485 C per mole-e−)
55
m
exponent for electron transfer efficiency based on spontaneity of pyruvate creation (-)
56
TE D
M AN U
SC
50
n
number of electrons transferred at the bioanode (-)
58
V
volume of MEC chamber (L)
59
Xanode
volatile biomass attached on the bioanode (g-VSS)
60
Yobserved
observed yield coefficient (g-VSS g-COD-1)
61
Ytrue
true yield coefficient (g-VSS g-COD-1)
62
∆Ga
63
∆Gc°ʹ
64
AC C
EP
57
free energy released from the electron acceptor reaction (kJ per mole-e−) free energy required to convert pyruvate to cellular carbon under standard conditions at pH 7 (kJ per mole-e−)
3
ACCEPTED MANUSCRIPT
65
∆Gd
free energy released from the electron donor reaction (kJ per mole-e−)
66
∆Gn°ʹ
free energy required to create ammonia from other nitrogen sources under standard
∆Gp°ʹ
free energy required to convert the substrate to pyruvate under standard conditions at pH 7 (kJ per mole-e−)
69
∆Gr
free energy that the microorganism obtains from the electron donor and acceptor reactions (kJ per mole-e−)
71
72
SC
70
∆Gs°ʹ
free energy required for cell synthesis under standard conditions at pH 7 (kJ per
M AN U
68
RI PT
conditions at pH 7 (kJ per mole-e−)
67
mole-e−)
73
ε
electron transfer efficiency (-)
75
τ
average time period of each fed-batch cycle (d)
76
Superscripts
77
an
anolyte or anode chamber
78
cat
catholyte or cathode chamber
79
Subscripts
80
eff
81
in
AC C
EP
TE D
74
effluent
influent
82
83
4
ACCEPTED MANUSCRIPT
84
1. Introduction Bioelectrochemical systems are an emerging method for wastewater treatment and
86
simultaneous recovery of energy and valuable resources (Tice and Kim, 2014a; Zhang and
87
Angelidaki 2015a; Zhang and Angelidaki 2015b; Kim et al., 2015; Qin et al., 2015). In
88
bioelectrochemical systems, various reduction reactions at the cathode (e.g., hydrogen evolution,
89
oxygen reduction, biofuel production, and heavy metal reduction) are coupled with oxidation of
90
organic substrates at the bioanode (Logan et al., 2006; Logan et al., 2008; Modin et al., 2012;
91
Nilges and Schröder, 2013). Exoelectrogenic bacteria, growing on bioanode surfaces, oxidize
92
organic substrates and transfer electrons directly to the bioanode, creating electric current. The
93
created electric current can be beneficially utilized in engineered bioelectrochemical systems.
94
For instance, microbial fuel cells (MFCs) are used to generate electric power (Logan et al., 2006);
95
microbial electrolysis cells (MECs) produce H2 gas (Logan et al., 2008); microbial desalination
96
cells (MDCs) utilize the electric current to separate charged ions from salt water (Cao et al.,
97
2009); and bioanodes can also be used as water quality sensors to monitor toxic contaminants
98
(Ahn and Schröder, 2015). In addition to these beneficial aspects, bioelectrochemical systems
99
were found in a recent study to produce substantially small amounts of waste sludge in
101
SC
M AN U
TE D
EP
wastewater treatment (Brown et al., 2015).
AC C
100
RI PT
85
In conventional activated sludge systems, approximately 60% of organic substrates are
102
converted into heterotrophic cells on the COD (chemical oxygen demand) basis (Rittmann and
103
McCarty, 2001; Grady et al., 2011). This high yield coefficient (0.6 g-COD g-COD-1) of
104
heterotrophic bacteria results in substantial amounts of waste sludge in wastewater treatment.
105
The waste sludge is further treated in anaerobic digesters and mechanical dewatering processes
106
before sludge cakes are disposed of in landfills or burnt in incinerators. The sludge treatment and 5
ACCEPTED MANUSCRIPT
final disposal require high costs and especially the cost for the final disposal has dramatically
108
increased as wet wastes (including wastewater sludge cakes) are regulated in landfill disposal. As
109
a result, the cost for sludge treatment and disposal is one of the main expenses for wastewater
110
treatment; thus, reduction in waste sludge will allow cost-effective wastewater treatment.
111
RI PT
107
In a recent study, bioelectrochemical systems were found to produce a substantially small amount of waste sludge (Brown et al., 2015); however, the yield coefficient of exoelectrogenic
113
bacteria has not been quantified. Exoelectrogenic bacteria grow on anaerobic respiration as they
114
originally use ferric iron or elemental sulfur as a terminal electron acceptor (Rotaru et al., 2011).
115
Anaerobic microorganisms are known to have a relatively small yield coefficient (0.04 to 0.1 g-
116
COD g-COD-1 at 35°C) compared to aerobic microorganisms (0.67 g-COD g-COD-1 for
117
heterotrophs and 0.24 g-COD g-N-1 for autotrophs at 20°C) (Batstone et al., 2002; Henze, 2000).
118
Thus, the exoelectrogenic yield is expected to be small within the typical range for anaerobic
119
microorganisms. In addition, the energy level that is applied to exoelectrogenic bacteria can be
120
easily regulated by controlling the bioanode potential during bioelectrochemical system
121
operation. For instance, when a new bioelectrochemical system is started, the yield coefficient
122
will be maximized for a rapid start-up of the system. However, when exoelectrogenic biofilms
123
are fully developed on the bioanode, the yield coefficient will be minimized to reduce the
124
production of waste sludge.
M AN U
TE D
EP
AC C
125
SC
112
The rate of cell decay in biological wastewater treatment also affects the amount of waste
126
sludge production. The rate of cell decay is known to be linearly proportional to the microbial
127
population in bioreactors (Rittmann and McCarty, 2001; Grady et al., 2011). Thus, attached
128
growth bioreactors (e.g., trickling filters, rotating biological contactors, moving bed biofilm
129
reactors) generate small amounts of waste sludge compared to suspended growth bioreactors 6
ACCEPTED MANUSCRIPT
(e.g., activated sludge) because relatively dense microbial populations in biofilms (>10,000 mg-
131
volatile biomass L-1) induce high rates of cell decay (Grady et al., 2011). Note that the microbial
132
population in suspended growth bioreactors is usually an order smaller, ranging 1,000 to 4,000
133
mg-VSS (volatile suspended solid) L-1. Since exoelectrogenic bacteria form biofilms on the
134
bioanode, the rate of cell decay is expected to be high and thus bioelectrochemical systems are
135
expected to generate a small amount of waste sludge. However, none of previous studies have
136
reported kinetic information on cell decay of exoelectrogenic bacteria in bioelectrochemical
137
systems.
SC
M AN U
138
RI PT
130
One of the main objectives of this study is to demonstrate a theoretical method to estimate the yield coefficient of exoelectrogenic bacteria in bioelectrochemical systems by
140
modifying McCarty’s free energy-based method (Rittmann and McCarty, 2001). By using the
141
developed method, various operational factors (i.e., bioanode potential, pH, substrate
142
concentration, and bicarbonate concentration) were examined to explain how they affect the rate
143
of exoelectrogenic growth. Another important objective of this study is to determine the
144
observed yield coefficient of the bioanode in a lab-scale experiment. By comparing the observed
145
yield coefficient from the experiment with the theoretical true yield coefficient, the cell decay
146
coefficient was approximated to quantify the contribution of cell decay to overall sludge
147
production in bioelectrochemical systems.
EP
AC C
148
TE D
139
149
2. Microbial energetics for cell yield estimation
150
2.1 Energy from electron donor and acceptor reactions (∆Gr)
7
ACCEPTED MANUSCRIPT
151
In bioelectrochemical systems, exoelectrogenic bacteria on the bioanode obtain energy from oxidation of organic substrates (i.e., electron donor reaction) and transfer of electrons to the
153
bioanode (i.e., electron acceptor reaction) (Rittmann and McCarty, 2001).
154
RI PT
152
∆Gr = ε(∆Gd – ∆Ga)
(1)
∆Gr is the free energy that bacteria obtain from the electron-donor and -acceptor reactions per
156
mole of electron transfer. The electron transfer efficiency (ε) was assumed to be 0.6 in this study
157
(Rittmann and McCarty, 2001) unless otherwise noted. With acetate as an organic substrate, the
158
free energy of the donor reaction (∆Gd) can be found based on the acetate oxidation half reaction
159
(Rittmann and McCarty, 2001):
160
HCO
161
∆ = ∆ ° +
M AN U
SC
155
+ H + e ↔ CH COO + H O {CH3 COO− } /
(2)
(3)
TE D
{ !"#$ } /%
∆Gd°ʹ = 26.87 kJ/mole-e−
In bioelectrochemical systems, the electron acceptor reaction occurs at the bioanode as
163
exoelectrogens directly transfer electrons to electrically conductive bioanode surfaces. Thus, the
164
free energy of the electron acceptor reaction (∆Ga) is determined by the bioanode potential as: ∆Ga = −nFEan
(4)
AC C
165
EP
162
166
F is the Faraday constant (96485 C per mole-e−), Ean is the bioanode potential in volts, and n is
167
the number of electrons transferred by exoelectrogens in the bioanode reaction. In this study, n
168
was assumed to be unity since most enzymes in bacterial electron transfer chains carry a single
169
charge (Madigan et al., 2005). For instance, the cytochrome c enzyme is known to play a key
170
role in the exoelectrogenic capability of Geobacter and Shewanella species in bioelectrochemical
8
ACCEPTED MANUSCRIPT
systems (Richter et al., 2012; Malvankar et al., 2012) and the enzyme carries a single electron
172
depending on the oxidation state of its central metal (Madigan et al., 2015).
173
2.2 Energy required for cell synthesis (∆Gs)
174
RI PT
171
In microbial energetics, new cells are synthesized in two steps: from a carbon source to pyruvate; and from pyruvate to the cellular carbon (Rittmann and McCarty, 2001). Also, a
176
certain amount of energy (∆Gn°ʹ) is required to create ammonia (i.e., readily available nitrogen
177
source) from other nitrogen sources (e.g., nitrate, nitrogen gas). Thus, the free energy required
178
for cell synthesis (∆Gs°ʹ) can be written as: ∆& ° =
∆'( °) *+
+
∆', °) *
+ ∆- °
M AN U
179
SC
175
(5)
∆Gp°ʹ is the free energy to covert a given carbon source to pyruvate under standard conditions at
181
pH 7 (∆Gp°ʹ = 7.57 kJ per mole-e− for acetate as the carbon source, Rittmann and McCarty,
182
2001). If the carbon source-to-pyruvate reaction is thermodynamically spontaneous, the exponent
183
m is −1 while it is +1 if the reaction is non-spontaneous (Rittmann and McCarty, 2001). ∆Gc°ʹ is
184
the free energy required to convert pyruvate into new microbial cells under standard states at pH
185
7 (∆Gc°ʹ = 18.81 kJ per mole-e−, Rittmann and McCarty, 2001). Since a sufficient amount of
186
ammonia was provided in the feed solution, ∆Gn°ʹ was assumed to be zero in this study.
187
2.3 Energy balance to determine yield coefficient
EP
AC C
188
TE D
180
In McCarty’s microbial energetics model, if (A+1) moles of electrons are released in the
189
electron donor reaction, one mole of electrons are used for cell synthesis and A moles of
190
electrons are transferred to the terminal electron acceptor though the electron acceptor reaction
191
(Rittmann and McCarty, 2001). The total amount of energy released from the electron donor
9
ACCEPTED MANUSCRIPT
reaction is also split by the same ratio. At steady state, the energy from the electron-donor and -
193
acceptor reactions (Aε∆Gr) is identical to that required for cell synthesis (∆Gs°ʹ). Thus, the
194
fraction constant A can be found as:
195
.=−
RI PT
192
∆'/ °)
(6)
∆'0
By finding the fraction of the electron flow to cell synthesis (1/(A+1)), the true (theoretical) yield
197
coefficient was further calculated using C5H7O2N for the chemical formula of new cells
198
(Rittmann and McCarty, 2001).
M AN U
199
200
3. Experimental Method
201
3.1 MEC construction
In lab-scale experiments, the microbial electrolysis cell (MEC) was constructed using
TE D
202
SC
196
polypropylene blocks with a cylindrical interior chamber (3.81 cm in diameter). The reactor
204
consisted of an anode and a cathode chambers (70 and 30 mL, respectively) which were
205
separated by a cation exchange membrane (Selemion CMV, Asahi Glass, Japan). Note that the
206
separation was necessary so that the biomass produced in the anode chamber was not affected by
207
potential microbial activities in the cathode chamber by hydrogenotrophic methanogens (Tice
208
and Kim, 2014b) and sulfate reducing bacteria (Rabaey et al., 2005). A graphite fiber brush was
209
used as the bioanode which was pretreated in a muffle furnace at 505°C for 2 hours (Wang et al.,
210
2009). The cathode was a piece of stainless steel mesh without any catalyst applications (200 ×
211
200 mesh; Type 304; McMaster Carr, USA). A gas collection tube was attached on top of the
212
cathode chamber using epoxy glue. The constructed MEC was inoculated using primary effluent
AC C
EP
203
10
ACCEPTED MANUSCRIPT
from a local wastewater treatment facility and operated about 6 weeks before the main part of the
214
experiment was initiated.
215
3.2 Feed solution preparation
216
RI PT
213
The feed solution was prepared with 60 mM phosphate buffer solution (10.38 g L-1
Na2HPO4-7H2O; 2.55 g L-1 NaH2PO4; 0.36 g L-1 NH4Cl; 0.15 g L-1 KCl). Approximately 0.02
218
mM NaOH was added into the phosphate buffer solution and the resulting pH ranged 9.0 to 9.5.
219
This high pH condition was necessary to avoid potential acidification in the anode chamber that
220
can limit the exoelectrogenic growth and activity on the bioanode (He et al., 2008). The prepared
221
solution was directly used as the catholyte solution while the anolyte solution was then further
222
prepared through the addition of 1 g L-1 sodium acetate as well as trace vitamins and minerals
223
(Cheng et al., 2009).
224
3.3 Reactor operation
M AN U
TE D
225
SC
217
Three independent MECs were operated in a fed-batch mode and each fed-batch cycle lasted between 3 and 5 days (2 cycles per week). The MECs were operated at an applied voltage
227
(Eap) of 1.0 V for the bioanode yield and Coulombic efficiency measurement while three
228
different applied voltages (1.0, 0.75, and 0.5 V) were examined when the anode potential was
229
monitored using an Ag/AgCl reference electrode (MW-2030; BASi, USA). The voltage
230
application, bioanode potential monitoring, and current measurement were conducted using
231
multi-channel potentiostat (MPG2, Bio-Logic Science Instruments; France). All experiments
232
were performed in an air conditioned laboratory with a stationary temperature of 21.2 ± 0.8°C.
233
3.4 Experimental measurements
AC C
EP
226
11
ACCEPTED MANUSCRIPT
234
For the solid analysis, 60 mL was taken from the anolyte effluent (70 mL) and acidified using HCl before it was divided into two 30 mL samples which were individually centrifuged at
236
9000 RPM (rotations per min) for 20 min to separate suspended biomass. Approximately 22.5
237
mL of the supernatant was poured off without disturbing the centrifuged biomass adhered to the
238
wall of the centrifuge tube. The remaining sample was sonicated briefly to dislodge the biomass
239
and then poured into a ceramic crucible. The crucible was kept in a 103°C oven for at least 12 hr
240
and heated to 505°C for 2 hr in a muffle furnace to determine volatile and total suspended solids
241
(VSS and TSS). Note that a small amount of dissolved solids in the remaining sample (7.5 mL)
242
was included in the measurement of VSS and TSS. It should be emphasized that the standard
243
methods for VSS and TSS using filter disks (Eaton et al., 2005) was not sensitive enough to
244
detect the biomass production in the MECs since the observed yield of the bioanode was
245
extremely small (~0.02 g-VSS g-COD-1).
SC
M AN U
The influent and effluent solutions were analyzed for pH and conductivity (SevenMulti;
TE D
246
RI PT
235
Mettler-Toledo International Inc., USA). The anolyte pH decreased from 9.2 to 6.3 while the
248
catholyte pH increased from 9.3 to 12.1 since the anode and cathode chambers were separated by
249
a cation exchange membrane. The anolyte conductivity decreased from ~10.43 to 6.96 mS cm-1
250
while the catholyte conductivity increased from ~10.04 to 19.21 mS cm-1 because the anode
251
chamber loses cations through the cation exchange membrane (Strathmann, 2004).
AC C
252
EP
247
After 99 days of the MEC operation, the reactors were dissembled and the carbon fiber
253
brush bioanodes were dried at 105° and burnt at 505°C to determine the amount of volatile
254
biomass growing on the bioanode.
255
3.5 COD removal and Coulombic efficiency
12
ACCEPTED MANUSCRIPT
In a separate experiment, the effluent anolyte and catholyte as well as the influent anolyte
257
were taken and acidified using phosphoric acid (3% v/v). The acidified samples were analyzed in
258
FID-GC (flame ionization detector-gas chromatography) to determine acetate concentration
259
(Varian CP3800, USA). In the FID-GC analysis, the Stabilwax-DA column was used to separate
260
individual organic fatty acids (Restek, USA). It should be noted that acetate was detected in the
261
effluent catholyte at low concentration (16.1 ± 7.9 mg L-1 as sodium acetate; n = 6) due to non-
262
ideality of the cation exchange membrane. The chemical oxygen demand removal (∆COD) was
263
determined by performing the mass balance on sodium acetate:
264
88=8> ∆COD = 0.78(7 8- 9:− 7 8- 9;<< − 7 =8> 9;<< )
M AN U
SC
RI PT
256
(7)
The factor 0.78 is for the conversion from mg-sodium acetate into mg-COD, c is the sodium
266
acetate concentration measured in the FID-GC analysis, V is the volume with the superscript an
267
for anolyte and cat for catholyte. The subscript in is for influent and eff for effluent.
269
270
The Coulombic efficiency (CE) is the charge-based ratio between the amount of electric current (i) generated and the amount of COD removed during MEC fed-batch cycles: @A =
C :> D∆!"E
(8)
EP
268
TE D
265
The measured CE was 107.9 ± 5.1% (n = 6) and this high CE result indicates that the majority of
272
provided acetate was oxidized by the bioanode. Thus, the biomass produced in the anode
273
chamber was mainly contributed by the excess growth of exoelectrogenic bacteria on the
274
bioanode. After confirming consistently high CE, COD was not further monitored in the
275
bioanode yield experiment and ∆COD was determined by integrating electric current with time
276
by assuming CE of 100%.
AC C
271
13
ACCEPTED MANUSCRIPT
278 279
280
3.6 Decay coefficient estimation The cell decay coefficient (b) was calculated by comparing the true yield coefficient (Ytrue) and observed yield coefficient from the experiment (Yobserved). F=
RI PT
277
(GH0IJ GKL/J0MJN )O PQ ∆!"E
(9)
RPQKNJ S
Xanode is the volatile biomass attached on the bioanode and τ is the average time period of the fed-
282
batch cycle.
SC
281
M AN U
283
284
4. Results and Discussion
285
4.1 Anode potential and pH governing bioanode cell yield
The anode potential was found to govern the excess cell yield on the bioanode. The yield
287
coefficient sharply increased with increasing anode potential from −0.4 to 0 V vs. SHE (standard
288
hydrogen electrode) (Fig 1A). A more positive anode potential corresponds to a greater amount
289
of energy available for bacterial growth. The equilibrium anode potential of the bioanode is
290
−0.293 V vs. SHE (10 mM acetate; 10 mM bicarbonate; pH = 7; 25°C) using the Nernst equation
291
(Logan et al., 2008). This equilibrium anode potential corresponds to a zero cell yield of
292
exoelectrogenic bacteria (i.e., x-intercept of the pH 7 curve in Fig 1A), indicating that the
293
developed method for the yield coefficient is consistent with the prediction based on
294
electrochemistry equilibrium. Based on the slope of the yield vs. anode potential curve, there is
295
an increase of approximately 0.1 g-VSS g-COD-1 in the yield coefficient per 0.1 V increase in
296
the anode potential. The anode potential in MFCs and MECs can be wide but usually in the range
297
of –0.1 to –0.3 V vs. SHE (Aelterman et al., 2008; Torres et al., 2009; Wagner et al., 2010; Lee
AC C
EP
TE D
286
14
ACCEPTED MANUSCRIPT
and Rittmann, 2010; Nam et al., 2011; Sun et al., 2012). Assuming the anode potential of –0.1 V
299
vs. SHE under neutral to slightly basic conditions (pH 7 to 9), the theoretical yield coefficient of
300
exoelectrogenic bacteria will range 0.1 to 0.2 g-VSS g-COD-1.
301
RI PT
298
A change in pH also resulted in a sensitive response in the yield coefficient as an increase in pH by two units raised the yield coefficient by ~0.1 g-VSS g-COD-1 (Fig 1A). It should be
303
emphasized that very small cell yield coefficients were observed at pH 5 (e.g., 0.05 g-VSS g-
304
COD-1 at −0.1 V vs. SHE). This observation indicates that the exoelectrogenic growth is
305
seriously inhibited even at the mildly acidic pH condition. This finding is consistent with
306
experimental results reported in the previous studies that the bioanode practically stops
307
generating electric current at pH of 5 or lower (He et al., 2008).
M AN U
308
SC
302
Exoelectrogenic bacteria form biofilms on bioanode surfaces, and thus the local pH and electric potential vary in the exoelectrogenic biofilm. For instance, the local pH is lower and the
310
electric potential is more positive at a deeper location of the biofilm (Babauta et al., 2012),
311
leading to a decrease and an increase in the microbial yield, respectively (Fig 1A). In addition,
312
such variations in the local pH and electric potential are more pronounced at higher current
313
conditions (Babauta et al., 2012). Thus, the yield coefficient can be slightly different at different
314
locations in exoelectrogenic biofilms. While the scope of this study does not include such micro-
315
scale environmental factors on the yield coefficient, this interesting aspect can be further
316
investigated in future studies.
317
4.2 Substrate and bicarbonate concentration
318 319
AC C
EP
TE D
309
The substrate (acetate) concentration has relatively minor effects on the biomass production. The yield coefficient increased gradually (~10% of its magnitude) with the
15
ACCEPTED MANUSCRIPT
increasing substrate concentration up to 20 mM acetate or 1.28 g-COD/L (Fig 1B) because high
321
substrate concentration decreases the amount of energy required for new cell synthesis, leaving a
322
greater amount of energy for cell growth. Conversely, an increase in bicarbonate concentration
323
increases the energy required for cell synthesis, resulting in a smaller yield coefficient. However,
324
the result shows that both acetate and bicarbonate concentrations have relatively minor effects on
325
the bioanode yield coefficient. Note that there was only ~10% decrease in the yield coefficient
326
with an increase in bicarbonate concentration by an order of magnitude (Fig 1B).
327
4.3 Sensitivity analysis on electron transfer efficiency
SC
M AN U
328
RI PT
320
While the electron transfer efficiency (ε) is commonly assumed to be 0.6, its precise estimation is difficult without a clear understanding of proton motive force generation in the
330
electron transport chain (McCarty, 2007). Thus, we examined the sensitivity of the yield
331
coefficient to the electron transfer efficiency. As the electron transfer efficiency increased, the
332
microbial yield coefficient increased (Fig 1C) because the bacteria obtain a greater amount of
333
energy from substrate utilization (∆Gr in Eq. 1) while they need a smaller amount of energy for
334
cell synthesis (∆Gs°ʹ in Eq. 5) with a higher electron transfer efficiency. Note that ∆Gp°ʹ was
335
always positive unless bicarbonate concentration is extremely low (e.g., [HCO3-] ≤ 0.001 mM),
336
indicating that the reaction from acetate to pyruvate is usually non-spontaneous. When the
337
bioanode potential was more positive, the sensitivity of the yield coefficient to the electron
338
transfer efficiency was more pronounced (Fig 1C). This observation can be explained by more
339
negative free energy for the electron acceptor reaction (∆Ga in Eq. 4) when the bioanode
340
potential is more positive, leading to a greater amount of energy available for microbial growth.
341
4.4 Observed yield coefficient in experiments
AC C
EP
TE D
329
16
ACCEPTED MANUSCRIPT
342
The observed yield coefficient obtained from the MEC experiment was substantially small at about 0.020 g-VSS g-COD-1 (Table 1). In the experiment, the anolyte pH ranged from
344
9.2 to 6.3 and the anode potential was stable about −0.1 V vs. SHE for Eap of 1.0 V when the
345
bioanode is active in generating electric current with acetate (Fig 2). For these given conditions,
346
the true yield coefficient of the bioanode is expected to decrease from 0.2 to 0.1 g-VSS g-COD-1
347
with the decreasing pH from 9.2 to 6.3 (Fig 1A). When compared with this theoretical
348
estimation, the observed yield coefficient is smaller by an order of magnitude. For the
349
consecutive 6 fed-batch cycles, the electric current was high with the peak current ranging 0.6 to
350
3.2 mA (Fig 3), indicating that the bioanode was healthy and thus actively oxidizing the organic
351
substrate (acetate) over the course of the experiments. Thus, the substantially small observed
352
yield coefficient was not contributed by any potential malfunctions of the bioanode during the
353
experiment.
SC
M AN U
Since the MEC reactors were operated at a fed-batch mode, acetate concentration
TE D
354
RI PT
343
decreased with time and thus low acetate concentration near the end of fed-batch cycles could
356
potentially have resulted in the small observed yield coefficient. However, for the 5th and 6th
357
fed-batch cycles, the electric current was high about 1 mA or higher when the fed-batch
358
operation was terminated at 18 and 21 d, respectively (Fig 3). This high current indicates that
359
acetate concentration was sufficiently high at the end of these two fed-batch cycles so that the
360
bioanode actively oxidized acetate and thus created the high electric current throughout the fed-
361
batch cycles. The observed yield coefficient for the 5th and 6th cycles was 0.032 and 0.016 g-
362
VSS g-COD-1, respectively while the average observed yield coefficient over 6 fed-batch cycles
363
was 0.020 g-VSS g-COD-1 (Table 1). Thus, potentially low substrate concentration near the end
AC C
EP
355
17
ACCEPTED MANUSCRIPT
of fed-batch cycles cannot explain the substantial difference between the observed and
365
theoretical yield coefficients.
366
4.5 Cell decay on bioanodes
367
RI PT
364
The cell decay at the bioanode can explain the substantial difference between the
observed yield coefficient in the experiment (0.020 g-VSS g-COD-1) and the theoretical true
369
yield coefficient (0.1 to 0.2 g-VSS g-COD-1). At the end of the 3-month MEC operation, the
370
amount of volatile biomass on the bioanode was measured to be 126.7 ± 8.7 mg VSS per
371
bioanode. Based on this biomass measurement, the decay coefficient was plotted using Eq. 9 as a
372
function of true yield coefficient (Fig 4). The decay coefficient was zero when the true yield
373
coefficient is equal to the observed yield coefficient (0.020 g-VSS g-COD-1) and it increased
374
linearly with the increasing true yield coefficient (Fig 4). During the MEC operation, the anode
375
potential was −0.1 V vs. SHE (Fig 2) and the average pH in the anode chamber was
376
approximately 8. Thus, the resulting true yield coefficient (Ytrue) was 0.2 g-VSS g-COD-1 (Fig
377
1A) and consequently the decay coefficient can be estimated to be 0.013 ± 0.002 d-1 (Fig 4). This
378
estimated decay coefficient is similar to the suggested value for anaerobic microorganisms in
379
Anaerobic Digestion Model No. 1 (0.02 d-1 at 35°C) (Batstone et al., 2012). Thus, the rate of cell
380
decay in bioelectrochemical systems is comparable to that in anaerobic digestion.
M AN U
TE D
EP
AC C
381
SC
368
Using the estimated decay coefficient (0.013 d-1), the amount of exoelectrogenic cells
382
decayed was found to be 5.78 mg VSS per fed-batch cycle (0.013 d-1 × 126.7 mg VSS × 3.5 d).
383
The created volatile biomass using the true yield coefficient was 6.42 mg VSS per cycle (0.2 mg-
384
VSS mg-COD-1 × 458.4 mg-COD L-1 × 0.070 L). Thus, the amount of exoelectrogenic cells
385
decayed was approximately 90% of the amount of newly created cells on the bioanode. This
18
ACCEPTED MANUSCRIPT
386
comparison explains the substantially reduced waste sludge production in bioelectrochemical
387
wastewater treatment that was demonstrated in a recent study (Brown et al., 2015).
389
RI PT
388
5. Conclusions
In this study, we estimated the true (theoretical) cell yield coefficient of exoelectrogenic
391
bacteria in bioelectrochemical systems by modifying McCarty’s free energy-based method. The
392
theoretical yield coefficient ranged 0.1 to 0.3 g-VSS g-COD-1 (pH 7 – 9; anode potential between
393
−0.2 and 0 V vs. SHE; ε = 0.6), which is similar to that of most anaerobic microorganisms
394
growing in anaerobic digesters (Batstone et al., 2012) but much smaller than that of aerobic
395
microorganisms in activated sludge systems (Henze, 2000). The true yield coefficient of
396
exoelectrogenic bacteria was greatly affected by pH and as a result, even a mildly acidic
397
condition (e.g., pH 5) seriously inhibited the bacterial growth on bioanodes. This finding is
398
consistent with previous study results where the bioanode practically stops creating electric
399
current at pH 5 or lower (He et al., 2008). The exoelectrogenic yield was also governed by the
400
anode potential as the true yield coefficient increased with the increasing (i.e., more positive)
401
anode potential. However, the reactant (substrate) and product (bicarbonate) concentration had
402
relatively minor effects on the yield coefficient of exoelectrogenic bacteria. While the electron
403
transfer efficiency is commonly assumed to be 0.6, the sensitivity study result found that the
404
estimation of the yield coefficient is greatly affected by the electron transfer efficiency. This
405
finding indicates that precise estimation of the electron transfer efficiency is necessary in future
406
studies when the electron transfer chain in exoelectrogenic cells are clearly understood.
AC C
EP
TE D
M AN U
SC
390
19
ACCEPTED MANUSCRIPT
407
In the experiment using lab-scale MECs, the observed yield coefficient was found to be 0.020 ± 0.008 g-VSS g-COD-1, which is smaller than the true yield coefficient by an order
409
magnitude. After the 3-month experiment was completed, the total amount of biomass on the
410
bioanode was measured and 126.7 ± 8.7 mg volatile solids were found to be attached on each
411
bioanode. Based on this measurement, the rate of cell decay was obtained from the difference
412
between the true and observed yield coefficients. The estimated decay coefficient was 0.013 ±
413
0.002 d-1 at 21.2°C, which is similar to that of anaerobic microorganisms in anaerobic digesters
414
(0.02 d-1 at 35°C) ( Batstone et al., 2012). Using the obtained decay coefficient, the amount of
415
cell decayed over the fed-batch cycle was 5.78 mg VSS, which was 90% of the created cell (6.42
416
mg VSS), explaining the substantially small observed yield coefficient.
M AN U
SC
RI PT
408
The found yield and decay coefficients will be used in model developments and
418
simulations of bioelectrochemical systems to predict their performance of wastewater treatment
419
and resources recovery. The substantially small observed yield coefficient of exoelectrogenic
420
bacteria (0.020 g-VSS g-COD-1) confirmed a strong potential of bioelectrochemical systems for
421
future wastewater treatment with greatly reduced sludge generation as recently demonstrated
422
(Brown et al., 2015). Considering that sludge treatment and disposal are responsible for major
423
expenses in wastewater treatment, bioelectrochemical systems can effectively reduce the cost for
424
wastewater treatment.
EP
AC C
425
TE D
417
426
Acknowledgements
427
This study was supported by Discovery Grants (435547-2013, Natural Sciences and Engineering
428
Research Council of Canada), Canada Research Chairs Program (950-2320518, Government of
20
ACCEPTED MANUSCRIPT
Canada), Leaders Opportunity Fund (31604, Canada Foundation for Innovation), Ontario
430
Research Fund-Research Infrastructure (31604, Ontario Ministry of Economic Development and
431
Innovation), and Undergraduate Student Research Awards (Natural Sciences and Engineering
432
Research Council of Canada). The authors thank Ms. Anna Robertson and Mr. Peter Koudys for
433
their help on equipment operation and reactor construction.
SC
434
RI PT
429
References
436 437 438
Aelterman, P., Freguia, S., Keller, J., Verstraete, W., Rabaey, K., 2008. The anode potential regulates bacterial activity in microbial fuel cells. Applied Microbiology Biotechnology, 78(3), 409-418.
439 440
Ahn, Y., Schröder, U., 2015. Microfabricated, continuous-flow, microbial three-electrode cell for potential toxicity detection. BioChip Journal, 9(1), 27-34.
441 442 443 444
Babauta, J. T., Nguyen, H. D., Harrington, T. D., Renslow, R., Beyenal, H., 2012. pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer. Biotechnology Bioengineering, 109(10), 26512662.
445 446 447
Batstone, D. J., Keller, J., Angelidaki, I., Kalyuzhny, S. V., Pavlostathis, S. G., Rozzi, A., Sander, W. T. M., Siegrist, H., Vavilin, V. A., 2002. Anaerobic digestion model no. 1 (ADM1). IWA publishing, London.
448 449 450
Brown, R. K., Harnisch, F., Dockhorn, T., Schröder, U., 2015. Examining sludge production in bioelectrochemical systems treating domestic wastewater. Bioresource Technology, 198, 913-917.
451 452 453
Cao, X., Huang, X., Liang, P., Xiao, K., Zhou, Y., Zhang, X., Logan, B. E., 2009. A new method for water desalination using microbial desalination cells. Environmental Science Technology, 43(18), 7148-7152.
454 455 456
Cheng, S., Xing, D., Call, D. F., Logan, B. E., 2009. Direct biological conversion of electrical current into methane by electromethanogenesis. Environmental Science Technology, 43(10), 3953-3958.
457 458
Eaton, A.D., Clesceri, L.S., Rice, E.W., Greenberg, A.E., 2005. Standard methods for examination of water and wastewater. 21st ed. American Public Health Association.
AC C
EP
TE D
M AN U
435
21
ACCEPTED MANUSCRIPT
Grady, C.P.L., Daigger, G.T., Love, N.G., Filipe, C.D.M., 2011. Biological Wastewater Treatment, 3rd ed. Taylor and Francis Group, Boca Raton.
461 462 463
He, Z., Huang, Y., Manohar, A. K., Mansfeld, F., 2008. Effect of electrolyte pH on the rate of the anodic and cathodic reactions in an air-cathode microbial fuel cell. Bioelectrochemistry, 74(1), 78-82.
464 465
Henze, M. (Ed.), 2000. Activated sludge models ASM1, ASM2, ASM2d and ASM3. IWA Publishing, London.
466 467
Kim, T., An, J., Jang, J. K., Chang, I. S., 2015. Coupling of anaerobic digester and microbial fuel cell for COD removal and ammonia recovery. Bioresource Technology, 195, 217-222.
468 469
Lee, H. S., Rittmann, B. E., 2010. Characterization of energy losses in an upflow single-chamber microbial electrolysis cell. International Journal of Hydrogen Energy, 35(3), 920-927.
470 471 472
Logan, B. E., Hamelers, B., Rozendal, R., Schröder, U., Keller, J., Freguia, S., Aelterman, P., Verstraete, W., Rabaey, K., 2006. Microbial fuel cells: methodology and technology. Environmental Science Technology, 40(17), 5181-5192.
473 474 475
Logan, B. E., Call, D., Cheng, S., Hamelers, H. V., Sleutels, T. H., Jeremiasse, A. W., Rozendal, R. A., 2008. Microbial electrolysis cells for high yield hydrogen gas production from organic matter. Environmental Science Technology, 42(23), 8630-8640.
476 477
Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., Stahl, D. A., Brock biology of microorganisms. 2015. 14th Ed., Pearson Education, Glenview.
478 479 480
Malvankar, N. S., Mester, T., Tuominen, M. T., Lovley, D. R., 2012. Supercapacitors Based on c‐Type Cytochromes Using Conductive Nanostructured Networks of Living Bacteria. ChemPhysChem, 13(2), 463-468.
481 482
McCarty, P. L., 2007. Thermodynamic electron equivalents model for bacterial yield prediction: modifications and comparative evaluations. Biotechnology Bioengineering, 97(2), 377-388.
483 484
Modin, O., Wang, X., Wu, X., Rauch, S., Fedje, K. K., 2012. Bioelectrochemical recovery of Cu, Pb, Cd, and Zn from dilute solutions. Journal of Hazardous Materials, 235, 291-297.
485 486 487
Nam, J. Y., Tokash, J. C., Logan, B. E., 2011. Comparison of microbial electrolysis cells operated with added voltage or by setting the anode potential. International Journal of Hydrogen Energy, 36(17), 10550-10556.
488 489 490
Nilges, P., Schröder, U., 2013. Electrochemistry for biofuel generation: production of furans by electrocatalytic hydrogenation of furfurals. Energy Environmental Science, 6(10), 29252931.
491 492 493
Qin, M., Molitor, H., Brazil, B., Novak, J. T., He, Z., 2016. Recovery of nitrogen and water from landfill leachate by a microbial electrolysis cell–forward osmosis system. Bioresource Technology, 200, 485-492.
AC C
EP
TE D
M AN U
SC
RI PT
459 460
22
ACCEPTED MANUSCRIPT
Rabaey, K., Clauwaert, P., Aelterman, P., Verstraete, W., 2005. Tubular microbial fuel cells for efficient electricity generation. Environmental Science Technology, 39(20), 8077-8082.
496 497 498
Richter, K., Schicklberger, M., Gescher, J., 2012. Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration. Applied Environmental Microbiology, 78(4), 913-921.
499 500
Rittmann, B. E., McCarty, P. L., 2001. Environmental biotechnology: principles and applications. McGraw-Hill Higher Education, New York.
501 502 503
Rotaru, D. E. H., Franks, A. E., Orellana, R., Risso, C., Nevin, K. P. (2011). Geobacter: the microbe electric’s physiology, ecology, and practical applications. Advances in Microbial Physiology, 59, 1-100.
504
Strathmann, H., 2004. Ion-exchange membrane separation processes. Elsevier.
505 506 507
Sun, D., Call, D. F., Kiely, P. D., Wang, A., Logan, B. E., 2012. Syntrophic interactions improve power production in formic acid fed MFCs operated with set anode potentials or fixed resistances. Biotechnology Bioengineering, 109(2), 405-414.
508 509
Tice, R. C., Kim, Y., 2014a. Energy efficient reconcentration of diluted human urine using ion exchange membranes in bioelectrochemical systems. Water Research, 64, 61-72.
510 511
Tice, R. C., Kim, Y., 2014b. Methanogenesis control by electrolytic oxygen production in microbial electrolysis cells. International Journal of Hydrogen Energy, 39(7), 3079-3086.
512 513 514 515
Torres, C. I., Krajmalnik-Brown, R., Parameswaran, P., Marcus, A. K., Wanger, G., Gorby, Y. A., Rittmann, B. E., 2009. Selecting anode-respiring bacteria based on anode potential: phylogenetic, electrochemical, and microscopic characterization. Environmental Science Technology, 43(24), 9519-9524.
516 517
Wagner, R. C., Call, D. F., Logan, B. E., 2010. Optimal set anode potentials vary in bioelectrochemical systems. Environmental Science Technology, 44(16), 6036-6041.
518 519 520
Wang, X., Cheng, S., Feng, Y., Merrill, M. D., Saito, T., Logan, B. E., 2009. Use of carbon mesh anodes and the effect of different pretreatment methods on power production in microbial fuel cells. Environmental Science Technology, 43(17), 6870-6874.
521 522
Zhang, Y., Angelidaki, I., 2015a. Bioelectrochemical recovery of waste-derived volatile fatty acids and production of hydrogen and alkali. Water Research, 81, 188-195.
523 524
Zhang, Y., Angelidaki, I., 2015b. Recovery of ammonia and sulfate from waste streams and bioenergy production via bipolar bioelectrodialysis. Water Research, 85, 177-184.
AC C
EP
TE D
M AN U
SC
RI PT
494 495
525 526
23
ACCEPTED MANUSCRIPT
527
Tables
528
Table 1. Summary of experimental results to find the observed yield coefficient.
RI PT
Fed-batch cycle 1 2 3 4 5 6 Mean ± SD -1 VSS (mg L ) 6.59 10.86 5.84 9.16 13.22 8.41 9.01 ± 2.73 ∫idt (C) 401.1 447.2 381.9 307.6 345.1 439.3 387.0 ± 54.1 ∆COD (mg L-1)* 475.1 529.7 452.4 364.4 408.7 520.4 458.4 ± 64.1 Observed yield 0.0139 0.0205 0.0129 0.0251 0.0323 0.0162 0.0202 ± 0.0075 (g-VSS g-COD-1) * Coulombic efficiency of 100% was assumed as demonstrated in a separate experiment.
SC
529
AC C
EP
TE D
M AN U
530
24
ACCEPTED MANUSCRIPT
0.1
0.2
0.1
0.0
0.0
(C)
−0.1 V vs. SHE −0.2 V vs. SHE −0.3 V vs. SHE
0.2
0.1
0.0
0
5
10 15 Acetate (mM)
20
M AN U
‐0.4 ‐0.2 0 Anode potential (V vs. SHE)
0.3
RI PT
0.2
0.1 mM HCO₃ˉ 1.0 mM HCO₃ˉ 10.0 mM HCO₃ˉ
(B)
Yield (g‐VSS/g‐COD)
0.3
(A)
SC
pH = 9 pH = 7 pH = 5
Yield (g‐VSS/g‐COD)
Yield (g‐VSS/g‐COD)
0.3
0
0.2 0.4 0.6 Transfer efficiency
0.8
Figure 1. The true yield coefficient estimation. (A) Effect of anode potential and pH (10 mM acetate; 10 mM bicarbonate; ε = 0.6; 25°C) (B) Effect of acetate and bicarbonate concentration (pH = 7; anode potential = −0.1 V vs. SHE; ε = 0.6; 25°C) (C) Sensitivity
AC C
EP
bicarbonate; 25°C).
TE D
analysis results on the electron transfer efficiency (pH = 7; 10 mM acetate; 10 mM
ACCEPTED MANUSCRIPT
-0.1
RI PT
MEC-1 MEC-2 MEC-3
SC
-0.2
-0.3
-0.4 0.25
TE D
0
M AN U
Anode potential (V vs SHE)
0
0.5 Eap (V)
0.75
1
AC C
EP
Figure 2. Relationship between the applied voltage (Eap) and anode potential (Ean).
MEC-1 MEC-2 MEC-3
SC
2
1
0 5
10 15 Time (day)
TE D
0
M AN U
Electric current (mA)
3
RI PT
ACCEPTED MANUSCRIPT
20
Figure 3. Electric current during the apparent yield coefficient measurement over 6 fed-batch
AC C
EP
cycles.
ACCEPTED MANUSCRIPT
ΔCOD = 458 mg/L ΔCOD = 394 mg/L
SC
0.02
RI PT
ΔCOD = 523 mg/L
0.01
M AN U
Decay coefficient (d-1)
0.03
0.00
0.1 0.2 0.3 True yield coefficient (g-VSS/g-COD)
TE D
0
Figure 4. Decay coefficient estimation using Eq. 9. (ΔCOD = 458.4 ± 64.1 mg-COD L-1 (Table 1); Apparent yield coefficient = 0.02 g-VSS g-COD-1; anolyte volume = 70 mL; average
AC C
EP
fed-batch cycle period = 3.5 d)
ACCEPTED MANUSCRIPT
Highlights The true yield coefficient of exoelectrogenic bacteria was 0.1 to 0.3 g-VSS g-COD-1. More positive anode potential and higher pH resulted in greater cell yield.
RI PT
The apparent yield coefficient of exoelectrogens was 0.020 ± 0.008 g-VSS g-COD-1. The decay coefficient of exoelectrogens was estimated to be 0.013 ± 0.002 d-1.
AC C
EP
TE D
M AN U
SC
Waste sludge production in bioelectrochemical systems was substantially small.
S0043-1354(16)30113-0
DOI:
10.1016/j.watres.2016.02.054
Reference:
WR 11870
To appear in:
Water Research
Received Date: 1 December 2015 Revised Date:
25 February 2016
Accepted Date: 26 February 2016
Please cite this article as: Wilson, E.L., Kim, Y., The yield and decay coefficients of exoelectrogenic bacteria in bioelectrochemical systems, Water Research (2016), doi: 10.1016/j.watres.2016.02.054. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
pH = 9 & anode = −0.1 V vs. SHE
Bioelectrochemical systems
Cell yield
Organic substrate
Exoelectrogen
ACCEPTED MANUSCRIPT
SC TE D
M AN U
Cathode
EP
Bioanode
AC C
CO2
RI PT
pH = 5 & anode = −0.3 V vs. SHE Cell yield
ACCEPTED MANUSCRIPT
1 2
Submitted to: Water Research Date of submission: Feb. 25, 2016 (Original submission: Nov. 28, 2015)
5
Erica L. Wilson & Younggy Kim*
6 7 8 9
Department of Civil Engineering, McMaster University 1280 Main St. W., JHE 301, Hamilton, Ontario, L8S 4L7, Canada *corresponding author: [email protected]; 1-905-525-9140 ext.24802
RI PT
4
The yield and decay coefficients of exoelectrogenic bacteria in bioelectrochemical systems
3
Abstract
11
In conventional wastewater treatment, waste sludge management and disposal contribute the
12
major cost for wastewater treatment. Bioelectrochemical systems, as a potential alternative for
13
future wastewater treatment and resources recovery, are expected to produce small amounts of
14
waste sludge because exoelectrogenic bacteria grow on anaerobic respiration and form highly
15
populated biofilms on bioanode surfaces. While waste sludge production is governed by the yield
16
and decay coefficient, none of previous studies have quantified these kinetic constants for
17
exoelectrogenic bacteria. For yield coefficient estimation, we modified McCarty’s free energy-
18
based model by using the bioanode potential for the free energy of the electron acceptor reaction.
19
The estimated true yield coefficient ranged 0.1 to 0.3 g-VSS (volatile suspended solids) g-COD-1
20
(chemical oxygen demand), which is similar to that of most anaerobic microorganisms. The yield
21
coefficient was sensitively affected by the bioanode potential and pH while the substrate and
22
bicarbonate concentrations had relatively minor effects on the yield coefficient. In lab-scale
23
experiments using microbial electrolysis cells, the observed yield coefficient (including the effect
24
of cell decay) was found to be 0.020 ± 0.008 g-VSS g-COD-1, which is an order of magnitude
25
smaller than the theoretical estimation. Based on the difference between the theoretical and
26
experimental results, the decay coefficient was approximated to be 0.013 ± 0.002 d-1. These
AC C
EP
TE D
M AN U
SC
10
1
ACCEPTED MANUSCRIPT
findings indicate that bioelectrochemical systems have potential for future wastewater treatment
28
with reduced waste sludge as well as for resources recovery. Also, the found kinetic information
29
will allow accurate estimation of wastewater treatment performance in bioelectrochemical
30
systems.
RI PT
27
31
Keywords
33
Wastewater sludge; microbial electrolysis cells; microbial fuel cells; microbial kinetics and
34
energetics; yield coefficient; cell decay coefficient
M AN U
SC
32
35
Nomenclature
37
Abbreviations
38
COD
chemical oxygen demand
39
FID-GC
flame ionization detector-gas chromatography
40
MDC
microbial desalination cell
41
MEC
microbial electrolysis cell
42
MFC
43
RPM
44
SHE
standard hydrogen electrode
45
TSS
total suspended solids
AC C
EP
TE D
36
microbial fuel cell rotations per min
2
ACCEPTED MANUSCRIPT
46
VSS
volatile suspended solids
47
Symbols
48
A
RI PT
number of moles of electrons transferred to terminal electron acceptor out of A+1 moles (-)
49
b
cell decay coefficient (d-1)
51
c
sodium acetate concentration (mg L-1)
52
CE
Coulombic efficiency (-)
53
Ean
bioanode potential (V vs. SHE)
54
F
Faraday constant (96485 C per mole-e−)
55
m
exponent for electron transfer efficiency based on spontaneity of pyruvate creation (-)
56
TE D
M AN U
SC
50
n
number of electrons transferred at the bioanode (-)
58
V
volume of MEC chamber (L)
59
Xanode
volatile biomass attached on the bioanode (g-VSS)
60
Yobserved
observed yield coefficient (g-VSS g-COD-1)
61
Ytrue
true yield coefficient (g-VSS g-COD-1)
62
∆Ga
63
∆Gc°ʹ
64
AC C
EP
57
free energy released from the electron acceptor reaction (kJ per mole-e−) free energy required to convert pyruvate to cellular carbon under standard conditions at pH 7 (kJ per mole-e−)
3
ACCEPTED MANUSCRIPT
65
∆Gd
free energy released from the electron donor reaction (kJ per mole-e−)
66
∆Gn°ʹ
free energy required to create ammonia from other nitrogen sources under standard
∆Gp°ʹ
free energy required to convert the substrate to pyruvate under standard conditions at pH 7 (kJ per mole-e−)
69
∆Gr
free energy that the microorganism obtains from the electron donor and acceptor reactions (kJ per mole-e−)
71
72
SC
70
∆Gs°ʹ
free energy required for cell synthesis under standard conditions at pH 7 (kJ per
M AN U
68
RI PT
conditions at pH 7 (kJ per mole-e−)
67
mole-e−)
73
ε
electron transfer efficiency (-)
75
τ
average time period of each fed-batch cycle (d)
76
Superscripts
77
an
anolyte or anode chamber
78
cat
catholyte or cathode chamber
79
Subscripts
80
eff
81
in
AC C
EP
TE D
74
effluent
influent
82
83
4
ACCEPTED MANUSCRIPT
84
1. Introduction Bioelectrochemical systems are an emerging method for wastewater treatment and
86
simultaneous recovery of energy and valuable resources (Tice and Kim, 2014a; Zhang and
87
Angelidaki 2015a; Zhang and Angelidaki 2015b; Kim et al., 2015; Qin et al., 2015). In
88
bioelectrochemical systems, various reduction reactions at the cathode (e.g., hydrogen evolution,
89
oxygen reduction, biofuel production, and heavy metal reduction) are coupled with oxidation of
90
organic substrates at the bioanode (Logan et al., 2006; Logan et al., 2008; Modin et al., 2012;
91
Nilges and Schröder, 2013). Exoelectrogenic bacteria, growing on bioanode surfaces, oxidize
92
organic substrates and transfer electrons directly to the bioanode, creating electric current. The
93
created electric current can be beneficially utilized in engineered bioelectrochemical systems.
94
For instance, microbial fuel cells (MFCs) are used to generate electric power (Logan et al., 2006);
95
microbial electrolysis cells (MECs) produce H2 gas (Logan et al., 2008); microbial desalination
96
cells (MDCs) utilize the electric current to separate charged ions from salt water (Cao et al.,
97
2009); and bioanodes can also be used as water quality sensors to monitor toxic contaminants
98
(Ahn and Schröder, 2015). In addition to these beneficial aspects, bioelectrochemical systems
99
were found in a recent study to produce substantially small amounts of waste sludge in
101
SC
M AN U
TE D
EP
wastewater treatment (Brown et al., 2015).
AC C
100
RI PT
85
In conventional activated sludge systems, approximately 60% of organic substrates are
102
converted into heterotrophic cells on the COD (chemical oxygen demand) basis (Rittmann and
103
McCarty, 2001; Grady et al., 2011). This high yield coefficient (0.6 g-COD g-COD-1) of
104
heterotrophic bacteria results in substantial amounts of waste sludge in wastewater treatment.
105
The waste sludge is further treated in anaerobic digesters and mechanical dewatering processes
106
before sludge cakes are disposed of in landfills or burnt in incinerators. The sludge treatment and 5
ACCEPTED MANUSCRIPT
final disposal require high costs and especially the cost for the final disposal has dramatically
108
increased as wet wastes (including wastewater sludge cakes) are regulated in landfill disposal. As
109
a result, the cost for sludge treatment and disposal is one of the main expenses for wastewater
110
treatment; thus, reduction in waste sludge will allow cost-effective wastewater treatment.
111
RI PT
107
In a recent study, bioelectrochemical systems were found to produce a substantially small amount of waste sludge (Brown et al., 2015); however, the yield coefficient of exoelectrogenic
113
bacteria has not been quantified. Exoelectrogenic bacteria grow on anaerobic respiration as they
114
originally use ferric iron or elemental sulfur as a terminal electron acceptor (Rotaru et al., 2011).
115
Anaerobic microorganisms are known to have a relatively small yield coefficient (0.04 to 0.1 g-
116
COD g-COD-1 at 35°C) compared to aerobic microorganisms (0.67 g-COD g-COD-1 for
117
heterotrophs and 0.24 g-COD g-N-1 for autotrophs at 20°C) (Batstone et al., 2002; Henze, 2000).
118
Thus, the exoelectrogenic yield is expected to be small within the typical range for anaerobic
119
microorganisms. In addition, the energy level that is applied to exoelectrogenic bacteria can be
120
easily regulated by controlling the bioanode potential during bioelectrochemical system
121
operation. For instance, when a new bioelectrochemical system is started, the yield coefficient
122
will be maximized for a rapid start-up of the system. However, when exoelectrogenic biofilms
123
are fully developed on the bioanode, the yield coefficient will be minimized to reduce the
124
production of waste sludge.
M AN U
TE D
EP
AC C
125
SC
112
The rate of cell decay in biological wastewater treatment also affects the amount of waste
126
sludge production. The rate of cell decay is known to be linearly proportional to the microbial
127
population in bioreactors (Rittmann and McCarty, 2001; Grady et al., 2011). Thus, attached
128
growth bioreactors (e.g., trickling filters, rotating biological contactors, moving bed biofilm
129
reactors) generate small amounts of waste sludge compared to suspended growth bioreactors 6
ACCEPTED MANUSCRIPT
(e.g., activated sludge) because relatively dense microbial populations in biofilms (>10,000 mg-
131
volatile biomass L-1) induce high rates of cell decay (Grady et al., 2011). Note that the microbial
132
population in suspended growth bioreactors is usually an order smaller, ranging 1,000 to 4,000
133
mg-VSS (volatile suspended solid) L-1. Since exoelectrogenic bacteria form biofilms on the
134
bioanode, the rate of cell decay is expected to be high and thus bioelectrochemical systems are
135
expected to generate a small amount of waste sludge. However, none of previous studies have
136
reported kinetic information on cell decay of exoelectrogenic bacteria in bioelectrochemical
137
systems.
SC
M AN U
138
RI PT
130
One of the main objectives of this study is to demonstrate a theoretical method to estimate the yield coefficient of exoelectrogenic bacteria in bioelectrochemical systems by
140
modifying McCarty’s free energy-based method (Rittmann and McCarty, 2001). By using the
141
developed method, various operational factors (i.e., bioanode potential, pH, substrate
142
concentration, and bicarbonate concentration) were examined to explain how they affect the rate
143
of exoelectrogenic growth. Another important objective of this study is to determine the
144
observed yield coefficient of the bioanode in a lab-scale experiment. By comparing the observed
145
yield coefficient from the experiment with the theoretical true yield coefficient, the cell decay
146
coefficient was approximated to quantify the contribution of cell decay to overall sludge
147
production in bioelectrochemical systems.
EP
AC C
148
TE D
139
149
2. Microbial energetics for cell yield estimation
150
2.1 Energy from electron donor and acceptor reactions (∆Gr)
7
ACCEPTED MANUSCRIPT
151
In bioelectrochemical systems, exoelectrogenic bacteria on the bioanode obtain energy from oxidation of organic substrates (i.e., electron donor reaction) and transfer of electrons to the
153
bioanode (i.e., electron acceptor reaction) (Rittmann and McCarty, 2001).
154
RI PT
152
∆Gr = ε(∆Gd – ∆Ga)
(1)
∆Gr is the free energy that bacteria obtain from the electron-donor and -acceptor reactions per
156
mole of electron transfer. The electron transfer efficiency (ε) was assumed to be 0.6 in this study
157
(Rittmann and McCarty, 2001) unless otherwise noted. With acetate as an organic substrate, the
158
free energy of the donor reaction (∆Gd) can be found based on the acetate oxidation half reaction
159
(Rittmann and McCarty, 2001):
160
HCO
161
∆ = ∆ ° +
M AN U
SC
155
+ H + e ↔ CH COO + H O {CH3 COO− } /
(2)
(3)
TE D
{ !"#$ } /%
∆Gd°ʹ = 26.87 kJ/mole-e−
In bioelectrochemical systems, the electron acceptor reaction occurs at the bioanode as
163
exoelectrogens directly transfer electrons to electrically conductive bioanode surfaces. Thus, the
164
free energy of the electron acceptor reaction (∆Ga) is determined by the bioanode potential as: ∆Ga = −nFEan
(4)
AC C
165
EP
162
166
F is the Faraday constant (96485 C per mole-e−), Ean is the bioanode potential in volts, and n is
167
the number of electrons transferred by exoelectrogens in the bioanode reaction. In this study, n
168
was assumed to be unity since most enzymes in bacterial electron transfer chains carry a single
169
charge (Madigan et al., 2005). For instance, the cytochrome c enzyme is known to play a key
170
role in the exoelectrogenic capability of Geobacter and Shewanella species in bioelectrochemical
8
ACCEPTED MANUSCRIPT
systems (Richter et al., 2012; Malvankar et al., 2012) and the enzyme carries a single electron
172
depending on the oxidation state of its central metal (Madigan et al., 2015).
173
2.2 Energy required for cell synthesis (∆Gs)
174
RI PT
171
In microbial energetics, new cells are synthesized in two steps: from a carbon source to pyruvate; and from pyruvate to the cellular carbon (Rittmann and McCarty, 2001). Also, a
176
certain amount of energy (∆Gn°ʹ) is required to create ammonia (i.e., readily available nitrogen
177
source) from other nitrogen sources (e.g., nitrate, nitrogen gas). Thus, the free energy required
178
for cell synthesis (∆Gs°ʹ) can be written as: ∆& ° =
∆'( °) *+
+
∆', °) *
+ ∆- °
M AN U
179
SC
175
(5)
∆Gp°ʹ is the free energy to covert a given carbon source to pyruvate under standard conditions at
181
pH 7 (∆Gp°ʹ = 7.57 kJ per mole-e− for acetate as the carbon source, Rittmann and McCarty,
182
2001). If the carbon source-to-pyruvate reaction is thermodynamically spontaneous, the exponent
183
m is −1 while it is +1 if the reaction is non-spontaneous (Rittmann and McCarty, 2001). ∆Gc°ʹ is
184
the free energy required to convert pyruvate into new microbial cells under standard states at pH
185
7 (∆Gc°ʹ = 18.81 kJ per mole-e−, Rittmann and McCarty, 2001). Since a sufficient amount of
186
ammonia was provided in the feed solution, ∆Gn°ʹ was assumed to be zero in this study.
187
2.3 Energy balance to determine yield coefficient
EP
AC C
188
TE D
180
In McCarty’s microbial energetics model, if (A+1) moles of electrons are released in the
189
electron donor reaction, one mole of electrons are used for cell synthesis and A moles of
190
electrons are transferred to the terminal electron acceptor though the electron acceptor reaction
191
(Rittmann and McCarty, 2001). The total amount of energy released from the electron donor
9
ACCEPTED MANUSCRIPT
reaction is also split by the same ratio. At steady state, the energy from the electron-donor and -
193
acceptor reactions (Aε∆Gr) is identical to that required for cell synthesis (∆Gs°ʹ). Thus, the
194
fraction constant A can be found as:
195
.=−
RI PT
192
∆'/ °)
(6)
∆'0
By finding the fraction of the electron flow to cell synthesis (1/(A+1)), the true (theoretical) yield
197
coefficient was further calculated using C5H7O2N for the chemical formula of new cells
198
(Rittmann and McCarty, 2001).
M AN U
199
200
3. Experimental Method
201
3.1 MEC construction
In lab-scale experiments, the microbial electrolysis cell (MEC) was constructed using
TE D
202
SC
196
polypropylene blocks with a cylindrical interior chamber (3.81 cm in diameter). The reactor
204
consisted of an anode and a cathode chambers (70 and 30 mL, respectively) which were
205
separated by a cation exchange membrane (Selemion CMV, Asahi Glass, Japan). Note that the
206
separation was necessary so that the biomass produced in the anode chamber was not affected by
207
potential microbial activities in the cathode chamber by hydrogenotrophic methanogens (Tice
208
and Kim, 2014b) and sulfate reducing bacteria (Rabaey et al., 2005). A graphite fiber brush was
209
used as the bioanode which was pretreated in a muffle furnace at 505°C for 2 hours (Wang et al.,
210
2009). The cathode was a piece of stainless steel mesh without any catalyst applications (200 ×
211
200 mesh; Type 304; McMaster Carr, USA). A gas collection tube was attached on top of the
212
cathode chamber using epoxy glue. The constructed MEC was inoculated using primary effluent
AC C
EP
203
10
ACCEPTED MANUSCRIPT
from a local wastewater treatment facility and operated about 6 weeks before the main part of the
214
experiment was initiated.
215
3.2 Feed solution preparation
216
RI PT
213
The feed solution was prepared with 60 mM phosphate buffer solution (10.38 g L-1
Na2HPO4-7H2O; 2.55 g L-1 NaH2PO4; 0.36 g L-1 NH4Cl; 0.15 g L-1 KCl). Approximately 0.02
218
mM NaOH was added into the phosphate buffer solution and the resulting pH ranged 9.0 to 9.5.
219
This high pH condition was necessary to avoid potential acidification in the anode chamber that
220
can limit the exoelectrogenic growth and activity on the bioanode (He et al., 2008). The prepared
221
solution was directly used as the catholyte solution while the anolyte solution was then further
222
prepared through the addition of 1 g L-1 sodium acetate as well as trace vitamins and minerals
223
(Cheng et al., 2009).
224
3.3 Reactor operation
M AN U
TE D
225
SC
217
Three independent MECs were operated in a fed-batch mode and each fed-batch cycle lasted between 3 and 5 days (2 cycles per week). The MECs were operated at an applied voltage
227
(Eap) of 1.0 V for the bioanode yield and Coulombic efficiency measurement while three
228
different applied voltages (1.0, 0.75, and 0.5 V) were examined when the anode potential was
229
monitored using an Ag/AgCl reference electrode (MW-2030; BASi, USA). The voltage
230
application, bioanode potential monitoring, and current measurement were conducted using
231
multi-channel potentiostat (MPG2, Bio-Logic Science Instruments; France). All experiments
232
were performed in an air conditioned laboratory with a stationary temperature of 21.2 ± 0.8°C.
233
3.4 Experimental measurements
AC C
EP
226
11
ACCEPTED MANUSCRIPT
234
For the solid analysis, 60 mL was taken from the anolyte effluent (70 mL) and acidified using HCl before it was divided into two 30 mL samples which were individually centrifuged at
236
9000 RPM (rotations per min) for 20 min to separate suspended biomass. Approximately 22.5
237
mL of the supernatant was poured off without disturbing the centrifuged biomass adhered to the
238
wall of the centrifuge tube. The remaining sample was sonicated briefly to dislodge the biomass
239
and then poured into a ceramic crucible. The crucible was kept in a 103°C oven for at least 12 hr
240
and heated to 505°C for 2 hr in a muffle furnace to determine volatile and total suspended solids
241
(VSS and TSS). Note that a small amount of dissolved solids in the remaining sample (7.5 mL)
242
was included in the measurement of VSS and TSS. It should be emphasized that the standard
243
methods for VSS and TSS using filter disks (Eaton et al., 2005) was not sensitive enough to
244
detect the biomass production in the MECs since the observed yield of the bioanode was
245
extremely small (~0.02 g-VSS g-COD-1).
SC
M AN U
The influent and effluent solutions were analyzed for pH and conductivity (SevenMulti;
TE D
246
RI PT
235
Mettler-Toledo International Inc., USA). The anolyte pH decreased from 9.2 to 6.3 while the
248
catholyte pH increased from 9.3 to 12.1 since the anode and cathode chambers were separated by
249
a cation exchange membrane. The anolyte conductivity decreased from ~10.43 to 6.96 mS cm-1
250
while the catholyte conductivity increased from ~10.04 to 19.21 mS cm-1 because the anode
251
chamber loses cations through the cation exchange membrane (Strathmann, 2004).
AC C
252
EP
247
After 99 days of the MEC operation, the reactors were dissembled and the carbon fiber
253
brush bioanodes were dried at 105° and burnt at 505°C to determine the amount of volatile
254
biomass growing on the bioanode.
255
3.5 COD removal and Coulombic efficiency
12
ACCEPTED MANUSCRIPT
In a separate experiment, the effluent anolyte and catholyte as well as the influent anolyte
257
were taken and acidified using phosphoric acid (3% v/v). The acidified samples were analyzed in
258
FID-GC (flame ionization detector-gas chromatography) to determine acetate concentration
259
(Varian CP3800, USA). In the FID-GC analysis, the Stabilwax-DA column was used to separate
260
individual organic fatty acids (Restek, USA). It should be noted that acetate was detected in the
261
effluent catholyte at low concentration (16.1 ± 7.9 mg L-1 as sodium acetate; n = 6) due to non-
262
ideality of the cation exchange membrane. The chemical oxygen demand removal (∆COD) was
263
determined by performing the mass balance on sodium acetate:
264
88=8> ∆COD = 0.78(7 8- 9:− 7 8- 9;<< − 7 =8> 9;<< )
M AN U
SC
RI PT
256
(7)
The factor 0.78 is for the conversion from mg-sodium acetate into mg-COD, c is the sodium
266
acetate concentration measured in the FID-GC analysis, V is the volume with the superscript an
267
for anolyte and cat for catholyte. The subscript in is for influent and eff for effluent.
269
270
The Coulombic efficiency (CE) is the charge-based ratio between the amount of electric current (i) generated and the amount of COD removed during MEC fed-batch cycles: @A =
C :> D∆!"E
(8)
EP
268
TE D
265
The measured CE was 107.9 ± 5.1% (n = 6) and this high CE result indicates that the majority of
272
provided acetate was oxidized by the bioanode. Thus, the biomass produced in the anode
273
chamber was mainly contributed by the excess growth of exoelectrogenic bacteria on the
274
bioanode. After confirming consistently high CE, COD was not further monitored in the
275
bioanode yield experiment and ∆COD was determined by integrating electric current with time
276
by assuming CE of 100%.
AC C
271
13
ACCEPTED MANUSCRIPT
278 279
280
3.6 Decay coefficient estimation The cell decay coefficient (b) was calculated by comparing the true yield coefficient (Ytrue) and observed yield coefficient from the experiment (Yobserved). F=
RI PT
277
(GH0IJ GKL/J0MJN )O PQ ∆!"E
(9)
RPQKNJ S
Xanode is the volatile biomass attached on the bioanode and τ is the average time period of the fed-
282
batch cycle.
SC
281
M AN U
283
284
4. Results and Discussion
285
4.1 Anode potential and pH governing bioanode cell yield
The anode potential was found to govern the excess cell yield on the bioanode. The yield
287
coefficient sharply increased with increasing anode potential from −0.4 to 0 V vs. SHE (standard
288
hydrogen electrode) (Fig 1A). A more positive anode potential corresponds to a greater amount
289
of energy available for bacterial growth. The equilibrium anode potential of the bioanode is
290
−0.293 V vs. SHE (10 mM acetate; 10 mM bicarbonate; pH = 7; 25°C) using the Nernst equation
291
(Logan et al., 2008). This equilibrium anode potential corresponds to a zero cell yield of
292
exoelectrogenic bacteria (i.e., x-intercept of the pH 7 curve in Fig 1A), indicating that the
293
developed method for the yield coefficient is consistent with the prediction based on
294
electrochemistry equilibrium. Based on the slope of the yield vs. anode potential curve, there is
295
an increase of approximately 0.1 g-VSS g-COD-1 in the yield coefficient per 0.1 V increase in
296
the anode potential. The anode potential in MFCs and MECs can be wide but usually in the range
297
of –0.1 to –0.3 V vs. SHE (Aelterman et al., 2008; Torres et al., 2009; Wagner et al., 2010; Lee
AC C
EP
TE D
286
14
ACCEPTED MANUSCRIPT
and Rittmann, 2010; Nam et al., 2011; Sun et al., 2012). Assuming the anode potential of –0.1 V
299
vs. SHE under neutral to slightly basic conditions (pH 7 to 9), the theoretical yield coefficient of
300
exoelectrogenic bacteria will range 0.1 to 0.2 g-VSS g-COD-1.
301
RI PT
298
A change in pH also resulted in a sensitive response in the yield coefficient as an increase in pH by two units raised the yield coefficient by ~0.1 g-VSS g-COD-1 (Fig 1A). It should be
303
emphasized that very small cell yield coefficients were observed at pH 5 (e.g., 0.05 g-VSS g-
304
COD-1 at −0.1 V vs. SHE). This observation indicates that the exoelectrogenic growth is
305
seriously inhibited even at the mildly acidic pH condition. This finding is consistent with
306
experimental results reported in the previous studies that the bioanode practically stops
307
generating electric current at pH of 5 or lower (He et al., 2008).
M AN U
308
SC
302
Exoelectrogenic bacteria form biofilms on bioanode surfaces, and thus the local pH and electric potential vary in the exoelectrogenic biofilm. For instance, the local pH is lower and the
310
electric potential is more positive at a deeper location of the biofilm (Babauta et al., 2012),
311
leading to a decrease and an increase in the microbial yield, respectively (Fig 1A). In addition,
312
such variations in the local pH and electric potential are more pronounced at higher current
313
conditions (Babauta et al., 2012). Thus, the yield coefficient can be slightly different at different
314
locations in exoelectrogenic biofilms. While the scope of this study does not include such micro-
315
scale environmental factors on the yield coefficient, this interesting aspect can be further
316
investigated in future studies.
317
4.2 Substrate and bicarbonate concentration
318 319
AC C
EP
TE D
309
The substrate (acetate) concentration has relatively minor effects on the biomass production. The yield coefficient increased gradually (~10% of its magnitude) with the
15
ACCEPTED MANUSCRIPT
increasing substrate concentration up to 20 mM acetate or 1.28 g-COD/L (Fig 1B) because high
321
substrate concentration decreases the amount of energy required for new cell synthesis, leaving a
322
greater amount of energy for cell growth. Conversely, an increase in bicarbonate concentration
323
increases the energy required for cell synthesis, resulting in a smaller yield coefficient. However,
324
the result shows that both acetate and bicarbonate concentrations have relatively minor effects on
325
the bioanode yield coefficient. Note that there was only ~10% decrease in the yield coefficient
326
with an increase in bicarbonate concentration by an order of magnitude (Fig 1B).
327
4.3 Sensitivity analysis on electron transfer efficiency
SC
M AN U
328
RI PT
320
While the electron transfer efficiency (ε) is commonly assumed to be 0.6, its precise estimation is difficult without a clear understanding of proton motive force generation in the
330
electron transport chain (McCarty, 2007). Thus, we examined the sensitivity of the yield
331
coefficient to the electron transfer efficiency. As the electron transfer efficiency increased, the
332
microbial yield coefficient increased (Fig 1C) because the bacteria obtain a greater amount of
333
energy from substrate utilization (∆Gr in Eq. 1) while they need a smaller amount of energy for
334
cell synthesis (∆Gs°ʹ in Eq. 5) with a higher electron transfer efficiency. Note that ∆Gp°ʹ was
335
always positive unless bicarbonate concentration is extremely low (e.g., [HCO3-] ≤ 0.001 mM),
336
indicating that the reaction from acetate to pyruvate is usually non-spontaneous. When the
337
bioanode potential was more positive, the sensitivity of the yield coefficient to the electron
338
transfer efficiency was more pronounced (Fig 1C). This observation can be explained by more
339
negative free energy for the electron acceptor reaction (∆Ga in Eq. 4) when the bioanode
340
potential is more positive, leading to a greater amount of energy available for microbial growth.
341
4.4 Observed yield coefficient in experiments
AC C
EP
TE D
329
16
ACCEPTED MANUSCRIPT
342
The observed yield coefficient obtained from the MEC experiment was substantially small at about 0.020 g-VSS g-COD-1 (Table 1). In the experiment, the anolyte pH ranged from
344
9.2 to 6.3 and the anode potential was stable about −0.1 V vs. SHE for Eap of 1.0 V when the
345
bioanode is active in generating electric current with acetate (Fig 2). For these given conditions,
346
the true yield coefficient of the bioanode is expected to decrease from 0.2 to 0.1 g-VSS g-COD-1
347
with the decreasing pH from 9.2 to 6.3 (Fig 1A). When compared with this theoretical
348
estimation, the observed yield coefficient is smaller by an order of magnitude. For the
349
consecutive 6 fed-batch cycles, the electric current was high with the peak current ranging 0.6 to
350
3.2 mA (Fig 3), indicating that the bioanode was healthy and thus actively oxidizing the organic
351
substrate (acetate) over the course of the experiments. Thus, the substantially small observed
352
yield coefficient was not contributed by any potential malfunctions of the bioanode during the
353
experiment.
SC
M AN U
Since the MEC reactors were operated at a fed-batch mode, acetate concentration
TE D
354
RI PT
343
decreased with time and thus low acetate concentration near the end of fed-batch cycles could
356
potentially have resulted in the small observed yield coefficient. However, for the 5th and 6th
357
fed-batch cycles, the electric current was high about 1 mA or higher when the fed-batch
358
operation was terminated at 18 and 21 d, respectively (Fig 3). This high current indicates that
359
acetate concentration was sufficiently high at the end of these two fed-batch cycles so that the
360
bioanode actively oxidized acetate and thus created the high electric current throughout the fed-
361
batch cycles. The observed yield coefficient for the 5th and 6th cycles was 0.032 and 0.016 g-
362
VSS g-COD-1, respectively while the average observed yield coefficient over 6 fed-batch cycles
363
was 0.020 g-VSS g-COD-1 (Table 1). Thus, potentially low substrate concentration near the end
AC C
EP
355
17
ACCEPTED MANUSCRIPT
of fed-batch cycles cannot explain the substantial difference between the observed and
365
theoretical yield coefficients.
366
4.5 Cell decay on bioanodes
367
RI PT
364
The cell decay at the bioanode can explain the substantial difference between the
observed yield coefficient in the experiment (0.020 g-VSS g-COD-1) and the theoretical true
369
yield coefficient (0.1 to 0.2 g-VSS g-COD-1). At the end of the 3-month MEC operation, the
370
amount of volatile biomass on the bioanode was measured to be 126.7 ± 8.7 mg VSS per
371
bioanode. Based on this biomass measurement, the decay coefficient was plotted using Eq. 9 as a
372
function of true yield coefficient (Fig 4). The decay coefficient was zero when the true yield
373
coefficient is equal to the observed yield coefficient (0.020 g-VSS g-COD-1) and it increased
374
linearly with the increasing true yield coefficient (Fig 4). During the MEC operation, the anode
375
potential was −0.1 V vs. SHE (Fig 2) and the average pH in the anode chamber was
376
approximately 8. Thus, the resulting true yield coefficient (Ytrue) was 0.2 g-VSS g-COD-1 (Fig
377
1A) and consequently the decay coefficient can be estimated to be 0.013 ± 0.002 d-1 (Fig 4). This
378
estimated decay coefficient is similar to the suggested value for anaerobic microorganisms in
379
Anaerobic Digestion Model No. 1 (0.02 d-1 at 35°C) (Batstone et al., 2012). Thus, the rate of cell
380
decay in bioelectrochemical systems is comparable to that in anaerobic digestion.
M AN U
TE D
EP
AC C
381
SC
368
Using the estimated decay coefficient (0.013 d-1), the amount of exoelectrogenic cells
382
decayed was found to be 5.78 mg VSS per fed-batch cycle (0.013 d-1 × 126.7 mg VSS × 3.5 d).
383
The created volatile biomass using the true yield coefficient was 6.42 mg VSS per cycle (0.2 mg-
384
VSS mg-COD-1 × 458.4 mg-COD L-1 × 0.070 L). Thus, the amount of exoelectrogenic cells
385
decayed was approximately 90% of the amount of newly created cells on the bioanode. This
18
ACCEPTED MANUSCRIPT
386
comparison explains the substantially reduced waste sludge production in bioelectrochemical
387
wastewater treatment that was demonstrated in a recent study (Brown et al., 2015).
389
RI PT
388
5. Conclusions
In this study, we estimated the true (theoretical) cell yield coefficient of exoelectrogenic
391
bacteria in bioelectrochemical systems by modifying McCarty’s free energy-based method. The
392
theoretical yield coefficient ranged 0.1 to 0.3 g-VSS g-COD-1 (pH 7 – 9; anode potential between
393
−0.2 and 0 V vs. SHE; ε = 0.6), which is similar to that of most anaerobic microorganisms
394
growing in anaerobic digesters (Batstone et al., 2012) but much smaller than that of aerobic
395
microorganisms in activated sludge systems (Henze, 2000). The true yield coefficient of
396
exoelectrogenic bacteria was greatly affected by pH and as a result, even a mildly acidic
397
condition (e.g., pH 5) seriously inhibited the bacterial growth on bioanodes. This finding is
398
consistent with previous study results where the bioanode practically stops creating electric
399
current at pH 5 or lower (He et al., 2008). The exoelectrogenic yield was also governed by the
400
anode potential as the true yield coefficient increased with the increasing (i.e., more positive)
401
anode potential. However, the reactant (substrate) and product (bicarbonate) concentration had
402
relatively minor effects on the yield coefficient of exoelectrogenic bacteria. While the electron
403
transfer efficiency is commonly assumed to be 0.6, the sensitivity study result found that the
404
estimation of the yield coefficient is greatly affected by the electron transfer efficiency. This
405
finding indicates that precise estimation of the electron transfer efficiency is necessary in future
406
studies when the electron transfer chain in exoelectrogenic cells are clearly understood.
AC C
EP
TE D
M AN U
SC
390
19
ACCEPTED MANUSCRIPT
407
In the experiment using lab-scale MECs, the observed yield coefficient was found to be 0.020 ± 0.008 g-VSS g-COD-1, which is smaller than the true yield coefficient by an order
409
magnitude. After the 3-month experiment was completed, the total amount of biomass on the
410
bioanode was measured and 126.7 ± 8.7 mg volatile solids were found to be attached on each
411
bioanode. Based on this measurement, the rate of cell decay was obtained from the difference
412
between the true and observed yield coefficients. The estimated decay coefficient was 0.013 ±
413
0.002 d-1 at 21.2°C, which is similar to that of anaerobic microorganisms in anaerobic digesters
414
(0.02 d-1 at 35°C) ( Batstone et al., 2012). Using the obtained decay coefficient, the amount of
415
cell decayed over the fed-batch cycle was 5.78 mg VSS, which was 90% of the created cell (6.42
416
mg VSS), explaining the substantially small observed yield coefficient.
M AN U
SC
RI PT
408
The found yield and decay coefficients will be used in model developments and
418
simulations of bioelectrochemical systems to predict their performance of wastewater treatment
419
and resources recovery. The substantially small observed yield coefficient of exoelectrogenic
420
bacteria (0.020 g-VSS g-COD-1) confirmed a strong potential of bioelectrochemical systems for
421
future wastewater treatment with greatly reduced sludge generation as recently demonstrated
422
(Brown et al., 2015). Considering that sludge treatment and disposal are responsible for major
423
expenses in wastewater treatment, bioelectrochemical systems can effectively reduce the cost for
424
wastewater treatment.
EP
AC C
425
TE D
417
426
Acknowledgements
427
This study was supported by Discovery Grants (435547-2013, Natural Sciences and Engineering
428
Research Council of Canada), Canada Research Chairs Program (950-2320518, Government of
20
ACCEPTED MANUSCRIPT
Canada), Leaders Opportunity Fund (31604, Canada Foundation for Innovation), Ontario
430
Research Fund-Research Infrastructure (31604, Ontario Ministry of Economic Development and
431
Innovation), and Undergraduate Student Research Awards (Natural Sciences and Engineering
432
Research Council of Canada). The authors thank Ms. Anna Robertson and Mr. Peter Koudys for
433
their help on equipment operation and reactor construction.
SC
434
RI PT
429
References
436 437 438
Aelterman, P., Freguia, S., Keller, J., Verstraete, W., Rabaey, K., 2008. The anode potential regulates bacterial activity in microbial fuel cells. Applied Microbiology Biotechnology, 78(3), 409-418.
439 440
Ahn, Y., Schröder, U., 2015. Microfabricated, continuous-flow, microbial three-electrode cell for potential toxicity detection. BioChip Journal, 9(1), 27-34.
441 442 443 444
Babauta, J. T., Nguyen, H. D., Harrington, T. D., Renslow, R., Beyenal, H., 2012. pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer. Biotechnology Bioengineering, 109(10), 26512662.
445 446 447
Batstone, D. J., Keller, J., Angelidaki, I., Kalyuzhny, S. V., Pavlostathis, S. G., Rozzi, A., Sander, W. T. M., Siegrist, H., Vavilin, V. A., 2002. Anaerobic digestion model no. 1 (ADM1). IWA publishing, London.
448 449 450
Brown, R. K., Harnisch, F., Dockhorn, T., Schröder, U., 2015. Examining sludge production in bioelectrochemical systems treating domestic wastewater. Bioresource Technology, 198, 913-917.
451 452 453
Cao, X., Huang, X., Liang, P., Xiao, K., Zhou, Y., Zhang, X., Logan, B. E., 2009. A new method for water desalination using microbial desalination cells. Environmental Science Technology, 43(18), 7148-7152.
454 455 456
Cheng, S., Xing, D., Call, D. F., Logan, B. E., 2009. Direct biological conversion of electrical current into methane by electromethanogenesis. Environmental Science Technology, 43(10), 3953-3958.
457 458
Eaton, A.D., Clesceri, L.S., Rice, E.W., Greenberg, A.E., 2005. Standard methods for examination of water and wastewater. 21st ed. American Public Health Association.
AC C
EP
TE D
M AN U
435
21
ACCEPTED MANUSCRIPT
Grady, C.P.L., Daigger, G.T., Love, N.G., Filipe, C.D.M., 2011. Biological Wastewater Treatment, 3rd ed. Taylor and Francis Group, Boca Raton.
461 462 463
He, Z., Huang, Y., Manohar, A. K., Mansfeld, F., 2008. Effect of electrolyte pH on the rate of the anodic and cathodic reactions in an air-cathode microbial fuel cell. Bioelectrochemistry, 74(1), 78-82.
464 465
Henze, M. (Ed.), 2000. Activated sludge models ASM1, ASM2, ASM2d and ASM3. IWA Publishing, London.
466 467
Kim, T., An, J., Jang, J. K., Chang, I. S., 2015. Coupling of anaerobic digester and microbial fuel cell for COD removal and ammonia recovery. Bioresource Technology, 195, 217-222.
468 469
Lee, H. S., Rittmann, B. E., 2010. Characterization of energy losses in an upflow single-chamber microbial electrolysis cell. International Journal of Hydrogen Energy, 35(3), 920-927.
470 471 472
Logan, B. E., Hamelers, B., Rozendal, R., Schröder, U., Keller, J., Freguia, S., Aelterman, P., Verstraete, W., Rabaey, K., 2006. Microbial fuel cells: methodology and technology. Environmental Science Technology, 40(17), 5181-5192.
473 474 475
Logan, B. E., Call, D., Cheng, S., Hamelers, H. V., Sleutels, T. H., Jeremiasse, A. W., Rozendal, R. A., 2008. Microbial electrolysis cells for high yield hydrogen gas production from organic matter. Environmental Science Technology, 42(23), 8630-8640.
476 477
Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., Stahl, D. A., Brock biology of microorganisms. 2015. 14th Ed., Pearson Education, Glenview.
478 479 480
Malvankar, N. S., Mester, T., Tuominen, M. T., Lovley, D. R., 2012. Supercapacitors Based on c‐Type Cytochromes Using Conductive Nanostructured Networks of Living Bacteria. ChemPhysChem, 13(2), 463-468.
481 482
McCarty, P. L., 2007. Thermodynamic electron equivalents model for bacterial yield prediction: modifications and comparative evaluations. Biotechnology Bioengineering, 97(2), 377-388.
483 484
Modin, O., Wang, X., Wu, X., Rauch, S., Fedje, K. K., 2012. Bioelectrochemical recovery of Cu, Pb, Cd, and Zn from dilute solutions. Journal of Hazardous Materials, 235, 291-297.
485 486 487
Nam, J. Y., Tokash, J. C., Logan, B. E., 2011. Comparison of microbial electrolysis cells operated with added voltage or by setting the anode potential. International Journal of Hydrogen Energy, 36(17), 10550-10556.
488 489 490
Nilges, P., Schröder, U., 2013. Electrochemistry for biofuel generation: production of furans by electrocatalytic hydrogenation of furfurals. Energy Environmental Science, 6(10), 29252931.
491 492 493
Qin, M., Molitor, H., Brazil, B., Novak, J. T., He, Z., 2016. Recovery of nitrogen and water from landfill leachate by a microbial electrolysis cell–forward osmosis system. Bioresource Technology, 200, 485-492.
AC C
EP
TE D
M AN U
SC
RI PT
459 460
22
ACCEPTED MANUSCRIPT
Rabaey, K., Clauwaert, P., Aelterman, P., Verstraete, W., 2005. Tubular microbial fuel cells for efficient electricity generation. Environmental Science Technology, 39(20), 8077-8082.
496 497 498
Richter, K., Schicklberger, M., Gescher, J., 2012. Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration. Applied Environmental Microbiology, 78(4), 913-921.
499 500
Rittmann, B. E., McCarty, P. L., 2001. Environmental biotechnology: principles and applications. McGraw-Hill Higher Education, New York.
501 502 503
Rotaru, D. E. H., Franks, A. E., Orellana, R., Risso, C., Nevin, K. P. (2011). Geobacter: the microbe electric’s physiology, ecology, and practical applications. Advances in Microbial Physiology, 59, 1-100.
504
Strathmann, H., 2004. Ion-exchange membrane separation processes. Elsevier.
505 506 507
Sun, D., Call, D. F., Kiely, P. D., Wang, A., Logan, B. E., 2012. Syntrophic interactions improve power production in formic acid fed MFCs operated with set anode potentials or fixed resistances. Biotechnology Bioengineering, 109(2), 405-414.
508 509
Tice, R. C., Kim, Y., 2014a. Energy efficient reconcentration of diluted human urine using ion exchange membranes in bioelectrochemical systems. Water Research, 64, 61-72.
510 511
Tice, R. C., Kim, Y., 2014b. Methanogenesis control by electrolytic oxygen production in microbial electrolysis cells. International Journal of Hydrogen Energy, 39(7), 3079-3086.
512 513 514 515
Torres, C. I., Krajmalnik-Brown, R., Parameswaran, P., Marcus, A. K., Wanger, G., Gorby, Y. A., Rittmann, B. E., 2009. Selecting anode-respiring bacteria based on anode potential: phylogenetic, electrochemical, and microscopic characterization. Environmental Science Technology, 43(24), 9519-9524.
516 517
Wagner, R. C., Call, D. F., Logan, B. E., 2010. Optimal set anode potentials vary in bioelectrochemical systems. Environmental Science Technology, 44(16), 6036-6041.
518 519 520
Wang, X., Cheng, S., Feng, Y., Merrill, M. D., Saito, T., Logan, B. E., 2009. Use of carbon mesh anodes and the effect of different pretreatment methods on power production in microbial fuel cells. Environmental Science Technology, 43(17), 6870-6874.
521 522
Zhang, Y., Angelidaki, I., 2015a. Bioelectrochemical recovery of waste-derived volatile fatty acids and production of hydrogen and alkali. Water Research, 81, 188-195.
523 524
Zhang, Y., Angelidaki, I., 2015b. Recovery of ammonia and sulfate from waste streams and bioenergy production via bipolar bioelectrodialysis. Water Research, 85, 177-184.
AC C
EP
TE D
M AN U
SC
RI PT
494 495
525 526
23
ACCEPTED MANUSCRIPT
527
Tables
528
Table 1. Summary of experimental results to find the observed yield coefficient.
RI PT
Fed-batch cycle 1 2 3 4 5 6 Mean ± SD -1 VSS (mg L ) 6.59 10.86 5.84 9.16 13.22 8.41 9.01 ± 2.73 ∫idt (C) 401.1 447.2 381.9 307.6 345.1 439.3 387.0 ± 54.1 ∆COD (mg L-1)* 475.1 529.7 452.4 364.4 408.7 520.4 458.4 ± 64.1 Observed yield 0.0139 0.0205 0.0129 0.0251 0.0323 0.0162 0.0202 ± 0.0075 (g-VSS g-COD-1) * Coulombic efficiency of 100% was assumed as demonstrated in a separate experiment.
SC
529
AC C
EP
TE D
M AN U
530
24
ACCEPTED MANUSCRIPT
0.1
0.2
0.1
0.0
0.0
(C)
−0.1 V vs. SHE −0.2 V vs. SHE −0.3 V vs. SHE
0.2
0.1
0.0
0
5
10 15 Acetate (mM)
20
M AN U
‐0.4 ‐0.2 0 Anode potential (V vs. SHE)
0.3
RI PT
0.2
0.1 mM HCO₃ˉ 1.0 mM HCO₃ˉ 10.0 mM HCO₃ˉ
(B)
Yield (g‐VSS/g‐COD)
0.3
(A)
SC
pH = 9 pH = 7 pH = 5
Yield (g‐VSS/g‐COD)
Yield (g‐VSS/g‐COD)
0.3
0
0.2 0.4 0.6 Transfer efficiency
0.8
Figure 1. The true yield coefficient estimation. (A) Effect of anode potential and pH (10 mM acetate; 10 mM bicarbonate; ε = 0.6; 25°C) (B) Effect of acetate and bicarbonate concentration (pH = 7; anode potential = −0.1 V vs. SHE; ε = 0.6; 25°C) (C) Sensitivity
AC C
EP
bicarbonate; 25°C).
TE D
analysis results on the electron transfer efficiency (pH = 7; 10 mM acetate; 10 mM
ACCEPTED MANUSCRIPT
-0.1
RI PT
MEC-1 MEC-2 MEC-3
SC
-0.2
-0.3
-0.4 0.25
TE D
0
M AN U
Anode potential (V vs SHE)
0
0.5 Eap (V)
0.75
1
AC C
EP
Figure 2. Relationship between the applied voltage (Eap) and anode potential (Ean).
MEC-1 MEC-2 MEC-3
SC
2
1
0 5
10 15 Time (day)
TE D
0
M AN U
Electric current (mA)
3
RI PT
ACCEPTED MANUSCRIPT
20
Figure 3. Electric current during the apparent yield coefficient measurement over 6 fed-batch
AC C
EP
cycles.
ACCEPTED MANUSCRIPT
ΔCOD = 458 mg/L ΔCOD = 394 mg/L
SC
0.02
RI PT
ΔCOD = 523 mg/L
0.01
M AN U
Decay coefficient (d-1)
0.03
0.00
0.1 0.2 0.3 True yield coefficient (g-VSS/g-COD)
TE D
0
Figure 4. Decay coefficient estimation using Eq. 9. (ΔCOD = 458.4 ± 64.1 mg-COD L-1 (Table 1); Apparent yield coefficient = 0.02 g-VSS g-COD-1; anolyte volume = 70 mL; average
AC C
EP
fed-batch cycle period = 3.5 d)
ACCEPTED MANUSCRIPT
Highlights The true yield coefficient of exoelectrogenic bacteria was 0.1 to 0.3 g-VSS g-COD-1. More positive anode potential and higher pH resulted in greater cell yield.
RI PT
The apparent yield coefficient of exoelectrogens was 0.020 ± 0.008 g-VSS g-COD-1. The decay coefficient of exoelectrogens was estimated to be 0.013 ± 0.002 d-1.
AC C
EP
TE D
M AN U
SC
Waste sludge production in bioelectrochemical systems was substantially small.