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Critical Review
Methane production and conductive materials: a critical review Gilberto Martins, Andreia Filipa Salvador, Luciana Pereira, and Maria Madalena Alves Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01913 • Publication Date (Web): 17 Aug 2018 Downloaded from http://pubs.acs.org on August 19, 2018
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Methane production and conductive materials: a critical review
2 Gilberto Martins†*, Andreia F. Salvador†, Luciana Pereira† and M. Madalena Alves†
3 4
†
Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
5 6
Abstract
7
Conductive materials (CM) have been extensively reported to enhance methane production in
8
anaerobic digestion processes. The occurrence of direct interspecies electron transfer (DIET) in
9
microbial communities, as an alternative or complementary to indirect electron transfer (via
10
hydrogen or formate), is the main explanation given to justify the improvement of methane
11
production. Not disregarding that DIET can be promoted in the presence of certain CM, it surely
12
does not explain all the reported observations. In fact, in methanogenic environments DIET was
13
only unequivocally demonstrated in cocultures of Geobacter metallireducens with
14
Methanosaeta harundinacea or Methanosarcina barkeri and frequently Geobacter sp. are not
15
detected in improved methane production driven systems. Furthermore, conductive carbon
16
nanotubes were shown to accelerate the activity of methanogens growing in pure cultures,
17
where DIET is not expected to occur, and hydrogenotrophic activity is ubiquitous in full-scale
18
anaerobic digesters treating for example brewery wastewaters, indicating that interspecies
19
hydrogen transfer is an important electron transfer mechanism in those systems. This paper
20
presents an overview of the effect of several iron-based and carbon-based CM in bioengineered
21
systems, focusing on the improvement in methane production and in microbial communities’
22
changes. Control assays, as fundamental elements to support major conclusions in reported
23
experiments, are critically revised and discussed.
24 25 26 27 *
Corresponding author: Gilberto Martins, Tel.:(+351)253601986; email:
[email protected] 1 ACS Paragon Plus Environment
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Abstract art
29 30 31 32
1. Introduction
33
Methane is a renewable energy source that can be produced in controlled bioengineered systems
34
from a wide range of organic substrates including diluted industrial wastewater, animal manure
35
or the organic fraction of municipal solid waste, through a process generally called anaerobic
36
digestion (AD). Fundamental knowledge and technology developments of AD processes have
37
evolved significantly and in parallel in the last decades. The fact that the process relies on the
38
activity of slow growing anaerobic microorganisms1 results in low nutrient requirements and
39
low amounts of sludge produced, which are strong advantages of the anaerobic treatment
40
process. These microorganisms grow slowly because the energy gain from the anaerobic
41
metabolism is low and has to be divided by different trophic groups, i.e., the bacteria
42
performing hydrolytic, acidogenesis and acetogenesis reactions, and the methanogens
43
converting intermediary degradation products into methane.2
44
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Syntrophic interactions between bacteria and methanogens are the basis to maintain an AD
46
system working efficiently. These microorganisms, with distinct, but complementary metabolic
47
capabilities, exchange electrons for energy purposes, normally through the transfer of small
48
soluble chemical compounds, such as hydrogen or formate, that act as electron shuttles. This
49
interspecies hydrogen/formate transfer process is very important since the overall
50
thermodynamics depends on the capacity of the microbial communities to maintain a low
51
hydrogen partial pressure.3 Thus, diffusion limitations of these metabolites, between anaerobic
52
bacteria and methanogenic archaea, can be important bottlenecks in the anaerobic conversion
53
process.4,5
54 55
Recent studies proposed that interspecies electron transfer (IET) can also be performed directly
56
between bacteria and methanogenic archaea, or with the aid of conductive materials (CM),
57
being potentially a more energy conserving approach, and thus improving the rate of
58
methanogenesis.6,7 However, clear evidence of direct interspecies electron transfer (DIET) was
59
only observed in cocultures of electroactive bacteria, namely Geobacter species, and in
60
cocultures of G. metallireducens with Methanosaeta harundinacea3,7 or Methanosarcina
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barkeri.8 Moreover, DIET seems to require outer membrane c-type cytochromes and pili,6,7 but
62
traditional syntrophic fatty acid-degrading bacteria (e.g.: Syntrophomonas wolfei and
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Syntrophus aciditrophicus)9 and most methanogens (e.g.: all members of the orders
64
Methanopyrales, Methanococcales, Methanobacteriales and Methanomicrobiales)10 lack the
65
genes for these cell components. Another indication that not all syntrophic bacteria are capable
66
of DIET is the case of Pelobacter carbinolicus, a known syntrophic ethanol oxidizing
67
bacterium, that could only establish syntrophic interactions with Geobacter sulfurreducens via
68
interspecies hydrogen transfer or interspecies formate transfer,11 although it has been reported to
69
contain c-type cytochromes.12
70 71
Notwithstanding these facts, in the past five years, many studies have reported the improvement
72
of methane production in anaerobic reactors amended with CM such as magnetite, granular 3 ACS Paragon Plus Environment
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activated carbon (GAC), carbon nanotubes (CNT), and biochar, among others.1,8,21–27,13–20 In
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general, these CM are highly stable, have large surface area, good adsorption capacity and high
75
electric conductivity.28–30 Some of these materials may act as redox mediators in microbial
76
catalysis of compounds with electrophilic groups, such as dyes.30
77 78
The role of DIET is discussed in recent papers on anaerobic digestion processes amended with
79
CM.31–33 The authors of these papers interpret the enhancement of methane production as a
80
direct consequence of DIET promoted by CM. However, exploring the results collected from a
81
significant number of studies on this topic, turned clear that the effect of CM goes beyond the
82
stimulation of DIET.
83 84
In this review, the recent findings on the effect of CM in AD bioengineered systems is
85
summarized, exploring how these materials affect methane production rate and the structure of
86
anaerobic microbial communities. This is a recent research field with a relevant impact in
87
engineered and natural anaerobic microbial processes. Little is known about the mechanisms
88
behind the improvement of methane production rates when CM are present. Apart from DIET,
89
other elements of knowledge should be considered, and main interpretations of the researchers
90
contributing to the knowledge in this field are herein addressed and discussed.
91 92
2. Electron transfer mechanisms in methanogenic environments
93
Anaerobic digestion is a biological process, where complex organic compounds are sequentially
94
converted to simple compounds, by a wide variety of microorganisms. The rate and the route
95
governing electrons transfer among the microbial community, determines the entire process
96
efficiency.34–37 Mineralization of waste to methane is dependent on the activity of hydrolytic,
97
acidogenic, and acetogenic bacteria, which generate the substrates (i.e., hydrogen, formate and
98
acetate) for methanogenic archaea, which ultimately produce methane in AD processes.37–40 IET
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efficiency between syntrophic partners dictates the rates of conversion bioprocesses when
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methanogenic activity is guaranteed. In methanogenic systems microorganisms can transfer 4 ACS Paragon Plus Environment
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electrons between each other: (1) via soluble chemical compounds (e.g., hydrogen, formate and
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acetate) which act as electron shuttles; (2) via other chemicals (e.g., humic substances); (3) via
103
direct electron transfer by electrically conductive pili or direct contact; and (4) by direct electron
104
transfer mediated by CM (Figure 1). In the following subsections the fundamentals and
105
mechanisms of IET are briefly exposed.33,41–43
106
107 108
Figure 1. Possible interspecies electron transfer mechanisms in methanogenic environments.42–
109
44
110 111 112
2.1. Interspecies electron transfer via soluble molecules
113
The most studied and well known mechanism of electron transfer in methanogenic communities
114
is the indirect interspecies electron transfer via hydrogen or formate.9,44–46 Syntrophic bacteria
115
produce hydrogen or formate as a way to dissipate the reducing power, i.e., the electrons formed
116
during the degradation of organic compounds and, in turn, methanogens utilize those molecules
117
as electron donors to reduce CO2 to methane (Figure 1A). Therefore, hydrogen/formate act as
118
shuttles
119
methanogens. At high hydrogen concentrations (>10 Pa), the hydrogenase activity is inhibited
120
and consequently the metabolism of syntrophic bacteria is inhibited as well, while that of the
between
hydrogen/formate-forming
bacteria
and
hydrogen/formate-utilizing
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methanogens is stimulated, and vice versa.3,47–49 Formate formation has been detected
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particularly in cocultures growing on proteins,50 or fatty acids like propionate and butyrate.3,51,52
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Under certain conditions, interspecies formate transfer may prevail because formate has a higher
124
diffusion coefficient, comparing to hydrogen.53,54 Syntrophic interactions involving hydrogen or
125
formate as electron shuttles are well described in cocultures degrading common compounds
126
formed during the AD process such as butyrate,55 propionate,56 ethanol,57 and acetate.58
127 128
2.2. Interspecies electron transfer via extracellular compounds
129
In numerous anaerobic environments, the interspecies electron transfer can be mediated also by
130
insoluble compounds, such as humic acids present in humus.59–62 Unlike soluble electron
131
shuttles, such as hydrogen or formate, that can diffuse in and out of the cell,63 insoluble
132
compounds do not penetrate the cell surface. Lovley and co-workers64 demonstrated that humus
133
can mediate electron transfer between humics-reducing and humics-oxidizing microorganisms
134
(Figure 1B). The electron acceptor properties have been related mainly with the redox active
135
quinone moieties present in humic substances.65 Several microorganisms were found to reduce
136
humic acids or anthraquinone-2,6-disulphonate (AQDS) using hydrogen as electron donor (e.g.,
137
the halorespiring bacterium, Desulfitobacterium PCE1, the sulfate-reducing bacterium,
138
Desulfovibrio G11 and the methanogenic archaea, Methanospirillum hungatei JF1) or lactate
139
(Desulfitobacterium dehalogenans and Desulfitobacterium PCE1).59
140 141
Humus can also be re-oxidized and act as an electron donor. For example, humic acids can be
142
redox mediators in the anaerobic substrate oxidation coupled to the abiotic reduction of metal
143
oxides such as Fe(III) and Mn(IV), being re-oxidized and participating in many cycles.59 Some
144
bacteria of the genus Geobacter have been reported as quinone-reducing microorganisms using
145
Fe(III) as the terminal electron acceptor66–69 but other microorganisms share this ability, such as
146
some Shewanella, Desulfitobacterium, Desulphuromonas, Geospirillum, Wolinella and
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Geothrix69 and the methanogenic archaea Methanopyrus kandleri, Methanobacterium
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thermoautotrophicum.70 The anaerobic oxidation of lactate and hydrogen by Desulfitobacterium
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dehalogenans was obtained with AQDS as mediator, associated to the reduction of goethite.59
150 151
2.3. Direct interspecies electron transfer
152
Recently, DIET has been described in anaerobic environments, involving the formation of an
153
electric current between electron-donating and electron-accepting microbes (Figure 1C)42,43,71,72
154
and without the need to produce and exchange electron carriers (i.e., hydrogen and formate).
155
DIET is analogous to direct extracellular electron transfer, which consists in the electron
156
transfer between cells and a solid-state electron acceptor such as iron and manganese oxides or
157
electrodes.73 DIET is well studied in bacteria belonging to the genera Shewanella and
158
Geobacter,42,43,71,72 which are highly efficient in dealing with solid extracellular electron
159
acceptors. DIET was first described in defined cocultures of G. metallireducens, an ethanol
160
oxidizing bacteria, and G. sulfurreducens, a fumarate reducing bacteria.71
161
microorganisms establish a syntrophic relationship, via hydrogen interspecies electron transfer,
162
with G. metallireducens converting ethanol to hydrogen and G. sulfurreducens using hydrogen
163
as electron donor for fumarate reduction. The ability of this culture for performing DIET was
164
discovered when cocultures formed with G. sulfurreducens strains lacking the hyb gene (thus
165
unable to utilize hydrogen), were able to oxidize ethanol and to reduce fumarate. Under these
166
conditions, interspecies electron exchange between G. metallireducens and G. sulfurreducens
167
occurred directly via conductive pili and without the formation of soluble intermediates.71
These
168 169
More recently, it was shown that DIET can occur in cocultures of Geobacter species and
170
acetoclastic methanogens (i.e., Methanosaeta and Methanosarcina species).6,8 Rotaru and co-
171
workers6 showed that Methanosaeta harundinacea, a strictly acetoclastic methanogen, can
172
receive electrons directly from G. metallireducens to produce methane. The idea that
173
Methanosaeta species are acetoclastic specialists, only producing methane from acetate,
174
changed from this point on, since it seems to be able to activate the CO2 reduction pathway for
175
methane production. In this context, the electrons released by Geobacter species are transferred, 7 ACS Paragon Plus Environment
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via pili, directly to Methanosaeta, but the cell machinery involved in electrons uptake by the
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methanogen is not yet known.6 These findings gave a new perspective on the interspecies
178
interactions taking place in anaerobic bioreactors producing methane.
179 180
2.4. DIET promoted by conductive materials
181
The presence of CM such as GAC, carbon cloth and biochar appears to promote DIET via a
182
conduction-based mechanism, in which electrons migrate through the CM from electron-
183
donating to electron-accepting cells.20,22,74
184 185
Surprisingly, it was observed that the lack of pili and other cell component involved in the
186
exogenous electron transfer can be compensated by the presence of CM, namely GAC,8 carbon
187
cloth74 and biochar.22 This was verified in defined cocultures of pilA-deficient strains of G.
188
metallireducens with Methanosarcina barkeri, which could not convert ethanol to methane
189
unless in the presence of CM. This methanogenic coculture in the presence of biochar were able
190
to utilize 86% of the electrons released from ethanol oxidation for methane production, but
191
without biochar no ethanol was consumed and no methane was produced.22 Similarly, the lack
192
of the pilin-associated c-type cytochrome (OmcS), necessary for extracellular electron transfer
193
in Geobacter species, could be compensated by magnetite, another conductive material.75
194
Without magnetite Geobacter strains lacking genes for OmcS were ineffective in forming viable
195
cocultures,71 but in the presence of magnetite, the OmcS-deficient mutants performed similarly
196
to the wild type.75
197 198
2.5. Hydrogen and formate IET versus DIET
199
An interesting approach towards the clarification of the importance of DIET in methanogenesis
200
was presented by Storck and co-workers73 who proposed a mechanistic framework that enabled
201
the direct assessment of the relative feasibility of DIET, and mediated interspecies electron
202
transfer (MIET) in a thermodynamically restricted syntrophic system (propionate conversion to
203
acetate and methane), through mathematical modelling. They found that DIET could be more 8 ACS Paragon Plus Environment
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favorable than hydrogen-MIET, but substantially less favorable than formate-MIET (1 order of
205
magnitude rate difference), assuming a default parameter set based on literature data. The model
206
results also suggested that DIET may be a thermodynamically more feasible alternative to
207
MIET for disperse communities limited by diffusion, which is contrary to experimental
208
observations where nanowire-DIET is commonly observed in dense aggregates,6,8,71 possibly
209
indicating that co-evolution and co-metabolism are more important than external limitations in
210
the simulated system.73 These authors also suggested that CM reduce resistivity, and leave only
211
activation losses, making long-range transport even more feasible.
212 213 214
3. Effect of conductive materials on methane production
215
3.1. CM improving methane production
216
Several strategies have been implemented to improve AD. Empirical modification of process
217
design, operational conditions, and application of substrate pre-treatments, such as
218
microaeration, thermal and mechanical treatments are some examples.1,76,77 Recently, several
219
authors have been reporting the enhancement of methane production by the use of CM (Figure
220
2a), which is now considered a strategy to improve methane production efficiency and stability
221
of anaerobic reactors.7,20 The type of conductive material, the microbial substrates, the mode of
222
operation and the main results reported in scientific communications are summarized in Table 1
223
and Table S1. In general, lag phases preceding methane production are reduced and methane
224
production rates increase when CM are added to batch experiments.8,16,18–20,27,74,78,79 In
225
continuous anaerobic digesters, CM also accelerate methane production and contribute for a
226
more stable operation, allowing higher applied organic loading rates while maintaining high
227
COD removal rates.21,80–83
228 229
Table 1. Summary of the studies reporting the enhancement of methane production by CM Conductive material
Particle size / µm
Applied concentration / g/L
Common substrates
Methane improvement*
Reference
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Conductive material
Particle size / µm
Applied concentration / g/L
Common substrates
Methane improvement*
Ferric oxyhydroxide
1 to 2
1.8
Real dairy wastewater
2.0
1
Magnetite
0.01 to 0.3
0.01 to 25
1.3 to 3.6
24, 25, 27, 78,
Iron oxide nanoparticles Ferroferric oxide Ferrihydrite
0.02
0.8
0.01 to 0.3 ~0.03
10 3.4 to 4.2
Hematite
0.02 to 0.3
0.022 to 10.9
Iron powder Nanoscale zero valent iron
0.001 to 0.1
10
Acetate, Ethanol, Benzoate, Butyrate, Propionate, Real and synthetic dairy wastewater Beet sugar industrial wastewater Synthetic wastewater Acetate, Ethanol Acetate, Ethanol, Benzoate, Sludge Sludge
0.02 to 10.9
Red mud
-
Reference
1, 16, 17, 23,
83, 84, 85, 86
1.3
87
1.2 to 1.8 1.1
14, 88
1.3 to 2.2
24, 25, 84, 89
1.4
90
Sludge
1.3 to 2.2
89, 90
20
Activated sludge from municipal WWTP
1.4
91
24
500 to 2000 0.07 to 1.5
25.7
Synthetic wastewater
1.3
92
2 to 8
Synthetic wastewater
4.4
93
0.25 to 3
1.2
Sucrose
2.0
94
-
0.03 to 2.0
1.2 to 1.5
95, 96
Multi-walled CNT
0.010 to 0.2
0.1 to 5.0
Ethanol, Synthetic wastewater H2/CO2, Acetate, Butyrate, Beet sugar industrial wastewater
1.1 to 17
16, 18, 87
Single-walled CNT
0.001
1.0
Sucrose, Glucose
1.8 to 2
79, 97
Graphite
6 to 25
12 to 132 graphite rods
1.0 to 1.3
13, 80
GAC
841 to 2000
3.3 to 50
Synthetic complex waste and wastewater Acetate, Ethanol, Butyrate, Propionate, Glucose, Synthetic brewery wastewater, Synthetic dairy wastewater, Activated sludge, Organic fraction of municipal solid waste, Synthetic complex waste
1.1 to 18
20, 21, 79, 83,
Powered activated carbon
149 to 177
5
Synthetic brewery wastewater
n.a.
21
Carbon felt
-
3 to 30 pieces
Synthetic wastewater (glucose), and Synthetic complex waste
1.1
13, 101
Stainless steel Manganese Polyaniline nanorods Graphene
230 231
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Ethanol, Synthetic wastewater, Leachate from municipal solid waste incineration, Carbon cloth 10 to 20 pieces Organic fraction of municipal solid, waste, Synthetic complex waste Ethanol, Butyrate, Propionate, Biochar 60 to 700 0.3 to 42.7 Sludge, Synthetic wastewater * – Number of times that methane production increases relative to the control
8, 13, 15, 19,
98, 99, 100
13, 19, 74, 80,
1.1 to 10
81, 102
22, 80, 82, 103,
1.2 to 1.3
104
232 233
Only few studies used direct methanogenic substrates, such as acetate or H2/CO2,18,24,84,85,98
234
while most studies used volatile fatty acids, such as butyrate and propionate, and alcohols as
235
substrates to promote the syntrophic metabolism within microbial communities.8,16–18,20,22–
236
24,78,79,82,85,96,100
More complex organic matter, such as dairy wastewater,1,83,86 brewery 10 ACS Paragon Plus Environment
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wastewater,20 beet sugar wastewater,87 food waste,13 municipal solid waste,19 waste activated
238
sludge89,90,99,103,104 or leachate81 have also been investigated. When compared to control
239
conditions, the addition of CM to bioreactors treating these complex wastewaters resulted in
240
less accumulation of VFAs, thus improving the efficiency of the AD process.
241 242
GAC and magnetite were the most common CM applied in AD systems, followed by CNT,
243
carbon cloth and biochar (Figure 2/Table 1).
244
245 246
Figure 2. Number of studies reporting the effect of different types of CM on methane
247
production.
248 249 250
3.2. CM inhibiting methane production
251
From all CM tested, magnesium oxide, silver nanoparticles, ferrihydrite and carbon black
252
(amorphous carbon) were the only ones inhibiting methanogenesis (Table 2).
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Table 2. Summary of the studies reporting the inhibition of methane production by CM Conductive material
Particle size / µm
Applied concentration / g/L
Substrate
Methane inhibition*
Ferrihydrite
~0.03
2.2 to 4.2
Acetate, Ethanol
1.9 to 4.3
24, 84
Magnesium oxide
