Methane Production and Conductive Materials: A Critical Review

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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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Environmental Science & Technology

1

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]

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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

61

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

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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

99

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

122

particularly in cocultures growing on proteins,50 or fatty acids like propionate and butyrate.3,51,52

123

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

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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

147

Geothrix69 and the methanogenic archaea Methanopyrus kandleri, Methanobacterium


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thermoautotrophicum.70 The anaerobic oxidation of lactate and hydrogen by Desulfitobacterium

149

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



Conductive material

Particle size / µm


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


Substrate

Methane inhibition*

Ferrihydrite

~0.03

2.2 to 4.2

Acetate, Ethanol

1.9 to 4.3

24, 84

Magnesium oxide

Methane Production and Conductive Materials: A Critical Review

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