pretreatment of microalgae to improve biogas production: a review
TRANSCRIPT
Accepted Manuscript
Review
Pretreatment of microalgae to improve biogas production: a review
Fabiana Passos, Enrica Uggetti, Hélène Carrère, Ivet Ferrer
PII: S0960-8524(14)01230-9DOI: http://dx.doi.org/10.1016/j.biortech.2014.08.114Reference: BITE 13877
To appear in: Bioresource Technology
Received Date: 30 June 2014Revised Date: 25 August 2014Accepted Date: 26 August 2014
Please cite this article as: Passos, F., Uggetti, E., Carrère, H., Ferrer, I., Pretreatment of microalgae to improve biogasproduction: a review, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.08.114
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1
Pretreatment of microalgae to improve biogas production: a review 1
2
Fabiana Passos1, Enrica Uggetti
1, Hélène Carrère
2 and Ivet Ferrer
1* 3
4
1 GEMMA - Group of Environmental Engineering and Microbiology, Department of 5
Hydraulic, Maritime and Environmental Engineering, Universitat Politècnica de 6
Catalunya·BarcelonaTech, c/ Jordi Girona 1-3, Building D1, E-08034 Barcelona, Spain 7
8
2 INRA, UR0050, Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, 9
Narbonne F-11100, France 10
11
E-mails addresses: [email protected]; [email protected]; 12
[email protected]; [email protected] 13
14
* Corresponding author: 15
E-mail: [email protected]; Tel.: +34.934016463 16
17
2
Abstract 18
Microalgae have been intensively studied as a source of biomass for replacing conventional 19
fossil fuels in the last decade. The optimization of biomass production, harvesting and 20
downstream processing is necessary for enabling its full-scale application. Regarding 21
biofuels, biogas production is limited by the characteristics of microalgae, in particular the 22
complex cell wall structure of most algae species. Therefore, pretreatment methods have been 23
investigated for microalgae cell wall disruption and biomass solubilization before undergoing 24
anaerobic digestion. This paper summarises the state of the art of different pretreatment 25
techniques used for improving microalgae anaerobic biodegradability. Pretreatments were 26
divided into 4 categories: i) thermal; ii) mechanical; iii) chemical and iv) biological methods. 27
According to experimental results, all of them are effective at increasing biomass 28
solubilisation and methane yield, pretreatment effect being species dependent. Pilot-scale 29
research is still missing and would help evaluating the feasibility of full-scale implementation. 30
31
Keywords: 32
Anaerobic digestion; Bioenergy; Biogas; Biofuel; Methane; Microalgae 33
34
3
1. Introduction 35
The recent interest for microalgae lies in their potential utilization to produce biomass for 36
biofuels and high-value chemicals. Nowadays, algae are mainly produced at large-scale 37
within the human food and animal feed industries to obtain high-value products, such as 38
omega-3 and carotenoids; and also pharmaceutics and cosmetics. Notwithstanding, in the last 39
decade new efforts have been drawn for studying microalgal biofuels. Microalgae were first 40
studied as substrate for biofuel production in the 1950’s. However, investigation on this new 41
energy source was intensively encouraged during the oil crisis of the 1970’s and, in the last 10 42
years great advances on microalgae cultivation in raceway ponds and biofuel production were 43
made. 44
These organisms have been investigated for many biofuel products, such as biodiesel, 45
biogas, bioethanol and bio-hydrogen. According to numerous studies, the main benefits of 46
using algae for bioenergy production are the following: i) they consume less water for growth 47
than terrestrial crops and can be cultivated in saline water, brackish water or wastewater; ii) 48
they can be cultivated in non-arable lands and, therefore, without competing with food crops; 49
iii) they can contribute to carbon dioxide mitigation, since they may grow in high CO2 50
concentrations from coal-fired power stations; and iv) they have a high photosynthetic 51
efficiency compared to terrestrial crops, which may lead to high productivities (Li et al., 52
2008; Wiley et al., 2011). 53
In spite of the aforementioned advantages, biofuel production from microalgae 54
biomass is still unviable at large-scale. For meeting algal biofuels next level, new strategies 55
on cultivation techniques, harvesting methods and downstream processes are required. 56
Accordingly, new approaches should combine technological (such as reactor design and 57
process control) and strain (such as genetic engineering) improvements (Wijffels and 58
Barbosa, 2010). Novel research on the field highlights the algae biorefinery concept, which 59
4
comprises the use of all compounds of algal biomass and aggregates all technological 60
processes from cultivation to final product generation. 61
Most on-going research on microalgae production for biofuel purposes is focused on 62
biodiesel. However, full-scale biodiesel production still has many limitations; for instance the 63
necessity of massive microalgae cultivation and energy consuming technologies for biomass 64
harvesting and processing. As an alternative, biogas generation through anaerobic digestion of 65
microalgal biomass has been pointed out as a more energetically-favourable process (Wiley et 66
al., 2011). In this case, wet biomass can be used and, therefore, no extensive drying methods 67
are required. Indeed, the energy input for operating anaerobic reactors is very low; meaning 68
that net energy production may be more feasible. In respect to infrastructure, anaerobic 69
digestion is a consolidated technology already available for sewage sludge, biowaste and 70
agricultural residues treatment in full-scale facilities. Furthermore, microalgae biodiesel 71
production has been shown viable only linked to anaerobic digestion of residual biomass after 72
lipid extraction (Sialve et al., 2009). 73
74
2. Anaerobic digestion of microalgae 75
Anaerobic digestion consists of organic carbon degradation into organic acids and biogas. 76
Biogas mainly consists of methane (around 65%), which is carbon most reduced state, and 77
carbon dioxide (around 35%), which is its most oxidized state. Other gases (normally less 78
than 1%), such as nitrogen, nitrogen oxides, hydrogen, ammonia and hydrogen sulphide are 79
also formed (Angelidaki and Sanders, 2004). Anaerobic digestion consists in 4 main steps: 80
hydrolysis, acidogenesis, acetogenesis and methanogenesis, leading to the production of 81
biogas, a renewable and versatile bioenergy source used for electricity and heat cogeneration, 82
or if upgraded (> 90% CH4) for biofuel production or natural gas grid injection. Among the 83
5
main advantages of biogas production are the low amount of biomass produced during the 84
process and the potential reuse of the digestate as soil conditioner (Wiley et al., 2011). 85
Anaerobic digestion of microalgal biomass was first studied in the 1950’s (Golueke et 86
al., 1956). In the last decades, two main approaches have been evaluated to produce biogas 87
from microalgae: i) anaerobic digestion of the whole biomass, and ii) anaerobic digestion of 88
lipid extracted biomass for biodiesel production. 89
In the review by Sialve et al. (2009), the theoretical microalgae methane yield was 90
estimated as 0.48-0.80 L CH4/g volatile solids (VS). For instance, Phaeodactylum 91
tricornutum methane yield was expected to be 100% higher (0.27 L CH4/g VS) as compared 92
to Scenedesmus obliquus (0.13 L CH4/g VS) (Zamalloa et al., 2011). Indeed, the methane 93
potential is species-specific (González-Fernández et al., 2011; Mussgnug et al., 2010). In 94
practice, microalgae have lower methane yield in respect to theoretical values. Experimental 95
results have so far been limited to 0.05-0.31 L CH4/g VS (González-Fernández et al., 2011). 96
The anaerobic digestion of Chlorella vulgaris achieved 0.24 L CH4/g VS and 51% COD 97
removal at 28 days hydraulic retention time (HRT) (Ras et al., 2011); while microalgal 98
biomass cultivated in wastewater treatment raceway ponds attained 0.17 L CH4/g VS and 99
31% COD removal at 20 days HRT (Passos et al., 2014). On the other hand, the methane 100
yield of marine microalgae Tetraselmis sp. reached 0.31 L CH4/g VS at 14 days HRT (San 101
Marzano et al., 1982). 102
Such a high variability is related to two main aspects: i) the macromolecular 103
composition, and ii) the cell wall characteristics of each microalgae species. The difference in 104
anaerobic biodegradability due to the macromolecular composition lies on the methane 105
potential of different organic compounds in microalgae cells. Organic matter composition can 106
be converted stoichiometrically into methane for calculating the theoretical methane yield. 107
Thus, lipids (1.014 L/g VS), followed by proteins (0.851 L/g VS) and carbohydrates (0.415 108
6
L/g VS) have the highest theoretical methane yield (Sialve et al., 2009). Indeed, inducing a 109
particular macromolecule accumulation in microalgae cells has proven to successfully 110
increase the methane yield. Research conducted with carbohydrate-enriched cyanobacteria 111
Arthrospira platensis by phosphorus limitation attained a methane yield of 0.203 L/g COD 112
when biomass had 60% of carbohydrates in respect to 0.123 L/g COD when the carbohydrate 113
content was 20% (Markou et al., 2013). According to the authors, carbohydrate-enrichment is 114
a promising technique for improving anaerobic digestion performance, since carbohydrates 115
are accumulated as non-structural storage compounds (e.g. starch) rather than as structural 116
carbohydrate compounds (e.g. cellulose) in the cell wall. Although carbohydrates have a 117
lower methane potential compared to lipids and proteins, when they are present as non-118
structural storage compounds, they may be more readily available to anaerobic bacteria than 119
glycoproteins and lipids forming part of microalgae cell wall structure. Experimental results 120
on microalgae methane yield increase after lipid accumulation are still unavailable, since all 121
research on the technique is focused on biodiesel production. 122
An important issue when dealing with microalgae anaerobic digestion is ammonia 123
toxicity. Since microalgae cells have a high protein content (~ 50-60%), hydrolysis may lead 124
to high ammonium concentration, which might be toxic to methanogens. Mesophilic reactors 125
have shown concentrations below toxicity with HRT of 20-30 days and OLR of 1-2 kg 126
VS/m3·day (Passos and Ferrer, 2014; Passos et al., 2014; Schwede et al., 2013). Nonetheless, 127
this aspect could be more critical under thermophilic conditions. 128
Regarding the cell wall characteristics, it is mostly composed of organic compounds 129
with slow biodegradability and/or bioavailability, such as cellulose and hemicellulose. This 130
tough cell wall hinders the methane production, since organic matter retained in the cytoplasm 131
is not easily accessible to anaerobic bacteria. This is not an isolated case for microalgae, many 132
other organic substrates, such as waste activated sludge and lignocellulosic biomass consist of 133
7
a complex structure, which hampers the hydrolysis rate in the anaerobic digestion process 134
(Carrère et al., 2010; Carlsson et al., 2012; De la Rubia et al., 2013 Hendriks and Zeeman, 135
2009; Monlau et al. 2013). For this reason, pretreatment techniques have been used to 136
solubilize particulate biomass and improve the anaerobic digestion rate and extent. 137
138
3. Pretreatment of microalgae 139
Pretreatment techniques were pointed out as a necessary step for microalgae cell disruption 140
and biogas production by Chen and Oswald (1998). The effectiveness of pretreatment 141
methods on biogas production depends on the characteristics of microalgae, i.e., the toughness 142
and structure of the cell wall, and the macromolecular composition of cells. For instance, 143
Scenedesmus sp. has one of the most resistant cell walls, since it is composed by multilayers 144
of cellulose and hemicellulose on the inside, and sporopolenun and politerpene on the outside 145
(González-Fernández et al., 2011). Microalgae complex cell wall structure confers a 146
resistance to biological attack. In fact, species without cell wall (e.g. Dunaliella sp. and 147
Pavlova_cf sp.) or containing a glycoprotein cell wall (e.g. Clamydomonas sp., Euglena sp. 148
and Tetraselmis sp.) showed higher methane yields than those with a more complex cell wall, 149
containing recalcitrant compounds (e.g. Scenedesmus sp. and Chlorella sp.) (Mussgnug et al., 150
2010; Bohutskyi et al., 2014). Chlorella sp. and Nannochloropsis sp. exhibited the lowest 151
methane yield compared to other microalgae species, which may be due to the polysaccharide 152
component of the cell wall (Bohutskyi et al., 2014), although the wall composition is still 153
poorly understood and can vary widely within a genus (Gerken et al., 2012). 154
Pretreatment methods can be divided into four categories: thermal, mechanical, 155
chemical and biological processes (Fig. 1). Up to date, thermal and mechanical pretreatments 156
are regarded as the most effective for microalgae cell disruption. Thermal pretreatments have 157
been the most widely studied, already in continuous reactors and leading to net energy 158
8
production (Passos and Ferrer, 2014; Schwede et al., 2013). Mechanical pretreatments were 159
less dependent on microalgae species, but required a higher energy input if compared with 160
chemical, thermal and biological methods (Lee et al., 2012). Chemical pretreatments have 161
been proved successful, particularly when combined with heat (Mendez et al., 2014a). 162
However, the use of chemicals may contaminate downstream products. Enzymatic 163
pretreatments seem to improve microalgae hydrolysis (Ehimen et al, 2013), which is 164
promising due to its low energy input. The following sections summarise the state of the art 165
on pretreatment techniques for improving microalgae biogas production. 166
167
3.1 Thermal pretreatments 168
Thermal pretreatments are those where microalgae biomass is solubilized by applying heat. 169
They have long been used for enhancing particulate organic matter disintegration at 170
temperatures from 50 to 270 ºC (Carrère et al., 2010; Hendriks and Zeeman, 2009). However, 171
the optimal temperature range depends on the substrate characteristics. In the case of sewage 172
sludge, temperatures above 180 ºC may lead to the formation of recalcitrant compounds, 173
reducing biomass anaerobic biodegradability (Wilson and Novak, 2009). On the other hand, 174
lignocellullosic biomass starts solubilizing at temperatures above 150-180oC, and only 175
temperatures above 250 oC are to be avoided (Hendriks and Zeeman, 2009). Temperatures 176
from 55 to 170 oC have been applied to increase microalgae methane yield (Alzate et al., 177
2012; González-Fernández et al., 2012a,b; Keymar et al., 2013; Passos et al., 2013a; Passos 178
and Ferrer, 2014; Schwede et al., 2013). In this review, thermal pretreatments are sub-divided 179
into three categories: thermal pretreatment, hydrothermal pretreatment and thermal 180
pretreatment with steam explosion (Table 1). Thermal pretreatment consists of biomass 181
heating at temperatures below 100 ºC under atmospheric pressure. Hydrothermal consists in 182
applying heat at temperatures above 100 ºC, with gradual pressure release after pretreatment. 183
9
Steam explosion consists of a sudden pressure drop after pretreatment at temperatures above 184
100 ºC. Each of them will be described in the following sections. 185
186
3.1.1 Thermal pretreatment 187
Thermal pretreatment at temperatures below 100 ºC and under atmospheric pressure is 188
sometimes referred to as low temperature pretreatment. The main advantage is the low energy 189
demand for biomass heating as compared to high temperature pretreatment. Indeed, energy 190
requirements may be fulfilled using waste heat from cogeneration engines fuelled by biogas. 191
Furthermore, the pretreatment at thermophilic and hyper-thermophilic temperatures (50-70 192
oC) can be applied to promote the activity of thermophilic and hyper-thermophilic bacteria in 193
the first reactor of a two-step process, followed by a mesophilic (35 ºC) or thermophilic (55 194
oC) digester (Lu et al., 2008). 195
Pretreatment performance may be influenced by both temperature and exposure time. 196
Inasmuch, temperature seems to be the most influencing factor on biomass disintegration and 197
anaerobic biodegradability. The first study on microalgae thermal pretreatment already 198
mentioned that temperature was the dominant factor affecting the anaerobic biodegradability 199
in respect to exposure time and biomass concentration, explaining 50% of the pretreatment 200
effectiveness in a model analysis (Chen and Oswald, 1998). Likewise, the solubilization of 201
microalgal biomass grown in wastewater was higher when applying higher temperatures (95-202
75 vs. 55 ºC) than when applying longer exposure times (15 vs. 10h). Indeed, the soluble 203
volatile solids (VSs) per VS ratio (VSs/VS) increased by 4-fold when biomass was pretreated 204
at 55 ºC, 10.6-fold at 75 ºC and 11.8-fold at 95 ºC (Passos et al., 2013a). Microscopic 205
techniques revealed different effects depending on the pretreatment temperature. For instance, 206
Sytox staining of Scenedesmus sp. showed how cells remained intact and alive after 70 oC 207
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pretreatment, while after 90 oC pretreatment biomass was mostly composed of dead cells with 208
damaged membrane (González-Fernández et al., 2012a). 209
Regarding the methane yield (Table 1), results obtained after pretreatment at 50-70 ºC 210
varied a between 13% decrease to 12% increase as compared to non-pretreated microalgae 211
(Alzate et al., 2012; González-Fernández et al., 2012a,b; Cho et al., 2013; Passos et al., 212
2013a). On the other hand, when higher temperatures of 80-100 ºC were applied, microalgae 213
methane yield increased by 60-220% (González-Fernández et al., 2012a,b; Passos et al., 214
2013a). Depending on the species, lower values were attained, like 14% increase after 215
Chlorella sp. and Scenedesmus sp. pretreatment at 80 ºC (Cho et al., 2013). The variability on 216
the methane yield increase may be attributed to the pretreatment temperature, exposure time, 217
inoculum acclimation and microalgae species. Indeed, experimental results suggest that at 218
relatively low temperatures (< 70 ºC) organic matter solubilisation may increase due to 219
exopolymers and other extracellular compounds; while at high temperatures (> 80 o
C) it could 220
be due to cell disruption and release of intracellular macromolecules to the soluble phase 221
(González-Fernández et al., 2012a; Passos and Ferrer, 2014). However, the optimal 222
temperature depends on the microalgae species investigated. For instance, a previous study 223
showed how Stigeoclonium sp. was not digested without pretreatment, while it was damaged 224
and partly disrupted after thermal pretreatment, and degraded after anaerobic digestion. On 225
the other hand, the diatom Nitzschia sp. was not digested even after pretreatment (Passos and 226
Ferrer, 2014). 227
228
3.1.2 Hydrothermal pretreatment 229
Hydrothermal pretreatment is applied at temperatures higher than 100 ºC, with gradual 230
pressure release after pretreatment. Under this temperature range, generally shorter times (15-231
30 min) are used in comparison with low temperature pretreatment (3-24 hours). For example, 232
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the methane yield increased by 60% after pretreatment at 80-100 oC for 3-10 hours 233
(González-Fernández et al., 2012a; Passos et al., 2013a), while it increased by 60-120% after 234
pretreatment at 120-140 oC for 15-30 minutes (Alzate et al., 2012; Cho et al., 2013). Longer 235
exposure time was assayed with Nannochloropsis salina (100-120 ºC for 2 hours) increasing 236
the methane yield from 0.13 to 0.27 L CH4/g VS (108% increase) in continuous anaerobic 237
reactors. Furthermore, transmission electronic microscopic (TEM) images indicated that 238
microalgae cells were partly damaged after pretreatment (Schwede et al., 2013). Similarly, 239
microalgal biomass mainly composed by Oocystis sp. was pretreated at 130 ºC for 15 min, 240
reaching a methane yield of 0.17 L CH4/g VS, 42% higher than the control. Furthermore, 241
TEM images showed that the cell wall external layer was disrupted after hydrothermal 242
pretreatment (Passos and Ferrer, submitted). In both studies, microalgae cell wall disruption 243
increased soluble organic matter concentration, enhancing the hydrolysis and methane 244
production. 245
246
3.1.3 Thermal pretreatment with steam explosion 247
Pretreatment pressure increases along with temperature, especially above 160 oC. After 248
pretreatment, pressure can be released gradually or rapidly; the sudden pressure drop to 249
ambient conditions is defined as steam explosion. Thermal pretreatment with steam explosion 250
is industrially known as thermal hydrolysis, where biomass is placed in a vessel and steam is 251
applied at high temperature (~ 160 oC) and pressure (~ 6 bars) for a few minutes (10-30 252
minutes); afterwards, steam is flashed and biomass is quickly cooled down in another vessel 253
(Keymer et al., 2013; Hendriks and Zeeman, 2009). The sudden pressure drop leads to cell 254
wall rupture and biomass disintegration. This technology is already available in full-scale 255
wastewater treatment plants prior to sludge anaerobic digestion, increasing by 50-100% the 256
biogas production (Kepp et al., 2000). As for microalgae, it is under investigation at 257
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laboratory-scale and it has only been tested in BMP tests. High temperature with steam 258
explosion at 140-180 oC and 3-10 bars enhanced organic matter solubilization, particularly 259
carbohydrates, which increased by a factor of 4-6, and proteins, which increased by a factor of 260
1-2 (Mendez et al., 2014b). In terms of methane yield, increases between 40 and 80% were 261
attained for different microalgae species (Table 1). 262
263
3.2 Mechanical pretreatments 264
Mechanical pretreatments act by directly breaking cells through a physical force. Most studies 265
using mechanical methods have been used for improving lipid extraction for biodiesel 266
production. In such cases, mechanical methods are usually preferred, since they are less 267
dependent on microalgae species and less likely to contaminate the lipid product, in 268
comparison with chemical pretreatments (Lee et al., 2012). However, the main disadvantage 269
is high electricity consumption. Regarding biogas production, ultrasounds and microwave 270
pretreatments have already shown positive results (Table 2). 271
272
3.2.1 Ultrasound 273
Ultrasounds consist in rapid compression and decompression cycles of sonic waves. The 274
continuing cycles generate cavitation, which is the formation of regions with liquid vapor 275
inside the cell, so-called microbubbles. These microbubbles are formed by the movement of 276
liquid molecules by acoustic waves. Depending mainly on the ultrasound intensity, they are 277
compressed to their minimum and they implode, producing heat, free radicals, high pressure, 278
shockwaves and, finally, damaging the cell wall (Kim et al., 2013). 279
Ultrasound can be applied at low (< 50 kHz) and high (> 50 kHz) frequencies. Low 280
frequencies favour mechanical effects, while high frequencies favour the formation of free 281
radicals. Once frequency is fixed, controllable parameters influencing ultrasound pretreatment 282
13
are output power and exposure time. The specific energy is calculated as the applied power 283
times the exposure time, divided by the biomass concentration (i.e. total solids). Therefore, 284
biomass concentration may also influence the pretreatment effect. Biomass concentration 285
should be high enough for increasing the probability of contact between cells; but not as high 286
as to increase biomass viscosity (Lee et al., 2012; Kim et al., 2013). Temperature may also 287
play an important role, since it affects the vapour pressure inside the cell; the lower the 288
temperature, the lower the pressure and effectiveness of pretreatment (Lee et al., 2012; Kim et 289
al., 2013). 290
Ultrasound pretreatment promotes microalgae cell wall disruption and organic matter 291
solubilization; however the results depend on the microalgae species and pretreatment 292
conditions, namely the applied specific energy. Indeed, the methane yield increase did not 293
exceed 20% with an applied specific energy below 75 MJ/kg TS, (Alzate et al., 2012; 294
Gonzalez-Fernandez et al., 2012b); whereas it increased by 80-90% with an applied specific 295
energy of 100-200 MJ/kg TS (Gonzalez-Fernandez et al., 2012b). For microalgal biomass 296
grown in wastewater treatment open ponds, a linear correlation was found between the 297
applied specific energy and methane yield increase, reaching 33% increase after pretreatment 298
at 67 MJ/kg TS (Passos et al., in press). Although experimental results indicate that the higher 299
the specific energy, the higher the methane yield increase, the high electricity demand for 300
ultrasonication may unbalance the energy input and output; especially for experimental scale 301
devices, which generally have poor energy efficiency in comparison with pilot and full-scale 302
equipment. For instance, with an ultrasound device pretreating sewage sludge (6 kWh/m3) 303
while it was fed to a pilot digester (100 L), a positive energy balance was attained despite the 304
TS concentration; with a net energy generation of 3-10 kW per kW of energy consumed 305
(Perez-Elvira et al., 2009). 306
307
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3.2.2 Microwave 308
Microwaves are short waves of electromagnetic energy varying in frequency from 300 MHz 309
to 300 GHz. Generally, microwave frequencies are around 2450 MHz. It is a consequence of 310
the rapidly oscillating electric field of a polar or dielectric material, which induces heat by the 311
frictional forces of molecules in movement. The increase of kinetic energy leads water to a 312
boiling state. The quantum energy applied by microwave irradiation is not capable of breaking 313
down chemical bonds; however hydrogen bonds are or can be broken. In this manner, 314
induction heating and dielectric polarization result in changes in the secondary and tertiary 315
structure of proteins (Kaatze, 1995). 316
Similarly to ultrasounds, the main controllable parameters influencing microwave 317
pretreatment are output power and exposure time (i.e. applied energy). Irradiation quick 318
penetration into biomass is the main advantage of this technique (Kim et al., 2013), while the 319
main disadvantage is high electricity consumption, which depends on the biomass 320
concentration. Temperature may also be an important parameter, since biomass is heated by 321
water molecules movement. 322
Experimental results indicated that pretreatment effect on biomass solubilisation and 323
methane increased with the applied specific energy, regardless of the output power and 324
exposure time. In BMP tests, optimal pretreatment conditions corresponded to the highest 325
applied specific energy (65.4 MJ/kg TS), with 8% biomass solubilisation and 78% methane 326
yield increase (Passos et al., 2013b). In continuous reactors operating at 20 days HRT, the 327
methane yield was 60% higher after microwave pretreatment (0.27 L CH4/g VS) compared to 328
the control (0.17 L CH4/g VS) (Passos et al., 2014). Furthermore, optic microscope and 329
transmission electronic microscope (TEM) images revealed that some cell walls were 330
disrupted after the pretreatment step; yet others remained intact. Nevertheless, cell organelles 331
were damaged beyond repair, which most probably improved the anaerobic biodegradability, 332
15
since intracellular constituents were more readily available to anaerobic bacteria (Passos et 333
al., 2014). 334
335
3.3 Chemical pretreatments 336
Chemical pretreatments have been by far less used than thermal and mechanical ones. Among 337
chemical methods, mostly alkali pretreatments have been applied to microalgae, often 338
combined with heat. Acid and alkali reagents are commonly used to solubilize polymers, 339
favouring the availability of organic compounds for enzymatic attacks (Bohutskyi and 340
Bouwer, 2013). The small amount of residual alkali remaining in pretreated biomass may be 341
helpful to prevent pH reduction during subsequent acidogenesis step. However, it should be 342
mentioned that some of the solubilized compounds may induce the formation of potentially 343
toxic by-products for methanogens. 344
The effect of thermochemical pretreatment with alkali and acid addition was studied 345
for Chlorella vulgaris samples. Results showed that thermal pretreatment at 120 ºC for 40 346
minutes without chemical addition attained the highest methane yield increase (93%). 347
Nevertheless, the thermo-alkali pretreatment with NaOH resulted in higher carbohydrates and 348
protein solubilisation increase (7- and 2-fold, respectively), compared to the solubilisation 349
increase after the thermal pretreatment (4.5-fold and 8%, respectively). The explanation for 350
the higher solubilisation, but lower methane yield increase after thermo-alkali pretreatment, 351
was the release of unidentified side-products, which may have hindered the anaerobic 352
biodegradability (Mendez et al., 2014a). 353
Similarly, 5 microalgae species (Chlorella sp., Nannochloropsis sp., Thalassiosira 354
weissflogii, Tetraselmis sp., and Pavlova_cf sp. CCMP459) were pretreated with different 355
NaOH concentrations (from 0 to 21 g/L). According to the results, alkali pretreatment was 356
ineffective, while thermochemical pretreatment increased 30-40% the methane yield by of 357
16
Chlorella sp. and Nannochloropsis sp. (Bohutskyi et al., 2014). Moreover, alkali pretreatment 358
of Chlorella sp. and Scenedesmus sp. mixed biomass had negative results with pH of 11 and 359
13, while it increased the methane yield by 8% with pH of 9 (Cho et al., 2013). 360
In summary, results on chemical pretreatments are scarce and sometimes 361
contradictory. Even so, the combination of thermal and chemical pretreatments seems more 362
promising than chemical ones. 363
364
3.4 Biological pretreatments 365
Enhancing the enzymatic hydrolysis of microalgae cell walls and other biopolymers is a 366
promising alternative to energy-consuming pretreatments (Bohutskyi and Bouwer, 2013). 367
Moreover, enzymatic pretreatment does not involve inhibitory compounds. Hydrolytic 368
enzymes convert compounds of microalgae cell wall, such as cellulose and hemicellulose, to 369
compounds with lower molecular weight, which are more readily available for anaerobic 370
bacteria. The most important parameters influencing the pretreatment effect are enzyme dose, 371
temperature and exposure time. Pretreament temperature and pH are set within the optimal 372
activity range of each enzyme. However, the enzyme to substrate specificity, the enormous 373
diversity of microalgae cell wall composition and structure, and the enzyme production costs 374
are among the major drawbacks that need to be addressed before applying this technology in 375
the biofuel industry. 376
Up to date, literature in this field is very scarce. The enzyme endo-b-1,4-glucanase 377
from Cellulomonas sp. YJ5 hydrolyzed Chlorella sorokiniana cell wall and caused cell lysis 378
after 60–180 min of treatment (Fu et al., 2010). Likewise, a combined mechanical blending 379
(<0.1 mm length) and enzymatic pretreatment (a-amylase, protease, lipase, xylanase (endo-380
1,4-), cellulase complex) was applied to Rhizoclonium sp. biomass. The combined enzymatic 381
pretreatment improved the methane yield over 20% as compared to the mechanical 382
17
pretreatment alone. Among single enzyme pretreatments, cellulase showed the best results 383
(0.133 L CH4/g TS); however the combination of all enzymes reached the highest methane 384
yield (0.145 L CH4/g TS) (Ehimen et al., 2013). This is due to chain behaviour, where the 385
hydrolysis of one compound enhanced the bioavailability of another one, which may be 386
subsequently hydrolysed. 387
388
4. Comparison between pretreatment methods 389
Comparing pretreatment techniques is not an easy task, since most research has been 390
conducted with different microalgae species and under different conditions. However, some 391
authors applied different pretreatment techniques to the same microalgae species. For 392
instance, Alzate et al. (2012) compared thermal pretreatment at 55, 110, 140 and 170 ºC and 393
ultrasound pretreatment at 10, 27, 40 and 57 MJ/kg TS effects on three mixtures of microalgal 394
biomass. Thermal pretreatment with steam explosion (170 ºC and 6.4 bars) was the most 395
effective method for biomass composed by Clamydomonas sp., Scenedesmus sp. and 396
Nannocloropsis sp. and for biomass composed by Acutodesmus obliquus and Oocystis sp.; 397
achieving a methane yield increase of 46% and 57%, respectively. However, a different 398
behaviour was found for biomass composed by Microspora sp., which attained the highest 399
increase after hydrothermal pretreatment at 110 ºC and 1 bar (62% increase). This was 400
attributed to the formation of recalcitrant compounds in Maillard reactions. In all cases, 401
thermal pretreatment at 55 ºC and ultrasound pretreatment showed a low methane yield 402
increase (Alzate et al., 2012). Cho et al. (2013) compared thermal (50 and 80 ºC), 403
hydrothermal (120 ºC), ultrasound (39, 117 and 234 MJ/kg VS) and alkali pretreatment with 404
NaOH (9, 11 and 13 pH) on Chlorella sp. and Scenedesmus sp. biomass. In this case, 405
hydrothermal pretreatment at 120 ºC for 30 min was the best method, reaching a methane 406
yield increase of 20.5%.González-Fernández et al. (2012b) compared ultrasound and thermal 407
18
pretreatment of Scenedesmus biomass, observing the highest methane yield increase after 408
ultrasound pretreatment with a specific energy higher than 100 MJ/kg TS (75-88%). 409
The relation between organic matter solubilisation and methane yield increase after 410
different pretreatments is represented in Fig. 2. Since the effect of pretreatments on 411
microalgae anaerobic digestion depends not only on the pretreatment conditions, but also on 412
the species, the comparison included only mixed microalgal biomass and is shown separately 413
for each pretreatment technique (Alzate et al., 2012; Cho et al., 2013; Passos et al., 2013a; 414
Passos et al., 2013b; Passos et al., in press; Passos and Ferrer, submitted). As can be observed, 415
for microwave pretreatment the relation between solubilisation and methane yield increase 416
showed a steep trend, which means that with low solubilisation (10%) the methane yield 417
increase was already very high (80%). It was followed by thermal pretreatment, where a 418
solubilisation of 20% led to a methane yield increase around 60%. For hydrothermal 419
pretreatment and thermal pretreatment with steam explosion, for a solubilisation between 20 420
and 60% a methane yield increase of 60% was attained. Finally, ultrasound pretreatment 421
showed an almost constant trend between solubilisation and methane yield increase, with 422
solubilisation of 80-100% and 20-30% methane yield increase. 423
As shown in Fig. 2, each pretreatment technique has a different effect on microalgae 424
solubilisation, and consequently, on the anaerobic biodegradability. This is due to the 425
mechanisms taking place in each pretreatment method, which differ in the impact on 426
microalgae cell structure and in the bioavailability of different organic compounds. When the 427
pretreatment technique increases biomass solubilisation, but methane yield remains similar to 428
non-pretreated biomass, one possible explanation is the formation of recalcitrant compounds 429
in the hydrolysis step, which hinders the anaerobic digestion process. On the other hand, 430
when the pretreatment technique results in low solubilisation degree, but high methane yield, 431
19
non-soluble organic compounds may have been modified and possibly are more available to 432
anaerobic bacteria. 433
The main pros and cons of microalgae pretreatment methods are summarised in Table 434
3. As can be seen, thermal pretreatment seems effective at increasing biogas production, while 435
energy demand is low compared to mechanical ones. Nevertheless, biomass thickening or 436
dewatering is crucial. Scalability may be a handicap for microwave pretreatment. Regarding 437
thermo-chemical pretreatment, studies have shown positive results on microalgae 438
biodegradability increase; however further studies should evaluate the risk of contamination 439
in continuous bench and pilot-scale reactors. 440
On the whole, the comparison of pretreatment methods is more reliable when using the 441
same microalgal biomass, since pretreatment effects are species-specific and difficult to 442
extrapolate. In addition, microscopic analysis of pretreated biomass would help understanding 443
the effect of each pretreatment on the cell structure. 444
445
5. Energy assessment 446
Laboratory-scale research has shown how pretreatments improve microalgae anaerobic 447
biodegradability and biogas production. However, energy and environmental aspects are 448
crucial for full-scale implementation of the process. Since input energy for the pretreatment 449
step accounts for the highest environmental impact, optimal pretreatment conditions must 450
balance the biogas production increase with energy consumption (Caballa et al., 2011). In this 451
review, the energy assessment of pretreatment methods was calculated by comparing the 452
energy input required for the pretreatment step and the energy output from the increment in 453
biogas production. Therefore, the extra energy produced should at least cover the energy 454
requirement for the pretreatment step. Results may be expressed in two ways: as the energy 455
balance between the energy output (Eo) and energy input (Ei) (Eo - Ei), or as the energy ratio 456
20
between the Eo and Ei (Eo / Ei). In this way, a positive energy balance or an energy ratio higher 457
than 1 indicate net energy production. 458
In regards to microalgae pretreatment, mechanical techniques are very effective at 459
disrupting cell wall and improving biofuel production, but also highly energy inefficient 460
(Carlsson et al., 2012). Based on lab-scale experiments, mechanical pretreatments result in 461
negative energy balances, partly due to the low solids concentration in pretreated biomass 462
(Cho et al., 2013; Passos et al., 2014). On the other hand, thermal pretreatments result in 463
positive energy balances (Schwede et al., 2013; Passos and Ferrer, 2014). For the sake of 464
comparison, energy ratios (Eo/Ei) were here calculated for microalgae anaerobic digestion in 465
continuous reactors following thermal and mechanical (i.e. microwave) pretreatments based 466
on experimental results (Table 4). Other pretreatment techniques could not be considered due 467
to the lack of experimental data from continuous digesters. 468
The energy input for the thermal pretreatment was calculated as the amount of heat 469
required to raise microalgal biomass temperature from ambient conditions (Ta) to 470
pretreatment temperature (Tp), considered as the main energy consumption in this case. Ta 471
was defined as 20 ºC and Tp as 75, 95, 100, 120 or 130 ºC (according to experimental 472
conditions) (Chen and Oswald, 1998; Schwede et al., 2013; Passos and Ferrer, 2014; Passos 473
and Ferrer, submitted). Since the reactor was operated under mesophilic conditions (35 ºC), 474
no extra energy was needed to heat biomass from pretreatment to digestion temperature (Td). 475
In fact, heat could be recovered when cooling down biomass from pretreatment to digestion 476
temperature by means of a heat exchanger, with an efficiency ϕ of 85% (Lu et al., 2008). 477
Therefore, input heat was calculated as the energy required to raise influent biomass 478
temperature from ambient to pretreatment temperature, but subtracting the energy recovered 479
by cooling down biomass from pretreatment to digestion temperature (Eq. 1). In order to 480
21
normalize the results, the energy input was divided by the volatile solids (VS) content of 481
pretreated biomass (g VS). 482
Ei = [(ρ V γ (Tp - Ta)) – (ρ V γ (Tp - Td) ϕ)] / g VS (1) 483
where: Ei: energy input (kJ/g VS); �: density (kg/L); V: biomass volume (L); �: specific heat (kJ/kg·ºC); Tp: 484
pretreatment temperature (75, 95, 100, 120 or 130 ºC); Td: anaerobic digestion temperature (35 ºC) and Ta: 485
ambient temperature (20 ºC). 486
The energy input for microwave pretreatment was determined by the applied energy 487
under the studied conditions, i.e. 900 W and 3 min (Passos et al., 2014). Again, the energy 488
input was divided by the VS content for normalizing the results (Eq. 2). 489
Ei = Pt / g VS (2) 490
where: Ei: energy input (kJ/g VS); P: output power (kW) and t: exposure time (s). 491
The energy output was always calculated from the difference between the methane 492
yield of pretreated and non-pretreated microalgae (Eq. 3). The lower heating value of methane 493
(ξ) was defined as 35800 kJ/m3 CH4 (Metcalf and Eddy, 2003). An efficiency of 90% on 494
energy conversion was considered (η). 495
Eo = ∆YCH4 ξ η (3) 496
where: Eo: energy output (kJ/g VS); ∆YCH4: difference between the methane yield of pretreated and non-497
pretreated microalgal biomass (m3 CH4/g VS); ξ: lower heating value of methane (35800kJ/m
3 CH4) and η: : 498
energy conversion efficiency (90%). 499
As can be observed (Table 4), only hydrothermal pretreatment of dewatered 500
microalgae attained net energy production (Eo/Ei 7.78). For the other cases, Eo/Ei ratio was 501
below 1. For hydrothermal pretreatment Eo/Ei ranged between 0.19 and 7.78, for thermal 502
pretreatment Eo/Ei between 0.56 and 0.59, and for microwave pretreatment Eo/Ei it was 0.05. 503
As can be seen, microwave pretreatment required much more energy (i.e. electricity) than the 504
extra energy produced. As already mentioned, the highest energy ratio was obtained for the 505
hydrothermal pretreatment of dewatered microalgae, meaning that the energy output from the 506
22
extra biogas production was 7.78 times higher than the energy input (i.e. heat) for the 507
pretreatment. The reason for this is the total solids (TS) concentration of Nannocloropsis 508
salina, which was dewatered with a centrifuge reaching between 16 and 30% TS during the 509
studied period (Schwede et al., 2013). To some extent, the energy ratio would decrease if the 510
energy required for centrifugation was also accounted for. The water content in pretreated 511
biomass increases energy consumption with respect to energy production per reactor volume, 512
decreasing the energy balance of the process (Passos et al., 2014). 513
Therefore, the energy ratio was recalculated for each pretreatment considering a range 514
of VS concentration (Fig. 3). Again, thermal pretreatment outcompeted electricity consuming 515
mechanical pretreatment. For microwave pretreatment, the energy ratio was below 1 even 516
with a VS concentration of 200 g/L, which is unrealistic at full-scale. On the other hand, for 517
thermal pretreatment energy ratios were always higher than 1, showing an increasing trend 518
with the concentration of VS. For a concentration of 5% VS, Eo/Ei values were between 1.17 519
and 1.94 for hydrothermal pretreatment and between 4.45 and 5.61 for thermal pretreatment. 520
In most full-scale anaerobic digestion plants, biogas is valorised through cogeneration 521
to produce electricity and heat. In some cases, heat is produced in excess and, therefore, it 522
may be consumed for pretreatment. For instance, the energy assessment of a sewage sludge 523
treatment plant (100,000 population equivalent) with cogeneration from biogas showed that 524
the main factors influencing the energy balance were heat recovery from hot steam and sludge 525
concentration. In this case, the required TS concentration was 8%. Sludge pretreatment at 526
high temperature with steam explosion improved biogas production by 24 and 33% at 17 and 527
9 days HRT, respectively; while economic benefits were 87,600 and 132,373 €/year, 528
respectively (Perez-Elvira and Fdz-Polanco, 2012). 529
To summarise, the scalability of pretreatments relies on the energy balance of the 530
process. In this respect, thermal pretreatments seem more feasible than mechanical 531
23
techniques, since energy consumption for biomass heating is much lower than that of electric 532
devices. For the latter case, biomass thickening seems imperative for reaching net energy 533
production. 534
535
6. Conclusions and Recommendations 536
To date, microalgae pretreatment for biogas production has not been as extensively studied as 537
other organic substrates, like sewage sludge and lignocelluosic biomass. Few pretreatment 538
methods have been explored, most of them only in BMP tests. The main parameters used for 539
evaluating pretreatment efficiency are biomass solubilisation and methane yield increase; in 540
this manner the mechanisms taking place and how they affect the cell wall structure are not 541
yet well understood. Research efforts should focus on understanding pretreatment 542
mechanisms and should focus on investigating pretreatment techniques in pilot-scale reactors 543
to obtain more reliable information on the scalability of the technology. 544
545
Acknowledgements 546
The authors want to thank the Spanish Ministry of Economy and Competitiveness for 547
financial support to this project (BIOALGAS CTM2010-17846). Fabiana Passos appreciates 548
her PhD scholarship funded by the Coordination for the Improvement of Higher Level 549
Personal (CAPES) from the Brazilian Ministry of Education. 550
551
References 552
1. Alzate, M. E., Muñoz, R., Rogalla, F., Fdz-Polanco, F., Perez-Elvira, S. I., 2012. Biochemical methane 553
potential of microalgae: Influence of substrate to inoculum ratio, biomass concentration and pretreatment. 554
Bioresour. Technol. 123, 488-494. 555
24
2. Alzate, M. E., Muñoz, R., Rogalla, F., Fdz-Polanco, F., Perez-Elvira, S. I., 2014. Biochemical methane 556
potential of microalgae biomass after lipid extraction. Chem. Eng. Journal 243, 405-410. 557
3. Angelidaki, I., Sanders, W., 2004. Assessment of the anaerobic digestion biodegradability of macropollutants. 558
Reviews Environ. Sci. Biotechnol. 3, 114-129. 559
4. Bohutskyi, P., Bouwer, E., 2013. Biogas Production from Algae and CyanobacteriaThrough Anaerobic 560
Digestion: A Review, Analysis, and Research Needs. in: Advanced Biofuels and Bioproducts, (Ed.) J.W. Lee, 561
Springer New York, pp. 873- 975. 562
5. Bohutskyi, P., Betenbaugh, M.J., Bouwer, E.J., 2014. The effects of alternative pretreatment strategies on 563
anaerobic digestion and methane production from different algal strains. Biores. Technol. 155, 366-372. 564
6. Carballa, M., Duran, C., Hospido, A., 2011. Should we pretreat solid waste prior to anaerobic digestion? An 565
assessment of its environmental cost. Environ. Sci. Technol. 45, 10306-10314. 566
7. Carlsson, M., Lagerkvist, A., Morgan-Sagastume, F., 2012. The effects of substrate pre-treatment on 567
anaerobic digestion systems: A review. Waste Manag. 32, 1634-1650. 568
8. Carrère, H., Dumas, C., Battimelli, A., Batstone, D. J., Delgenes, J. P., Steyer, J. P., Ferrer, I., 2010. 569
Pretreament methods to improve sludge anaerobic degradability: A review. J. Hazard. Mat. 183, 1-15. 570
9. Chen, P. H., Oswald, W. J., 1998.Thermochemical treatment for algal fermentation. Environ Int. 24, 889-897. 571
10. Cho, S., Park, S., Seon, J., Yu, J., Lee, T., 2013. Evaluation of thermal, ultrasonic and alkali pretreatments on 572
mixed-microalgal biomass to enhance anaerobic methane production. Bioresour. Technol. 143, 330-336. 573
11. De la Rubia, M. A., Fernández-Cegrí, V., Raposo, F., Borja, R., 2013. Anaerobic digestion of sunflower oil 574
cake: A current overview. Wat. Sci. Technol. 67, 410-417. 575
11. Ehimen, E. A., Sun, Z. F., Carrington, C. G., Birch, E. J., Eaton-Rye, J. J., 2011. Anaerobic digestion of 576
microalgae residues resulting from the biodiesel production process. Appl. Energy 88, 3454-3463. 577
12. Ehimen, E. A., Holm-Nielsen, J. B., Poulsen, J. B., Boelsmand, J. E., 2013. Influence of different pre-578
treatment routes on the anaerobic digestion of a filamentous algae. Renewable Energy 50, 476-480. 579
25
13. Fu, C.C., Hung, T.C., Chen, J.Y., Su, C.H., Wu, W.T., 2010. Hydrolysis of microalgae cell walls for 580
production of reducing sugar and lipid extraction. Bioresour Technol 101(22):8750–8754. 581
14. Golueke, C. G., Oswald, W. J., Gotaas, H. B., 1956. Anaerobic digestion of algae. Appl. Microbiol., 47-55. 582
15. González-Fernández, C., Sialve, B., Bernet, N., Steyer, J. P., 2011. Impact of microalgae characteristics on 583
their conversion to biofuel. Part II: Focus on biomethane production. Biofuels, Bioproducts and Biorefining 6, 584
205-218. 585
16. González-Fernández, C., Sialve, B., Bernet, N., Steyer, J. P., 2012a. Thermal pretreatment to improve 586
methane production of Scenedesmus biomass. Biomass and Bioenergy 40, 105-111. 587
17. González-Fernández, C., Sialve, B., Bernet, N., Steyer, J. P., 2012b. Comparison of ultrasound and thermal 588
pretreatment of Scenedesmus biomass on methane production. Bioresour. Technol. 110, 610-616. 589
18. Hendriks, A. T. W. M., Zeeman, G., 2009. Pretreaments to enhance the digestibility of lignocellulosic 590
biomass. Bioresour. Technol. 100, 10-18. 591
19. Kaatze, U.D.O., 1995. Fundamentals of microwaves. Radiation Physics and Chemistry 45, 539–548. 592
20. Keymar, P., Ruffell, I., Pratt, S., Lant, P., 2013. High pressure thermal hydrolysis as pre-treatment to 593
increase the methane yield during anaerobic digestion of microalgae. Bioresour. Technol. 131, 128-133. 594
21. Kepp, U., Machenbach, I., Weisz, N., Solheim, O. E., 2000. Enhanced stabilisation of sewage sludge through 595
thermal hydrolysis – three years of experience with full-scale plant. Wat. Sci. Technol. 42, 89-96 596
22. Kim, J., Yoo, G., Lee, H., Lim, J., Kim, K., Kim, C. H., Park, M. S., Yang, J-W., 2013. Methods of 597
downstream processing for the production of biodiesel from microalgae. Biotechnol. Adv. 31, 862-876. 598
23. Lee, A. K., Lewis, D. A., Ashman, P. J., 2012. Disruption of microalgal cells for the extraction of lipids for 599
biofuels: Processes and specific energy requirements. Biomass and Bioenergy 46, 89-101. 600
24. Li, Y., Horsman, M., Wu, N., Lan, C. Q., Dubois-Calero, N., 2008. Biofuels from microalgae. Biotehnol. 601
Prog. 24, 815-820. 602
25. Lu, J., Gavala, H.N., Skiadas, I.V., Mladenovska, Z., Ahring, B.K., 2008. Improving anaerobic sewage 603
26
sludge digestion by implementation of a hyperthermophilic prehydrolisis step. J. Environ. Manag. 88, 881-889. 604
26. Markou, G., Angelidaki, I., Georgakakis, D., 2013. Carbohydrate-enriched cyanobacterial biomass as 605
feedstock for bio-methane production through anaerobic digestion. Fuel 111, 872-879. 606
27. Mendez, L. Mahdy, A., Timmers, R. A., Ballesteros, M., González-Fernández, C., 2014a. Enhancing 607
methane production of Chlorella vulgaris via thermochemical pretreatments. Bioresource Technology 149, 136-608
141. 609
28. Mendez, L., Mahdy, A., Demuez, M., Ballesteros, M., González-Fernández, C., 2014b. Effect of high 610
pressure thermal pretreatment on Chlorella vulgaris biomass: Organic matter solubilisation and biochemical 611
methane potential. Fuel 117, 674-679. 612
29. Metcalf & Eddy, Tchobanoglous, G., Burton, F. L., Stensel, H. D., 2003. Wastewater Engineering, 613
Treatment and Reuse, 4th Edition, McGraw Hill Education. 614
30. Monlau F, Barakat A, Trably E, Dumas C, Steyer J P, Carrere H., 2013. Lignocellulosic materials into 615
biohydrogen and biomethane: impact of structural features and pretreatment. Crit Rev Env Sci Tech 43(3), 260-616
322 617
31. Mussgnug, J. H., Klassen, V., Schüter, A., Kruse, O., 2010. Microalgae as substrates for fermentative biogas 618
production in a combined biorefinery concept. J. of Biotechnol. 150, 51-56. 619
32. Park, K. Y., Kweon, J., Chantrasakdakul, P., Lee, K., Cha, H. Y., 2013. Anaerobic digestion of microalgal 620
biomass with ultrasonic disintegration. Int. Biodeter.Biodegrad. 85, 598-602. 621
33. Passos, F., Garcia, J., Ferrer, I., 2013a. Impact of low temperature on the anaerobic digestion of microalgal 622
biomass. Bioresour. Technol. 138, 79-86. 623
34. Passos, F., Sole, M., Garcia, J., Ferrer, I., 2013b. Biogas production from microalgae grown in wastewater: 624
Effect of microwave pretreatment. Appl. Energy 108, 168-175. 625
35. Passos, F., Hernandez-Marine, M., Garcia, J., Ferrer, I., 2014. Long-term anaerobic digestion of microalgae 626
grown in HRAP for wastewater treatment. Effect of microwave pretreatment. Water Res. 49, 351-359. 627
27
36. Passos, F., Ferrer, I., 2014. Microalgae conversion to biogas: thermal pretreatment contribution on net energy 628
production. Environmental Science and Technology 48, 7171-7178. 629
37. Passos, F., Astals, S., Ferrer, I., in press. Anaerobic digestion of microalgal biomass after ultrasound 630
pretreatment. Waste Management. 631
38. Passos, F., Ferrer, I., submitted. Influence of hydrothermal pretreatment on microalgal biomass anaerobic 632
digestion and bioenergy production. Water Research. 633
39. Perez-Elvira, S. I., Fdz-Polanco, M., Plaza, F. I., Garralón, G., Fdz-Polanco, F., 2009. Ultrasonic pre-634
treatment for anaerobic digestion improvement. Wat. Sci. Technol. 60, 1525-1532. 635
40. Perez-Elvira, S. I., Fdz-Polanco, F., 2012. Continuous thermal hydrolysis and anaerobic digestion of sludge. 636
Energy integration study. Wat. Sci. Technol. 65, 1839-1846 637
41. Ramon-Suárez, J. L., Carreras, N. 2014. Use of microalgae residues for biogas production. Chemical 638
Engineering Journal 242, 86–95. 639
42. Ras, M., Lardon, L., Sialve, B., Bernet, N., Steyer, J. P., 2011. Experimental study on a coupled process of 640
production and anaerobic digestion of Chlorella vulgaris. Bioresour. Technol. 102, 200-206. 641
43. San Marzano A., C., Legros, A., Naveau, H., Nyns, E., 1982. Biomethanation of the marine algae 642
Tetraselmis. International Journal of Sustainable Energy 1, 263-272. 643
44. Schwede, S., Rehman, Z-U., Gerber, M., Theiss, C., Span, R., 2013. Effects of thermal pretreatment on 644
anaerobic digestion of Nannocloropsis salina biomass. Bioresour. Technol. 143, 505-511 645
45. Sialve, B., Bernet, N., Bernard, O., 2009. Anaerobic digestion of microalgae as a necessary step to make 646
microalgal biodiesel sustainable. Biotechnol. Adv. 27, 409-416. 647
46. Wijffels, R. H., Barbosa, M. J., 2010. An outlook on microalgal biofuels. Science 329, 796-799. 648
47. Wilson, C. A., Novak, J. T., 2009. Hydrolysis of macromolecular components of primary and secondary 649
wastewater sludge by thermal hydrolytic pretreatment. Water Res. 43, 4489-4498. 650
48. Wiley, P. E., Campbell, J. E., McKuin, B., 2011. Production of biodiesel and biogas from algae: A review of 651
process train options. Water Environ. Res. 83, 326-338. 652
28
49. Zamalloa, C., Vulsteke, E., Albrecht, J., Verstraete, W., 2011. The techno-economic potential of renewable 653
energy through the anaerobic digestion of microalgae. Bioresour. Technol. 102, 1149-1158. 654
29
Figure 1. Pretreatments for improving microalgae biogas production.
30
Organic matter solubilisation (%)
0 20 40 60 80 100 120
Me
tha
ne
yie
ld in
cre
ase
(%
)
-20
0
20
40
60
80
100
120
Thermal pretreatment
Hydrothermal pretreatment
Thermal pretreatment with steam explosion
Ultrasound pretreatment
Microwave pretreatment
Figure 2. Organic matter solubilisation vs. methane yield increase after mixed microalgal
biomass pretreament using different techniques (Alzate et al., 2012; Cho et al., 2013; Passos
et al., 2013a; Passos et al., 2013b; Passos and Ferrer, submitted; Passos et al., in press).
31
Biomass concentration (g VS/L)
40 60 80 100 120 140 160 180 200 220
Energ
y r
atio
(E
o/E
i)
0
5
10
15
20
25
30
Thermal pretreatment (75 ºC)
Thermal pretreatment (95 ºC)
Hydrothermal pretreatment (120 ºC)
Thermal pretreatment (100 ºC)
Hydrothermal pretreatment (130 ºC)
Microwave pretreatment (70 MJ/kg VS)
Figure 3. Energy ratio of microalgae anaerobic digestion after thermal and mechanical
pretreatments, at increasing VS concentration. (Data source: Schwede et al., 2013; Passos et
al., 2014; Passos and Ferrer, 2014; Passos and Ferrer, submitted)
32
Table 1. Thermal pretreatments for microalgae biogas production.
Microalgae species Reactor conditions Methane yield
(L CH4/g VS)
Pretreatment
conditions
Solubilisation
increase
Methane yield
increase
References
Thermal pretreatment
Microalgal biomass1 CSTR, 28 days HRT 0.270
2 100
oC; 8 h n.d. 33% Chen and Oswald, 1998
Scenedesmus biomass BMP 0.0763 70, 90
oC; 3 h 7, 11-fold 12, 220% González-Fernández et al., 2012a
Scenedesmus biomass BMP 0.0823 70, 80
oC; 25 min 1.9, 2.3-fold 9, 57% González-Fernández et al., 2012b
Clamydomonas sp.,
Scenedesmus sp. and
Nannocloropsis sp.
BMP 0.272 55 oC; 12-24 h 11, 9% -4, -7% Alzate et al., 2012
Acutodesmus obliquus
and Oocystis sp.
BMP 0.198 55 oC; 12-24 h 21, 19% -2, -13% Alzate et al., 2012
Microspora sp. BMP 0.255 55 oC; 12-24 h 29, 29% 4% Alzate et al., 2012
Chlorella sp. and
Scenedesmus sp.
BMP 0.336 50, 80 oC; 30 min 2, 17% 4, 14% Cho et al., 2013
Microalgal biomass1 BMP 0.105-0.111 55-95
oC; 5-15 h 5-21% 11, 48, 60% Passos et al., 2013a
Microalgal biomass1 CSTR, 20 days HRT 0.180 75, 95 ºC; 10 h n.d. 67, 72% Passos and Ferrer, 2014
Hydrothermal
pretreatment
Clamydomonas sp.,
Scenedesmus sp. and
Nannocloropsis sp.
BMP 0.272 110, 140 oC ; 1-1.2
bar; 15 min
9, 16% 19, 33% Alzate et al., 2012
33
Acutodesmus obliquus
and Oocystis sp.
BMP 0.198 110, 140 oC ; 1-1.2
bar; 15 min
22, 37% 11, 31% Alzate et al., 2012
Microspora sp. BMP 0.255 110, 140 oC ; 1-1.2
bar; 15 min
38, 39% 62, 50% Alzate et al., 2012
Chlorella sp. and
Scenedesmus sp.
BMP 0.336 120 oC; 30 min 29% 20% Cho et al., 2013
Nannocloropsis salina CSTR, 120 days HRT 0.130 100-120 oC ; 2 h n.d. 108% Schwede et al., 2013
Chlorella vulgaris BMP 0.139 120 ºC; 40 min 54%4 93% Mendez et al., 2014b
Microalgal biomass1 BMP 0.122 110, 130 ºC; 15-30
min
8, 9, 15, 13% 24, 17, 39, 33% Passos and Ferrer, submitted
Microalgal biomass1 CSTR, 20 days HRT 0.120 130 ºC; 15 min n.d. 42% Passos and Ferrer, submitted
Thermal pretreatment
with steam explosion
Clamydomonas sp.,
Scenedesmus sp. and
Nannocloropsis sp.
BMP 0.272 170 oC ; 6 bar; 15
min
32% 46% Alzate et al., 2012
Acutodesmus obliquus
and Oocystis sp.
BMP 0.198 170 oC ; 6 bar; 15
min
63% 55% Alzate et al., 2012
Microspora sp. BMP 0.255 170 oC ; 6 bar; 15
min
40% 41% Alzate et al., 2012
Scenedemus biomass BMP 0.180 170 oC; 8 bar; 30
min
10-fold 81% Keymer et al., 2013
Chlorella vulgaris BMP 0.156 160 oC ; 20 min 4.5-fold
4 65% Mendez et al., 2014b
Note: 1Corresponds to microalgal biomass grown in wastewater treatment open ponds;
2Data expressed in L biogas/g VS;
3Data expressed in L CH4/g COD;
4Data from
solubilisation of carbohydrates.
34
Table 2. Mechanical pretreatments for microalgae biogas production.
Microalgae species Reactor conditions Methane yield
(L CH4/g VS)
Pretreatment
conditions
Solubilisation
increase
Methane yield
increase
References
Ultrasound
Clamydomonas sp.,
Scenedesmus sp. and
Nannocloropsis sp.
BMP 0.272 10, 27, 40, 57 MJ/kg TS 14, 28, 30, 32% 14, 14, 14, 12% Alzate et al., 2012
Acutodesmus obliquus
and Oocystis sp.
BMP 0.198 10, 27, 40, 57 MJ/kg TS 24, 48, 53, 60% 6, 8, 13, 13% Alzate et al., 2012
Microspora sp. BMP 0.255 10, 27, 40, 57 MJ/kg TS 30, 56, 57, 62% 23, 18, 18, 22% Alzate et al., 2012
Scenedesmus biomass BMP 0.0821 100-130 MJ/kg TS 2.2-3.1-fold 75-90% González-Fernández et al.,
2012b
Chlorella sp. and
Scenedesmus sp.
BMP 0.336 39, 117 and 234 MJ/kg
VS
2, 8, 13% 6, 10, 15% Cho et al., 2013
Microalgal biomass BMP 0.148 8-34 MJ/kg TS; 19
gTS/L
16-101% 6-33% Passos et al., in press
Microwave
Microalgal biomass3 BMP 0.117 21.8, 43.6, 65.4 MJ/kg
TS
3-8% 12-78% Passos et al., 2013b
Microalgal biomass3 CSTR, 20 days HRT 0.170 900 W; 3 min (70
MJ/kg VS); 26 gTS/L
n.d. 60% Passos et al., 2014
Note: 1Data expressed in L CH4/g COD;
2Data expressed in biogas;
3Corresponds to microalgal biomass grown in wastewater treatment open ponds.
35
Table 3. Comparison of pretreatment methods for increasing microalgae anaerobic biodegradability.
Pretreatment Control parameters Biomass solubilisation Methane yield increase Pros Cons
Thermal (<100°C) Temperature; exposure time √√√ √√ Low energy demand; Scalability High exposure time
Hydrothermal
(>100°C) Temperature; exposure time √√√ √√ Scalability
High heat demand;
Thickened or dewatered
biomass; Risk of formation
of refractory compounds
Thermal with steam
explosion
Temperature; exposure time;
pressure √√√ √√√ Scalability
High heat demand;
Thickened or dewatered biomass; Risk of formation
of refractory compounds
Investment cost
Microwave Power; exposure time √√ √√ -
High electricity demand;
scalability; Biomass
dewatering
Ultrasound Power; exposure time √ √ Scalability High electricity demand;
Biomass dewatering
Chemical Chemical dose; exposure time √ √ Low energy demand
Chemical contamination;
Risk of formation of
inhibitors; Cost
Thermo-chemical Chemical dose; exposure time;
temperature √√√ √√ Low energy demand
Chemical contamination; Risk of formation of
inhibitors; Cost
Enzymatic Enzyme dose; exposure time; pH,
temperature √ √ Low energy demand Cost, sterile conditions
36
Table 4. Energy assessment of pretreatment methods for improving microalgae anaerobic digestion.
Pretreatment Optimal
condition
VS concentration
(g/L)
Methane yield increase
(L CH4/g VS)
Energy input
(kJ/g VS)
Energy output
(kJ/g VS)
Energy ratio
(Eo/Ei)
Experimental data
source
Thermal 75 ºC ; 10 h 13.5 0.120 6.50 3.87 0.59 Passos and Ferrer,
2014
Thermal 95 ºC ; 10 h 13.5 0.130 7.43 4.19 0.56 Passos and Ferrer,
2014
Hydrothermal 100 oC; 8 h 37 0.080 2.80 2.58 0.92 Chen and Oswald,
1998
Hydrothermal 120 oC; 2 h 200 0.140 0.58
4.51 7.78 Schwede et al.. 2013
Hydrothermal 130 ºC ; 15
min
14.6 0.050 8.37 1.61 0.19 Passos and Ferrer,
submitted
Microwave 900 W; 3 min 9.8 0.100 70.13 3.22 0.05 Passos et al., 2014
37
Highlights
Pretreatments are mostly focused on microalgae biodegradability increase in BMP test
Studies with the same biomass are necessary for comparing different techniques
Pretreatment and anaerobic digestion effectiveness depend on algae characteristics
Research on continuous reactors is crucial for evaluating the process scalability