Bio-ethanol from lignocellulosic biomass: current findings

1
Bio-ethanol from lignocellulosic biomass: current findings determine
2
research priorities
3
1,2*
4
5
6
7
8
9
Qian Kang, 2 Lise Appels, 1 Tianwei Tan, 2 Raf Dewil
1
Beijing University of Chemical Technology, School of Life Science and
Biotechnology, Beijing, China
2
KU Leuven, Dpt. Chemical Engineering, Process and Environmental Technology
Lab, Sint-Katelijne-Waver, Belgium
*
Author of correspondence
e-mal: [email protected]
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12
ABSTRACT
13
14
Bio-ethanol is considered an important renewable fuel to partly replace fossil-derived
15
16
fuels. “Second generation” bio-ethanol, with lignocellulose material as feedstock, is a
promising alternative for first generation bio-ethanol. This paper provides an
17
overview of the current status and reveals the bottlenecks that hamper its
18
implementation: the production process requires additional processing steps,
especially in pre-treatment and hydrolysis; the production yield is low; and the energy
19
20
21
22
consumption of distillation is higher due to the lower ethanol concentration of the
broth. Current literature specifies a conversion of biomass to bio-ethanol of 30 to
~50% only. Novel processes increase the conversion yield to about 92 % of the
24
theoretical yield. However, this conversion yield strongly depends upon the source of
biomass, due to the largely different proportion of inert lignin, due to the difficult
25
accessibility of cellulose and hemicellulose towards hydrolysis, and due to the
26
27
specific yield of C5 and C6 sugars. Methods that are widely applicable to a diversity
of raw materials, and to the joint C5 and C6 fermentation are hence important in the
28
further development of biomass-to-bioethanol, and adapted microorganisms for C5
29
30
and C6 sugar fermentation are discussed in the paper.
New combined processes reduce both the number of operational steps and the
31
production of inhibitors. Recent advances in genetically engineered microorganisms
32
are promising for higher alcohol tolerance and conversion efficiency. By combining
advanced systems and by intensive add itional research to eliminate current
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33
34
35
bottlenecks, second generation bio-ethanol could surpass the traditional first
generation processes.
36
37
Keywords:
Lignocellulosics;
Biomass;
38
microorganisms, C5 and C6 fermentation.
Bio-ethanol
production;
Adapted
1
39
1. INTRODUCTION
40
1.1 Bio-ethanol as sustainable fuel
41
42
With the global increasing demand for energy, energy shortage will be a global
problem. Bio-ethanol is considered as an important renewable fuel to partly replace
43
fossil-derived fuels. The world production of bio-ethanol increased from 50 million
44
m3 in 2007 to over 100 million m3 in 2012 [1]: Brazil and the United States represent
approximately 80% of the world supply, mostly using corn or sugarcane. In
45
47
developing economies, food-related feedstock is preferably replaced by non-food raw
materials, such as sweet sorghum or cassava. The use of common biomass could
48
significantly increase the bio-ethanol production, and lignocellulose-based bio-ethanol
49
is therefore the topic of the present review paper. The current technological
development and bottlenecks define the short and medium term research priorities.
46
50
51
53
Industrial ethanol is mainly produced petrochemically through the acid-catalyzed
hydration of ethylene. Ethanol for use in alcoholic beverages, and the vast majority of
54
ethanol for use as bio- fuel, is produced by fermentation where certain species of yeast
55
(e.g., Saccharomyces cerevisiae) or bacteria (e.g. Zymomonas mobilis) metabolize
sugars in oxygen- lean conditions to produce ethanol and carbon dioxide.
52
56
57
58
59
The main reasons for the enhanced development of bio-ethanol are its use as a
favourable and near carbon-neutral renewable fuel, thus reducing CO 2 emissions and
61
associated climate change; its use as octane enhancer in unleaded gasoline; and its use
as oxygenated fuel- mix for a cleaner combustion of gasoline, hence reducing tailpipe
62
pollutant emissions and improving the ambient air quality. The largest single use of
63
ethanol is as engine fuel and fuel additive, with common types of available fuel- mixes
listed in Table 1.
60
64
65
66
Table 1. Common ethanol-petrol mixtures [1, 2]
Code
Composition
Countries
Comments
E5
max. 5% anhydrous ethanol, min. 95% petrol
Western Europe
E10
max. 10% anhydrous ethanol, min. 90% petrol
USA, Europe
Blends for regular
E15
max. 15% anhydrous ethanol, min. 85% petrol
USA, cars >2000
cars
E25
max. 25% anhydrous ethanol, min. 75% petrol
Brazil
E85
max. 85% anhydrous ethanol, min. 15% petrol
USA, Europe
E100
hydrous ethanol (~ 5.3 wt% water)
Brazil
Flex-Fuel vehicles
67
2
68
Ethanol has appropriate properties for spark ignition IC engines. Its octane numbers,
69
70
motor octane number (MON) and research octane number (RON), are 90 and 109,
respectively, on average 99 compared to 91 for regular gasoline. Due to its low cetane
71
number, ethanol does not burn efficiently by compression ignition, and is moreover
72
not easily miscible with diesel fuel. To improve the use of ethanol in
compression- ignition (CI) engine vehicles, measures can be taken, such as the
73
75
addition of an emulsifier in order to increase the ethanol-diesel miscibility; the
addition of ethylhexylnitrate or diterbutyl peroxide to enhance the cetane number ; the
76
use of a dual fuel operation in which ethanol and diesel are introduced separately into
77
the cylinder; or the modification of diesel engines in order to adapt their
characteristics of auto- ignition [3].
74
78
79
80
81
1.2 Bio-ethanol from different feedstock
Fermentation of sugar-based raw materials is referred to as "first generation"
83
bio-ethanol, whereas the use of lignocellulose raw materials is commonly called
"second generation" bio-ethanol. The "third generation" of algal bio-ethanol is at an
84
early stage of investigation. The first generation processes were discussed in detail by
85
Kang et al. [1]. Since the present paper deals with second generation bio-ethanol,
relevant and recent (> 2010) literature is summarized in Table 2. The 2nd. generation
bio-ethanol processes will use cellulose-released sugars, despite the cost of the
82
86
87
89
required enzymes to hydrolyse cellulose. Development of this technology could deal
with a number of cellulose-containing agricultural by-products, such as e.g. straw,
90
wood trimmings, sawdust, bamboo and others.
88
91
92
Table 2. Recent literature about second generation bio-ethanol production
Reference
Objectives
[4]
Optimal industrial symbiosis
system
to
improve
bioethanol production
[5]
[6]
Main Results
-Reduced bioethanol production and logistic
costs;
nd
-2 generation biomass should be used for
bioethanol production.
Bio-ethanol production from
dilute acid pre-treated Indian
-Bio-ethanol yield of 1.76% (v/v) with an
efficiency of 41.69%;
bamboo variety by separate
-Bamboo can be used as feedstock for the
hydrolysis and fermentation
production of bio-ethanol.
-An ethanol yield based on total sugar of 480 g
Fuel ethanol production
kg-1 was obtained;
from sweet sorghum bagasse
-Ethanol produced on marginal land at 0.252
using microwave irradiation
3
-1
m ton biomass.
3
[7]
Ultrasonic-assisted
simultaneous
SSF
of
pre-treated oil palm fronds
-Maximal bioethanol concentration (18.2 g/l)
and yield (57.0%).
for bio-ethanol production
[8]
[9]
Convert
sucrose
and
homocelluloses in sweet
sorghum stalks into ethanol
pulsed
-Increase the production of bio-ethanol from
increase
lignocellulosic biomass to 52±16%.
bio-ethanol production
process
for
bio-ethanol production from
pre-treated olive pruning
biomass
Bio-ethanol production from
[11]
[12]
water hyacinth Eichhornia
crassipes
Enhanced saccharification of
biologically
pre-treated
wheat
straw for
ethanol
production
Fermentation of biologically
[13]
[14]
[15]
stalk
into
Low-intensity
ultrasound
to
Different
configurations
[10]
-All sugars in sweet sorghum
lignocellulose
were
hydrolysed
fermentable sugars.
pre-treated wheat straw for
ethanol production
Integration of pulp and paper
technology with bio-ethanol
production
Production of bio-ethanol by
fermentation of lemon peel
wastes
pre-treated
steam explosion
with
-Ethanol concentration of 3.7 vol% was
obtained.
-Yeast
Saccharomyces cerevisiae
produced ethanol at 9.6 ± 1.1 g/l.
TY2
-Increase of the sugar yield from 33 to 54%,
and reduction of the quantity of enzymatic
mixture by 40%.
-The highest
overall ethanol yield
was
obtained
with
the yeast Pachysolen
tannophilus: yielded 163 mg ethanol per gram
of raw wheat straw (23 and 35% greater).
-Re-use existing assets to the maximum extent;
-Keep the process as simple as possible;
-Match the recalcitrance of the biomass with
the severity of the pre-treatment.
-Reduces the residual content of essential oils
below 0.025% and decreases the hydrolytic
enzyme requirements;
-Obtained ethanol production in excess of 60
L/1000 kg fresh lemon peel biomass.
4
-The maximum glucose yield obtained was
91.28% of the theoretical yield and the
maximum amount of glucose obtained was
[16]
Ultrosonic-assisted
enzymatic saccharification
38.4 g/L (MTCC 7450);
-The hydrolyte obtained was 91.22% of the
of sugarcane bagasse for
bio-ethanol production
theoretical ethanol yield (MTCC 89);
-Decreases the reaction time;
-The application of low intensity ultrasound
enhanced the enzyme release and intensified
the enzyme-catalysed reaction.
-The major barriers for the commercialization
of 2nd generation ethanol production are the
Status
[17]
and
advanced
technologies
barriers
of
biofuel
high costs of pre-treatment, enzymes used in
hydrolysis, and conversion of C5 sugars to
ethanol;
-The residues need to be processed for
by-products through biorefinery to improve the
economics of the whole process.
[18]
[19]
Sugarcane
hydrolysis
bagasse -This enzyme extract promoted the conversion
using
yeast of approximately 32% of the cellulose;
cellulolytic enzymes
Pre-treatment of unwashed
-C. laurentii is a good β-glucosidase producer.
water-insoluble solids of
reed straw and corn stover
-Obtain a high ethanol concentration of 56.28
g/L (reed straw) and 52.26 g/L (corn stover);
pre-treated with liquid hot
water to
obtain
high
-Ethanol yield reached a maximum of 69.1%
(reed straw) and 71.1% (corn stover).
concentrations of bioethanol
[20]
Waste paper sludge as a
potential
biomass
for
bio-ethanol production
Assessment of combinations
[21]
between pre-treatment and
conversion
configurations
for bio-ethanol production
-SSF using cellulase produced by A.
cellulolyticus gave ethanol yield 0.208 (g
ethanol/g PS organic material);
-Consolidated biomass processing
technology gave ethanol
ethanol/g Solka floc).
yield
(CBP)
0.19
(g
-The process based on dilute acid
pre-treatment and enzymatic hydrolysis and
co-fermentation combination shows the best
economic potential;
-The cellulose
hydrolysis
based
on
an
enzymatic process showed the best energy
efficiency.
5
[22]
Combined use of gamma ray
-Increasing
enzymatic
hydrolysis
after
combined pre-treatment is resulting from or
and
decrease in crystallinity of cellulose, loss of
dilute
acid
for
bio-ethanol production
Ethanol
[23]
[24]
production
lignocellulosic
(exergy analysis)
hemicelluloses, and removal or modification
of lignin.
from
biomass
Alkaline pretreatment
sugarcane
bagasse
on
for
bio-ethanol production
[25]
Influence of dual salt
pretreatment of sugarcane
bagasse for bio-ethanol
production
-Lowest
environmental
impact
for
second-generation bio-ethanol production;
-Highest exergy efficiency (Steam Explosion
Pre-treatment + SSF + Dehydration) reaching
79.58%.
-The lowest lignin content (7.16%) was
obtained;
-Cellulose content increased after alkaline
pretreatment.
-Better performance was observed using H2 O2
with MnSO4 ·H2 O and ZnO;
-The inhibitor formation was limited;
-The maximum theoretical ethanol yield of
84.32% (13.1g/L, 0.184g/g sugarcane bagasse)
was achieved during the fermentation.
-MG-60 produced cellulose and xylanese
[26]
Bio-ethanol production from
alkaline pretreated sugarcane
rapidly
(CBP);
bagasse using Phlebia sp.
MG-60
-The maximum theoretical ethanol yield of
65.7% (4.5g/L) was achieved during the
fermentation.
Integrated
fungal
fermentation of sugarcane
[27]
bagasse for bio-ethanol
production by Phlebia sp.
MG-60
[28]
Furfural
and
xylose
production from sugarcane
bagasse
production
in
ethanol
during
consolidated
bioprocessing
-75% moisture content was suitable for
subsequent ethanol production;
-Some additives improved delignification in
integrated fungal fermentation (IFF);
2+
2+
-Some inorganic chemicals (e.g. Fe , Mn ,
Cu2+) increased the ethanol production.
-The furfural yield and xylose yield were 6 and
15.5 g/g of sugarcane bagasse respectively;
-Ethanol was produced from the residual solid
materials obtained from furfural and xylose at
87.4% and 89.3% respectively.
93
95
The specificities of using lignocellulosic raw materials will be dealt with in Section
1.3. Whether first, second or third generation feed stock is used, fermentation
96
produces an alcohol- lean broth only, as such unusable in industrial and fuel
94
6
97
applications. The ethanol must hence be purified. Fractional distillation can
98
99
concentrate ethanol to 95.6vol% (89.5 mol%), corresponding to the azeotropic
composition with a boiling point of 78.2 °C. Further ethanol enrichment by common
100
distillation is impossible, but different alternatives have been investigated, as reported
101
by Kang et al. [29].
102
104
1.3 Lignocellulosic biomass
1.3.1 Sources
105
Available biomass can be categorized into primary sources, produced as either crop or
106
key product, e.g., sugar cane, short rotation energy plantations; secondary sources, as
residues from the production processes, e.g., bagasse, rice husks, straw; and tertiary
103
107
109
sources, as residues produced during and at post-end application, e.g., the organic
fraction of municipal solid waste (MSW), sewage treatment sludge, wood trimmings,
110
etc [30]. In general the final availability of organic wastes and residues may fluctuate
111
112
and is affected by market growth, although climate and other factors have influences,
especially when considering the primary sources. The energy potential of biomass
113
residues and organic wastes depends on the yield, the total land area available and the
114
type of production.
The organic fraction of municipal solid waste (MSW) is an inexpensive source of
108
115
117
biomass and covers domestic and industrial waste collected in a specific area. The
overall potential of the organic fraction of municipal waste and its waste wood
118
fraction is strongly reliant on economic growth, consumption and the use of
119
biomaterials. It is estimated at between 5 and 50 EJ/year [30]. The unit EJ (eta-joule)
is equal to 1018 J.
116
120
121
123
Agro- industrial biomass residues are by-products of agriculture or its related industry,
including cotton stalks, wheat and rice straw, coconut shells, maize cobs, jute sticks,
124
rice husks, etc. [31]. The agricultural residues are produced decentralised and have a
125
128
low density. Due to the high transportation cost, it is expensive to apply agricultural
residues as the main fuel in power stations. The potential of agriculture residues varies
between 15 up to 70 EJ/year [30], as a function of regional production, harvesting
processing factors and recoverability factors. Within the agro- industrial residues,
129
dried manure is considered as a tertiary source. A total worldwide estimate is difficult
130
to make, and given as 5 to 55 EJ/year, with the lower estima te due to the current use
as fertiliser, while the higher estimate considers the total technical potential [31].
122
126
127
131
132
134
Forestry residues include biomass, not harvested or removed from sorting regions in
commercial hardwood and softwood production, through forest management
135
operations such as pre-commercial thinning and removal of dead and dying trees.
133
7
136
Forestry waste includes wood chips, sawdust and bark. It can provide 65% of the
137
138
biomass energy potential [32,33]. The extraction costs and the required transportation
to centralized processing plants make forest fuels expensive. Several studies have
139
focused on their use for energy production at a district level, applying appropriate
140
designs of decentralized smaller plants e.g. Malinen et al. [34] and Demirbas [35].
The energy potential of the world's forests is again difficult to estimate. The possible
141
143
contribution by 2050 is estimated at 98 EJ/year of excess natural forest growth, and
32-52 EJ/year of processing residues. Although these lignocellulosic biomass
144
resources represent a significant energy value, ～150 EJ/year, only part of this
145
resource can be used as feedstock for bio-ethanol, for reasons given below.
142
146
147
1.3.2 Composition
148
Lignocellulose, the principal component of the plant cell walls, is mainly composed of
cellulose (40-60% of the total dry weight), hemicellulose (20-40%) and lignin
149
151
(10-25%). Cellulose consists of long chains of β-glucose monomers gathered into
micro- fibril bundles. The hemicelluloses, mostly xyloglucans or xylans, are linked to
152
the micro-fibrils by hydrogen bonds. Lignins are phenolic compounds which are
153
formed by polymerisation of three types of monomers (p-coumatyl, coniferyl and
synapyl alcohols). Lignin adds compressive strength and stiffness to the cell wall [36].
Once the lignocellulosic biomass is pre-treated and hydrolysed, the released sugars
150
154
155
157
can be fermented and the down-stream process is similar to that of first generation
feed stock [1]. Potential lignocellulosic feedstocks and their composition are
158
summarized in Table 3.
156
159
160
Table 3. Potential lignocellulosic biomass sources and compositions (% dry weight) [37,38]
Raw material
Hemicelluloses
Cellulose
Lignin
Others (i.e., ash)
Agricultural residues
25-50
37-50
5-15
12-16
Hardwood
25-40
45-47
20-25
0.80
Softwood
25-29
40-45
30-60
0.50
Grasses
35-50
25-40
—a
2-5
12-20
50-70
6-10
2
Newspaper
25-40
40-55
18-30
5-8
Switch grass
30-35
40-45
12
4-5
Waste papers from
chemical pulps
161
a
Not present or Not available
8
162
164
High lignin and/or high ash concentrations are unfavorable for bio-ethanol production.
Especially softwood can hence be excluded. The extensive hydrogen linkages among
165
cellulose molecules lead to a crystalline and strong matrix structure [39]. Although
166
167
starches require temperatures of only 60-70°C to be converted from crystalline to
amorphous texture, cellulose requires 320°C as well as high pressures (up to 25 MPa)
168
to transform the rigid crystalline structure into an amorphous structure in water [40].
169
Cotton, flax and chemical pulps represent the purest sources of cellulose, while soft
and hardwoods contain less than 50% of cellulose, as shown in Table 4.
163
170
172
Hemicellulose is an amorphous structure formed of different heteropolymers
including hexoses (D-glucose, D- galactose and D- mannose) as well as pentose
173
(D-xylose and L-arabinose). It may contain sugar acids (uronic acids) [41]. Its
174
175
backbone chain is primarily composed of xylan- linkages including α-xylose (~ 90%)
and L-arabinose (~ 10%) [42]. The degree of branching and the xylan composition
176
vary with the nature and the source of raw materials. To be totally hydrolysed into
177
free monomers, hemicellulose requires a wide range of enzymes in view of the
diversity of its sugars.
171
178
179
181
Lignin is an aromatic and rigid biopolymer, covalently bonded to hemicellulosic
xylans and responsible for the rigidity and high level of compactness of the plant cell
182
wall [43]. Lignin is composed of monomers of phenyl propionic alcohol, i.e.,
183
coumaryl, coniferyl and sinapyl alcohol. The lignin fraction in biomass sources varies
considerable, as illustrated in Table 4. Lignin components are gaining importance
180
184
185
186
because of their dilution effect on the processes of hydrolysis and fermentation
[44,45]. The phenolic groups, formed from the degradation of lignin, substantially
189
deactivate cellulolytic enzymes and hence hamper enzymatic hydrolysis. Chen et al.
[45] however demonstrated that lignin modification via genetic engineering could
considerably reduce lignin formation and improve ethanol yield. This could however
190
be problematic as lignin components serve as the major plant defence system to
191
192
pathogens and insects and its modification could disrupt the plants' natural protection
[46]. Retaining the lignin could moreover benefit the energy-economy of the process,
193
since once recovered, it can be applied in a combined heat and power unit (CHP), thus
194
195
being a potential energy self-sustaining source of the process. Biomass feedstock with
a high lignin content is not readily applicable as raw material for the bio-ethanol
196
fermentation. This certainly eliminates most of the soft woods.
187
188
197
198
The different composition of biomass feedstock (dry) is also reflected in its elemental
199
composition. The exact composition is largely dependent on the biomass sources. C,
9
200
H and O are the key components of biomass and largely determine their calorific
201
202
value. Some typical values of the C, H, O-content, as well as other essential data are
summarized in Table 4, as a result of an extensive literature survey [35,47,48] and
203
own analyses.
204
205
Table 4. Ultimate and proximate analys is of different biomass (wt%)
Wood and woody biomass
Ultimate analysis-(wt% on dry basis)
Proximate analys is
Sample
C
O
H
N
S
VM
M
FC
A
Pine
54.5
38.7
5.9
0.5
0.42
46.1
37.8
12.9
3.2
Eucalyptus bark
48.7
45.3
5.7
0.3
0.05
68.7
12
15.1
4.2
Forest residue
52.7
41.1
5.4
0.7
0.10
34.5
56.8
7.3
1.4
Land clearing wood
50.7
42.8
6
0.4
0.07
35.4
49.2
7
8.4
Olive wood
49
44.9
5.4
0.7
0.03
74.3
6.6
16.1
3
Pine chips
52.8
40.5
6.1
0.5
0.09
66.9
7.6
20
5.5
Pine sawdust
51
42.9
6
0.1
0.01
70.4
15.3
14.2
0.1
Poplar
51.6
41.7
6.1
0.6
0.02
79.7
6.8
11.5
2
Mixed Sawdust
49.8
43.7
6
0.5
0.02
55.1
34.9
9.3
0.7
Spruce wood
52.3
41.2
6.1
0.3
0.10
75.7
6.7
17.1
0.5
Willow
49.8
43.4
6.1
0.6
0.06
74.2
10.1
14.3
1.4
Herbaceous and agriculture biomass
Bamboo
52
42.5
5.1
0.4
0.04
71
13
15.2
0.8
Miscanthus grass
49.2
44.2
6
0.4
0.15
71.9
11.4
14
2.7
Sweet sorghum
49.7
43.7
6.1
0.4
0.09
71.8
7
16.8
4.4
Switchgrass
49.7
43.4
6.1
0.7
0.11
70.8
11.9
12.8
4.5
Corn straw
48.7
44.1
6.4
0.7
0.08
67.4
7.4
17.8
7.1
Rice straw
50.1
43
5.7
1
0.16
59.4
7.6
14.4
18.6
Wheat straw
49.4
43.6
6.1
0.7
0.17
67.2
10.1
16.3
6.4
Coconut shell
51.1
43.1
5.6
0.1
0.1
70.5
4.4
22
3.1
Cotton husks
50.4
39.8
8.4
1.4
0.01
73
6.9
16.9
3.2
10
Corn stover
42.5
42.6
5
0.8
NA
78.1
10.6
17.6
3.7
Groundnut shell
50.9
40.4
7.5
1.2
0.02
68.1
7.9
20.9
3.1
Hazelnut shell
51.5
41.6
5.5
1.4
0.04
71.5
7.2
19.9
1.4
Olive husks
50
42.1
6.2
1.6
0.04
73.7
6.8
17.4
2.1
Rice husks
49.3
43.7
6.1
0.8
0.22
56.1
10.6
17.2
16.1
Soya husks
45.4
46.9
6.7
0.9
0.08
69.6
6.3
19
5.1
Bagasse
49.8
43.9
6
0.2
0.08
76.6
10.4
11.1
1.9
Sunflower husks
50.4
43
5.5
1.1
0.1
69.1
9.1
19
2.8
Tea wastes
48.6
42.2
5.4
3.8
70.3
7.26
18.57
3.88
Other biomass sources
206
Chicken litter
60.5
25.3
6.8
6.2
1.2
43.3
9.3
13.1
34.3
Agricultural residue
52.4
41.2
6
0.4
0.04
54.7
30.3
12.7
2.3
Mixed waste paper
52.3
40.2
7.2
0.2
0.08
76.8
8.8
6.8
7.6
Refuse-derived fuel
53.8
36.8
7.8
1.1
0.47
70.3
4.2
0.5
25
Sewage sludge
50.9
33.4
7.3
6.1
2.33
45
6.4
5.3
43.3
Wood yard waste
52.2
40.4
6
1.1
0.3
40.9
38.1
8.4
12.6
VM: volatile matter; M: moisture; FC: fixed carbon; A: ash
207
208
The ash content of biomass sources is generally low, a s illustrated in Tables 5. Its
209
210
composition should, however, be taken into consideration, since most of the ash will
concentrate in the lignin residue, thus possibly hampering further energy generation
211
by fouling or sintering. Biomass contains a significant content of K, Cl, and Si as well
212
215
as lower concentrations of Ca, Mg, Al, Fe and Na. The ash content and its chemical
composition are a strong function of the biomass species. The elements in the ash are
O, Ca, K, Si, Mg, Al, S, Fe, P, Cl, Na, Mn, and Ti [49]. An extensive literature survey
was provided by several authors [47,50,51] and some examples are summarized in
216
Table 5.
213
214
217
218
Table 5. Elemental ash composition of different biomass
Wood and woody biomass
Sample
SiO2
CaO
K2 O
P2 O5
Al2O3
MgO
Fe2 O3
SO3
Na2 O
TiO2
Eucalyptus
10.04
57.74
9.29
2.35
3.1
10.91
1.12
3.47
1.86
0.12
11
bark
Poplar bark
1.86
77.31
8.93
2.48
0.62
2.36
0.74
0.74
4.84
0.12
Willow
6.1
46.09
23.4
13.01
1.96
4.03
0.74
3
1.61
0.06
Wood residue
53.15
11.66
4.85
1.37
12.64
3.06
6.24
1.99
4.47
0.57
Herbaceous and agriculture biomass
Bamboo whole
9.92
4.46
53.38
20.33
0.67
6.57
0.67
3.68
0.31
0.01
Miscanthus
56.42
10.77
19.75
5.54
0.79
3.01
0.94
2.28
0.47
0.03
Sorghum grass
73.21
7.02
8.97
4.43
1.83
2.21
0.95
1.11
0.25
0.02
Sweet sorghum
66.85
10.41
4.49
3.47
0.81
3.12
0.58
3.47
1.47
0.06
Switchgrass
66.25
10.21
9.64
3.92
2.22
4.71
1.36
0.83
0.58
0.28
Wheat straw
50.35
8.21
24.89
3.54
1.54
2.74
0.88
4.24
3.52
0.09
Rice husks
94.48
0.97
2.29
0.54
0.21
0.19
0.22
0.92
0.16
0.02
46.79
4.91
6.95
3.87
14.6
4.56
11.12
3.57
1.61
2.02
23.66
15.31
28.53
7.13
8.75
7.33
4.27
4.07
0.8
0.15
Sugar cane
bagasse
Sunflower
husks
Other biomass varieties
Chicken litter
Mixed waste
paper
Refuse-derived
fuel
Sewage sludge
Wood yard
waste
5.77
56.85
12.19
15.4
1.01
4.11
0.45
3.59
0.6
0.03
28.62
7.63
0.16
0.2
53.53
2.4
0.82
1.73
0.54
4.37
38.67
26.81
0.23
0.77
14.54
6.45
6.26
3.01
1.36
1.9
33.28
13.04
1.6
15.88
12.91
2.49
15.7
2.05
2.25
0.8
60.1
23.92
2.98
1.98
3.08
2.17
1.98
2.46
1.01
0.32
219
220
2. PROCESSING OF BIOMASS-TO-ETHANOL
221
2.1 Generalitics
222
Once the feedstock is delivered to the ethanol plant, it needs to be carefully stored and
223
conditioned to prevent early fermentation and bacterial contamination. Through
224
225
pre-treatment, simple sugars are made available in proportions depending on the type
of biomass used and the pre-treatment process. The main steps are summarized in
226
Figure 1, providing a general production flow sheet.
227
228
229
12
230
231
232
233
234
235
236
237
238
239
240
241
242
Fig.1 Second generation biomass-to-ethanol production (ST: steam addition)
243
244
2.2 Pre-treatment and first stage hydrolysis
245
Pre-treatment involves delignification of the feedstock [52] in order to make cellulose
246
more accessible in the hydrolysis step, using physical, physico-chemical, chemical
247
and biological treatment (Table 6). Carbonic acid and alkaline extraction have the best
performance. However, the most common methods are steam explosion and dilute
248
249
250
acid pre-hydrolysis, which are followed by enzymatic hydrolysis. Sulphuric acid or
carbon dioxide are often added in order to reduce the production of inhibitors and
253
improve the solubilisation of hemicellulose [43]. Steam explosion has a few
limitations since the lignin-carbohydrate matrix is not completely broken down;
degradation products are generated that reduce the efficie ncy of the hydrolysis and
254
fermentation steps; and a portion of the xylan fraction is destroyed.
251
252
255
256
Table 6. Assessment of selected pre-treatment processes [43,53-61]
Yield of
Pre-treatment process
fermentable
Wastes
Investment
sugars
Physical or Physico-chemical
-Mechanical
Low
Very Low
Low
-Steam explosion
High
Low
High
Moderate
Very Low
High
-Ammonia fiber explos ion (AFEX)
13
-Carbonic Acid
Very High
Very Low
Low
Chemical
-Dilute acid
Very High
High
Moderate
-Concentrated acid
Very High
High
High
-Alkaline extraction
Very High
High
Low
High
Low
Low
Very High
Low
Very High
-Wet oxidation
-Organosolv
257
258
The use of dilute sulphuric acid (0.5-1%; 433-463K for 10 minutes) has the
259
preference of the US National Renewable Energy Laboratory [62]: hemicellulose is
260
261
largely hydrolysed releasing different simple sugars (xylose, arabinose, mannose and
galactose) but also other compounds of the cellulosic matrix, that can however inhibit
262
the enzymatic hydrolysis and fermentation. Part of the acetic acid, much of the
263
sulphuric acid and other inhibitors produced during the degradation of the materials
need to be removed, and neutralisation is performed before fermentation.
264
266
Pre-treatment is a costly separation, accounting for approximately 33% of the total
cost [63]: the economy needs to be improved, and the release of microbial and
267
chemical contamination that possibly reduces the overall yield, needs further
268
attention.
265
269
270
2.3 Second stage hydrolysis
271
272
In the second stage hydrolysis, the released cellulose of the biomass is converted into
glucose, which is again catalysed by dilute acid, concentrated acid or preferably by
273
cellulase enzymes, either produced in a separate reactor or bought externally from
274
industrial suppliers [64-67].
275
276
The conversion of cellulose and hemicellulose can be expressed by the reaction of
277
glucan (for hexoses) and xylan (for pentose) with water:
278
(C6 H 10O5 ) n  nH 2O  nC6 H 12O6
279
(C5 H 8O4 )n  nH 2O  nC5 H10O5
280
The maximum theoretical yield of hexoses and pentoses is 1.136 kg and 1.111 kg per
281
kg of glucan and xylan respectively.
(1)
(2)
282
283
To overcome inhibition by hydrolyte components, membrane techniques have been
284
investigated [2,29]. Chandel et al. [68] investigated the strategies that have been
285
adopted to detoxify lignocellulosic hydrolysates and their effects on the chemica l
14
286
composition of the hydrolysates to improve the ferme ntability of lignocellulosics.
287
288
Hydrolysis of myco-LB (LB after fungal pretreatment) has been recognized as a
promising approach to avoid fermentation inhibitors and improve total sugar recovery.
289
Genetic manipulation could modify the metabolic routes to produce bioethanol or
290
other value-added compounds in an efficient manner. Further research is certainly
required, as described in Section 3.
291
292
293
2.4 Fermentation
294
Contrarily to the conversion of disaccharides and starch to ethanol, which are mature
295
296
technologies, modern lignocellulose-to-ethanol processes are at pilot and
demonstration stage: NREL (USA) [62], Iogen Corporation (Canada) [69], and ETEK
297
(Sweden) [70], have built pilot plants capable to produce a few hundred thousand
298
litres of ethanol per year.
299
301
Fermentation is the biological process to convert the hexoses and pentoses into
ethanol by a variety of microorganisms, such as bacteria, yeast or fungi. The
302
conversion reaction for hexoses (C6) and pentoses (C5) is as follows:
300
303
304
C6 H 12O6  2C2 H 5OH  2CO2
(3)
305
3C6 H 10O5  5C2 H 5OH  5CO2
(4)
306
The theoretical maximum yield of broth hexoses and pentoses is 0.511 kg ethanol and
0.489 kg CO 2 per kg sugar. The overall theoretical ethanol yield (at 20 °C) hence
307
308
309
becomes 0.719 and 0.736 litrer per kg of glucan (or/and other 6C structures) and
xylan (or/and other 5C structures) respectively.
310
312
S. cerevisiae, the yeast commonly used for first generation ethanol production, cannot
metabolize xylose. Other yeasts and bacteria are under investigation to ferment xylose
313
and other pentoses into ethanol.
314
Genetically engineered fungi that produce large volumes of cellulase, xylanase, and
hemicellulase enzymes, are under investigation. These could convert agricultural
311
315
317
residues (e.g. corn stover, straw, sugar cane bagasse) and energy crops (e.g.
switchgrass) into fermentable sugars [69,71]. Additional research tried to find
318
microorganisms which can effectively ferment both types of sugars into ethanol with
319
Escherichia coli, Klebisiella oxytoca and Zymomonas mobilis, as promising
320
candidates [72,73].
316
321
322
15
323
When using enzymatic hydrolysis, different integration methods of hydrolysis and
324
325
fermentation steps are proposed. In the Separate Hydrolysis and Fermentation (SHF),
the liberated cellulose is treated in a different reactor for hydrolysis and subsequent
326
fermentation than the hydrolysed hemicellulose and lignin. Although this facilitates
327
both the optimization of each separate reactor, and the selection of sugar-appropriate
micro-organisms to ferment the different sugars, the higher investment costs for two
328
330
separate reactors and the inhibition of the high glucose concentration to fermenting
organisms are major disadvantages [74,75]. Separate Hydrolysis and Co-Fermentation
331
(SHCF) and Simultaneous Saccharification and Co- Fermentation (SSCF) are possible
332
333
alternatives: co-fermenting both C5 and C6 sugars by a single strain of
micro-organisms in the same reactor, significantly improves the process economics
334
and enhances the commercial production of lignocellulosic ethanol in the short term
335
[75-77].
329
336
338
A novel development, the Consolidated BioProcessing (CBP), proceeds by producing
all required enzymes and ethanol using a single type of micro-organisms in a single
339
reactor. CBP is considered as the ultimate evolution of biomass-to-bio-ethanol
340
conversion technology, since it implies neither capital nor operating costs for
dedicated enzyme production together with a reduced consumption of s ubstrate for
enzyme production. Unfortunately, it is predicted that it will take several years of
337
341
342
343
344
research to determine such microorganisms or compatible combinations of
micro-organisms [77].
345
346
With bio-ethanol production from lignocellulosic biomass, chemical inhibition is a
347
more severe problem than encountered in first generation raw materials. Pre-treatment
348
349
and hydrolysis of lignocellulosics release specific inhibitors, e.g., furans such as
furfural and 5-hydroxymethylfurfural (5-HMF), and phenols such as
350
4-hydroxybenzaldehyde (4-HB), vanillin, and syringaldehyde, that need to be dealt
351
with to operate hydrolysis and fermentation under optimum conditions and maximum
conversion.
352
353
354
355
To increase the critical ethanol- inhibition concentration, adapted yeasts or bacteria
can be used. The most commonly used yeast is Saccharomyces cerevisiae, with a
357
moderate yield of fermentation. Research has been done on more promising yeasts
and bacteria: Zymomonas mobilis succeeds to survive higher ethanol concentrations in
358
the fermenter up to 16 vol%. Not only this advantage, but also a moderate tolerance
359
360
for acids and sugars, typical inhibitors present in biomass hydrolysis makes this a very
popular yeast for industrial application. The fermentation rate is also higher with
361
Zymomonas mobilis in comparison to Saccharomyces cerevisiae [1]. An interesting
356
16
362
characteristic of Z. mobilis is indeed that its plasma membrane contains hopanoids,
363
364
pentacyclic compounds similar to eukaryotic sterols, thus providing an extraordinary
tolerance to ethanol in its environment, around 16 wt%. However, in spite of these
365
attractive advantages, its substrate range is limited to glucose, fructose and sucro se. It
366
cannot ferment C5 sugars like xylose and arabinose which are important components
of lignocellulosic hydrolytes. Unlike yeast, Z. mobilis cannot tolerate toxic inhibitors
367
369
present in lignocellulosic hydrolytes such as acetic acid. Concentration of acetic acid
in lignocellulosic hydrolytes can be as high as 1.5wt%, well above the tolerance
370
threshold of Z. mobilis. Several attempts have been made to engineer Z. mobilis to
371
372
overcome its inherent deficiencies by metabolic engineering, mutagenesis or adaptive
mutation to produce acetic acid resistant strains of Z. mobilis [78,79]. However, when
373
these engineered strains metabolize mixed sugars in the presence of inhibitors, the
374
yield and productivity are much lower, thus preventing their industrial applicatio n.
368
375
377
To overcome inhibition by hydrolyte components, membrane techniques have been
investigated, although further research is certainly required, as described in Section 3
378
below.
376
379
380
381
382
383
384
385
2.5 Purification
Typical ethanol concentrations are in the range of 3-6 vol% only, very low in
comparison with 12 to 15 vol% obtained from 1 st generation feed stock [1]. Due to the
higher water content of the broth, additional distillation efforts are required. Different
process improvements, including energy pinch, very high gravity fermentation and
hybrid processes are described in detail in Kang et al [1].
386
388
2.6 Steam and electricity generation
The bottom product of the first distillation column (stillage) contains mainly lignin
389
and water next to unconverted cellulose and hemice llulose. This insoluble fraction is
390
391
dewatered by a pressure filter and sent to a fluidized bed combustor system for steam
and electricity generation. This system allows the plant to be self sufficient in energy
392
supply, reduces solid waste disposal cost and generates additional revenue trough
393
sales of excess electricity [75,80]. Burning the solid residues for steam and power
production is the most beneficial option and meets the energy demand of the plant.
387
394
395
396
3. CURRENT RESEARCH PRIORITIES IN BIOMASS-TO-ETHANOL
397
From the previous process assessment, several bottlenecks emerge. Biomass to
398
399
bio-ethanol will only be a technical and economic viable alternative to first generation
bio-ethanol, if appropriate solutions are developed. Current production problems
400
hence determine immediate and future research priorities.
17
401
Pre-treatment, as the first step accounts for about 33% of the total cost [63]. Better
402
403
and cost-efficient pre-treatment techniques need further investigation, together with
methods to reduce or eliminate microbial and chemical contaminants that can reduce
404
the yields. It was already stated that membrane techniques could help to overcome
405
some of the problems, with microfiltration (suspe nded solids) and ultrafiltration,
nanofiltration or reverse osmosis dealing with dissolved contaminants. The possible
406
407
408
application of microfiltration to eliminate suspended solids has recently been
confirmed by Kang et al. [29].
409
411
In Ultrafiltration (UF), solutes of high molecular weight are retained in the so-called
retentate, while water and low molecular weight solutes pass through the
412
semi-premeable membrane in the permeate. Ultrafiltration is used in industry and
413
research for purifying and concentrating macromolecular (10 3 -106 Da) solutions,
especially protein solutions. Removal of suspended solids prior to feeding the
410
414
415
416
membrane is essential to prevent damage to the membrane and minimize the effects of
fouling which greatly reduce the separation efficiency.
417
418
419
420
Nanofiltration and Reverse Omosis are high-pressure membrane filtration processes
used most often with low total dissolved solids water (surface water and fresh
groundwater), for softening (polyvalent cation removal) and removal of disinfection
422
by-product precursors such as natural organic matter and synthetic organic matter.
These membrane separation technologies have been examined in different stages of
423
the bio-ethanol production.
421
424
425
Enhancing ethanol production by pre-treatment involving fungi (e.g. T. reesei and
426
427
Basidiomycetes) with appropriate lignocellulolytic properties at low pH and high
temperatures is also a promising and added- value step in SSF ethanol bioconversion.
428
While fungi act slowly, potential lignocellulolytic fungi have been produced by
429
430
mutagenesis, gene expression and co-culturing [81]. Some genera, such as Candida,
Pichia and Dekkera were isolated from sugarcane molasses, but resulted in low
431
ethanol concentrations and produced acetic acid, an inhibitor of the fermentative yeast
432
[82]. Some natural wild yeast species appear capable of replacing S. cerevisiae in
second- generation bioethanol [83], but their low bio-ethanol yield and poor survival
433
435
in the fermenter need further improvement. As described before, some groups of
bacteria such as Zymomonas mobilis can convert sugars into ethanol [83], but they are
436
more vulnerable to chemical inhibition than S. cerevisiae.
434
437
438
The
development
of
genetically
modified
fermentative
and
cellulolytic
439
microorganisms is recommended to increase the ethanol yield and productivity under
18
440
the stress conditions of high production bio-ethanol processes [83]. Simultaneous
441
442
saccharification and fermentation (SSF), simultaneous saccharification and combined
fermentation (SSCombF) of the enzymatic hydrolyzate, and CBP are also considered
443
to be cost-effective whilst reducing end-product inhibition. Genetic engineering has
444
succeeded in altering the conventional S. cerevisiae's capacity to ferment glucose and
pentose sugars simultaneously [84]. Almaida et al. [85] investigated a modified S.
445
447
cerevisiae, capable of co-fermenting saccharides but can also generating less furfural
inhibitors. As mentioned before, CBP combines hydrolysis and fermentation
448
operations in a single reactor, by using genetically modified microorganisms that
449
450
produce cellulase enzyme to ferment sugars in a single step. This avoids the costs
related to the purchase of cellulolytic enzymes [86]. Lignin should be considered as a
451
valuable energy source, used as a fuel in a CHP and being capable of supplying the
452
power and heat requirements of the complete conversion process.
446
453
455
Genetic engineering, as a powerful biotechnological tool, is required to design new
strategies for increasing the ethanol fermentation performance. Upregulation of stress
456
tolerance genes by recombinant DNA technology can be a useful approach to
457
overcome inhibitory situations [87]. Ge et al. [88] obtained three recombinants:
HDY-ZMYWBG1, HDY-ZMYWBG2 and HDY- ZMYWBG3 using the lithium
acetate transformation method into the S. cerevisiae cells. The ethanol yield for
454
458
459
460
461
HDY-ZMYWBG1 and HDY- ZMYWBG3 are 0.368 g/g and 0.365 g/g, respectively.
The resulting consortium was demonstrated to utilize phosphoric acid swollen
463
cellulose (PASC) for growth and ethanol production. The final ethanol production of
1.25 g/L corresponded to 87% of the theoretical value and was 3-fold higher than a
464
similar yeast consortium secreting only the three cellulases [89]. Reconstitution of the
465
466
N. crassa cellodextrin transport system in Saccharomyces cerevisiae promotes
efficient growth of this yeast on cellodextrins [90,91]. The engineered yeast strains
467
more rapidly convert cellulose to ethanol when compared with yeast lacking this
468
469
system. Ha et al. [92] engineered yeasts to coferment mixtures of xylose and
cellobiose. It improved ethanol yield when compared to fermentation with either
470
cellobiose or xylose as sole carbon sources. This is a critical step towards enabling
471
economic biofuel production.
462
472
474
Since fermentative microorganisms must be capable of surviving the high
temperatures of SSF/SSCombF/CBP processes, further research is required: high
475
temperature ethanol fermentation is an emerging technology provided appropriate
476
477
microorganisms can be developed. Such high temperature operations do not require
cooling and cellulase addition [93]. The thermo-tolerant yeast, K. marxianus has been
478
documented as a candidate for its ability to co-ferment both hexose and pentose
473
19
479
sugars and survive temperatures of 42-45 °C [94]. K. marxianus was moreover
480
481
genetically modified to exhibit T. reesei and Aspergillus aculeatus cellulolytic
activities allowing direct and continuous conversion of cellulosic β-glucan into
482
ethanol at 48 °C, yielding 0.47 g/g ethanol, i.e. 92.2% of the theoretical yield and
483
proving to be an ideal gene modified organism (GMO) for CBP processes [94].
484
486
The industrial potential for S. cerevisiae fermentation has already been proven for first
generation large-scale bio-ethanol production. Its genetic improvement is gaining
487
increasing research, especially with respect to the CBP option [95,96], where
488
489
hydrolysis and substrate fermentation are possible in a single step.
Z. mobilis remains an attractive candidate due to its high ethanol yield and resistance
490
to temperatures in the range of 40 °C [72]. Numerous genes have been introduced and
491
heterologous expression has been incorporated into Z. mobilis to extend its
effectiveness toward other substrates namely, xylose and arabinose [97]. Both the
485
492
493
494
gene engineered Z. mobilis and S. cerevisiae
adaptability [98].
have proven a high ethanol yield and
495
496
497
498
Further research is certainly required in optimizing biological pretreatment involving
fungi (e.g. T. reesei and a Basidiomyctes) that exhibit lignocellulolytic properties at
low pH levels and high temperature.
499
500
The use of GMOs is questionable, since their introduction into large-scale
501
502
fermentation operations can pose risks of environmental dissemination and potential
exposure risks to public health. Industrial operations using antibiotics to control
503
microbial contaminants in fermenters or as strain markers would generate and release
504
antibiotic resistant organisms and offer another potential enviro nmental and public
health risk.
505
506
508
Improvement in each of these individual aspects is required to achieve high
conversion and cost-effective biomass-to-bio-ethanol operations. This needs to be
509
complemented by a comprehensive systems approach, encompassing the different
510
individual steps, and accounting for all inputs and outputs during the entire operation
regardless of modifications in any of these individual steps.
507
511
512
513
4. CONCLUSIONS
514
The cellulosic bio-ethanol production process involves specific processing steps,
515
especially in the pre-treatment and hydrolysis. Fermentation of C5 and C6 sugars
needs adapted microorganisms, still to be further investigated.
516
517
20
518
New combined processes reduce both the number of operation steps and the
519
520
production of chemical inhibitors. Recent advances in genetically engineered S.
cerevisiae and Z. mobilis are promising for higher alcohol tolerance and conversion
521
efficiency. The bottlenecks of the present processing that second generation
522
bio-ethanol provided are removed and the best combination of advanced systems is
used.
523
524
525
ACKNOWLEDGEMENTS
526
This work was supported by Chinese Science Council, in support of the delegation of
527
the principal author to the KU Leuven.
528
529
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530
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