Bietanol Segunda Generación Review 2014

Review
Received: 23 October 2013
Revised: 1 September 2014
Accepted article published: 5 September 2014
Published online in Wiley Online Library: 1 October 2014
(wileyonlinelibrary.com) DOI 10.1002/jctb.4544
The potential of microbial processes

for lignocellulosic biomass conversion
to ethanol: a review
Davide Dionisi,a* James A. Anderson,a,b Federico Aulenta,c Alan McCueb
and Graeme Patond
Abstract
BACKGROUND: This paper assesses the feasibility of a single- or multi-stage process entirely based on microbial cultures, with no
or minimal non-biological pretreatment and with no external enzyme addition, for the conversion of lignocellulosic materials
into ethanol. The process considered involves three distinct microbial processes, which can possibly combined in one single
reaction stage: (a) lignin hydrolysis; (b) cellulose and hemicelluloses hydrolysis; and (c) glucose fermentation to ethanol. This
paper critically reviews the literature on the three microbial processes and compares the rates of microbial processes with those
of the alternative physico-chemical pretreatment processes.
RESULTS: There is a large number of microbial species that can perform each of the three processes required for the conversion of
lignocellulosic biomass to ethanol, although only one species has been unquestionably reported, so far, to be able to hydrolyse
lignin under anaerobic conditions; another challenge is controlling the anaerobic fermentation of glucose to ethanol with mixed
cultures; the rates of the microbial processes reported so far in the literature are generally lower than the rates obtained with
physico-chemical pretreatments.
CONCLUSIONS: While in principle the whole process from lignocellulosic biomass to bioethanol can be carried out with existing,
non-engineered microorganisms, there is a need for further research to obtain rates and yields which are commercially
attractive.
2014 Society of Chemical Industry
Keywords: bioethanol; cellulose; lignin; microbial hydrolysis; mixed cultures
INTRODUCTION
366
Second generation bioethanol produced from lignocellulosic

biomass is gaining increasing interest, since it can avoid the
fuel vs. food competition which is intrinsic in rst generation
bioethanol produced from starch- or sugar-based materials such
as corn or sugarcane.1 Lignocellulosic materials such as the organic
fraction of municipal solid wastes, agricultural wastes, forestry
residues, etc. represent an almost unlimited resource, which could
potentially be used for bioethanol production.2 However, production of bioethanol from lignocellulosic materials poses signicant
technical and economic challenges. According to a UN panel of
experts,3 in 2012 biofuels production from lignocellulosic feedstock accounted for some 140 million litres per year, only 0.15%
of the total production. The worlds rst commercial scale cellulosic ethanol plant is considered to be the one in Crescentino, Italy,
by Beta Renewables, with a full capacity of 75 million litres a year,
which was started up in 2013.4
The bioethanol production process from lignocellulosic materials usually consists of several steps: pretreatment to break the
lignin structure and make cellulose (and/or hemicellulose) available for hydrolysis, hydrolysis to hydrolyse cellulose to glucose,
fermentation to convert glucose into ethanol and separation
processes for ethanol purication.5,6 As far as lignin hydrolysis is concerned, the most widely adopted pretreatments are
J Chem Technol Biotechnol 2015; 90: 366383
steam explosion, acid hydrolysis, or ammonia bre expansion

(AFEX).7 As an example, the Iogen demonstration plant (Canada),
which has an average productivity of about 300 m3 ethanol yr-1
receives wheat straw, corn stover and bagasse as feedstocks
and uses a modied steam explosion process to make cellulose
more accessible for the following steps (Iogen, (http://www.
iogen.ca/technology/cellulosic-ethanol.html)). These chemical
or physical pretreatment processes usually give good lignin
hydrolysis and they make cellulose and hemicelluloses free for
Correspondence to: Davide Dionisi, Materials and Chemical Engineering Group,

School of Engineering, University of Aberdeen, Aberdeen, AB24 3UE, UK. E-mail:
davidedionisi@abdn.ac.uk
a Materials and Chemical Engineering Group, School of Engineering, University

of Aberdeen, Aberdeen, AB24 3UE, UK
b Surface Chemistry and Catalysis Group, School of Natural and Computing
Sciences, University of Aberdeen, Aberdeen, AB24 3UE, UK
c Water Research Institute, National Research Council (CNR-IRSA), Via Salaria km
29.300 C.P. 10, 00015, Monterotondo (RM), Italy
d Department of Plant and Soil Science, University of Aberdeen, Cruickshank
Building, St. Machar Drive, Aberdeen, AB24 3UU, UK
www.soci.org
Conversion of lignocellulosic biomass into ethanol
www.soci.org
the next hydrolysis stages. However, they require conditions

of high temperature and/or high pressure, or the addition of
signicant amounts of chemicals. These conditions make these
processes expensive and limit their economic attractiveness at
commercial scale. Also, the severe operating conditions used in
these processes may generate toxic substances which inhibit
the following stages and therefore a detoxication stage is often
necessary.8 The subsequent step of the process is cellulose and
hemicellulose hydrolysis to generate glucose and other sugars.
This step is usually carried out enzymatically, using commercially
available or on-site produced cellulase enzymes. For example,
the Iogen demonstration plant mentioned above and the Abengoa demonstration plant (Abengoa, http://www.abengoa.com/
web/en/innovacion/casos_exito/), which uses wheat and barley
straw as feedstock, both use enzymatic hydrolysis. Finally, ethanol
production from glucose is usually carried out using pure cultures
of selected species, usually the yeast Saccharomyces cerevisiae.
While this yeast allows a very high ethanol yield and high ethanol
productivity, the use of pure cultures has some disadvantages:
pure cultures usually have a very narrow substrate spectrum, e.g.
non-engineered S. cerevisiae cannot metabolise xylose, the main
component in hemicellulose, and this limits the range of feedstock
that can be used; pure cultures of fermentative microorganisms
usually cannot hydrolyse cellulose, and this requires the addition
of the cellulose hydrolysis stage; the use of pure cultures requires
sterilisation of the fermentation vessel and of all the process lines
leading to them, and this causes additional costs.
All of these factors contribute to the high cost of ethanol production from lignocellulosic biomass. This paper investigates,
by critically analysing the relevant literature, the feasibility of an
alternative process for ethanol production from lignocellulosic
biomass. In contrast to the physico-chemical pretreatment and

hydrolysis processes described above, the process investigated
in this paper is entirely, or almost entirely, based on microbial
processes, i.e. based on the physical contact of microorganisms
with the substrate. In the investigated process, the three distinct
processes required to convert lignocellulose to ethanol, i.e. lignin
hydrolysis, cellulose and hemicellulose hydrolysis and glucose and
other sugars fermentation, are all carried out by dierent microorganisms, which could co-exist in the same reactor, or could be
present in dierent reactors in sequence. Obviously, the option of
a single reactor where all the dierent microbial species co-exist
would be preferable from an economic point of view. This would
constitute an open (undened) mixed culture, where the microorganisms responsible for the various processes are selected from
a mixed-culture inoculum due to the applied process conditions
(e.g. nature of the feedstock, residence time, pH, temperature). The
process investigated in this study has many aspects in common
with simultaneous saccharication and cofermentation (SSCF)
and consolidated bioprocessing (CBP) processes, described by
Lynd et al.9 In SSCF an aerobic reactor is used for cellulase production, and the cellulases produced are then used in a subsequent
anaerobic reactor where cellulose hydrolysis and sugars (both
hexoses and pentoses) fermentation to ethanol takes place. In
CBP the production of cellulases and the fermentation of sugars
all take place in the same anaerobic reactor. However, there are
two main dierences between SSCF and CBP process and the
process investigated in this study (Fig. 1): SSCF and CBP are usually
thought to come after a chemical-physical pretreatment step
for lignin hydrolysis, while the process considered here includes
microbial lignin pretreatment; SSCF and CBP are usually thought
of as pure culture processes, using native microorganisms or
A
Lignocellulosic
biomass
Combined reactor for lignin

Ethanol to purification
hydrolysis, cellulose
hydrolysis and carbohydrates
fermentation (anaerobic)
B
Lignocellulosic
biomass
Lignin
cellulose
hydrolysis
Cellulose carbohydrates
hydrolysis
Fermentation
Ethanol to
purification
C
Lignocellulosic
biomass
Chemical or
physical
pretreatments
cellulose
Cellulase
production
cellulase
Cellulose Ethanol to
hydrolysis purification
and
fermentation
D
Lignocellulosic
biomass
Chemical or cellulose
physical
pretreatments
Combined reactor for

cellulose hydrolysis and
carbohydrates fermentation
(anaerobic)
Ethanol to
purification
wileyonlinelibrary.com/jctb
367
Figure 1. Possible schemes for conversion of lignocellulosic biomass to ethanol. (A) and (B) are the entirely microbial processes considered in this study.
Scheme (C) is SSCF, scheme (D) is CBP. See text for denitions of abbreviations. Not all the material ows are shown in the schemes and cellulose also
includes hemicellulose.
www.soci.org
D Dionisi et al.
Table 1. Approximate composition (% of dry weight) for various lignocellulosic materials. Minor components such as ash, proteins, etc are not
included in the table
Cellulose and hemicelluloses
Material
Lignin
glucose
Corn stover
Wheat straw
Rice straw
Leaves
Paper
Newspaper
Switchgrass
Poplar
Eucaliptus
Pine
Spruce
Angiosperms
Conifers
21
15
10
0
015
1830
23
29
28
28
28
1824
2732
40
32
41
xylose
22
3540
15
3
48
5
other carbohydrates
1
48
2
8599
20
15
11
6
7
1226
510
LIGNOCELLULOSIC BIOMASS
368
The main components of lignocellulosic biomass are lignin, cellulose and hemicelluloses. Lignin is an aromatic polymer in which
105
106,107
45
106
106
6080
32
40
50
45
45
4252
4346
Ref
106
95100
genetically engineered ones, while the process studied in this

paper is aimed at using open mixed microbial cultures.
Microbial processes based on open mixed cultures are gaining
increasing interest due to their lower cost and higher exibility
compared with the traditional pure culture processes.10 A well
known industrial process involving the use of open mixed cultures
is anaerobic digestion of organic materials to methane,11 whereas
more recently the use of mixed cultures to produce hydrogen,12
ethanol,13 and biodegradable plastics14 16 has been investigated.
An interesting mixed culture process is the MixAlco process,17
which converts biomass to carboxylic acids salts, which are then
chemically converted to hydrocarbon fuels.
An entirely microbial process, if proven feasible, would have
obvious cost advantages compared with existing processes for
lignocellulosic ethanol production, due to the use of atmospheric
pressure, temperature close to ambient, and no addition of expensive chemicals or enzymes. The aim of this paper is to review the
literature on the individual processes that are necessary to obtain
microbial conversion of lignocellulosic materials into bioethanol,
i.e. microbial lignin and cellulose hydrolysis and glucose and
xylose fermentation by mixed cultures. This review for the rst
time reports rate values from the literature studies and critically
discusses the eect of process parameters, in order to help identify
the conditions that maximize the rates and yields of the microbial
processes.
In principle, an alternative microorganism-based approach to
convert lignocellulosic materials into ethanol is to use genetically
modied microorganisms, as opposed to open mixed cultures of
naturally occurring species. The rationale behind this is that the
ability to hydrolyse lignin and cellulose/hemicelluloses and to ferment sugars to ethanol with high yields could be introduced into
microorganisms using metabolic engineering techniques. Without
attempting to review the vast area of metabolic engineering for
ethanol production, which has been reviewed elsewhere,18,19 this
paper compares the use of open mixed cultures and of genetically
modied microorganisms for lignin and cellulose hydrolysis and
for glucose and xylose fermentation to ethanol.
arabinose
4
1
<1
2
1
0.50.6
0.52
<1
2
2
14
15
24
914
105
105
108
108
109
110
110
the substituents are connected by ether and carbon-carbon linkages. The main building blocks in lignin are p-coumaryl alcohol
(p-hydroxyphenyl propanol), coniferyl alcohol (guaiacyl propanol)
and sinapyl alcohol (syringyl propanol).20 The molecular weight
of lignin is variable but is typically very high, 6001000 kDa.21
Cellulose is a polysaccharide composed of (14) linked D-glucose
units, with molecular weight up to >500 kDa.22 Hemicellulose is
a polysaccharide composed of various carbohydrate monomers,
mainly xylan, arabinose, mannose and glucose, present in dierent
ratios in the various materials. The molecular weight of hemicelluloses is usually lower than that of cellulose.23 The composition
of various lignocellulosic materials, potential feedstock for ethanol
production, is reported in Table 1. Values in Table 1 are to be considered as orientative values only, because the measured lignin
content in a given biomass species is highly dependent on the
biomass history and on the measurement method used.24
The hemicellulose content in the feedstock is important due
to the fact that hemicellulose hydrolysis generates monosaccharides other than glucose, which need to be converted to
ethanol in order to avoid yield loss in the process. The conversion of non-glucose sugars to ethanol poses signicant challenges as discussed later in this paper. However, hemicellulose
hydrolysis is not considered to be a signicant challenge, since
hemicellulose is hydrolysed more easily than cellulose,25 and it
is expected that a mixed microbial culture that hydrolyses cellulose would also be able to hydrolyse hemicelluloses.9 Therefore, in this paper only the hydrolysis of lignin and cellulose is
discussed.
MICROBIAL DEGRADATION
OF LIGNOCELLULOSIC BIOMASS
The aim of this section is to identify, on the basis of the existing
literature, whether there are microorganisms that are able to
catalyse the various steps required for lignocellulosic biomass
conversion to ethanol, i.e. lignin hydrolysis, cellulose hydrolysis
and sugars fermentation to ethanol. Both pure culture and mixed
cultures of naturally existing microorganisms and genetically
modied microorganisms are considered. The rates of microbial processes are reported and compared with the rates of the
alternative chemical-physical processes.
www.soci.org
Lignin
Anaerobic conditions
High molecular weight lignin is generally considered to be nonbiodegradable, or only biodegradable at insignicant rates, in the
absence of oxygen,21,26,27 even though there is some evidence to
the contrary.28,29 On the other hand, there is enough evidence
to suggest that the various lignin building blocks, constituted
by the various aromatic compounds as monomers or oligomers,
are readily metabolized under anaerobic conditions.30,31 The very
limited evidence of anaerobic lignin biodegradation reported in
the literature can be attributed21 to non-lignin components or
to low-molecular weight materials (<600 Da). However, Benner
et al.29 using mixed cultures, reported anaerobic lignin biodegradation rates of up to 37% of the aerobic rates, for several lignocellulosic marine or wetland plants. Silanikove and Brosh28 reported
4558% anaerobic metabolization of lignin in wheat-straw, by the
rumen bacteria in the goats gastrointestinal tract.
Only very recently, convincing evidence of lignin degradation under anaerobic conditions has been presented.32 The
authors observed that the majority (85%) of insoluble switchgrass
biomass, that had not previously been chemically treated, was
converted at 78 C by the anaerobic bacterium Caldicellulosiruptor bescii. Interestingly, the glucose/xylose/lignin ratio did not
substantially change over the incubation period (3 successive
cycles of 5 days each) providing an indication that the three
major biomass components, including lignin, were solubilized
and/or metabolized at comparable rates. A mass balance revealed
that lignin was not assimilated, only carbohydrates served as
carbon and energy sources. Lignin degradation was conrmed
by gas-chromatographymass spectrometry analyses which
revealed the presence of lignin-derived aromatic compounds,
such as syringylglycerol, guaiacylglycerol, and phenolic acids, in
the spent culture broth.
Indirect evidence of lignin degradation/hydrolysis under anaerobic conditions can be obtained observing anaerobic digestion
studies, which are always carried out using open mixed microbial
cultures, with lignocellulosic materials as feedstock (Table 2). In
general, methane production has been reported with many lignocellulosic materials, even though the possible lignin degradation
and its extent are not usually measured. Methane production has
been reported even with feedstocks with high lignin content, such
as Mirabilis and Ipomoea stulosa leaves33 and woody biomass
such as poplar.34 Several studies investigated the eect of lignin
content on the anaerobic degradability of lignocellulosic biomass.
Triolo et al.35 observed that the higher the lignin content in the raw
material the lower the methane production. However, Tong et al.36
found only a poor correlation between the lignin content of various lignocellulosic substrates and their methane production rate.
This indicates that biodegradation of lignocellulosic substrates is
not only aected by the lignin content, but also by other factors
such degree of association between lignin and carbohydrates, cellulose crystallinity and others.37 According to Turick et al.,34 one of
the reasons why many investigators observed only poor methane
production from woody or other highly lignied biomass is that
the time length of the tests is not long enough. These authors
carried out tests for 100 days and observed substantial methane
production from woody biomass. Interestingly, they observed that
most of the methane production occurred after more than 50 days
from the start of the test, and they explained this behaviour by
the need of the microorganisms to become able to degrade lignin,
making the cellulosic materials initially shielded by lignin available.
Overall, even though so far only limited evidence for anaerobic
lignin metabolization or solubilization is available, it is apparent
that a signicant fraction of the cellulose or hemicellulose carbohydrates in lignocellulosic materials becomes available during anaerobic digestion, and this indicates that lignin hydrolysis probably
occurs under anaerobic conditions.
Aerobic conditions
In contrast to anaerobic conditions, there is wide literature evidence that lignin is biodegraded under aerobic conditions. Various
species of bacteria and fungi have been reported to biodegrade
lignin (Table 3). Fungi, in particular white-rot fungi, have been studied more extensively than bacteria for their lignin biodegradation
ability, and they are generally considered more interesting than
bacteria as a pretreatment of lignocellulosic materials at industrial
scale.38 Aerobic biodegradation of lignin has also been reported
Table 2. Evidence of anaerobic biodegradation of lignocellulosic materials by mixed cultures
Feedstock
8.6
10
11
20
25
17
10
27
Time
(days)
Measure of degradation
0.180.22 m3 CH4 kg-1 VS, 3070% neutral detergent bres reduction
0.160.25 m3 CH4 kg-1 VS, 2638% cellulose reduction
0.13 (average) m3 CH4 kg-1 VS
0.250.33 m3 CH4 kg-1 VS
0.300.33 m3 CH4 kg-1 VS (7078% of TBMP)
0.36 m3 CH4 /kg VS (84% of TBMP)
0.29 m3 CH4 kg-1 VS (66% of TBMP)
0.270.31 m3 CH4 kg-1 VS (7080% of cellulose control)
0.270.29 m3 CH4 kg-1 VS (7074% of cellulose control)
0.27 m3 CH4 kg-1 VS (70% of cellulose control)
0.32 m3 CH4 kg-1 VS (82% of cellulose control)
0.190.21 m3 CH4 kg-1 VS
65 days
8 weeks
8 weeks
8 weeks
8 weeks
25 weeks
1736 days
70 days
70 days
70 days
100 days
100 days
100 days
100 days
Ref
49
33
33
33
33
111
112
36
36
36
34
34
34
34
113
369
Sisal bre waste

Wheat straw
Rice straw
Mirabilis leaves
Ipomoea stulosa leaves
Lignocellulosic (woody) biomass
Wheat straw
Wheat straw
Corn stover
Wood grass
Salix eriocephala (pussy willow)
Salix lucida (shining willow)
Populus sp. (hybrid poplar)
Platanus occidentalis (sycamore)
Water hyacinth
Lignin content in
the feedstock
(% of dry weight)
www.soci.org
D Dionisi et al.
Table 3. Microorganisms reported to degrade lignin under aerobic conditions

Microbial species
Bacteria
Pseudomonas spp.
Acinetobacter spp.
Pseudomonas spp.
Xanthomonas spp.
Mixed culture
Pseudomonas spp.
Streptomyces badius
Streptomyces viridosporous
Streptomyces cyaneus
Thermomonospora mesophila
Fungi
Pleurotus ostreatus
Phanerochaete chrysosporium
Echinodontium taxodii 2538
Trametes versicolor spp.
Trametes ochracea spp.
Ganoderma spp.
Ceriporia lacerata
Stereum hirsutum
Polyporus brumalis
Substrate
Extent of degradation (%)
Kraft lignin
Poplar wood
Poplar wood
Poplar wood
Wood our
Wood our
Indulin lignin
Indulin lignin
Barley straw
Barley straw
Cotton stalks
Cotton stalks
Cotton stalks
Bamboo culms
Bamboo culms
Bamboo culms
Bamboo culms
Synthetic lignin
Red pine
Red pine
Red pine
Time (days)
Ref
39
4757
4052
3948
80
20
34
34
2952
3648
52
30
30
30
4060
4060
35
35
21
21
114
40
60
28
24
924
519
516
Up to 38
13
15
12
30
30
14
28
28
42
115
115
115
41
41
116
116
27
48
42
47
43
43
43
43
40
35
56
56
56
45
45
45
Table 4. Evidence of aerobic biodegradation of lignocellulosic materials by mixed cultures

Substrate
Ryegrass straw
Horse manure, wheat straw
Canola residue (Brassica campestris)
Wheat leaves
Spruce groundwood
Sewage sludge and green plant waste
Agricultural organic waste
Wheat straw, root vegetables residues, bran and soild
Olive-mill wastewaters and wheat straw
Rice straw
% lignin (initial)
12
20
11
6
2327
25
10
370
in mixed culture studies, usually carried out in composting environments (Table 4). While substantial lignin degradation has been
reported for a wide range of lignocellulosic substrates, usually
the treatment times are quite long and lignin degradation is not
complete.
The literature evidence so far indicates that lignin cannot be
used as a sole carbon and energy source, but requires an additional
substrate to support microbial growth.21 The growth substrate can
be the glucose or carbohydrates units contained in the cellulose
or hemicellulose inside the lignin matrix,39 or can be externally
added. This is important from the process point of view, since it is
expected that microbial lignin depolymerization may require the
use of part of the cellulose and hemicellulose as growth substrate
for the microorganisms, making it not available for conversion to
ethanol. However, in a mixed culture environment with real lignocellulosic biomass as feedstock, other carbon and energy sources
than polysaccharides may be available (e.g. proteins) and so the
loss of carbohydrates for ethanol production may be avoided.
727% lignin degradation
CO2 evolution 1040% of the maximum
80% straw degradation
Time (days)
45
47
154
224
4569
135
45
50
90
9
Ref
117
118
119
120
121
122
123
124
125
102
Eect of pH
The optimum pH for aerobic lignin degradation by fungi is approx.
4.04.5 and signicantly lower biodegradation rates are observed
when the pH is lower than 4 or above 5.21,40 pH in the range 45
is considered the optimum for the growth of most white-rot fungi.
For bacteria, no dierence in lignin degradation rate was observed
in the pH range 5.37.8, for a mixed culture.41 These studies
seem to indicate that the optimum pH for lignin biodegradation
coincides with the usual optimum pH for microorganism growth
on carbon substrates.
Eect of feedstock particle size
The biodegradation of lignin occurs extracellularly and by decreasing the particle size it is expected that the surface to volume
ratio of lignocellulosic biomass increases, so an increase in lignin
biodegradation rate is expected. Limited experimental investigation has been carried out, however, on the eect of feedstock
www.soci.org
Table 5. Lignin degradation rates, calculated by the authors of this paper based on literature data
Microorganism
Degradation rate (g L-1 h-1 )
Substrate
Irpex lacteus
Irpex lacteus
Trametes versicolor MrP 1
Trametes versicolor MrP 1
Streptomyces cyaneus
Thermonospora mesophila
Pleurotus ostreatus
Echinodontium taxodii 2538
Trametes versicolor G20
Ganoderma sp En3
Acinetobacter spp.
Wood chips of Pinus strobes

Wood chips of Liriodendron tulipifera
Wood chips of Pinus strobes
Wood chips of Liriodendron tulipifera
Barley straw
Barley straw
Cotton stalks
Cotton stalks
Bamboo culms
Bamboo culms
Bamboo culms
Cotton stalks
Poplar wood
Ref
126
0.007
0.014
0.004
0.020
0.0045
0.004
0.05
0.1
0.04
0.04
0.03
0.040.06
0.00010.0002
126
126
126
48
48
42
42
43
43
43
47
115
Table 6. Rates of lignin degradation with non-biological pretreatments reported in the literature 7
Pretreatment technology
Pretreatment conditions
Temperature ( C)
Chemical loading
Reaction time (min)
Lignin degradation rate (g L-1 h-1 )
Dilute acid
140
1% H2 SO4
40
4.8
SO2 -enhanced
steam explosion
180
0.05 gSO2 g-1 biomass
10
22
particle size on lignin biodegradation and usually studies are carried out with a single feedstock size, which is in the mm42 44 or
cm45,46 range or not reported.47 Zimmerman and Broda48 observed
higher lignin degradation for straw pre-treated with a vibratory
ball mill (particle size 25 m) than for straw ground with a blender
(particle size 0.51 mm). Even though still limited, investigation
of the eect of particle size has been carried out in the area of
anaerobic digestion of lignocellulosic biomass. Mshandete et al.49
observed that total bre degradation during anaerobic digestion
to methane increased from 31% to 70% when the feedstock was
ground to 2 mm bres compared with untreated bres (larger than
100 mm). Sharma et al.33 observed that the quantity of biogas produced increased when the feedstock particle size was reduced. The
range of sizes in their study was 0.130 mm. However, they did not
specically investigate lignin degradation.
200
None
10
23
Aqueous
ammonia
Lime
160
15% NH4 OH
60
11.9
120
1 gCa(OH)2 + 100 psi O2
240
1.6
It is worth comparing the lignin degradation rates reported in

Table 5 with the rates obtained with non-biological pretreatment
stages reported in the literature7 (Table 6). It is evident that the
lignin degradation rates obtained with fungi are in general at least
one order of magnitude lower than those obtained with chemical
pretreatments. However, the process conditions required by the
chemical pretreatments are much more severe, with much higher
temperature and usually (with the exception of the hot water
treatment) with the addition of chemicals, which obviously cause
higher process costs.
Lignin hydrolysis by genetically modied microorganisms
While the focus of genetic engineering for lignin hydrolysis
has been on genetically manipulating lignin biosynthesis in
plants in order to reduce lignin content and make its hydrolsysis
easier,50,51 there are so far no reported attempts to genetically
modify microorganisms in order to make them capable of lignin
hydrolysis.52 The reason for this is probably the large number
of enzymes which are potentially dedicated to lignin hydrolysis
in naturally occurring lignin-hydrolysing microorganisms, such
as the white-rot fungus Phanerochaete chrysosporium.53,54 It is
possible, however, that only a few out of the whole spectrum of
lignases might be needed in industrial processes,52 therefore making genetic engineering of microorganisms for lignin hydrolysis a
more feasible option.
Cellulose
Unlike the case of lignin, a wide range of bacteria and fungi have
been reported to hydrolyse cellulose under anaerobic or aerobic
conditions.
371
Lignin degradation rates

The main limitation of microorganisms-based pretreatment
processes for lignocellulosic biomass conversion to ethanol is
considered to be the low rate. However, very limited direct information on the rate of lignin hydrolysis under aerobic conditions
is present in the literature. Table 5 reports lignin hydrolysis rates
using fungi under aerobic conditions, calculated by the authors
of this paper on the basis of literature data. The maximum rate
is approx 0.1 g L-1 h-1 . Under anaerobic conditions, the only evidence of lignin degradation32 gives a lignin degradation rate of
0.012 g L-1 h-1 . It is important to observe that the rates reported in
Table 5 have been obtained at lab scale with very small volumes
and under non-optimized conditions. Several variables could, in
principle, be optimized to maximize the lignin degradation rate:
biomass concentration, oxygen concentration, pH and particle
size of the feedstock.
Liquid hot
water
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D Dionisi et al.
Table 7. Microorganisms reported to hydrolyse cellulose under anaerobic conditions
Microorganism
Extent of
degradation (%)
Substrate
Ruminococcus albus
Clostridium thermocellum
Ruminococcus avefaciens
Clostridium cellulolyticum
Caldicellulosiruptor bescii
Bacteroides succinogenes + Selenomonas ruminantium
Clostridium thermocellum + Clostridium thermohydrosulfuricum
Fibrobacter Succinogenes
Avicel PH-105
MN300
Sigmacell 20
MN301
MN300
Switchgrass
Ball milled Whatman no.1 Filter paper
SW40
MN300
Sigmacell 20
3070
100
5487
2075
45
85
100
80
100
5479
Time
(days)
0.52.5
4
0.32
0.53
4
15
NR
5
5
0.53
Ref
127
128
55
129
130
32
131
132
132
56
Table 8. Anaerobic degradation of cellulose by mixed cultures

Substrate
Filter paper
BW200
Paper (hardwood and softwood pulp)
Cellulose
Sigmacell 50
Cellulose powder
99% (as CH4 production compared to glucose)
100% (as CH4 production compared to glucose)
62% reduction in cellulose
Anaerobic conditions
Table 7 summarizes bacteria and fungi that have been reported
to hydrolyse cellulose under anaerobic conditions. Unlike lignin,
complete cellulose hydrolysis can be obtained under anaerobic
conditions, provided that the contact or residence time is adequate. Table 8 reports literature evidence for cellulose degradation
by mixed cultures under anaerobic conditions, conrming that virtually complete cellulose hydrolysis is possible. Most of the data in
Table 8 refer to batch tests with an unacclimated inoculum, and
this explains the long time required for cellulose degradation.
The enzyme groups responsible for cellulose hydrolysis are very
similar under anaerobic and aerobic conditions but the spatial
arrangement of the enzymes can be dierent.9 Under anaerobic
conditions cellulolytic enzymes are often bound to the external
membrane of the cell, even though in some cases they are present
as free enzymes in the liquid medium. Under aerobic conditions
the enzymes are usually excreted in the liquid medium and are not
attached to the cell membrane.
Time (days)
133
69
13
9 (HRT)
20
5
Ref
36
36
133
134
135
136
Shi and Weimer55 found an optimum pH of 6.5 for cellulose hydrolysis with Ruminococcus avefaciens, and Weimer56 with Fibrobacter succinogenes found very little inuence of pH on cellulose
hydrolysis in the pH range 6.16.8. Using ruminal microbes under
anaerobic conditions Hu et al.57,58 found no cellulose hydrolysis at
pH < 5.5 and a very low rate at pH < 6.0, the optimum pH being
7.07.5. Berquist et al.59 reviewed various cellulolytic thermophilic
bacteria, employing either aerobic or anaerobic conditions, and
reported an optimum pH in the range 7.08.1 in most cases. This
reported evidence is consistent with a study60 on the anaerobic
hydrolysis of organic waste, partially composed of lignocellulosic
biomass, where an approximately 3-fold increase in the hydrolysis
rate was observed when the pH was increased from 5 to 7.
372
Aerobic conditions
Table 9 summarizes microorganisms that have been reported to
hydrolyse cellulose under aerobic conditions. Consistent with ndings for anaerobic conditions, virtually complete cellulose hydrolysis can be obtained under aerobic conditions. Similar evidence is
obtained for mixed cultures studies (Table 10), which usually refer
to composting environments. An interesting observation is that
usually the rate of cellulose hydrolysis is comparable under anaerobic and aerobic conditions.9 In terms of maximizing the rate of
cellulose hydrolysis, this means that no preference should be given
to aerobic compared with anaerobic conditions.
Eect of particle size

In general, as cellulose hydrolysis is dependent on the contact
between the solid cellulose and either the microorganisms or
the excreted cellulolytic enzymes, it is expected that the rate
of cellulose hydrolysis should increase as cellulose particle size
decreases, since the area per unit volume increases. However, the
benecial eect of feedstock particle size reduction is expected to
depend on the substrate-to-microorganisms ratio, as well as on the
particle size.61 A few experimental studies have been conducted
on the eect of particle size on cellulose hydrolysis rate. Weimer
et al.62 observed a decrease in the hydrolysis rate and an increase
in the induction time when the cellulose particle size increased.
Similarly, Hu et al.57 reported faster cellulose hydrolysis rate for 50
than for 100 m sized particles. Similar evidence was obtained in
continuous studies. Chyi and Dague63 observed a faster hydrolysis
rate with 20 than with 50 m particles.
Eect of pH
For anaerobic and aerobic bacteria the optimum pH for cellulose
hydrolysis is usually in the range 6.58.0. For anaerobic bacteria,
Cellulose hydrolysis rates

Table 11 summarizes microbial cellulose hydrolysis rates, calculated by the authors of this paper on the basis of literature data.
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Table 9. Microorganisms reported to hydrolyse cellulose under aerobic conditions

Microorganism
Substrate
Bacteria
Cellulomonas uda JC3
Cellulomonas fermentans
Cytophaga sp. LX-7
Fungi
Trichoderma viride
Trichoderma reesei
Extent of degradation (%)
Avicel
Solka-Floc
CC31 (Whatman)
Filter paper (Whatman no 1)
MN300
Amorphous cellulose
Whatman for Chromatography
MN300
Whatman CF11
Ball milled wood cellulose (BW 200)
Solka Floc 200
Table 10. Aerobic degradation of cellulose by mixed cultures
Substrate
Avicel
Avicel
Cellulose
Avicel
Avicel
Cellulose lter paper
Filter paper
97% of theoretical CO2
84% of theoretical CO2
87% of initial cellulose
95% of theoretical cellulose
79% weight loss
Time
(days)
47
45
3
100
70
72
4
Ref
142
143
144
145
146
147
102
The reported rates for microbial cellulose hydrolysis are, in general,

higher than the corresponding rates for lignin hydrolysis (Table 5),
therefore indicating that the critical stage in the process is the
lignin hydrolysis. Cellulose hydrolysis rates up to about 0.3 g cellulose L-1 h-1 have been reported, both under aerobic and anaerobic conditions. As observed for lignin degradation rates, the rates
reported in Table 11 have usually been obtained with very low volume systems and have not been optimized. Important factors that
can increase the rates of microbial cellulose hydrolysis are: biomass
concentration, cellulose concentration, reactor dilution rate, temperature, pH and cellulose particle size, as discussed in previous
sections. One of the most successful technologies for cellulose
hydrolysis is enzymatic hydrolysis, where the cellulolytic enzymes
are externally generated and added to the liquid mixture. Table 12
reports typical rates for enzymatic cellulose hydrolysis. It is evident that enzymatic hydrolysis is generally faster than microbial
hydrolysis. However it has to be taken into account that enzymatic
hydrolysis has a higher capital and/or operational cost than microbial hydrolysis due to the need for external generation or purchase
of the enzymes.
Ref
15
20
30
35
45
75
70
60
100
5
5
5
5
5
5
5
28
4
137
5075
100
0.51.2
7
140
137
137
137
137
137
137
138
139
141
Saccharomyces cerevisiae67 and others. However, while the results

are in general promising and encourage further research in this
area, there is still no evidence that a cellulose hydrolysis capability at rates which are high enough for a commercial process has
been engineered in genetically modied microorganisms. Most
of the engineered microorganisms reported so far have gained
the capability of hydrolysing cellulose derivatives but not native
or crystalline cellulose. Genetically modied K. oxytoca exhibited
good capability to hydrolyse soluble carboxy-methyl cellulose65 or
phosphoric acid-swollen Avicel,66 but very limited ability to hydrolyse crystalline Sigmacell 50.65 The yeast S. cerevisiae, expressing
cellulases from Bacillus species, has been reported67 to have activity on lter paper but was not able to grow in the absence of
externally-added cellulases. The same yeast has been engineered
to hydrolyse phosphoric acid-swollen cellulose. 68,69
Fermentation of carbohydrates to ethanol by mixed microbial
cultures
Once cellulose and hemicellulose have been hydrolysed,
monomeric sugars need to be fermented to ethanol. The main
sugars that are present in the hydrolysis products of lignocellulosic
biomass are glucose, present in cellulose and in minor fractions in
hemicellulose, and xylose, which is often the main component of
hemicellulose (Table 1). The fermentation of glucose and xylose by
mixed microbial cultures is reviewed in following sections, which
also cover the use of genetically modied microorganisms. Other
sugars are also present in the hydrolysis products of lignocellulosic
biomass, e.g. arabinose, mannose, galactose, but usually in lower
amounts than glucose and xylose and their fermentation is not
discussed here. It is worth observing that various studies have
been carried out on the anaerobic fermentation of arabinose by
mixed cultures, however they were mainly aimed at hydrogen and
not ethanol production.70,71 Fermentation of arabinose to ethanol
has been mainly investigated by means of genetically modied
microorganisms.72,73
Fermentation of glucose
In an anaerobic mixed microbial culture glucose can be fermented
to several dierent end products, as summarized in Fig. 2. Ethanol
can be produced directly from glucose, and then converted to
acetate or organic acids, which can then be converted to methane.
Lactate, butyrate and acetate can also be produced directly from
373
Cellulose hydrolysis by genetically modied microorganisms

In recent years there has been a considerable interest in engineering microorganisms in order to make them capable of hydrolysing
cellulose.9 Particular attention has been given to adding the
cellulose-hydrolysis capability in microorganisms which are naturally able to ferment glucose to ethanol with high yields. Several
cellulase-encoding genes have been expressed in various bacteria and yeasts, such as Zymomonas mobilis,64 Klebsiella oxytoca,65,66
Time (days)
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D Dionisi et al.
Table 11. Cellulose degradation rates calculated by the authors of the present paper on the basis of literature data
Microorganism
Substrate
Rumen microorganisms
Rumen microorganisms
Cellulomonas fermentans
Fibrobacter succinogenes
Ruminococcus avefaciens
Clostridium straminisolvens
Trichoderma reesei
Clostridium cellulolyticum
Trichoderma viride
Ruminococcus albus
Mixed culture
MN300
Avicel PH102
Avicel PH101
MN300
Sigmacell 20
Sigmacell 20
Avicel
MN300
MN300
Solka Floc 200
MN301
BW200
CC31
Avicel
Solka Floc
Filter paper Whatman no. 1
MN300
Whatman for chromatography
Avicel PH105
Avicel
Filter paper
Avicel
Table 12. Cellulose hydrolysis rates with enzymatic hydrolysis

reported in the literature
Cellulose
type
Avicel
Solka Floc SW40
Avicel
Enzyme loading
15 FPU g-1 cellulose
17.6E-3 IU mL-1
60 FPU g-1 cellulose
Cellulose hydrolysis
rate (g L-1 h-1 )
0.23
0.08
1.3
Anaerobic
Anaerobic
Anaerobic
Anaerobic
Anaerobic
Anaerobic
Anaerobic
Anaerobic
Anaerobic
Aerobic
Anaerobic
Aerobic
Aerobic
Anaerobic
Anaerobic
Anaerobic
Anaerobic
Degradation
rate (g L-1 h-1 )
0.005
0.12
0.05
0.005
0.050.20
0.060.27
0.03
0.07
0.04
0.30
0.030.05
0.120.33
0.05
0.04
0.044
0.055
0.105
0.25
0.160.33
0.001
0.0025
0.10
Ref
128
58
57
138
56
55
148
132
130
141
129
140
137
127
149
150
in the liquid medium. However, much less focus has been given to
the mixed culture fermentation to ethanol. In the next sections, the
available information on the eect of process operating conditions
on the anaerobic fermentation of glucose to ethanol is reviewed.
Ref
151
152
153
glucose through microbial action under anaerobic conditions

(Fig. 2). Conversely, propionate typically derives from the conversion of lactate; under methanogenic conditions propionate, once
produced, can be further converted into acetate and H2 (provided
that methanogens keep the H2 partial pressure below 10-5 atm).
Table 13 lists some bacterial or fungal species that are able to
convert glucose to ethanol and some species that are able to
convert ethanol to organic acids. The stoichiometry of the key
reactions hereafter discussed, i.e. glucose fermentation to ethanol
and ethanol conversion to acetic acid, are reported below:
ethanol production from glucose C6 H12 O6 2C2 H5 OH + 2CO2
ethanol conversion to acetic acid C2 H5 OH + H2 O CH3 COOH
+ 2H2
374
If ethanol is the desired product, the operating conditions of

the fermentation should be chosen in order to maximize the
rate of the ethanol-producing reactions and to minimize the rate
of ethanol-consuming reactions. The anaerobic fermentation by
mixed cultures to methane is a well known process and is widely
used in industry.74 Also, relatively wide attention has been given
to anaerobic fermentation to organic acids such as acetate, propionate and butyrate, since they are often found as intermediates
in the fermentation to methane and can, undesirably, accumulate
Aerobic/anaerobic
Eect of pH. A few studies have investigated the eect of pH on

the anaerobic fermentation of glucose to ethanol and, while it
seems that pH has an important eect on ethanol production,
there is still no clear evidence on the optimum pH range to drive
the fermentation process towards ethanol, rather than acetate and
methane. Based on thermodynamic considerations, Rodriguez
et al.75 predicted that acidic pH values, below 5.5, should favour
ethanol production, while at pH values higher than 6.5, acetate
should be the only product in the liquid medium. In agreement
with the theory that conversion to ethanol is favoured by acid pH
values, Ren et al.76 found that in a continuous reactor in the pH
range 4.34.9 ethanol concentration increased at lower pH, and in
this pH range ethanol and acetate were always the main fermentation products. In the same paper, in a batch study in the pH range
3.05.5, the authors reported the highest ethanol concentration
at pH 5.0, observing a much lower ethanol production at pH 5.5. In
a continuous study in the pH range 4.07.0,77 the highest ethanol
concentration was found at pH 6.0, but in this case, ethanol was not
the main fermentation product, the main products being butyrate
and acetate. Hwang et al.78 found that acetate and ethanol were
the main fermentation products at pH 4.55.0, while at pH 5.06.0
propionate and acetate were the main products.
However, other studies found higher ethanol yield at neutral or
basic pH values. Temudo et al.13 investigated anaerobic fermentation of glucose with mixed cultures in a chemostat in the range
pH 48.5. They found that in the pH range 6.258.5 acetate and
ethanol were the main fermentation products, in approximately
equal molar ratio, while in the pH range 45.5 very little ethanol
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Glucose
(24 e-eq/mol)
Butyrate
(20 e-eq/mol)
2 x Ethanol
(12 e-eq/mol)
2 x Lactate
(12 e-eq/mol)
2 x H2
(2 e-eq/mol)
2/3 x
2x
2 x Acetate
(8 e-eq/mol)
2x
4x
4 x H2
(2 e-eq/mol)
2x
4/3 x Propionate
(14 e-eq/mol)
4/3 x
4x
Acetate
4 x H2
(2 e-eq/mol)
(8 e-eq/mol)
CH4
(8 e-eq/mol)
Figure 2. Possible products of anaerobic fermentation of glucose in a mixed culture environment with associated electron ow. Electron equivalents
(e-eq) represent the moles of electrons which would be released upon complete oxidation.
was produced and acetate and butyrate were the main fermentation products. However, in this study the dilution rate at pH 45.5
was also dierent from the one at pH 6.258.5 and this could also
have aected the results. In general agreement with their ndings, Zoetemeyer et al.79 found that acetate and ethanol were the
main products of anaerobic fermentation at pH 8.0, while at pH values below 7, the main product became butyrate. In a chemostat
study in the pH range 58,80 the highest ethanol concentration
was found at pH 8, but in this study ethanol was a minor fermentation product, the main ones being acetate and propionate.
Overall, analysis of the literature indicates that further study is
needed to address the eect of pH on ethanol yield from anaerobic
fermentation of glucose.
Microorganism
Glucose to ethanol
Clostridium thermohydrosulfuricum
Thermoanaerobium brockii
Sarcina ventriculi
Thermoanaerobacter ethanolicus
Ruminococcus albus
Saccharomyces cerevisiae
Zymomonas mobilis
Aspergillus spp.
Fusarium spp.
Penicillum spp.
Schizosaccharomyces pombe
Kluyveromyces marxianus
Ethanol to organic acids
Desulfotomaculum nigricans
Pelobacter acetylenicus
Desulfovibrio spp.
Desulfobulbus propionicus
Pelobacter propionicus
Microorganism
type
Bacterium
Bacterium
Bacterium
Bacterium
Bacterium
Bacterium
Yeast
Bacterium
Fungus
Fungus
Fungus
Yeast
Yeast
Bacterium
Bacterium
Bacterium
Bacterium
Bacterium
375
Eect of temperature. The eect of temperature on glucose fermentation to ethanol by mixed cultures is potentially particularly
interesting. In general, the rates of all microorganism-mediated
processes increase with temperature, up to the maximum temperature which is tolerable by the microorganisms. Microorganisms used in anaerobic fermentations can be classied as either
mesophilic (optimum temperature <45 C) or thermophilic (optimum temperature >45 C). An interesting advantage of thermophilic over mesophilic bacteria when using mixed cultures for
ethanol production is that among thermophilic bacteria there are
many microorganisms that are able to convert glucose to ethanol
but only very few that are able to oxidise ethanol to acetate
or other organic acids.81 Therefore, it is expected that higher
Table 13. Microorganisms which are able to convert glucose to

ethanol and ethanol to acetate under anaerobic conditions (adapted
from81,82,89,154 159 )
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ethanol yields and rates might be obtained under thermophilic
than mesophilic conditions.
Other advantages of thermophilic conditions (adapted from
Wiegel)81 are the following:
(a) lower use of the substrate for biomass production, therefore
increasing ethanol yield;
(b) pathogens do not grow at temperature higher than 60 C;
(c) since microbial processes generate heat, higher temperatures
may be easier to maintain than lower ones;
(d) ethanol can be continuously distilled from the fermentation
vessel by using a moderate vacuum.
On the other hand, thermodynamic calculations82 show that
at higher temperatures the reactions that generate hydrogen
become more favourable. Since glucose oxidation to acetate or
butyrate and ethanol oxidation to acetate generate hydrogen,
these reactions become more favourable at higher temperatures,
potentially leading to higher ethanol loss.
The most comprehensive study on the eect of temperature on
the acidogenic fermentation of glucose has been carried out by
Zoetemeyer et al.83 The authors operated a chemostat at pH 5.8
in the temperature range 2060 C. At temperatures up to 50 C,
butyrate and acetate were the main products, and the ethanol
yield was quite low (0.100.20 mol ethanol mol-1 glucose). At
55 C, on the other hand, ethanol was the main fermentation
product, with a yield of 0.8 mol mol-1 glucose.
In general, analysis of the literature shows that the eect of
temperature on anaerobic fermentation to ethanol is potentially
very important and deserves further investigation.
376
Eect of hydrogen partial pressure. Hydrogen concentration in the

liquid phase or hydrogen partial pressure in the gas phase (the two
are proportional via Henrys law), which can be manipulated by
sparging with an inert gas or by changing the process pressure, is
an important variable that can aect the spectrum of product distribution in anaerobic fermentation. The eect of hydrogen partial
concentration is twofold: (a) hydrogen levels aect the NADH/NAD
ratio and therefore the feasibility of the biochemical pathways that
determine product formation;84 (b) certain fermentation reactions
which generate hydrogen (Fig. 2) are close to the thermodynamic
equilibrium and hydrogen concentration (as well as pH) can determine whether they are feasible or not.
The eect of hydrogen concentration on methane formation is
well known: hydrogen concentration has to be maintained at very
low values in order for the conversion of organic acids to acetate
to occur, which is thermodynamically unfeasible at high hydrogen concentrations, and this require a close syntrophy between
hydrogen-producing and hydrogen-consuming microorganisms.
However, when methane is not the desired product, very little
is known about the eect of hydrogen concentration on the spectrum of product distribution. The biochemical model by Rodriguez
et al.84 predicts that hydrogen partial pressures above approx
0.4 atm should lead to butyrate as the main fermentation product,
while lower hydrogen pressures would give acetate. In those simulations, carried out at pH 7, no ethanol formation was predicted,
since the model predicted ethanol formation only at acidic pH values. Considering the conversion of ethanol to acetate, this reaction
is thermodynamically feasible, at pH 7, only for a hydrogen partial pressure lower than approx 0.15 atm.85 Therefore, hydrogen
pressures higher than this value should prevent ethanol oxidation
to acetate and therefore decrease ethanol losses. The experimental study by Mizuno et al.86 showed that a reduction in hydrogen
D Dionisi et al.
partial pressure from 0.5 to 0.05 atm increased the rate of hydrogen production by more than 50%, however very little eect was
observed on the composition of the liquid euent, the main products being acetic and butyric acids, with much lower amounts of
ethanol.
Eect of solids retention time. The solids retention time is a critical
parameter for glucose fermentation with mixed cultures. It is well
known that the end-product of glucose fermentation by mixed
cultures is methane, if the digestion time or residence time is long
enough.74,87,88 Therefore, glucose fermentation to ethanol has to
be carried out at relatively short residence times. However, within
the region of relatively short residence times, little systematic
study has been carried out to investigate whether the residence
time aects the distribution of fermentation products. Zoetemeyer
et al.79 investigated the eect of residence time in the range
1.510 h (at 30 C) and they reported ethanol proles for pH values
of 5.69 and 6.44. They found that ethanol yield tended to increase
with longer residence times at pH 5.69 (up to 0.3 mol mol-1 ), while
it tended to increase with shorter residence times at pH 6.44 (up to
approx 0.2 mol mol-1 ).
Rates and yields. Table 14 summarizes ethanol production rates
and yields in glucose fermentation studies with mixed cultures.
Only studies where the main target was ethanol or acids production are considered here. It is evident that with mixed cultures ethanol yields up to 0.8 mol ethanol mol-1 glucose have
been obtained. The maximum theoretical yield of ethanol on glucose is 2 mol ethanol mol-1 glucose, assuming that all glucose is
fermented to ethanol. However, this maximum yield achievable
in practice is lower than this, owing to the fact that some glucose is inevitably used for biomass growth. For the yeast Saccharomyces cerevisiae the ethanol yield on glucose is typically in the
range 1.61.9 mol ethanol mol-1 glucose.89 The lower ethanol yield
obtained with mixed cultures is because part of the glucose is fermented to other products, mainly acetate and in some cases other
acids such as propionate and butyrate. In order to develop commercial processes for ethanol production with mixed cultures, the
challenge is to determine process conditions that direct glucose
fermentation to ethanol, minimizing both glucose and ethanol
conversion to organic acids. In this regard, it is important to understand the causes for the observed variability in ethanol yield
under similar process conditions. As an example, at a residence
time of 8 h, 30 C, pH 6.25, Temudo et al.13 observed an ethanol
yield on glucose higher than 0.6 mol mol-1 , while under similar
conditions (residence time about 7 h, 30 C, pH 6.44) Zoetemeyer
et al.79 found negligible ethanol yield. The reasons for the dierent
behaviour could be the presence or absence of nitrogen sparging,
the use of dierent inocula, the start-up procedure, the glucose
concentration in the feed, etc.
In terms of ethanol productivity, high ethanol production rates
up to 1.5 g L-1 h-1 have been reported with mixed cultures. This
value is lower than ethanol productivity on glucose for Saccharomyces cerevisiae, 318 g L-1 h-1 .89 However, considering that the
literature studies reported in Table 14 were not specically aimed
at maximizing ethanol productivity, and that ethanol productivity
could easily be increased simply by increasing glucose concentration in the feed, it seems that, with more lab- or pilot-scale investigation, ethanol productivity from glucose with mixed cultures
could reach the same or higher productivities currently obtained
with industrial processes.
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Table 14. Glucose fermentation to ethanol by mixed cultures in chemostat studies

Residence
time (h)
8
512
6
34
72
1.510
a
pH
6.258.5
8
47
8
5
5.8
Ethanol yield
(mol/mol glucose)
Temp. ( C)
30
37
36
30
35
20-60
Ethanol rate
(g L-1 h-1 )
0.550.70
0.10.25
0.080.17
0.7
0.18
0.1-0.8
0.070.09
0.020.08
0.0250.05
0.5
0.02
1.5
Other main
productsa
acetate
acetate, propionate
acetate, butyrate
acetate
acetate
acetate, butyrate
Ref
13
80
77
79
78
83
In all the studies the main products in the gas phase were hydrogen and carbon dioxide
Fermentation of xylose
Fermentation of xylose is much less well known than glucose fermentation, in particular as far as mixed cultures are concerned.
In principle, the spectrum of substrates that can be obtained by
anaerobic fermentation of xylose is similar to that which can be
obtained from glucose (Fig. 2), even though the quantitative distribution of the products and the microbial species involved may
be dierent. The stoichiometry of xylose conversion to ethanol is
the following:90
C5 H10 O5 1.67CH3 CH2 OH + 1.67CO2
The theoretical maximum yield of ethanol from xylose is 1.67 mol
ethanol mol-1 xylose, i.e. virtually the same yield as glucose if
expressed in mass terms (0.51 g ethanol g-1 xylose).
Table 15 reports several species of microorganisms that have
been reported to convert xylose into ethanol. Certain microbial
species are able to produce ethanol from xylose with almost
maximum yield, while others always generate other co-products,
mainly acetate.91,92 In terms of the process considered with mixed
cultures, operating conditions have to be found that maximize
ethanol yield, minimizing the formation of other fermentation
by-products. However, while several recent studies have investigated the eect of operating conditions on xylose fermentation
to hydrogen,93 96 the only study that has investigated xylose conversion to ethanol by mixed cultures is the one by Temudo et al.97
They compared chemostat cultures grown on xylose or glucose
as only carbon sources comparing ethanol and acids production
with the two substrates. They observed that the culture grown on
xylose produced much less ethanol than the one grown on glucose
(0.05 mol ethanol mol-1 xylose vs. 0.24 mol ethanol mol-1 glucose),
the other main products being in both cases acetate and butyrate.
However, interestingly, ethanol yield on xylose increased very signicantly when xylose concentration in the feed was increased
from 4 to 10 g L-1 , from 0.05 to 0.69 mol ethanol mol-1 xylose (the
yield of butyrate was correspondingly much lower), but the reason
for this is not known. The authors also observed that the mixed
culture grown solely on xylose was immediately able to metabolize glucose when this substrate was added, indicating that in
a mixed culture with complex substrates such as real wastes, the
same microorganisms may be able to metabolize both glucose and
xylose.
Microorganism
Microorganism type
Bacillus macerans
Clostridium thermohydrosulfuricum
Thermoanaerobacter ethanolicus
Aerobacter aerogenes
Fusarium oxysporum
Aeromonas hydrophila
Bacillus polymixa
Aerobacter indologenes
Brettanomyces spp.
Candida shehatae
Pachysolen tannophilus
Pichia stipitis
Monilia spp.
Mucor spp.
Neurospora spp.
Paecilomyces spp.
Polyporus spp.
Rhizopus spp.
Bacterium
Bacterium
Bacterium
Bacterium
Fungus
Bacterium
Bacterium
Bacterium
Yeast
Yeast
Yeast
Yeast
Fungus
Fungus
Fungus
Fungus
Fungus
Fungus
essentially to increase the range of substrates that can potentially

be converted to ethanol at high yield by a single microorganism. Indeed, native strains of the yeast Saccharomyces cerevisiae
are not able to utilize pentoses such as xylose, therefore limiting
the range of feedstock that can be used for ethanol production.
Other microorganisms such as Escherichia coli are, on the other
hand, able to metabolize a wider range of substrates but the native
strains dont produce ethanol as main fermentation product.
The enteric microorganism Klebsiella oxytoca M5A1 converts
xylose to ethanol, but also produces organic acids (acetic, lactic,
succinic). By metabolic engineering Ohta et al.98 increased the
molar fraction of ethanol in the products of xylose fermentation from 62% to 90%. Similarly, using metabolic engineering
on E. Coli KO11, Yomano et al.99 obtained almost stoichiometric
conversion of xylose to ethanol, with very minor production of
organic acids. Other researchers used metabolic engineering to
increase the ethanol yield from glucose in Lactobacillus sp.100 Since
the most common microorganism used in industrial bioethanol
production is the yeast Saccharomyces cerevisiae, considerable
eort has been dedicated to engineering this microorganism to
metabolize xylose.101 In general, good success has been obtained,
however, the volumetric productivity obtained with recombinant
S. cerevisiae on xylose is still signicantly lower than that of the
377
Fermentation of glucose and xylose to ethanol by genetically

modied microorganisms
In general, the reason behind metabolic engineering of microorganisms in order to produce ethanol from glucose and xylose is
Table 15. Microorganisms which are able to convert xylose to

ethanol under anaerobic conditions
www.soci.org
D Dionisi et al.
Table 16. Glucose and xylose fermentation to ethanol by genetically modied microorganisms
Microorganism
Glucose
Erwinia sp. SR38
Klebsiella oxytoca M5A1
Lactobacillus casei 686
Lactobacillus plantarum
Xylose
Escherichia coli LY160
Saccharomyces cerevisiae 424A
Saccharomyces cerevisiae MA-R5
Saccharomyces cerevisiae DA24-16
Saccharomyces cerevisiae ADAP8
pH
6.0
6.0
6.0
6.0
6.5
Temp. ( C)
Ethanol rate (g L-1 h-1 )
30
30
37
37
1.9
2.0
0.8
0.9
0.7
2.1
0.18
0.02
30
30
37
30
30
30
30
1.6
1.7
1.6
0.7
1.2
1.3
1.11.4
2.0
1.3
0.9
0.13
0.50
1.3
0.030.07
native strain on glucose.19 So far the maximum ethanol productivity obtained for recombinant S. cerevisiae on xylose is 0.5 g L-1 h-1
(lab scale study). Table 16 reports ethanol production rates and
yields from glucose and xylose by genetically modied microorganisms in selected literature studies. In general, volumetric
productivities of up to 2 g ethanol L-1 h-1 and almost quantitative
conversions of glucose and xylose to ethanol have been obtained,
so indicating the success of genetic engineering in generating
microorganisms able to convert multiple sugars to ethanol at high
rate and yield.
TOWARDS AN INTEGRATED MICROBIAL

PROCESS TO CONVERT LIGNOCELLULOSIC
BIOMASS TO ETHANOL: CHALLENGES
AND RESEARCH OPPORTUNITIES
The literature reviewed in this paper shows that there are microorganisms able to catalyse each of the three steps required for the
conversion of lignocellulosic biomass into ethanol: lignin hydrolysis, cellulose hydrolysis and glucose and xylose fermentation
to ethanol. Therefore, an entirely microbial process converting
lignocellulosic biomass to ethanol can, at least in principle, be
considered. Compared with the alternative processes which use
high-pressure/high temperature conditions for lignin hydrolysis
and the addition of external enzymes for cellulose hydrolysis, an
entirely microbial process at ambient pressure and relatively low
temperature would clearly give an important reduction in process
costs.
An integrated (or single stage or consolidated) process which
uses untreated lignocellulosic biomass as feedstock and converts
it to ethanol is clearly the most desirable option. This process
could be obtained using two alternative approaches: use of an
open mixed culture, where many dierent naturally-occurring
microorganisms co-exist and carry out the various steps, or use
of a pure culture of a genetically modied microorganism which
is able to carry out all the required process steps. However, either
approach is still far from becoming reality. In this section the main
challenges and research opportunities for the two approaches will
be discussed.
378
Open mixed cultures

The main challenges to be overcome for the development of a
mixed culture process are the following:
Ethanol yield (mol mol-1 sugar)
Ref
160
98
161
100
98
162
99
163
164
165
166
Low rates of lignin and cellulose hydrolysis. The rates of lignin

and cellulose hydrolysis reported so far for microbial processes
are lower than for chemical-physical or enzymatic processes;
Control of the anaerobic fermentation of sugars (mainly glucose
and xylose) to ethanol. In a mixed culture environment, fermentation of sugars can lead to many dierent products, in addition
to ethanol, i.e. other alcohols, volatile fatty acids (acetic, propionic, butyric, etc), hydrogen or methane;
Co-existence of lignin- and cellulose-hydrolysing and of
ethanol-producing microorganisms in the same vessel. For
an integrated process the microbial populations responsible for
lignin and cellulose hydrolysis and those responsible for sugars
fermentation should co-exist in the same vessel. This might be
possible or not, depending on the operating conditions of the
process and on the growth rate of the various microorganisms.
Some strictly interlinked research opportunities which may
address the challenges above are discussed below.
Enrichment studies: as discussed earlier, microbial hydrolysis of
lignin is usually considered to be dicult and slow and a factor
that may limit the rate of hydrolysis is adaptation of the microorganisms to the substrate. It is possible to hypothesize that once
mixed cultures have become adapted to a lignocellulosic substrate and have synthesized the enzymes required for its hydrolysis, then the rate of hydrolysis should proceed faster. Therefore
a process can be envisaged, where a mixed culture is previously
acclimated to the lignocellulosic substrate (slow process) and
then transferred in a continuous reactor, or semi-continuous
reactor such a sequencing batch reactor, with a continuous, or
semi-continuous, feed of the substrate (fast process). Having
been previously acclimated, the microbial culture should be
able to remove the substrate at high rate. Investigation of this
process at lab-scale is possible but very few studies have been
carried out. An example is the study by Haruta et al.102 where a
stable microbial community able to degrade various cellulosic
and lignocellulosic substrates was generated from composting
microorganisms by acclimation on lter paper.
Particle size reduction: reducing the feedstock particle size is
expected to give higher rates of hydrolysis, but the quantitative
evidence for this eect is rather limited, especially as far as
ethanol production is concerned. Lab-scale studies specically
targeted at exploring and quantifying the possible rate increase
obtainable by particle size reduction are required.
www.soci.org
Reactor conguration and process parameters: the reactor used

for the integrated process can be operated under various
congurations, e.g. continuous ow with or without biomass
recycle, sequencing batch reactor, etc. For each congurations,
various process parameters have to be specied, e.g. temperature, pH, hydraulic retention time, solids retention time, length
of cycle, length of the feed (the latter two only apply to sequencing batch reactors). The choice of these parameters can aect
both the hydrolysis rate and the spectrum of product distribution of sugars fermentation. It can be expected, in principle,
that in a sequencing batch reactor the hydrolysis rate should be
higher than in a continuous-ow reactor due to the higher substrate concentration at the start of the cycle, which is expected
to give a higher reaction rate. However, no experimental proof
of this in the context of lignocellulosic biomass hydrolysis has
been reported. Similarly, only limited experimental investigation has been carried out regarding the eect of process parameters on products distribution of sugars fermentation; examples
are the studies by Temudo et al.13,97 All these aspects deserve
systematic investigation at lab scale.
An interesting alternative to open mixed cultures is the use
of selected mixed cultures, where only selected species, responsible for dierent stages of the lignocellulosic biomass conversion to ethanol, are inoculated in the reactor. A successful study
using this approach has been published very recently.103 The
authors obtained 67% ethanol yield from pretreated (dilute acid)
wheat straw using a microbial culture composed of three naturally
occurring strains: Trichoderma reesei, Saccharomyces cerevisiae and
Scheersomyces stipitis. The fungus T. reesei was responsible for cellulose and hemicellulose hydrolysis, while the yeasts were responsible for ethanol production from glucose (S. cerevisiae) or pentoses (S. stipitis). The authors utilized a biolm membrane reactor
with the presence in the same reactor of aerobic, microaerophilic
and anaerobic conditions, therefore allowing the co-existence of
the three dierent species.
introduction of the lignin hydrolysis capability into microorganisms that are naturally able to hydrolyse cellulose, or, as opposite
strategy, introduction of the cellulose hydrolysis capability into
microorganisms that are naturally able to hydrolyse lignin;
improvement in the ability to hydrolyse crystalline cellulose
with microorganisms that are native ethanol producers;
increase in the ethanol yield for microorganisms that are naturally able to hydrolyse cellulose.
CONCLUSIONS
This paper has reviewed the existing literature on microbial processes for lignin hydrolysis, cellulose hydrolysis and glucose fermentation to ethanol. The main evidence from this study is the
following:
there is a wide range of microorganisms that can perform each
of the three steps required for lignocellulosic biomass conversion into ethanol, i.e. lignin hydrolysis, cellulose hydrolysis and
glucose, or xylose, fermentation to ethanol;
while there are many reported fungi species that are able to
hydrolyse lignin under aerobic conditions, there is only one
recent study in the literature giving clear evidence of lignin
hydrolysis under anaerobic conditions. However, many mixed
culture studies give indirect evidence that lignin can be at least
partially degraded under anaerobic conditions. In principle,
if anaerobic lignin hydrolysis can be achieved, a single-stage
process with mixed microbial cultures including lignin and
cellulose hydrolysis and glucose fermentation to ethanol can be
envisaged;
cellulose and hemicelluloses hydrolysis can be carried out by
many dierent microbial species, both under aerobic and anaerobic conditions. Interestingly, the literature evidence collected
so far indicates no signicant dierences in the cellulose hydrolysis rate under aerobic or anaerobic conditions;
regarding anaerobic fermentation of sugars to ethanol, literature studies with mixed cultures specically targeted at ethanol
production have been very limited and they have reported a
maximum yield of 0.8 mol ethanol mol-1 glucose, compared
with the 2 mol ethanol mol-1 glucose which is the theoretical
maximum yield;
metabolic engineering has been successful in generating
microorganisms able to convert a wider range of sugars to
ethanol with high yields, however, much more limited success
has been obtained by engineering microorganisms in order to
combine cellulose hydrolysis and high ethanol yield.
An integrated (or consolidated) process converting untreated
lignocellulosic biomass to ethanol can, at least in principle, be
conceived according to two dierent approaches: use of open
mixed cultures of existing microorganisms or use of a pure culture
of a genetically modied microorganism. Regarding the use of
open mixed cultures, the main challenges to be overcome are: low
rates of lignin and cellulose hydrolysis, control of the anaerobic
fermentation of sugars to ethanol and co-existence of dierent
microbial populations in the same reactor. Possible research areas
that can help address these challenges are: enrichment studies
with microbial adaptation to the lignocellulosic substrate, investigation of the eect of particle size reduction on the hydrolysis
rates and investigation of the eect of reactor conguration and
379
Genetically modied microorganisms

Similarly to the use of open mixed cultures, the approach of
using a single, genetically modied, microorganism to convert
lignocellulosic biomass to ethanol is still far from becoming
reality.
So far no attempt has been reported to introduce in microorganisms the ability to hydrolyse/break down lignin and this is probably due to the complexity of the genome of the native lignin
degrading species. Therefore, so far the concept of metabolic engineering for bioethanol production has been focused on the use
of chemically or physically pretreated feedstocks, where lignin has
been hydrolysed and cellulose is available for microbial attack.
Considering metabolic engineering for cellulose hydrolysis, the
ability to hydrolyse pretreated cellulose has been introduced in
various microbial strains, but so far very little success has been
reported with untreated crystalline cellulose. More success has
been reported in the increase of ethanol yield in microorganisms that are naturally able to hydrolyse cellulose, but even in
this case the rates are in the majority of cases very low.104 So far,
the main success of genetic engineering for bioethanol production has been the development of microorganisms able to convert
multiple sugars to ethanol with high yields. While this is an important step forward, the main issues of lignin and cellulose hydrolysis
are still far from being solved by means of genetically modied
microorganisms.
Interesting research opportunities lie ahead in the following

areas:
www.soci.org
operating parameters. Regarding the use of genetically modied
microorganisms the main challenges are the development of
microorganisms able to hydrolyse lignin and crystalline cellulose
and convert the sugars produced to ethanol.
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Review

Received: 23 October 2013

Revised: 1 September 2014

Accepted article published: 5 September 2014

Published online in Wiley Online Library: 1 October 2014

(wileyonlinelibrary.com) DOI 10.1002/jctb.4544

The potential of microbial processes

Second generation bioethanol produced from lignocellulosic

steam explosion, acid hydrolysis, or ammonia bre expansion

Correspondence to: Davide Dionisi, Materials and Chemical Engineering Group,

a Materials and Chemical Engineering Group, School of Engineering, University

2014 Society of Chemical Industry

Conversion of lignocellulosic biomass into ethanol

the next hydrolysis stages. However, they require conditions

biomass. In contrast to the physico-chemical pretreatment and

Combined reactor for lignin

Combined reactor for

J Chem Technol Biotechnol 2015; 90: 366383

2014 Society of Chemical Industry

genetically engineered ones, while the process studied in this

2014 Society of Chemical Industry

J Chem Technol Biotechnol 2015; 90: 366383

Conversion of lignocellulosic biomass into ethanol

Table 2. Evidence of anaerobic biodegradation of lignocellulosic materials by mixed cultures

J Chem Technol Biotechnol 2015; 90: 366383

2014 Society of Chemical Industry

Sisal bre waste

Table 3. Microorganisms reported to degrade lignin under aerobic conditions

Extent of degradation (%)

Table 4. Evidence of aerobic biodegradation of lignocellulosic materials by mixed cultures

2014 Society of Chemical Industry

J Chem Technol Biotechnol 2015; 90: 366383

Conversion of lignocellulosic biomass into ethanol

Degradation rate (g L-1 h-1 )

Wood chips of Pinus strobes

J Chem Technol Biotechnol 2015; 90: 366383

It is worth comparing the lignin degradation rates reported in

2014 Society of Chemical Industry

Lignin degradation rates

Table 7. Microorganisms reported to hydrolyse cellulose under anaerobic conditions

Table 8. Anaerobic degradation of cellulose by mixed cultures

Eect of particle size

Cellulose hydrolysis rates

2014 Society of Chemical Industry

J Chem Technol Biotechnol 2015; 90: 366383

Conversion of lignocellulosic biomass into ethanol

Table 9. Microorganisms reported to hydrolyse cellulose under aerobic conditions

Extent of degradation (%)

Table 10. Aerobic degradation of cellulose by mixed cultures

The reported rates for microbial cellulose hydrolysis are, in general,

J Chem Technol Biotechnol 2015; 90: 366383

Saccharomyces cerevisiae67 and others. However, while the results

2014 Society of Chemical Industry

Cellulose hydrolysis by genetically modied microorganisms

Table 12. Cellulose hydrolysis rates with enzymatic hydrolysis

glucose through microbial action under anaerobic conditions

If ethanol is the desired product, the operating conditions of

Eect of pH. A few studies have investigated the eect of pH on

2014 Society of Chemical Industry

J Chem Technol Biotechnol 2015; 90: 366383

Conversion of lignocellulosic biomass into ethanol

J Chem Technol Biotechnol 2015; 90: 366383

2014 Society of Chemical Industry

Table 13. Microorganisms which are able to convert glucose to