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Received: 23 October 2013

Revised: 1 September 2014

Accepted article published: 5 September 2014

Published online in Wiley Online Library: 1 October 2014

( 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
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
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Keywords: bioethanol; cellulose; lignin; microbial hydrolysis; mixed cultures



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. 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:

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

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Conversion of lignocellulosic biomass into ethanol

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,
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


Combined reactor for lignin

Ethanol to purification
hydrolysis, cellulose
hydrolysis and carbohydrates
fermentation (anaerobic)




Cellulose carbohydrates


Ethanol to

Chemical or




Cellulose Ethanol to
hydrolysis purification

Chemical or cellulose

Combined reactor for

cellulose hydrolysis and
carbohydrates fermentation

Ethanol to

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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.

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



Corn stover
Wheat straw
Rice straw





other carbohydrates





The main components of lignocellulosic biomass are lignin, cellulose and hemicelluloses. Lignin is an aromatic polymer in which







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
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.





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

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.

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Conversion of lignocellulosic biomass into ethanol

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




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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
0.240.36 m3 CH4 kg-1 VS, 3448% cellulose reduction
0.290.34 m3 CH4 kg-1 VS, 3439% cellulose reduction
0.390.43 m3 CH4 kg-1 VS, 4247% 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

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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



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)

D Dionisi et al.

Table 3. Microorganisms reported to degrade lignin under aerobic conditions

Microbial species
Pseudomonas spp.
Acinetobacter spp.
Pseudomonas spp.
Xanthomonas spp.
Mixed culture
Pseudomonas spp.
Streptomyces badius
Streptomyces viridosporous
Streptomyces cyaneus
Thermomonospora mesophila
Pleurotus ostreatus
Phanerochaete chrysosporium
Phanerochaete chrysosporium
Echinodontium taxodii 2538
Trametes versicolor spp.
Trametes ochracea spp.
Ganoderma spp.
Phanerochaete chrysosporium
Ceriporia lacerata
Stereum hirsutum
Polyporus brumalis


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)





Up to 38







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

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)



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
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.

Measure of degradation
727% lignin degradation
1243% lignin degradation
17% lignin degradation
83% lignin degradation
CO2 evolution 1040% of the maximum
37% lignin degradation
25% lignin degradation
26% lignin degradation
70% lignin degradation
80% straw degradation

Time (days)


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

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Conversion of lignocellulosic biomass into ethanol

Table 5. Lignin degradation rates, calculated by the authors of this paper based on literature data

Degradation rate (g L-1 h-1 )


Irpex lacteus
Irpex lacteus
Trametes versicolor MrP 1
Trametes versicolor MrP 1
Streptomyces cyaneus
Thermonospora mesophila
Pleurotus ostreatus
Phanerochaete chrysosporium
Echinodontium taxodii 2538
Trametes versicolor G20
Ganoderma sp En3
Phanerochaete chrysosporium
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




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
1% H2 SO4

SO2 -enhanced
steam explosion
0.05 gSO2 g-1 biomass

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.

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15% NH4 OH

1 gCa(OH)2 + 100 psi O2

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.
Unlike the case of lignin, a wide range of bacteria and fungi have
been reported to hydrolyse cellulose under anaerobic or aerobic

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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

D Dionisi et al.

Table 7. Microorganisms reported to hydrolyse cellulose under anaerobic conditions


Extent of
degradation (%)


Ruminococcus albus
Clostridium thermocellum
Ruminococcus avefaciens
Clostridium cellulolyticum
Clostridium thermocellum
Caldicellulosiruptor bescii
Bacteroides succinogenes + Selenomonas ruminantium
Clostridium thermocellum + Clostridium thermohydrosulfuricum
Clostridium thermocellum + Clostridium thermohydrosulfuricum
Fibrobacter Succinogenes

Avicel PH-105
Sigmacell 20
Ball milled Whatman no.1 Filter paper
Sigmacell 20




Table 8. Anaerobic degradation of cellulose by mixed cultures

Filter paper
Paper (hardwood and softwood pulp)
Sigmacell 50
Cellulose powder

Measure of degradation
99% (as CH4 production compared to glucose)
100% (as CH4 production compared to glucose)
62% reduction in cellulose
68% reduction in cellulose
80% reduction in cellulose
3858% 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)
9 (HRT)


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.


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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Conversion of lignocellulosic biomass into ethanol

Table 9. Microorganisms reported to hydrolyse cellulose under aerobic conditions



Cellulomonas uda JC3
Cellulomonas uda JC3
Cellulomonas uda JC3
Cellulomonas uda JC3
Cellulomonas uda JC3
Cellulomonas uda JC3
Cellulomonas uda JC3
Cellulomonas fermentans
Cytophaga sp. LX-7
Trichoderma viride
Trichoderma reesei

Extent of degradation (%)

CC31 (Whatman)
Filter paper (Whatman no 1)
Amorphous cellulose
Whatman for Chromatography
Whatman CF11
Ball milled wood cellulose (BW 200)
Solka Floc 200

Table 10. Aerobic degradation of cellulose by mixed cultures

Cellulose lter paper
Filter paper

Measure of degradation
97% of theoretical CO2
84% of theoretical CO2
87% of initial cellulose
83% of initial cellulose
95% of theoretical cellulose
100% of initial cellulose
79% weight loss



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.

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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
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
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

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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)

D Dionisi et al.

Table 11. Cellulose degradation rates calculated by the authors of the present paper on the basis of literature data



Clostridium thermocellum
Rumen microorganisms
Rumen microorganisms
Cellulomonas fermentans
Fibrobacter succinogenes
Ruminococcus avefaciens
Clostridium straminisolvens
Clostridium thermocellum + Clostridium thermohydrosulfuricum
Clostridium thermocellum
Trichoderma reesei
Clostridium cellulolyticum
Trichoderma viride
Cellulomonas uda JC3

Ruminococcus albus
Mixed culture
Clostridium thermocellum

Avicel PH102
Avicel PH101
Sigmacell 20
Sigmacell 20
Solka Floc 200
Solka Floc
Filter paper Whatman no. 1
Whatman for chromatography
Avicel PH105
Filter paper

Table 12. Cellulose hydrolysis rates with enzymatic hydrolysis

reported in the literature
Solka Floc SW40

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 )



rate (g L-1 h-1 )




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.


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


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


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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Conversion of lignocellulosic biomass into ethanol

(24 e-eq/mol)

(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


2 x Acetate
(8 e-eq/mol)



4 x H2
(2 e-eq/mol)


4/3 x Propionate
(14 e-eq/mol)
4/3 x


4 x H2
(2 e-eq/mol)

(8 e-eq/mol)


(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.

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Glucose to ethanol
Clostridium thermocellum
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

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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 )
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.


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
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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Conversion of lignocellulosic biomass into ethanol

Table 14. Glucose fermentation to ethanol by mixed cultures in chemostat studies

time (h)


Ethanol yield
(mol/mol glucose)

Temp. ( C)

Ethanol rate
(g L-1 h-1 )



Other main
acetate, propionate
acetate, butyrate
acetate, butyrate


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

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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.


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

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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

D Dionisi et al.

Table 16. Glucose and xylose fermentation to ethanol by genetically modied microorganisms
Erwinia sp. SR38
Klebsiella oxytoca M5A1
Lactobacillus casei 686
Lactobacillus plantarum
Klebsiella oxytoca M5A1
Klebsiella oxytoca M5A1
Escherichia coli LY160
Saccharomyces cerevisiae 424A
Saccharomyces cerevisiae MA-R5
Saccharomyces cerevisiae DA24-16
Saccharomyces cerevisiae ADAP8




Temp. ( C)

Ethanol rate (g L-1 h-1 )







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.


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
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.


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)



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.

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Conversion of lignocellulosic biomass into ethanol

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.

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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.

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
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
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

2014 Society of Chemical Industry


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
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

Interesting research opportunities lie ahead in the following

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.



1 Soccol CR, Faraco V, Karp S, Vandenberghe LPS, Thomaz-Soccol V,

Woiciechowski A and Pandey A, Lignocellulosic bioethanol: current status and future perspectives, in Biofuels: Alternative Feedstocks and Conversion Processes, ed by Pandey A, Larroche C,
Ricke SC, Dussap CG, and Gnansounou E. Elsevier, Amsterdam,
pp. 101122 (2011).
2 Bohlmann GM, Process economic considerations for production of
ethanol from biomass feedstocks. Ind Biotechnol 2:1420 (2006).
3 High Level Panel of Experts on Food Security and Nutrition (HLPE),
Biofuels and Food Security. Committee on World Food Security,
Rome (2013).
4 Pool R, Brewing a better biofuel. Eng Technol 9:5659 (2014).
5 Balat M, Production of bioethanol from lignocellulosic materials via
the biochemical pathway: a review. Ener Conv Manage 52:858875
6 Walker GM, Fuel alcohol: current production and future challenges. J
Inst Brew 117:322 (2011).
7 Wyman CE, Balan V, Dale BE, Elander RT, Falls M, Hames B, Holtzapple
MT, Ladisch MR, Lee Y and Mosier N, Comparative data on eects
of leading pretreatments and enzyme loadings and formulations
on sugar yields from dierent switchgrass sources. Bioresource
Technol 102:1105211062 (2011).
8 Olsson L and Hahn-Hgerdal B, Fermentation of lignocellulosic
hydrolysates for ethanol production. Enzyme Microbial Technol
18:312331 (1996).
9 Lynd LR, Weimer PJ, Van Zyl WH and Pretorius IS, Microbial cellulose
utilization: fundamentals and biotechnology. Microbiol Molec Biol
Rev 66:506577 (2002).
10 Kleerebezem R and van Loosdrecht M, Mixed culture biotechnology
for bioenergy production. Curr Opin Biotechnol 18:207212 (2007).
11 Alatriste-Mondragon F, Samar P, Cox HH, Ahring BK and Iranpour R,
Anaerobic codigestion of municipal, farm, and industrial organic
wastes: a survey of recent literature. Water Environ Res 78:607636
12 Li C and Fang HH, Fermentative hydrogen production from wastewater and solid wastes by mixed cultures. Crit Rev Environ Sci Technol
37:139 (2007).
13 Temudo MF, Kleerebezem R and van Loosdrecht M, Inuence of the
pH on (open) mixed culture fermentation of glucose: a chemostat
study. Biotechnol Bioeng 98:6979 (2007).
14 Dionisi D, Majone M, Vallini G, Gregorio SD and Beccari M, Eect
of the length of the cycle on biodegradable polymer production
and microbial community selection in a sequencing batch reactor.
Biotechnol Prog 23:10641073 (2007).
15 Villano M, Beccari M, Dionisi D, Lampis S, Miccheli A, Vallini G and
Majone M, Eect of pH on the production of bacterial polyhydroxyalkanoates by mixed cultures enriched under periodic feeding.
Proc Biochem 45:714723 (2010).
16 Reis M, Seram L, Lemos P, Ramos A, Aguiar F and Van Loosdrecht M,
Production of polyhydroxyalkanoates by mixed microbial cultures.
Bioprocess Bios Eng 25:377385 (2003).
17 Pham M, Holtzapple M and El-Halwagi M, Techno-economic analysis
of biomass to fuel conversion via the MixAlco process. J Ind
Microbiol Biotechnol 37:11571168 (2010).
18 Dien B, Cotta M and Jeries T, Bacteria engineered for fuel ethanol
production: current status. Appl Microbiol Biotechnol 63:258266
19 Matsushika A, Inoue H, Kodaki T and Sawayama S, Ethanol production from xylose in engineered Saccharomyces cerevisiae strains:
current state and perspectives. Appl Microbiol Biotechnol 84:3753
20 Jeries TW, Biodegradation of Lignin and Hemicelluloses, ed by
Ratledge C. Kluwer Academic Publishers, The Netherlands,
233277 (1994).
21 Kirk TK and Farrell RL, Enzymatic combustion: the microbial degradation of lignin. Ann Rev Microbiol 41:465505 (1987).
22 Kraemer EO, Molecular weights of celluloses and cellulose derivates.
Ind Eng Chem 30:12001203 (1938).

D Dionisi et al.

23 Prez J, Munoz-Dorado J, De la Rubia T and Martinez J, Biodegradation and biological treatments of cellulose, hemicellulose and
lignin: an overview. Int Microbiol 5:5363 (2002).
24 Hateld R and Fukushima RS, Can lignin be accurately measured?
Crop Sci 45:832839 (2005).
25 Brigham J, Adney W and Himmel M, Hemicelluloses: diversity and
applications. Handbook on Bioethanol: Production and Utilization,
/ed by Wyman CE. Taylor and Francis, pp. 119142 (1996).
26 Field JA, Limits of anaerobic biodegradation. Wat Sci Technol 45:918
27 Zimmermann W, Degradation of lignin by bacteria. J Biotechnol
13:119130 (1990).
28 Silanikove N and Brosh A, Lignocellulose degradation and subsequent metabolism of lignin fermentation products by the desert
black bedouin goat fed on wheat straw as a single-component
diet. Br J Nutr 62:509520 (1989).
29 Benner R, Maccubin AE and Hodson RE, Anaerobic biodegradation
of the lignin and polysaccharide components of lignocellulose
and synthetic lignin by sediment microora. Appl Environ Microbiol
47:9981004 (1984).
30 Chen W, Ohmiya K, Shimizu S and Kawakami H, Degradation of
dehydrovanillin by anaerobic bacteria from cow rumen uid. Appl
Environ Microbiol 54:12541257 (1985).
31 Fuchs G, Anaerobic metabolism of aromatic compounds. Ann N Y
Acad Sci 1125:8299 (2008).
32 Kataeva I, Foston MB, Yang S, Pattathil S, Biswal AK, Poole FL,
Basen M, Rhaesa AM, Thomas TP and Azadi P, Carbohydrate and
lignin are simultaneously solubilized from unpretreated switchgrass by microbial action at high temperature. Energy Environ Sci
6:21862195 (2013).
33 Sharma SK, Mishra IM, Sharma MP and Saini JS, Eect of particle
size on biogas generation from biomass residues. Biomaterials
17:251263 (1988).
34 Turick CE, Peck MW, Chynoweth DP, Jerger DE, White EH, Zsua L
and Andy Kenney W, Methane fermentation of woody biomass.
Bioresource Technol 37:141147 (1991).
35 Triolo JM, Sommer SG, Moller HB, Weisbjerg MR and Jiang XY, A
new algorithm to characterize biodegradability of biomass during
anaerobic digestion: inuence of lignin concentration on methane
production potential. Bioresource Technol 102:93959402 (2011).
36 Tong X, Smith LH and McCarty PL, Methane fermentation of selected
lignocellulosic materials. Biomaterials 21:239255 (1990).
37 Nallathambi Gunaseelan V, Anaerobic digestion of biomass for
methane production: a review. Biomater Bioeng 13:83114 (1997).
38 Malherbe S and Cloete TE, Lignocellulose biodegradation: fundamentals and applications. Rev Environ Sci Biotechnol 1:10514
39 Leonowicz A, Matuszewska A, Luterek J, Ziegenhagen D,
Wojtas-Wasilewska M, Cho N, Hofrichter M and Rogalski J,
Biodegradation of lignin by white rot fungi. Fung Gen Biol
27:175185 (1999).
40 Kirk TK, Schultz E, Connors W, Lorenz L and Zeikus J, Inuence
of culture parameters on lignin metabolism by Phanerochaete
chrysosporium. Arch Microbiol 117:277285 (1978).
41 Srensen H, Decomposition of lignin by soil bacteria and complex
formation between autoxidized lignin and organic nitrogen compounds. J Gen Microbiol 27:2134 (1962).
42 Kerem Z, Friesem D and Hadar Y, Lignocellulose degradation during
solid-state fermentation: Pleurotus ostreatus versus Phanerochaete
chrysosporium. Appl Environ Microbiol 58:11211127 (1992).
43 Zhang X, Yu H, Huang H and Lio Y, Evaluation of biological pretreatment with white rot fungi for the enzymatic hydrolysis of bamboo
culms. Int Biodeter Biodegrad 60:159164 (2007).
44 Keller FA, Hamilton JE and Nguyen QA, Microbial pretreatment of
biomass. Appl Biochem Biotechnol 105:2741 (2003).
45 Lee J, Gwak K, Park J, Park M, Choi D, Kwon M and Choi I, Biological
pretreatment of softwood pinus densiora by three white rot
fungi. J Microbiol 45:485491 (2007).
46 Hwang SS, Lee SJ, Kim HK, Ka JO, Kim KJ and Song HG, Biodegradation and saccharication of wood chips of Pinus strobus and
Liriodendron tulipifera by white rot fungi. J Microbiol Biotechnol
18:18191825 (2008).
47 Shi J, Chinn MS and Sharma-Shivappa RR, Microbial pretreatment of
cotton stalks by solid state cultivation of Phanerochaete chrysosporium. Bioresource Technol 99:65566564 (2008).

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






thermophilic anaerobic mixed cultures. Biotechnol Biofuels 5:6

Li J, Ren N, Li B, Qin Z and He J, Anaerobic biohydrogen production
from monosaccharides by a mixed microbial community culture.
Bioresource Technol 99:65286537 (2008).
Deanda K, Zhang M, Eddy C and Picataggio S, Development of an
arabinose-fermenting Zymomonas mobilis strain by metabolic
pathway engineering. Appl Environ Microbiol 62:44654470
Wisselink HW, Toirkens MJ, del Rosario Franco Berriel M, Winkler AA,
van Dijken JP, Pronk JT and van Maris AJ, Engineering of Saccharomyces cerevisiae for ecient anaerobic alcoholic fermentation of
L-arabinose. Appl Environ Microbiol 73:48814891 (2007).
Batstone DJ, Keller J, Angelidaki I, Kalyuzhnyi S, Pavlostathis S, Rozzi
A, Sanders W, Siegrist H and Vavilin V, The IWA anaerobic digestion
model no 1(ADM 1). Water Sci Technol 45:6573 (2002).
Rodriguez J, Lema JM and Kleerebezem R, Energy-based models
for environmental biotechnology. Trends Biotechnol 26:366374
Ren N, Wang B and Huang J, Ethanol-type fermentation from carbohydrate in high rate acidogenic reactor. Biotechnol Bioeng
54:428433 (1997).
Fang HH and Liu H, Eect of pH on hydrogen production from glucose
by a mixed culture. Bioresource Technol 82:8793 (2002).
Hwang MH, Jang NJ, Hyun SH and Kim IS, Anaerobic bio-hydrogen
production from ethanol fermentation: the role of pH. J Biotechnol
111:297309 (2004).
Zoetemeyer RJ, Vandenheuvel JC and Cohen A, pH inuence on
acidogenic dissimilation of glucose in an anaerobic digester. Water
Res 16:303311 (1982).
Horiuchi J, Shimizu T, Tada K, Kanno T and Kobayashi M, Selective production of organic acids in anaerobic acid reactor by pH control.
Bioresource Technol 82:209213 (2002).
Wiegel J, Formation of ethanol by bacteria. a pledge for the use
of extreme thermophilic anaerobic bacteria in industrial ethanol
fermentation processes. Experen 36:14341446 (1980).
Stams AJM, Metabolic interactions between anaerobic bacteria
in methanogenic environments. Ant van Leeuwen 66:271294
Zoetemeyer R, Arnoldy P, Cohen A and Boelhouwer C, Inuence of
temperature on the anaerobic acidication of glucose in a mixed
culture forming part of a two-stage digestion process. Water Res
16:313321 (1982).
Rodriguez J, Kleerebezem R, Lema JM and van Loosdrecht MCM,
Modelling product formation in anaerobic mixed culture fermentations. Biotechnol Bioeng 93:593606 (2006).
Majone M, Aulenta F, Dionisi D, DAddario EN, Sbardellati R, Bolzonella D and Beccari M, High-rate anaerobic treatment of
FischerTropsch wastewater in a packed-bed biolm reactor.
Water Res 44:27452752 (2010).
Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL and Noike T, Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresource Technol 73:5965 (2000).
Kalyuzhnyi S and Davlyatshina M, Batch anaerobic digestion of glucose and its mathematical modeling. I. kinetic investigations. Bioresource Technol 59:7380 (1997).
Kalyuzhnyi S, Batch anaerobic digestion of glucose and its mathematical modeling. II. Description, verication and application of model.
Bioresource Technol 59:249258 (1997).
Cardona CA, Sanchez OJ and Gutierrez LF, Process Synthesis for Fuel
Ethanol Production. CRC Press, Boca Raton, USA, Chapter 5 (2010).
Zhao C, Karakashev D, Lu W, Wang H and Angelidaki I, Xylose fermentation to biofuels (hydrogen and ethanol) by extreme thermophilic
(70 C) mixed culture. Int J Hydr Energy 35:34153422 (2010).
McMillan JD, Xylose fermentation to ethanol: a review.
NREL/TP-421-4944, National Renewable Energy Lab, Golden,
CO, USA (1993).
Rosenberg S, Fermentation of pentose sugars to ethanol and other
neutral products by microorganisms. Enzyme Microbiol Technol
2:185193 (1980).
Lin CY and Cheng CH, Fermentative hydrogen production from
xylose using anaerobic mixed microora. Int J Hydrogen Energy
31:832840 (2006).
Lin CY, Wu CC and Hung CH, Temperature eects on fermentative
hydrogen production from xylose using mixed anaerobic cultures.
Int J Hydrogen Energy 33:4350 (2008).

2014 Society of Chemical Industry


48 Zimmermann W and Broda P, Utilization of lignocellulose from barley

straw by actinomyycetes. Appl Microbiol Biotechnol 30:103109
49 Mshandete A, Bjrnsson L, Kivaisi AK, Rubindamayugi MST and
Mattiasson B, Eect of particle size on biogas yield from sisal bre
waste. Renew Energy 31:23852392 (2006).
50 Hisano H, Nandakumar R and Wang Z, Genetic modication of lignin
biosynthesis for improved biofuel production. In Vit Cell Develop
Biol - Plant 45:306313 (2009).
51 Fu C, Mielenz JR, Xiao X, Ge Y, Hamilton CY, Rodriguez M, Chen
F, Foston M, Ragauskas A and Bouton J, Genetic manipulation
of lignin reduces recalcitrance and improves ethanol production
from switchgrass. Proc Nat Acad Sci 108:38033808 (2011).
52 Weng J, Li X, Bonawitz ND and Chapple C, Emerging strategies of
lignin engineering and degradation for cellulosic biofuel production. Curr Opin Biotechnol 19:166172 (2008).
53 Wymelenberg AV, Sabat G, Mozuch M, Kersten PJ, Cullen D and
Blanchette RA, Structure, organization, and transcriptional
regulation of a family of copper radical oxidase genes in the
lignin-degrading basidiomycete Phanerochaete chrysosporium.
Appl Environ Microbiol 72:48714877 (2006).
54 Martinez D, Larrondo LF, Putnam N, Gelpke MDS, Huang K, Chapman
J, Helfenbein KG, Ramaiya P, Detter JC and Larimer F, Genome
sequence of the lignocellulose degrading fungus Phanerochaete
chrysosporium strain RP78. Nat Biotechnol 22:695700 (2004).
55 Shi Y and Weimer P, Response surface analysis of the eects of pH and
dilution rate on Ruminococcus avefaciens FD-1 in cellulose-fed
continuous culture. Appl Environ Microbiol 58:25832591 (1992).
56 Weimer PJ, Eects of dilution rate and pH on the ruminal cellulolytic
bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture. Arch Microbiol 160:288294 (1993).
57 Hu Z, Yu H and Zhu R, Inuence of particle size and pH on anaerobic
degradation of cellulose by ruminal microbes. Int Biodeter Biodegrad 55:233238 (2005).
58 Hu Z, Wang G and Yu H, Anaerobic degradation of cellulose by rumen
microorganisms at various pH values. Biochem Eng J 21:5962
59 Bergquist PL, Gibbs MD, Morris DD, Teo V, Saul DJ and Morgan
HW, Molecular diversity of thermophilic cellulolytic and hemicellulolytic bacteria. FEMS Microbiol Ecol 28:99110 (1999).
60 Veeken A, Kalyuzhnyi S, Schar H and Hamelers B, Eect of pH
and VFA on hydrolysis of organic solid waste. J Environ Eng
126:10761081 (2000).
61 Dionisi D, Analysis of the eect of cellulose particle size on the rate
of microbial hydrolysis for bioethanol production. Energy Technol
1:675684 (2013).
62 Weimer P, Lopez-Guisa J and French A, Eect of cellulose ne structure on kinetics of its digestion by mixed ruminal microorganisms
in vitro. Appl Environ Microbiol 56:24212429 (1990).
63 Chyi YT and Dague RR, Eects of particulate size in anaerobic acidogenesis using cellulose as a sole carbon source. Water Environ Res
66:670678 (1994).
64 Brestic-Goachet N, Gunasekaran P, Cami B and Baratti JC, Transfer and expression of an Erwinia chrysanthemi cellulase gene in
Zymomonas mobilis. J Gen Microbiol 135:893902 (1989).
65 Zhou S, Davis F and Ingram L, Gene integration and expression
and extracellular secretion of Erwinia chrysanthemi endoglucanase
CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2.
Appl Environ Microbiol 67:614 (2001).
66 Zhou S and Ingram L, Simultaneous saccharication and fermentation of amorphous cellulose to ethanol by recombinant Klebsiella oxytoca SZ21 without supplemental cellulase. Biotechnol Lett
23:14551462 (2001).
67 Myung K and Yoo YJ, Novel SSF process for ethanol production from
microcrystalline cellulose using the -integrated recombinant
yeast, Saccharomyces cerevisiae L2612GC. J Microbiol Biotechnol
9:340345 (1999).
68 Den Haan R, Rose SH, Lynd LR and van Zyl WH, Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces
cerevisiae. Metab Eng 9:8794 (2007).
69 Yamada R, Yamakawa S, Tanaka T, Ogino C, Fukuda H and Kondo
A, Direct and ecient ethanol production from high-yielding rice
using a Saccharomyces cerevisiae strain that express amylases.
Enzyme Microbiol Technol 48:393396 (2011).
70 Abreu AA, Karakashev D, Angelidaki I, Sousa DZ and Alves MM, Biohydrogen production from arabinose and glucose using extreme


95 Calli B, Schoenmaekers K and van broekhoven KLD, Dark fermentative H2 production from xylose and lactose-eects of on-line pH
control. Int J Hydrogen Energy 32:522530 (2008).
96 Lo YC, Chen WM, Hung CH, Chen SD and Chang JS, Dark H2 fermentation from sucrose and xylose using H2 producing indigenous bacteria: feasibility and kinetic studies. Water Res 42:827842 (2008).
97 Temudo MF, Mato T, Kleerebezem R and van Loosdrecht MC, Xylose
anaerobic conversion by open-mixed cultures. Appl Microbiol
Biotechnol 82:231239 (2009).
98 Ohta K, Beall D, Mejia J, Shanmugam K and Ingram L, Metabolic
engineering of Klebsiella oxytoca M5A1 for ethanol production
from xylose and glucose. Appl Environ Microbiol 57:28102815
99 Yomano L, York S, Zhou S, Shanmugam K and Ingram L,
Re-engineering Escherichia coli for ethanol production. Biotechnol
Lett 30:20972103 (2008).
100 Liu S, Nichols NN, Dien BS and Cotta MA, Metabolic engineering of a
Lactobacillus plantarum double ldh knockout strain for enhanced
ethanol production. J Ind Microbiol Biotechnol 33:17 (2006).
101 Hahn-Hgerdal B, Wahlbom CF, Grdonyi M, van Zyl WH, Otero RRC
and Jnsson LJ, Metabolic Engineering of Saccharomyces cerevisiae
for Xylose Utilization. Springer, pp. 5384 (2001).
102 Haruta S, Cui Z, Huang Z, Li M, Ishii M and Igarashi Y, Construction
of a stable microbial community with high cellulose-degradation
ability. Appl Microbiol Biotechnol 59:529534 (2002).
103 Brethauer S and Studer MH, Consolidated bioprocessing of lignocellulose by a microbial consortium. Energy Environ Sci 7:14461453
104 Olson DG, McBride JE, Joe Shaw A and Lynd LR, Recent progress
in consolidated bioprocessing. Curr Opin Biotechnol 23:396405
105 Esteghlalian A, Hashimoto AG, Fenske JJ and Penner MH, Modelling
and optimization of the dilute sulfuric-acid pretreatment of corn
stover, poplar and switchgrass. Bioresource Technol 59:129136
106 Sun Y and Cheng J, Hydrolysis of lignocellulosic materials for ethanol
production: a review. Bioresource Technol 83:111 (2002).
107 Sun R, Lawther JM and Banks W, Fractional and structural characterization of wheat straw hemicelluloses. Carbohydr Polym
29:325331 (1996).
108 Hamelinck CN, van Hooijdonk G and Faaij APC, Ethanol from lignocellulosic biomass: techno-economic performance in short-, middleand long-term. Biomater Bioenergy 28:384410 (2005).
109 Galbe M and Zacchi G, Pretreatment of lignocellulosic materials
for ecient bioethanol production, in Biofuels. Springer, Berlin,
pp. 4165 (2007).
110 Timell TE, Recent progress in the chemistry of wood hemicelluloses.
Wood Sci Technol 1:4570 (1967).
111 Shiralipour A and Smith PH, Conversion of biomass into methane gas.
Biomater 6:8592 (1984).
112 Badger D, Bogue M and Stewart D, Biogas production from crops
and organic wastes. 1. Results of batch digestions. New Zeal J Sci
22:1120 (1979).
113 Chynoweth D, Turick C, Owens J, Jerger D and Peck M, Biochemical
methane potential of biomass and waste feedstocks. Biomater
Bioenergy 5:95111 (1993).
114 Forney LJ and Reddy CA, Bacterial degradation of kraft lignin. Devel
Ind Microbiol 20:163175 (1980).
115 Odier E, Janin G and Monties B, Poplar lignin decomposition
by gram-negative aerobic bacteria. Appl Environ Microbiol
41:337341 (1981).
116 Giroux H, Vidal P, Bouchard J and Lamy F, Degradation of kraft indulin
lignin by Streptomyces viridosporus and Streptomyces badius. Appl
Environ Microbiol 54:30643070 (1988).
117 Horwath W and Elliott LF, Ryegrass straw component decomposition
during mesophilic and thermophilic incubations. Biol Fert Soils
21:227232 (1996).
118 Waksam SA, Cordon T and Hulpoi N, Inuence of temperature upon
the microbiological population and decomposition processes in
composts of stable manure. Soil Sci 47:83114 (1939).
119 Franzluebbers A, Arshad M and Ripmeester J, Alterations in canola
residue composition during decomposition. Soil Biol Biochem
28:12891295 (1996).
120 Robinson CH, Dighton J, Frankland JC and Roberts J, Fungal communities on decaying wheat straw of dierent resource qualities. Soil
Biol Biochem 26:10531058 (1994).

D Dionisi et al.

121 Tuomela M, Hatakka A, Raiskila S, Vikman M and Itvaara M, Biodegradation of radiolabelled synthetic lignin (14C-DHP) and mechanical pulp in a compost environment. Appl Microbiol Biotechnol
55:492499 (2001).
122 Jouraiphy A, Amir S, El Gharous M, Revel J and Hadi M, Chemical
and spectroscopic analysis of organic matter transformation during composting of sewage sludge and green plant waste. Int Biodeterior Biodegrad 56:101108 (2005).
123 Yu H, Zeng G, Huang H, Xi X, Wang R, Huang D, Huang G and Li
J, Microbial community succession and lignocellulose degradation during agricultural waste composting. Biodegrad 18:793802
124 Huang D, Zeng G, Feng C, Hu S, Lai C, Zhao M, Su F, Tang L and
Liu H, Changes of microbial population structure related to lignin
degradation during lignocellulosic waste composting. Bioresource
Technol 101:40624067 (2010).
125 Tomati U, Galli E, Pasetti L and Volterra E, Bioremediation of olive-mill
wastewaters by composting. Waste Manage Res 13:509518
126 Hwang SS, Lee SJ, Kim HK, Ka JO, Kim KJ and Song HG, Biodegradation and saccharication of wood chips of Pinus strobus and
Liriodendron tulipifera by white rot fungi. J Microbiol Biotechnol
18:18191826 (2008).
127 Pavlostathis SG, Miller TL and Wolin MJ, Fermentation of insoluble cellulose by continuous cultures of Ruminococcus albus. Appl Environ
Microbiol 54:26552659 (1988).
128 Ng T, Weimer P and Zeikus J, Cellulolytic and physiological properties
of Clostridium thermocellum. Arch Microbiol 114:17 (1977).
129 Desvaux M, Guedon E and Petitdemange H, Carbon ux distribution and kinetics of cellulose fermentation in steady-state continuous cultures of Clostridium cellulolyticum on a chemically dened
medium. J Bacteriol 183:119130 (2001).
130 Weimer P and Zeikus J, Fermentation of cellulose and cellobiose
by Clostridium thermocellum in the absence and presence of
Methanobacterium thermoautotrophicum. Appl Environ Microbiol
33:289297 (1977).
131 Scheinger C and Wolin MJ, Propionate formation from cellulose and
soluble sugars by combined cultures of Bacteroides succinogenes
and Selenomonas ruminantium. Appl Microbiol 26:789795 (1973).
132 Ng TK, Ben-Bassat A and Zeikus J, Ethanol production by thermophilic
bacteria: fermentation of cellulosic substrates by cocultures of
Clostridium thermocellum and Clostridium thermohydrosulfuricum.
Appl Environ Microbiol 41:13371343 (1981).
133 Siegert I and Banks C, The eect of volatile fatty acid additions on
the anaerobic digestion of cellulose and glucose in batch reactors.
Process Biochem 40:34123418 (2005).
134 Yang Y, Tsukahara K, Yagishita T and Sawayama S, Performance of
a xed-bed reactor packed with carbon felt during anaerobic
digestion of cellulose. Bioresource Technol 94:197201 (2004).
135 OSullivan CA, Burrell PC, Clarke WP and Blackall LL, Structure of a cellulose degrading bacterial community during anaerobic digestion.
Biotechnol Bioeng 92:871878 (2005).
136 Ueno Y, Kawai T, Sato S, Otsuka S and Morimoto M, Biological production of hydrogen from cellulose by natural anaerobic microora. J
Ferment Bioeng 79:395397 (1995).
137 de Coninck-Chosson J, Aerobic degradation of cellulose and adsorp
tion properties of cellulases in Cellulomonas uda JC3: eects of
crystallinity of substrate. Biotechnol Bioeng 31:495501 (1988).
138 Bagnara C, Gaudin C and Belaich JP, Physiological properties of Cellulomonas fermentans, a mesophylic cellulolytic bacterium. Appl
Microbiol Biotechnol 26:170176 (1987).
139 Li X and Gao P, Isolation and partial properties of
cellulose-decomposing strain of Cytophaga sp. LX-7 from soil.
J Appl Microbiol 82:7380 (1997).
140 Peitersen N. Continuous cultivation of Trichoderma viride on cellulose.
Biotechnol Bioeng 19:337348 (1977).
141 Velkovska S, Marten MR and Ollis DF, Kinetic model for batch cellulase
production by Trichoderma reesei RUT C30. J Biotechnol 54:8394
142 Degli-Innocenti F, Tosin M and Bastioli C, Evaluation of the biodegradation of starch and cellulose under controlled composting conditions. J Environ Pollut Degrad 6:197202 (1998).
143 Pagga U, Beimborn D, Boelens J and De Wilde B, Determination of
the aerobic biodegradability of polymeric material in a laboratory
controlled composting test. Chemosphere 31:44754487 (1995).

2014 Society of Chemical Industry

J Chem Technol Biotechnol 2015; 90: 366383

Conversion of lignocellulosic biomass into ethanol

144 Hurwitz E, Beck A, Sakellariou E and Krup M, Degradation of cellulose by activated sludge treatment. J Water Pollut Control Fed
33:10701075 (1961).
145 Mezzanotte V, Bertani R, Degli Innocenti F and Tosin M, Inuence of
inocula on the results of biodegradation tests. Polym Degrad Stab
87:5156 (2005).
146 Bellia G, Tosin M, Floridi G and Degli-Innocenti F, Activated vermiculite, a solid bed for testing biodegradability under composting
conditions. Polym Degrad Stab 66:6579 (1999).
147 Mohee R, Unmar G, Mudhoo A and Khadoo P, Biodegradability of
biodegradable/degradable plastic materials under aerobic and
anaerobic conditions. Waste Manage 28:16241629 (2008).
148 Kato S, Haruta S, Cui ZJ, Ishii M and Igarashi Y, Eective cellulose
degradation by a mixed-culture system composed of a cellulolytic
clostridium and aerobic non-cellulolytic bacteria. FEMS Microbiol
Ecol 51:133142 (2004).
149 Lo YC, Bai MD, Chen WM and Chang JS, Cellulosic hydrogen production with a sequencing bacterial hydrolysis and dark fermentation
strategy. Bioresource Technol 99:82998303 (2008).
150 Lynd LR, Grethlein HE and Wolkin RH, Fermentation of cellulosic substrates in batch and continuous culture by Clostridium thermocellum. Appl Environ Microbiol 55:31313139 (1989).
151 Yang B and Wyman CE, BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechnol Bioeng
94:611617 (2006).
152 Huang X and Penner MH, Apparent substrate inhibition of the Trichoderma reseei cellulase system. J Agric Food Chem 39:20962100
153 Yang B, Willies DM and Wyman CE, Changes in the enzymatic hydrolysis rate of Avicel cellulose with conversion. Biotechnol Bioeng
94:11221128 (2006).
154 Claassen P, Van Lier J, Lopez Contreras A, Van Niel E, Sijtsma L, Stams
A, De Vries S and Weusthuis R, Utilisation of biomass for the supply
of energy carriers. Appl Microbiol Biotechnol 52:741755 (1999).
155 Kdr Z, Szengyel Z and Rczey K, Simultaneous saccharication
and fermentation (SSF) of industrial wastes for the production of
ethanol. Ind Crops Prod 20:103110 (2004).

156 Bullock G, Ethanol from Sugarcane. Sugar Research Institute, Australia

157 South C, Hogsett D and Lynd L, Continuous fermentation of cellulosic biomass to ethanol. Appl Biochem Biotechnol 39:587600
158 Zeikus J, Chemical and fuel production by anaerobic bacteria. Ann
Rev Microbiol 34:423464 (1980).
159 Schink B, Phelps TJ, Eichler B and Zeikus JG, Comparison of ethanol
degradation pathways in anoxic freshwater environments. J Gen
Microbiol 131:651660 (1985).
160 Beall D and Ingram L, Genetic engineering of soft-rot bacteria for ethanol production from lignocellulose. J Ind Microbiol
11:151155 (1993).
161 Nichols NN, Dien BS and Bothast RJ, Engineering lactic acid bacteria
with pyruvate decarboxylase and alcohol dehydrogenase genes
for ethanol production from Zymomonas mobilis. J Ind Microbiol
Biotechnol 30:315321 (2003).
162 Burchhardt G and Ingram L, Conversion of xylan to ethanol by
ethanologenic strains of Escherichia coli and Klebsiella oxytoca.
Appl Environ Microbiol 58:11281133 (1992).
163 Govindaswamy S and Vane LM, Kinetics of growth and ethanol production on dierent carbon substrates using genetically engineered xylose-fermenting yeast. Bioresource Technol 98:677685
164 Matsushika A, Inoue H, Watanabe S, Kodaki T, Makino K and
Sawayama S, Ecient bioethanol production by a recombinant occulent Saccharomyces cerevisiae strain with a
genome-integrated NADP-dependent xylitol dehydrogenase
gene. Appl Environ Microbiol 75:38183822 (2009).
165 Ha SJ, Galazka JM, Kim SR, Choi JH, Yang X, Seo JH, Glass NL, Cate
JH and Jin YS, Engineered Saccharomyces cerevisiae capable of
simultaneous cellobiose and xylose fermentation. Proc Nat Acad Sci
USA 108:504509 (2011).
166 Madhavan A, Tamalampudi S, Srivastava A, Fukuda H, Bisaria VS
and Kondo A, Alcoholic fermentation of xylose and mixed sugars
using recombinant Saccharomyces cerevisiae engineered for xylose
utilization. Appl Microbiol Biotechnol 82:10371047 (2009).


J Chem Technol Biotechnol 2015; 90: 366383

2014 Society of Chemical Industry