Fermentation of Lignocellulosic PDF

ELSEVIER
Fermentation of lignocellulosic
hydrolysates for ethanol production
Lisbeth Olsson and B~irbel Hahn-H~igerdal
Applied Microbiology, University o f Lund/Lund Institute o f Technology, Lund, Sweden
Ethanol production from lignocellulosic hydrolysates in an economically feasible process requires microorganisms that produce ethanol with a high yield from all sugars present (hexoses as well as pentoses) and have a high
ethanol productivity in lignocellulosic hydrolysates, i.e., can withstand potential inhibitors. Different fermentation organisms among bacteria, yeasts, and fungi (natural as well as recombinant) are reviewed with emphasis
on their performance in lignocellulosic hydrolysates. Depending on the type of lignocelluIosic hydrolysate, the
composition of inhibitors will differ and their influence on the microorganisms and the fermentation performance
will consequently vary. The inhibition may be partly overcome by the removal of inhibitors, i.e., detoxification.
Microbial constraints on parameters such as pH, temperature, and nutrient supplementation are discussed in
relation to their implication on the process economy. Not only are the properties of the microorganism of
importance in the process, but also the choice of fermentation strategies such as batch culture, continuous culture
with cell recycling and in situ ethanol removal. For the realization of the ethanol production from lignocellulosic
materials, the fermentation step has to be integrated with the rest of the process. These aspects are also discussed.
Keywords:Lignocellulosichydrolysates; inhibitors; detoxification; pentose fermentation; process integration; economics;

environmental parameters; analysis
Introduction
Ethanol has attracted interest as an alternative liquid fuel,
especially for transportation, for two reasons. Firstly, the oil
crisis in the mid 70s stressed the dependence on the supply
of petroleum which can be reduced by the use of alternative
fuels from renewable resources such as ethanol produced
from lignocellulosic materials. Secondly, if the ethanol production process only uses energy from renewable energy
sources, no net carbon dioxide is added to the atmosphere,
making ethanol an environmentally beneficial energy
source. In addition, the exhaust emissions from and toxicity
of ethanol are lower than those of petroleum.l.2 Lignocellulosic materials constitute an abundant and cheap feed-
Address reprint requests to Professor BO.rbelHahn-H~igerdal,Applied Microbiology, University of Lund/Lund Institute of Technology, P.O. Box
124, S-221 00 Lund, Sweden
The present address of Dr. Lisbeth Olsson is the Department of Biotechnology, Building 223, Technical University of Denmark, DK-2800
Lyngby, Denmark
Received 7 February 1995; revised 4 July 1995; accepted 8 February,1995
Enzyme and Microbial Technology 18:312-331, 1996

1996 by Elsevier Science Inc.
655 Avenue of the Americas, New York, NY 10010
stock, but the processing techniques required for ethanol

production are presently extensive and cosrly. The cost of
ethanol produced from lignocellulosic materials with currently available technology and under the present economic
conditions is not competitive with the cost of gasoline.
Comprehensive process development and optimization are
still required to make the process economically viable. In
reality, environmental considerations and energy and tax
policies will determine the extent of fuel ethanol utilization
in the future. 3
Ethanol production from l~gnocellulosic materials comprises the following steps: degradation of the lignocellulosic
structure to a fermentable substrate followed by fermentation and distillation of the fermentation broth to obtain 95%
ethanol (Figure 1). The ethanol yield in the process is an
important parameter with regard to economy both because
the cost of the raw material constitutes a major part of the
total production cost and also because the processing costs
are typically associated with the amount of material passing
through the process and not the amount of product made. 4
This review discusses the fermentation of lignocellulosic
hydrolysates, i.e., the process step in which ethanol is ac-
0141-0229/96/$15.00
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Ethanol production from lignocellulosic hydrolysate: L. Olsson and B. Hahn-H&gerdal

Lignocellulosic Prehydrolysis
material
hemicellulose
kignin <
terials. 4-7 Economically feasible ethanol production from

lignocellulosic materials requires: (i) the development of a
functional process organism, and (ii) the development of
process techniques such as fermentation strategies and integration of the fermentation step to the rest of the process.
Hydrolysed
1
Fermentation ]
I Hydrolysis
(pentose-rich)
Hydrolysed
cellulose ~(
Fermentation
(hexose-rich)
1
Distillation
Ethanol
Figure 1 A general flowchart for ethanol production from Iignocellulosic materials
tually produced. The main problems encountered in the efficient conversion of the lignocellulosic hydrolysates to
ethanol are twofold. Firstly after pretreatment, the hydrolysate contains not only fermentable sugars, but also a broad
range of compounds having inhibitory effects on the microorganisms used for fermentation. The composition of these
compounds depends upon the type of lignocellulosic material used and the chemistry and nature of the pretreatment
process. Secondly, the hemicellulose hydrolysates contain
not only hexoses but also pentoses. Hexoses can easily be
fermented by Saccharomyces cerevisiae with well-known
process techniques. The pentoses are more difficult to ferment. Several economic evaluations have illustrated that
efficient fermentation of pentoses is important for the overall economy of ethanol production from lignocellulosic ma-
Composition of lignocellulosic materials

Lignocellulosic materials are composed of mainly cellulose,
hemicellulose, and lignin. Cellulose is a linear, crystalline
polymer of [3-D-glucose units. The structure is rigid and
harsh treatment is required to break it down. Hemicellulose
is composed of linear and branched heteropolymers of L--arabinose, D-galactose, D-glucose, D-mannose, and D-xylose.
Methyl or acetyl groups are attached to the carbon chain to
various degrees. The composition of hemicellulose varies
with the origin of the lignocellulosic material. The structure
is not crystalline and is therefore easier to hydrolyze. Lignocellulosic materials considered for ethanol production are
hardwood, softwood, forestry residues, agricultural residues, and municipal solid waste (MSW). The composition
of the raw material depends on the source (Table 1).
Both cellulose and hemicellulose, which typically constitute 36-61% and 13-39% of the total dry matter, respectively (Table 1), can be used for ethanol production. The
pentose content in the raw material is of importance as
pentoses are difficult to ferment to ethanol. They constitute
typically 6-28% of the total dry matter (Table 1). To
achieve maximum ethanol yield, all monosaccharides have
to be fermented. Softwood hemicellulose contains a high
proportion of mannose units and more galactose units than
hardwood hemicellulose whereas hardwood hemicellulose
contains a high proportion of pentoses (Table ]).z4 In the
latter case, efficient fermentation of the pentose fraction is
of special importance.
Production of lignocellulosic hydrolysates

The degradation of the lignocellulosic structure often requires two steps--:first, the prehydrolysis in which the
Table 1 Composition of different lignocellulosic materials

Cellulose ( % ) ~
Lignin ( % ) a
Hemicellulose ( % ) a
Pentoses (%)~
References
Wheat straw
Corn cob
36
36
29
-
28
28
24
28
8
9
Spruce
Pine
43
44
29
29
26
26
6
8
8
10
40
37
51
21
21
16
39
23
29
25
14
16
10
(Theander, unpublished data)
11
43
47
61
6
12
21
13
25
16
n.d.
n.d.
n.d.
12
13
13
Material
Agricultural waste
Softwood
Hardwood
Birch
Willow
Aspen
MSW
Paper-based
Processed
Newspaper
n.d. means not determined

a% of total dry material. Ash and extractives are not included in the figures
E n z y m e M i c r o b . T e c h n o l . , 1996, v o l . 18, A p r i l
313
Papers
hemicellulose structure is broken down, and second, the
hydrolysis of the cellulose fraction in which lignin will remain as a solid by-product. The two hydrolyzed streams are
fermented to ethanol either together or separately, whereafter they are mixed together and distilled (Figure 1). During
the degradation of the lignocellulosic structure, not only
fermentable sugars are released, but a broad range of compounds, some of which might inhibit the fermenting microorganism.
The prehydrolysis process can be performed by physical,
chemical, or biological methods such as steam pretreatment,
milling, freeze explosion, acid treatment (hydrochloric acid,
phosphoric acid, sulfuric acid, sulfur dioxide), alkaline
treatment (sodium hydroxide, ammonia), or treatment with
organic solvents (ethanol, ethylene glycol) or white rot
fungi.15-! 7 In the prehydrolysis step, the hemicellulose is
liquified resulting in a mixture of mono- and oligosaccharides.
The hydrolysis of the cellulose is usually performed by
weak acids ~8 or by enzymes. 19-21 Concentrated hydrochloric acid has also been utilized and in this case, the prehydrolysis and hydrolysis are carried out in one step. 22 Generally, acid hydrolysis procedures give rise to a broad range
of compounds in the resulting hydrolysate, some of which
might negatively influence the subsequent steps in the process. A weak acid hydrolysis process is often combined with
a weak acid prehydrolysis.
Three of the most commonly considered hydrolysis processes, the concentrated hydrochloric acid process, the twostep dilute acid hydrolysis, and enzymatic hydrolysis have
been compared in an economical analysis. 23 None of the
three ethanol production processes could be excluded as!ess
economical than the others. The cost of the enzymatic process was most unclear due to the uncertainty in the cost of
the enzyme. The technology in the enzymatic process also
has the best potential for improvement.
Pentose fermentation
The pentose fraction in hemicellulose consists mainly of
xylose, but depending on the raw material origin, the arabinose fraction may be substantial. Efficient xylosefermenting microorganisms have been found among bacteria, yeasts, and fungi (natural as well as recombinant). During the last fifteen years, research has been focused on
finding these xylose-fermenting microorganisms and understanding the xylose metabolism 24-26 while less research has
been concerned with the arabinose metabolism. Typical
ethanol yields and total volumetric ethanol productivities
for batch fermentations with these microorganisms in laboratory medium using xylose as the carbon source are summarized in Table 2. The yields are based on the initial xylose
concentration as those are economically relevant opposed to
consumed xylose concentrations frequently reported in the
literature. The theoretical yield for ethanol production from
glucose is 0.51 g ethanol g-i glucose (2 mol mol-1). The
theoretical ethanol yield from xylose is generally considered
to be 0.51 g ethanol g-1 xylose (1.67 mol mol-1), which is
also assumed here. Other suggestions for the ethanol yield
from xylose have ranged from 0.31-0.61 g g-l.XT,X8
314
Enzyme Microb. Technol., 1996, vol. 18, April
The total volumetric ethanol productivity, referred to as

the productivity in this review, is expressed in grams per
liter per hour and is defined as the final ethanol concentration divided by the total fermentation time. To compare the
fermentation capacity of different microorganisms, the specific productivity, i.e., the amount of ethanol produced per
hour per gram of cell mass, is the most appropriate measurement since the volumetric productivity is a function of
the cell density. However, in the case of batch fermentations, it is difficult to calculate the specific productivity as
the cell mass concentration changes during the course of the
fermentation, and therefore the volumetric productivities
are reported here.
Among naturally occurring bacteria, the highest yield has
been reported to be 0.39 g g-1 for Clostridium thermohydrosulfuricum 39E (Table 2). Generally, bacteria have a
broad substrate range, but ethanol is seldom the only product which is reflected in the low yields; 0.16-0.39 g g-l. Not
only can monosaccharides be fermented by bacteria but also
cellulose and other biopolymers. 62 Zymomonas mobilis is an
exceptional bacterium in the sense that it produces ethanol
with high yield and productivity, but the carbon sources it
can utilize for ethanol production are restricted to glucose,
fructose, and sucrose. 63,64 T w o operons encoding xylose
assimilation (xylose isomerase and xylulokinase) and pentose phosphate pathway enzymes (transaldolase and transketolase) has been constructed and transformed into Z. mobilis in order to broaden its substrate utilization. 65 This recombinant Z. mobilis grew only on xylose as the carbon
source and produced ethanol with a productivity of 0.57 g
1-1 h -1 and a yield of 0.44 g g-1 xylose.
Genetic engineering has been used to improve the ethanol yield of bacteria, resulting in recombinant bacteria
showing high yields; 0.44-0.52 g g-1 and high productivities; 0.18-0.96 g 1-1 h -1 (Table 2). In Escherchia coli, the
metabolism was redirected toward ethanol production by
inserting the genes for pyruvate decarboxylase and alcohol
dehydrogenase from Z. mobilis, thereby increasing the flux
through this pathway. 66'67 Additional changes were made in
order to minimize the acid production. 36 The metabolic
fluxes through the ethanol-producing pathway were enhanced in recombinant E. coli compared with the wild type
strain when fermenting in a laboratory medium. 68 This resulted in higher rates of both cell mass and ethanol production compared with celbmass and mixed acid production of
the wild type strain. Also, when lignocellulosic hydrolysates
were fermented, increased metabolic fluxes were observed
(Olsson et al., unpublished data). The initial sugar consumption rate was 0.02 g g-1 h -1 for E. coli ATCC 11303
(the wild type strain) and 0.16 g g-i h-1 for recombinant E.
coli KO 11 when fermenting spent sulfite liquor (SSL), and
0.06 g g-1 h-i and 0.65 g g-1 h-a, respectively, when fermenting an enzymatic hydrolysate of willow under comparable conditions. In addition to xylose, recombinant E. coli
can also ferment arabinose, glucose, galactose, and mannose 69, i.e., all monosaccharides present in the hydrolysates.
Hexoses are the preferred carbon source for recombinant E.
coli; the problem of "xylose sparring", i.e., incomplete
fermentation of xylose, has been found when hexoses domi-
Ethanol production from lignocellulosic hydrolysate: L. Olsson and B. Hahn-H#gerdal

Table 2
Performance of ylose-fermenting microorganisms
Strain
Bacteria: naturally occurring

Bacillus macerans DMS 1574
Bacteroides po/ypragmatus NRCC 2288
C/ostridium saccharolyticum ATCC 35040
C. thermohydrosu/furicum 39E
Erwinia chrysanthemi B374
Thermoanaerobacte# ethano/icus ATCC 31938
Bacteria: recombinant b
Erwinia chrysanthemi B374 (pdc)
Escherichia coil B, pLOI297 (pc/c, adhB)
E. coil B K O l l (pdc, adhB, frd-)
K/ebsiel/a oxytoca M5A1 (pdc, ac/hB)
K. p/antico/a SDF20 (pde, pf/ )
Zymomonas mobi/is CP4 (pZB5)
Yeasts: naturally occurring
Candida b/ankii ATCC 18735
C. famata
C. fructus JCM-1513
C. guil/iermondii ATCC 22017
C. shehatae CBS 4705
C. shehatae CSIR-Y492
C. sp. CSIR-62 A/2
C. tenius CBS 4435 (11) d
C. tropica/is KY 5014 (2)
C/avispora sp. UWO(PS) 83-877-1 (11)
K/uyveromyces ce//obiovorus KV 5199 (3)
K. marxianus
Pachysolen tannophi/us NRRL Y-2460
P. tannophi/us RL 171
Pichia segobiensis CBS 6857
P. stipitis CBS 5773 (5)
P. stipitis CBS 5776
Schizosaccharomyces pombe ATCC 2478 (8)
Yeasts: recombinant e
Saccharomyces cerevisiae (XYL 1, XYL 2)
S. cerevisiae TJ 1 (XYL I, XYL 2)
S. cerevisiae H560 (XYL 1, XYL 2)
Schizosaccharomyces pombe (xy/ A)
Fungi
Aeurobasiclium pu//u/ans (2)
Fusarium avenaceum VTT-D-80146 (5)
F. c/amydosporum VTT-D-77055
F. cu/morum VTT-D-80148 (3)
F. graminearum VTT-D-79129 (4)
F. lycopersici ATCC 16417
F. oxysporum VTT-D-80134 (3)
F. sambucium VTT-D-77056
F. solani VTT-D-77057 (2)
F. tricinetum VTT-D-80139 (2)
Moni/ia sp.
Mucor 105 (2)
Neurospora crassa NCIM 870
Paecilomyces sp. NFI ATCC 20766
Xylose
(g i-1)
Ethanol
(g i-1)
Yield
(g g-l)
Productivity
(g 1-1 h -1)
20
44
25
5
5
4
3.3
6.5
5.2
2.0
0.03
0.09
0.05
1.5
0.16
0.15
0.21
0.39
0.23 a
0.36
80
80
100
17
25
39.2
41.6
46.0
7.7
11
0.44a
0.49
0.52
0.46
0.44
0.44
0.70 c
0.87
0.96
0.18
0.57
33
35
36
37
38
65
50
20
20
40
50
90
50
20
20
20
20
20
20
50
20
20
50
50
5.1
3.9
4.7
4.5
24.0
26.2
20.1
6.4
2.8
5.9
4.4
5.6
6.2
13.8
5.0
5.9
22.3
5.0
0.10
0.20
0.24
0.11
0.48
0.29
0.40
0.32
0.14
0.30
0.22
0.28
0.31
0.28
0.25
0.30
0.45
0.10
0.07
0.07
0.02
0.04
0.19
0.66
0.42
0.03
0.06
0.11
0.09
0.10
0.06
0.28
0.02
0.02
0.34
0.07
39
40
41
42
43
44
45
46
47
48
47
49
50
51
46
46
52
39
21.7
50
49.2
50
1.6
2.7
0.3
21.0
0.07
0.05
0.01
0.42
0.07
0.02
0.01
0.19
53
54
55
56
4.2
12.0
11.0
12.0
11.0
16.0
25.0
13.0
11.0
7.0
12.6
8.0
6.9
39.8
0.21
0.24
0.22
0.24
0.22
0.32
0.50
0.26
0.22
0.14
0.25
0.16
0.35
0.40
0.09
0.07
0.07
0.07
0.07
0.17
0.17
0.08
0.07
0.04
0.08
0.08
0.04
0.24
40
57
57
57
57
58
57
57
57
57
59
58
60
61
20
50
50
50
50
50
50
50
50
50
50
50
20
100
References
29
30
31
32
33
34
Adapted from Tables 10. 1, 10.8, and 10.9 in Ref 26. Reprinted with the permission of the publisher and authors
ag ethanol g-1 xylose consumed
bThe relevant genotype is given in parentheses, pdc, pyruvate decarboxylase; pfl, pyruvate formate lyase; adhB, alcohol dehydrogenase II; frd, fumarate reductase, pZB5 carries the genes for xylose isomerase, xylulokinase, transketolase, and transaldolase
CMaximum volumetric productivity
dFigures in parentheses denote number of strains investigated (if more than one)
eThe relevant genotype is given in parentheses. XYL 1, xylose reductase; XYL 2, xylitol dehydrogenase; xyl A, xylose isomerase
nate in the medium. 7-72 Hexoses and pentoses can be utilized simultaneously by ethanologenic E. coli, but exercise
mutual inhibition. 73,74
Candida shehatae, Pachysolen tannophilus, and Pichia

stipitis are the most thoroughly investigated and most efficient naturally occurring xylose fermenters among the
315
Papers
yeasts. The yields for these microorganisms are average to

high (0.28-0.48 g g-~), and the productivities reasonable
(0.02-0.66 g 1-1 h -1) (Table 2). For optimal performance,
these organisms need carefully controlled oxygenation.75,76
The two main reasons for yields below the theoretical values are (i) the production of xylitol especially by P. tannophilus, and (ii) the reassimilation of ethanol. 77'78 Mutants
of P. tannophilus which showed reduced growth on ethanol
have been selected. 79 These mutants gave an increased ethanol yield on xylose. Other yeasts and yeast genera have also
been reported to produce ethanol at high yields; Candida,
0.10-0.40 g g-l; Clavispora sp., 0.30 g g-l; Kluyveromyces, 0.22-0.28 g g-l; and Pichia segobiensis, 0.25 g
g-1 (Table 2).

When ethanol is produced from hexoses, S. cerevisiae is
the most widety used process organism providing high
yields and productivities in addition to a remarkable ethanol
tolerance. The unusual properties of this yeast are the result
of adaptation to efficient ethanol production from hexose
sugars during thousands of years. However, S. cerevisiae is
not able to utilize xylose for ethanol production whereas the
isomer of xylose (xylulose) can be fermented. Therefore,
genetically engineered xylose-fermenting S. cerevisiae are
being developed (Table 2). So far, only low ethanol yields
have been demonstrated from xylose. 53-55 The limited success has been ascribed to redox imbalances and limitations
in existing pathways. The fermentation rate of xylulose by
S. cerevisiae is 10 times less than the fermentation rate of
glucose. 8 The genetically engineered Schizosaccharomyces
pombe showed a yield of 0.42 g g-~ and a productivity of
0.19 g 1-1 h-1. 56 However, the recombinant S. pombe was
dependent on nutrient supplementation and without yeast
extract, malt extract, and peptone, the yield was only 0.15
g g=l.
Presently, fungi exhibit too low productivities; 0.04-0.24
g 1-t h-1 on xylose (Table 2) to be of commercial interest.
Fungi can utilize a broad range of substrates including not
only monosaccharides, but also disaccharides, cellulose,
and xylan. This property would be especially beneficial in a
process where the hydrolysis of the lignocellulosic material,
i.e., saccharification and fermentation, take place simultaTable 3
neously. Several species have shown high yields in laboratory media. With Fusarium oxysporum VTT-D-80134,
Neurospora crassa NCIM 870, and Paecilomyces sp. NFI
ATCC 20766, yields of 0.50, 0.35, and 0.40 g g-l, respectively, have been achieved.
Yet another way of producing ethanol from xylose would
be to first convert xylose to xylulose using the enzyme
xylose isomerase, and then ferment xylulose to ethanol.
Candida tropicalis ATCC 20240, Hansenula anomala
ATCC 34080, Kluyveromyces fragilis ATCC 12424, Saccharomyces uvarum ATCC 24556, and Schizosaccharomyces pombe ATCC 2476 all produced ethanol with high
yields (0.41-0.47 g g-~ xylulose). 39 The equilibrium between xylose and xylulose is temperature dependent and lies
on the xylose side. sl A process where the two steps are
performed simultaneously has the advantage of pulling the
isomerase reaction towards xylulose. Under these conditions, a compromise must be made between optimum conditions for the enzyme and the microorganism. The xylulose-fermenting yeasts are mesophilic and have an acidic
pH optimum. Xylose isomerase, on the other hand, has a pH
optimum in the range of 7-9 and a temperature optimum of
50-80C. 82 Xylose isomerase from Lactobacillus brevis has
been investigated for xylose fermentation due to its comparably low pH optimum 6-7, and it was found to be active at
pH 5. 83 Therefore, this xylose isomerase would be better
suited for simultaneous isomerization and fermentation. An
alternative approach to the problem of the enzyme and the
microorganisms having different pH optima is the construction of a bilayered immobilized enzyme pellet. 84 The inner
layer consists of xylose isomerase and the outer of urease.
Urea was added to the fermentation broth and was converted to ammonia by the enzyme urease, creating a pH of
7.0-8.0 at the isomerization site when the pH was 4.0-5.0 in
the fermentation broth.
The best results achieved for xylose fermentation with
xylose isomerase and yeasts are summarized in Table 3. S.
cerevisiae and S. pombe were the most successful microorganisms for this application, giving yields in the range of
0.15-0.37 g g-1 and productivities of 0.10-0.67 g 1-1 h -1
(Table 3). The highest reported yield was 0.50 g g-1 and the
Xylose fermentation with xylose isomerase and yeasts
Strain
Enzyme
Candida shehatae ATCC 22984 (2) a

C. tropicalis NRRL Y-11860 (3)
Pichia stipitis NRRL Y-17104 (2)
Saccharomyces cerevisiae ATCC 24860
Sweetzyme Q
Sweetzyme Q
Sweetzyme Q
S. cerevisiae ATCC 24860

S. cerevisiae ATCC 24860 (2)
S. cerevisiae ATCC 24860
S. cerevisiae, Bakers' yeast b
Streptomyces sp. ATCC 21175
Ethanol
(g i-1)
Yield
(g g-l)
Productivity
(g 1-1 h -1)
References
60
60
60
10.2
7.9
12.7
0.17
0.13
0.21
0.11
0.08
0.13
85
85
85
60
60
60
60
48
60
40
60
18.1
19.5
9.2
20.3
24.0
16.0
14.8
20.4
0.30
0.32
0.15
0.34
0.50
0.27
0.37
0.34
0.38
0.41
0.10
0.42
1.00
0.17
0.37
0.67
86
86
85
86
87
85
88
88
Actinoplanes missouriensis
ATCC 14538
Schizosaccharomyces uvarum ATCC 26192

S. pombe NRRL Y-164
S. pombe NRRL Y-164
Xylose
(g i-1)
Sweetzyme Q
Taka-sweet
Optisweet-P
Sweetzyme Q
Sweetzyme Q
Sweetzyme Q
Adapted from Table 10.3 in Ref 26. Reprinted with the permission of the publisher and authors
~Figures in parentheses are the number of strains investigated (if more than one)
bUnder anaerobic conditions with azide present (46 mM)
316
E t h a n o l p r o d u c t i o n f r o m l i g n o c e l l u l o s i c h y d r o l y s a t e : L, Olsson a n d B. H a h n - H ~ g e r d a l
productivity was 1.0 g 1-1 h -1 when S. cerevisiae was used

together with Optisweet-P, a commercially available xylose
isomerase, s7 In this case, an extremely high cell mass concentration of 75 g 1-1 dry weight was used. It is uncertain to
what extent the addition of azide influenced the results.
drolysis and hydrolysis due to the degradation of sugars. S.

cerevisiae can metabolize furfural; after it has been consumed, the inhibitory effect disappears. 13 S. cerevisiae can
be adapted to the presence of sugar degradation products
(furfural, maltol, and 5-hydroxymethyl furfural).104 The nutrient content of the medium was found to be important for
their degree of inhibition. Thirdly, lignin degradation products are produced in prehydrolysis and hydrolysis. This
group of inhibitors includes a wide range of aromatic and
polyaromatic compounds with a variety of substituents.
Fourthly, products from the fermentation process, such as
ethanol, acetic acid, glycerol, and lactic acid, inhibit the
microorganism; the influence of these compounds will be
especially evident in recirculating systems. 95 Finally, metals
released from the equipment and additives such as SO2 will
also inhibit fermentation. 15 Sulfites are widely used in the
food industry as a preservative. 16 The inhibitory effect of
SO 2 is, like acetic acid, pH dependent. For instance, 0.3 g
1-1 SO 2 inhibited P. stipitis CBS 5773 at pH values below
5.1 The pH dependence of the inhibitory action of acetic
acid is, however, more pronounced than that of SO 2. The
inhibitors not only reduced the fermentation rate but also
influenced the product pattern. For example, sulfite present
in SO2-pretreated hydrolysates and in SSL can increase the
glycerol production of S. cerevisiae, lO7,1o8 In contrast to its
inhibitory action, sulfite may have a positive influence on
the fermentability in connection with its use in detoxificafion (see below).
Thus, the hydrolysates comprise a complex mixture of
components. For instance, when a hemicellulose hydrolysate of steam-pretreated birchwood was analyzed with capillary GC, more than 60 compounds were separated. 19 The
literature does not offer a clear picture of which inhibitors
have the most pronounced effect, but acetic acid and lignin
degradation products are suggested to be most inhibitory
(Table 4).11 The potential inhibition of a component should
Inhibiting compounds in
lignocellulosic hydrolysates
In the actual process, tire liquid streams must be recirculated
to minimize the requirement for fresh water and the production of wastewater, s9 The consequence of recirculation
is an accumulation of nonmetabolizable compounds in the
hydrolysate. These components are referred to as inhibitors
since they may have an inhibitory effect on the process
organism. Therefore, knowledge about inhibitors and how
to minimize their effects is of the utmost importance for
efficient fermentation in real process situations.
The amount and nature of inhibiting compounds depends
on the raw material, the prehydrolysis and hydrolysis procedures, and the extent of recirculation in the process. Fermentation inhibitors in lignocellulosic hydrolysates can be
divided into several groups depending on their origin (Table
4). Firstly, substances released during prehydrolysis and hydrolysis include acetic acid (released when the hemicellulose structure is degraded) and extractives. The extractives
comprise terpenes, alcohols, and aromatic compounds such
as tannins. 9s The inhibitory effect of acetic acid is pH dependent (Table 4) since it is the undissociated acetic acid
which penetrates the cell membrane and dissociates intracellularly due to the higher intracellular pH. The intracellular pH thereby decreases. 99 The fermentability of a lignocellulosic hydrolysate can be improved by raising the
pH. 1,101 Secondly, a group of inhibitors (furfural, 5-hydroxymethyl furfural, laevulinic acid, formic acid, and humic substances) 12 is produced as by-products in prehy-
Table 4
Effects of inhibiting compounds on fermentation
Group of inhibitors
Compounds released
during pretreatment
Sugar degradation
products
Lignin degradation
products
Fermentation products
Remaining
Inhibitor
acid
acid
acid
acid
Furfural
5-hydroxymethyl furfural
Cinnamaldehyde
p-hydroxybenzaldehyde
p-hyd roxybenzaldehyde
Syringaldehyde
Syringaldehyde
Acetaldehyde
Ethanol
Formic acid
Lactic acid
Chromium
Copper
Iron
Acetic
Acetic
Acetic
Acetic
Nickel
Concentration
(gl 1)
Microorganism
1.4
4.3
8.0
8.0
1.0
3.0
1.0
0.4
1.0
0.5
0.22
5.0
120
2.7
38
0.1
0.04
0.5
0.05
Saccharomyces cerevisiae
S, cerevisiae
Pichia stipitis
P. stipitis
P. stipitis
P. stipitis
S. cerevisiae
Klebsiella pneumoniae
S. cerevisiae
K. pneumoniae
P. stipitis
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
Pachysolen tannophilus
P. tannophilus
P. tannophilus
P. tannophilus
% inhibition of
growth (g) or
fermentation (f)
References
50% (f); pH = 4.5

50% (f); pH = 5.5
98% (f); pH = 5.1 "
25% (f); pH = 6.5
47% (g); 71% (f)
69% (g); 90% (f)
100% (f)
68% (g)
48% (f)
40% (g)
72% (f)
80% (g)
100% (g)
80% (g)
80% (g)
95% (f)
29% (f)
45% (f)
92% (f)
90
90
91
91
92
92
93
94
93
94
52
95
96
95
95
97
97
97
97
317
Papers
be considered in relation to its concentration in the hydrolysate.
Analysis of lignocellulosic hydrolysates

The comparison of fermentation organisms and fermentation modes requires reliable analytical methods for the determination of substrate consumption and product formation. The analysis of fermentation broths based on lignocellulosic materials is not straightforward due to the complex
sample matrix, i.e., the diversity of constituents in the hydrolysates (see above) will interfere. In addition, the ethanol
produced during the process increases the solubility of hydrophobic substances in the broth so that the composition of
the matrix continuously changes during fermentation. 111
Samples from the fermentation of SSL and the fermentation of enzymatic lignocellulose hydrolysates were analyzed on two different chromatographic set-ups; ligandexchanged-based separation coupled to refractive index detection and an anion-exchange-based system coupled to
pulsed amperometric detection. The carbohydrate concentrations determined by the two methods were compared and
found to agree within 95% in only 30% of the cases. T M
Sugar utilization and solvent production in several fermentations of lignocellulosic hydrolysates were determined in
two different ways: the sample components were determined using single column liquid chromatography (CLC)
analysis and with a combination of gas chromatographic
analysis and colorimetric methods. 112 The concentrations
determined by the two methods were compared; the results
for 15 out of 19 samples gave results that agreed within
80%, but only 5 samples agreed within 95%. Substantial
differences in analysis results (a relative standard deviation
of 12% in total sugar content) were also found when different laboratories altogether made 18 analyses of the same
corn cob hydrolysates. 113 One reason for the difference in
analysis results is the lack of selectivity in the detection
method employed. During the development of analysis
methods, careful evaluation of the selectivity is necessary.
The use of two different detection principles may indicate
impurities in the individual chromatographic peaks. 114 Diode array detection and mass spectrometry, which both give
spectra over each peak resulting from the CLC separation, ~1 will give detailed information on the peak composition. In line with discussions of suitable fermentation alternatives, awareness of the difficulties and uncertainties in
the analysis methods used for the determination of the fermentation characteristics is necessary.
Detoxification and adaptation

Several methods of detoxication, i.e., the removal of inhibitors from lignocellulosic hydrolysates have been proven to
increase their fermentability. The addition of activated charcoal, 115 extraction with organic solvents, 9sA16'117 ionexchange, 98,12,116 ion exclusion, t9 molecular sieves, 52
overliming,11s.119 and steam stripping 12o have been investigated. Detoxification increases the cost of a process; it
would be better if it were not required.
Overliming has been the most widely used detoxification
318
method and can be performed in various "ways. Calcium

hydroxide (or some other hydroxide) is added to the medium until the pH reaches 10-10.5. After mixing, the resulting precipitate is removed. The precipitate consists
mainly of calcium salts of low solubility dominated by calcium sulfate. This treatment can be combined with heat,
because at elevated temperatures the solubility of calcium
sulfate decreases and, in addition, volatile compounds such
as furfural are stripped off. T M Calcium sulfate precipitates
acidic compounds. Sulfite is often added at some stage of
the detoxification--before or after overtiming. Sulfite functions as a reducing agent and it has been suggested that the
redox potential of the fermentation broth is of importance
for the fermentability. 118 The results of the detoxification
depend, for example, on the temperature, the hydroxide
used, and the conditions of when and how much sutfite was
added. 74,1t9,121A22 Different detoxification methods have
various effects on the hydrolysates (Table 5), but in most
instances the inhibitors are only partly removed. ~23 The
choice of detoxification method will depend both on the
type of hydrolysate to be detoxified (regarding the efficiency of the detoxification method on that kind of hydrolysate) and the fermentation organism employed (regarding
which inhibitors need to be removed).
Comparing the efficiency of several detoxification methods, it was found that a non-fermentable lignocellulosic
hydrolysate, without treatment, could be fermented as
quickly as a comparable glucose solution after ion-exchange
treatment or charcoal treatment. 126 When a hydrolysate of
steam-pretreated willow was fermented by E. coli KO11,
the productivity in undetoxified hydrolysate was 0.03 g 1-1
h -a compared with 0.42 g 1-1 h -1 in a hydrolysate that had
been overlimed and supplemented with sodium sulfite. TM
The yield was 0.51 g g-1 in both cases. An economic evaluation for the use of E. coli K O l l in the fermentation of a
pentose-rich hydrolysate of steam-pretreated willow, which
was detoxified by overliming, has been made.la7 The cost
analysis indicated potentially good economy, although the
detoxification constituted 22% of the total ethanol production cost. Consequently, considerable cost savings could be
made if new, specific, efficient, and cheap detoxification
methods were available. In order to facilitate the development of such methods, the key inhibitors must be identified;
for instance, by fractionating the hydrolysate, fermenting
the fractions, and thereafter identifying the components in
the fractions
which are most inhibiting to the microorganI
isms. Identification of all the components in lignocellulosic
hydrolysates requires a combination of several analysis
methods. In a few studies, attempts have been made to
obtain a more complete picture of these components. 111.128
This knowledge should enable not only the development of
custom-made detoxification methods, but also modifications of the prehydrolysis and hydrolysis processes to minimize the formation of the most potent inhibitors. ~29
An alternative approach to detoxification is to adapt the
microorganism to the lignocellulosic hydrolysate. Adaptation has been shown to increase the ability of a broad range
of yeast strains to grow in lignocellulosic hydrolysates. 116
Adaptation of P. stipitis CBS 5776 by repeated recycling in
an acid hydrolysate from aspen resulted in an increase in

Table 5 Detoxification procedures and their effect
Procedure
Steam stripping
Neutralization with CaO, NaOH, KOH; activated
carbon; filtration
Neutralization to pH 6.5 or overtitration to pH 10
with Ca(OH)2, CaO, or KOH; removal of
precipitate; H2SO4 to pH 6.5
Ion exclusion chromatography (cation exchange
resin)
Ether extraction
Evaporation under vacuum
Ethylacetate extraction
Molecular sieve
Mixed bed ion resins
Anion/cation exchange resins
Effect of detoxification
Removal of volatiles (furfural, phenols) including
acetic acid
Reduction of acetic acid concentration
References
115, 121
115
Precipitation of acetate, heavy metals, furfurals,

tannins, terpenes, phenolics
115,121,119,124
Removal of aromatic monomers and dimers
Removal of furfural
Removal of acetic acid
Removal of ligNn-degradation products
Partial removal of acetic acid, furfural, and
soluble lignin
Partial removal of acetic acid, furfural, and
soluble lignin
Partial removal of acetic acid, removal of metal
ions
109
125
117
117
52
52
97
Adapted from Table 6 in Ref 123. Reprinted with permission of the publisher and the authors
productivity from 0.60-0.85 g 1-1 h -I and an increase in

yield from 0.32-0.45 g g-1.13o In another study, P. stipitis
CBS 5776 was adapted to a detoxified hydrolysate (red oak
acid hydrolysate) and was then able to ferment an undetoxifled hydrolysate with a larger inoculum giving a yield of
0.30 g g-~.52 The unadapted P. stipitis could not ferment the
undetoxified hydrolysate at all.
At Nanjing Forestry University, China, extensive work
on the adaptation of C. shehatae to SSL has been carried
out, resulting in strains that can tolerate high temperature
(38C), high acetic acid concentration (15 g 1-1), and a high
pentose fraction (70%) (Prof. S. Yu, personal communication). With this strain, SSL containing 15 g 1-1 acetic acid
could be fermented at a pH of 5.2 giving a yield of 0.41 g
g 1. In addition, the adapted C. shehatae was bigger than the
wild type strain, enabling easier separation. The ability to
adapt S. cerevisiae to lignocellulosic hydrolysates has been
shown to be strain dependent. 131 The adapted strains are
seldom deposited in culture collections; they have only been
used in individual laboratories, thereby making verification
of their performance difficult. In addition, the stability of
the adapted strains may present a problem.
Isolation of strains from natural or industrial habitants
has been shown to be a useful technique for finding strains
with suitable properties for use in lignocellulosic hydrolysates. Xylose-fermenting yeast strains have been found in
black knots, insect frass, and tree exudates. 4 A strain of S.
cerevisiae isolated from a SSL fermentation plant was
shown to be able to utilize glucose and galactose simultaneously in the presence of acetic acid 132 in contrast to the
behavior of Bakers' yeast. As an alternative to adaptation
and isolation from harsh environments, genetic engineering
might improve the microorganism to better withstand a specific inhibitor; however, this can only be attempted if the
inhibiting mechanism is known.
Fermentation of lignocellulosic hydrolysates

The performance of several of the pentose-fermenting microorganisms described above has been evaluated in differ-
ent lignocellulosic hydrolysates containing varying amounts

of hexoses and pentoses. The sensitivity of the microorganism to the hydrolysate varies with strain and fermentation
conditions (temperature, pH, aeration, and nutrient supplementation). Microorganisms shown to be efficient ethanol
producers from xylose in laboratory media will not necessarily ferment the lignocellulosic hydrolysates efficiently.
Therefore, studies of the fermentative performance of microorganisms in lignocellulosic hydrolysates are a useful
way of finding suitable strains. 64,1,133 The raw material
and process steps before fermentation will strongly influence the composition and the fermentability of the hydrolysate. In order to achieve optimum total production efficiency; process integration, i.e., studies of how optimization
of one unit operation will influence related process steps, is
of importance. Until more is known about inhibitors and
how they are generated, accurate predictions of optimal process conditions are impossible and process optimization is a
matter of trial and error. Some illustrative examples of batch
fermentations in lignocellulosic hydrolysates have been
compiled in Table 6.
Among the bacteria used for ethanol production from
lignocellulosic hydrolysates, recombinant E. coli gave the
best yields (0.36-0.51 g g-1 in a range of different hydrolysates, Table 6). However, in order to achieve productivities comparable to those in a laboratory medium (Table 2),
detoxification was necessary (Table 6). In an interlaboratory
comparison of recombinant E. coli, P. stipitis, recombinant
S. cerevisiae, S. cerevisiae in combination with xylose
isomerase, and F. oxysporum, the recombinant E. coli KO11
was the best choice for the fermentation of an acid hydrolysate of corn cob. 113 With the addition of nutrients and
detoxification of the hydrolysate by overliming, the yield
was 0.54 g g-1 and the maximum productivity 1.32 g 1-1 h -1.
C. shehatae and P. stipitis were the two pentose-fermenting
yeasts that fermented lignocellulosic hydrolysates most successfully (Table 6). The adapted strains, called R-strains,
have shown yields in the range of 0.34-0.45 g g-1 and
productivities of 0.14-1.27 g 1-1 h -1. Fermentation in steamE n z y m e M i c r o b . T e c h n o l . , 1996, v o l . 18, A p r i l
319
Papers
Table 6
Fermentation of lignocellulosic hydrolysates
Microorganism
Raw
material
Pretreatment
Ethanol
(g i-1)
Yield
(g g-l)
Productivity
(g I-lh -1)
References
27.8g, 9.7x
8.3
0.22
0.09
134
Sugars a
(g 1-1)
Detoxification
Pentose-fermenting
microorganisms
Bacteria
Bacteroides
polypragmatus
NRCC 2288
B. polypragmatus
NRCC 2288
Aspen
Weak acid
Aspen
Steam + acid
Clostridium
thermosacchrolyticum
ATCC 31925
Escherichia coli B
(pLOI 297)
E. coli B (pLOI 297)
Sawdust
Acid
Aspen
Softwood
E. coli KO11
E. coli KO11
CaO, pH = 7.0
filtered,
adaptation b
Ion exchange,
evaporation,
adaptation
-
3.5g, 34.5x
6.1
0.16
0.06
134
7.5x
1.2
0.16
0.05
135
Sulfur
dioxide
SSLc
Sulfur
dioxide
SSL
Overliming
31.0x
14.9
0.48
0.26
124
2.7g, 5.9m, 21.7x

8.7g, 19.8m, 10x
10.9
14.5
0.36
0.38
0.07
0.25
136
137
13.7
0.39
0.33
136
Pine
Sulfur
dioxide
Overliming
sulfite
32.0
0.43
0.67
7O
Willow
hemicellulose
Willow
hemicellulose
Steam
6.3g, 3.7ga,
17.0m, 8.2x
2.9a, 20.0g,
5.0ga, 30.7m,
15.0x
0.7a, 2.3g, 6.0x
4.6
0.51
0.03
74
Steam
Overliming
sulfite
0.7a, 2.3g, 6.0x
4.6
0.51
0.42
74
Softwood
SSL
0.37
0.36
138
SSL
15.2
0.45
1.27
120
Hardwood
hemicellulose
Wheat straw
Acid
2.9a, 4.7g, 6.5ga,

14m, 7.0x
1.6a, 3.3g, 4.4ga,
16.2m, 7.8x
5a, 4g, 44x
13.0
Softwood
Steam
stripped
Steam stripped
21.2
0.40
0.44
139
Steam + acid
Ether extraction
48.9x
6.0
0.12
0.04
140
Wheat straw
TFA d
Evaporation
46x
13.3
0.29
0.08
141
Aspen
Acid
5.0
0.21
0.14
142
P. stipitis CBS 5776 (R)
Aspen
Acid
Steam stripped
8.9
0.39
0.37
142
Softwood
SSL
15,7
0.34
0.33
142
Softwood
SSL
Steam stripped
18.8
0.44
0.78
142
P. stipitis NRRL Y1724
Pinus radiata
Acid
Sulfite, pH = 5,
autoclaved,
filtered,
adaptation
7.6
0.34
0.18
143
Wheat straw,
Acid
hemicellulose
Wheat straw,
Acid
hemicellulose
1.8a, 1.3g, 11x
3.0
0.21
0.02
125
1.9a, 1.2g, 10.1x
3.6
0.27
0.04
125
E. coli KO11
Hardwood
Newsprint
Yeasts
Candida shehatae
ATCC 22984
C. shehatae ATCC
22984 (R)
Candida sp. XF217
Pachysolen
tannophilus NRRL
Y2460
P. tannophflus N R R L
Y2460
Ether extracted
0.9a, 6.2g, 0.8ga,

1.2m, 15.0x
0.8a, 5.9g, 0.8ga,
1~1m, 14.4x
2.1a, 8.5g, 3.4ga,
22.6m, 9.7x
2.0a, 7.9g, 3.2ga,
21.1m, 9.1x
2.0g, 0.3ga,
1.3m, 0.9x
Y e a s t in c o m b i n a t i o n
w i t h Xl
Saccharomyces
cerevisiae isolate 3 +
Lactobacillus brevis
Xl
S. cerevisiae, Baker's
yeast + Maxazyme
S. cerevisiae, Baker's
yeast + Maxazyme
320
Softwood
SSL
1.9a,
4.6g, 5.6ga,
17.7m, 11.2x
16.4
0.40
0.11
132
Softwood
SSL
16.8
0.41
0.37
133
Straw
Acid (HF)
4.0a, 4.8g, 7.1ga,

15.0m, 10.5x
2.3a, 7.9g, 1.6ga,
0.8m, 9.6x
16.8
0.40
0.33
133
Enzyme Microb. Technol.,
1996, v o l . 18, A p r i l
Ethanol production from lignocellulosic hydrolysate: L. Olsson and B. Hahn-Hggerdal

Table 6
Continued
Microorganism
Raw
material
Pretreatment
Detoxification
Sugars a
(g i-1)
Ethanol
(g i-1)
Yield
(g g-l)
Productivity
(g I-lh -1)
References
8.6a, 14.5g, 44.6x
19.5
0.29
0.16
58
49.9g
23.8
0.48
1.50
144
52g
25.0
0.48
100
144
49.9g
24.2
0.48
3.50
144
Fungi
Mucor sp. 105
Sugar cane
bagasse
Acid
Hexose-fermenting
microorganisms
S. cerevisiae
Aspen
Z. mobilis NRRL 14023
Aspen
_7. mobilis NRRL 14023
Aspen
Sulfur
dioxide
+ acid
Sulfur
dioxide
+ enzymatic
Sulfur
dioxide
+ acid
aa, arabinose; g, glucose; ga, galactose; m, mannose; x, xylose

bThe strain has been adapted to fermentation in lignocellulosic hydrolysate or ethanol-rich broths
cSpent sulfite liquor, waste liquor from sulfite pulping
dTrifluoroacetic acid
stripped hydrolysates showed improved yields and productivities (Table 6) probably because acetic acid was removed.
In some lignocellulosic hydrolysates, P. stipitis did not perform well; in SSL, a yield of 0.17 g g-1 was obtained 133 and
in corn cob hydrolysate, the yield ranged from 0.18-0.33 g
g-1 depending on whether or not detoxification was employed. 1~3 In another investigation, C. shehatae ATCC
22984 showed a low yield (0.05 g g-l) when employed in an
acid hydrolysate of red oak.145 The only fungus included in
Table 6, Mucor sp. 105, gave a yield of 0.29 g g-1. Fusarium oxysporum, which was shown to give high yields in a
laboratory medium-(Table 2), did not function in lignocellulosic hydrolysates due to its sensitivity to the inhibitors
present. 1~3,146 Fermentation of lignocellulosic hydrolysates
with yeast in combination with xylose isomerase has proven
to be successful in SSL and HF-hydrolyzed straw with
yields of 0.40-0.41 g g-1 (Table 6). The productivity was
0.11-0.37 g 1-1 h -~. However, in order to achieve the highest productivity, a cell mass concentration of 75 g 1-1 dry
weight was necessary. Although detoxification was not employed, the fermentative performance in lignocellulosic hydrolysates was comparable to that in a laboratory medium
(Tables 2 and 6). This fermentation alternative was not successful when tested on a corn cob hydrolysate. The yield
was only 0.06 g g-i as a result of the poor xylose fermentation. 113 This is probably because efficient xylose fermentation requires the presence of fermentable hexoses.
Comparing the performances of pentose- and hexosefermenting microorganisms in lign0cellulosic hydrolysates,
it is clear that the latter exhibit much higher yields (0.48 g
g-l) and productivities (t.5-100 g 1-1 h-l; Table 6). The
hexose-fermenting organisms fermented only lignocellulosic hydrolysates containing only glucose whereas the pentose-fermenting organisms fermented lignocellulosic hydrolysates comprised of mixtures of monosaccharides (Table
6). The rapid glucose fermentation is not surprising since
glucose utilization represents the "basic" metabolism in
many microorganisms. Although S. cerevisiae is considered
to be tolerant to harsh conditions, i.e., lignocellulosic hydrolysates, its fermentation rate was lower in SSL than in
laboratory medium. 147 In addition, it has been observed that
S. cerevisiae had a slower specific growth rate in SSL than
in a laboratory medium. 148 However, the specific product
formation rate was the same in SSL as in laboratory medium
in that study.
Prehydrolysis and hydrolysis of the hydrolysate will influence the fermentability of the hydrolysate, i.e., affect the
amount and character of inhibitors present in the hydrolysate. The fermentability of enzymatically hydrolyzed steampretreated aspen wood and enzymatically hydrolyzed biologically delignified aspen wood was compared. It was
found that in hydrolysate where biological delignification
had been employed, the fermentability was better, but the
hydrolysis resulted in a lower sugar yield. 149 In another
study, aspen was prehydrolyzed by steam and then either
hydrolyzed enzymatically or by acid. P. stipitis CBS 5776
was used to ferment both hydrolysates, and a yield of 0.41
g g-1 and a productivity of 0.08 g 1-1 h -1 were found in the
enzymatic hydrolysate whereas a yield of 0.29 g g-~ and a
productivity of 0.05 g 1-1 h -1 were found in the acid hydrolysate.117 When the fermentation performances of S. cidri,
S. cerevisiae, Lactobacillus brevis, Lactococcus lactis ssp.
lactis, E. coli, and Z. mobilis were compared in spent sulfite
liquor and an enzymatic hydrolysate of steam-pretreated
willow, the enzymatic hydrolysate was found to be easier to
ferment with all strains. 64
The development of more efficient microorganisms can
follow two direction; (i) making P. stipitis, C. shehatae, and
recombinant E. coli more resistant to inhibitors, or (ii) genetically engineering S. cerevisiae for xylose fermentation.
For genetic engineering purposeg, one should look for suitable hosts for recombinant constructions, i.e., microorganisms which can ferment lignocellulosic hydrolysates at low
pH and are suitable for genetic engineering. 64 It is not
known whether the use of recombinant microorganisms on
a large scale will be regulated in the future and whether
321
Papers
there will be different restrictions depending on the host. In
addition, the handling of excess cell mass may present a
problem. Perhaps the excess cell mass could be used as a
nutrient source for the subsequent fermentation. A more
futuristic suggestion is that the recombinant cell mass could,
in addition to ethanol production, also be used to produce
valuable enzymes as coproducts. 15
Effect of environmental parameters

In addition to the choice of microorganism, constraints on
environmental parameters such as temperature, pH, ethanol
and substrate concentration, and nutrient supplementation
must be considered. However, industrial processes generally do not permit optimal conditions for the microorganism
since environmental parameters are determined by technical
or economic considerations.
Temperature and p H
Among the pentose-fermenting microorganisms, bacteria
that are thermophiles or mesophiles are found. These ferment in the neutral pH range. The pentose-fermenting yeasts
and fungi are mesophilic; they ferment in the acidic pH
range. Generally, an elevated temperature, i.e., the use of
thermophilic bacteria, 151-a53 or an acidic fermentation pH
(yeast and fungi) is preferred because the risk of contamination is lower. Lactic acid-forming bacteria are the main
contaminants in a distillery; contamination is best avoided
at pH values below 5. TM When using pentose-fermenting
yeast for the fermentation of lignocellulosic hydrotysates,
the pH employed is usually in the upper region of the optimum pH range; otherwise, the acetic acid inhibition would
severely retard the fermentation rate. Bacterial contamination was seen to appear after 3-5 days of continuous fermentation with P. stipitis CBS 5773 in SSL at pH 5.7. l
In simultaneous enzymatic saccharification and fermentation (SSF), it is necessary to compromise between the
optimal conditions for the two reactions. Enzymatic hydrolysis is performed in the pH range 4-5 and at temperatures
of 40-50C. Thermotolerant yeasts, such as Candida acidothermophilum ATCC 20831, Candida brassicae ATCC
32196, and S. uvarum ATCC 26602, are of special importance for SSF. 155 It has also been shown that the fermentation results with yeasts at elevated temperatures were improved if more nutrients were added or higher cell mass
concentrations were employed. 156 Generally, an elevated
temperature enhances the inhibition by ethanol. It has been
suggested that there is a link between the three forms of
stress resistance (osmo-, thermo-, and ethanol tolerance),
i.e., if a strain has a higher temperature tolerance compared
with other strains of that microorganism, it will also be
comparably more osmo- and ethanol tolerant.157
Ethanol and substrate concentration

Distillation of the fermentation broth is energy demanding
and for ethanol concentrations below 2% (w/w), the energy
demand rises steeply. 158 Therefore, high substrate concentrations, i.e., high concentrations of fermentable carbohydrates and thus high final ethanol concentrations, are pre-
322
ferred. Microorganisms generally have a limited tolerance

to either the substrate or ethanol, or both, and the sensitivity
differs between genera. The ethanol tolerance of thermophilic bacteria is 30-80 g 1-1 ethanol; 159 for recombinant E.
coli, the maximum achieved ethanol concentration was 68 g
1-1; 16 and for pentose-fermenting yeasts, ethanol tolerance
up to 100 g 1-1 has been reported? 61 Zymomonas mobilis
produces up to 120 g 1-a ethanol whereas growth ceased at
lower ethanol concentrations.162 S. cerevisiae has the highest ethanol tolerance, growing up to 120 g 1-I and fermenting over 300 g 1-1 ethanol. 96
Candida shehatae ATCC 22984 and ethanologenic E.
coli showed unchanged yields up to substrate concentrations
of 140-200 g 1-1. 69'161 The productivity often increases up
to a substrate concentration where substrate inhibition becomes significant. The productivity of ethanol fermentation
by C. shehatae ATCC 22984 increased from 0.47-0.68 g 1-1
h -~ when the xylose concentration increased from 30-90 g
1-1.163 When the xylose concentration was further increased
to 200 g 1-1, the productivity decreased to 0.42 g 1-1 h -1. In
the case of Fusarium oxysporum VTT-D-80134, the productivity was unchanged up to 200 g 1-1 xylose, but the
maximum achievable ethanol concentration was 40 g 1-1.164
The restricted ethanol tolerance of several microorganisms may impose a limitation in some fermentation
schemes. Process design may reduce the influence of ethanol inhibition. In the case of C. shehatae ATCC 22984,
growth was more sensitive to ethanol than fermentation.
Therefore, a two-stage continuous process was designed.165
In the first reactor where the ethanol concentration was low,
cell mass was produced and the cells were then fed into the
next reactor where fermentation took place. The productivity of this system was 0.96 g 1-1 h -1 compared with 0.46 g
1-1 h -1 in a batch fermentation. In situ ethanol removal from
the fermentation process can be utilized to enhance the fermentation rate. Several methods for in situ removal of ethanol have been suggested such as vacuum fermentation,
flashing off the ethanol, stripping it off with CO 2, or using
solvent extraction. 166
Nutrient supplementation
The medium components added to the hydrolysate influence
the process kinetics and economy. Medium components
should primarily satisfy the basic requirements of the microorganism such as carbon, oxygen, nitrogen, phosfor, and
sulfur. Additionally, metals, growth factors, and vitamins
may be useful. Fermenting lignocellulosic hydrolysates
places other demands on the microorganism than when fermenting a laboratory medium. Therefore, the optimization
of nutrient additions should be performed in the actual hydrolysate.
The literature on pentose fermentation provides mainly
data from experiments in laboratory media or lignocellulosic hydrolysates to which complex nutrients, such as yeast
extract and peptone, have been added. Since ethanol is a
low-value product, there will only be little leeway for nutrient cost. Complex nutrients, such as peptone, can rarely
be added due to their high cost, but it may be possible to
identify a limiting factor, e.g., a vitamin, and add that separately. P. stipitis CSIR-Y633 can ferment xylose with no
Ethanol production from lignocellulos& hydrolysate: L. Olsson and B. Hahn-H#gerdal

vitamins added. 167 However, the fermentation rate was improved threefold when biotin and thiamin were added; the
addition of other vitamins did not further improve the fermentation performance. When fermenting with ethanologenic E. coli B (pLOI 297), a lag phase of 24 hours was
avoided by using a complex medium instead of a mineral
salt medium. 124
Oxygen is required for oxidative metabolism and is
needed for optimal ethanol production by the pentoseutilizing yeasts. S. cerevisiae can grow anaerobically, but a
small amount of molecfllar oxygen is required for fatty acid
and sterol synthesis. It may be necessary to add sterols to the
medium if no oxygen is provided, a68,169 In contrast, the
pentose-fermenting yeasts need carefully controlled low
levels of oxygenation which might be difficult to maintain
uniformally in large fermenters. Recombinant E. coli
showed decreased ethanol yield from glucose and xylose
when the fermenter was aerated, since more carbon was
used for cell mass production. 17
It has been shown that periodic additions of glucose in
xylose fermentation with P. tannophilus NRRL Y-2460 improved the yield from 0.28 to 0.41 g g-~ xylose. 171 It was
suggested that the effect was caused either by repression of
ethanol respiration or stimulation of a more efficient operation of the pentose phosphate shunt. Alternatively during
xylose fermentation, glycolysis may not be active enough to
allow full activation of pyruvate decarboxylase which may
reduce the ethanol production rate. 172 The addition of glucose has also been shown to improve the yield in the fermentation of lignocellutosic hydrolysates. Improvements in
yield from 0.30 to 0.40 g g-~ have been demonstrated when
feeding cellulose hydrolysate intermittently to an acid hardwood hemicellulose hydrolysate in fermentation with P.
tannophilus. 173
Fermentation strategies
Fermentation can be performed as a batch, fed-batch, or
continuous process. The most suitable choice will depend
on the kinetic properties of the microorganism in addition to
aspects of process economics. Immobilization and recirculation of cells are ways of increasing the cell mass concentration in the fermenter which leads to a higher productivity.
The higher the productivity, the smaller the fermenter required and therefore, the capital cost is usually lower. As
lignocellulosic hydrolysates contain several monosaccharides, cocultuting two microorganisms has been suggested
since one microorganism cannot ferment all substrates optimally.
Traditionally, ethanol has been produced batchwise. In
batch fermentation, the microorganism works in a high substrate concentration initially and a high product concentration finally. Generally, batch fermentations are characterized by low productivity and they are labor intensive. 166
Continuous operation offers ease of control and is less labor
intensive than batch operation. However, contamination is
more serious in continuous operation since the process must
be interrupted, all the equipment cleaned, and the operation
started again with the growth of a new inoculum. It has been
claimed that for a fermentation with S. cerevisiae, continuous operation is more resistant to contamination due to the
high ethanol concentration and the low substrate concentration in the fermentation b r o t h . 174 In the fermentation of
pentose-tich substrates, the situation is somewhat different
because a higher fermentation pH is required regardless of
whether recombinant bacteria, pentose-fermenting yeasts or
S. cerevisiae in combination with xylose isomerase are
used. Additionally, the final ethanol concentration will be
lower than in a S. cerevisiae fermentation due to the limited
ethanol tolerance of the other microorganisms. Ultimately,
the organism with the lowest K S under the given conditions
(the cultivated organism or the contaminant) will take over
the fermentation.
Continuous fermentation can be performed in different
kinds of bioreactors--stirred-tank reactors (single or in seties) or plug-flow reactors. 166 In a continuous stirred-tank
reactor (CSTR), the microorganism works at a low substrate
concentration and a high ethanol concentration all the time;
the ethanol tolerance of the microorganism may limit the
fermentation rate. Continuous fermentation often gives a
higher productivity than batch fermentation, but at dilution
rates offering the highest productivities, all the substrate is
not consumed and consequently the yield is lower. Additionally, a consistent product quality is achieved and high
peak loads on accessory equipment are avoided in continuous operation.
In fed-batch fermentation, the microorganism works at
low substrate concentrations with an increasing ethanol concentration during the course of the fermentation process. In
such a fermentation with P. tannophilus RL171, ethanol
was produced from xylose with a yield of 0.41 g g-1 compared with 0.29 g g-t in a batch system. 5~ It was concluded
that the optimum level of xylose in the medium was 5-8 g
1-1, since higher xylose concentrations enhanced xylitol production and lower concentrations increased acetate production.
In addition to a high yield, it is desirable to achieve as
high a productivity as possible. Increasing the cell density
has been shown in several cases to be a suitable way of
increasing the volumetric productivity (Table 7). When increasing the cell mass, the specific productivity (the amount
produced by each cell) usually decreases. In batch fermentation with C. shehatae ATCC 22984, ethanol was produced
at a productivity of 0.28 g 1-~ h -1 at a cell density of 0.72 g
1-~. When the cell density was increased tenfold to 7.6 g 1-1,
the productivity only increased 2.5 times to 0.69 g t-1
h-a. 175 High cell density helps to overcome thetoxicity of
the lignocellulosic hydrolysate by overcoming an unfavorable oxidation-reduction potential. 126 There are two general
ways of achieving a high cell density--either by cell recycling or immobilization of the cells. Using a high celt density means diverting more carbon to the production of cell
mass initially and less to ethanol production. The carbon
needed for maintenance increases with increasing cell mass
and the requirement of nutrients will also be higher. Cell
recycling or retention makes it possible to achieve higher
dilution rates in continuous operation than the growth rate
permits. Several kinds of bioreactors have been suggested to
achieve cell retention. 166 Alternatively, immobilized cells
can be used, but the immobilization procedure adds extra
costs to the ethanol production. On the other hand, low
323
Papers
Table 7 Fermentation with high cell density
Cell status
Microorganism
Laboratory
medium
Suspended cells
Candida shehatae
Pichia stipitis
Pachyso/en
tannophi/us
immobilized,
growing
Immobilized
P. tannophi/us
Immobilized
Cell recycling
C. shehatae
Ethanol b
(g 1-1)
References
0.42
0.28
0-37.0
175
Xylose
Batch
0.42
0.60
0-21.1
176
Xylose
Continuous,
packed bed
0.27
0.71
17.7
177
Xylose
0.41
1.08
26.9
177
Xylose
Continuous,
packed bed
CSTR
0.33
2.72
16.5
178
Xylose
Plug-flow reactor
0.39
1.75
19.5
178
Xylose
Continuous
0.26
4.40
23.4
175
Xylose
Continuous
0.35 a
0.42
14
179
Xylose
Continuous
0.32 a
2.43
12.8
179
Glucose
Continuous
0.35
35
180
Acid hydrolysate
of pine
Acid hydrolysate
of coniferous
wood, steam
stripped
Acid hydrolysate
of aspen
cellulose
Acid hydrolysate
of pine
Softwood SSL
Batch
0.35
0.21
0-7.0
143
Batch
0.44
0.42
0-10.2
142
Batch
0.48
3.5
0-24.2
144
CSTR
0.50
0.27
1~1.3
143
Continuous, rotating
fiber disc fermenter
0.42
1.30
18.0
142
0.40
1.80
9.2
142
0.45
2.60
10.3
142
Continuous rotating,
0.48
24.0
144
Continuous rotating,
0.45
21.8
144
Continuous
0.44
55.0
181
CBS 4044
CBS 4044
P. stipitis NRRL
Y-7124
P. stipitis NRRL
"(-7124
Productivity
(g 1-1 h-1)
Batch
NRRL -7124
Immobilized,
nongrowing
Yield
(g g-l)
Xylose
ATCC 22984
Suspended cells
Fermentation
conditions
Fermentation
substrate
ATCC 22984
Cell recycling,
nongrowing
Cell recycling,
growing
Cell recycling
P. tannophi/us
NRRL Y-2460
P. tannophi/us
NRRL Y-2460
Saccharomyces
cerevisiae
170
NRRL-Y132
Lignocellulosic
hydrolysates
Suspended cells
P, stipitis NRRL
1724
Suspended cells
P, stipitis R
Suspended cells
Zyrnomonas
mobilis NRRL
Immobilized
P. stipitis NRRL
14023
1724
Immobilized
P. stipitis R
Immobilized
P. stipitis R
Immobilized
P. stipitis R
Immobilized
Z. mobilis NRRL
14023
Immobilized
Z. rnobilis NRRL
14023
Cell recycling
Flocculating
yeast IR-2
Acid hydrolysate
of aspen,
steam stripped
Acid hydrolysate
of coniferous
wood, steam
stripped
Enzymatic
hydrolysate of
aspen cellulose
Acid hydrolysate
of aspen
cellulose
Sugar cane juice
73
5.20
34.6
CSTR means continuously stirred tank reactor

ag g-1 xylose consumed
bEthanol concentration at which the productivity has been achieved
equipment expenditure has been claimed.182 Immobilization

by adhesion to a surface (electrostatic or covalent), entrapment in polymeric matrices, or retention by membranes
have been successful for ethanol production from hexoses. la3 A flocculent strain of S. pombe underwent morphological changes from soft flocs to heavy particles (1-3 mm
324
diameter) after continuously being fed with fresh substrate.

The cells behaved like immobilized cells, a84
Results obtained with different fermentation methods for
S. cerevisiae have been summarized in relation to the fermentation method used.18'182 In batch fermentation, a productivity of 2.2-4.0 g 1-l h-~ was obtained; in continuous

fermentation with suspended cells, 4.1-7.0 g 1-~ h - 1 w a s
observed. The productivity for the other fermentation methods was even higher; for continuous culture with immobilized cells, 25-100 g 1-~ h -1 was recorded. Continuous culture with cell recycling gave 29-170 g 1-1 h -1, and continuous culture with cell recycling and vacuum removal of the
ethanol produced 82 g 1-a h -1. Different pentose-fermenting
yeasts fermented a laboratory medium with productivities
up to 4.4 g l-a h -~ (C. shehatae), 2.4 g 1-1 h -a (P. tannophilus), and 2.7 g 1-1 h -~ (P. stipitis) when high cell densities
were employed (Tabl~ 7). In batch fermentation, the productivities were 0.28-0.60 g 1-l h -1. In both cell recycling
and immobilization of P. tannophilus, the productivities
were better when the cells were growing than when they
were not (Table 7). Immobilization has been employed in
lignocellulosic hydrolysates with a maximum productivity
of 2.6 g 1-~ h -~ and a yield of 0.45 g g-1 reported for P.
stipitis R (Table 7). The productivity and yield in batch
fermentation oCfhe same lignocellulosic hydrolysate for P.
stipitis R were 0.42 g 1-~ h -1 and 0.44 g g-~, respectively. In
the case of immobilized P. stipitis R, steam stripping which
removed the acetic acid was required for optimal performance. For P. stipitis NRRL 1724, continuous fermentation
with immobilized cells only marginally increased the productivity compared with batch fermentation, but the yield
increased from 0.35-0.50 g g-1 (Table 7). When applying
cell mass concentrations as high as 22 and 44 g 1-1 dry
weight, E. coli pLOI297 showed a total volumetric productivity of 14 and 20 g 1-1 h -1, respectively. 35
In a laboratory medium, hexose fermentation with S.
cerevisiae NRRL Y-132 was more efficient than pentose
fermentation in high cell-density systems (Table 7). When
fermenting in such systems and lignocellulosic hydrolysates
containing hexoses, Z. mobilis NRRL 14023 reached productivities of 5.2-73 g 1-1 h-a; a flocculating yeast gave a
productivity of 34.6 g 1-a h -1 whereas P. stipitis fermenting
both hexoses and pentoses only reached productivities of
0.27-2.6 g 1-1 h -1 (Table 7). The improvements in productivity for pentose-fermenting microorganisms achieved by
using more efficient fermentation strategies were 10- to 20fold (Table 7) compared with a S. cerevisiae fermentation
where the improvements were 50-fold. 18,a82 The productivities compiled in Table 7 have been achieved mostly at
low ethanol concentrations. In order to minimize the distillation cost, high final ethanol concentrations are required,~ 58
but then the productivities are supposed to be lowered due
to ethanol inhibition (see above).
Different fermentation strategies have been economically evaluated for ethanol production from molasses by S.
cerevisiae. 185 The processes resulting in the highest cell
densities (cell recycling, CSTR, plug-flow, APV tower) also
resulted in the highest productivities and the lowest equipment costs. The final ethanol cost ranged from 48.7-53.0
cents 1-~ (U.S.) without in situ ethanol removal. The cost of
raw material (molasses) accounted for 70% of the ethanol
cost, but further savings could be made by using more efficient fermentation strategies.185 Potentially, in situ ethanol
removal would account for the highest savings, although the
result is dependent on how much the separator, which was
not included in the cost analysis, influences the final cost.
When in situ ethanol removal is employed, the most volatile

by-products will also be removed while the others accumulate in the fermentation broth. In this study, the influence of
acetic acid accumulation on the fermentation kinetics was
included in the calculations.
The main objective in using a coculture is to use the most
efficient microorganisms for hexose and pentose fermentations. A coculture of S. cerevisiae and P. stipitis was shown
to be impossible since several P. stipitis strains exhibit a
killer character towards S. cerevisiae, ls6 However, S. cerevisiae and P. stipitis were successfully coimmobilized for
simultaneous conversion of a glucose/xylose mixture, ls7 In
the core of the beads used, the glucose concentration was
low and therefore, xylose could be fermented by P. stipitis.
However, the conditions in the core were anaerobic; consequently, only xylitol was produced. A prerequisite for a
successful coculture is that the optimal fermentation conditions for both microorganisms coincide. Aeration may present a problem if Z mobilis, which needs anaerobic conditions, is cocultured together with pentose-fermenting
yeasts) 88 A coculture of a respiratory-deficient mutant of
Saccharomyces diastaticus and P. stipitis was used for ethanol production under continuous culture conditions. 189 At a
dilution rate of 0.2 h -1 ethanol was produced at a yield of
0.43 g g-i and a productivity of 4.3 g 1-i h -1 from 35 g 1-1
glucose and 15 g 1-1 xylose. The reasons for the successful
results were twofold. Firstly, the respiratory deficiency of S.
diastaticus enabled optimization of the oxygenation required by P. stipitis. Secondly, the continuous culture condition resulted in minute amounts of glucose in the fermenter; therefore, xylose was fermented by P. stipitis.
Long-term stability of the coculture needs to be demonstrated for applications in lignocellulosic hydrolysates. A
coculture of Z. mobilis ATCC 29191 and Clostridium saccharolyticum (pyruvate-negative mutant) was used to ferment a hydrogen fluoride hydrolysate of aspen. 19 The hydrolysate consisted of 34 g 1-1 glucose and 15 g 1-1 xylose.
Ethanol was produced with a yield of 0.47 g g-1 and a
productivity of 0.24 g 1-1 h -1.
Process integration
In parallel with the ongoing development of pentosefermenting microorganisms, pentose fermentation must be
considered as an integrated part of the rest of the process.
The suitability of different fermentation strategies should be
investigated in relation to the whole process. B y studying
the interrelation between different process steps, the areas
that need special attention in order to make the pentose
fermentation process economically feasible should be identified. To further improve the economy of ethanol production, energy integration of the ethanol-producing plant to
already existing plants, such as paper and pulp plants, is
necessary. The cost of producing ethanol from pine with a
dilute acid hydrolysis process was calculated to be 3.22
SEK 1-1 in a stand-alone plant while an integrated plant
could produce ethanol at a cost of 2.54 SEK 1-1.TM These
costs can be compared with a world market price of 2.7
SEK 1-l.
The fermentation of lignocellulosic hydrolysates involves only one or two of several process steps that are
325
Papers
In the overall process scheme shown (Figure 2), hexose
and pentose fermentation is performed separately mainly
due to the fact that the pentose-fermenting organisms ferment both pentoses and hexoses slower than the hexosefermenting organisms ferment hexoses. Additionally, pentose-fermenting microorganisms are generally more sensitive to inhibitors and the ethanol produced which further
reduces the fermentation rate and may necessitate detoxification of the hemicellulose hydrolysate. A smaller volume
of hydrolysate has to be detoxified if the hemicellulose and
the cellulose hydrolysate are fermented separately. For the
pentose-fermenting microorganisms, hexoses are the preferred substrates in a mixture of hexoses and pentoses. If the
fermentation time is insufficient, the pentoses will remain.
Mixed substrate utilization is observed in laboratory experiments under continuous culture conditions in contrast to
batch fermentation. However, there have been no reports on
the preference of substrate utilization in continuous culture
of lignocellulosic hydrolysates and therefore, it is not
known whether hexoses and pentoses can be cofermented.
On the flow sheet of the ethanol production process (Figure 2), enzymatic hydrolysis and hexose and pentose fermentation have been presented as separate operations. Another possibility would be to perform the hydrolysis together with the fermentation in a SSF operation. TM Glucose
and cellobiose would thus be continuously removed and the
inhibition of the hydrolysis enzymes reduced. This results in
lower amounts of enzymes being necessary for the hydrolysis. In addition, the risk of contamination is lower since
required for ethanol production. The complexity of ethanol

production from lignocellulosic materials is illustrated in an
overall process scheme for an enzymatic hydrolysis process
(Figure 2). The process steps--prehydrolysis with steam,
delignification, recovery of delignification chemicals, enzyme production, enzyme hydrolysis, and recovery of enzymes, hexose (cellulose hydrolysate) and pentose (hemicellulose hydrolysate) fermentation, distillation, steam generation, and waste treatment are all interrelated. For pentose
fermentation, the conditions at which prehydrolysis is performed determine the composition of the hemicellulose hydrolysate. The formation of inhibitors is of special importance. Additionally, the need for recirculation to minimize
the fresh water consumption results in a buildup of byproducts in the process, s9 The stiltage can, for example, be
recirculated to the washing stage preceding the delignification. In the ethanol production process, the cell mass produced in the fermentation step is recirculated to various
degrees depending on the need in the fermentation. Excess
cell mass may be used as a nutrient source, thereby reducing
nutrient costs in the process. The amount of excess cell mass
for disposal will be minimized. Further investigations need
to determine whether excess cell mass is a suitable nutrient
source and develop any necessary technology. The process
conditions that give the best fermentation performance are
not always optimal from an economical point of view for
the simple reason that it costs more to achieve those conditions than what is gained by the improved ethanol production.
Feed
~/~/\/\/\I
- ~ Prehydrolysis
i Recoveryof
Delicghnifi~ca~n
Washing
Delignification
P
Washing
Hydrolysis
Enzyme
Production
FimatorfS,al
;Enzyme
Recovery
Hexose
Fermentation
Ugnin ~
326
Fermentation
I Distilation
Stil!age
Figure2
Solidresidue
Schematic flow sheet for an ethanol production process based on enzymatic hydrolysis
Enzyme M i c r o b . Technol., 1996, vol. 18, A p r i l
Pentose
_ Ethanol
Preconcen~ation
t
Steam
Generation
I PrecI
Preconcentration

the glucose and cellobiose produced are immediately converted to ethanol. The equipment cost can be reduced due to
the fact that the process is performed in one vessel instead
of two, and the increased productivity enables smaller vessels to be used. The conditions for SSF have to be a compromise between the optimal conditions for hydrolysis and
fermentation. The cellulases work optimally at pH 4-5 and
temperatures of 40-50C whereas S. cerevisiae (hexose fermentation) ferments optimally at 30C and pH 4-5, and the
pentose-fermenting organisms work optimally at 30-70C
and pH 5-7. If SSF includes pentose fermentation, the substrate concentrations will enable production of ethanol concentrations higher than most pentose-fermenting organisms
tolerate. A coculture of P. stipitis R and Brettanomyces
clausenii has been utilized for SSF including pentose fermentation. 192 In 48 hours, 369 1 ethanol were produced per
t0nne of aspen at 38C and pH 4.8. Another alternative
would be to utilize a system for simultaneous saccharification, xylose iso~erization, and fermentation (with S. cerevisiae). 193 No application of SSF has demonstrated a
method of recirculating the cell mass after the process, because of the difficulty in separating the cells from the solid
residue. Consequently, it appears necessary to produce new
cell mass for each SSF batch.
With the present knowledge, two areas that require more
effort in order to achieve an economically viable ethanol
production process will emerge. Firstly, we know today so
much about the fermentation of lignocellulosic hydrolysates
that an integration of fermentation with the rest of the process should be investigated. Furthermore, it is important to
evaluate its effect on the complete process. Secondly, further development of the fermentation step is required with
regard to the inhibitors especially since their presence will
be increased due to recirculation in a closed process. It is an
advantage if detoxification is not required and therefore,
there is a requirement for robust microorganisms.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Acknowledgments
The research on xylose fermentation at the Department of
Applied Microbiology is supported financially by NUTEK
(Swedish National Board for Industrial and Technical Development), NI (Nordic Fund for Technology and Industrial
Development), SSEU (Swedish Ethanol Development
Foundation), NFR (Swedish Natural Science Research
Council), and the Knut and Alice Wallenberg Foundation.
Our special thanks go to Prof. Michael Ladisch and the
members of the Ethanol Group at the University of Lund for
their valuable suggestions.
20.
21.
22.
23.
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ELSEVIER

Keywords:Lignocellulosichydrolysates; inhibitors; detoxification; pentose fermentation; process integration; economics;

Enzyme and Microbial Technology 18:312-331, 1996

stock, but the processing techniques required for ethanol

Ethanol production from lignocellulosic hydrolysate: L. Olsson and B. Hahn-H&gerdal

terials. 4-7 Economically feasible ethanol production from

Composition of lignocellulosic materials

Production of lignocellulosic hydrolysates

Table 1 Composition of different lignocellulosic materials

n.d. means not determined

Enzyme Microb. Technol., 1996, vol. 18, April

The total volumetric ethanol productivity, referred to as

Ethanol production from lignocellulosic hydrolysate: L. Olsson and B. Hahn-H#gerdal

Performance of ylose-fermenting microorganisms

Bacteria: naturally occurring

Candida shehatae, Pachysolen tannophilus, and Pichia

Enzyme Microb. Technol., 1996, vol. 18, April

yeasts. The yields for these microorganisms are average to

g-1 (Table 2).

Xylose fermentation with xylose isomerase and yeasts

Candida shehatae ATCC 22984 (2) a

S. cerevisiae ATCC 24860

Streptomyces sp. ATCC 21175

Schizosaccharomyces uvarum ATCC 26192

productivity was 1.0 g 1-1 h -1 when S. cerevisiae was used

drolysis and hydrolysis due to the degradation of sugars. S.

Effects of inhibiting compounds on fermentation

50% (f); pH = 4.5

Analysis of lignocellulosic hydrolysates

Detoxification and adaptation

Enzyme Microb. Technol., 1996, vol. 18, April

method and can be performed in various "ways. Calcium

Ethanol production from lignocellulosic hydrolysate: L. Olsson and B. Hahn-H#gerdal

Precipitation of acetate, heavy metals, furfurals,

productivity from 0.60-0.85 g 1-1 h -I and an increase in

Fermentation of lignocellulosic hydrolysates

ent lignocellulosic hydrolysates containing varying amounts

Fermentation of lignocellulosic hydrolysates

E. coli B (pLOI 297)

2.7g, 5.9m, 21.7x

0.7a, 2.3g, 6.0x

2.9a, 4.7g, 6.5ga,

P. stipitis CBS 5776 (R)

P. stipitis CBS 5776 (R)

P. stipitis CBS 5776 (R)

P. stipitis NRRL Y1724

P. stipitis NRRL Y7124

1.8a, 1.3g, 11x

1.9a, 1.2g, 10.1x

P. stipitis NRRL Y7124

0.9a, 6.2g, 0.8ga,

4.0a, 4.8g, 7.1ga,

Enzyme Microb. Technol.,

Ethanol production from lignocellulosic hydrolysate: L. Olsson and B. Hahn-Hggerdal

8.6a, 14.5g, 44.6x

Z. mobilis NRRL 14023

_7. mobilis NRRL 14023

aa, arabinose; g, glucose; ga, galactose; m, mannose; x, xylose

Enzyme Microb. Technol., 1996, vol. 18, April

Effect of environmental parameters

Ethanol and substrate concentration

Enzyme Microb. Technol., 1996, vol. 18, April

ferred. Microorganisms generally have a limited tolerance