Table Of Content27 Advances ni Biochemical Engineering/
Biotechnology
Managing Editor: ~ A. Fieehter
Co-Editor" Th. W. Jeffries
ISBN 3-540-12182-X Springer-Verlag Berlin Heidelberg New York
ISBN O-387-12182-X Springer-Verlag New York Heidelberg Berlin
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215213020-543210
sesotneP dna ningiL
With Contributions by
Y .K Chan, A. Fiechter, Ch.-Sh. Gong,
.N .B Jansen, H. Janshekar, Th.W. ,seirffeJ
.C .P Kurtzman, .R Maleszka, L.D. McCracken,
.L Neirinck, .H Schneider, T ,ynsezczS
.G .T Tsao, I.A.Veliky, ,ykseloV.B .P .Y Wang
With 64 Figures and 64 Tables
r
Springer-Verlag
Berlin Heidelberg NewYork Tokyo
3891
Managing Editor
Professor Dr. A. Fiechter
Institut f/Jr Biotechnologie
Eidgen6ssische Technische Hochschule,
H6nggerberg,
CH-8093 Zfirich
Co-Editor: Th. W. Jeffries
Editorial Board
Prof. Dr. S. Aiba Department of Fermentation Technology, Faculty of
Engineering, Osaka University, Yamada-Kami, Suita-
Shi, Osaka 565, Japan
Prof. Dr. B. Atkinson University of Manchester, Dept. Chemical Engineering,
Manchester/England
Prof. Dr. E. Bylinkina Head of Technology Dept., National Institute of
Antibiotika. 3a Nagatinska Str., Moscow M-105/USSR
Prof. Dr. Ch. L. Cooney Massachusetts Institute of Technology,
Department of Chemical Engineering,
Cambridge, Massachusetts 02139/USA
Prof. Dr. H. Dellweg Techn. Universit~it Berlin, Lehrstuhl fiir
Biotechnologie, SeestraBe ,31 D-1000 Berlin 56
Prof. Dr. A. L. Demain Massachusetts Institute of Technology, Dept. of
Nutrition & Food Sc., Room 56-125,
Cambridge, Mass. 02139/USA
Prof. Dr. S. Fukui Dept. of Industrial Chemistry, Faculty of
Engineering, Sakyo-Ku, Kyoto 606, Japan
Prof. Dr. K. Kieslich Wissenschaftl. Direktor, Ges. ri./f Biotechnolog.
Forschung mbH, Mascheroder Weg ,1
D-3300 Braunschweig
Prof. Dr. R. M. Lafferty Techn. Hochschule Graz, Institut fiir
Biochem. Teehnol., Schl6gelgasse 9, A-8010 Graz
Prof. Dr. K. Mosbach Biochemical Div., Chemical Center, University of Lund,
S-22007 Lurid/Sweden
Prof. Dr. H. L. Rehm Westf. Wilhelms Universit/it, Institut ftir
Mikrobiologie, Tibusstrale 7-15, D-4400 Miinster
Prof. Dr. P. L. Rogers School of Biological Technology, The University
of New South Wales. PO Box ,1
Kensington, New South Wales, Australia 2033
Prof. Dr. H. Sahm Institut fiir Biotechnologie, Kernforschungsanlage
Jiilieh, D-5170 J/ilich
Prof. Dr. K. Schiigerl Institut fiir Technische Chemic, 'Universit~it Han nover,
CallinstraBe ,3 D-3000 Hannover
Prof. Dr. H. Suomalainen Director, The Finnish State Alcohol Monopoly, Alko,
P.O.B. 350, 00101 Helsinki 10/Finland
Prof. Dr. S. Suzuki Tokyo Institute of Technology,
Nagatsuta Campus, Research Laboratory of Resources
Utilization
4259, Nagatsuta, Midori-ku, Yokohama 227/Japan
Prof. Dr. H.Taguchi Faculty of Engineering, Osaka University, Yamada-kami,
Suita-shi, Osaka 565/Japan
Prof. Dr. G. T. Tsao Director, Lab. of Renewable Resources Eng., A. A. Potter
Eng. Center, Purdue University, West Lafayette,
IN 47907/USA
Table of Contents
Utilization of Xylose by Bacteria, Yeasts, and Fungi
Th. W. Jeffries . . . . . . . . . . . . . . . . . . . . .
D-Xylose Metabolism by Mutant Strains of Candida sp.
L. D. McCracken, Ch.-Sh. Gong . . . . . . . . . . . . 33
Ethanol Production from D-Xylose and Several Other
Carbohydrates by nelosyhcaP sulihponnat and Other Yeasts
H. Schneider, R. Maleszka, L. Neirinck, I. A. Veliky,
P. Y. Wang, Y. K. Chan . . . . . . . . . . . . . . . . 57
Biology and Physiology of the D-Xylose Fermenting Yeast
nelosyhcaP snlihponnat
C. P. Kurtzman . . . . . . . . . . . . . . . . . . . . 73
Bioconversion of Pentoses to 2,3-Butanediol by alleisbelK
eainomnenp
N. B. Jansen, G. T. Tsao . . . . . . . . . . . . . . . . 85
Bacterial Conversion of Pentose Sugars to Acetone and Butanol
B. Volesky, T. Szczesny . . . . . . . . . . . . . . . . 101
Lignin: Biosynthesis, Application, and Biodegradation
H. Janshekar, A. Fiechter . . . . . . . . . . . . . . . 911
Author Index Volumes 1-27 . . . . . . . . . . . . . . . 971
Utilization of Xylose yb Bacteria, Yeasts, dna ignuF
Thomas W. Jeffries*
Microbiologist. Forest Products Laboratory, U.S. Dept. of Agriculture,
P.O. Box 5130, Madison, Wisconsin 53705, U.S.A.
1 Introduction ..................................................................... 2
1.1 Distribution of Pentoses in Lignocellulosic Residues ............................... 3
2.1 Recovery of Hemicellulosic Sugars .............................................. 6
2 D-Xylose Metabolism ............................................................. 7
1.2 Transport .................................................................... 8
2.1.1 Bacteria ..................................... .. .......................... 8
2.1.2. Yeasts and Fungi ........................................................ 9
2.2 Conversion of D-Xylose to D-Xylulose-5-Phosphate ......................... ....... 01
2.2.1 Isomerization ..... " ....................................................... 11
2.2.2 Reduction, Oxidation, and Polyol Formation ................................ 21
2.2.3 Phosphorylation ......................................................... 31
2.3 The Pentose Phosphate Pathway ................................................ 41
2.4 Phosphoketolase ......................................... 2 .................... 51
3 Regulation of D-Xylose Metabolism ................................................. 61
1.3 Aerobic and Anaerobic Utilization of D-Xylose ................................... 71
3.2 D-Glucose-6-Phosphate Dehydrogenase .......................................... 02
3.3 Nutritional Factors ............................................................ 12
4 Utilization of D-Xylose, D-Xylulose, and Xylitol by Yeasts and Fungi ................... 22
1.4 D-Xylose ..................................................................... 22
4.2 D-Xylulose ................................................................... 32
4.3 Xylitol ....................................................................... 24
4.4 Sugar Mixtures ............................................................... 24
4.5 Hydrolysates of HemiceUulose .................................................. 52
5 Depolymerization and Fermentation ................................................ 26
6 Implications for Strain Selection and Process Design .................................. 26
7 Acknowledgements ............................................................... 82
8 References ....................................................................... 82
Hemicellulosic sugars, especially D-xylose, are relatively abundant in agricultural and forestry residues.
Moreover, they can be recovered from the hemicelluloses by acid hydrolysis more readily and in
better yields than can D-glucose from cellulose. These factors favor hemicellulosic sugars as a
feedstock for production of ethanol and other chemicals. Unfortunately, D-xylose is not so readily
utilized as D-glucose for the production of chemicals by microorganisms. The reason may lie in the
biochemical pathways used for pentose and hexose metabolism. Different pathways are employed by
prokaryotes and eukaryotes in the initial stages of pentose assimilation. Transport and pbosphoryla-
tion possibly limit the overall rate of D-xylose utilization. The intermediary steps of pentose metabo-
lism are generally similar for both bacteria and fungi, but substantial variations exist. Phosphoketolase
is present in some yeasts and bacteria able to use pentoses. Regulation of the oxidative pentose
phosphate pathway occurs at D-glucose-6-phosphate dehydrogenase by the intracellular concentra-
tion of NADPH. Regulation of nonoxidative pentose metabolism is not well understood. In some
* Maintained in cooperation with the University of Wisconsin.
2 T.W. Jeffries
yeasts and fungi, conversion of D-xylose to ethanol takes place under aerobic or anaerobic conditions
with rates and yields generally higher in the former than in the latter. Xylitol and acetic acid are
major byproducts of such conversions. Many yeasts are capable of utilizing D-xylose for the production
of ethanol. Direct conversion of D-xylose to ethanol is comPared with two-stage processes employing
yeasts and D-xylose isomerase.
I Introduction
Hemicellulosic sugars in acid hydrolysates of hardwoods and agricultural residues
could become important feedstocks for the production of ethanol and other chemicals
by microbial processes. Several factors favor their use: they are relatively abundant
in a variety of common lignocellulosic residues; they can be recovered by mild acid
hydrolysis; and new microbiological processes are being developed for their conver-
sion.
Within the past 2 years, several significant findings have advanced the prospects
for PrOduction of ethanol and other d':micals from D-xylose. First, yeasts, which
were previously considered unable to ferment 1 5-carbon sugars, have now been shown
HIGH-FRUCTOSE
CORN ~ CORN SYRUP
Wet Glucose
milling
STARCH J ESOCULG j is~
,
OIL Amylase Ferman- -
+ tation
GLUTEN ETHANOL, YEAST
HARDWOODS OR ~ XYLULOSEI
AGRICULTURAL RESIDUES Glucose ~ I
I ~ isomerase
-~ Acid = XYLOSE + Fermentation
prehydrotysis GLUCOSE 1
CELLULOSE Fermentation
+ ,= HEAT, FEED ETHANOL,
LIGNIN ~ OTHER CHEMICALS,
YEAST, ANIMAL
FEED
secnavdA .i Sio. Eng.
Fig. .1 Comparison of ethanol production from grain and lignocellulosic residues. (M 151671). For
definition see footnote
1 The term "fermentation" and its various derivatives is used herein to refer to dissimilatory metabolic
processes through which an organic substrate is converted into oxidized and reduced products
without a net overall change in the oxidation state.
noitazilitU of esolyX by ,airetcaB ,stsaeY dna Fungi 3
to utilize the pentulose D-xylulose under anaerobic conditions x-5). Since D-xylulose
can be formed from D-xylose through the action of glucose isomerase (actually
xylose isomerase) ,6 ,)7 processes have been developed employing two-stage isomeriza-
tion and fermentation 8-12). Second, several yeasts, particularly those belonging to
the genera Pachysolen )51-ax and Candida 16- la), have been shown to convert D-xylose
to ethanol under aerobic and anaerobic conditions. Besides these recent findings, it
is known that certain fungi, particularly Fusarium lini ,9-23), are capable of converting
D-xylose to ethanol; and various bacteria can form several potentially useful products
such as ethanol, acetic acid, 2,3-butanediol, acetone, isopropanol, and n-butanol
from D-xylose ~-29). Finally, unlike the fermentation of sugar from grains, utilization
of pentoses derived from forestry and agricultural residues for the production of
chemicals does not decrease food supplies; indeed, by virtue of the production of
microbial biomass and unutilized sugars, such processes can supplement animal feed
resources (Fig. .)1
This review attempts to examine recent microbiological findings in relation to the
previous understanding of D-glucose fermentation and pentose metabolism. It briefly
examines the availability of hemicellulosic sugars -- particularly D-xylose -- in
lignocellulosic residues, reviews aspects of pentose metabolism and metabolic regula-
tion of fermentative processes, and discusses some recent research progress on aerobic
and anaerobic conversions of D-xylose to ethanol by yeasts and bacteria. The 2,3-
butanediol fermentation and the butanol/acetone/ethanol fermentations are reviewed
in other chapters of this volume.
1.1 Distribution of Pentoses in Lignocellulosie Residues
Hemicelluloses are widely distributed, major components of lignocellulosic materials
comprised of neutral sugars, uronic acids, and acetal groups, all present as their
respective anhydrides (e.g., the anhydride of D-xylose is xylan). As the anhydrides,
hemicellulosic sugars average about 26 ~ of the dry weight of hardwoods, 2 and 22
of softwoods and about 25 ~ of several major agricultural residues. Pectin, ash, and
protein account for variable fractions in lignocellulosic materials, whereas cellulose
(anhydro o-glucose) and lignin make up the balance (Table .)1
The xylan and arabinan contents of hemicelluloses vary with the plant species.
The xylan content of hardwoods is generally much higher than that of softwoods,
ranging between 11 ~ and 25 ~ in the former and between 3 ~ and 8 ~ in the lat-
ter 3o-a2). Hemicelluloses in hardwoods contain appreciable amounts of D-xylose,
D-mannose, acetyl, and uronic acid. The acetyl content ranges between 3 ~ and 4.5 o~
in hardwoods and between 1 ~ and 5.1 ~ in softwoods; uronic acid (as the anhydride)
ranges between 3 o~ and 5 ~ in both hardwoods and softwoods .)oa In conifers, the
predominant hemicellulosic sugar is o-mannose, which, as mannan, averages about
11, ~ of the total dry weight .)23 Whereas the xylan content of softwoods is lower
than in hardwoods, the lignin content is higher. The predominant hemicellulosic
sugar of agricultural residues is D-xylose. The xylan content of corn residues varies
2 ehT word "hardwoods" srefer to defael-daorb trees )smrepsoigna( dna sah nothing to do with eht
ssendrah of eht .sdoow ,ylralimiS "softwoods" refers to suorefinoc trees .)smrepsonmyg(
4 T.W. seirffeJ
from about 17 ~ in the leaves and stalks to 13 ~ in the cobs, but, on the average, it
comprises about 24~ of the total dry weight of corn stover (L. H. KruU, personal
communication). The chemistry of the hemicelluloses of grasses has been reviewed
recently )53
Table .1 Proximate composition of various biomass resources
of total dry weight
Glu- Galac- Man- Arabi- Xylan Hemi- Hemi- Cellu- Lignin a Ref.
can tan nan nan cellu- cellu- esol ~
losic esol b
sugars a
Hardwoods 05 8.0 5.2 5.0 4.71 2.62 43 54 12 )23-03
sdoowtfoS 64 4.1 2.11 0.1 7.5 3.22 82 34 92 )53-23
Wheat
straw 53 7.0 4.0 4.4 91 5.82 -- 13 41 )43-sa
Corn
stalks 5.63 1.1 6.0 1.2 2.71 5.72 -- 03 -- )43-33
Soybean
residue 83 8.1 4.2 0.1 5.21 7.81 -- 73 -- )43-33
a Reported as anhydrides;
b Includes acetyl- and uronic-acid residues;
c Residual glucan following acid prehydrolysis;
d Analyzed as Klason lignin (acid-insoluble)
Overall, it would appear that the high hexose (D-glucose plus D-mannose) content
of softwoods would favor their utilization as fermentation feedstocks. Presently,
however, most softwood residues find their way into pulping operations because of
the favorable fiber characteristics of conifer species. In contrast, hardwood residues
have much less value for paper production and are generally burned for the generation
Table .2 Estimated and projected total agri-
cultural residues in the United States ,63 )Ta
Material Quantity
0891 0002
601 ODT
Corn residue I00 241
Wheat straw 101 78
Soybean residue 89 951
Other grains 75 47
Other
agricultural products 82 53
Totals 583 794
