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Enzyme and Microbial Technology 31 (2002) 5366

Strategies for enhancing fermentative production


of glycerola review
Mohammad J. Taherzadeh a , Lennart Adler b , Gunnar Lidn a,
a
b

Department of Chemical Engineering II, Lund Institute of Technology, P.O. Box 124, 221 00 Lund, Sweden
Department of Cell and Molecular Biology, Gteborg University, P.O. Box 462, 405 30 Gteborg, Sweden
Received 24 July 2001; received in revised form 28 January 2002; accepted 6 February 2002

Abstract
The present paper reviews the metabolic basis of different methods for fermentative glycerol production. The most important microbial
production organism is the yeast Saccharomyces cerevisiae but other yeast species, as well as molds, algae, and bacteria are of potential
interest for glycerol production. A large variety of methods have been applied to increase the fermentative glycerol yield. The first methods
were based on physiological control, e.g. chemically induced overproduction of glycerol through NADH entrapment by the addition
of chemical steering agents (such as bisulfite). More recently, genetic engineering of the glycolytic pathway has been used to improve
production, involving modulated function of e.g. triose phosphate isomerase, phosphoglycerate mutase, PDC or alcohol dehydrogenase.
Direct intervention in the glycerol pathway, such as overexpression of G3P dehydrogenase, has also been tried. The applied strategies can
be divided into three principal groups; (a) deactivation or down-regulation of NADH oxidation sites alternative to G3P dehydrogenase, (b)
increase of NADH generation or, (c) direct changes in the carbon flux to glycerol. 2002 Published by Elsevier Science Inc.
Keywords: Yeast; Glycerol; Osmoregulation; Redox; NADH; Genetic engineering

1. Introduction
Glycerol is a widely used chemical with many commercial
applications, presently finding its largest use in the manufacture of drugs and oral care products including toothpaste,
mouthwash and oral rinses. In addition, glycerol is used in
foods and cosmetics, tobacco, wrapping and packaging materials, lubricants, urethane polymers, gaskets, cork products, cement compounds, soldering compounds, compasses,
cleaning materials, detergents, wetting agents, emulsifiers,
skin protectives, asphalt, ceramics, photographic products,
leather and wood treatment and adhesives [1]. The current world production of glycerol amounts to 600,000 t/year.
Bulk production of glycerol can be achieved by any of
three different principal methods [14]: (a) glycerol is recovered as a by-product in fat and oil industries. The spent
lyes resulting from current soap making processes generally contain 815% glycerol. Sweet waters from hydrolysis of fats contain as much as 20% glycerol. (b) Glycerol
can be synthesized from propylene by a variety of methods. (c) Glycerol can be produced by fermentation, which is
the focus of this review. The glycerol formation accompa

Corresponding author. Tel.: +46-222-0862; fax: +46-46-14-91-56.


E-mail address: gunnar.liden@chemeng.lth.se (G. Liden).

0141-0229/02/$ see front matter 2002 Published by Elsevier Science Inc.


PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 0 6 9 - 8

nying ethanol production in fermentation of sugars has been


described since the days of Pasteur [5]. However, no major attempts were made to produce glycerol in a dedicated
process until the First World War. At that time, a process
was established based on research conducted by Neuberg
[6,7]. The process was short-lived and could not compete
with post-war methods for synthetic glycerol production.
The drawbacks of the fermentative processes have primarily
been the low yield of glycerol from carbohydrates, and difficulties in the recovery of glycerol from the fermentation
broth.
In the past decades, the significant advances in biotechnology have led to a renewed interest in the production
of chemicals from renewable sources of carbohydrates,
so-called green chemistry. Fermentative production of glycerol is one example of a process of interest in this respect.
A large number of articles and patents, reporting improvements of fermentative glycerol yield or addressing the
issues of downstream processing, have been published in
the past two decades. A few reviews on the subject have
also appeared, describing selection of microorganisms, old
processes, and the choice of bioreactor system for the fermentative production of glycerol [4,810]. In the current
review, the metabolic rationale of suggested improvements
of glycerol production is reviewed, and reported studies

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M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

concerning enhancement of glycerol production are classified according to their underlying principle.

2. Production microorganisms
Glycerol is a well-known metabolite formed by many microorganisms including bacteria, yeasts, molds, and algae
[8,9,11]. Consequently, there are a number of microorganisms, which are potential candidates for glycerol production
(cf. Table 1). Since glycerol often serves the function of
an osmolyte, balancing external osmotic pressure (e.g. cf.

[1214]), osmophilic organisms are of particular interest for


glycerol production.
Most of the studies concerning glycerol formation have
been carried out using bakers yeast, S. cerevisiae. There
are several reasons for the interest in glycerol production
by this organism: (a) glycerol is a predominant by-product
of the fermentative metabolism of S. cerevisiae [1519], (b)
glycerol is a desirable end product in wine, providing the
quality of mouth-feel [2024], (c) glycerol is typically an
unwanted by-product in the production of distilled ethanol
[2527].

3. The metabolic basis of glycerol production in yeast


Table 1
Some microorganisms in which glycerol production has been studied
Genera

Strain

Reference

Yeast

Saccharomyces cerevisiae
Saccharomyces ellipsoideus
Zygosaccharomyces rouxii
Saccharomyces mellis
Saccharomyces formonensis
Saccharomyces uvarum
Zygosaccharomyces acidifaciens
Torulopsis magnoliae
Candida stellata
Candida boidinii
Candida kefyr
Candida pseudotropicali
Candida veratilis
Pichia angusta
Pichia anomala
Pichia farinosa
Pichia miso
Kluyveromyces bulgaricus
Kluyveromyces lactis
Kluyveromyces marxianus
Hanseniaspora guilliermondii
Kloeckera apicula
Pachysolen tannophilus

[110,174,175]
[127,128,176179]
[73,170,180182]
[181,182]
[183]
[171,184]
[185]
[169]
[20]
[120,163,164]
[95]
[95]
[95]
[120]
[120]
[165,168,170,186]
[187]
[95]
[95]
[95]
[188]
[188]
[189]

Mold

Debaryomyces mogii
Rhizopus nigricans
Rhizopus javanicus
Botrytis cinerea
Aspergillus niger

[187]
[190]
[162]
[190]
[190]

Algae

Penicillum
Dunaliella
Dunaliella
Dunaliella
Dunaliella

[190]
[191,192]
[193,194]
[192]
[195]

Protozoa

Dunaliella bardawil
Trypanosoma cruzi
Leishmania mexic

[67]
[196]
[196]

Bacteria

Crithidia fasciculata
Bacillus subtilis
Bacillus coli
Bacillus welchii
Bacterium orleanense
Bacterium ascendens
Bacterium pasteurianum
Lactobacillus lycopersici

[196]
[197]
[113]
[113]
[198]
[198]
[198]
[199]

italicum
tertiolecta
salina
viridis
bioculata

A simplified scheme of the metabolism in S. cerevisiae is


shown in Fig. 1. Glycerol is known to serve at least two important functions in yeast: (a) as a sink for the excess NADH
which is produced by anabolic reactions during anaerobic
conditions [2832], and (b) as an osmolyte balancing a high
external osmotic pressure during e.g. salt stress [13,14].
3.1. The role of glycerol in cellular redox balance
Concerning the redox regulation, it is evident that S.
cerevisiae possesses a very efficient means of redox regulation, since the cells grow rapidly under aerobic as well
as anaerobic conditions. For an eukaryotic organism, this
is a unique feature [33]. The fermentation of glucose to
ethanol is by itself redox neutral, the NADH generated
by glycolytic oxidation of glyceraldehyde-3-phosphate
to 1,3-diphosphoglycerate being reoxidized by reduction of acetaldehyde to ethanol. Interestingly, the enzymes catalyzing these two NAD-dependent steps:
glyceraldehyde-3-phosphate dehydrogenase (TDH) and
alcohol dehydrogenase (ADH) are very well-represented
proteins, one of the isoforms of TDH (Tdh3p) being the
most abundant protein of S. cerevisiae [34]. Although this
major pathway is redox inert, excess NADH is formed due
to the drainage of glycolytic intermediates as precursors
(as shown in Fig. 1) for amino acids, and by the generation of NADH in the biosynthetic pathways of some
amino acids [25,32]. Furthermore, aerobic oxidation of
pyruvatevia pyruvate dehydrogenase and the TCA cycle
enzymesproduces NADH in the mitochondrial matrix.
This NADH can be reoxidized in the respiratory chain via
the internal NADH dehydrogenase (Ndi1p). As NADH
cannot pass the inner membrane of the mitochondrion, excess cytosolic NADH can be oxidized by the respiratory
chain only by transferring reducing equivalents via the two
shuttle systems described for S. cerevisiae: the FAD dependent glycerol 3-phosphate dehydrogenase (Gut2p) system
[35,36] or the ethanolacetaldehyde shuttle [37]. However,
unlike mammalian cells, yeast also possesses an external
NADH dehydrogenase [38], whose catalytic site faces the
mitochondrial intermembrane space. This makes possible

M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

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Fig. 1. The sugar assimilation in glycolysis and TCA cycle in S. cerevisiae. NAD(P)+ , ADP, H+ , Pi , and CoA are not included in the picture for clarity.
Enzyme notations are:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21

Glucokinase (GLK) and Hexokinase (HXK)


Phosphoglucose isomerase (PGI)
Phosphofructokinase (PFK)
Aldolase (ALD)
Triosephosphate isomerase (TPI)
Glycerol-3-phosphate dehydrogenase (GPD)
Glycerol-3-phosphatase (GPP)
Triosephosphate dehydrogenase (TDH)
Phosphoglycerate kinase (PGK)
Phosphoglycerate mutase (GPM)
Enolase (ENO)
Pyruvate kinase (PYK)
Pyruvate decarboxylase (PDC)
Alcohol dehydrogenase (ADH)
Aldehyde dehydrogenase (AlDH)
Acetyl-CoA synthetase (ACS)
Probably via nonspecific decarboxylation of acetolactate formed in valine biosynthesis
Butanediol dehydrogenase
Glucose-6-phosphate dehydrogenase
Lactonase
6-Phospho-gluconate dehydrogenase

22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41

Ribose-5-phosphate keto-isomerase (RKI)


Ribulose-5-phosphate epimerase (RPE)
Transketolase (TKL)
Transketolase (TKL)
Transaldolase (TAL)
Pyruvate carboxylase (PYC)
Pyruvate dehydrogenase (PDH)
Citrate synthase (CS)
Aconitase
Isocitrate dehydrogenase (IDH, IDP1, IDP2)
2-Oxoglutarate dehydrogenase (OGDH)
Succinyl-CoA synthetase
Succinate dehydrogenase
Fumarate reductase (cytosolic, FRDS)
Fumarase
Malate dehydrogenase
Glutamate dehydrogenase (GDH)
Glutamine synthetase (GS)
Phosphoglucomutase (PGM)
Phosphomannoisomerase (PMI)

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M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

reoxidation of cytosolic NADH by direct delivery of reducing power to the respiratory chain. A comprehensive treatment of the importance of compartmentalization of NADH
metabolism was recently given by Bakker et al. [39].
Under anaerobic conditions the principal redox sink
in S. cerevisiae is the NADH coupled reduction of
dihydroxyacetone-phosphate (DHAP) to glycerol-3-phosphate
(G3P). This was first demonstrated by redox and carbon
balance studies [28,40] and given firm genetic evidence by
mutant studies [41,42]. The DHAP reduction is catalyzed
by NAD dependent glycerol-3-phosphate dehydrogenase
encoded by the two iso-genes, GPD1 and GPD2 [43,44].
The produced G3P is further dephosphorylated via a highly
specific phosphatase encoded by the iso-genes GPP1 and
GPP2 [45,46]. The isoenzymes of the glycerol pathway
appear to have distinct physiological roles [42,46]. For
example, only one iso-gene from each pair (GPD2 and
GPP1) is induced by shift to anaerobic conditions. Deletion
of either of these two genes results in defective anaerobic
growth, whereas deletion of either of the other iso-genes
(GPD1 or GPP2) is without anaerobic effects. Deletion of
both GPD1 and GPD2 or GPP1 and GPP2 leads to blocked
glycerol production and a completely inhibited anaerobic
growth. Norbeck and Blomberg [34] have demonstrated
the presence of enzymes/genes allowing for an alternative
glycerol pathway in S. cerevisiae, the dihydroxyacetone
(DHA) pathway. This route involves several genes (e.g.
GCY1), encoding a putative glycerol dehydrogenase and
two characterized genes, DAK1/DAK2, for dihydroxyacetone kinase, suggesting that the pathway functions in glycerol catabolism. Whether the pathway also has a role for
glycerol biosynthesis remains an open question.

3.2. The role of glycerol as an osmolyte


Most cells possess mechanisms that maintain the intracellular osmolarity higher than that of the extracellular medium.
The consequent osmotic gradient across the cell membrane
allows for influx of water to promote cell expansion and
development of turgor pressure (e.g. cf. [47]). Many organisms use osmolytes, or organic solutes, to adjust their intracellular osmolarity [13,48]. Intracellular accumulation of
osmolytes that are compatible with protein and membrane
structure, allows for osmotic adjustment without detriment
to cellular functions. Since the selected organic solutes used
for this purpose tend to stabilize cell structures during adverse conditions, increased synthesis can be triggered not
only by dehydration stress, but also by other stresses such
as heat stress. In yeasts and filamentous fungi, glycerol is
repeatedly found as the major osmolyte [14]. The protective nature of glycerol seems related to the fact that it interacts unfavorably with hydrophobic regions of proteins
(the so-called solvophobic effect), thereby favoring the native state of the proteins in which their hydrophobic regions
are buried [49]. This is an important aspect of a compatible

solute since it can be accumulated to high concentrations.


In yeasts exposed to strong osmotic stress the intracellular
glycerol concentration can reach molar levels [50,51]. For
S. cerevisiae, [42,43], Schizosaccharomyces pombe [52] and
the strongly osmotolerant (so-called osmophilic) yeast Debaryomyces hansenii [53], mutants having defective glycerol
production are unable to grow in media of high osmolarity.
Hence, there is good genetic evidence that glycerol serves
as the major osmolyte in these organisms. The increased intracellular accumulation of glycerol during osmotic stress is
due to both increased production of glycerol and enhanced
retention within the cells. In S. cerevisiae, the glycerol efflux
is partly controlled by FPS1, encoding a glycerol facilitator
channel that closes during hyperosmotic stress and opens
during hypoosmotic stress [54]. Mutants having a constitutively open Fps1p channel show increased osmosensitivity,
but appear to compensate for glycerol loss by increased production [55]. Osmotolerant yeasts, like D. hansenii [56,57],
Zygosaccharomyces rouxii [58] and Pichia sorbitophila [59]
are able to concentrate glycerol by uptake against a concentration gradient, using transport systems exhibiting Na+ or
H+ symport. In a study of 42 yeast species, only the most
salt tolerant yeasts possessed transport system that could accumulate glycerol by uptake from the surrounding medium
[60]. In five of these strains the glycerol uptake was mediated
by H+ /glycerol symport. In S cerevisiae, two genes, GUP1
and GUP2, encode membrane proteins that are involved in
uptake of glycerol, driven by electrogenic proton symport.
These transporters facilitate growth of S. cerevisiae on glycerol but may also have a role for glycerol retention of cells
exposed to hyperosmotic stress [61]. Turning to the other
important aspect of glycerol accumulation, the osmostress
induced glycerol production in S. cerevisiae and S. pombe
is in part due to increased activity of the GPD enzyme encoded by GPD1 [62] and gpd1 [52], respectively. The increased expression of these genes result from activation of a
mitogen activated protein (MAP) kinase cascade controlling
adaptation to high osmolarity. In both yeasts, the pathway is
activated by hyperosmotic stress, which promotes changed
expression of a number of target genes [63,64].
Intracellular accumulation of glycerol as an osmoregulatory strategy to counteract cell dehydration is used also by
salt tolerant unicellular algae of the genus Dunaliella [65].
These algae, which lack a rigid polysaccharide wall, thrive
in media containing from 0.1 M NaCl up to a saturated NaCl
solution. On changes in the osmolarity of the surrounding
medium the cells behave instantaneously as osmometers;
they shrink under hypertonic or swell under hypotonic conditions. Following this rapid volume change, synthesis or elimination of glycerol continues until the cell volume returns to
its original value [66]. On exposure to hypertonic stress the
algae convert stored polysaccharides and Calvin cycle intermediates to DHAP, which is then reduced to G3P and dephosphorylated to glycerol via GPD and a specific glycerol
3-phosphatase, as in S. cerevisiae. When subjected to hypotonic stress the glycerol is thought to be oxidized via glycerol

M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

dehydrogenase to DHA and then phosphorylated to DHAP


via dihydroxyacetone kinase. DHAP is thereafter probably
converted to polysaccharides by classical pathways. Hence,
these algae appear to efficiently reutilize glycerol used for
osmoregulatory purposes. Since Dunaliella has the potential of converting solar energy to glycerol, experiments for
large-scale outdoor cultivation have been conducted [67,68].
A productivity of 8 g glycerol per meter square and day was
achieved in short-term experiments in pilot plants, whereas
long term experiments resulted in about half this value.
Besides glycerol, most osmotolerant yeasts produce a variety of polyhydroxy alcohols [69,70] and may convert as
much as 60% of the utilized sugar to a mixture of polyols
[11]. The physiological role of the higher polyols, among
which erythritol, ribitol, arabinitol, xylitol, mannitol, dulcitol and heptitols [70,71] are reported, remains to be established. The osmotolerant yeast D. hansenii produces glycerol
and arabinitol as the main polyols. When cultured at high
salinity, arabinitol is significantly accumulated only when
the glycerol content is declining as the cells are reaching
the stationary phase [72]. It was therefore suggested that
higher polyols may serve as substitute compatible solutes
for glycerol in nongrowing cells exposed to hyperosmotic
stress, to allow for more efficient retention of the protective
solute under such conditions [14]. Commercial production
of glycerol from osmotolerant yeasts would require aerobic
conditions since these yeasts have an aerobic metabolism.
An advantage with such a process is that the high sugar or
sodium chloride concentrations minimize the risks of microbial contamination. However, although yields can be high
(a glycerol yield as high as 43% was reported for Z. rouxii
cultured at 18% NaCl [73]) conversion rates are severely
retarded in media with high osmolarities [74,75].

4. Strategies for optimizing glycerol production


With respect to optimizing glycerol production, it is important to keep in mind that the overall cost of 1 mol of
glycerol produced is 0.5 mol of glucose, 1 mol of NADH

57

and 1 mol of ATP (cf. Fig. 1). Regardless of the strategies appliedwhether this involves genetically engineering
strains or merely physiological controlthis cost must
somehow be paid. Obviously, a changed carbon flux necessarily involves changes in NADH formation/consumption
and ATP usage. Conversely, a change in NADH formation
will give a changed carbon flux. Nevertheless, strategies for
increasing glycerol production have taken different starting
points, as schematically described in Fig. 2. Genetic engineering was not used in the original processes from the
beginning of the century, although the basic ideas in these
studies were not fundamentally different from the ideas of
more recent genetic engineering work. (One of the oldest
strategies, i.e. the sulfite method introduced by Neuberg, is
based on the restriction of the NADH oxidation that occurs
in the reduction of acetaldehyde to ethanol, whereas one of
the most interesting new approaches, the work by Overkamp
et al. [76], is based on decreasing oxidation of cytosolically
generated NADH by deleting the genes encoding external
mitochondrial dehydrogenases NDE1/NDE2).
The strategy behind most of the applied methods for enhancing the production of glycerol is in a sense indirect,
since it aims at creating conditions during which the NADH
generation in metabolism is maximized. The consequent
carbon flux redistribution is caused by the necessity for
NAD regeneration, giving increased glycerol production. A
second strategy relies on direct interference with the carbon
flux, for example by blocking the isomerization reaction
between DHAP and GAP or by inhibiting the later part of
glycolysis. In this case, the redox balance will be distorted
causing a redistribution of fermentation products in order to
meet the need for generation of NADH. The latter strategy
can be accomplished for example by overexpression of enzymes in the glycerol pathway, such as glycerol-3-phosphate
dehydrogenase (GPD) and/or down regulation of enzymes
in the later part of glycolysis, such as ADH. In this case, the
increased glycerol formation results in a need for increased
NADH production, which has to be met by an increased
production of oxidized compounds, e.g. carboxylic acids.
The use of hyperosmotic conditions may represent a third

Fig. 2. Different strategies for improving fermentative glycerol yields.

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M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

Table 2
Genetic engineering targets for improved glycerol production
Target enzyme

Known genes

Comment

Reference

Triose phosphate isomerase (EC 5.3.1.1)

TPI1

[79,80]

Phosphoglycerate mutase (EC 5.4.2.1)


Pyruvate decarboxylase (EC 4.1.1.1)
Alcohol dehydrogenase (EC 1.1.1.1)
Glycerol-3-P dehydrogenase (EC 1.1.1.8)
Glycerol-3-phosphatase
Mitochondrial NADH-dehydrogenase

GPM1, GPM2, GPM3


PDC1, PDC2, PDC3, PDC5, PDC6
ADH1, ADH2, ADH3, ADH4, ADH5
GPD1, GPD2
GPP1, GPP2
NDE1, NDE2

Deletion of TPI1 tested. Only


aerobic growth possible
Deletion of GPM1 tested
Repression of PDC tested
Repression of ADH tested
Overexpression of GPD tried

strategy, in which the signal transduction pathways are involved in the redirection of carbon and NADH flux as a
response to the direct cellular need of glycerol production.
The discussed strategies can be accomplished by genetic
engineering, by physiological controlthat is by influencing the cellular physiology, e.g. by the choice of growth
medium and growth conditions, and/or addition of steering
chemicalsor by a combination of genetic engineering and
physiological control.
4.1. Genetic engineering
As seen in Fig. 1, there are several potential target genes in
the glycolytic, glycerol, and the respiratory pathways. Some
of the targets that have been examined are given in Table 2.
4.1.1. Glycolytic targets
4.1.1.1. Triose phosphate isomerase. The glycolytic
enzyme triose-phosphate-isomerase (TPI) catalyzes the
conversion of dihydroxyacetone-phosphate (DHAP) to
glyceraldehyde-3-phosphate (Fig. 1). The isomerase is encoded by the TPI1 gene in S. cerevisiae. A deficiency in
TPI activity has been shown to give rise to accumulation of DHAP [77,78]. In a TPI-mutant strain, completely
lacking TPI activity, the net energy gain from glycolysis
would be zero. Therefore, the strain will only be able to
grow by respiratory metabolism. The maximum theoretical
yield of glycerol from glucose in this strain is 1 mol/mol
(corresponding to 0.51 g/g). Compagno et al. [79] used a
TPI-mutant strain to produce glycerol from glucose, and
reported a glycerol yield as high as 85% of the theoretical
yield. However, a concentration of glucose >0.1 M was
needed for the conversion to proceed [80]. Interestingly,
iron limitation is reported to result in increased glycerol
production [81,82] and was observed to decrease the stability of the TP11 mRNA, leading to an about three-fold
decreased gene expression [81].
4.1.1.2. Phosphoglycerate mutase. Phosphoglycerate mutase (GPM) converts 3-phosphoglycerate into 2-phosphoglycerate in the later part of the glycolytic pathway (Fig. 1).

Deletion of NDE1 and NDE2 tried also


in combination with deletion of GUT2

[83]
[84]
[85]
[90]
[34]
[38,76]

As expected, no growth on glucose is possible in the absence


of this enzyme. The lack of gluconeogenesis also makes
respiratory growth on ethanol impossible. Neither is growth
on a medium containing both glucose and ethanol possible
due to glucose repression of respiration. However, Michnik
and Salmon [83] screened for derepressed gpm1 strains,
which would allow respiratory ethanol utilization even in the
presence of glucose. In the strains isolated by these investigators, glycerol was produced from glucose at very high
yields, up to 0.75 g/g (1.47 mol/mol), which is 73.5% of the
theoretical yield of 2 mol/mol. Obviously, ethanol was also
consumed to provide NADH and ATP for glycerol production. The authors did not report the glycerol yield on ethanol
due to problems of quantifying the ethanol evaporated.
4.1.1.3. Pyruvate decarboxylase. Pyruvate decarboxylase
(PDC) catalyzes the decarboxylation of pyruvate to acetaldehyde (Fig. 1). Nevoigt and Stahl [84] studied mutant strains
of S. cerevisiae in which the PDC activity was only 19%
of that of the wild type. In the mutant strains a 4.7-fold
increased glycerol yield was found under anaerobic conditions. The by-product yields also changed, with an increased
formation of, in particular, pyruvate, whereas the biomass
and ethanol yields decreased.
4.1.1.4. Alcohol dehydrogenase. S. cerevisiae possesses
several genes encoding alcohol dehydrogenases: ADH1,
ADH2 and ADH3. The protein encoded by ADH1 is believed to be the enzyme responsible for anaerobic cytosolic
reduction of acetaldehyde to ethanol. A lowered activity
of different ADH isoenzymes, especially Adh1p, results in
enhancement of the glycerol production [8587]. Johansson
and Sjstrm [85] reported a six- to seven-fold increased
glycerol production for a strain in which the specific activity of ADH was decreased to 6.7% of that of the wild-type
strain.
4.1.2. Targets in the glycerol formation pathway
4.1.2.1. Glycerol-3-phosphate dehydrogenase. Glycerol3-phosphate dehydrogenase (GPD) has been suggested
to be a limiting enzyme for glycerol production by

M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

S. cerevisiae [88,89]. Michnick et al. [90] increased the glycerol formation by a factor of 4 from 0.069 to 0.275 mol/mol
(0.0350.14 g/g), by overexpression of GPD1 in S. cerevisiae at the expense of ethanol. A similar study was carried
out by Remize et al. [21], where overexpression of Gpd1p
was analyzed in nine strains of S. cerevisiae. Glycerol
production increased between 1.5- and 2.5-fold in the examined strains. The biomass and ethanol yield decreased,
whereas the yields of pyruvate, acetate, acetaldehyde,
2,3-butanediol, succinate and acetoin increased. Notably,
by-product formation was considerably strain dependent.
An even higher overexpression of GPD1 was achieved in
experiments by Nevoigt and Stahl [84] who reported up to
6.5 times increased glycerol yield. Somewhat surprisingly,
overexpression of GPP1 or GPP2, resulting in an up to
50-fold increased glycerol-3-phosphatase activity, did not
appear to significantly affect glycerol production [46].
4.1.3. Targets in the respiratory pathway
4.1.3.1. External NADH dehydrogenases and glycerol utilization enzymes. Glycerol is perhaps most often considered as being an anaerobic end product. However, high
yields of glycerol can also be attained under aerobic conditions. One glucose molecule catabolized via glycolysis
and the TCA cycle produces 10 NADH molecules (cf.
Fig. 1), some of which could be used for DHAP reduction.
However, even when neglecting the limitation caused by
compartmentalization, all this NADH cannot be utilized for
DHAP reduction, since the ATP demand for glycerol formation must be met. This requires that part of the NADH
formed is oxidized in the respiratory chain. The necessary
amount will depend on the P/O ratio, i.e. the ratio between
moles of ATP formed and moles of oxygen (O) consumed.
A simple calculation (assuming the same ATP yield from
FADH and NADH) gives a maximum theoretical glycerol yield of 1.582 mol/mol (0.81 g/g), for a P/O ratio of
1 and 1.6 mol/mol (0.82 g/g) for a P/O ratio of 2. More
importantly, however, the inner mitochondrial membrane is
impermeable to NADH, which will impose a more severe
restriction on the maximum theoretical glycerol yield, since
only cytosolic NADH is available for DHAP reduction.
Overkamp et al. [76] conducted aerobic chemostat studies
of strains of S. cerevisiae, in which the two genes encoding the external mitochondrial NADH dehydrogenases,
NDE1 and NDE2, had been deleted. The NADH generated
in the cytosol could thus not be oxidized via the action
of these dehydrogenases, and an increased glycerol yield
might be expected from the need to regenerate cytosolic
NAD+ . However, up to a dilution rate of 0.1/h, very little formation of glycerol was found, suggesting that the
glycerol utilization pathway involving the mitochondrial
glycerol-3-phosphate dehydrogenase encoded by the GUT2
gene, was active. At higher dilution rates, the glycerol yield
increased, reaching a maximum of about 0.33 mol/mol
(0.17 g/g) at a dilution rate of about 0.2/h. Above the

59

critical dilution rate, the glycerol yield decreased, due to


the onset of ethanol formation, which consumed cytosolic
NADH. In a triple mutant strain, in which also the GUT2
gene was deleted, a glycerol yield as high as 0.58 mol/mol
(0.3 g/g) was obtained at a dilution rate of 0.1/h [39]. The
authors found it likely that the ethanolacetaldehyde shuttle was responsible for partly reoxidizing cytosolic NADH,
which suggests that an even higher glycerol yield might be
possible.
4.2. Physiological control
4.2.1. Steering chemicalsthe sulfite process
The most well-known production method of glycerol is
probably the sulfite method, which was used for large-scale
glycerol production during both the First and Second World
War (the so-called Protol process) [91]. In this process,
sulfiteor a mixture of sulfite and bisulfiteis added to the
medium. The underlying mechanism is the formation of a
complex between bisulfite and acetaldehyde, preventing the
reduction of acetaldehyde to ethanol. Glycerol formation results from the cellular need to reduce the NADH formed
together with the acetaldehyde [92]. Consequently, glycerol
should be formed in amounts equimolar to the quantity of
acetaldehyde bound to the sulfite [93,94]. The distribution
between sulfite and bisulfite ions in the medium is governed
by the pH value, due to the chemical equilibria:
H2 SO3 HSO3 + H+ SO3 2 + 2H+
The pKa is 1.8 for the first and 6.9 for the second dissociation. The dissociation constant for the bisulfiteacetaldehyde
complex is in the order of 2 106 mol/l [95], giving a very
strong complex at pH values below 7.
The sulfite method (Neubergs second form of fermentation) was first developed by Neuberg, Connstein and
Ldecke around the year 1910 [7,96], and was early on applied for glycerol production in Germany [97] and England
[98]. A good description of the process has been presented
by Harris [99]. The maximum theoretical yield of glycerol
from glucose in the absence of products other than acetaldehyde and CO2 is 1 mol/mol (corresponding to 0.51 g/g).
However, in practice some ethanol formation is needed to
provide extra ATP for sustaining glycolysis [39]. In addition
to the formation of the bisulfiteacetaldehyde complex, various forms of sulfites interact in the cellular metabolism in
several different ways for example by reacting with Vitamin
B1 [100,101], by deactivation of pyruvate decarboxylase or
TDH [102105] and by inhibiting cell division [106]. For
this reason, sulfite should be added gradually to maintain
a sufficient production rate. Although a certain amount of
glycerol must be formed to balance NADH formed in connection to biomass synthesis and carboxylic acid formation
[31,107], any product formation besides acetaldehyde will
give a decreased glycerol yield.

60

M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

Optimization of the sulfite process has been the subject


of a number of investigations. Addition of NaHSO3 together with Na2 SO3 can substantially increase the glycerol
yield [94,108,109]. Furthermore, use of sparingly soluble
forms of sulfite e.g. MgSO3 , or CaSO3 , or use of SO2 ,
(NH4 )2 SO3 , or K2 SO3 instead of Na2 SO3 , have given positive effects [7,110117]. Another way of minimizing the
required amount of sulfite is to operate the fermentation under vacuum conditions, or with continuous sparging of CO2
[118,119]. In this way acetaldehyde can be stripped-off during fermentation. Actual glycerol yields in the sulfite process
seldom exceed about 0.39 mol/mol (0.2 g/g) of the utilized
sugar [39], although yields of up to 0.68 mol/mol (0.35 g/g)
have been reported [120]. Steering chemicals other than sulfite may, in principle, also be used to trap metabolic intermediates e.g. hydroxylamine, hydrazine or other compounds
having a free amino-group [121]. However, the toxic effects
of these compounds make them less attractive for use in
large-scale operation.
4.2.2. The alkaline process
The alkaline process, also known as Neubergs third form
of fermentation, is probably as old as the sulfite process
[6,122]. In this method, the alkalinity of the medium is
maintained by consecutive additions of Na2 CO3 . The enhanced production of acetate, which is caused by the high
pH values, is considered the main mechanism behind the increased glycerol formation [9,122]. Importantly, the acetic
acid that is excreted from the cells cannot diffuse back into
the cells, since the ionic form of acetic acid predominates
at high pH [27]. Acetate formation is accompanied by formation of NADH (cf. Fig. 1), which must be balanced by
glycerol formation during anaerobic conditions. The maximum theoretical glycerol yield is 1 mol/mol (0.51 g/g), as
with the sulfite process but, besides glycerol, the main fermentation products of the alkaline process are ethanol, acetate and CO2 . The pH value is maintained at 78 during
most of the process and is only brought to 67 towards
the end of the fermentation [123,124]. Growth of S. cerevisiae is possible between pH values 2.7 and 6.5 [27,125].
However, the fermentation capacity decreases by more than
30% when the pH is outside the range 36 [126]. The optimal concentration of Na2 CO3 was reported to be 5%, with
lower concentrations giving a decreased glycerol yield and
higher concentrations resulting in an inhibition of the fermentation [127,128]. As in the case of sulfite, sodium carbonate should thus be added gradually, to avoid inhibition.
Glycerol yields in the same range as for the sulfite process
(0.2 g/g) have been reported [129]. In order to maximize
the glycerol yield, the acetaldehyde dehydrogenase responsible for oxidization of acetaldehyde to acetate should be
NAD+ (and not NADP+ ) dependent. Several genes encode
aldehyde dehydrogenases in S. cerevisiae. The ALD2 and
ALD3 genes are known to encode NAD+ coupled aldehyde
dehydrogenases, whereas ALD5 and ALD6 encode NADP+
dependent aldehyde dehydrogenases [130133]. Apart from

sodium carbonate other salts such as Na, K, or NH4 carbonate, bicarbonate, acetate, phosphate and hydroxide have also
been used as buffers to increase the pH value for alkaline
glycerol production [96,134136].
4.2.3. The effects of the nitrogen source
Albers et al. [25] reported a large effect of the nitrogen
source on the anaerobic glycerol production by S. cerevisiae.
Use of ammonium sulphate as nitrogen source resulted in
a higher yield of glycerol, compared to the use of glutamate or a mixture of amino acids. This difference can be
explained from the NADH formation that is associated with
amino acid synthesis [25]. It has also been reported that the
activity of ADH is decreased when amino acids rather than
ammonium salts are used as nitrogen source [137], resulting
in an increased glycerol yield.
4.2.4. Osmotic stress
A vital feature of the events that are initiated by yeast cells
in response to hyperosmotic stress is the osmostress-induced
accumulation of glycerol [138,139]. In S. cerevisiae, this
response is dependent on the stress-activated HOG pathway
[64,140]. This signaling pathway senses the stress condition,
evaluates its severity and adjusts expression of appropriate
genes to counteract the toxic stress effects. At the very heart
of this pathway is a MAP kinase module consisting of a cascade of kinases, that when activated, phosphorylates and activates the MAP kinase, Hog1p [141,142]. The high osmolarity stress is sensed by two distinct plasma membrane sensor
systems that converge via different signal transduction systems at the MAP kinase activator protein, Pbs2p. One of the
branches is defined by the Sln1pYpd1pSsk1p multicomponent system [143], the other by the Sho1p plasma membrane sensor protein [142]. In the activated pathway Pbs2p
phosphorylates Hog1p [141], which is then rapidly transferred to the nucleus to promote activation of gene expression [144]. Since Hog1p is a well-conserved stress-activated
MAP kinase, with counterparts in many other organisms
[145], it is likely that similar osmosensing and signalling systems may operate in other organisms. This is supported by
the recently described and analogous Sty1p pathway in the
distantly related S. pombe (reviewed in [139]). In an effort to
identify the target genes of the HOG pathway, genome-wide
transcriptional responses of wild type and hog1p mutants
have been analyzed following exposure of the cells to high
osmolarity [146,147]. The complex nature of the response
is clearly indicated by the fact that a shift to 0.4 M NaCl resulted in a more than five-fold transient induction of about
7% of the genes encoded in the entire yeast genome. The
HOG pathway was required for full induction of many but
not all of the responding genes, whereas the most strongly responsive were highly or completely dependent on this pathway for their activation. Among the genes that were most
sharply induced by osmotic stress were GPD1 and GPP2 of
the glycerol pathway, but also genes encoding plasma membrane sugar transporters such as SLT1 and HXT10 as well

M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

as genes encoding glucose phosphorylating enzymes such


HXK2 and GLK1. It is also interesting to note that the ALD3
and ALD2 genes, encoding the NAD+ -dependent cytosolic
and mitochondrial aldehyde dehydrogenases, respectively,
were among the most strongly induced. Hence, novel strategies for increasing glycerol production by S. cerevisiae may
well make use of components in the signalling pathway
rather than the direct brute-force overexpression of target
genes.
4.2.5. Heat shock
A heat shock treatment of S. cerevisiae, by a temperature
increase to 45 C for 1 h, has been reported to increase the
glycerol production by 1525%, [23,148]. Omori et al. [22]
applied heat shock treatment (45 C, 20 min) to screen for
heat shock resistant (HSR) mutants and reported an about
two-fold increase of glycerol production in such mutants as
compared to the parental strain. The increased expression of
GPD1 following heat shock [149] is the most likely reason of
the observed increase in glycerol production [23,148]. The
combined effects of salt stress and cultivation temperature
were studied by Carvalheiro et al. [150], who reported the
highest glycerol production by cultivation at 44 C in the
presence of 0.75 M NaCl.

5. Industrial carbon sources


Industrial production of glycerol by fermentation demands cheap carbon sources in order to compete with other
production methods. Some of the carbon sources, other than
glucose, that have been studied are listed in Table 3. Molasses from beet sugar or sugar cane are among the prime
choices for glycerol production [119,151155]. Starch is
another potential carbon source for production of glycerol
[18,156,157]. Figard [156] hydrolyzed starch with 0.1N sulfuric acid at 1.7 bar in 2 h, which yielded 96% monomeric
glucose. The glucose was subsequently converted using the

Table 3
Industrial carbon sources for glycerol production
Carbon source
Beet sugar molasses
Sugar cane molasses
Cane juice
Grape juice

Starch
Lignocellulose
Xylose
Whey permeate
Enzyme-hydrolyzed lactose
Methanol

Microorganism used
S. cerevisiae
A. niger
P. italicum
R. nigricans
B. cinerea
Z. acidifaciens
S. cerevisiae
R. javanicus
K. marxianus
P. farinosa
C. boidinii

Reference
[200]
[118,201]
[183]
[110,190]

[156,157]
[158]
[162]
[95]
[95]
[163,164]

61

sulfite process. Lignocellulosic materials have also been of


interest for the production of glycerol. These materials can
be pulped by a conventional sulfite process, followed by
a dilute-acid hydrolysis [158,159], steam treatment [160]
or enzymatic hydrolysis [161] to prepare a solution of
monomeric sugars for conversion to glycerol.
Sugars other than glucose have also been used for glycerol
production. A glycerol yield on xylose of up to 0.21 mol/mol
(0.13 g/g) was reported using Rhizopus javanicus [162].
However, high sugar concentrations and a high aeration
rate were needed. Conversion of lactose in whey permeate
to glycerol was investigated by Rapin et al. [95]. Different
strains of Candida and Kluyveromyces species were used,
and it was found that Kluyveromyces marxianus gave the
best results in terms of growth rate (0.15/h) and glycerol
yield (0.1 g/g) at 37 C and pH 7. Another potential raw
material for glycerol is methanol [163,164]. The yeast Candida boidinii can grow on methanol, but the genes encoding
glycerol kinase should be deleted, or the enzyme should be
inhibited, in order to obtain production of glycerol.

6. Fermentation technology
Batch fermentation using shake flask cultivations
(semi-aerobic by using cotton-plugged flasks or anaerobic
by using loop-traps) is most commonly used in laboratory
experiments due to its simplicity. One obvious drawback
with these systems is that temperature is usually the only
variable that can be accurately controlled. Use of a bioreactor gives the added possibility of accurately controlling
the pH value, which has indeed been found to be an important parameter in a number of genetic and physiological
approaches of glycerol enhancement.
Fed-batch operation is experimentally somewhat more
demanding, but gives certain advantages. The most important advantage of fed-batch compared to batch cultivation
is that effects of inhibitors, such as steering chemicals, can
be minimized since their concentration in the medium can
be kept low [15,165168]. In addition, by using fed-batch
technique, it is possible to maintain limitation of a certain
substrate component, such as phosphate in the cultivation of
the osmophilic yeast Torulopsis magnoliae [169]. Very high
glycerol concentrations can be obtained using fed-batch
cultivation [165].
Continuous cultivation shares several advantages with
fed-batch cultivation, although the risk of contamination is
increased. A continuous process based on the sulfite method
was found to work satisfactorily [99]. However, the low
concentration of biomass, which decreases the volumetric
glycerol productivity, may be considered a serious drawback
of continuous cultivation. Cell immobilization or recycling
has been applied in order to provide a high cell density in the
bioreactor. Centrifuging and recycling of the cells resulted
in an increased production rate of the glycerol [170], and
cell immobilization was reported to significantly increase

62

M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

the glycerol production rate in batch [20], fed-batch [15]


and continuous cultivation [17,155,171,172]. Various supports such as agarose [171], Ca-alginate and polyacrylamide
hydrazide [173], sintered glass [19,172] and -carrageenan
[174] have been used. Obviously, the benefits of increased
volumetric productivity must be weighed against the added
cost for cell recycling or immobilization.

7. Conclusion
Fermentative production of glycerol has a long and varied
history. With the exception of wartimes, fermentative glycerol production has normally not been economically competitive with other production methods. Technological progress
in terms of downstream processing, cultivation technology
and genetic engineering, in combination with increased consumer requirements of green chemistry can be expected
to change that situation in the near future.
References
[1] Morrison LR. Glycerol. In: Encyclopedia of chemical technology.
New York: Wiley, 1994. p. 68194.
[2] Jakobson G, Kathagen FW, Klatt M. Glycerol. In: Ullmanns
encyclopedia of industrial chemistry. Weinheim: VCH, 1989.
p. 44789.
[3] Foster EG. Glycerine, synthetic. In: Encyclopedia of chemical
processing and design. New York: Marcel Dekker Inc., 1986.
p. 38293.
[4] Rehm H. Microbial production of glycerol and other polyols. In:
Biotechnology. Weinheim: VCH, 1988. p. 519.
[5] Pasteur L. Production constante de glycrine dans la fermentation
alcooliqe. Compt Rend Acad Sci 1858;46:857.
[6] Neuberg C, Reinfurth E. About the process of the alcoholic
fermentation with alkaline reaction. I. Cellfree fermentation in
alkaline solutions. Biochem Z 1917;78:23863.
[7] Neuberg C, Reinfurth E. Further investigations on the correlative
formation of acetaldehyde and glycerol in the scission of sugar and
new contributions for the theory of the alcoholic fermentation. Ber
Dtsch Cem Ges 1919;52B:1677703.
[8] Vijaikishore P, Karanth N. Glycerol production by fermentationa
review. Process Biochem 1986;21:547.
[9] Rehm H. Microbial production of glycerol and other polyols. In:
Biotechnology. Weinheim: VCH, 1996. p. 20527.
[10] Agarwal GP. Glycerol. Adv Biochem Eng Biotechnol 1990;41:95
128.
[11] Spencer JFT. Production of polyhydric alcohols by yeasts. Prog Ind
Microbiol 1968;7:142.
[12] Sunder S, Singh AJ, Gill S, Singh B. Regulation of intracellular
level of Na+ , K+ and glycerol in Saccharomyces cerevisiae under
osmotic stress. Mol Cell Biochem 1996;158:1214.
[13] Brown AD. Compatible solutes and extreme water stress in
eukaryotic microorganisms. Adv Microb Physiol 1978;17:181242.
[14] Blomberg A, Adler L. Physiology of osmotolerance in fungi. Adv
Microb Physiol 1992;33:145212.
[15] Bisping B, Hecker D, Rehm H. Glycerol production by
semicomtinuous fed-batch fermentation with immobilized cells of
Saccharomyces cerevisiae. Appl Microbiol Biotechnol 1989;32.
[16] Andr L, Hemming A, Adler L. Osmoregulation in Saccharomyces
cerevisiae. Studies on the osmotic induction of glycerol production
and glycerol-3-phosphate dehydrogenase (NAD+ ). FEBS Lett
1991;286:137.

[17] Benito G, Ozores M, Pea M. Continuous glycerol production in


a packed-bed bioreactor with immobilized cells of Saccharomyces
cerevisiae. Bioresource Technol 1994;49:20912.
[18] Brumm PJ, Hebeda RE. Glycerol production in industrial alcohol
fermentations. Biotechnol Lett 1988;10:67782.
[19] Bisping B, Rehm H. Glycerol production of cells by Saccharomyces
cerevisiae immobilized in sintered glass. Appl Microbiol Biotechnol
1986;23:1749.
[20] Ciani M, Ferraro L. Enhanced glycerol content in wines made
with immobilized Candida stellata cells. Appl Environ Microbiol
1996;62:12832.
[21] Remize F, Roustan J, Sablayrolles J, Barre P, Dequin S. Glycerol
overproduction by engineered Saccharomyces cerevisiae wine yeast
strains leads to substantial changes in by-product formation and to
a stimulation of fermentation rate in stationary phase. Appl Environ
Microbiol 1999;65:1439.
[22] Omori T, Umemoto Y, Ogawa K, Kajiwara Y, Shimoda M, Wada
H. A novel method for screening high glycerol- and ester-producing
brewing yeasts (Saccharomyces cerevisiae) by heat shock treatment.
J Ferment Bioeng 1997;83:649.
[23] Omori T, Ogawa K, Umemoto Y, Yuki K, Kajihara Y, Shimoda
M, et al. Enhancement of glycerol production by brewing yeast
(Saccharomyces cerevisiae) with heat shock treatment. J Ferment
Bioeng 1996;82:18790.
[24] Scanes KT, Hohmann S, Prior BA. Glycerol production by the yeast
Saccharomyces cerevisiae and its relevance to wine: a review. S
Afr J Enol Vitic 1998;19:1724.
[25] Albers E, Larsson C, Lidn G, Niklasson C, Gustafsson L. The
influence of the nitrogen source on the anaerobic growth and product
formation of Saccharomyces cerevisiae. Appl Environ Microbiol
1996;62:318795.
[26] Converti A, Zilli M, Rovatti M, Borghi M. Effect of glycerol on
alcohol fermentation. Inhibition mechanism and diffusion limitation.
Bioprocess Eng 1995;13:25763.
[27] Taherzadeh MJ, Niklasson C, Lidn G. Acetic acidfriend or foe in
anaerobic batch conversion of glucose to ethanol by Saccharomyces
cerevisiae? Chem Eng Sci 1997;52:26539.
[28] Nordstrm K. Yeast growth and glycerol formation. Acta Chem
Scand 1966;20:101625.
[29] Lagunas R, Gancedo JM. Reduced pyridine-nucleotides balance
in glucose-growing Saccharomyces cerevisiae. Eur J Biochem
1973;37:904.
[30] Oura E. Reaction products of yeast fermentations. Process Biochem.
1977;12:1921,35.
[31] Taherzadeh MJ, Lidn G, Gustafsson L, Niklasson C. The effect
of pantothenate deficiency and acetate addition on anaerobic
batch fermentation of glucose by Saccharomyces cerevisiae. Appl
Microbiol Biotechnol 1996;46:17682.
[32] Albers E, Lidn G, Larsson C, Gustafsson L. Anaerobic redox
balance and nitrogen metabolism in Saccharomyces cerevisiae.
Recent Res Dev Microbiol 1998;2:25379.
[33] ter Linde JJ, Liang H, Davis RW, Steensma HY, van Dijken
JP, Pronk JT. Genome-wide transcriptional analysis of aerobic
and anaerobic chemostat cultures of Saccharomyces cerevisiae. J
Bacteriol 1999;181:740913.
[34] Norbeck J, Blomberg A. Metabolic and regulatory changes
associated with growth of Saccharomyces cerevisiae in 1.4 M NaCl.
Evidence for osmotic induction of glycerol dissimilation via the
dihydroxyacetone pathway. J Biol Chem 1997;272:554454.
[35] Rnnow B, Kielland-Brandt MC. GUT2 a gene for mitochondrial
glycerol 3-phosphate dehydrogenase of Saccharomyces cerevisiae.
Yeast, 11:40718 1993;9:112130.
[36] Larsson C, Phlman I-L, Ansell R, Rigoulet M, Adler L, Gustafsson
L. The importance of the glycerol-3-phosphate shuttle during
aerobic growth of Saccharomyces cerevisiae. Yeast, 11:40718
1998;14:34757.

M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366


[37] Von Jagow G, Klingenberg M. Pathways of hydrogen in
mitochondria of Saccharomyces carlsbergensis. Eur J Biochem
1970;12:58392.
[38] Luttik MA, Overkamp KM, Kotter P, de Vries S, van Dijken JP,
Pronk JT. The Saccharomyces cerevisiae NDE1 and NDE2 genes
encode separate mitochondrial NADH dehydrogenases catalyzing
the oxidation of cytosolic NADH. J Biol Chem 1998;273:2452934.
[39] Bakker BM, Overkamp KM, van Maris AJA, Kotter P, Luttik
MAH, van Dijken JP, et al. Stoichiometry and compartmentation of
NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiol
Rev 2001;25:1537.
[40] Holzer H, Bernhardt W, Schneider S. Zur Glycerinbildung in
Bckerhefe. Biochem Z 1963;329:1372.
[41] Bjrkqvist S, Ansell R, Alder R, Lidn G. Glycerol-3-phosphate
dehydrogenase mutants of Saccharomyces cerevisiae grown under
aerobic and anaerobic conditions. Appl Environ Microbiol
1997;63:12832.
[42] Ansell R, Granath K, Hohmann S, Thevelein JM, Adler L. The
two isoenzymes for yeast NAD+ -dependent glycerol 3-phosphate
dehydrogenase encoded by GPD1 and GPD2 have distinct roles in
osmoadaptation and redox regulation. Embo J 1997;16:217987.
[43] Larsson K, Ansell R, Eriksson P, Adler L. A gene encoding
sn-glycerol 3-phosphate dehydrogenase (NAD+ ) complements an
osmosensitive mutant of Saccharomyces cerevisiae. Mol Microbiol
1993;10:110111.
[44] Eriksson P, Andre L, Ansell R, Blomberg A, Adler L. Cloning
and characterization of GPD2, a second gene encoding sn-glycerol
3-phosphate dehydrogenase (NAD+ ) in Saccharomyces cerevisiae
and its comparison with GPD1. Mol Microbiol 1995;17:95107.
[45] Norbeck J, Pahlman AK, Akhtar N, Blomberg A, Adler
L. Purification and characterization of two isoenzymes
of d,l-glycerol-3-phosphatase from Saccharomyces cerevisiae.
Identification of the corresponding GPP1 and GPP2 genes and
evidence for osmotic regulation of Gpp2p expression by the
osmosensing mitogen-activated protein kinase signal transduction
pathway. J Biol Chem 1996;271:1387581.
[46] Phlmann AK, Granath K, Ansell R, Hohmann S, Adler L. The
yeast glycerol 3-phosphatatases gpp1p and gpp2p are required for
glycerol biosynthesis and differentially involved in the cellular
responses to osmotic, anaerobic and oxidative stress. J Biol Chem
2001;276:355563.
[47] Le Rudulier D, Strom AR, Dandekar AM, Smith LT, Valentine RC.
Molecular biology of osmoregulation. Science 1984;224:10648.
[48] Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN.
Living with water stress: evolution of osmolyte systems. Science
1982;217:121422.
[49] Timasheff SN. Solvent stabilization of protein structure. Methods
Mol Biol 1995;40:25369.
[50] Larsson C, Morales C, Gustafsson L, Adler L. Osmoregulation of
the salt-tolerant yeast Debaryomyces hansenii grown in a chemostat
at different salinities. J Bacteriol 1990;172:176974.
[51] lz R, Larsson K, Adler L, Gustafsson L. Energy flux and
osmoregulation of Saccharomyces cerevisiae grown in chemostats
under sodium chloride stress. J Bacteriol 1993;175:220513.
[52] Ohmiya R, Yamada H, Nakashima K, Aiba H, Mizuno T.
Osmoregulation of fission yeast: cloning of two distinct genes
encoding glycerol-3-phosphate dehydrogenase, one of which
is responsible for osmotolerance for growth. Mol Microbiol
1995;18:96373.
[53] Andr L, Nilsson A, Adler L. The role of glycerol in
osmotolerance of the yeast Debaryomyces hansenii. J Gen Microbiol
1988;134:66977.
[54] Luyten K, Albertyn J, Skibbe WF, Prior BA, Ramos J, Thevelein
JM, et al. Fps1, a yeast member of the MIP family of channel
proteins, is a facilitator for glycerol uptake and efflux and is inactive
under osmotic stress. Embo J 1995;14:136071.

63

[55] Tamas MJ, Luyten K, Sutherland FC, Hernandez A, Albertyn J,


Valadi H, et al. Fps1p controls the accumulation and release of the
compatible solute glycerol in yeast osmoregulation. Mol Microbiol
1999;31:1087104.
[56] Adler L, Blomberg A, Nilsson A. Glycerol metabolism and
osmoregulation in the salt-tolerant yeast Debaryomyces hansenii. J
Bacteriol 1985;162:3006.
[57] Lucas C, Da Costa M, Van Uden N. Osmoregulatory
active sodium-glycerol co-transport in the halotolerant yeast
Debaryomyces hansenii. Yeast, 11:40718 1990;6:18791.
[58] van Zyl PJ, Kilian SG, Prior BA. The role of an
active transport mechanism in glycerol accumulation during
osmoregulation by Zygosaccharomyces rouxii. Appl Microbiol
Biotechnol 1990;34:2315.
[59] Lages F, Lucas C. Characterization of a glycerol/H+ symport
in the halotolerant yeast Pichia sorbitophila. Yeast, 11:40718
1995;11:11119.
[60] Lages F, Silva-Graca M, Lucas C. Active glycerol uptake is a
mechanism underlying halotolerance in yeasts: a study of 42 species.
Microbiology (UK) 1999;145:257785.
[61] Holst B, Lunde C, Lages F, Oliveira R, Lucas C, Kielland-Brandt
MC. GUP1 and its close homologue GUP2, encoding
multimembrane-spanning proteins involved in active glycerol uptake
in Saccharomyces cerevisiae. Mol Microbiol 2000;37:10824.
[62] Albertyn J, Hohmann S, Thevelein JM, Prior BA. GPD1, which
encodes glycerol-3-phosphate dehydrogenase, is essential for growth
under osmotic stress in Saccharomyces cerevisiae, and its expression
is regulated by the high-osmolarity glycerol response pathway. Mol
Cell Biol 1994;14:413544.
[63] Banuett F. Signalling in the yeasts: an informational cascade
with links to the filamentous fungi. Microbiol Mol Biol Rev
1998;62:24974.
[64] Gustin MC, Albertyn J, Alexander M, Davenport K. MAP kinase
pathways in the yeast Saccharomyces cerevisiae. Microbiol Mol
Biol Rev 1998;62:1264300.
[65] Avron M. The osmotic components of halotolerant algae. Trends
Biochem Sci 1986;11:56.
[66] Ben-Amotz A, Sussman I, Avron M. Glycerol production by
Dunaliella. Experientia 1982;38:4952.
[67] Ben-Amotz A, Avron M. Glycerol and -carotene metabolism in
the halotolerant alga Dunaliella: a model system for biosolar energy
conversion. Trends Biochem Sci 1981;6:2979.
[68] Ben-Amotz A, Avron M. Accumulation of metabolites by
halotolerant algae and its industrial potential. Ann Rev Microbiol
1983;37:95119.
[69] Spencer JFT, Spencer DM. Production of polyhydroxy alcohols
by osmotolerant yeasts. In: Econ Microbiology. London: Academic
Press, 1978. p. 39325.
[70] Onishi H. Osmophilic yeasts. Adv Food Res 1963;12:5394.
[71] Onishi H, Perry MB. The production of meso-glycero-ido-heptitol
and d-glycero-d-ido-heptitol by Pichia miso. Can J Microbiol
1965;11:92934.
[72] Adler L, Gustafsson L. Polyhydric alcohol production and
intracellular amino acid pool in relation to halotolerance of the
yeast Debaryomyces hansenii. Arch Microbiol 1980;124:12330.
[73] Onishi H. Glycerol. US Patent No. 3,012,943 (1961).
[74] Onishi H. Osmophilic yeasts. II. Factors affecting growth of soy
yeasts and others in the environment of a high concentration of
sodium chloride. Bull Agric Chem Soc Jpn 1957;21:14350.
[75] Onishi H. Studies on osmophilic yeasts. VI. Glycerol production
by the salt-tolerant yeasts in the medium with high concentrations
of sodium chloride. Bull Agric Chem Soc Jpn 1959;23:35968.
[76] Overkamp KM, Bakker BM, Kotter P, van Tuijl A, de Vries S, van
Dijken JP, et al. In vivo analysis of the mechanisms for oxidation
of cytosolic NADH by Saccharomyces cerevisiae mitochondria. J
Bacteriol 2000;182:282330.

64

M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

[77] Ciriacy M, Breitenbach I. Physiological effects of seven different


blocks in glycolysis in Saccharomyces cerevisiae. J Bacteriol
1979;139:15260.
[78] Ationu A, Humphries A, Bellingham A, Layton M. Metabolic
correction of triose phosphate isomerase deficiency in vitro by
complementation. Biochem Biophys Res Commun 1997;232:528
31.
[79] Compagno C, Boschi F, Ranzi BM. Glycerol production in a triose
phosphate isomerase deficient mutant of Saccharomyces cerevisiae.
Biotechnol Prog 1996;12:5915.
[80] Compagno C, Boschi F, Ranzi B. Factors affecting glycerol
production by a bioconversion process with a triose phosphate
isomerase deficient mutant of Saccharomyces cerevisiae. Biocat
Biotransform 1998;16:13543.
[81] Krieger K, Ernst JF. Iron regulation of triosephosphate isomerase
transcript stability in the yeast Saccharomyces cerevisiae.
Microbiology 1994;140(5):107984.
[82] Ansell R, Adler L. The effect of iron limitation on glycerol
production and expression of the iso-genes for NAD+ -dependent
glycerol 3-phosphate dehydrogenase in Saccharomyces cerevisiae.
FEBS Lett 1999;461:1737.
[83] Michnick S, Salmon J. Glycerol production from sugars
with phosphoglycerate mutase-deficient Saccharomyces cerevisiae
partially resistant to glucose repression. J Ind Microbiol 1994;13:17
23.
[84] Nevoigt E, Stahl U. Reduced pyruvate decarboxylase and
increased glycerol-3-phosphate dehydrogenase [NAD+ ] levels
enhance glycerol production in Saccharomyces cerevisiae. Yeast,
11:40718 1996;12:13317.
[85] Johansson M, Sjstrm JE. Enhanced production of glycerol in an
alcohol dehydrogenase (ADH I) deficient mutant of Saccharomyces
cerevisiae. Biotechnol Lett 1984;6:4954.
[86] Drewke C, Thielen J, Ciriacy M. Ethanol formation in adh0 mutants
reveals the existence of a novel acetaldehyde-reducing activity in
Saccharomyces cerevisiae. J Bacteriol 1990;172:390917.
[87] Ohbuchi K, Kanda A, Hamachi M, Nunokawa Y. Breeding higher
glycerol-productive yeast strains and brewing sake with them.
Hakkokogaku Kaishi-J Soc Ferment Technol 1991;69:2039.
[88] Blomberg A, Adler L. Role of glycerol and glycerol-3-phosphate
dehydrogenase (NAD+ ) in acquired osmotolerance of
Saccharomyces cerevisiae. J Bacteriol 1989;171:108792.
[89] Rodrguez-Acosta F, Regalado CM, Torres NV. Nonlinear
optimization of biotechnological processes by stochastic algorithms:
application to the maximization of the production rate of
ethanol, glycerol and carbohydrates by Saccharomyces cerevisiae.
J Biotechnol 1999;68:1528.
[90] Michnick S, Roustan J, Remize F, Barre P, Dequin S. Modulation
of glycerol and ethanol yields during alcoholic fermentation
in Saccharomyces cerevisiae strain overexpressed or disrupted
for GPD1 encoding glycerol 3-phosphate dehydrogenase. Yeast,
11:40718 1997;13:78393.
[91] Freeman GG, Donald GMS. Fermenation process leading to
glycerol. I. The influcence of certain variables on glycerol formation
in the presence of sulfite. Appl Microbiol 1957;5:197210.
[92] Tomoda Y. Production of glycerol by fermentation. IV. Dissociation
of acetaldehdyesodium bisulfite complex in alkaline solution. J
Soc Chem Ind 1927;30:74759.
[93] Zerner E. The chemistry of alcoholic fermentation. Ber Dtsch Cem
Ges 1920;53B:32534.
[94] Schwenk E. Glycerol by fermentation and its theroretical basis.
Seifenfabr 1920;40:4950.
[95] Rapin JD, Marison IW, von Stockar U, Reilly PJ. Glycerol
production by yeast fermentation of whey permeate. Enzyme Microb
Technol 1994;16:14350.
[96] Connstein W, Ldecke K. Obtaining glycerol by fermentation. Ber
Dtsch Cem Ges 1919;52:138591.
[97] Connstein W, Ldecke K. Process of manufacturing of propantriol
from sugar. US Patent No. 1,368,023 (1921).

[98] Cocking AT, Lilly CH. Glycerol production by fermentation. US


Patent No. 1,425,838 (1922).
[99] Harris J, Hajny G. Glycerol production: a pilot plant investigation
for continuous fermentation and recovery. J Biochem Microbiol
Technol Eng 1960;2:924.
[100] Williams RR, Waterman RE, Keresztesy JC, Buchman ER. Studies
of crystalline Vitamin B1. III. Cleave of vitamin with sulfite. J Am
Chem Soc 1935;57:5367.
[101] Buchman ER. Studies of crystalline Vitamin B1. XIV. Sulfite
cleavege. J Am Chem Soc 1936;58:18035.
[102] Fukui S, Kishibe T. Relation between the glycerol fermentation
and yeast cocarboxylase. I. The changes of yeast cocarboxylase
during the sulfite process fermentation. J Ferment Technol (Japan)
1950;28:41822.
[103] Schimz KL, Holzer H. Rapid decrease of ATP content in intact
cells of Saccharomyces cerevisiae after incubation with low
concentrations of sulfite. Arch Microbiol 1979;121:2259.
[104] Hinze H, Holzer H. Effect of sulfite or nitrite on the ATP content
and the carbohydrate metabolism in yeast. Z Lebensm Unters Forsch
1985;181:8791.
[105] Hinze H, Holzer H. Analysis of the energy metabolism after
incubation of Saccharomyces cerevisiae with sulfite or nitrite. Arch
Microbiol 1986;145:2731.
[106] Sacchetti M, Turtura GC. Alcoholic fermentation shunted to glycerol
production. Ind Sac Ital 1968;61:33641.
[107] Verduyn C, Postma E, Scheffers WA, van Dijken JP. Physiology of
Saccharomyces cerevisiae in anaerobic glucose-limited chemostat
cultures. J Gen Microbiol 1990;136:395403.
[108] Cocking AT, Lilly CH. Improvements in the production of glycerine
by fermentation. Britain Patent No. 164,034 (1919).
[109] Lilly CH. Glycerol production by fermentation. US Patent No.
1,987,260 (1934).
[110] Gardner N, Rodrigue N, Champagne CP. Combined effects of
sulfites, temperature, and agitation time on production of glycerol in
grape juice by Saccharomyces cerevisiae. Appl Environ Microbiol
1993;59:20228.
[111] Lipska I. The preparation of glycerol by the fermentation process.
Roczniki Farmacji 1927;5:804.
[112] Tomoda Y. Production of glycerol by fermentation. VII. The
velocity of fermentation in the presence of sulfite. J Soc Chem Ind
1929;32:22930.
[113] Neuberg C, Nord FF. Application of the fixation method in bacterial
fermentation I. Acetaldehyde as an intermediate product in the
fermentation of sugar, mannitol, and glycerol by Bacillus coli,
dysentery and gas gangrene organisms. Biochem Z 1919;96:13358.
[114] Barbet EA. Glycerol. Britain Patent No. 282,917 (1926).
[115] Duchenne JO. Manufacture of glycerol from sugar by fermentation.
Proc. sixteenth Ann. Congr. S. African Sugar Technol. Assoc.
1942;1:457.
[116] Fulmer EI, Underkofler LA, Hickey RJ. Glycerol production by
fermentation process. US Patent No. 2,416,745 (1947).
[117] Zagrodski S, Jaworowska I, Jaworowski T. Glycerol fermentation
in presence of potassium salts. Przemyst Chem 1952;8:13541.
[118] Kalle GP, Naik SC, Lashkari BZ. Improved glycerol production
from cane molasses by the sulfite process with vacuum or continuous
carbon dioxide sparging during fermentation. J Ferment Technol
(Japan) 1985;63:2317.
[119] Virkar P, Panesar M. Glycerol production by anaerobic vacuum
fermentation of molasses on pilot scale. Biotechnol Bioeng
1987;29:7734.
[120] Petrovska B, Winkelhausen E, Kuzmanova S. Glycerol production
by yeasts under osmotic and sulfite stress. Can J Microbiol
1999;45:6959.
[121] Egorov AS, Visnevskaya GL, Skirstymonskii AI. Glycerol. USSR
Patent No. 104,880 (1957).
[122] Neuberg C, Hirsch J. Alcoholic fermentation in an alkaline medium.
II. Fermentation with living yeast in an alkaline medium. Biochem
Z 1919;96:175203.

M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366


[123] Gaerings-Industri DAS. Glycerol and alcohol by fermentation.
Denmark Patent No. 62,582 (1944).
[124] Hodge HM. Glycerol fermentation. Britain Patent No. 569,683
(1945).
[125] Eroshin VK, Utkin IS, Ladynichev SV, Samoylov VV, Kuvshinnikov
VD, Skryabin GK. Influence of pH and temperature on the substrate
yield coefficient of yeast growth in a chemostat. Biotechnol Bioeng
1976;18:28995.
[126] Buzs Z, Dallmann K, Szajani B. Influence of pH on the growth
and ethanol production of free and immobilized Saccharomyces
cerevisiae cells. Biotechnol Bioeng 1989;34:8824.
[127] Adams AB. The production of glycerol from sugar by fermentation.
Chem Trade J 1919;64:3856.
[128] Eoff JR, Linder WV, Beyer GF. Production of glycerol from sugar
by fermentation. J Ind Eng Chem 1919;11:8425.
[129] Eoff JR. Glycerol from fermentation of sugars. US Patent No.
288,393 (1918).
[130] Costanzo MC, Hogan JD, Cusick ME, Davis BP, Fancher
AM, Hodges PE, et al. The yeast proteome database (YPD)
and Caenorhabditis elegans proteome database (WormPD):
comprehensive resources for the organization and comparison of
model organism protein information. Nucl Acids Res 2000;28:736.
[131] Navarro-Avino JP, Prasad R, Miralles VJ, Benito RM, Serrano
RA. Proposal for nomenclature of aldehyde dehydrogenases
in Saccharomyces cerevisiae and characterization of the
stress-inducible ALD2 and ALD3 genes. Yeast, 11:40718
1999;15:82942.
[132] Meaden PG, Dickinson FM, Mifsud A, Tessier W, Westwater J,
Bussey H, et al. The ALD6 gene of Saccharomyces cerevisiae
encodes a cytosolic, Mg2+ -activated acetaldehyde dehydrogenase.
Yeast, 11:40718 1997;13:131927.
[133] Miralles VJ, Serrano RA. Genomic locus in Saccharomyces
cerevisiae with 4 genes up-regulated by osmotic-stress. Mol
Microbiol 1995;17:65362.
[134] Eoff JR. Glycerol. Britain Patent No. 133,374 (1918).
[135] Vereinigte CWA. Glycerol. Britain Patent No. 138,099 (1920).
[136] Brooks BT. Recovery of glycerol from fermented molasses. US
Patent No. 2,614,964 (1952).
[137] Omori T, Takashita H, Omori N, Shimoda M. High glycerol
production amino acid analogue-resistant Saccharomyces cerevisiae
mutant. J Ferment Bioeng 1995;80:21822.
[138] Attfield PV. Stress tolerance: the key to effective strains of industrial
bakers yeast. Nat Biotechnol 1997;15:13517.
[139] Toone WM, Jones N. Stress-activated signalling pathways in yeast.
Genes Cells 1998;3:48598.
[140] Hohmann S. Shaping up: the response of yeast to osmotic stress.
In: Yeast stress responses New York: Chapman & Hall, 1997.
p. 10145.
[141] Brewster JL, De Valoir T, Dwyer ND, Winter E, Gustin MC.
An osmosensing signal transduction pathway in yeast. Science
1993;259:17603.
[142] Maeda T, Takekawa M, Saito H. Activation of yeast PBS2 MAPKK
by MAPKKKs or by binding of an SH3-containing osmosensor.
Science 1995;269:5548.
[143] Maeda T, Wurgler-Murphy SM, Saito H. A two-component system
that regulates an osmosensing MAP kinase cascade in yeast. Nature
1994;369:2425.
[144] Ferrigno P, Posas F, Koepp D, Saito H, Silver PA. Regulated
nucleo/cytoplasmic exchange of HOG1 MAPK requires the
importing beta homologs NMD5 and XPO1. Embo J 1998;17:5606
14.
[145] Widmann C, Gibson S, Jarpe MB, Johnson GL. Mitogen-activated
protein kinase: conservation of a three-kinase module from yeast
to human. Physiol Rev 1999;79:14380.
[146] Rep M, Krantz M, Thevelein JM, Hohmann S. The transcriptional
response of Saccharomyces cerevisiae to osmotic shock. Hot1p
and Msn2p/Msn4p are required for the induction of subsets of

[147]

[148]

[149]

[150]

[151]
[152]
[153]
[154]
[155]

[156]
[157]
[158]
[159]

[160]

[161]
[162]
[163]

[164]

[165]
[166]

[167]

[168]

[169]

[170]

65

high osmolarity glycerol pathway-dependent genes. J Biol Chem


2000;275:8290300.
Posas F, Chambers JR, Heyman JA, Hoeffler JP, De Nadal E, Arino
J. The transcriptional response of yeast to saline stress. J Biol Chem
2000;275:1724955.
Kajiwara Y, Ogawa K, Takashita H, Omori T. Enhanced glycerol
production in Shochu yeast by heat shock treatment is due to
prolonged transcription of GPD1. J Biosci Bioeng 2000;90:1213.
Siderius M, Van Wuytswinkel O, Reijenga KA, Kelders M,
Mager WH. The control of intracellular glycerol in Saccharomyces
cerevisiae influences osmotic stress response and resistance to
increased temperature. Mol Microbiol 2000;36:138190.
Carvalheiro F, Roseiro J, Grio F. Interactive effects of
sodium chloride and heat shock on trehalose accumulation and
glycerol production by Saccharomyces cerevisiae. Food Microbiol
1999;16:54350.
Owen W, Levey H, Owen W. The production of glycerin by
fermentation. Int Sugar J 1940;42:24850.
Bolcato V. Glycerol fermentation under particular yeasting
conditions. Ann Chim Appl 1948;38:6670.
Leon MC. Obtaining glycerol by fermentation of final molasses.
Anales Asoc Peruana Tecnol Azucareros 1951;2:20514.
Mostafa N. Kinetic studies on glycerol production from molasses
and akalona hydrolyzate. Energy Convers Manage 1997;38:14459.
Mostafa N, Magdy Y. Utilization of molasses and akalona
hydrolyzate for continuous glycerol production in a packed bed
bioreactor. Energy Convers Manage 1998;39:6717.
Figard P. Glycerol fermentation of starch. Iowa State College J Sci
1951;25:20810.
Schwarcz H. Glycerol. US Patent No. 3,158,550 (1964).
Thomsen AM. Glycerol manufacture from lignified cellulose. US
Patent No. 3,003,924 (1954).
Taherzadeh MJ, Eklund R, Gustafsson L, Niklasson C, Lidn G.
Characterization and fermentation of dilute-acid hydrolyzates from
wood. Ind Eng Chem Res 1997;36:465965.
Boussaid A, Robinson J, Cai YJ, Gregg DJ, Saddler
JR. Fermentability of the hemicellulose-derived sugars from
steam-exploded softwood (Douglas fir). Biotechnol Bioeng
1999;64:2849.
Wright JD. Ethanol from biomass by enzymatic hydrolysis. Chem
Eng Prog 1988;84:6274.
Lu Z, Yang C, Tsao G. Fermentation of xylose to glycerol by
Rhizopus javanicus. Appl Biochem Biotechnol 1995;51/52:8395.
Yamada K, Kuwae S, Tani Y. Production of glycerol from methanol
by a mutant strain of Candida boidinii no. 2201. Agric Biol Chem
1989;53:5413.
Yamada K, Yosheida M, Hori T, Tani Y. Glycerol production
from methanol by a respiration deficient mutant strian of a
methylotrophic yeast Candida boidinii no. 2201. Biosci Biotech
Biochem 1993;57:348.
Vijaikishore P, Karanth N. Glycerol production by fermentation: a
fed-batch approach. Biotechnol Bioeng 1987;30:3258.
Li Y, Yin Y, Bi G, Yan X. Glycerol production by reutilization yeast
fermentation. Hubei Daxue Xuebao, Ziran Kexueban 1998;20:187
8.
Hecker D, Bisping B, Rehm HJ. Glycerol production with
immobilized cells of Saccharomyces cerevisiae in semicontinuous
and continuous culture. DECHEMA Biotechnol Conf 1989;3:589
92.
Bisping B, Baumann U, Rehm H. Production of glycerol
by immobilized Pichia farinosa. Appl Microbiol Biotechnol
1990;32:3806.
Button DK, Garver JC, Hajny GJ. Pilot plant glycerol production
with a slow-feed osmophilic yeast fermentation. Appl Microbiol
1966;14:2924.
Vijaikishore P, Karanth N. Glycerol production by fermentation.
Appl Biochem Biotechnol 1984;9:24353.

66

M.J. Taherzadeh et al. / Enzyme and Microbial Technology 31 (2002) 5366

[171] Yamade K, Nakata O, Fukushima S. Continuous glycerol production


with yeast cells, Saccharomyces uvarum, immobilized with agarose.
Hakko Kogaku Kaishi 1989;67:16772.
[172] Hecker D, Bisping B, Rehm H. Continuous glycerol production
by the sulphite process with immobilized cells of Saccharomyces
cerevisiae. Appl Microbiol Biotechnol 1990;32:62732.
[173] Bisping B, Rehm HJ. Production of glycerol by immobilized yeast
cells. Eur Congr Biotechnol 1984;2:12531.
[174] Bisping B, Rehm HJ. Glycerol production by immobilized cells
of Saccharomyces cerevisiae. Eur J Appl Microbiol Biotechnol
1982;14:1369.
[175] Olama Z, Temsah S. Glycerol production by Saccharomyces
cerevisiae under solute stress. Alex Sci Exch 1995;16:41929.
[176] Chapman AC. Microorganisms and some of their industrial relations.
J Roy Soc Arts 1921;69:581619.
[177] Sala JP, de Ayala EB. Production of glycerol by fermentation.
France Patent No. 1,178,479 (1959).
[178] Sala JP, Fuster TG. Preparation of glycerol by fermentation. Spain
Patent No. 213,353 (1954).
[179] Lees T. The fermentative production of glycerol. Iowa State College.
J. Sci. 1944;19.
[180] Onishi H. Glycerol. Japan Patent No. 12,450 (1962).
[181] Lavin O, Holloway JW. Glycerol fermentation. Britain Patent No.
823,740 (1959).
[182] Spencer JFT, Roxburgh JM, Sallans HR. Glycerol and d-arabitol
by fermentation. US Patent No. 2,793,981 (1957).
[183] Iwata Y. Fermentation process for obtaining glycerol from cane
juice. Rep Govt Sugar Exp Sta, Tainan, Formosa 1936;3:
7692.
[184] Gutierrez LE. Glycerol production by strains of Saccharomyces
during alcoholic fermentation. An Esc Super Agric, Luiz de
Queiroz,Univ. Sao Paulo 1991;48:5569.
[185] Nickerson WJ, Carrol WR. On the metabolism of Zygosaccharomyces. Arch Biochem 1945;7:25771.
[186] Vijaikishore P, Karanth NG. Glycerol production from glucose in
alkaline medium. Biotechnol Lett 1984;6:1038.
[187] Onishi H. Simultaneous production of d-arabinol, erythritol, and
glycerol by fermentation. US Patent No. 2,986,495 (1961).

[188] Romano P, Suzzi G, Comi G, Zironi R, Maifreni M. Glycerol


and other fermentation products of apiculate wine yeasts. J Appl
Microbiol 1997;82:6158.
[189] Ligthelm ME, Prior BA, du Preez JC. The oxygen requirement of
yeasts for the fermentation of d-xylose and d-glucose to ethanol.
Appl Microbiol Biotechnol 1988;28:638.
[190] Ravji R, Rodriguez S, Thornton R. Glycerol production by four
common grape molds. Am J Enol Vitic 1988;39:7782.
[191] Wegmann K. Osmotic regulation of photosynthetic glycerol
production in Dunaliella. Biochim Biophys Acta 1971;234:31723.
[192] Borowitzka LJ, Kessly DS, Brown AD. The salt relations of
Dunaliella. Further observations on glycerol production and its
regulation. Arch Microbiol 1977;113:1318.
[193] Lustigman B, McCormick JM, Dale G, McLaughlin JJ. Effect of
increasing copper and salinity on glycerol production by Dunaliella
salina. Bull Environ Contam Toxicol 1987;38:35962.
[194] Thakur A, Kumar H. Effect of different nitrogen source on growth
and glycerol production by Dunaliella salina. Cytobios 1999;97:79
86.
[195] Ahmed AM, Zidan MA. Glycerol production by Dunaliella
bioculata. J Basic Microbiol 1987;27:41925.
[196] Cazzulo J, Arauzo S, de Cazzulo B, Cannata J. On the production
of glycerol and l-alanine during the aerobic fermentation of glucose
by trypanosomatids. FEMS Microbiol Lett 1988;51:18792.
[197] Neish AC, Ledingham GA, Blackwood AC. Glycerol. US Patent
No. 2,432,032 (1947).
[198] Neuberg C, Nord FF. Application of the fixation method in bacterial
fermentation. II. The establishment of an aldehyde stage in acetic
acid fermentation. Biochem Z 1919;96:15875.
[199] Nelson ME, Werkman CH. Dissimilation of glucose by
heterofermentative lactic acid bacteria. J Bacteriol 1935;30:5745.
[200] Kampen W. Process for manufacturing ethanol, glycerol, succinic
acid, lactic acid, betaine, potassium sulfate, l-pyroglutamic acid,
and free flowing distillers dry grain and solubles or a solid fertilizer.
European Patent No. EP0411780 (1991).
[201] Kalle GP, Naik SC. Effect of controlled aeration on glycerol
production in a Sulphite process by Saccharomyces cerevisiae.
Biotechnnol Bioeng 1987;29:11735.