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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,

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

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

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E-mail address: (G. Liden).

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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
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


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

3. The metabolic basis of glycerol production in yeast

Table 1
Some microorganisms in which glycerol production has been studied




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



Debaryomyces mogii
Rhizopus nigricans
Rhizopus javanicus
Botrytis cinerea
Aspergillus niger






Dunaliella bardawil
Trypanosoma cruzi
Leishmania mexic



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



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


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:

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
6-Phospho-gluconate dehydrogenase


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)
Isocitrate dehydrogenase (IDH, IDP1, IDP2)
2-Oxoglutarate dehydrogenase (OGDH)
Succinyl-CoA synthetase
Succinate dehydrogenase
Fumarate reductase (cytosolic, FRDS)
Malate dehydrogenase
Glutamate dehydrogenase (GDH)
Glutamine synthetase (GS)
Phosphoglucomutase (PGM)
Phosphomannoisomerase (PMI)


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


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.


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



Triose phosphate isomerase (EC



Phosphoglycerate mutase (EC

Pyruvate decarboxylase (EC
Alcohol dehydrogenase (EC
Glycerol-3-P dehydrogenase (EC
Mitochondrial NADH-dehydrogenase



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 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]. 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


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. 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. 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
4.1.2. Targets in the glycerol formation pathway 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 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


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
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.


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
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

Whey permeate
Enzyme-hydrolyzed lactose

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




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


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.
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