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Zymomonas mobilis: A bacterium for ethanol production
Biotech. Adv. Vol. 4, pp. 95-115, 1986
0734-9750/86 $0.00 + .50 Copyright © Pergamon Journals Ltd
Printed in Great Britain. All Rights Reserved.
ZYMOMONAS MOBILIS: A B A C T E R I U M FOR ETHANOL PRODUCTION JACQUES C. BARATTI* and J. D. BU'LOCKt *Laboratoire de Chimie Bactdrienne du CNRS, 13277 Marseille Cedex 9, France ~'Microbiology Laboratory, Department of Chemistry, University of Manchester, Manchester M13 9PL, UK
l.lntroduction.
Zy1~monas bacterium first the African
mobilis
isolated
palm w i n e ,
is
a
in tropical
facultative countries
anaerobic
from a l c o h o l i c
t h e Mexican e p u l q u e " and a l s o
gram
negative
beverages like
as a contaminant of
cider ("cider sickness") or beer in the European countries. It is one of the few facultative anaerobic bacteria degrading glucose by the Entner-Voudoroff pathway usually
found in
strictly aerobic
microorganisms.
Some work was
devoted to this bacterium in the 50s and 60s and was reviewed by Swings and De Ley in their classical paper published in 1977 (122).
During the 7Os
there was very little work on the bacterium until 1979 and the first report by the Australian group of P.L.
Rogers on the great potentialities of Z.
mobilis for ethanol production.
At that time the petroleum crisis had led
the
developed
ressources. bacterium
countries
to
search
for
alternative
fuel
from
renewable
The Australian group clearly demonstrated the advantages of the compared
to
the
yeasts
traditionally
used
for
alcoholic
fermentation. As a result, there was a considerable "burst" in the Zymomonas literature which
started
from
nearly
95
zero in the late 7Os to attain
70
96
J.C. BARATTIANDI. D. BU'LOCK
papers published
in the field in 1984 (Fig I).
reducing slowly,
By now the "Z)momonia" is
mainly because the easiest things have been done and the
more difficult and challenging work started.
¢n 7 5 I,g Z gJ nr LU
u. 5 0 uJ
LI.
o w 25,.a :E :D z 0
I 1970
1975
1980
1985 YEAR
Number of references on Z.mobilis found in the Chemical
Fig I. Abstract file.
The state of the art was reviewed in 1982 by Rogers (I05) and we have selected,
in this report,
papers published from 1982 to 1986,
Recent
developments by the Australian group (106,119) or by an American group (59) have been already reported.
2 . G e n e r a l b i o c h e m i s t r ~ and m e t a b o l i s m . Wild type strains sources : been
glucose,
done on
of Z.
mobilis can utilize only three carbon
fructose and sucrose. Most of the fermentation work has
glucose but
fermentations
of
sucrose,
fructose
or natural
polymers like cellulose or starch after hydrolysis have been also reported.
2.l.Netabolism preatreated
and
hydrogen-fluoride culture
using
o f sugars o t h e r than g l u c o s e .
hydrolyzed solvolysis
Z.mobilis
for
by
Trichoderma
enzymes
Cellulose is first
(88,107)
or
by
(95) and t h e n f e r m e n t e d t o e t h a n o l by a mixed the
fermentation
of h e x o s e
and Saccharomyces
Z Y M O M O N A S MOBIUS
cerevisiaie (95,79).
or C l o s t r i d i u m
saccharolyticum
A ~-glucosidase,
97
for t h e
fermentation of xylose
coimmobilized with whole cells
of Z.mobilis
allowed the conversion of cellobiose to ethanol (75). Starch can be converted to ethanol by Z.sobilis after hydrolysis to glucose either by industrial enzymes from NOVO (74,104) or by the use of an amylolytic yeast in coculture (41). Fructose
conversion
to
ethanol
is
comparable
to
glucose
(105,125). When the fructose comes from enzymatically hydrolyzed inulin (the polymer found in Jerusalem artichoke),
ethanol production was also observed
(46). The f e r m e n t a t i o n
of
sucrose
poses
several
difficult
problems
m a i n l y due t o t h e f o r m a t i o n o f h i g h amounts o f b y p r o d u c t s ( s e e below) l i k e levan or
sorbitol.
It
may be possible to control the fermentation to
eliminate the formation of levan (at 35oC) or sorbitol (at pH 6.4) In that medium.
Thus
the
fermentation
of sucrose
production of ethanol plus fructose, or ethanol plus sorbitol (38). most
(38,39).
latter case fructose accumulate in great amounts in the culture
abundant
concentrations
industrial which
inhibit
by Z.mobilis
leads
to the
or ethanol plus fructose and sorbitol,
A second difficulty is that molasses,
source
of
sucrose,
the
growth
of
contains
Z.mobilis
high
(128).
the salt
Desalted
molasses sustain much better growth than crude (103) and resistant mutants have been selected which can grow faster than the wild type on molasses (128). However no sucrose process has been yet developed. 2.2. Z.mbilis acetoin
Products
other
than
ethanol.
produces byproducts like glycerol, and b u t a n e d i o l
(2,62).
When grown
succinate,
on
acetate,
glucose lactate,
The amount o f t h e s e b y p r o d u c t s i s much l e s s
in the bacterium than in the yeast
S.bayanus
(2) w i t h a r e s u l t a n t
increase
in the ethanol yield. When s u c r o s e i s Levan f o r m a t i o n recently
the
the substrate
from s u c r o s e reduction
the situation
by Z . m o b i l i s
of
fructose
is to
well
is
quite
different.
known ( 1 0 1 , 1 2 2 ) .
sorbitol
was
More
demonstrated
(10,129,130).
I t o c c v r s d u r i n g t h e f e r m e n t a t i o n o f s u c r o s e , when g l u c o s e and
fructose
both present
are
in
the
culture
medium a s
a result
of a faster
hydrolysis of sucrose than sugar utilization. In these conditions, the first enzyme
of
fructose
metabolism,
fructokinase,
is inhibited
by glucose
98
I C BARATTIANDI D BU'LOCK
reducing the fructose phosphorylation. Thus, fructose is reduced to sorbitol by
a
polyol
dehydrogenase
with
a
concomitant
oxidation
of
glucose
to
gluconic acid (22,78). The synthesis of polyholosides have also been detected.
They are
formed of two fructose units (II) or one mole of glucose and one to four moles of fructose (131).
2.3. Cells components.
The cell membranes o f s t r a i n
shows an unusual lipid composition pentacyclic
triterpenes
(the
(9,127).
ZM1 and ZM4
The major neutral lipids are
hopanoids)
which
are
the
prokaryotic
equivalents of sterols in eukaryotes. The phospholipid composition is almost similar to that of E.coli with phosphatidylethanolamine and
lesser
amounts
of
phosphtidylethanolamine absent
in
E.coli.
phosphatidylglycerol,
cardiolipin
and phosphatidylcholine.
The
fatty
acids
are
as a major component ,
This latter
mainly
dimethyl
compound
cis-vaccenic
acid
is
(70~),
myristic acid and palmitic acid. Ethanol and glucose causes a decrease in phosphatidylethanolamine and
phosphatidylglycerol
contents.
and
an
increase
in
an
phosphatidylcholine
Ethanol causes a reduction in the lipid to protein ratio (26) and
an increase in the cis-vaccenic acid and hopanoid content composition
of
concentrations. interaction
Z.mobilis Lipids
of
may
represent
an
adaptation
from E.coli have been
liposomes
(90).
The
LPS
(21). to
transfered
contains
no
The lipid
high
in
ethanol
Z.mobilis
by
heptose
nor
KDO,
~-hydroxyfattyacids which are usually found in gram negative bacteria (127). The protein purified
cytoplasmic
polyacrylamide affects
gel
pattern of the whole cell membranes and
outer
the protein pattern The n u t r i t i o n a l
that of yeast (82).
membranes
electrophoresis.
(5)
have
Incubation
been
in
(91) or of the characterized
presence
of
on
ethanol
(91). value of Z , m o b i l i s c e l l s
compared f a v o u r a b l y w i t h
The protein content is high (62-68~) with similar DNA
and RNA content and the aminoacids profile is equilibrated (80). The stereospecific,
transport
of
D-glucose
occurs
able to transport fructose and xylose (34). mM,
compared
intrace]lular
to the usual concentrations accumulation.
glucose phosphorylation rate limiting.
by
a
constitutive,
carrier-mediated facilitated diffusion system which is also The Km for glucose is low, 5-15 found in nature and there is no
The maximal velocity is high compared
by glucokinase,
thus glucose
transport
to the
cannot
be
99
Z Y M O M O N A S MOB~IS
2.4.
Growth requirements.
Most of the reported experiments on
ethanol fermentation by Z.mobilis were done in a medium containing 5-]0 g/l of yeast extract,
used
bacterium (122).
to provide the Ca-pantothenate required by the
Synthetic media were first designed for genetic analysis
(55) and further used for fermentation (7,29,52).
Under suitable conditions
these minimum media gave similar fermentation kinetics to the rich medium
(8,50,97,124)
and
therefore
are
of
great
interest
for
industrial
fermentations.
2.5.
Inhibition.
is sensitive to the
The growth and ethanol production of Z.mobilis
presence of chemicals
in
the culture
instance compounds found in wood acid hydrolysate, propionic
acids,
furfural,
of 2-20 g/1 (48). high
substrate
production
Consequently,
For
formic or
phenol are inhibitory in a concentration range
Oxygen shows i n h i b i t o r y e f f e c t s concentration
and
medium.
like acetic,
enhances
(125).
the
Carbon
residual
(20,100) e s p e c i a l l y at
dioxide
glucose
inhibits
biomass
concentration
(96).
cultures with high partial pressure of carbon dioxide show
lower ethanol productivities. The strongest inhibitor is ethanol itself (24,59). analysis describes recently,
the effect
(134),
The kinetic
but very little was known,
until
about the mechanism of this inhibition. It was known that ethanol
inhibits reversibly some enzymes of the Entner-Doudoroff pathway (92) and alcohol dehydrogenase (58),
but the major breakthrough came recently when
the Ingram group showed that the first effect of ethanol is to increase the permeability of the plasma membrane (99). of essential cofactors,
Consequently,
there is a leakage
coenzymes and possibly of intermediary metabolites
from the cells, thus reducing the rate of sugar conversion to ethanol. Evidence were also given recently that ethanol or temperature stress which
induces might
be
the synthesis of specific proteins (heat shock proteins) involved
in
ethanol
tolerance
(89).
An
antibacterial
substance has been detected (117) which resembles bacteriocins.
Protection
against ethanol inhibition has been observed, during the fermentation, after addition of soy flour (63).
2.6.
Enzyles. The p u r i f i c a t i o n and p r o p e r t i e s of s e v e r a l enzymes
from Z . m o b i l i s have been r e p o r t e d .
Most of the work concerns the enzymes of
the
gluco- and f r u c t o - k i n a s e s
F~tner-Voudoroff
glucose-6-phosphate
pathway l i k e dehydrogenase
(3,112),
gluconate
(35,36,112),
kinase
(136),
100
J.C, BARATT1ANDI. D. BU'LOCK
2-keto-3-deoxy-6-phosphogluconate dehydratase
(III),
aldolase
(109),
6-phosphogluconate
6-phosphogluconolactonase
decarboxylase (58).
(II0)
and
pyruvate
An NMR study has shown that the rate limiting steps in
the pathway are the conversion of glucose 6-phosphate to 6-phosphogluconate and of 3-phosphoglycerate
to 2-phosphoglycerate
(12).
In the presence
of
ethanol, inhibition of phosphoglycerate kinase and pyruvate carboxylase were detected (92). Two alcohol dehydrogenases
are present in Z.mobilis (66,58) with
different molecular weights and enzymatic properties.
One of these enzymes
is iron activated (108) and may contain ferrous ions instead of Zinc at the active site.
One of the alcohol dehydrogenase may be coded by a plasmld
(43). The o e c u r e n c e o f a l e v a n s u c r a s e i n Z.mobil_is i s w e l l known and the enzyme has been partially purified (85), presence of a separate invertase
There are some reports on the
(94) but its existence and involvement in
sucrose utilization is not yet clarified.
At high sucrose concentration the
hydrolysis is higher than the sugar uptake. inhibits
sucrose
liberated
hydrolysis
glucose
(16)
and fructose
with
a
In these conditions, resulting
and remaining
assimilation
of non
utilized
glucose of
the
sucrose.
A
glucose isomerase has also been reported (25).
3. H a b i t and m o r p h o l o g y .
New s t r a i n s or
found as
o f Z . m o b i l i s have been i s o l a t e d
a contaminant of
Some o f t h e new s t r a i n s s i m p l e method f o r t h e
the
alcoholic
from s u g a r c a n e
f e r m e n t a t i o n by y e a s t s
from s u g a r cane j u i c e a r e h i g h l y f l o c c u l e n t d e t e r m i n a t i o n o f t h e taxonomic p o s i t i o n
of
(30)
(133). (73). A strains
have been d e s c r i b e d ( 3 3 ) . Although mutants
have
been
not
classified
isolated
in
as
flocculent,
continuous
stable
culture
(47).
flocculating The a t t a c h m e n t
between cells seems to involve the formation of cellulose by the bacterium (119) but the exact mechanism is still not known. The
morphology
culture conditions. 2 to
6 pm
differences
long with from
of
Z.mobilis
is
quite
changing
depending
on
Swings and De Ley (122) described the bacteria as "rods an
unusual
this morphology
glucose concentrations
cell have
width been
of
1
observed.
to
1.4 For
~m". instance
Strong high
lead to large cells wall vesicles or bleb formation
101
ZYMOMONAS MOBIHS
(40, 47)- High carbon d i o x i d e o r e t h a n o l c o n c e n t r a t i o n s cause t h e a p p e a r a n c e o f extensive slime and granular layers around the cell
(37).
Very long
filaments can be formed (until 64 ~m long) under inhibitory conditions (48) or in reactor (32). Cell division occurs by septation (14).
4- K i n e t i c s a s p e c t s The mathematical modelling of growth and ethanol production have been studied using unstructured models (62),
the available electron balance
(44,98) and substrate and product measurement (18,77). All the models allows the calculation of kinetic and yield parameters. The value and importance of maintenance coefficient determination
is
has
proposed
been
pointed out
:
Temperature
and
(15).
a
new
method
ethanol
for
its
concentration
increase the maintenance coefficient (51). Kinetic conditions:
the
and
concentration (72,125), these parameters
yield
effect
of
parameters pH
and
were
measured
temperature
(65),
in
different
of
substrate
of medium composition (124) or of a combination of
(69) as well as £ntracellular ethanol accumulation have
been described (71).
5- Fermentation. Numerous works have been devoted to the conditions of ethanol production by Z.mobiI_is. The data focus on the comparison with yeast (64) or increase in biomass concentration by cell recycle (28,76).
For instance a
two stage reactor is used for the production of 100 g/l of ethanol with a yield of 94~ of theoretical and productivity of 18 g/l.h (28).
However,
cells are recycled by ultrafiltration and long term utilization of such a system poses problems of flow rate. The
most
important
breakthrough
was
obtained
by
the
use of
flocculent strains (49,73,102,113,114,121,126) in continuous reactors. high ethanol productivities were obtained with these systems, ethanol concentration around 50 g/l.
Very
usually at an
High cell concentration in the reactor
is obtained by recycling the flocculent cells with a simple internal or external settler.
Cell concentrations in the range 20-30 g/1 are obtained.
The reactors are continuous stirred tanks (CSTR) (49,73,126) or fluidized
102
J C. BARATT1ANDJ. D. BU'LOCK
beds ( 1 0 2 , 1 1 3 , 1 1 4 , 1 2 1 ) .
In c o n t i n u o u s c u l t u r e
dilution
h -1 c a n be o b t a i n e d w i t h o u t w a s h - o u t r e s u l t i n g o f c o n v e r s i o n , y i e l d and p r o d u c t i v i t y The t o w e r r e a c t o r and g i v e s t h e h i g h e s t
(102,113,114).
industrial scale.
a
settler
seems v e r y s u i t a b l e
However,
as both oxygen
o f g r o w t h and e t h a n o l p r o d u c t i o n ,
a i r n o r t h e g a s from t h e f e r m e n t o r c a n be u s e d f o r a CSTR w i t h
h i g h e r than 1.0
(50-100 g / 1 . h ) .
used for yeast fermentation
productivity
and c a r b o n d i o x i d e a r e i n h i b i t o r y
reason,
rates
in high performances i n term
seems
the
most
fluidizatlon.
promising
neither For t h i s
(126,49)
on an
The limitations of these processes appeared at high sugar
concentrations around 150 g/l where ethanol inhibition strongly affects the performances (8,28,73,113,114). Fed batch fermentation (115) and continuous ethanol extraction by solvent (87) were also tested.
There is only one report (19) on large scale
(50 m 3) ethanol fermentation
with Z.mobilis.
A continuous
culture was
operated for 39 days producing 60 g/l ethanol from glucose or hydrolyzed wheat starch. In these experiments conducted under non aseptic conditions 15 g/l of lactic acid was formed due to the presence of contaminant lactic bacteria.
On the laboratory scale, the susceptibility of Z.mobilis cultures
to contamination by LacZobacillus spp. was found (56) low in batch (addition of penicillin was effective) and very low in continuous cultures where only a transient contamination was observed.
6. Immobilized cells.
Cell immobilization (81,53) is an alternative way to obtain high cell concentration (and then high performances) in the reactors for ethanol production.
The
methods
used
are
K-carrageenan or calcium alginate.
rather
classical
:
entrapment
in
Comparison of immobilized yeasts and
Z.~bilis showed better performances for the bacteria (I) although long term operation was not possible because of catalyst inactivation in both cases (54).
This problem is overcome using immobilized growing cells in alginate
(86) or K-carrageenan (60,83) gels.
The cells are
grown in the gel by
repeated batches until concentrations up to 50 g/l of gel. shows
no
limitations
in
ethanol
production
by
This preparation
diffusional
limitations
(60,68) and can be used in a column reactor in long term operation. 55 days no loss
of activity was detected
Within
and the reactor produced
only
103
Z Y M O M O N A S MOBIUS
ethanol
with
concentration inhibition
no
biomass
than
I00
the
effluent
the
line
(61).
performance
was
At
higher
reduced
by
sugar ethanol
(6,61). Two-stage
single
in g/l,
stage
(67,84).
one
or multi-stage
and
An economic
production
costs
of
lead
to
higher
analysis
ethanol
reactors
is
are
ethanol
(83,84) expected
more e f f i c i e n t
concentrations
shows with
that an
a
than (70-80
reduction
immobilized
cell
the g/l)
in
the
system
compared t o a b a t c h p r o c e s s . C e l l i m m o b i l i z a t i o n by a d s o r p t i o n i s a l s o d e s c r i b e d In
this
type
of
reactor
impaired the reactor
important
cell
growth
occured
(4,
and
17, 7 0 ) .
flocculation
performances (70).
7.,~etic. A great
attention
is
now g i v e n t o
the
genetic
improvement o f
Z.mobi]Xs strains. The main goals are increasing the substrate utilization range
and
ethanol
tolerance.
Several
reviews
have
recently
appeared
(45,118,119). The natural plasmids of strains ZMI and ZM4 have been described in detail by several groups with results not always identical.
Strain ZM4
contained 2 large plasmids (size 70 and 32 kb approx£matly) (116,120). These plasmids were also found in strain ZMI (120) while several authors reported only the presence of the smaller one (42,123). contains at least three plasmids of low size, kb which are not
present
in Z~M
In addition,
strain ZM1
approx£matly 1.5, 1.9 and 2.5
(42,93,116,120,123).
There is a great
homology between these plasmids which are found in most Z°mobilis strains independently of their origin.
This is a strong argument for the existence
of vital functions coded by the plasmid DNA (106,119). The transfer of R-plasmids, like R68.45, in Z.mobilXs is possible but the frequency depends on the recipient strain. confirmed
since
segregation
of
markers
The stability is not yet
is observed
(106).
Conjugation
experiments are difficult with Z.mobilis for two reasons : a) growth is slow on
the mineral
antibacterial
medium used
substances
for selection
which often
(55),
kill the
b)
donor
Z.mobilJs produces strain
inhibitory strains have been isolated after mutagenesis (57), used in conjugation experiments.
(117).
Non
but not yet
104
J c BARATTI AND J D BU'LOCK Plasmids like R68 and RPI have been successfully
conjugation
in
Z.mobilis
(27,31).
The
frequency
of
transfered
tranfer
is
low
by but
expression of antibiotic resistance was observed.
In the latter case,
the
plasmid
The
and
contains
a
lactose
transposon
(Tn951).
~-galactosidase
permease from E.coli were both synthesized in Z.mobilis.
The proteins are
active and their biosynthesis induced by ONPG as in E.coli.
However,
the
resulting Z.mobilis conjugant was not able to grow on lactose (27). Cloning
of
the
glucoamylase
gene
from
&spergillus
niger
was
attempted but stable conjugants were not obtained in Z.mobilis (119). The 70 kb plasmid resistance
to
gentamicin,
of strain ZM4 is carrying the genes for the kanamycin
transmissible by conjugation in E.coli.
and
streptomycin
(132).
It
is
A strain of ZM1 was cured of the 3
kb plasmid and was found to have an impaired ethanol production (43). It may contain the structural gene for one of the two alcohol dehydrogenases known in Z.mobilis.
No other activity has been described as coded by the plasmid
DNA. A method for transformation in Z.mobilis has been described using a cointegrate plasmid between a 13 kb E.coli plasmid and the 15 kb cryptic plasmid of Z.mobilis (23).
However the method does not work with plasmids
like DBR 322 and thus needs to be generalized
before utilization
for gene
tranfer in Z.mobilis. An alternative way is the fusion of sphaeroplast which has been described for genetic recombination in Z.mobilis (135). Mutants
resistants
to
high
ethanol
isolated and used for ethanol production (106).
concentration
have
been
Thermotolerant mutants have
also been described (13).
R~CF~S
1. AMIN G. and H. VERACHTERT, (1982). Comparative study of ethanol production by immobilized cells systems using Zymomonas mobilis or Saccharomyces bayanus. Eur. J. Appl. M£crobiol. Biotechnol. 14,59-63. 2. AMIN G., E. VAN DEN EYNDE and H. VERACHTERT, (1983). Determination of by products formed during the ethanolic fementation using batch and immobilized cell systems of Zymomomms mobilis and Saccharomyces bayanus. Eur. J. Appl. bt£crobiol. Biotechnol. 18,1-5. 3. ANDERSON A.J. and E.A. DAWES, (198S). Regulation of glucose-6-phosphate dehydrogenase in Zymomonas mobil_is CP4. FEMS Microb£ol. Letters, 27723-27.
105
ZYMOMONAS MOBIUS
4. ARCURI E . J . , (1982). Continuous ethanol production and c e l l growth i n an immobilized cell bioreactor employing Zymononas mobilis. Biotechnol. Bioeng. 24,595-604. 5. AZOULAY T., G.P.F. MICHEL and J. STARKA, (1985). Separation of membrane fractions of ZymoBonas mobilis. FEMS Letters, 30,251-255. 6. BAJPAI B.K. and A. MARGARITIS, (1985). Kinetic of ethanol production by immobilized cells of ZymmMmas mohilis at varying D-glucose concentrations. Enzyme Microh. Technol. 7,462-464. 7. BAJPAI P.K. and A. MARGARITIS, (1984). Effect of calcium chloride on the growth and ethanol production by free cells of Zymolonas iobilis. Biotechnol. Letters, 6,673-676. 8. BARATTI J, R. VARMA and J.D. BU'LOCK, (1986). High productivity ethanol fermentation on a mineral medium using a flocculent strain of ZymomonaS mobilis. Biotechnol. Letters 8,175-180. 9. BARROW K.D., J.O. COLLINS, P.L. ROGERS and G.M. SMITH, (1983). Lipid composition of an ethanol tolerant strain of Zym~monas mobius. Biochim. Biophys. Acta, 753,324-330.
10. BARROW K.D., J.G. COLLINS, D.A. LEIGH, P.L. (1984). Sorbitol production by ZFuomonas iobilis. Appl. Microbiol. Biotechnol. 20,225-232.
ROGERS and R.O. WARR,
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F o r m a t i o n o f l e v a n and s o r b i t o l
from s u c r o s e by
Zymomonas mobilis. Appl. Microbiol. Biotechnol. 19,252-255. 130. VIIKARI L. , (1984). Formation of sorbitol by Zymomonas mobilis. Appl. bi£crobiol. Biotechnol. 20,118-123. 131. VIIKARI L. and M. LINKO, (1986). Rate and yield limiting factors in continuous fermentation of sucrose by ZymoQonas mobilis. Biotechnol. Letters, 8,139-144. 132. WALIA S.K., V.C. CAREY, B.P. ALL III and L.O. INGRAM, (1984). Self-transmissible plasmids in Zymomo~s mobilis carrying antibiotic resistance . Appl. Env. Microbiol. 47,198-200. 133. WARR, R.G., A.E. GOODMAN, P.L. ROGERS and M.L. SKOTNICKI, (1984). Isolation and characterization of highly productive strains of Zymomonas mobilis.
Microbios 160,71-78 134. WORDEN R.M., T.L. DONALDSON and S.E. SHUMATE, (1983). inhibition of Zymomonas mobilis : evidence for two inhibitors. Biotechnol. Bioeng. Symp. 13,265-269. 135. YANASE H., M.YASUI, T.MIYAZAKA and K. TONOMURA, (1985). spheroplasts and genetic recombination of Zymomonas mobilis. Agric. Biol. Chem. 49,133-140.
Product
Fusion of
ZYMOMONAS MOBILIS
115
136. ZACHARIOU, M. and R.K. SCOPES, (1985). Gluconate k i n a s e from Zy~omonas m o b i l i s : i s o l a t i o n and c h a r a c t e r i s t i c s . Biochem. I n t . 10,367-371.
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ZYMOMONAS MOBILIS: A B A C T E R I U M FOR ETHANOL PRODUCTION JACQUES C. BARATTI* and J. D. BU'LOCKt *Laboratoire de Chimie Bactdrienne du CNRS, 13277 Marseille Cedex 9, France ~'Microbiology Laboratory, Department of Chemistry, University of Manchester, Manchester M13 9PL, UK
l.lntroduction.
Zy1~monas bacterium first the African
mobilis
isolated
palm w i n e ,
is
a
in tropical
facultative countries
anaerobic
from a l c o h o l i c
t h e Mexican e p u l q u e " and a l s o
gram
negative
beverages like
as a contaminant of
cider ("cider sickness") or beer in the European countries. It is one of the few facultative anaerobic bacteria degrading glucose by the Entner-Voudoroff pathway usually
found in
strictly aerobic
microorganisms.
Some work was
devoted to this bacterium in the 50s and 60s and was reviewed by Swings and De Ley in their classical paper published in 1977 (122).
During the 7Os
there was very little work on the bacterium until 1979 and the first report by the Australian group of P.L.
Rogers on the great potentialities of Z.
mobilis for ethanol production.
At that time the petroleum crisis had led
the
developed
ressources. bacterium
countries
to
search
for
alternative
fuel
from
renewable
The Australian group clearly demonstrated the advantages of the compared
to
the
yeasts
traditionally
used
for
alcoholic
fermentation. As a result, there was a considerable "burst" in the Zymomonas literature which
started
from
nearly
95
zero in the late 7Os to attain
70
96
J.C. BARATTIANDI. D. BU'LOCK
papers published
in the field in 1984 (Fig I).
reducing slowly,
By now the "Z)momonia" is
mainly because the easiest things have been done and the
more difficult and challenging work started.
¢n 7 5 I,g Z gJ nr LU
u. 5 0 uJ
LI.
o w 25,.a :E :D z 0
I 1970
1975
1980
1985 YEAR
Number of references on Z.mobilis found in the Chemical
Fig I. Abstract file.
The state of the art was reviewed in 1982 by Rogers (I05) and we have selected,
in this report,
papers published from 1982 to 1986,
Recent
developments by the Australian group (106,119) or by an American group (59) have been already reported.
2 . G e n e r a l b i o c h e m i s t r ~ and m e t a b o l i s m . Wild type strains sources : been
glucose,
done on
of Z.
mobilis can utilize only three carbon
fructose and sucrose. Most of the fermentation work has
glucose but
fermentations
of
sucrose,
fructose
or natural
polymers like cellulose or starch after hydrolysis have been also reported.
2.l.Netabolism preatreated
and
hydrogen-fluoride culture
using
o f sugars o t h e r than g l u c o s e .
hydrolyzed solvolysis
Z.mobilis
for
by
Trichoderma
enzymes
Cellulose is first
(88,107)
or
by
(95) and t h e n f e r m e n t e d t o e t h a n o l by a mixed the
fermentation
of h e x o s e
and Saccharomyces
Z Y M O M O N A S MOBIUS
cerevisiaie (95,79).
or C l o s t r i d i u m
saccharolyticum
A ~-glucosidase,
97
for t h e
fermentation of xylose
coimmobilized with whole cells
of Z.mobilis
allowed the conversion of cellobiose to ethanol (75). Starch can be converted to ethanol by Z.sobilis after hydrolysis to glucose either by industrial enzymes from NOVO (74,104) or by the use of an amylolytic yeast in coculture (41). Fructose
conversion
to
ethanol
is
comparable
to
glucose
(105,125). When the fructose comes from enzymatically hydrolyzed inulin (the polymer found in Jerusalem artichoke),
ethanol production was also observed
(46). The f e r m e n t a t i o n
of
sucrose
poses
several
difficult
problems
m a i n l y due t o t h e f o r m a t i o n o f h i g h amounts o f b y p r o d u c t s ( s e e below) l i k e levan or
sorbitol.
It
may be possible to control the fermentation to
eliminate the formation of levan (at 35oC) or sorbitol (at pH 6.4) In that medium.
Thus
the
fermentation
of sucrose
production of ethanol plus fructose, or ethanol plus sorbitol (38). most
(38,39).
latter case fructose accumulate in great amounts in the culture
abundant
concentrations
industrial which
inhibit
by Z.mobilis
leads
to the
or ethanol plus fructose and sorbitol,
A second difficulty is that molasses,
source
of
sucrose,
the
growth
of
contains
Z.mobilis
high
(128).
the salt
Desalted
molasses sustain much better growth than crude (103) and resistant mutants have been selected which can grow faster than the wild type on molasses (128). However no sucrose process has been yet developed. 2.2. Z.mbilis acetoin
Products
other
than
ethanol.
produces byproducts like glycerol, and b u t a n e d i o l
(2,62).
When grown
succinate,
on
acetate,
glucose lactate,
The amount o f t h e s e b y p r o d u c t s i s much l e s s
in the bacterium than in the yeast
S.bayanus
(2) w i t h a r e s u l t a n t
increase
in the ethanol yield. When s u c r o s e i s Levan f o r m a t i o n recently
the
the substrate
from s u c r o s e reduction
the situation
by Z . m o b i l i s
of
fructose
is to
well
is
quite
different.
known ( 1 0 1 , 1 2 2 ) .
sorbitol
was
More
demonstrated
(10,129,130).
I t o c c v r s d u r i n g t h e f e r m e n t a t i o n o f s u c r o s e , when g l u c o s e and
fructose
both present
are
in
the
culture
medium a s
a result
of a faster
hydrolysis of sucrose than sugar utilization. In these conditions, the first enzyme
of
fructose
metabolism,
fructokinase,
is inhibited
by glucose
98
I C BARATTIANDI D BU'LOCK
reducing the fructose phosphorylation. Thus, fructose is reduced to sorbitol by
a
polyol
dehydrogenase
with
a
concomitant
oxidation
of
glucose
to
gluconic acid (22,78). The synthesis of polyholosides have also been detected.
They are
formed of two fructose units (II) or one mole of glucose and one to four moles of fructose (131).
2.3. Cells components.
The cell membranes o f s t r a i n
shows an unusual lipid composition pentacyclic
triterpenes
(the
(9,127).
ZM1 and ZM4
The major neutral lipids are
hopanoids)
which
are
the
prokaryotic
equivalents of sterols in eukaryotes. The phospholipid composition is almost similar to that of E.coli with phosphatidylethanolamine and
lesser
amounts
of
phosphtidylethanolamine absent
in
E.coli.
phosphatidylglycerol,
cardiolipin
and phosphatidylcholine.
The
fatty
acids
are
as a major component ,
This latter
mainly
dimethyl
compound
cis-vaccenic
acid
is
(70~),
myristic acid and palmitic acid. Ethanol and glucose causes a decrease in phosphatidylethanolamine and
phosphatidylglycerol
contents.
and
an
increase
in
an
phosphatidylcholine
Ethanol causes a reduction in the lipid to protein ratio (26) and
an increase in the cis-vaccenic acid and hopanoid content composition
of
concentrations. interaction
Z.mobilis Lipids
of
may
represent
an
adaptation
from E.coli have been
liposomes
(90).
The
LPS
(21). to
transfered
contains
no
The lipid
high
in
ethanol
Z.mobilis
by
heptose
nor
KDO,
~-hydroxyfattyacids which are usually found in gram negative bacteria (127). The protein purified
cytoplasmic
polyacrylamide affects
gel
pattern of the whole cell membranes and
outer
the protein pattern The n u t r i t i o n a l
that of yeast (82).
membranes
electrophoresis.
(5)
have
Incubation
been
in
(91) or of the characterized
presence
of
on
ethanol
(91). value of Z , m o b i l i s c e l l s
compared f a v o u r a b l y w i t h
The protein content is high (62-68~) with similar DNA
and RNA content and the aminoacids profile is equilibrated (80). The stereospecific,
transport
of
D-glucose
occurs
able to transport fructose and xylose (34). mM,
compared
intrace]lular
to the usual concentrations accumulation.
glucose phosphorylation rate limiting.
by
a
constitutive,
carrier-mediated facilitated diffusion system which is also The Km for glucose is low, 5-15 found in nature and there is no
The maximal velocity is high compared
by glucokinase,
thus glucose
transport
to the
cannot
be
99
Z Y M O M O N A S MOB~IS
2.4.
Growth requirements.
Most of the reported experiments on
ethanol fermentation by Z.mobilis were done in a medium containing 5-]0 g/l of yeast extract,
used
bacterium (122).
to provide the Ca-pantothenate required by the
Synthetic media were first designed for genetic analysis
(55) and further used for fermentation (7,29,52).
Under suitable conditions
these minimum media gave similar fermentation kinetics to the rich medium
(8,50,97,124)
and
therefore
are
of
great
interest
for
industrial
fermentations.
2.5.
Inhibition.
is sensitive to the
The growth and ethanol production of Z.mobilis
presence of chemicals
in
the culture
instance compounds found in wood acid hydrolysate, propionic
acids,
furfural,
of 2-20 g/1 (48). high
substrate
production
Consequently,
For
formic or
phenol are inhibitory in a concentration range
Oxygen shows i n h i b i t o r y e f f e c t s concentration
and
medium.
like acetic,
enhances
(125).
the
Carbon
residual
(20,100) e s p e c i a l l y at
dioxide
glucose
inhibits
biomass
concentration
(96).
cultures with high partial pressure of carbon dioxide show
lower ethanol productivities. The strongest inhibitor is ethanol itself (24,59). analysis describes recently,
the effect
(134),
The kinetic
but very little was known,
until
about the mechanism of this inhibition. It was known that ethanol
inhibits reversibly some enzymes of the Entner-Doudoroff pathway (92) and alcohol dehydrogenase (58),
but the major breakthrough came recently when
the Ingram group showed that the first effect of ethanol is to increase the permeability of the plasma membrane (99). of essential cofactors,
Consequently,
there is a leakage
coenzymes and possibly of intermediary metabolites
from the cells, thus reducing the rate of sugar conversion to ethanol. Evidence were also given recently that ethanol or temperature stress which
induces might
be
the synthesis of specific proteins (heat shock proteins) involved
in
ethanol
tolerance
(89).
An
antibacterial
substance has been detected (117) which resembles bacteriocins.
Protection
against ethanol inhibition has been observed, during the fermentation, after addition of soy flour (63).
2.6.
Enzyles. The p u r i f i c a t i o n and p r o p e r t i e s of s e v e r a l enzymes
from Z . m o b i l i s have been r e p o r t e d .
Most of the work concerns the enzymes of
the
gluco- and f r u c t o - k i n a s e s
F~tner-Voudoroff
glucose-6-phosphate
pathway l i k e dehydrogenase
(3,112),
gluconate
(35,36,112),
kinase
(136),
100
J.C, BARATT1ANDI. D. BU'LOCK
2-keto-3-deoxy-6-phosphogluconate dehydratase
(III),
aldolase
(109),
6-phosphogluconate
6-phosphogluconolactonase
decarboxylase (58).
(II0)
and
pyruvate
An NMR study has shown that the rate limiting steps in
the pathway are the conversion of glucose 6-phosphate to 6-phosphogluconate and of 3-phosphoglycerate
to 2-phosphoglycerate
(12).
In the presence
of
ethanol, inhibition of phosphoglycerate kinase and pyruvate carboxylase were detected (92). Two alcohol dehydrogenases
are present in Z.mobilis (66,58) with
different molecular weights and enzymatic properties.
One of these enzymes
is iron activated (108) and may contain ferrous ions instead of Zinc at the active site.
One of the alcohol dehydrogenase may be coded by a plasmld
(43). The o e c u r e n c e o f a l e v a n s u c r a s e i n Z.mobil_is i s w e l l known and the enzyme has been partially purified (85), presence of a separate invertase
There are some reports on the
(94) but its existence and involvement in
sucrose utilization is not yet clarified.
At high sucrose concentration the
hydrolysis is higher than the sugar uptake. inhibits
sucrose
liberated
hydrolysis
glucose
(16)
and fructose
with
a
In these conditions, resulting
and remaining
assimilation
of non
utilized
glucose of
the
sucrose.
A
glucose isomerase has also been reported (25).
3. H a b i t and m o r p h o l o g y .
New s t r a i n s or
found as
o f Z . m o b i l i s have been i s o l a t e d
a contaminant of
Some o f t h e new s t r a i n s s i m p l e method f o r t h e
the
alcoholic
from s u g a r c a n e
f e r m e n t a t i o n by y e a s t s
from s u g a r cane j u i c e a r e h i g h l y f l o c c u l e n t d e t e r m i n a t i o n o f t h e taxonomic p o s i t i o n
of
(30)
(133). (73). A strains
have been d e s c r i b e d ( 3 3 ) . Although mutants
have
been
not
classified
isolated
in
as
flocculent,
continuous
stable
culture
(47).
flocculating The a t t a c h m e n t
between cells seems to involve the formation of cellulose by the bacterium (119) but the exact mechanism is still not known. The
morphology
culture conditions. 2 to
6 pm
differences
long with from
of
Z.mobilis
is
quite
changing
depending
on
Swings and De Ley (122) described the bacteria as "rods an
unusual
this morphology
glucose concentrations
cell have
width been
of
1
observed.
to
1.4 For
~m". instance
Strong high
lead to large cells wall vesicles or bleb formation
101
ZYMOMONAS MOBIHS
(40, 47)- High carbon d i o x i d e o r e t h a n o l c o n c e n t r a t i o n s cause t h e a p p e a r a n c e o f extensive slime and granular layers around the cell
(37).
Very long
filaments can be formed (until 64 ~m long) under inhibitory conditions (48) or in reactor (32). Cell division occurs by septation (14).
4- K i n e t i c s a s p e c t s The mathematical modelling of growth and ethanol production have been studied using unstructured models (62),
the available electron balance
(44,98) and substrate and product measurement (18,77). All the models allows the calculation of kinetic and yield parameters. The value and importance of maintenance coefficient determination
is
has
proposed
been
pointed out
:
Temperature
and
(15).
a
new
method
ethanol
for
its
concentration
increase the maintenance coefficient (51). Kinetic conditions:
the
and
concentration (72,125), these parameters
yield
effect
of
parameters pH
and
were
measured
temperature
(65),
in
different
of
substrate
of medium composition (124) or of a combination of
(69) as well as £ntracellular ethanol accumulation have
been described (71).
5- Fermentation. Numerous works have been devoted to the conditions of ethanol production by Z.mobiI_is. The data focus on the comparison with yeast (64) or increase in biomass concentration by cell recycle (28,76).
For instance a
two stage reactor is used for the production of 100 g/l of ethanol with a yield of 94~ of theoretical and productivity of 18 g/l.h (28).
However,
cells are recycled by ultrafiltration and long term utilization of such a system poses problems of flow rate. The
most
important
breakthrough
was
obtained
by
the
use of
flocculent strains (49,73,102,113,114,121,126) in continuous reactors. high ethanol productivities were obtained with these systems, ethanol concentration around 50 g/l.
Very
usually at an
High cell concentration in the reactor
is obtained by recycling the flocculent cells with a simple internal or external settler.
Cell concentrations in the range 20-30 g/1 are obtained.
The reactors are continuous stirred tanks (CSTR) (49,73,126) or fluidized
102
J C. BARATT1ANDJ. D. BU'LOCK
beds ( 1 0 2 , 1 1 3 , 1 1 4 , 1 2 1 ) .
In c o n t i n u o u s c u l t u r e
dilution
h -1 c a n be o b t a i n e d w i t h o u t w a s h - o u t r e s u l t i n g o f c o n v e r s i o n , y i e l d and p r o d u c t i v i t y The t o w e r r e a c t o r and g i v e s t h e h i g h e s t
(102,113,114).
industrial scale.
a
settler
seems v e r y s u i t a b l e
However,
as both oxygen
o f g r o w t h and e t h a n o l p r o d u c t i o n ,
a i r n o r t h e g a s from t h e f e r m e n t o r c a n be u s e d f o r a CSTR w i t h
h i g h e r than 1.0
(50-100 g / 1 . h ) .
used for yeast fermentation
productivity
and c a r b o n d i o x i d e a r e i n h i b i t o r y
reason,
rates
in high performances i n term
seems
the
most
fluidizatlon.
promising
neither For t h i s
(126,49)
on an
The limitations of these processes appeared at high sugar
concentrations around 150 g/l where ethanol inhibition strongly affects the performances (8,28,73,113,114). Fed batch fermentation (115) and continuous ethanol extraction by solvent (87) were also tested.
There is only one report (19) on large scale
(50 m 3) ethanol fermentation
with Z.mobilis.
A continuous
culture was
operated for 39 days producing 60 g/l ethanol from glucose or hydrolyzed wheat starch. In these experiments conducted under non aseptic conditions 15 g/l of lactic acid was formed due to the presence of contaminant lactic bacteria.
On the laboratory scale, the susceptibility of Z.mobilis cultures
to contamination by LacZobacillus spp. was found (56) low in batch (addition of penicillin was effective) and very low in continuous cultures where only a transient contamination was observed.
6. Immobilized cells.
Cell immobilization (81,53) is an alternative way to obtain high cell concentration (and then high performances) in the reactors for ethanol production.
The
methods
used
are
K-carrageenan or calcium alginate.
rather
classical
:
entrapment
in
Comparison of immobilized yeasts and
Z.~bilis showed better performances for the bacteria (I) although long term operation was not possible because of catalyst inactivation in both cases (54).
This problem is overcome using immobilized growing cells in alginate
(86) or K-carrageenan (60,83) gels.
The cells are
grown in the gel by
repeated batches until concentrations up to 50 g/l of gel. shows
no
limitations
in
ethanol
production
by
This preparation
diffusional
limitations
(60,68) and can be used in a column reactor in long term operation. 55 days no loss
of activity was detected
Within
and the reactor produced
only
103
Z Y M O M O N A S MOBIUS
ethanol
with
concentration inhibition
no
biomass
than
I00
the
effluent
the
line
(61).
performance
was
At
higher
reduced
by
sugar ethanol
(6,61). Two-stage
single
in g/l,
stage
(67,84).
one
or multi-stage
and
An economic
production
costs
of
lead
to
higher
analysis
ethanol
reactors
is
are
ethanol
(83,84) expected
more e f f i c i e n t
concentrations
shows with
that an
a
than (70-80
reduction
immobilized
cell
the g/l)
in
the
system
compared t o a b a t c h p r o c e s s . C e l l i m m o b i l i z a t i o n by a d s o r p t i o n i s a l s o d e s c r i b e d In
this
type
of
reactor
impaired the reactor
important
cell
growth
occured
(4,
and
17, 7 0 ) .
flocculation
performances (70).
7.,~etic. A great
attention
is
now g i v e n t o
the
genetic
improvement o f
Z.mobi]Xs strains. The main goals are increasing the substrate utilization range
and
ethanol
tolerance.
Several
reviews
have
recently
appeared
(45,118,119). The natural plasmids of strains ZMI and ZM4 have been described in detail by several groups with results not always identical.
Strain ZM4
contained 2 large plasmids (size 70 and 32 kb approx£matly) (116,120). These plasmids were also found in strain ZMI (120) while several authors reported only the presence of the smaller one (42,123). contains at least three plasmids of low size, kb which are not
present
in Z~M
In addition,
strain ZM1
approx£matly 1.5, 1.9 and 2.5
(42,93,116,120,123).
There is a great
homology between these plasmids which are found in most Z°mobilis strains independently of their origin.
This is a strong argument for the existence
of vital functions coded by the plasmid DNA (106,119). The transfer of R-plasmids, like R68.45, in Z.mobilXs is possible but the frequency depends on the recipient strain. confirmed
since
segregation
of
markers
The stability is not yet
is observed
(106).
Conjugation
experiments are difficult with Z.mobilis for two reasons : a) growth is slow on
the mineral
antibacterial
medium used
substances
for selection
which often
(55),
kill the
b)
donor
Z.mobilJs produces strain
inhibitory strains have been isolated after mutagenesis (57), used in conjugation experiments.
(117).
Non
but not yet
104
J c BARATTI AND J D BU'LOCK Plasmids like R68 and RPI have been successfully
conjugation
in
Z.mobilis
(27,31).
The
frequency
of
transfered
tranfer
is
low
by but
expression of antibiotic resistance was observed.
In the latter case,
the
plasmid
The
and
contains
a
lactose
transposon
(Tn951).
~-galactosidase
permease from E.coli were both synthesized in Z.mobilis.
The proteins are
active and their biosynthesis induced by ONPG as in E.coli.
However,
the
resulting Z.mobilis conjugant was not able to grow on lactose (27). Cloning
of
the
glucoamylase
gene
from
&spergillus
niger
was
attempted but stable conjugants were not obtained in Z.mobilis (119). The 70 kb plasmid resistance
to
gentamicin,
of strain ZM4 is carrying the genes for the kanamycin
transmissible by conjugation in E.coli.
and
streptomycin
(132).
It
is
A strain of ZM1 was cured of the 3
kb plasmid and was found to have an impaired ethanol production (43). It may contain the structural gene for one of the two alcohol dehydrogenases known in Z.mobilis.
No other activity has been described as coded by the plasmid
DNA. A method for transformation in Z.mobilis has been described using a cointegrate plasmid between a 13 kb E.coli plasmid and the 15 kb cryptic plasmid of Z.mobilis (23).
However the method does not work with plasmids
like DBR 322 and thus needs to be generalized
before utilization
for gene
tranfer in Z.mobilis. An alternative way is the fusion of sphaeroplast which has been described for genetic recombination in Z.mobilis (135). Mutants
resistants
to
high
ethanol
isolated and used for ethanol production (106).
concentration
have
been
Thermotolerant mutants have
also been described (13).
R~CF~S
1. AMIN G. and H. VERACHTERT, (1982). Comparative study of ethanol production by immobilized cells systems using Zymomonas mobilis or Saccharomyces bayanus. Eur. J. Appl. M£crobiol. Biotechnol. 14,59-63. 2. AMIN G., E. VAN DEN EYNDE and H. VERACHTERT, (1983). Determination of by products formed during the ethanolic fementation using batch and immobilized cell systems of Zymomomms mobilis and Saccharomyces bayanus. Eur. J. Appl. bt£crobiol. Biotechnol. 18,1-5. 3. ANDERSON A.J. and E.A. DAWES, (198S). Regulation of glucose-6-phosphate dehydrogenase in Zymomonas mobil_is CP4. FEMS Microb£ol. Letters, 27723-27.
105
ZYMOMONAS MOBIUS
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