Enzyme and Microbial Technology 45 (2009) 210–217

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Enzyme and Microbial Technology

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A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient
ethanol production on glucose/xylose mixtures
Nan Fu, Paul Peiris ∗ , Julie Markham, John Bavor
School of Natural Sciences, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia

a r t i c l e i n f o a b s t r a c t

Article history: An efficient conversion of glucose and xylose is a requisite for a profitable process of bioethanol produc-
Received 9 February 2009 tion from lignocellulose. Considering the approaches available for this conversion, co-culture is a simple
Received in revised form 8 April 2009 process, employing two different organisms for the fermentation of the two sugars. An innovative fer-
Accepted 24 April 2009
mentation scheme was designed, co-culturing immobilized Zymomonas mobilis and free cells of Pichia
stipitis in a modified fermentor for the glucose and xylose fermentation, respectively. A sugar mixture of
Keywords:
30 g/l glucose and 20 g/l of xylose was completely converted to ethanol within 19 h. This gave a volumetric
Ethanol
ethanol productivity of 1.277 g/l/h and an ethanol yield of 0.49–0.50 g/g, which is more than 96% of the
Co-fermentation
Lignocellulose
theoretical value. Extension of this fermentation scheme to sugarcane bagasse hydrolysate resulted in a
Co-culture complete sugar utilisation within 26 h; ethanol production peaked at 40 h with a yield of 0.49 g/g. These
Zymomonas mobilis values are comparable to the best results reported. Cell interaction was observed between Z. mobilis and
Pichia stipitis P. stipitis. Viable cells of Z. mobilis inhibited the cell activity of P. stipitis and the xylose fermentation. Z.
mobilis showed evidence of utilising a source other than glucose for growth when co-cultured with P.
stipitis.
© 2009 Elsevier Inc. All rights reserved.

1. Introduction fermenting strain, the fermentation of xylose was often slow

The bioethanol industry has developed rapidly in recent years to
cope with the depletion of fossil fuel. The utilisation of edible starch
material, such as corn, for bioethanol production has caused undue
pressure on the global food supply [1,2]. Materials unsuitable for
human consumption are considered ideal substrates for bioethanol
production, e.g. lignocellulose. A complete and efficient conversion
of these hexose and pentose sugars present in the lignocellulosic
hydrolysates to ethanol is a prerequisite for maximizing the prof-
itability of an industrial process for bioethanol production [3,4].
Since there is no wild type microorganism that could efficiently
accomplish this process, the utilisation of two microorganisms and
the construction of genetically modified biocatalysts have been two
common approaches.
Glucose and xylose are two dominating sugars in the lignocellu-
losic hydrolysates. The main difficulty of using two microorganisms
for the co-fermentation of these two sugars is the inability to
provide optimal environmental conditions for the two strains
simultaneously [5]. A majority of previous studies on strain co-
cultures reported that while the fermentation of glucose in the
sugar mixture proceeded efficiently with a traditional glucose-
∗ Corresponding author. Tel.: +61 2 4570 1304; fax: +61 2 4570 1267.
E-mail address: p.peiris@uws.edu.au (P. Peiris).
and of low efficiency due to the conflicting oxygen requirements
between the two strains and/or the catabolite repression on the
xylose assimilation caused by the glucose [6–9]. Approaches in
both process engineering and strain engineering have been car-
ried out to circumvent these difficulties and to improve the system
efficiency. Examples of process engineering include continuous
culture [8,10,11], the immobilization of one of the strains [8], co-
immobilization of two strains [7,8,12], two stage fermentation in
one bioreactor (i.e. sequential culture) [13], and separate fermen-
tation in two bioreactors [14,15]. For the utilisation of engineered
strains, there have been reports of respiratory deficient mutants
of Saccharomyces cerevisiae [6,9,16], respiratory deficient mutants
of Saccharomyces diastaticus [10,11,17], a mutants of Pichia stipitis
showing restricted glucose catabolite repression [6] and a geneti-
cally modified ethanologenic Escherichia coli strain [18].
Some reports in the literature have shown promising results.
These include the co-culture of a respiratory deficient mutant of
S. cerevisiae with P. stipitis [16], a co-immobilization system of S.
cerevisiae and P. stipitis [12], and a co-culture of S. cerevisiae with a
recombinant E. coli strain [18]. It can be seen that the yeast genus
Saccharomyces is preferably used as the glucose-fermenting strain
in the co-culture with another xylose-fermenting strain. Though
the bacterium Zymomonas mobilis is known for better ethanol pro-
ductivity and tolerance compared to S. cerevisiae [19,20], it has
rarely been employed in such a co-culture process. A sequential
culture of Z. mobilis and Pachysolen tannophilus has been previously

0141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2009.04.006
 N. Fu et al. / Enzyme and Microbial Technology 45 (2009) 210–217
reported [13]. The present study reports for the first time the new
strain combination of Z. mobilis and P. stipitis, employing four dif-
ferent fermentation schemes including one with a novel modified
fermentor. The fermentation efficiencies of these schemes and the
strain interactions in the co-culture are discussed.
In this article “co-fermentation” is used to describe a process
that converts two sugars to ethanol in a single process, no mat-
ter how this process is achieved; “co-culture” is used when two
microorganisms are cultured together and simultaneously exist in
the medium, whereas “sequential culture” is used for a process
where two strains are employed successively.
2. Materials and methods
2.1. Microorganisms
Z. mobilis ATCC 10988 was obtained from the Microbiology Culture Collection
of the School of Natural Sciences, University of Western Sydney. P. stipitis CBS
5773 was kindly provided by the Westmead Clinical School, University of Sydney.
Cultures were maintained on agar plates at 4 ◦ C with subculture to fresh media
every 2 weeks. Glucose agar for Z. mobilis consisted of 20 g/l glucose, 10 g/l yeast
extract, 2 g/l KH2 PO4 and 15 g/l agar, and the composition of xylose agar for P. stipi-
tis was as previously described [13]. Incubation of both strains was at 30 ◦ C for
48 h.
2.2. Synthetic media
Inoculum medium consisted of 10 g/l yeast extract, 1 g/l MgCl2 , 1 g/l (NH4 )2 SO4 ,
1 g/l KH2 PO4 , with 50 g/l glucose for Z. mobilis and 50 g/l xylose for P. stipitis. Unless
otherwise stated, fermentation medium contained the same components as the
inoculum medium, with 30 g/l glucose and 20 g/l xylose as carbon source. Media
were sterilized at 121 ◦ C for 15 min and sugars were separately autoclaved from
yeast extract and inorganic salts (YEIS) solution.
2.3. Inoculum preparation
Inoculum size was expressed as vcells /vferm , i.e. volume of inoculum medium that
cells were from to volume of fermentation medium that cells were inoculated into.
Cells from the corresponding volume of inoculum medium were firstly centrifuged
to exclude the inoculum medium and concentrate the cells, and then the cell pel-
lets were re-suspended with the sterilized YEIS solution to be inoculated into the
bioreactor.
All inocula were incubated in 250 ml conical flasks with 50 ml of inoculum
medium at 30 ◦ C, with a 24-h stationary incubation for Z. mobilis and a 36–48 h
shaking incubation at 150 rpm for P. stipitis. Multiple flasks were simultaneously
cultured to get the desirable volume of inoculum medium and subculture up to
three times was carried out to ensure that all inoculum flasks contained an identical
culture developed from the same initial colony.
2.4. Immobilization
Z. mobilis was immobilized following a procedure of calcium alginate gel encap-
sulation as described by Becerra et al. [21]. Briefly, cells from 400 ml of inoculum
medium, which had been incubated as described above, were firstly centrifuged to
obtain the cell pellets. The pellets were re-suspended with 50 ml of 0.9% (w/v) saline
solution, and mixed with 50 ml of 4% (w/v) sodium alginate solution. This mixture
was extruded through a 10-ml syringe into 50 mM CaCl2 solution with mild agi-
tation. After reticulation for 30 min, beads were collected and washed with saline
and distilled water. The resultant beads were approximately 2–3 mm in diameter
and 100 ml in volume, containing cells from 400 ml Z. mobilis culture. When not in
use, beads were stored at 4 ◦ C in glucose storage medium comprising 10 g/l glucose,
2.5 g/l yeast extract and 5.55 g/l CaCl2 . Beads from the storage condition were cul-
tured in a small volume of glucose inoculum medium at 30 ◦ C for 4–6 h to restore
the biomass level prior to fermentation.
2.5. Inoculation and fermentation conditions
All fermentations were carried out with 800 ml fermentation medium in a 1-l
fermentor (Braun Biostat B) with pH monitoring. Unless otherwise stated, the air-
flow level was 80 cm3 /min and the inoculum sizes for both strains were 50% (cells
from 400 ml of inoculum medium after proper incubation). In the fermentations
involving immobilized Z. mobilis, a batch of freshly made beads also contained cells
from 400 ml inoculum, equivalent to the 50% inoculum size. Decreased inoculum
size of immobilized Z. mobilis to 1/2 or 1/4 batch referred to the use of 1/2 or 1/4
beads out of a full batch (w/w).
In all fermentations the temperature was 30 ◦ C and the stirring speed was
150 rpm. The change of medium pH was used to estimate the fermentation status.
211
During the glucose fermentation stage, the pH rapidly decreased due to the associ-
ated CO2 production, and the slowing of the pH change indicated the completion of
glucose fermentation and the beginning of xylose fermentation.
In sequential culture, free cells of Z. mobilis were firstly inoculated and inacti-
vated by autoclaving at 115 ◦ C for 5 min after the completion of glucose fermentation;
free cells of P. stipitis were then inoculated to ferment the residual xylose to ethanol.
In free cell co-culture, free cells of both strains were inoculated simultaneously to
commence the fermentation. In co-culture involving immobilized Z. mobilis, the
immobilized Z. mobilis beads and free cells of P. stipitis were simultaneously inocu-
lated. After the glucose fermentation was completed, the immobilized beads were
left in the fermentation medium till the end of the whole process. Finally, an inno-
vative fermentation scheme was developed to co-culture the immobilized Z. mobilis
and free cells of P. stipitis, and remove the immobilized beads from the fermenta-
tion medium after the completion of glucose fermentation. Fig. 1 shows a schematic
demonstration of how this process was achieved by the addition of a sieve plate
and a movable device to a normal fermentor. The added sieve plate prevented the
immobilized Z. mobilis beads from passing through but allowed P. stipitis cells to pass
through. After the glucose fermentation was completed, the sieve plate was moved
upwards, removing Z. mobilis cells entrapped inside the beads from the fermentation
medium, and the xylose fermentation could proceed undisturbed at the meantime.
Shake flask experiments were carried out in a 30-◦ C orbital shaker at 100 rpm
with 100 ml fermentation medium in 250 ml capacity conical flasks.
2.6. Hydrolysis of sugarcane bagasse
Sugarcane bagasse was treated with 2% (v/v) dilute sulphuric acid at a
solid:liquid ratio of 1:6 as described by Takahashi et al. [22]. After discarding the
liquid phase, the residual bagasse material was mixed with 0.04 M acetate buffer
(pH 5.0) in the same ratio. Enzymatic hydrolysis was carried out with Celluclast 1.5 L
and Novozyme 188 at a loading of 2% (v/w) in a 60-◦ C shaker with a shaking speed
of 200 rpm for 24 h. Both enzymes were generously provided by Novozymes Aus-
tralia Pty. Ltd. The liquid phase was recovered by manually squeezing the bagasse
mixture and filter sterilizing with a 0.45-␮m filter. The hydrolysate obtained con-
tained approximately 9 g/l glucose and 4 g/l xylose. For the fermentations, 600 ml of
hydrolysate was supplemented with 100 ml of concentrated sugar mixture (200 g/l
glucose and 120 g/l xylose) and 100 ml of concentrated YEIS solution (80 g/l yeast
extract, 8 g/l MgCl2 , 8 g/l (NH4 )2 SO4 , 8 g/l KH2 PO4 ), made up to 800 ml fermentation
medium. Cells of inoculum were firstly cultured as described in the section of inocu-
lum preparation, and then subcultured to the hydrolysate medium and incubated
under inoculum conditions as adaptation.
2.7. Analytical methods
Total reducing sugars were estimated by the DNS method [23] with xylose stan-
dards. Glucose concentration in the sugar mixture was determined by a modified
Glucose Oxidase Method [24]. A PerkinElmer HPLC system equipped with a Bio-
rad Aminex HPX-87P column and a RI detector was used to determine the glucose
and xylose concentrations in the bagasse hydrolysate; the mobile phase was filtered
milli-Q water degassed with Helium at 0.6 ml/min and the column temperature was
85 ◦ C.
Viable cells were determined by plate count. Colonies of P. stipitis and Z. mobilis in
the co-culture could be distinguished from each other on the glucose agar plates after
48 h incubation. In a co-culture process with immobilized Z. mobilis and free cells
of P. stipitis, the free cell concentration of Z. mobilis in the medium was sometimes
too low to be detected using this technique, due to the high concentration of P.
stipitis cells. The concentration was thus expressed as 1 × 105 cfu/ml, referred to as
“undetectable on 104 dilution plate”.
Ethanol concentration was measured by a GC system equipped with a flame
ionization detector and a capillary column, which was approximately 125 cm in
length by 0.2 mm in diameter. The injector temperature was 190 ◦ C and the oven
temperature was 40 ◦ C, with the Hydrogen as carrier gas and 1-propanol as internal
standard.
3. Results
3.1. Ethanol production using four different fermentation schemes
Results of co-fermentations with the four fermentation schemes
on 50 g/l of sugar mixture are shown in Table 1. The sequential
culture of the two microorganisms achieved a high fermentation
performance. Both the glucose fermentation by Z. mobilis and the
xylose fermentation by P. stipitis proceeded efficiently, giving an
overall ethanol yield of 0.47 g/g and a volumetric ethanol produc-
tivity of 0.83 g/l/h. In contrast, co-culture of free cells of the two
strains resulted in a poor fermentation performance. In all four pro-
cesses, the xylose in the medium was not fully utilised. Neither the
adjustment in aeration level nor the increase in the inoculum size of
212 N. Fu et al. / Enzyme and Microbial Technology 45 (2009) 210–217

Fig. 1. Schematic demonstration of the modified fermentor. (a) A normal fermentor; (b) addition of a sieve plate and a moving device; (c) inoculation of immobilized Z. mobilis
and free cells of P. stipitis; and (d) removal of the immobilized Z. mobilis beads from the fermentation medium without disturbing the on-going xylose fermentation.

P. stipitis significantly improved the fermentation efficiency. When
immobilized Z. mobilis beads were used to replace the free cells of Z.
mobilis in the co-culture, the efficiency of the xylose fermentation
was significantly improved. All the sugars were completely utilised
in 24 h with an ethanol yield of 0.44 g/g and a volumetric productiv-
ity of 0.868 g/l/h. However, the repeat culture with the same batch
of Z. mobilis beads under the same conditions led to a decreased
performance. The immobilized Z. mobilis beads showed increasing
bead damage during the repeat culture. Associated with this obser-
vation, the efficiency of xylose fermentation continually decreased
in the second and the third culture, resulting in an increased fer-
mentation time.
The modified fermentor and the removal of the immobilized
Z. mobilis during a co-fermentation process were photographed in
Fig. 2. As this fermentation scheme enabled the removal of the
immobilized Z. mobilis from the medium and consequently allevi-
ated the bead damage, the fermentation performance in the repeat
culture was greatly stabilized. Inoculum level 1 using a full batch
of Z. mobilis beads and 50% inoculum size of P. stipitis showed
similar results to the first culture in the unmodified fermentor,
because of the similar inoculum sizes and environmental condi-
tions used. A reduced inoculum size of immobilized Z. mobilis
(from 1 batch to 1/4 batch) and an increased inoculum size of P.
stipitis (from 50% to 100%) were both able to shorten the fermen-

Table 1
Results of co-fermentations of glucose/xylose sugar mixture by Z. mobilis and P. stipitis, using four fermentation schemes and with increased initial sugar level.

Fermentation conditions Sequential Co-culture

s0 = 75 g/l
culture
Free cells Immobilized Z. mobilis Modified fermentor

Air flow (cm3 /min) P. stipitisa Repeated culture Inoculum levelb Z. mobilis level

50 80 100 100% First Second Third 1 2 3 1 batch 1/2 batch

Kinetic parameters tp (h) 28.5c 47 35 46 44 24 29 37 25 22 19 45 42

Sugar used (%) 100 87 86 91 91 100 100 91 100 100 100 100 100
Yp/s (g/g) 0.47 0.38 0.43 0.40 0.42 0.44 0.42 0.43 0.45 0.50 0.49 0.42 0.46
Qp (g/l/h) 0.830 0.349 0.518 0.385 0.424 0.868 0.687 0.501 0.867 1.126 1.277 0.679 0.878

tp : ethanol peak time (h); Yp/s : ethanol yield (g/g); Qp : volumetric ethanol productivity (g/l/h); s0 : initial sugar concentration.
a
Inoculum size of P. stipitis increased to 100%, with air flow level of 80 cm3 /min.
b
Level 1 employed a full batch of immobilized Z. mobilis beads with 50% inoculum size of P. stipitis; level 2 employed 1/4 batch of immobilized Z. mobilis beads with 50%
inoculum size of P. stipitis; level 3 employed 1/4 batch of immobilized Z. mobilis beads with 100% inoculum size of P. stipitis. Results of each level were the average of duplicate
fermentations.
c
2.5 h for glucose fermentation and 26 h for xylose fermentation, with 12 h time interval to allow the fermentor cooling down from the autoclaving process.
 N. Fu et al. / Enzyme and Microbial Technology 45 (2009) 210–217 213

Fig. 2. Modified fermentor and the removal of immobilized Z. mobilis beads by the sieve plate. (a) Modified fermentor with the added sieve plate; (b) co-culture of immobilized
Z. mobilis and free cells of P. stipitis; (c) and (d) sieve plate was raised, separating the immobilized beads containing Z. mobilis and xylose fermentation continuing with the
beads away from the fermentation medium.

tation time and improve the efficiency. With the use of 1/4 batch
of Z. mobilis beads, the ethanol yield was significantly increased
to 0.49–0.50 g/g, which is 96–98% of the theoretical value. The
best fermentation performance was a fermentation time of 19 h
and a volumetric productivity of 1.277 g/l/h, achieved with a 1/4
batch of Z. mobilis beads and a 100% inoculum size of P. stipitis free
cells.
A comparison of the three co-culture fermentation schemes is
shown in Fig. 3. The three processes used for comparison were
the free cell co-culture at 80 cm3 /min air flow, the first culture

Fig. 3. Comparison of three processes using different co-culture schemes. (a) Sugar utilisation and ethanol production; and (b) viable cell concentration of two microorganisms
in the fermentation medium.
214 N. Fu et al. / Enzyme and Microbial Technology 45 (2009) 210–217
employing immobilized Z. mobilis and free cells of P. stipitis, and
a process in the modified fermentor with inoculum level 2, all of
which used an inoculum size of 50% for P. stipitis. As can be seen in
Fig. 3(a), the process carried out in the modified fermentor took a
longer time to completely convert the glucose to ethanol because of
less immobilized Z. mobilis beads inoculated. However, its reducing
sugar curve was smoother and the ethanol accumulation was more
significant during the xylose fermentation stage, indicating a more
efficient xylose fermentation in comparison to the other two pro-
cesses with a full batch of Z. mobilis beads. The corresponding viable
cell concentrations in these three processes are shown in Fig. 3(b)
in logarithmic scale. Despite the same inoculum size of P. stipitis
free cells used in all three processes, the P. stipitis level in the free
cells co-culture was remarkably lower than the other two processes
employing immobilized Z. mobilis. Correspondingly, the Z. mobilis
free cells in these two processes were lower than 1 × 108 cfu/ml,
indicating a low level of cell leakage from the immobilized beads
to the fermentation medium. Z. mobilis concentration in the free
cell co-culture showed an unusual trend. Cells decreased to a level
of 2 × 107 cfu/ml at 4 h due to the exhaustion of glucose; however,
a re-increase of cell concentration to a level of 3–4 × 108 cfu/ml
was observed during the following xylose fermentation stage. This
abnormal re-increase of Z. mobilis viable cells occurred in three
out of four processes using the fermentation scheme of free cell
co-culture.
Fermentations with an increased initial sugar level to 45 g/l
glucose and 30 g/l of xylose were carried out with the best fer-
mentation scheme, i.e. the immobilized Z. mobilis and free cells
of P. stipitis in the modified fermentor. As shown in Table 1,
while the inoculum size of P. stipitis remained 50%, again pro-
cesses with decreased Z. mobilis beads level to 1/2 batch showed
higher fermentation performance (ethanol yield of 0.46 g/g com-
pared to 0.42 g/g with a full batch Z. mobilis beads). The increased
sugar level led to a prolonged fermentation time of 42–45 h, and
Fig. 4. Conversion of sugarcane bagasse hydrolysate to ethanol using immobilized Z. mobilis and free cells of P. stipitis. (a) Sugar utilisation and ethanol production; and (b)
viable cell concentration of two microorganisms in the fermentation medium.
the resulting volumetric ethanol productivities were lower than
that achieved with 50 g/l initial sugars (0.6–0.8 g/l/h compared
to 1.1–1.2 g/l/h).
3.2. Conversion of bagasse hydrolysate to ethanol
After the supplementation with sugars and other media compo-
nents, the hydrolysate medium contained approximately 31–32 g/l
of glucose and 17–21 g/l of xylose. No inhibitors were detected at
any significant level by HPLC analysis after overliming. The two fer-
mentations employed a 50% inoculum size of P. stipitis together with
1/4 batch and 1/2 batch of immobilized Z. mobilis beads, adapted
in the hydrolysate medium twice and once respectively prior to
the fermentation. Results are shown in Fig. 4. With the use of 1/4
batch beads, the glucose in the medium was completed in 9 h, corre-
sponding to a steady increase in ethanol production; however, the
xylose was utilised slowly with no significant ethanol accumula-
tion. Meanwhile, P. stipitis cells in the medium gradually decreased
throughout the process, from a level of 8 × 108 to 3 × 107 cfu/ml.
Although the immobilized beads of Z. mobilis were removed by lift-
ing the sieve plate at 6 h, viable cells of Z. mobilis in the fermentation
medium showed an abnormal increase in an environment devoid
of a known external carbon source suitable for its use.
Increasing the inoculum size of immobilized Z. mobilis to 1/2
batch and reducing the adaptation to a single culture significantly
improved the fermentation performance. As shown in Fig. 4, the
glucose was consumed more efficiently and the xylose was com-
pletely utilised in 26 h. Ethanol production steadily increased in
both stages, giving a conversion rate of 0.43 g/g when xylose was
used up, and peaked at 40 h with a conversion rate of 0.49 g/g. The
corresponding biomass in this fermentation was a steady P. stipi-
tis cell concentration at 5–7 × 108 cfu/ml throughout the process,
together with an undetectable leakage of Z. mobilis cells most of
time in the fermentation medium.
 N. Fu et al. / Enzyme and Microbial Technology 45 (2009) 210–217
3.3. Interactions of Z. mobilis and P. stipitis during the co-culture
The co-fermentation processes described above suggested some
cell interaction between Z. mobilis and P. stipitis. The xylose fermen-
tation proceeded more efficiently when there were less Z. mobilis
viable cells in the medium. To better determine this interaction,
the shake flask experiments were carried out on both xylose and
glucose/xylose sugar mixture media, using increased levels of Z.
mobilis cells co-cultured with P. stipitis. Xylose medium consisted of
the same components as the sugar mixture fermentation medium,
with 50 g/l xylose as the sole carbon source. For each medium, five
flasks were inoculated with 50% (vcells /vferm ) P. stipitis cells and 10%,
20%, 30%, 40% and 50% of Z. mobilis cells, respectively. A control flask
with P. stipitis sole culture was prepared for the xylose medium, but
no control flask for the sugar mixture medium, since the glucose-
fermenting strain was Z. mobilis in this co-culture scheme. Results
are shown in Fig. 5.
Despite the same inoculum size used for all 11 flasks, P. stipi-
tis viable cell concentration in the five flasks on xylose medium
was lower than that in the control flask without Z. mobilis, but was
higher than that in the five flasks on the sugar mixture medium
(where Z. mobilis showed higher cell concentration). Meanwhile,
for each medium, the final concentration of P. stipitis tended to
decrease with the increase in the initial cell concentration of Z.
mobilis, although the lowest final concentration of P. stipitis was
obtained in both flasks with 20% Z. mobilis. Another significant trend
was the re-increase of Z. mobilis cells on the xylose medium at 36 h
in an environment devoid of a known external carbon source suit-
Fig. 5. Cell interaction of P. stipitis and Z. mobilis when co-cultured on the xylose and
glucose/xylose sugar mixture medium. (a) Viable cell concentration of P. stipitis; (b)
viable cell concentration of Z. mobilis; and (c) ethanol production.
215
able for its use. In all five flasks no Z. mobilis cells was detected at
24 h, but a re-increase to 107 –108 cfu/ml was observed at 36 h.
In accordance with the biomass trends, the final ethanol produc-
tion in the five flasks with the xylose medium was generally lower
than that in the control flask at 36 h. In the five flasks with the
glucose/xylose sugar mixture medium, ethanol production at 12 h
was high due to the glucose fermentation by Z. mobilis. However,
no significant ethanol accumulation was observed thereafter. Fur-
thermore, lower ethanol accumulation was observed in the flasks
containing higher levels of initial Z. mobilis cells.
4. Discussion
4.1. Conversion of glucose/xylose sugar mixture by Z. mobilis and
P. stipitis
Amongst the four different fermentation schemes carried out,
schemes using sequential culture and using the modified fermentor
efficiently converted the glucose/xylose sugar mixture to ethanol,
with a high ethanol yield (0.47–0.50 g/g) and a high volumet-
ric productivity (0.830–1.277 g/l/h). Both schemes provided each
organism an optimized environment by either the separate cul-
ture or the containment of Z. mobilis cells within the gel beads,
and therefore successfully solved the problem of different oxygen
requirements of Z. mobilis and P. stipitis. However, considering the
energy input to inactivate the bacterial cells and the prolonged fer-
mentation time due to the separate fermentation stages, sequential
culture of two microorganisms was considered to be less desirable
compared to the co-culture process.
Free cell co-culture of P. stipitis and Z. mobilis failed to utilise the
xylose in all cases. Co-culture of immobilized Z. mobilis and free cells
of P. stipitis was able to convert the sugar mixture efficiently, but the
performance deteriorated along with the repeat utilisation of the
same batch of Z. mobilis beads. The removal of the immobilized
Z. mobilis beads from the medium after the completion of glu-
cose fermentation successfully solved this problem and achieved
steady fermentation performance using a modified fermentor. By
further optimization of the ratio of the inoculated Z. mobilis and
P. stipitis, high fermentation efficiency was obtained. The complete
conversion of 30 g/l of glucose and 20 g/l of xylose in 19 h with an
ethanol yield of 0.49 g/g is the highest result so far reported for
sugar mixture co-fermentation in batch culture. Application of this
fermentation scheme to 75 g/l initial sugars resulted in a decreased
ethanol yield due to the ethanol reassimilation observed at the late
stage of both processes; however, the volumetric ethanol productiv-
ity of 0.878 g/l/h compares favourably with the reported values. The
fermentation of sugarcane bagasse hydrolysate with sugar supple-
ment also showed a high efficiency with a strain adaptation of once
in the hydrolysate medium. Sugar mixture comprising 32.14 g/l of
glucose and 21.42 g/l of xylose was completely utilised in 26 h with
an ethanol yield of 0.43 g/g and a volumetric ethanol productiv-
ity of 0.879 g/l/h. When ethanol peaked at 40 h, the ethanol yield
increased to 0.49 g/g, but the productivity decreased to 0.699 g/l/h.
The lower fermentation efficiency observed when P. stipitis was
twice adapted in the hydrolysate medium was considered to be
as a result of cell aging during the prolonged incubation time
(48 h incubation for inoculum followed by twice 24 h incubation
for adaptation).
There are a number of reports using strain co-culture for the co-
fermentation of glucose/xylose sugar mixture. The co-culture of a
respiratory deficient mutant of S. cerevisiae and P. stipitis on a mix-
ture of 50 g/l of glucose and 25 g/l of xylose resulted in a complete
sugar utilisation in 40 h with an ethanol yield of 0.50 g/g [16]. Co-
immobilization of S. cerevisiae and P. stipitis with different biomass
proportions successfully utilised 42 g/l glucose and 12 g/l xylose in
the lignocellulosic hydrolysates within 24 h. However, the ethanol
216 N. Fu et al. / Enzyme and Microbial Technology 45 (2009) 210–217
peaked at 40 h with an ethanol yield of 0.38 g/g [12]; the trend
of the delayed ethanol peak time was similar to the fermentation
of bagasse hydrolysate in this study. A recombinant E. coli strain
was used for the conversion of softwood hydrolysates to ethanol
together with S. cerevisiae [18]. An ethanol yield of 0.45 g/g was
reached in 24 h together with the full utilisation of 37.5 g/l sugar
mixture containing 75% of glucose.
Results of the current study also compare favourably with other
sugar mixture co-fermentation schemes using strain sole culture,
both in the wild type or with genetically modified microorganisms.
Fermentation of enzymatically hydrolysed wheat straw reported
an ethanol yield of 0.37 g/g in 39 h using a recombinant E. coli
strain FBR5 [25]. Another study investigating an adapted strain
of P. stipitis reported highest volumetric ethanol productivity of
0.54 and 0.66 g/l/h on wheat straw hydrolysate and synthetic
medium, respectively [26]. A high ethanol concentration up to
44.3 g/l was achieved by a recombinant strain of Z. mobilis on
rice straw hydrolysate in 24 h fermentation; however, only 80% of
xylose was consumed in this process [3]. The conversion of acid
hydrolysate of sugarcane bagasse using a recombinant E. coli strain
achieved an ethanol yield of 0.47 g/g with a complete utilisation of
all sugars in 48 h [22].
It can be thus seen that the results reported here with the
co-culture of Z. mobilis and P. stipitis on both synthetic sugar mix-
ture medium and bagasse hydrolysate medium is one of the best
reported so far. The efficient fermentation of xylose in this fermen-
tation scheme may be directly attributed to the large inoculum size
used (50% vcells /vferm ).
4.2. Potential cell interactions between Z. mobilis and P. stipitis
An observation from both the fermentation processes in fer-
mentor and the shake flask experiments was that Z. mobilis viable
cells inhibited the xylose fermentation by P. stipitis. For example,
none of the four free cell co-culture fermentations could completely
utilise the xylose by the end of process, whereas the entrapment
of Z. mobilis cells in the calcium alginate gel beads resulted in an
efficient xylose fermentation when co-cultured with P. stipitis free
cells. However, with the repeated usage of the same batch of Z.
mobilis beads, the fermentation performance significantly deterio-
rated, since bead damage became increasingly severe and more free
cells of Z. mobilis were released into fermentation medium. In the
shake flask experiments the five test flasks with xylose medium
yielded lower ethanol compared to the control flask. In the five
test flasks with glucose/xylose mixture medium, ethanol produc-
tion from xylose was considerably insignificant even though there
were 4–5 g/l xylose being consumed between 12 h and 24 h (sugar
values not included in Fig. 5).
It is considered that the inhibition of Z. mobilis cells on the xylose
fermentation was due to the inhibition of P. stipitis cell activity.
A possible explanation could be the oxygen deprivation by the Z.
mobilis cells. Similar observation has been made for S. cerevisiae
in the co-culture with P. stipitis [9,16]. It also has been accepted
that although wild type Z. mobilis is strictly fermentative, it pos-
sesses a complete redox enzyme system on its cell membrane and
is able to rapidly consume oxygen in an aerobic culture [20,27].
The ethanol consumption of Z. mobilis under aerobic conditions has
been previously reported [13]. Consequently, the reduced efficiency
of xylose fermentation could be caused by the oxygenation compe-
tition between Z. mobilis and P. stipitis. Assuming that Z. mobilis
could oxidize the produced ethanol, the increased ethanol yield to
0.49–0.50 g/g when the inoculum size of Z. mobilis was reduced to
1/4 batch in the co-culture processes in the modified fermentor
could be explained.
Another significant trend for the cell interaction was the abnor-
mal re-increase of Z. mobilis after the exhaustion of glucose. This
trend was observed in three processes out of four in the free
cell co-cultures, the fermentation of bagasse hydrolysate, and the
shake flask experiments. The temporary reduction of cells at 24 h
in the shake flask experiment and the re-increase at 36 h suggest
that Z. mobilis cells had access to some nutrients that stimulated
its growth. Since Z. mobilis cannot utilise xylose, the only pos-
sibility is that it utilised some intermediate metabolites in the
metabolism of xylose by P. stipitis. As reported previously [5,28],
the assimilated xylose in the P. stipitis cells is firstly converted
to glyceraldehydes-3-phosphate through the pentose phosphate
pathway which enters the glycolytic pathway; the same pathway is
then used to convert the glyceraldehydes-3-phosphate to ethanol
as in the Entner–Doudoroff pathway which is used for the glucose
metabolism by Z. mobilis cells. Though the specific mechanism is
still not clear, it is considered possible for the bacterium to utilise
some metabolites from P. stipitis for its own growth, which also
explains why the re-increase of Z. mobilis always occurred in the
late stage of the xylose fermentation. The utilisation of the inter-
mediate metabolites of xylose fermentation by Z. mobilis could be
another reason for the inhibited P. stipitis cell activity when directly
exposed to Z. mobilis cells.
Because of the inhibition by Z. mobilis cells on the xylose fer-
mentation of P. stipitis, the separation of Z. mobilis cells from the
P. stipitis cells is a prerequisite for a successful sugar mixture co-
fermentation by these two strains. Fermentation schemes reported
here could be also used for the co-culture involving S. cerevisiae
and any xylose-fermenting yeast. Micro-encapsulation proved to
be an effective approach for entrapping the facultative aerobes
into the gel beads and thus avoid the undesirable oxygen com-
petition with the xylose-fermenters. With a more durable matrix
rather than calcium alginate, the performance of the immobiliza-
tion could be improved, which would lead to a more efficient
co-culture. Further studies in the metabolic processes of the two
microorganisms could be significant for understanding the mech-
anisms of the cell interactions between P. stipitis and Z. mobilis in a
co-culture.
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