Low Carbon Economy, 2011, 2, 224-229
doi:10.4236/lce.2011.24028 Published Online December 2011 (http://www.SciRP.org/journal/lce)
1
Comparative Study on Microorganisms Used for
the Bioethanol Production
Corina Amalia Macarie1, Adina-Elena Segneanu1, Ionel Balcu1, Raluca Pop1, Georgeta Burtica2,
Vasile-Daniel Gherman2
1National Institute for Research and Development in Electrochemistry and Condensed Matter, Timisoara, Romania; 2The Faculty of
Civil Engineering, Politehnica University of Timisoara, Timisoara, Romania.
Received September 2nd, 2011; revised October 10th, 2011; accepted October 17th, 2011.
ABSTRACT
Two different methods, namely simultaneous saccharification and fermentation and two-stage hydrolysis and fermen-
tation have been used for the conversion of the cellulose into bioethanol. Both aerobic and anaerobic conditions were
employed in order to obtain two types of microorganisms—Trichoderma reesei and Zymomonas mobilis—that are used
for the production of cellulases. The aim of the paper is to investigate also the efficiency of a microorganism’s consor-
tium in the fermentation stage of the two processes and the action of this microorganism’s consortium on different con-
centrations of the cellulosic substrate, in order to determine the optimum parameters of the process. Good yields (45% -
70%) of the cellulose degradation into fermentable sugars have been obtained.
Keywords: Bioethanol, Lignocellulosic Biomass, Microorganism
1. Introduction
Due to the limitations of fossil energy, there is a conti-
nuous increasing interest in finding new alternatives for
oil-based fuels. Renewable resources like sugarcane or
agricultural crops have represented the major raw materi-
als for the bioethanol production [1]. In the last years,
lignocellulosic materials have gained much importance
for the obtaining of bioethanol, mostly due to their large
availability. The main disadvantage is represented by the
higher costs caused by the small yields and by the diffi-
culties encountered in the cellulose hydrolysis (deploy-
merization) in soluble sugars [2,3].
Chemical methods for the depolymerization of the cellu-
lose are the acid, basic or enzymatic hydrolysis. Among
them, enzymatic hydrolysis shows the advantages of mild
reactions conditions and of avoiding the corrosion prob-
lems [4-7].
Cellulose transformation into soluble sugars during the
enzymatic hydrolysis process occurs by the means of cel-
lulase, an enzyme wit h a high cost of production. Late ly , in
the biofuels industry, cellulases obtained from the Tricho-
derma reesei fungus are used, with good results regarding
the vegetal feedstock degradation [8,9]. This could enlarge
the possible biomass resources. Another microorganism
used for the bioethanol production is Zymomonas mobilis
that may be used only for su bstrates that contain glucose,
fructose and sucrose [10-12].
There are two main procedures used for the bioethanol
production starting from cellulose. The first one implies
two different stages, namely the hydrolysis of the cell-
ulosic substrate, followed by the fermentation of the obt-
ained sugars. The second method implies the simulta-
neous saccharification and fermentation [10,13,14].
The present paper presents the cellulose degradation
under the action of Trichoderma reesei and Zymomonas
mobilis, in order to determine th e optimum conditio ns for
the bioethanol production through the hydrolysis and
fermentation of cellulose-based materials. The hydrolysis
of the cellulosic substrate was performed using Tricho-
derma reesei, and for the separate fermentation of sugars
there were used enzymes from Zymomonas mobilis. For
the simultaneous saccharification and fermentation of a
cellulosic substrate a consortium of the two microrgan-
isms was employed. The results of the two fermentation
methods (separate and simultaneous) have been quan-
tified by means of the concentration of the unreacted fer-
mentable sugars that were not transformed into ethanol.
2. Experimental Part
Lyophilized strains of Trichoderma reesei (IHEM 5652)

Comparative Study on Microorganisms Used for the Bioethanol Production 225
and Zymomonas mobilis (ATCC 29191) were used, that
were reactivated at their transfer on the culture media.
2.1. The Obtaining of Cellulose and He micellulose
De g r ad i n g En zy m e s f ro m Micro-Organism
Cultures
Cellulases from Trichoderma reesei and Zymomonas mo-
bilis.
The cultures were incubated for several days at a tem-
perature of 30˚C, without shaking, both in anaerobic and
aerobic conditions. At various time intervals samples
were taken in order to analyze the cellulase activity on
the substrate (carboxymethylcellulose standard) and also
for the determination of the protein content.
2.2. Cellulose Degradation by Enzymes Obtained
from Trichoderma reesei
The Trichoderma strains were inoculated on a natural
culture medium that contained 100 mL potato extract, 2 g
glucose and 0.5 g, 0.75 g, 1.0 g, 1.5 g and 2.0 g Avicel,
respectively. The cultures were sterilized in an autoclave,
inoculated and thermostated for several days at 30˚C.
Samples of a few milliliters were taken in order to deter-
mine the total sug ar content.
2.3. Sugar Fermentation by Enzymes from
Zymomonas mobilis
Cultures of Zymomonas in a synthetic medium were used.
The synthetic culture medium was made by 0.5 g yeast
extract, 0.1 g (NH4)2SO4, 0.2 g K2HPO4, 0.1 g MgSO4
and 2.0 g, 4.0 g, 6.0 g and 8.0 g glucose, respectively.
The cultures were syntetized by the same way like in case
of Trichoderma reesei strains.
2.4. Cellulose Degradation by Enzymes Obtained
from Trichoderma reesei and Zymomonas
mobilis
Cultures of Trichoderma and Zymomonas on natural me-
dium were used. The natural culture medium contained
100 mL potato extract, 2 g glucose and 1.0 g, 1.5 g, 2.0 g,
3.0 g and 4.0 g Avicel, respectively. Sterylization proce-
dure of cultures is identical with the previous method.
2.5. Determination of the Total Sugar Content
The total sugar content was calculated using the colori-
metric method for the determination of the reducing sug-
ars with the DNS (3,5-dinitrosalycilic acid) reagent. In
order to calculate the total sugar content, Equation (1)
was used.

540
Total sugars
A 0.074471.22612dilution factor

 

(1)
where, A540-solution absorbance (extinction) at 540 nm
dilution factor-5.
The calibration curve used fo r the determination of the
total sugar content (as glucose) is presented in Figure 1.
3. Results and Discussions
The total protein content obtained from cultures of Tri-
choderma reesei and Zymomonas mobilis both in anae-
robic and aerobic is presented in Figures 2-3 and Tables
1-2.
The protein content in aerobic and anaerobic conditions
has been de termine d from the inf luenc e of the con cen trat ion
of oxygen on the development of Trichoderma reesei and
Zymomonas mobilis.
Figure 1. Glucose calibration curve using the colorimetric
method (Correlation coefficient R(2) = 0.9908).
Figure 2. The evolution in time of the cellulase activity pro-
duced by Trichoderma reesei and of the amount of glucose
obtained in anaerobic conditions.

Comparative Study on Microorganisms Used for the Bioethanol Production 226
The cellulosic substrate degradation with the enzymes
obtained from T. reesei was quantified by the determina-
tion of the sugar content of the culture medium at differ-
ent times. The Zymomonas strains present less efficien tly
in aerobic conditions (Table 2) then in anaerobic conditions
(Table 1), but in this case also, no cellulase was pr od uc ed.
We can conclude that it cannot be used to produce cel l ula se
in the given, studied, conditions.
The results are presented in Table 2 and, in a graphic
form, in Figure 3. As a comparis on, the th eoretica l quan-
tities that could be obtained from the cellulose degrada-
tion with a 100% yield were calculated (Table 3). Data
from the Tables 3 and 4 were used for the calculation of
the yield of the cellulose enzymatic hydrolysis to sugars,
results that are presented in Table 4 and Figure 4.
As it may be observed, best results are obtained for
the cultu re me d ia w i th a low co n ten t o f c e llu los e (high er
va l u es a r e obtaine d f o r the me t hods w here an i nitial con-
Figure 3. The evolution in time of the cellulase produced by
Trichoderma reesei and of the amount of glucose obtained in
aerobic conditions.
Table 1. The total protein content in Zymomonas mobilis
cultures in anaerobic conditions.
Time [h] Carbon source Protein [mg/ml]
24 0.971
48 0.771
144
Cellulose
5 g/L
0.918
Table 2. The total protein content in Zymomonas mobilis
cultures in aerobic conditions.
Time [h] Carbon source Protein [mg/ml]
24 0.482
48 0.438
144
Cellulose 5 g/L
0.524
Table 3. Maximum theoretical concentrations of total sugars
and ethanol, considering maximum e fficiency of the hyd rol ys is
and fermentation processes.
No. of
culture Initial cellulose concentration (g/L)Theoretical sugars concentration (g/L) Theoretical ethanol concentration (g/L)
1 5 43.21 22.08
2 7.5 45.41 23.21
3 10 47.61 24.33
4 15 52.01 26.58
5 20 56.41 28.83
Table 4. Results of the cellulose hydrolysis into fermentable
sugars by means of enzymes obtained from Trichoderma
reesei.
No. of
culture Initial cellulose conc. (g/L) Time (h) Total sugars concentration (g/L)Sugars yield (%)
24 22.19 51.3
48 24.84 57.5
72 30.19 69.9
1 5
96 28.17 65.2
24 28.05 64.9
48 29.60 68.5
72 30.23 70.0
2* 5
96 28.20 65.3
24 25.91 57.1
48 28.08 61.8
72 31.36 69.1
3 7.5
96 29.87 65.8
24 27.55 60.7
48 26.12 57.5
72 29.44 64.8
4* 7.5
96 29.11 64.1
24 29.70 62.4
48 28.08 59.0
72 28.64 60.2
5 10
96 31.01 65.1
24 28.08 59.0
48 23.35 49.0
72 32.32 67.9
6* 10
96 28.89 60.7
24 20.11 38.7
48 19.83 38.1
72 26.40 50.8
7 15
96 23.74 45.6
24 25.15 48.4
48 23.53 45.2
72 31.04 59.7
8* 15
96 30.49 58.6
24 34.18 60.6
48 30.85 54.7
72 30.0 53.2
9 20
96 31.13 55.2
24 25.05 44.4
48 25.18 44.6
72 28.16 49.9
10* 20
96 29.54 52.4
*There were used five cultures (in duplicate) of Trichoderma reesei.

Comparative Study on Microorganisms Used for the Bioethanol Production 227
Figure 4. Yields of total sugars obtained from cellulose (best
results of each duplicate culture are presented).
centration of 5 g/L, 7.5 g/L and 10 g/L cellulose was
used). The maximum yield of the cellulose in glucose
transformation is 70% and it is obtained for a concentra-
tion of the cellulosic substrate of 5 g/L and a reaction
time of 72 h. An increase of the reaction time leads (ex-
cept the case of the substrate with the greatest cellulose
content) to better results in concentrations of the fer-
mentable sugars.
The fermentation process of the glucose using Zymo-
monas mobilis resulted in small amounts of unfermented
sugars. Results presented in Table 5 and Figure 5 show
that only small concentrations of unfermented sugars
were obtained. It may also be observed that the best re-
sults are obtained during a longer reaction time (48 h - 96
h) and when shaking is applied.
Table 5 shows the untransformed sugar contents after
the glucose fermentation in th e presence of Z. mobilis and,
starting from these experimental values, theoretical etha-
nol yield was calculated. The results presented in Figure
5 show that, independent from the substrate concentration,
the best results are obtained after an enzyme action time
of 72 - 96 h.
A series of cellulosic substrates (Table 6) were sub-
jected to the simultaneous action of Trichoderma reesei
and Zymomonas mobilis for a 30 days period. The results
are presented in Table 6 and Figure 6.
The results show no significant differences among the
content of untransformed total sugars, independent of the
initial cellu lose con centration s. It app ears that, in this cas e,
higher initial concentrations of cellulose have positive in-
fluence on the total amount s of fermented sugars.
4. Conclusions
Trichoderma reesei cultures showed a lower growth of
the microorganism under aerobic conditions than under
anaerobic condition s. Cellulase was not detected after the
first day, but after 48 hours it was formed in sufficiently
Table 5. The total sugar content experimentally resulted
and the calculated theoretical ethanol concentration at sug-
ars fermentation with Zymomonas.
No. of
culture Initial glucose conc. (g/L)Time (h) Total sugars conc. (g/L) Theoretical ethanol conc. (g/L)
24 2.21 9.09
48 1.07 9.67
72 0.45 9.99
1* 20
96 0.29 10.02
24 0.23 10.10
48 0.42 10.00
72 0.46 10.00
2 20
96 0.28 10.02
24 2.17 19.33
48 0.79 20.04
72 0.72 20.08
3* 40
96 0.74 20.06
24 0.35 20.26
48 0.58 20.15
72 0.43 20.22
4 40
96 0.36 20.26
24 0.89 30.21
48 1.03 30.14
72 1.03 30.14
5* 60
96 0.94 30.19
24 2.29 29.50
48 0.65 30.33
72 0.62 30.35
6 60
96 0.52 30.40
24 2.80 39.50
48 1.09 40.33
72 1.47 40.14
7* 80
96 1.30 40.22
24 2.17 39.78
48 0.49 40.64
72 0.46 40.65
8 80
96 0.45 40.65
*shaken at 150 rot/mi n.
Figure 5. Total sugar content a fter glucose fermentation with
Zymomonas mobilis.

Comparative Study on Microorganisms Used for the Bioethanol Production 228
Table 6. The total sugar content at the action of a mixture of
T. reesei and Z. mobilis on cellulosic substrates.
No. of
culture Initial cellulose conc. (g/L) Time (days) Total sugar content (g/L) Theoreticalethanol conc. (g/L)
1 10.0 30 1.077 23.78
2 15.0 30 1.091 26.03
3 20.0 30 1.110 28.77
4 30.0 30 1.112 32.76
5 40.0 30 1.065 37.28
Figure 6. Total content of untransformed sugars after the
action of a mixture of T. reesei and Z. mobilis.
large amounts. after 6 days, it disappeared almost comp-
letely from the medium. Cellulase can be also biosynthe-
sised under aerobic conditions from Trichoderma reesei
cultures, but it is very important to know the optimum
time for stopping the culture if the cellulase activity is
elevated. Zymomonas strain presented a lower growth
under aerobic conditions than under the anaerobic ones,
but cellulase was not produced in none of the cases. It
may be said that the studied Zymomonas strain cannot
produce cellulase in the culture media that were used in
our experiments.
The results of the study show that T. reesei action on
cellulose led to the obtaining of sugars (determined as
glucose) in 45% - 70% yield. Higher content of sugars
are obtained for an initial cellu lose concen tration of 5 g/L
- 10 g/L. Cellulose concentrations of 15 g/L - 20 g/L
show an inhibitory effect on the cellulose degradation.
The experimental values of the total sugar conten t after
the Z. mobilis action on glucose show that only small
sugar amounts had not been transformed. Similar values
of untransformed sugars are obtained after the action of a
mixture of T. reesei and Z. mobilis on cellulosic subst-
rates. As results from the values presented in Table 6 and
Figure 6, higher cellulose concentration of the synthetic
culture medium does not have negative consequences on
the cellulose degradation process.
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