Comparative Study on Microorganisms Used for the Bioethanol Production

doi:10.4236/lce.2011.24028

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Low Carbon Economy, 2011, 2, 224-229

doi:10.4236/lce.2011.24028 Published Online December 2011 (http://www.SciRP.org/journal/lce)

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.

Email: [email protected]

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.

REFERENCES

[1] M. Balat, H. Balat and C. Oz, “Progress in Bio-Ethanol

Processing,” Progress in Energy and Combustion Science,

Vol. 34, No. 5, 2008, pp. 551-573.

doi:10.1016/j.pecs.2007.11.001

[2] F. Abd El-Zaher and M. Fadel, “Production of Bioethanol

via Enzymatic Saccharification of Rice Straw by Cellu-

lase Produced by Trichoderma reesei under Solid State

Fermentation,” New York Science Journal, Vol. 3, 2010, p.

72.

[3] E. A. Bayer, R. Lamed and E. Himmel, “The Potentia l of

Cellulases and Cellulosomes for Cellulosic Waste Man-

agement,” Current Opinion in Biotechnology, Vol. 18, No.

3, 2007, pp. 237-245. doi:10.1016/j.copbio.2007.04.004

[4] H. Golias, G. J. Dumsday, G. A. Stanley and N. B. Pam-

ment, “Evaluation of a Recombinant Klebsiella oxytoca

Strain for Ethanol Production from Cellulose by Simulta-

neous Saccharification and Fermentation: Comparison

with Native Cellobiose-Utilising Yeast Strains and Per-

formance in Co-Culture with Thermotolerant Yeast and

Zymomonas mobilis,” Journal of Biotechnology, Vol. 96,

No. 2, 2002, pp. 155-168.

doi:10.1016/S0168-1656(02)00026-3

[5] S. Kumar, S. P. Singh, I. M. Mishra and D. K. Adhikari,

“Recent Advances in Production of Bioethanol from Lig-

nocellulosic Biomass,” Chemical Engineering & Tech-

nology, Vol. 32, No. 4, 2009, pp. 517-526.

doi:10.1002/ceat.200800442

[6] Y. Sun and J. Cheng, “Hydrolysis of Lignocellulosic Ma-

terials for Ethanol Production: A Review,” Bioresource

Technology, Vol. 83, No. 1, 2002, pp. 1-11.

doi:10.1016/S0960-8524(01)00212-7

[7] H. C. Vogel and C. L. Todaro, “Fermentation and Bioche-

mical Engineering Handbook,” Noyes Publications, New

Jersey, 1997.

[8] T. Fujii, X. Fang, H. Inoue, K. Murakami and S. Sa-

wayama, “Enzymatic Hydrolyzing Performance of Acre-

monium cellulolyticus and Trichoderma reesei against

Three Lignocellulosic Materials,” Biotechnology for Bio-

fuels, Vol. 2, 2009, p. 24.

doi:10.1186/1754-6834-2-24

[9] B. Nidetzky, W. Steiner, M. Hayn and M. Claeyssens,

“Cellulose Hydrolysis by the Cellulases from Trichoderma

reesei: Adsorptions of Two Cellobiohydrolases, Two Endo-

cellulases and Their Core Proteins on Filter Paper and Their

Relation to Hydrolysis,” Biochemical Journal, Vol. 298,

1994, p. 705.

[10] V. Ferreira, M. Faber, S. Mesquita and N. Pereira Jr.,

“Simultaneous Saccharification and Fermentation Process

of Different Cellulosic Substrates Using a Recombinant

Saccharomyces cerevisiae Harbouring the β-Glucosidase

gene,” Electronic Journal of Biotechnology, Vol. 13, No.

2, 2010, p. 1.

[11] J. H. Lee, R. J. Pagan and P. L. Rogers, “Continuous Si-

multaneous Saccharification and Fermentation of Starch

using Zymomonas mobilis,” Biotechnology and Bioengi-

neering, Vol. 25, No. 3, 1983, pp. 659-669.

Comparative Study on Microorganisms Used for the Bioethanol Production

229

doi:10.1002/bit.260250304

[12] H. Nellaiah, T. Karunakaran and P. Gunasekaran, “Etha-

nol Fermentation of Cassava Starch by Zymomonas mobi-

lis NRRL B-4286,” Biomass, Vol. 15, No. 3, 1988, pp.

201-207. doi:10.1016/0144-4565(88)90085-6

[13] S. H. Krishna, T. J. Reddy and V. Chowdary, “Simulta-

neous Saccharification and Fermentation of Lignocellu-

losic Wastes to Ethanol Using a Thermotolerant Yeast,”

Bioresource Technology, Vol. 77, No. 2, 2001, pp. 193-

196. doi:10.1016/S0960-8524(00)00151-6

[14] Zs. Kadar, Zs. Szengyel and K. Reczey, “Simultaneous

Saccharification and Fermentation (SSF) of Industrial

Wastes for the Production of Ethanol,” Industrial Crops

and Products, Vol. 20, No. 1, 2004, pp. 103-110.

doi:10.1016/j.indcrop.2003.12.015