10-Contenido Del Artículo Con Resumen, Palabras Claves y Referencias-26-1-10-20210627

Revista Carácter, diciembre 2020, Vol. 8, No.
1
e-ISSN: 2602-8476, ISSN: 1390-7662
www.upacifico.edu.ec/revistacaracter
https://doi.org/10.35936/caracter.v8i1.72
BIOETHANOL PRODUCTION FROM AGRICULTURAL RESIDUE BIOMASS BY

Zymomonas mobilis IMMOBILIZED AND FREE CELLS
PRODUCCIÓN DE BIOETANOL CON CÉLULAS LIBRES E INMOVILIZADAS DE
Zymomonas mobilis A PARTIR DE BIOMASA RESIDUAL AGRICOLA
Jaime Oliver Santos Pinargote1

José David Villalta Pastuzo2
Recibido: septiembre 2020 / Aceptado: Diciembre 2020 / Publicado: diciembre 2020
Abstract:
The objective of the present research was to evaluate the fermentation activity of Zymomonas mobilis
UGB-SA2001 strain for the production of bioethanol, for this purpose free and immobilized cells were used.
As substrates, three products of agricultural origin were used: Sugar beet pulp, banana pseudostem and
molasses 75%, 50% and 25%. Acid and alkaline pretreatments were also carried out before fermentation, to
the different types of residual lignocellulosic material Furthermore, the consumption of total reducing sugars
was evaluated. The assays with free cells and immobilized cells highest total reducing sugars consumption
of up to 50% in molasses culture at 75%. Ethanol production was determined by spectrophotometry,
obtaining the highest ethanol concentration and productivity in 50% and 75% molasses medium, with a
maximum concentration 30.8±0.2 g·L-1 and 0.26±0.01 g·h-1/L-1 respectively. Furthermore, it was observed
that the cells without immobilization obtained the best yields in the tests with banana pseudostem and sugar
beet pulp 16±0.3 g·L-1 and 5.5±0.01 g·L-1 respectively, with acid pretreatments. The Z. mobilis strain was
determined to be able to resist high concentrations of total reducing sugars up to 75%. This strain is also
within the normal standards for the production of ethanol.
Keywords: Acid, alkaline, banana, beet, molasses
1
Universidad de Guayaquil, Facultad de Ciencias Naturales, Laboratorio de Biotecnología. Ecuador. Universidad Agraria del Ecuador, Facultad de
Ciencias Agrarias j.santos2387@gmail.com
2
Universidad de Guayaquil, Facultad de Ciencias Naturales, Laboratorio de Biotecnología. Ecuador
Bioethanol Production from Agricultural Residue Biomass by Zymomonas Mobilis Immobilized
And Free Cells
Revista Carácter, diciembre 2020, Vol. 8, No.1
e-ISSN: 2602-8476, ISSN: 1390-7662
Introduction
Zymomonas mobilis is a promising organism for lignocellulosic biofuel production as it
can efficiently produce ethanol from simple sugars using unique metabolic pathways. Z. mobilis
displays what is known as the “uncoupled growth” phenomenon, meaning cells will rapidly
convert sugars to ethanol regardless of their energy requirements for growth. This makes Z.
mobilis attractive not only for ethanol production, but for alternative product synthesis as well
(Lynn, 2015).
Lignocellulosic biomass is one of the most abundant biological resources in nature with a
high energy content, which is why it has been considered a good option for the production of
biofuels. In addition, it has the advantage of being outside the human food chain, it is also an
attractive and relatively more economical raw material (Pereira et al., 2012). Another advantage
of using agricultural waste as raw materials for ethanol production is its low cost, easy availability,
avoiding conflicts with its use for food and the potential production of fuel from lignin (Liu et al.,
2012).
Many of the processes used to obtain biofuels are mainly based on the use of starches.
Therefore, much research has focused on exploring new carbon sources, such as lignocellulosic
biomass (Yang et al., 2016). Z. mobilis has used a variety of materials for the production of ethanol
(sugar cane, sugar beet, carob, sweet potato, and sweet sorghum), energy plants (eg, rod grass),
industrial waste (sugar flour) soybeans, a co-product of soybean oil and cornmeal production),
food waste, agricultural residues (corn cob residues, rice bran, sweet sorghum stalk, sugar cane
molasses, bamboo residues and sludge of waste paper), as well as biomass from Spirogyra hyalina
algae. (Behera et al., 2012; He et al., 2013; Saharkhiz et al., 2013; Yang et al., 2013; Peralta -
Jaime Oliver Santos Pinargote

José David Villalta Pastuzo 2
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e-ISSN: 2602-8476, ISSN: 1390-7662
Contreras et al., 2014; Todhanakasem et al., 2014; Gu et al., 2015; Ma et al., 2015; Sulfahri et al.,
2016).
The main function of the hydrolysis pretreatment of the residual plant biomass, be it in a
chemical or physical way, is to eliminate or destroy the lignin and other residues that prevent
access to sugars by Z. mobilis; and thus obtain a higher yield at the time of fermentation due to the
release of sugars such as: glucose, xylose, arabinose; As well as a series of minor compounds,
much research has been carried out regarding pretreatments that optimize the fermentation process
(Harmsen et al., 2010; Mood et al., 2013), physical (Mosier et al., 2005) physical-chemical (Sun
and Cheng, 2002; Wyman et al., 2005) and biological pretreatment used (Wyman et al., 2005;
Sindhu et al., 2016). The focus on pretreatment and conditioning research improves digestibility,
solid concentrations, and ethanol production from lignocellulosic feedstocks (Esteghlalian et al.,
1997; Mohagheghi et al., 2004; Mosier et al., 2005; Kumar et al., 2009).
The cell immobilization has an important role during the fermentation processes (He et al.,
2011; Panesar et al., 2011) because it can offer many advantages, for example the high productivity
and good cell stability (Argyrios et al., 1983; Genisheva et al., 2011). A variety of support materials
have been tested for cell immobilization, including inorganic and organic material (Avinash et al.,
2001; Akdogan and Pazarlioglu, 2015; Banerjee and Ghoshal, 2011).
The objective of this research was to evaluate the fermentation activity of Z. mobilis UGB-
SA2001 to produce bioethanol with free and immobilized cells using three products of agricultural
residue; sugar beet pulp (SBP), banana pseudostem (BPS) and molasses. Furthemore, emphasis is
placed on the evaluation of the banana pseudostem as it is an agricultural waste that is abundant in
Ecuador as it is a banana exporting country. In addition to not finding studies that have evaluated

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this type of biomass with Z. mobilis.
Materials and methods
Microorganism and culture condition
The bacteria strain used was isolated from Agave spp. (Obire, 2005; Fawole and Oso,
2004), of the ecuadorian highlands, this strain is conserved in the Biotechnology Laboratory of the
Faculty of Natural Sciences-University of Guayaquil under the code UGB-SA2001. With the
following geographical location: W 79º54´59.04´´ - S 2º08´57.12´´.
Z. mobilis was cultured in growth synthetic medium consisting of Sucrose 150 g·L-1, yeast
extract 2.0 g·L-1, peptone 2.0 g·L-1, KH2PO4 2.0 g·L-1, (NH4)2SO4 2.0 g·L-1 and
MgSO4·7H2O 2.0 g·L-1. Z. mobilis was inoculated into a 50 mL Erlenmeyer flask at 30 °C for
24 hours and pH7 (Ronald, 2010).
Immobilization Z. mobilis
After 24 hours of growth, the Z. mobilis biomass was centrifuged at 8000 rpm for 10
minutes. The pellet was mixed with the 16% sodium alginate solution trying to maintain a ratio of
0.5 g of biomass per gram of alginate, then mixed with 4% calcium chloride solution.
Lignocellulosic biomass
Molasses, SBP and BPS were used. In the case of molasses, it was not subjected to any
pre-treatment and it was evaluated three concentrations for fermentation 25%, 50% and 75%. The
beet bagasse and banana pseudostem were cut and dried in an oven (Memmert) at 50 °C for 72
hours.
Sugar beet pulp and Banana pseudostem hydrolysis
Alkaline hydrolysis

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A suspension Calcium hydroxide 5 % w/v was added to the SBP and BPS keeping a 10:1
liquid/solid ratio and hydrolyzed in an oven at 80 °C for 72 hours. After the hydrolysis, the samples
were washed successively with distilled water until the excess calcium hydroxide was removed
and neutralized with 20% citric acid for subsequent fermentation.
Acid hydrolysis
40 g of SBP and BPS were added individually in Erlenmeyer flask containing 1 L-1 of
sulfuric acid 50% (v/v). The samples were homogenized for five minutes using a shaker at low
speed and then hydrolyzed at 90°C for 72 hours. After hydrolysis, the samples were washed
successively with distilled water until excess sulfuric acid was removed and neutralized with 1M
potassium hydroxide.
Fermentation culture media
Fermentation culture media were prepared in triplicate in Erlenmeyer flask at 1000 mL of
final volume composed yeast extract 2.0 g·L-1, 2.0 g·L-1 KH2PO4, 2.0 g·L-1 (NH4)2SO4 and
2.0 g·L-1 MgSO4·7H2O. These media were added 25%, 50% and 75% (v/v) of molasses. Also,
beet bagasse 70 g·L-1 (p/v) and banana pseudostem 70 g·L-1 (p/v). Total reducing sugars control
was maintained every 24 hours during fermentation at 32±2 °C for 120 hours. A synthetic medium
was used as a control.
Total reducing sugars analysis
For the determination of total Reducing sugars (TRS), the spectrophotometric method
(Thermo Scientific GENESYS 20vis) specified by Miller was used (Miller, 1959). Various
volumes of samples solutions were delivered into the quartz cuvettes and the volume was
completed to 100 µL with distilled water. As blank, water was used. Standards were also included,

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ranging from 0 µg·mL−1 to 100 mg·mL−1 concentration of glucose. DNS reagent (100 µL) was
added to each sample, mixed well and were kept for 4 minutes. Finally, the samples were
absorbance reader at 540 nm.
Ethanol Determination
Firstly, a process of separating ethanol from the fermentation medium by means of
evaporation using a rotavapor (BUCHI Rotavapor ™ R-100) was carried out, then the
quantification of ethanol was carried out using the method proposed by Caputi Ueda, and Brown
(1968). In several flasks, 25 mL of sulfochromic solution and 1 mL of ethanol were added at
different concentrations (0.2%, 0.4%, 0.6%, 0.8% and 1%) to prepare the calibration curve and it
was used 1mL of the distillate for the sample and 1 mL of distilled water as blank. They were then
incubated in a water bath for 15 minutes at 80 °C. Then it was allowed to cool and the
absorbance at 600 nm was measured.
Data analysis
The research design used was CRD (Completely Randomized Design). This study was
conducted three times. The parameters measured were total reducing sugars and ethanol levels.
Data were analyzed statistically using analysis of variance (ANOVA) with level of confidence
95%. Analysis was carried out to determine the effect of the alkaline and acid hydrolysis process
of on total sugar production.
Results and discussion
Effect of hydrolysis process
The results show that obtaining reducing sugars will depend on the type of biomass and
the treatment they receive. This is how the pseudostem banana present higher concentration of

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TRS 140 g·L-1 in alkaline hydrolysis while sugar beet obtained in acid hydrolysis 110 g·L-1
(Figure 1).
This difference is explained in researches carried out previously, where it is analyzed the
degradation of hemicellulose and lignin with alkaline, acid and peroxide as pre-treatments SBP
and BPS for alcoholic fermentation.
Figure 1
Effect of acid and alkaline hydrolysis on lignocellulosic material
Detailed evaluation of the effects of different pretreatments on BPS showed that all severe
pretreatments resulted in low solid recovery, in agreement with the high amount of hemicellulose
and lignin removal from these samples. Acid pretreatment was particularly effective at removing
hemicellulose, while alkaline and peroxide pretreatments removed both hemicellulose and lignin
effectively. The removal of biomass components exposed cellulose, increasing its accessibility to
the TRS. However, the most severe pretreatments with acid and peroxide resulted in cellulose
degradation, increasing biomass recalcitrance relative to lower severity conditions (Lange et al.,
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2018; Brienzo et al., 2017; Junior, Milagres, and Ferraz, 2013; Kusmiyati and Ryzka, 2018).
In the case of SBP, the lignin portion may be removed or simply disrupted and in some
cases the hemicellulose fraction may also be degraded during pretreatment
(Hendriks and Zeeman, 2009; Kaar and Holtzapple, 2000). The highest levels of TRS was
found to be 42.4 g·L-1, which resulted from a lime loading of 0.4 g·g SBP and a pretreatment time
of 36 hours at 40 °C. With similar conditions for lime loading and pretreatment time but with the
highest temperature (70 °C), TRS was much lower at 6.3 g·L-1, but it is not clear why the increased
temperature had this effect (Dredge et al., 2009). What agrees with the present study, due to the
temperature used for the alkaline hydrolysis for SBP was 80 °C obtaining a low concentration of
TRS.
Furthermore, dilute sulfuric acid pretreatment and enzymatic hydrolysis of SBP have been
investigated. Mass balances on cellulose, hemicellulose, pectin, and protein were performed and
sugar degradation products such as 5-hydroxymethylfurfural (HMF), furfural and acetic acid were
monitored. Acid pretreatment increased the enzymatic digestibility of SBP from 33% (raw) to 93%
(treated). Pretreatment at optimum conditions (temperature = 120 °C, acid concentration = 0.66%
and solid loading = 6%) (Zheng et al., 2012). Likewise, the present investigation showed that acid
hydrolysis has a better yield for obtaining TRS at elevated temperatures for SBP with a yield of
110 g·L-1.
TSR consumption in free and immobilized cells
In the fermentation period, the treatments that were performed with 75% (v/v) molasses
presented the highest TRS consumption with immobilized and free cells, until 50% (425±1.5 g·L-
1) for 120 hours of fermentation (Figure 2). This is in agreement with studies carried out with Z.

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mobilis in immobilized systems using different types of matrices such as luffa sponge discs,
sodium and calcium alginate gel beads.
In which decreases of up to 47.05% and 47.93% in the total sugar concentration on the
initial content have been reported during 24 h of fermentation (Shuvashish, Rama, and Ramesh,
2012).
Figure 2.
TSR consumption by Z. mobilis.
Note. M: molasses; SBPC: Sugarbeet pulp treated with whit alkaline hydrolysis; SBPA: Sugar
beet pulp acid-treated; BPSA: Banana pseudostem whit acid hydrolysis; BPSC: Banana
pseudostem whit alkaline hydrolysis; MC: medium control.
Likewise, the consumption of reducing sugars in the tests with banana pseudostem and beet
bagasse was higher with immobilized cells 122.22±0.2 g·L-1 and 44.12±0.4 g·L-1 respectively.
The experiment with banana pseudostem was the one with the highest consumption with

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approximately 82% during 120 hours. Research carried out with Saccharomyces cereviseae in
banana pseudostem show that it is able to reduce sugars from alkaline hydrolysis up to 84% during
a period of 72 hours (Ingale, Sanket, and Gupte, 2014). In the case of the low consumption of
reducing sugars from acid solutions 3.27 g·L-1 in BPS this may be due to the generation of a
certain quantity of inhibitors during pre-treatment with diluted acid, therefore detoxification may
be needed by washing with water to reduce or remove inhibitors. Furthermore, a large amount of
protein was hydrolyzed in SBP during acid pretreatment, suggesting that the addition of nitrogen
(i.e. trypton and / or yeast extract) could be reduced or eliminated in the SSF process (Zheng et al.,
2012).
Ethanol production
During the production of ethanol with free cells and immobilized cells there is no
statistically significant difference, since the P-value 0.6936 is greater than 0.05. Free cells during
the fermentation period obtained the highest values, with molasses media at 50% and 75%
obtaining ethanol concentrations of 30.8 g·L-1 and productivity of 0.26 g·L·h-1 in both cases
(Table 1). In other studies, Z. mobilis have produced 9.3% (v/v) of bioethanol when using 16 g per
each 100 mL of sugar with a fermentation efficiency of 92.5% (Khoja et al., 2018). The low
production shown in this work could be due to an inhibition of cell growth at high concentrations
of molasses. This clearly demonstrates the negative effect of increasing molasses concentration on
fermentation kinetics and yield (Damilios et al., 2005).
In contrast to the results observed in TSR consumption where free cells have the lower
sugar consumption than with immobilized cells. The decrease in sugar reserve might be also due
to its utilization in part, for initial growth and metabolism of the bacteria in addition to its

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conversion into ethanol (Behera et al., 2010).
The test with BPS and SBP obtained a low productivity and ethanol concentration between
0.01 and 0.05 g·L·h-1 and 1.7-16 g·L-1 (Table 1). Despite the results obtained, the production
ranges are among the standards reported in similar studies, where they report concentrations of
around 4.32 g·L-1 from banana pseudostem pretreated with a 2N NaOH solution (Ingale, Sanket,
and Gupte, 2014). Furthermore, ethanol concentrations of up to 9.2 L per 100 kg have been
obtained with beet sugar, the fermenting organism being Saccaromyces cereviseae (Gumienna et
al., 2014)
Table 1.
Zymomonas mobilis ethanol production parameters with free cells.
SUBSTRAT EO: ET: ETc: Yp/st Yp/sc %E P

E g·L-1 g·L-1 g·L-1 g·L -1
g·L -1
g·h /L-1
-1
Molasses 30,8±0. 433,50±0 216,75±0 0,04±0.0 0,07±0.0 14,21±0. 0,26±0.0

75% 2 .1 .2 01 1 2 1
Molasses 30,8±0. 247,71±0 55,04±0. 0,06±0.0 0,29±0.0 55,95±0. 0,26±0.0
50% 2 .5 3 01 1 4 1
Molasses 19,1±0. 123,42±0 36,72±0. 0,08±0.0 0,27±0.0 52,02±0. 0,16±0.0
25% 1 .1 1 03 3 1 2
BPS (Acid) 16±0.3 76,98±0. 19,18±0. 0,04±0.0 0,16±0.0 30,76±0. 0,05±0.0
1 1 01 1 7 1
BPS 3,9±0.0 72,68±0. 43,10±0. 0,03±0.0 0,19±0.0 9,05±0.1 0,03±0.0
(Alkaline) 1 2 1 05 5 01
SBP (Acid) 5,5±0.0 48,00±0. 10,50±0. 0,05±0.0 0,27±0.0 52,38±0. 0,05±0.0
1 2 1 01 1 1 02
SBP 1,7±0.1 4,34±0.5 1,67±0.2 0,20±0.0 0,52±0.0 95,94±0. 0,01±0.0
(Alkaline) 02 5 5 01
Control 7,2±0.1 78,82±0. 57,14±0. 0,05±0.0 0,06±0.0 12,60±0. 0,06±0.0
1 2 02 1 1 1
Note. EO: ethanol obtained. ET: theoretical ethanol by TSR concentration, ETc: theoretical
ethanol by TSR consumed, Yp/st: yield per total substrate, Yp/sc: yield per consumed substrate,
% E: percentage of conversion efficiency and P: productivity.

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The immobilized cells produced a maximum concentration of ethanol of 19.2±0.02 g·L-1
in the molasses medium (Table 2). This result infers with the values showed by Z .mobilis
(ATCC® 2601) with a production of 50 g·L-1. Despite having been suspended in the same type
of material with the difference that (ATCC® 2601) was suspended and mixed with a 4% sodium
alginate solution (1:1 v:v) to obtain a mixture of 2% microorganism–alginate suspension (Ruiz et
al., 2016).
Table 2.
Zymomonas mobilis ethanol production parameters with free immobilized
SUBSTRAT EO: ET: ETc: Yp/st Yp/sc %E P

E g·L-1 g·L-1 g·L-1 g·L-1 g·L-1 g·h-1/L-1
Molasses 18,1±0. 433,50±0 216,75±0 0,02±0.0 0,04±0.0 8,35±0. 0,15±0.0
75% 01 .5 .4 01 01 1 1
Molasses 19,2±0. 247,71±0 103,21±0 0,04±0.0 0,09±0.0 18,60±0 0,16±0.0
50% 02 .2 .7 02 03 .4 1
Molasses 8±0.01 123,42±0 32,18±0. 0,03±0.0 0,13±0.0 24,86±0 0,07±0.0
25% .6 1 01 1 .3 01
BPS (Acid) 5.9±0.0 75,38±0. 22,85±0. 0,11±0.0 0,36±0.0 70,03±1 0,13±0.0
3 1 1 3 1 .0 2
BPS 1±0.01 75,39±0. 62,33±0. 0,01±0.0 0,02±0.0 1,60±0. 0,01±0.0
(Alkaline) 1 5 01 01 01 01
SBP (Acid) 4,8±0.0 52,50±0. 22,50±0. 0,05±0.0 0,11±0.0 21,33±0 0,04±0.0
1 3 1 02 5 .3 03
SBP 1,7±0.0 4,34±0.0 2,45±0.0 0,20±0.0 0,35±0.0 69,44±2 0,01±0.0
(Alkaline) 3 5 1 4 1 01
Control 19,6±0. 78,82±0. 52,14±0. 0,13±0.0 0,19±0.0 37,59±0 0,16±0.0
05 1 1 02 1 .2 4
Note. EO: ethanol obtained. ET: theoretical ethanol by TSR concentration, ETc: theoretical ethanol
by TSR consumed, Yp/st: yield per total substrate, Yp/sc: yield per consumed substrate, % E:
percentage of conversion efficiency and P: productivity.
The low yield in our strain could be due to the higher alginate sodium concentration used

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16%, probably avoiding further substrate-cell interaction. Despite having used sodium alginate in
immobilization, the yield percentage was higher than that the reported in research where the
immobilization was carried out with calcium alginate, where they obtained concentrations of
ethanol up to 7.28 g·L-1 in enriched synthetic medium (Musfil et al., 2010).
The present investigation in synthetic culture medium obtained up to 19.6±0.05 g·L -1 and
in culture media with molasses 50% and 75% with values between 18±0.01 and 19.2±0.02 g·L -1.
Which represents a high potential of the strain to withstand high concentrations of TSR in
immobilized conditions.
Conclusions
In the present research it was determined that the best fermentable substrates for the Z.
mobilis UGB-SA 2001 strain was molasses at 50% and 75%. The Z. mobilis strain was determined
to be able to resist high concentrations of TRS up to 75%. Ethanol produced by Z. mobilis UGB-
SA 2001 is among the normal ranges for Z. mobilis strains.
On the other hand, in the present work it was possible to determine that the best
pretreatment conditions for the banana stem was alkaline treatment obtaining a high yield, higher
conversion efficiency and productivity. These results mark a precedent to begin to use the banana
pseudostem as a carbon source for fermentation processes, thus giving additional value to this type
of agricultural waste.
In the case of beet bagasse, the most effective treatment was performed with acid, despite
releasing high concentrations of sugar, it did not have an effective alcoholic fermentation, but it is
possible to use this process for another type of fermentation and can thus be used this type of
sugars in other processes giving the opportunity to optimize the use of this type of waste.

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Bibliography
Akdogan, H. and Pazarlioglu, N. (2011). Fluorene biodegradation by P. osteratus–Part II:
Biodegradation by immobilized cells in a recycled packed bed reactor. Process Biochemistry.
46(4): 840-846 doi. http://acikerisim.pau.edu.tr:8080/xmlui/handle/11499/6048.
Argyrios, M. Fahar, J. and Abbott, B. (1983). Advances in Ethanol Production using Immobilized
Cell Systems, Critical Reviews in Biotechnology. 1 (4): 339-
393, DOI: 10.3109/07388558309084660
Avinash, M. Srinivas, N. Kulkarni, S. and Kaiser, J. (2001). Mesoporous molecular sieve (MCM-
41) as support material for microbial cell immobilization and transformation of 2,4,6-
trinitrotoluene (TNT): a novel system for whole cell immobilization, Journal of Molecular
Catalysis B: Enzymatic. 16(1): 17-26. https://doi.org/10.1016/S1381-1177(01)00040-6.
Banerjee, A. and Ghoshal, A. (2011). Phenol degradation performance by isolated Bacillus cereus
immobilized in alginate. International Biodeterioration & Biodegradation - INT
BIODETERIOR BIODEGRAD. 65: 1052-1060. DOI: 10.1016/j.ibiod.2011.04.011
Behera, S. Mohanty, R. and Ray, R. (2010). Ethanol fermentation of mahula (Madhuca latifolia)
flowers using free and immobilized bacteria Zymomonas mobilis MTCC 92. Biologia. 65:
416–421 https://doi.org/10.2478/s11756-010-0041-7.
Behera, S. Mohanty, R. and Ray, R. (2012). Ethanol fermentation of sugarcane molasses
by Zymomonas mobilis MTCC 92 immobilized in Luffa cylindrica L. sponge discs and Ca‐
alginate matrices. Braz J Microbiol 43: 1499–1507. [PMC free article] [PubMed] [Google
Scholar].

And Free Cells
e-ISSN: 2602-8476, ISSN: 1390-7662
Brienzo, M. Fikizolo, S. Benjamin, Y. Tyhoda, L. and Görgens, J. (2017). Influence of pretreatment
severity on structural changes, lignin content and enzymatic hydrolysis of sugarcane bagasse
samples. Renew. Energy. 104: 271–280. DOI: 10.1016/j.renene.2016.12.037
Caputi, A. Ueda, M. and Brown, J. (1968). Spectrophotometric determination of ethanol in wine.
Am. J. Enol. Viti. 19: 160-165.
Damilios, N. Buzato, J. Celligoi, M. and Oliveira, M., (2005). Optimization of ethanol production
by Zymomonas mobilis in sugar cane molasses fermentation Otimização da produção de
etanol por Zymomonas mobilis na fermentação do melaço de cana-de-Açúcar. Semina:
Ciências Exatas e Tecnológicas. 26(1):17-22
DOI: http://dx.doi.org/10.5433/1679-0375.2005v26n1p17
Dredge, R. Radloff, S. and Van Dyk, J. (2011). Lime pretreatment of sugar beet pulp and evaluation
of synergy between ArfA, ManA and XynA from Clostridium cellulovorans on the pretreated
substrate. Biotech. 1: 151–159 https://doi.org/10.1007/s13205-011-0019-3.
Esteghlalian, A. Hashimoto, A. Fenske, J. and Penner, M. (1997). Modeling and optimization of the
dilute‐sulfuric‐acid pretreatment of corn stover, poplar and switchgrass. Bioresour Technol
59: 129–136. [Google Scholar].
Fawole, M. and Oso, B. (2004). Characterization of Bacteria: Laboratory Manual of Microbiology.
4th Edn. Spectrum Book Ltd, Ibadan, Nigeria. 24-33.
Genisheva, Z. Mussatto, S. Oliveira, J. and Teixeira, J. (2011). Evaluating the potential of wine-
making residues and corn cobs as support materials for cell immobilization for ethanol
production Industrial Crops and Products. 34(1):979-985. DOI:
10.1016/j.indcrop.2011.03.006.

And Free Cells
e-ISSN: 2602-8476, ISSN: 1390-7662
Gu, H. Zhang, J. and Bao, J. (2015). High tolerance and physiological mechanism of Zymomonas
mobilis to phenolic inhibitors in ethanol fermentation of corncob residue. Biotechnol Bioeng
112: 1770–1782. [PubMed] [Google Scholar]
Gumienna, M. Szambelan, K. Jeleń, H. and Czarnecki, Z. (2014). Evaluation of ethanol fermentation
parameters for bioethanol production from sugar beet pulp and juice, J. Inst. Brew.
120: 543– 549. DOI: 10.1002/jib.181.
Harmsen, P. Huijgen, W. Bermudez, L. and Bakker, R. (2010). Literature review of physical and
chemical pretreatment processes for lignocellulosic. Report number: ECN‐E—10‐013.
Energy Research Centre of The Netherlands, Petten, The Netherlands. [Google Scholar]
He, S. Lin, Y. Hou, K. and Hwang, S. (2011). Degradation of dimethyl-sulfoxide-containing
wastewater using airlift bioreactor by polyvinyl-alcohol-immobilized cell beads. Bioresour
Technol. 102(10):5609‐5616. doi:10.1016/j.biortech.2011.02.030. Bioresource Technology
102: 5609–5616.
He, M. Li, Q. Liu, X. Hu, Q. Hu, G. Pan, K. (2013). Bio‐ethanol production from bamboo residues
with lignocellulose fractionation technology (LFT) and separate hydrolysis fermentation
(SHF) by Zymomonas mobilis. Am J Biomass Bioenergy 1: 1–10. [Google Scholar]
Hendriks, A. and Zeeman, G. (2009). Pretreatments to enhance the digestibility of lignocellulosic
biomass. Bioresour Technol. 100(1):10‐18. doi:10.1016/j.biortech.2008.05.027
Ingale, S. Sanket, J. and Gupte, A. (2014). Production of bioethanol using agricultural waste:
Banana pseudo stem Brazilian Journal of Microbiology. 45(3): 885-892DOI: 10.1590/S1517-
83822014000300018

And Free Cells
e-ISSN: 2602-8476, ISSN: 1390-7662
Junior, C. Milagres, M. and Ferraz, A. (2013). The effects of lignin removal and drying on the
porosity and enzymatic hydrolysis of sugarcane bagasse. Cellulose. 20: 3165–3177.
https://doi.org/10.1007/s10570-013-0032-2
Kaar, W. Holtzapple, M. (2000). Using lime pretreatment to facilitate the enzymic hydrolysis of
corn stover. Biomass Bioenerg. 18:189–199 DOI: 10.1016/S0961-9534(99)00091-4
Khoja, A. Mohidin, Y. Hamdya, S. Nawar, A. Ansari, A. and Qayyum, M. (2018). Fermentation of
Sugarcane Molasses Using Zymomonas Mobilis for Enhanced Bioethanol Production. Journal
of Advanced Research in Applied Sciences and Engineering Technology. 11 (1): 31-38.
Kumar, P. Barrett, D. Delwiche, M. and Stroeve, P. (2009). Methods for pretreatment of
lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind Eng Chem Res
48: 3713–3729. [Google Scholar]
Kusmiyati, and Ryzka, P. (2018). Pretreated of banana pseudo-stem as raw material for enzymatic
hydrolysis and bioethanol production. MATEC Web of Conferences 154, 01035.
https://doi.org/10.1051/matecconf/201815401035
Lange, S. Queiroz, M. Ciconello, P. Ranieri, B. Pagnoccac, F. De Souzad, W. Sant’Annab, C. and
Brienzo, M. (2018). Acid, alkali and peroxide pretreatments increase the cellulose
accessibility and glucose yield of banana pseudostem Industrial Crops & Products. 115:
62–68 DOI:10.1016/j.indcrop.2018.02.024
Liu, Y. Yang, C. Chen, W. and Wei, Y. (2012). Producing bioethanol from cellulosic hydrolyzate
via co-immobilized cultivation strategy. J Biosci Bioeng. 114(2):198‐203.
doi:10.1016/j.jbiosc.2012.03.005

And Free Cells
e-ISSN: 2602-8476, ISSN: 1390-7662
Lynn, K. (2015). Engineering Zymomonas mobilis for the production of biofuels and other value-
added products. Submitted in partial fulfillment of the requirements for the degree of Doctor
of Philosophy in Chemical Engineering in the Graduate College of the University of Illinois
at Urbana-Champaign. University of Illinois. Available from:
https://core.ac.uk/download/pdf/158311321.pdf
Ma, K. Ruan, Z. Shui, Z. Wang, Y. Hu, G. and He, M. (2015). Open fermentative production of
fuel ethanol from food waste by an acid‐tolerant mutant strain of Zymomonas mobilis .
Bioresour Technol 203: 295–302. [PubMed] [Google Scholar]
Miller, G. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar.
Anal.Chem. 31: 426-428. https://doi.org/10.1021/ac60147a030
Mohagheghi, A. Dowe, N. Schell, D. Chou, Y. Eddy, C. and Zhang, M. (2004). Performance of a
newly developed integrant of Zymomonas mobilis for ethanol production on corn stover
hydrolysate. Biotechnol Lett 26: 321–325. [PubMed] [Google Scholar]
Mood, S. Golfeshan, A. Tabatabaei, M. Jouzani, G. Najafi, G. Gholami, M. and Ardjmand, M.
(2013). Lignocellulosic biomass to bioethanol, a comprehensive review with a focus on
pretreatment. Renew Sust Energ Rev 27: 77–93. [Google Scholar]
Mosier, N. Wyman, C. Dale, B. Elander, R. Lee, Y. Holtzapple, M. and Ladisch, M.
(2005). Features of promising technologies for pretreatment of lignocellulosic
biomass. Bioresour Technol 96: 673–686. [PubMed] [Google Scholar]

And Free Cells
e-ISSN: 2602-8476, ISSN: 1390-7662
Musfil, A. Widjaj, T. Altway, A. and Darmawan, R. (2010). Ethanol Production from Molasses with
Immobilized Cells Technique in Packed Bed Bioreactor by Extractive. The Journal for
Technology and Science. 21: 29-32. DOI: 10.12962/j20882033.v21i1.26.
Obire, O. (2005). Activities of Zymomonas species in palm sap obtained from 3 areas in Edo State,
Nigeria. Journal of Applied science and Environmental management. 19(1): 2530 - 2539.
Panesar, R. Panesar, P. Singh, R. and Kennedy, J. (2011). Hydrolysis of milk lactose in a packed bed
reactor system using immobilized yeast cells. J. Chem. Technol. Biotechnol. 86: 42-46.
doi:10.1002/jctb.2481
Peralta‐Contreras, M. Aguilar‐Zamarripa, E. Perez‐Carrillo, E. Escamilla‐Garcia, E. and Serna‐
Saldivar, S. (2014). Ethanol production from extruded thermoplastic maize meal by high
gravity fermentation with Zymomonas mobilis . Biotechnol Res Int 2014: 654853. [PMC free
article] [PubMed] [Google Scholar]
Pereira, S. Špela, I. Evtuguin, D. and Serafim, L. (2012). Biological treatment of eucalypt spent
sulphite liquors: A way to boost the production of second generation bioethanol, Bioresource
Technology. 103(1);131-135. https://doi.org/10.1016/j.biortech.2011.09.095.
Ronald, M. (2010). Handbook of microbiological media Fourth Edition Atlas Washington, D.C.
Boca Raton London New York CRC Press. 1955.
Ruiz, A. Canedo, Y. Narváez, A. and Robles, J. (2016). Producción de etanol por Saccharomyces
cerevisiae y Zymomonas mobilis Coinmovilizadas: propuesta para el uso de desechos
orgánicos. Agrociencia [online]. 50 (5): 551-563. Available from:
<http://www.scielo.org.mx/scielo.php?script=sci_arttext&pid=S140531952016000500551&l
ng=en&nrm=iso>. ISSN 2521-9766.

And Free Cells
e-ISSN: 2602-8476, ISSN: 1390-7662
Saharkhiz, S. Mazaheri, D. and Shojaosadati, S. (2013). Evaluation of bioethanol production from
carob pods by Zymomonas mobilis and Saccharomyces cerevisiae in solid submerged
fermentation. Prep Biochem Biotechnol 43: 415–430. [PubMed] [Google Scholar]
Shuvashish, B. Rama, M. and Ramesh, C. (2012). Ethanol fermentation of sugarcane molasses by
Zymomonas mobilis MTCC 92 immobilized in Luffa cylindrica L. sponge discs and Ca-
alginate matrices. Braz. J. Microbiol. [Internet]. 43( 4 ): 1499-1507.
http://dx.doi.org/10.1590/S1517-83822012000400034.
Sindhu, R. Binod, P. and Pandey, A. (2016). Biological pretreatment of lignocellulosic biomass –
An overview. Bioresour Technol 199: 76–82. [PubMed] [Google Scholar]
Sulfahri, A. Sumitro, S. and Saptasari, M. (2016). Bioethanol production from algae Spirogyra
hyalina using Zymomonas mobilis . Biofuels. doi:10.1080/17597269.2016.1168028. [Google
Scholar]
Sun, Y. and Cheng, J. (2002). Hydrolysis of lignocellulosic materials for ethanol production: a
review. Bioresour Technol 83: 1–11. [PubMed] [Google Scholar]
Todhanakasem, T. Sangsutthiseree, A. Areerat, K. Young, G. and Thanonkeo, P. (2014). Biofilm
production by Zymomonas mobilis enhances ethanol production and tolerance to toxic
inhibitors from rice bran hydrolysate. N Biotechnol 31: 451–459. [PubMed] [Google
Scholar]

And Free Cells
e-ISSN: 2602-8476, ISSN: 1390-7662
Wyman, C. Dale, B. Elander, R. Holtzapple, M. Ladisch, M. and Lee, Y. (2005). Coordinated
development of leading biomass pretreatment technologies. Bioresour Technol 96: 1959–
1966. [PubMed] [Google Scholar]
Yang, S., Fei, Q., Zhang, Y., Contreras, L. M., Utturkar, S. M., Brown, S. D., Himmel, M. E., &
Zhang, M. (2016). Zymomonas mobilis as a model system for production of biofuels and
biochemicals. Microbial biotechnology, 9(6), 699–717. https://doi.org/10.1111/1751-
7915.12408
Yang, S. Pan, C. Tschaplinski, T. Hurst, G. Engle, N. Zhou, W. (2013). Systems biology analysis of
Zymomonas mobilis ZM4 ethanol stress responses. PLoS ONE 8: e68886. [PMC free article]
[PubMed] [Google Scholar]
Zheng, Y. Lee, C. Yu, C. Cheng, Y. Zhang, R. and Jenkins, B. (2012). Vander JS. Dilute acid
pretreatment and fermentation of sugar beet pulp to etanol. Applied Energy. 93: 168–175.


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Revista Carácter, diciembre 2020, Vol. 8, No.

BIOETHANOL PRODUCTION FROM AGRICULTURAL RESIDUE BIOMASS BY

Jaime Oliver Santos Pinargote1

Recibido: septiembre 2020 / Aceptado: Diciembre 2020 / Publicado: diciembre 2020

Keywords: Acid, alkaline, banana, beet, molasses

Zymomonas mobilis is a promising organism for lignocellulosic biofuel production as it

Jaime Oliver Santos Pinargote

2001; Akdogan and Pazarlioglu, 2015; Banerjee and Ghoshal, 2011).

Jaime Oliver Santos Pinargote

Materials and methods

Microorganism and culture condition

following geographical location: W 79º54´59.04´´ - S 2º08´57.12´´.

24 hours and pH7 (Ronald, 2010).

Sugar beet pulp and Banana pseudostem hydrolysis

Jaime Oliver Santos Pinargote

and neutralized with 20% citric acid for subsequent fermentation.

Fermentation culture media

Fermentation culture media were prepared in triplicate in Erlenmeyer flask at 1000 mL of

was used as a control.

Total reducing sugars analysis

Jaime Oliver Santos Pinargote

absorbance reader at 540 nm.

Firstly, a process of separating ethanol from the fermentation medium by means of

(1968). In several flasks, 25 mL of sulfochromic solution and 1 mL of ethanol were added at

absorbance at 600 nm was measured.

of on total sugar production.

Results and discussion

Effect of hydrolysis process

Jaime Oliver Santos Pinargote

and BPS for alcoholic fermentation.

Effect of acid and alkaline hydrolysis on lignocellulosic material

cases the hemicellulose fraction may also be degraded during pretreatment

TSR consumption in free and immobilized cells

Jaime Oliver Santos Pinargote

sodium and calcium alginate gel beads.

TSR consumption by Z. mobilis.

pseudostem whit alkaline hydrolysis; MC: medium control.

Jaime Oliver Santos Pinargote

fermentation kinetics and yield (Damilios et al., 2005).

Jaime Oliver Santos Pinargote

Zymomonas mobilis ethanol production parameters with free cells.

SUBSTRAT EO: ET: ETc: Yp/st Yp/sc %E P

Molasses 30,8±0. 433,50±0 216,75±0 0,04±0.0 0,07±0.0 14,21±0. 0,26±0.0

% E: percentage of conversion efficiency and P: productivity.

Jaime Oliver Santos Pinargote

alginate solution (1:1 v:v) to obtain a mixture of 2% microorganism–alginate suspension (Ruiz et

Zymomonas mobilis ethanol production parameters with free immobilized

SUBSTRAT EO: ET: ETc: Yp/st Yp/sc %E P

percentage of conversion efficiency and P: productivity.

Jaime Oliver Santos Pinargote

ethanol up to 7.28 g·L-1 in enriched synthetic medium (Musfil et al., 2010).

SA 2001 is among the normal ranges for Z. mobilis strains.

Jaime Oliver Santos Pinargote

Akdogan, H. and Pazarlioglu, N. (2011). Fluorene biodegradation by P. osteratus–Part II:

Biodegradation by immobilized cells in a recycled packed bed reactor. Process Biochemistry.

46(4): 840-846 doi. http://acikerisim.pau.edu.tr:8080/xmlui/handle/11499/6048.

Cell Systems, Critical Reviews in Biotechnology. 1 (4): 339-

393, DOI: 10.3109/07388558309084660

Catalysis B: Enzymatic. 16(1): 17-26. https://doi.org/10.1016/S1381-1177(01)00040-6.

immobilized in alginate. International Biodeterioration & Biodegradation - INT

BIODETERIOR BIODEGRAD. 65: 1052-1060. DOI: 10.1016/j.ibiod.2011.04.011

Behera, S. Mohanty, R. and Ray, R. (2012). Ethanol fermentation of sugarcane molasses

Jaime Oliver Santos Pinargote