Study On Ethanol Production From Sugar Cane Molasses by Using Irradiated Saccharomyces Cervisiae
Study On Ethanol Production From Sugar Cane Molasses by Using Irradiated Saccharomyces Cervisiae
Study On Ethanol Production From Sugar Cane Molasses by Using Irradiated Saccharomyces Cervisiae
ABSTRACT
In commercial ethanol production procedures often use sugar cane
molasses as a raw material due to their abundance and low costs. The most
employed microorganisms used for fermentation is Saccharomyces cerevisiae
yeasts due to its ability to hydrolyze sucrose from sugar cane molasses into
glucose and fructose, two easily assimilable hexoses.
The aim of this study was to evaluate the effect of gamma irradiation on
the activity of S. cerevisiae in the ethanol production yeast cells exposed to
different doses of gamma rays (0.05, 0.10, 0.2, 0.4, 0.6, 0.8 and 1.0 kGy. The
sugar cane substrate was optimized after maintaining deferent levels of sugar
concentrations (12-21%), medium pH (4.0-5.5), incubation temperature (25-
40oC) and rate of fermentation (24-168) h.
Data showed that rate of ethanol production was maximum by using the
irradiated S. cerevisiae cells at 0.1 kGy. dose at fermentation conditions as 15%
sugar concentration, initial pH 4.5, incubation temperature 30oC, fermentation
time 96 h at a fermentation medium volume 250 ml found in 500ml erlenmyer
flasks.
Keywords, Saccharomyces cerevisiae, Irradiation, Sugar cane molasses, ethanol.
INTRODUCTION
Ethanol is one of the most advanced liquid fuels because it is
environmental friendly. It is a clear, colorless liquid with a characteristic,
agreeable odor. In dilute aqueous solution, it has a sweet flavor, but in more
concentrated solutions it has a burning taste (Patil 1991). It is an alcohol, a
group of chemical compounds whose molecules contain an OH group, bonded
to a carbon atom. It melts at -114.1°C, boils at 78.5°C and has a density of
0.789 g/ml at 20°C (Kaur and Kocher 2002). Ethanol is produced by
fermentation: when certain species of yeast (notably Saccharomyces cerevisiae)
154 Botros, H.W et al., J. Rad. Res. Appl. Sci., Vol. 5, No. 2(2012)
metabolize sugar in the absence of oxygen, they produce ethanol and carbon
dioxide. Ethanol is particularly useful in industrial applications because of its
relatively high affinity for both water and organic compounds. The composition
of other alcohols limits their flexibility as compared to ethanol (Anxo et al.,
2008).
The molasses is obtained from different sources such as cane, beet and
citrus etc. It is a syrupy material left after the removal of sugar from the mother
syrup. The viscous material is composed of sucrose, glucose and fructose at
total carbohydrate concentration of 45-60% (w/v). The molasses is of three
types, the black strap, refinery and invert or high test molasses. Cane molasses
has less sucrose and more invert sugar, and lower content of nitrogen and
raffinose, more intense color and more buffer capacity (Wang et al., 1985;
Borzani et al., 1993; Borzani, 2001).
The production of raw cane sugar from sugarcane juice results in the
formation of molasses, a byproduct that contains minerals regarded as
impurities in raw sugar (Hugot and Jenkins 1986). However, this purification
process results in the loss of some high value disaccharides and
monosaccharides from the final raw sugar product that end up in the molasses.
The fermentable sugar content of molasses varies inversely with the purity of
the raw sugar produced at the factory. Molasses is a low value product that is
used as a cattle feed supplement, in specialized yeast propagation or as a
flavoring agent in some foods (Troiani, 2009a). Although the fermentable
sugars in the molasses cannot be further upgraded to raw sugar, they can be
converted to ethanol in a distillery. Hence, integrated sugarcane factories that
have sugar manufacturing co-located with an ethanol distillery can use molasses
as a feedstock for ethanol in addition to raw cane juice directly from the mill. A
significant number of sugarcane factories in Brazil and several hundreds of
others around the world are of this type (Szwarc, 2009).
Molasses is used in the baker’s yeast production, in the fermentation
technology for ethanol, citric, lactic and gluconic acids production, as well as
glycerol, butanol and acetone production, as an ingredient of mixed feeds or in
the production of amino acids (Belitz et al., 2009; Satyanarayana and Kunze,
2009). The fermentative yeast Saccharomyces cerevisiae is largely used in
ethanol production using such renewable biomass as sugar cane or sugar beet
molasses as the main carbon source (Echegaray et al., 2000; Sanchez and
Cardona, 2008). These types of S. cerevisiae were selected as production
Botros, H.W et al., J. Rad. Res. Appl. Sci., Vol. 5, No. 2(2012) 155
Methods:
Organism and culture maintenance:
Saccharomyces cervisiae maintained on yeast extract peptone glucose
(YPG) agar medium (pH 4.5), containing yeast extract (3.0 g/l), peptone (5.0
g/l), glucose (10.0 g/l), agar (20.0 g/l), The slants were incubated at 30°C for 1-
2 days for maximum growth.
Preparation of yeast cell suspension:
Sterilized distilled water (10 ml) was added to a 24-36 h old slant
culture of S. cerevisiae. The cells were scratched with a sterilized inoculating
needle and the tubes were shaken gently to form a homogeneous suspension.
The cell count was made using a Haemocytometer.
Development of inoculum:
Fifty milliliter of YPG medium was transferred to the individual 250ml
Erlenmeyer flasks. The flasks were cotton plugged, autoclaved and allowed to
cool at room temperature. One milliliter of cell suspension (2.74×106 CFU) was
added to each flask aseptically. The flasks were incubated in a rotary shaker
(160 rpm) at 30°C for 24h. Pre-grown culture of S. cerevisiae was centrifuged at
6000rpm for 15 min and yeast cells were separated out. The supernatant was
156 Botros, H.W et al., J. Rad. Res. Appl. Sci., Vol. 5, No. 2(2012)
discarded and the pellet was washed with saline water. It was re-centrifuged for
another 5 min to obtain the final pellet that was washed and then air-dried and
weighed. These were the free cells.
Pretreatment of molasses:
Pretreatment of molasses to reduce harmful compounds to the used
microorganisms (e.g. coloring substances, amino acids and heavy metals) that
may as inhibitors during fermentation. Sulfuric acid and activated carbon are
considered the best method for this purpose according to (Lazaridou et al.,
2002) by using molasses solution 20% total sugars were adjusted to pH 3 with
0.5M H2SO4. The solution was allowed to stand for 24 h and then centrifuged at
5000 g for 15min. The supernatant was also treated with 3% activated carbon by
stirred the mixture for 1h, then filtered through a Whatman no. 1 filter paper.
The pretreatment was repeated three the four times until the solution was almost
colorless and gave an absorbance reading at 490 nm.
Fermentation procedure and critical phases:
Three hundred milliliters of treated cane molasses with 15% (w/v) sugar
(initial pH 4.5) were taken into individual 500ml Erlenmeyer flasks. The flasks
were cotton plugged and steamed at 90°C in a water bath for 15-20 min. After
cooling to an ambient temperature, 2 g yeast cells were added to one flask and
placed in an incubator at 30°C for 120h. After the required incubation period,
the cells were separated out and stored for use of more experiments. The
fermented medium was used for estimation of ethanol and residual sugar
contents.
Assay methods:
The estimation of total reducing sugar was based on the dinitrosalicylic
acid (DNS) method. A double beam UV/VIS-scanning spectrophotometer was
used for measuring absorbance. Sugar contents in the supernatant were
determined by taking 1.0 ml of supernatant along with 2.0 ml of dinitrosalicylic
acid (DNS) reagent in a test tube. Blank containing 1.0 ml distilled water and
2.0 ml of DNS was run parallel. The tubes were heated in a boiling water bath
for 15 min. After cooling the tubes at room temperature, added 8 ml of distilled
water in each and absorbance was noted at 546nm using spectrophotometer.
Sugar concentration was determined from the standard curve of glucose.
Botros, H.W et al., J. Rad. Res. Appl. Sci., Vol. 5, No. 2(2012) 157
Ethanol estimation
Dichromate method:
Ethanol was also determined with good precision by oxidation with acid
dichromate solution (Kiransree et al., 2000). The ethanol in the known masses
of the solution was oxidized to acetic acid using a known mass of standard
potassium dichromate (0.1N) in the presence of sulfuric acid.
Irradiation of organism:
Slants of S. cerevisiae cells were exposed for doses of 0.05, 0.1, 0.2,
0.4, 0.6, 0.8 and 1.0 kGy of gamma rays using cobalt-60 gamma radiation cell at
the Nuclear Research Center, Inshas, Egypt. The dose rate at the time of
radiation treatment was 3.1 kGy/hr.
RESULTS AND DISCUSSION
Effect of gamma irradiation on growth of Saccharomyces cerevisiae cells:
ethanol production and 4.7% for sugar consume and the these data decreased by
increasing the pH value. Also, the value of ethanol production and sugar
consume increased by increasing the rate time of fermentation in all initial pH
values. Maximum production of ethanol yield was in yeast samples irradiated
for 0.1 kGy and the sugar consume was 16.1% at 96 h incubation at pH value
4.5 in initial cane molasses medium. These results showed that irradiation of
yeast cells at dose 0.1 kGy increased its activity and increase the yield of
ethanol production during fermentation process of cane molasses. So, data
showed that the rate of ethanol production by yeast cells is highly affected by
the pH of the fermentation medium. S. cerevisiae showed maximum growth
under acidic conditions and exhibited the maximum yield of ethanol (7.1%) at
pH 4.5 after 96h incubation time as similar to Rapeanu et al. (2000a). More
acidic and basic conditions, both retard the yeast metabolic pathways and hence
the growth of cells (Willaert and Viktor 2006). Productivity was decreased by
increase and decrease in pH due to the growth of other microbes of other
microbes as the fermentation was carried out without sterilization (Amutha and
Paramasamy 2001; Kourkoutas et al., 2004). In addition, pH of the surrounding
medium change the configuration and permeability of the cell membrane thus
reduced the rate of sugar fermented enzymes.
Table (4): Effect of the initial pH of cane molasses on ethanol production by
irradiated S. serevisiae cells (at 0.1 kGy) dose.
pH
Rate of 4.0 4.5 5.0 5.5
fermentation Ethanol Sugar Ethanol Sugar Ethanol Sugar Ethanol Sugar
(h) production consump- production consump- production consump- production consump-
ethanol production with irradiated (0.1 kGy) yeast cells. The temperature ranged
from 25-40oC and caused of fermentation was studied from 24-168 hours. Data
showed that ethanol yield increased with increasing the incubation period in
parallel with increasing in sugar consume. The optimal ethanol production was
obtained at 30oC by the irradiated cells giving ethanol yield 6.8% with 13.1%
sugar consumption at 96 hours of incubation. Changing in the incubation
temperature caused change in ethanol yield and sugar consumption, where at
slightly higher temperature growth rate may be adversely affected. Some strains
of S. cerevisiae have also been reported, capable of growing and fermenting
cane molasses at 40-45oC under batch conditions (Wang et al., 1985; Cachot
and Marie-Noelle 1991; Amutha and Paramasamy 2001).
Table (5): Effect of incubation temperature on ethanol production by irradiated S.
serevisiae cells (at 0.1 kGy) dose.
Incubation temperature oC
Rate of 25oC 30oC 35oC 40oC
fermentation Ethanol Sugar Ethanol Sugar Ethanol Sugar Ethanol Sugar
(h) production consump- production consump- production consump- production consump-
In the present study, on the basis of ethanol yield 96 hrs was found to be
optimal for maximal production by the irradiated cells. It might be due to the
fact that the time necessary to complete fermentation of sugar cane molasses to
ethanol is correlated with the initial sugar concentration and yeast cells (Kaur
and Kocher, 2002; Willaert and Viktor, 2006).
Table (6): Effect of different initial sugar concentration on ethanol production by
irradiated S. serevisiae cells (at 0.1 kGy) dose.
Sugar content %
Rate of 12% 15% 18% 21%
fermentation Ethanol Sugar Ethanol Sugar Ethanol Sugar Ethanol Sugar
(h) production consump- production consump- production consump- production consump-
CONCLUSION
The study showed that Saccharomy cerevisiae was able to produce
ethanol from cane molasses and fermentation process depends on sugar
concentration 15% and pH 4.5 and incubation temperature 30oC. Exposing S.
cerevisiae for (0.1 kGy) dose of gamma ray increased the ability of the yeast
cells for producing ethanol during the fermentation process of sugar cane at the
above conditions of fermentation.
REFERENCES
1. Amutha R. and Paramasamy G.; J. Biosci. Bioengi., 92: 560-564 (2001).
2. Anxo M.M., Lorenzo P., Jose A.V., Jesus M. and Maria P.G.;
Bioresourc. Technol., 99: 7255-7263 (2008).
3. Belitz, H.D., Grosch, W. and Schieberle P.; Food Chemistry, 4th edition,
Springer (2009).
4. Borzani W., Gerrab A., De-La-Higuera M.H., Pires R. and Piplovic N.;
World. J. Microbiol. Biotechnol., 9: 265-268 (1993).
5. Borzani W.; World. J. Microbiol. Biotechnol., 174: 253-258 (2001).
Botros, H.W et al., J. Rad. Res. Appl. Sci., Vol. 5, No. 2(2012) 163
24. Tyagi R.D. and Ghose T.K.; Biotechnol. Bioengin., 24: 781-795 (1982).
25. Wang L.H., Hsie M.C., Chang Y.C., Kuo S.L., Sang K. and Hsiao H.D.;
J. Bioengin., 28: 270-284 (1985).
26. Willaert R. and Viktor A.N.; J. Chem. Technol. Biotechnol., 81: 1353-
1367 (2006).
اﻟﻤﺆﺗﻤﺮ اﻟﺪوﻟﻲ اﻟﺜﺎﻟﺚ
ﻟﻠﻌﻠﻮم اﻹﺷﻌﺎﻋﯿﺔ وﺗﻄﺒﯿﻘﺎﺗﮭﺎ
١٦ – ١٢ﻧﻮﻓﻤﺒﺮ – ٢٠١٢اﻟﻐﺮدﻗﺔ -ﻣﺼﺮ
دراﺳﺔ ﻋﻠﻰ إﻧﺘﺎج اﻷﯾﺜﺎﻧﻮل ﻣﻦ ﻣﻮﻻس ﻗﺼﺐ اﻟﺴﻜﺮ ﺑﺎﺳﺘﺨﺪام ﺧﻼﯾﺎ اﻟﺨﻤﯿﺮة
اﻟﻤﺸﻌﻌﺔ
ھﺎﻧﺊ وھﯿﺐ ﺑﻄﺮس – أﺷﺮف ﺻﺒﺮى أﺣﻤﺪ – ﺳﻮزى ﺻﺒﺤﻰ ﻓﺮج – إﺳﻤﺎﻋﯿﻞ ﻋﺒﺪ اﻟﺤﻠﯿﻢ ﺣﺴﻦ
ﯾﺴﺘﺨﺪم ﻣﻮﻻس ﺳﻜﺮ اﻟﻘﺼﺐ ﻏﺎﻟﺒﺎً ﻓﻰ اﻹﻧﺘﺎج اﻟﺘﺠﺎرى ﻟﻜﺤﻮل اﻷﯾﺜﺎﻧﻮل ﻛﻤﺎدة ﺧﺎم ﻧﻈﺮًا
ﻟﺮﺧﺺ ﺛﻤﻨﮫ وﺗﻮاﻓﺮه .ﺧﻼﯾﺎ ﺧﻤﯿﺮة Saccharomyces cervisiaeﺗﺴﺘﺨﺪم أﺳﺎﺳﺎً ﻓﻰ إﻧﺘﺎج اﻷﯾﺜﺎﻧﻮل
ﻧﻈﺮاً ﻟﻘﺪرﺗﮭﺎ ﻋﻠﻰ ﺗﺤﻠﯿﻞ ﺳﻜﺮ اﻟﺴﻜﺮوز اﻟﻤﻮﺟﻮد ﻓﻰ ﻣﻮﻻس ﻗﺼﺐ اﻟﺴﻜﺮ وﺗﺤﻮﯾﻠﮫ إﻟﻰ ﺳﻜﺮ اﻟﺠﻠﻮﻛﻮز
واﻟﻔﺮﻛﺘﻮز ﺣﯿﺚ أﻧﮭﻤﺎ ﺳﻜﺮﯾﺎت ﺳﺪاﺳﯿﺔ ﺑﺴﯿﻄﺔ .ﺗﮭﺪف ھﺬه اﻟﺪراﺳﺔ ﻟﺘﻘﯿﯿﻢ إﺳﺘﺨﺪام أﺷﻌﺔ ﺟﺎﻣﺎ ﻋﻠﻰ ﻗﺪرة
ﺧﻼﯾﺎ اﻟﺨﻤﯿﺮة ﻓﻰ ﻋﻤﻠﯿﺔ إﻧﺘﺎج اﻷﯾﺜﺎﻧﻮل ﺣﯿﺚ ﺗﻢ ﺗﻌﺮﯾﻀﮭﺎ ﻟﺠﺮﻋﺎت )،٠٫٦ ،٠٫٤ ،٠٫٢ ،٠٫١ ،٠٫٠٥
(١٫٠ ،٠٫٨ﻛﯿﻠﻮﺟﺮاى ﻣﻦ أﺷﻌﺔ ﺟﺎﻣﺎ .ﺗﻤﺖ ﻋﻤﻠﯿﺔ ﺗﺨﻤﯿﺮ اﻟﻤﻮﻻس ﺗﺤﺖ ﻇﺮوف ﻣﺤﺘﻮى ﺳﻜﺮ )-١٢
،(%٢١وﺳﻂ ﺣﺎﻣﺾ ) ،(٥٫٥-٤ﺣﺮارة اﻟﺘﺤﻀﯿﻦ ) ( ٤٠-٢٥درﺟﺔ ﻣﺌﻮﯾﺔ وﻓﺘﺮات اﻟﺘﺤﻀﯿﻦ ﺗﺘﺮاوح
ﺑﯿﻦ ) (١٦٨-٢٤ﺳﺎﻋﺔ .أﻇﮭﺮت اﻟﻨﺘﺎﺋﺞ أن أﻋﻠﻰ ﻣﻌﺪل ﻹﻧﺘﺎج اﻷﯾﺜﺎﻧﻮل ﻛﺎن ﻋﻨﺪ ﺗﻌﺮﯾﺾ ﺧﻼﯾﺎ اﻟﺨﻤﯿﺮة
ﻟﺠﺮﻋﺔ ٠٫١ﻛﯿﻠﻮﺟﺮاى ﻣﻦ أﺷﻌﺔ ﺟﺎﻣﺎ ﻋﻨﺪ ﻧﺴﺒﺔ ﺳﻜﺮ ،%١٥درﺟﺔ ﺣﺎﻣﻀﯿﺔ ٤٫٥ﻋﻨﺪ درﺟﺔ ﺣﺮارة ٣٠
درﺟﺔ ﻣﺌﻮﯾﺔ وﻣﺪة ﺗﺤﻀﯿﻦ ٩٦ﯾﻮﻣﺎً.