Bioethanol Production

Although CO2 is the most important greenhouse gas (GHG), several studies show that it is
important to consider other GHGs as well. The continued use of fossil fuels to meet the
majority of the worlds energy demand is threatened by increasing concentrations of CO2
In the atmosphere and concerns over global warming. The combustion of fossil fuels is
responsible for 73% of the CO2 production. The heightened awareness of the global
warming issue has increased interest in the development of methods to mitigate GHG
emissions. Much of the current effort to control such emissions focuses on advancing
technologies that:
1) Reduce energy consumption.
2) Increase the efficiency of energy conversion or utilization.
3) switch to lower carbon content fuels,
4) enhance natural sinks for CO2,
5) Capture and store CO2.
Reducing use of fossil fuels would considerably reduce the amount of CO2 produced,
as well as reduce the levels of pollutants. To reduce the net contribution of GHGs to the
atmosphere, bioethanol has been recognized as a potential alternative to petroleumderived transportation fuels.

Overview
Ethanol is made from a variety of products such as grain, molasses, fruit, cobs, and shell; its
production, excluding that of beverages, has been declining since the 1930s because of the
low cost. With the oil crises of the 1970s, ethanol became established as an alternative fuel.
In 1975, only 76*106 lit of proof industrial ethanol was produced by fermentation
compared to 7.95*106 lit by synthesis corn ethanol production causes more soil erosion
than any other crop grown and uses more nitrogen fertilizer than any other crop grown.
These two environmental limitations also apply to sugar cane production in Brazil
properties

Properties of Bioethanol
Bioethanol has a higher octane number, broader flammability limits, higher flame
speeds and higher heats of vaporization than gasoline. These properties allow for a
higher compression ratio, shorter burn time and leaner burn engine, which lead to
theoretical efficiency advantages over gasoline in an internal combustion engine.
Disadvantages of bioethanol include its lower energy density than gasoline (bioethanol
has 66% of the energy that gasoline has), its corrosiveness, low flame luminosity, lower

vapor pressure (making cold starts difficult), miscibility with water, and toxicity to
ecosystems
Ethanol is an oxygenated fuel that contains 35% oxygen, which reduces particulate and
NOx emissions from combustion. Ethanol has a higher octane number (108), broader
flammability limits, higher flame speeds and higher heats of vaporization. These properties
allow for a higher
An oxygenate fuel such as bioethanol provides a reasonable antiknock value. Also, as it
contains oxygen, fuel combustion is more efficient, reducing hydrocarbons and particulates
in exhaust gases.
The presence of oxygen in bioethanol improves combustion and therefore reduces
hydrocarbon, carbon monoxide, and particulate emissions; but oxygenated fuels also tend
to increase nitrogen oxide emissions.

Blending of Ethanol with Gasoline

The most popular blend for light-duty vehicles is known as E85, and contains 85%
bioethanol and 15% gasoline. In Brazil, bioethanol for fuel is derived from sugar cane and
is used pure or blended with gasoline in a mixture called gasohol (24% bioethanol, 76%
gasoline)
In several states of the United States, a small amount of bioethanol (10% by volume) is
added to gasoline, known as gasohol or E10. Blends having higher concentrations of
bioethanol in gasoline are also used, e.g. in flexible-fuel vehicles that can operate on blends
of up to 85% bioethanolE85 [17]. Some countries have exercised biofuel program
involving both form bioethanolgasoline blend program, e.g. the United States (E10 and for
Flexible Fuel Vehicle (FFV) E85), Canada (E10 and for FFV E85), Sweden (E5 and for FFV
E85), India (E5), Australia (E10), Thailand (E10), China (E10), Columbia (E10), Peru (E10),
Paraguay (E7), and Brazil (E20, E25 and FFV any blend) [18].
Current status
In 2006, global production of bioethanol reached 13.5 billion gallons, up from 12.1 billion
gallons in 2005. Bioethanol currently accounts for more than 94% of global biofuel
production, with the majority coming from sugar cane. About 60% of global bioethanol
production comes from sugar cane and 40% from other crops. Brazil and the United States
are the world leaders, which exploit sugar cane and corn, respectively, and they together
account for about 70% of the world bioethanol production. However, the US and Brazil are
not oil independent countries. According to the Renewable Fuels Association
(RFA) 2007 figures. In the United States, 90% of bioethanol is derived from Corn

Bioethanol feedstock

Bioethanol feedstocks can be conveniently classified into three types:

1) Sucrose containing feedstock (e.g. sugar beet, sweet sorghum and sugar cane),
2) Starchy materials (e.g. wheat, corn, and barley),
3) lignocellulosic biomass (e.g. wood, straw, and grasses).

Two-third of world sugar production is from sugar cane and one-third is from sugar
Beet Compared to sugar cane, sugar beet requires 3540% less water and fertilizer.
The conversion of carbohydrates with 5 and 6 carbons into bioethanol is easier compared
to starchy materials and lignocellulosic biomass because previous hydrolysis of the
feedstock is not required since this disaccharide can be broken down by the yeast cells; in
addition, the conditioning of the cane juice or molasses favors the hydrolysis of sucrose

Reference
Mustafa Balat, Havva Balat, Cahide Oz. progress in Bioethanol processing, progress in
energy and combustion science 34 (2008) 551-578

Yeast Used for the production of Bioethanol

Zymomonas mobilis
Ethanol production from sugar cane molasses was analyzed under different culture
conditions using Z. mobilis in batch fermentation.
The total reducing sugars (TRS) concentrations in the molasses, temperature, and
agitation and culture time effects were studied simultaneously through factorial
design.
The best conditions for ethanol production were 200 g L-1 of total reducing sugars in the
molasses, temperature of 30 C and static culture and time of fermentation of 48 h,
achieving 55.8 g L-1. The pH of the medium was kept constant during the experiments,
showing that molasses presents a buffering effect.

Overview
Zymomonas mobilis, a Gram-negative bacterium, have been attracting increasing attention
for fuel ethanol. It is an osmo and ethanol tolerant bacterium and it has shown higher
specific rates of glucose uptake and ethanol production. Z. mobilis may have a greater
potential for industrial ethanol production from raw sugar, sugarcane juice and
sugarcane syrup.
Molasses is an agro-industrial by-product often used in alcohol distilleries due to the
presence of fermentative sugars, being an optimal carbon source for the microorganism
metabolism. 2.1. Microorganism and culture conditions the strain used were Z. mobilis
ATCC 29191.
Culture
The strain was maintained on agar plates containing (per liter): 200 g glucose, 10 g yeast
extract, 5 g peptone, 1 g (NH4)2SO4, 2 g KH2PO4, 0.5 g MgSO4 7H2O and 0.5 g FeSO4
(Merck). The culture medium was sterilized at 121 _C for 15 min. The cultures were
maintained at 4 C and renewed every five weeks.
The inoculum culture was grown composed with sucrose at 200 g L-1 and the components
mentioned previously. The cell concentration was standardized to 0.2 g L-1, determined by
turbidimetry at k = 605 nm. The batch fermentations were carried out in duplicate in the
sugar cane molasses culture medium, in the different culture conditions, according to the
experimental design. Analytical methods
After each fermentation, the culture was centrifuged (10, 0000 rpm for 15 min) and
the biomass concentration was determined by measuring the turbidity of diluted sample at

605 nm using a standard curve of absorbance against dry cell mass. The total reducing
sugars (TRS) were quantified
Ethanol was determined by Gas Chromatography (GC) Shimadzu, using a DBWAX column
(30.0 0.25 cm) with a flux of 40 ml min_1 and isopropanol as an internal standard. 3.
Results and discussion

Optimize the ethanol production

Four relevant factors for the fermentative process were selected. The variables, studied
simultaneously were: TRS concentration in sugar cane molasses (150 and 250 g /L),
temperature (25 and 35 C), agitation (180 oscillations per minute and static culture) and
culture time (12 and 24 h),
The main effects of the three factors concentration in sugar cane molasses,
temperature, and are all positives, and agitation rate is negative. The growth time
main effect is the most significant factorial design effect value for the production of
ethanol. The inclusion of agitation rate reduces the average ethanol production.
The results of the 3 variable showed that the condition of 200 g /L of TRS and temperature
30 C was the most favorable, achieving 54.83 g/L after a 48-hour-culture time.
The time was a decisive factor, once the ethanol production increased to more than 60%
from 24 to 48 h. By comparison reported a maximum ethanol concentration (55.3 g L_1) at
32.4 C, pH of 4.93 after 17.24 h from sago starch using Z. mobilis MTCC 92. Reported
similar values (54 g/L) for Z. mobilis ZM4 from hydrolysed waste starch stream.
In the central point, 250 g L_1 and 35 _C, ethanol production was an average of 31.15 g L_1.
The decrease in ethanol production at high sugar concentration occurred due to an
increase in the osmotic pressure that is one of the essential factors for by-products
synthesis such as sorbitol and levan. The molasses was an industrial sucrose containing
substrates that has been reported to contain substantial salt content. At 35 _C and 300 g
L_1 sugars concentration on molasses obtained maximum sorbitol production by Z. mobilis
ATCC 29191.
The temperature of 40 _C was negative for fermentative process, resulting in lower
productions, 4.6 g L_1. Numerous studies have shown that temperatures above 37 C are
detrimental for ethanol production. Based on the results of 23 factorial designs, it was
performed as a 22 factorial design, with central composite design, resulting in
11experiments (Table 3). In this stage the time was fixed in 48 h. With the central
composite design it was possible to confirm that maximum ethanol concentration occurred
at the central point, 55.8 g/L on average. These values are similar to the ones described for

ethanol production from sucrose and sago starch, which confirmed that the microorganism
showed an optimal adaptation to the non-treated molasses. The ethanol productivity was a
mean of 1.1 g L_1 h_1.
Low biomass production is normally observed in Z. mobilis, and cell growth and
fermentation are not linked. Approximately 2% of the carbon source is converted into
biomass. This occurs due to EntnerDoudoroff pathway used by this microorganism. This
pathway yields only a single mole of ATP per mole of sugar fermented, giving Zymomonas
the lowest molar growth yield reported for a bacterium.
The pH of the medium remained constant during the experiments, varying from 6.0 at the
beginning to 5.6, on average. The pH has also been described as a factor that strongly
interferes in the fermentative processes. However, according to molasses exhibits a
buffering effect. This regulatory action depends of molasses chemical composition. The
main stabilizer compounds of the pH are weak acids and amino acids that act in the acid
range, mainly between pH 3.0 and 5.0, or phosphates, whose buffering effects occur in the
range of 6.0 and 7.0.

Reference
Fermentation of molasses by Zymomonas mobilis:
Effects of temperature and sugar concentration on ethanol production
M.L. Cazetta a,*, M.A.P.C. Celligoi b, J.B. Buzato b, I.S. Scarmino c

Kluyveromyces Marxianus
Fuel ethanol production through yeast fermentation has generally not been found
economically viable in hot climates. This is because of the high energy input required to
maintain the fermentation process temperature at between 25 C and 35 C to maximize
ethanol production and prevent irreversible heat-inactivation of the yeast cells.
Yeasts, in general, grow at relatively low temperatures in comparison with bacteria, with
upper temperature limits for thermo tolerant and thermophilic yeasts at 42 C and 45C
respectively. Because of the advantages of thermo tolerant yeast in industrial
fermentations, strains capable of growth and ethanol production above 40C have been
actively sought, mainly through the screening of existing yeast strains a strain of
Kluyveromyces marxianus growing up to 49 C and capable of producing alcohol at
and above 40C. Members of the yeast genus Kluyveromyces were found to be more
thermotolerant than Saccharomyces or Candida.

Strains the selection and optimization of a Saccharomyces yeast strain suitable for
fermentation at a maximum temperature of 45 C but with incomplete sugar fermentation
and a maximum of 4.5% (w/v) ethanol production from 15 to 20% The enrichment and
isolation programme resulted in five thermo tolerant yeast cultures capable of growth
at temperatures up to 52 C being isolated. These cultures were designated as IMB1, IMB2,
IMB3, IMB4 and IMBS, and were believed to be different as they were selected according to
minor morphological differences on plates, liquid culture and microscopic examination.
The IDY Saccharomyces cerevisiae, in comparison, did not grow at temperatures above
40~

Molasses Fermentation
The five yeast cultures fermented molasses and produced appreciable amounts of alcohol.
The concentrations, however, were significantly less than that produced from glucose and
it took a longer time for the fermentation to stop, as indicated by CO2 release. Maximum
alcohol production of 5.6--6.0% (w/v) was at 40 C for all isolates.

However, little difference in alcohol concentration was observed at 37 C while a marked

decrease in alcohol production, to 3.0 to 3.5% (w/v), was detected at 45 C. The IDY culture,
in comparison, produced 6.0% (w/v) alcohol at both 37 C and 40 C and only 3.0% (w/v) at
45 C. Supplementation of molasses with P, K, Mg and Mn resulted in a significant increase
in the alcohol concentrations produced for the five thermo tolerant isolates at

temperatures of 37 C, 40 C, and 45 C.

Molasses, however, is not as good a substrate as glucose, which may be due to the
presence of some nonassimilatable reducing sugar complexes in the molasses or to
the lack of some essential nutrient or element required by these cultures at elevated
temperatures.

Supplement addition
This latter view is supported by the fact that higher ethanol concentrations were produced
by supplementing molasses with P, K, Mg and Mn. Similar increases in biomass yields on
supplementation with selected elements have been noticed by the authors when working
with thermophilic bacteria (data not reported). An increase in alcohol production and
complete utilization of 15% glucose by thermo tolerant yeast strains growing at 43 C only
when the other medium components were doubled to make them more readily available.

The requirement of magnesium at high temperatures for glycolytic enzyme production was
suggested as a possible explanation. A high requirement for potassium at higher growth
rates and temperatures in thermo tolerant yeasts.
Interestingly, in the case of the IDY strain of Saccharomyces cerevisiae, the results show
that it is capable of producing alcohol at the higher temperatures of 45 C and 50 C on both
glucose and molasses. However, it should be noted that this strain had no ability to grow at
these temperatures and it is only due to the fact that the inoculum were prepared at 30 C
that we detected such alcohol production. No alcohol production would be detected if cells
were required to grow aerobically at these temperatures before fermentation commenced.
The results presented in this paper show the good potential in using these thermo tolerant
IMB yeast cultures in industrial alcohol production and may create new areas of interest in
biotechnological and industrial fermentation applications, particularly in hot regions.

Reference
Isolation of thermotolerant, fermentative yeasts growing at 52 C and producing
ethanol at 45 C and 50 C
I.M. Banat*, P. Nigam and R. Marchant