O A

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Advances in Natural and Applied Sciences, 6(6): 802-818, 2012
ISSN 1995-0772
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Review paper Integration of Biodiesel and Bioethanol Processes: Convertion of Low
Cost Waste Glycerol to Bioethanol
1
Saifuddin Nomanbhay; 2Refal Hussain; 2Md. Mujibur Rahman; 2Kumaran Palanisamy
1
Department of Science and Mathematics; and
Department of Mechanical Engineering
2
College of Foundation Sciences and 2College of Engineering, Universiti Tenaga Nasional, Jalan IKRAMUNITEN 43000, Kajang Selangor, Malaysia
2
Saifuddin Nomanbhay; Refal Hussain; Md. Mujibur Rahman; Kumaran Palanisamy: Review paper
Integration of Biodiesel and Bioethanol Processes: Convertion of Low Cost Waste Glycerol to
Bioethanol
ABSTRACT
The once abundant petroleum reserves now dwindling and oil prices continue to rise; the search for
alternative fuels becomes more vigorous. The paper reviews current growing attention focusing on ethanol as a
renewable fuel to substitute and/or complement gasoline fuel. This trend is due to, among other aspects, the
growth of oil prices in the international market, and the pressure to reduce atmospheric emissions of CO2 to
mitigate the problem of global climate changes. Glycerol-rich streams generated in large amounts by during the
production of biodiesel, present an excellent opportunity to establish biorefineries. This review covers the
anaerobic fermentation of glycerol in microbes and the harnessing of this metabolic process to convert abundant
and low-priced glycerol streams into higher value products, thus creating a path to viability for the biofuels
industry. However, glycerol is rarely used as a carbon source in Escherichia coli fermentation because of its low
yield of products. A native, nonpathogenic strain of E. coli, able to ferment glycerol to useful products under
anaerobic condition is currently used by many researches. The key factor is not the type of strain, but rather on
the appropriate environment including an acidic pH, avoiding accumulation of fermentation gas hydrogen and
appropriate medium composition to achieve high yeilds. The process becomes increasingly attractive, using
biocatalyst like (Alcohol Dehydrogenase) to metabolize these chemicals anaerobically to produce ethanol.
Key words: Bioethanol; biodiesel; crude glycerol; Escherichia coli.
Introduction
A growing population, increasing per capita income, infrastructural development and rapid socio-economic
development has spurred an increase in energy consumption across all major sectors of the world economy. In
1895, a famous Swedish chemist, Svante Arrhenius, presented a paper to the Stockholm Physical Society titled
On the Influence of Carbonic Acid in the Air upon the Temperature of the Ground, in which he argued that the
use of fossil fuel for combustion would lead to global warming (Hoffert, et al., 2002; Somerville, 2006).
Although he was right, the widespread availability of inexpensive petroleum during the 20th century seemed to
disregard this theory. Today the earth must deal with the consequences of global climate change and somehow
meet expanding energy needs while limiting greenhouse gas emissions. Global warming, depletion of fossil
fuels and price rise of petroleum-based fuels are causing great concern, and the exigency of the situation has
encouraged the search for alternative, sustainable, renewable, efficient and cost-effective energy sources with
lesser greenhouse gas (GHG) emissions (Nigam and Singh, 2010). The transportation sector worldwide has
become has most entirely reliant on petroleum-based fuels and is responsible for 60% of the world oil
consumption (Balat & Balat, 2009). Furthermore, the transportation sector accounts for more than 70% of global
carbon monoxide (CO) emissions and 19% of global carbon dioxide (CO2) emissions (Goldemberg, 2008; Balat
& Balat, 2009). Around the world, there were about 806 million cars and light trucks in 2007 and the number is
expected to reach 1.3 billion by 2030 (Balat, 2011). This huge growth will influence the stability of ecosystems
and global climate as well as global oil reserves.
Corresponding Author: Saifuddin Nomanbhay, Department of Science and Mathematics; and Universiti Tenaga Nasional,
Jalan IKRAM-UNITEN 43000, Kajang Selangor, Malaysia
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Adv. in Nat. Appl. Sci., 6(6): 802-818, 2012
Energy security and environmental concerns have led to a concerted search for alternatives to fossils fuels.
Different types of renewable energy are currently being researched, namely solar, wind, geothermal,
hydrothermal and biofuels. Biofuels are liquid or gaseous fuels produced from renewable resources that absorb
carbon dioxide via photosynthesis as they grow. When biofuels are burned, only the carbon dioxide absorbed by
the biomass is released, so the net production of greenhouse gases (GHG) in total can be lower than that of
burning fossil fuels. Some of advantages of using biofuel as shown in figure 1 (IEA, 2011). This paper will
review the major issues concerning the technology development and challenges in production of bioethanol. It
will focus on the current status of glycerol fermentation using Escherichia coli to produce ethanol and other
value added products.
Sustainability
Environmental
Social




Employment
Land issues
Small holder integration
Food security




GHG emissions and air
quality
Soil quality
Water use and quality
Biodiversity
Economic



Energy security and
self-sufficiency
Balance of payments
Financing

Fuel cost
Fig. 1: Environmental, social and economic aspects of biofuel and bioenergy production (IEA, 2011).
Fuel Ethanol in the World:
Worldwide interest has increased in identifying, developing and commercializing technologies for
alternative renewable sources of energy. The most feasible biofuels for vehicles being considered globally are
biodiesel and bioethanol. Its market grew from less than a billion liters in 1975 to more than 39 billion liters in
2006, and is expected to reach 100 billion liters in 2015 (Mussatto, et al., 2010). Brazil and the United States
are the largest promoters of bio-fuels in the world. Between 2000 and 2009 fuel ethanol output experienced an
increase from 16.9 to 72.0 billion liters while biodiesel grew from 0.8 to 14.7 billion liters (Brown, 2009).
Figure 2 shows the annually ethanol and biodiesel production from 2000-2009 (Brown, 2009).
Pure ethanol is used rarely as a transportation fuel. Instead, it is mixed with gasoline, ranging from 10% to
85% in content, and is used mainly as a gasoline additive because it contains a high percentage of oxygen (35%)
and provides a high octane rating (113) (Dias De Oliveira, et al., 2005). Bioethanol can be produced from a
number of crops including sugarcane, corn, sorghum, grains, potatoes, etc. Biodiesel, however, is the fuel that
can be produced from vegetable oils-edible and non-edible, recycled waste vegetable oils, and animal fat
(Jianxin, et al., 2007; Agarwal, 2007). The transfer of crude oil-based refinery to biomass-based biorefinery has
attracted strong scientific interest which focuses on the development of cellulosic ethanol as an alternative
transportation fuel to petroleum fuels.
Fig. 2: World annual ethanol and biodiesel production, 1975–2009 (reproduced from Brown, 2009)
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Adv. in Nat. Appl. Sci., 6(6): 802-818, 2012
Global production of ethanol was about 17,335 million gallons (65,614 million liters) in 2008. The United
States led the world in production of fuel ethanol and produced 9000 million gallon (34,065 million liters).
Brazil was second with production of 6472 million gallon (24,497 million liters), followed by the European
Union (733 million gallon, or 2777 million liters) and China (501 million gallon, 1900 million liters)
(Renewable Fuels Associate, 2010). In the United States, more than 90% of fuel ethanol is derived from corn
feedstock, while sugarcane and molasses are the primary sources for ethanol production in Brazil (Rushing,
2008).
In 1970, Brazil set up a National Alcohol Program (ProAlcool) focusing on the production of ethanol from
sugar cane and today it is regarded as one of the more developed nation in ethanol production, being also one of
the world’s largest producer of this bio-fuel (RFA, 2010). Since the introduction of ProÁlcool policy in Brazil, a
substantial increase in the ethanol production has occurred, from 555 million liters in 1975/76 to more than 16
billion liters in 2005/06 (Orellana and Bonalume Neto, 2006). Brazil has continued its ethanol expansion plans,
by adding new sugar plantations and ethanol production capacity. Brazil’s ethanol is recognized as the most
price-competitive biofuel in the world. According to de Almeida, et al. (2008) the average production cost of
ethanol projects to be around US$0.37 per liter. These values would make ethanol competitive with oil prices at
about US$42 per barrel. Another estimate by Kojima and Johnson (2006) has put fuel ethanol average
production costs in Brazil at the range of between US$0.25 and US$0.29 per liter. Favorable climate conditions,
abundant and productive land allow Brazil to produce over 30 million tons of sugar and 20 billion liters of
ethanol annually (Cerqueira Leite, et al., 2009). Brazil’s low manufacturing expenses are the result of the
production feedstock, sugarcane, which is relatively cheap. High levels of land productivity combined with
almost no needs for irrigation gives cheap feedstock. In addition, the mills are able to satisfy almost all of their
energy needs through co-generation power plants based on bagasse, a by-product of sugarcane (de Almeida, et
al., 2008).
Similar to Brazil, the United States started to invest in the production of ethanol fuel as an alternative
source of energy to rely less on oil. The ethanol industry in United States has shown a fast growth and
development and USA capacity of ethanol production increased from 1.63 billion gallons in 2000, to 9 billion
gallons in 2008, representing a 5.5-fold increase. In 2006, ethanol production in the United States overtook the
Brazilian production, which was the world's largest producer for decades. The obligatory consumption of given
biofuel volumes was first implemented with the inclusion of Renewable Fuel Standards (RFS1) in the Energy
Policy Act of 2005. The objective was to employ 4 billion gallons of renewable in transport fuels in 2006 (RFA,
2010). The guiding principle of biofuel policies has been a reduction in the US’s dependency on oil. This speedy
growth in ethanol production is predicted to continue at least until 2012, when the United States intend to attain
an ethanol production of 28 billion liters/year (Solomon, et al., 2007). Currently, over 95% of ethanol
production in the United States comes from corn, with the rest made from wheat, barley, cheese whey, and
beverage residues (Solomon, et al., 2007).
The parliament of the European Union in April 2009 endorsed a minimum binding target of 10% for
biofuels in transport by 2020 as part of the EU Directive 2009/28/EC on renewable energy. (Directive
2003/30/EC, 2009) The directive also specified a minimum 35% reduction in GHG emissions to be achieved by
biofuels during their lifecycle, a target that is meant to increase to at least 50% starting from 2017. In several
countries in Europe, tax reductions or exemptions have been implemented in order to support production or
consumption of biofuels. In Europe, ethanol is mostly produced from wheat and sugar-beet. France, Germany
and Spain are Europe’s most strongly committed nations to ethanol production (Prieur-Vernat & His, 2006).
China’s biofuel policy focuses on ethanol production. The Ethanol Promotion Program was launched in
2002 in order to make use of excessive maize stock-piles. In August 2007 the National and Development
Reform Commission (NDRC) announced a Medium and Long Term Development Plan for Renewable Energy.
Renewable energy as a share of total primary energy consumption should rise to10% by 2010 and to 15% by
2020. Ethanol production in China is projected to reach 2 million tonnes by 2010 and 10 million tonnes by 2020
(Liu & Lin, 2009). Many projects recently have focused on sorghum bioethanol to replace the use of petroleum
based oil (Liu & Lin, 2009). China has also increased subsidies, tax and VAT exemptions in both biodiesel and
ethanol to promote this industry.
Energy Balance, Basic Properties and Green House Gas Emission of Ethanol Fuel:
Ethanol (Bioethanol; ethyl alcohol) is a clear, colourless, flammable oxygenated hydrocarbon, with the
chemical formula CH3–CH2–OH. It is a liquid biofuel produced from various biomass feed stocks and
conversion technologies. Bioethanol is an attractive alternative fuel because it is renewable resource oxygenated
fuel, thereby providing the potential to reduce particulate emissions in compression–ignition engines (Balat &
Balat, 2009). Much controversy over the energy balance of ethanol was created by Pimentel (1998), who
claimed that ethanol production from corn had a negative energy balance of 56300 British Thermal Units (BTU)
per gallon of ethanol produced. Table 1 summarizes the energy inputs and outputs of ethanol as mentioned in
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recent large-scale studies of ethanol production in the U.S. and Canada. Why Pimentel’s ethanol energy balance
is so different from other researches was well explained and rebutted by Graboski (2002). In light of the justified
criticisms of Pimentel’s work, and the consensus among other ethanol researchers, it is clear that ethanol does
have a positive energy balance.
Table 1: Comparison of Energy Balances from Recent Ethanol Studies (BTU/US Gallon) (Graboski, 2002)
Study
InputsInputsCo-Products
Total Inputs
Corn
Ethanol Manufacture
Credits
Growing
Pimentel (1998)
55,300
74,300
Nil
129600
Wang (2001)
21,896
41,400 (dry mill)
14 076
49220
40,300 (wet mill
12 493
49703
Graboski (2002)
21,268
48,539 (dry mill)
14 829
54978
60,658 (wet mill)
67097
Levelton (2000)
17,775
50415
14055
54135
Net
Energy
Balance
(53600)
26780
26297
21022
8903
21865
GHG emissions (%)
Besides being a renewable fuel, with high octane, ethanol is a much cleaner fuel than petrol. Ethanol blends
dramatically reduce emissions of hydrocarbons, (major sources of ground level ozone formation), cancercausing benzene and butadiene, sulfur dioxide and particulate matter. Moreover, ethanol blends can be used in
all petrol engines without modifications (Miller, 2003; Demirbas, 2008). On a life-cycle basis, not all biofuels
are equal in terms of environmental benefits. Figure 3 demonstrates the lower GHG emissions resulting from the
use of bioethanol from various sources compared to gasoline on a life-cycle basis. As Figure 3 shows, cornbased bioethanol offers rather limited benefits, as it reduces GHG emissions by only 18% compared to gasoline.
In contrast, sugarcane and cellulosic bioethanol result in almost 90% lower emissions (Philippidis, 2008).
To make sure that ‘‘good” bioethanol is produced, with reference to GHG benefits, the following demands
must be met (Börjesson, 2009): (1) bioethanol plants should use biomass and not fossil fuels, (2) cultivation of
annual feedstock crops should be avoided on land rich in carbon (above and below ground), such as peat soils
used as permanent grassland, (3) by-products should be utilized efficiently in order to maximize their energy
and GHG benefits, and (4) nitrous oxide emissions should be kept to a minimum by means of efficient
fertilization strategies, and the commercial nitrogen fertilizer utilized should be produced in plants which have
nitrous oxide gas cleaning. Disadvantages of bioethanol include lower energy density than gasoline (petrol)
which can reduce fuel economy as illustrated in Figure 4. However, ethanol has a higher octane rating than
petrol, potentially improving engine performance, and engine manufactures are developing engines that, by
taking advantage of ethanol’s octane advantage, will yield improved fuel economy when using E85. Other
disadvantages are low flame luminosity, lower vapor pressure (making cold starts difficult), and miscibility with
water, (Balat, et al., 2008). Some properties of alcohol fuels are shown in Table 2 (Demirbas, 2008).
100
80
60
40
20
0
Fig. 3: Reduction in GHG emissions, compared to gasoline, by bioethanol produced from a variety of
feedstocks (on a life-cycle basis) (Philippidis, 2008).
Another factor which is of importance in the field of fuel ethanol is the difference between anhydrous and
hydrous alcohol. Anhydrous alcohol is free of water and at least 99% pure. This ethanol may be used in fuel blends.
Hydrous alcohol on the other hand contains some water and usually has a purity of 96%. In Brazil, this ethanol is
being used as a 100% gasoline substitute in cars with dedicated engines. The distinction between anhydrous and
hydrous alcohol is of relevance not only in the fuel sector but may be regarded as the basic quality distinction in the
ethanol market. Anhydrous ethanol can be blended with gasoline (petrol) in various ratios for use in unmodified
gasoline engines; the most common blend of bioethanol with petrol (gasoline) 10% of ethanol to 90% of
gasoline (called E10) (Demirbas, 2008; Balat & Balat, 2009). Higher percentage of ethanol can be used in
blended fuels such as E85. (a blend of fuel mixed with 85% bioethanol and 15% gasoline) (De Oliviera, et al.,
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Adv. in Nat. Appl. Sci., 6(6): 802-818, 2012
2005). The biologically produced ethanol contains about 5% of water. This is an azotropic mixture. Therefore,
simple distillation is not enough to clean it. Hydrated ethanol is not completely mixed with gasoline or diesel
fuel. By using an appropriate emulsifier bioethanol could be mixed with diesel. A mixture of hydrated ethanol
with diesel oil with the emulsifier is called diesohol. Diesohol is blended in concentrations of 84.5% of diesel,
15% of hydrated ethanol and 0,5% of emulsifier (Demirbas, 2008). Table 3 shows the properties of gasoline fuel
blended with various percentages of ethanol (Tangka, et al., 2011).
Fig. 4: Volumetric energy density of fuels adjusted for engine efficiency
Table 2: Fuel properties of gasoline, methanol, ethanol.
Property
Chemical formula
Molecular weight
Lower heating value (MJ/Kg)
Stoichiometric AFR (Kg/Kg)
Density (Kg/m3)
Boiling point (oC at 1 atm)
Self-ignition temperature (oC)
Latent heat of evaporation (KJ/Kg)
Stoichiometric mixture heating value (KJ/m3 atm at 20oC)
RON
MON
Carbon (wt%)
Oxygen (wt%)
Hydrogen (wt%)
Gasoline
C4-C12
95-120
44
14.8
0.70-0.75
25-215
300-400
310-320
3750
90
81-89
85.5
0
14.5
Methanol
CH3OH
32
20.26
6.52
0.795
65
500
1100
3557
110
92
37.5
12.5
50
Ethanol
C2H5OH
46
27
9.05
0.79
78
420
862
3660
106
89
52.2
13
34.8
Table 3: Properties of gasoline fuel blended with various percentages of ethanol (Average values) (Tangka, et al., 2011).
Sample
Vapor
Flash
Energy
code
%
Auto
ignition pressure
Octane
% Ethanol
point
Density
Gasoline
temperature (°C)
(Kpa
at
number
(°C)
(MJ/L)
37.8°C)
E0
00
100
-65
246
36
34.2
91
E10
10
90
-40
260
38.9
33.182
93
E20
20
80
-20
279
39
32
94
E30
30
70
-15
281
38
31.5
95
E40
40
60
-13.5
294
35.6
30
97
E50
50
50
-5
320
34
29
99
E60
60
40
-1
345
31
28
100
E70
70
30
0.00
350
28
27
103
E80
80
20
5
362
24
26.5
104
E90
90
10
8.5
360
18
23.6
106
E100
100
00
12.5
365
9
23.5
129
Specific
gravity
0.7474
0.7508
0.7605
0.7782
0.7792
0.7805
0.7812
0.7823
0.7834
0.7840
0.7890
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Adv. in Nat. Appl. Sci., 6(6): 802-818, 2012
Major Issues Attributed to Fuel Ethanol Feedstock:
All plant and plant derived materials has great potential to provide renewable energy for the future. The
complexity of the production process depends on the feedstock. The spectrum of designed and implemented
technologies goes from the simple conversion of sugars by fermentation, to the multi-stage conversion of
lignocellulosic biomass into ethanol. Feed stocks for the production of bioethanol can be classified into three
categories namely; sucrose-containing feed stocks (e.g. sugar cane, sugar beet and fruits), starchy materials (e.g.
corn, wheat, rice, potatoes, cassava, sorghum, sweet potatoes and barley), and lignocellulose biomass(e.g. wood,
straw, and grasses, corn cobs and stalks) (Balat & Balat, 2009). Almost all fuel ethanol is currently produced is
the first‐generation ethanol (sugar and starch feed stock). About half the world’s bioethanol production uses
sugar crops as feedstock, mostly sugarcanes but also beets. The majority of the remaining ethanol is produced
from starch crops, mainly grains such as corn and wheat (Bai, et al., 2008). Estimates show that feedstock costs
can sometimes represent three quarters of bioethanol production costs (Gnansounou & Dauriat, 2005). The use
of herbicides, fertilizer, labor, firm machinery, electricity and water must be considered in the process of
feedstock cultivation. Table 4 highlights some comparisons of the yield and cost of some common feedstock
crops (Gnansounou & Dauriat, 2005).
There exist several reports on bioethanol production from lignocellulosic waste materials such as crop
residues (Kim and Dale, 2004), municipal solid waste (Mtui and Nakamura, 2005), forest products industry
wastes (Kadar, et al., 2004; Fan, et al., 2003), leaf and yard waste (Lissens, et al., 2004), as well as a few studies
involving dairy and cattle manures (Wen, et al., 2004; Chen, et al., 2003, 2004). Nonetheless, the feasibility of
using these materials as a feedstock is often limited by the low yield and the high cost of the hydrolysis process
based on current technologies. Lignocellulosic ethanol or also called as second‐generation ethanol, is produced
in almost the same way as first‐generation ethanol (sugar and starch feedstock). The pre‐treatment needed to
access the fermentable sugars in the ligno‐cellulosic plant materials, however, is much more difficult and may,
depending on the feedstock, require acid, pressurized steam, special enzymes or a combination of those. These
methods can result in undesirable toxins that inhibit the following fermentation process (Mann, 2004). Once
decomposed, the biomass requires a fermentation process in which both hemicellulose (C5) and cellulose (C6)
sugars must be processed. The major advantage of cellulosic ethanol is the low cost of feedstock, which as
mentioned can be agricultural or forestry residues or more dedicated energy crops such as willow and
switchgrass. Another advantage is that second‐generation production does not conflict, in the same way as
first‐generation ethanol, with production of human food. Unfortunately, the economics of cellulosic ethanol are
currently at a stage where the low cost of feedstock does not outweigh the high cost of production (Gnansounou
& Dauriat, 2005). Table 5 shows the costs of ethanol production that comes from corn and cellulosic materials.
Table. 4: The production cost and bioethanol yield of various crops
Annual
Conversion
Type
yield
rate to sugar
(ton/ha)
Or starch (%)
Sugar cane
70
12.5
Cassava
40
25
Sweet
35
14
Corn
5
69
Wheat
4
66
Conversion
rate to
ethanol (L/ton)
70
150
80
410
390
Annual
Yield
(kg/ha)
4900
6000
2800
2050
1560
Cost
(US$/m3)
~160
700
200-300
250-420
380-480
Table 5: Costs of ethanol production from corn and cellulosic feed stocks (Collins, 2007).
Corn ethanol cost US$ per gallon
Cellulosic ethanol cost currently US$ per gallon
Feedstock
1.17 (available at $3.22 per bushel)
1.00 (available at $60 per dry ton
Enzymes
0.04
0.40
Other costs
0.62
0.80
Capital costs
0.20
0.55
Total costs
2.03
2.75
By-product
-0.38
-0.10
Net costs
1.65
2.65
The most common concern related to the current first generation biofuel systems is that as production
capacities increase, the production of biofuels takes different dynamic in relation to fossil fuels, being based on
agricultural products, where land is the main input (Rathmann, et al., 2010). This represents a shift in land use
away from food production and poses a global dilemma, namely the need to feed humanity versus the greater
monetary returns to farmers through the incorporation of lands for agro-energy (Schmidhuber, 2006). A number
of studies have looked at the impact of bioethanol expansion on agricultural markets both at the national and
global levels (Elobeid, et al., 2007; Hill, et al., 2006; Secchi and Babcock, 2007; Tokgoz, et al., 2007). These
studies reveal that, in general, with increased ethanol expansion, the prices of both the agricultural feedstock
commodities and their competing crops rise with implications for land allocations, food prices, and the
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Adv. in Nat. Appl. Sci., 6(6): 802-818, 2012
environment. While there is consensus on the impact of the growth in bioethanol on the prices of agricultural
commodities, the debate as to whether the net effect on the economy and the environment is positive or negative
is still continuing. Doornbosch and Steenblik (2007), for example, contend that the contributions of bioethanol
to energy demands are very limited given their adverse effects on food prices and the environment.
Biodiesel Production:
Biodiesel is a suitable substitute for petroleum-derived diesel. It is biodegradable, almost sulfurless and a
renewable fuel, though still not produced by environmentally friendly routes. This alternative fuel consists of
methyl or ethyl esters, a result of either transesterification of triacylglycerides (TG) or esterification of free fatty
acids (FFAs) (Maa & Hannab, 1999). Currently, biodiesel mostly comes from transesterification of edible
resources such as animal fats, vegetable oils, and even waste cooking oils, under alkaline catalysis conditions
(Chhetri, et al., 2008; Zheng, et al., 2006; Kima, et al., 2004). Figure 5 shows transesterification of vegetable
oils or animal fats with the addition of alcohol.
Fig. 5: Transesterification of triglycerides with alcohol (Demirbas, 2009).
In general, the biodiesel has similar composition and characteristics when compared to petroleum-derived
diesel such as cetane number, energy content, viscosity and phase changes. Therefore, when blended with
petroleum-derived diesel, it can be used in any IC diesel engine without any modifications. Several of its distinct
advantages such as lower greenhouse gases emissions, higher lubricity and cetane ignition rating compare to
petroleum derived diesel have enabled biodiesel to become one of the most common biofuels in the world (Lim
& Teong, 2010). The transesterification is an equilibrium reaction and the transformation occurs essentially by
mixing the reactants. The presence of a catalyst (acid or base) could accelerate and control the equilibrium, to
achieve a high yield of the ester, however the alcohol has to be used in excess is used to increase the yield of the
alkyl esters and to allow its physical separation from the glycerol formed. Various other factors determine the
extent of the reaction such as type of catalyst (acid or base), alcohol to vegetable oil molar ratio, temperature,
purity of the reactants and free fatty acid content. The rise prices of vegetable oil and the occasional formation
of soaps, and the low yields, make biodiesel currently more expensive than petroleum-derived fuel (Haas, 2005).
The development and commercial use of biodiesel has been rapidly expanding in Europe and US for over
10 years. The superiority of biodiesel over petroleum diesel towards health and environment (free sulfur content,
low content of harmful emission, e.g. particulate matter, HC, CO, etc., better lifecycle of CO2 for global
warming alleviation) as well as engine performance (enhance lubricity, high cetane number for complete
combustion) (Pramanik and Tripathi, 2005; Bournay et al., 2005) has prompted Asia to use biodiesel as
alternative fuel. Figure 6 shows the world biodiesel production (Canakci, et al., 2009). In the EU, biodiesel is by
far the biggest biofuel and represents 82% of the biofuel production. Germany produced 1.9 billion liters or
more than half the world total. In February 2006, the European Union set the goal of fulfilling 5.75% of
transportation-fuel needs with biofuels in all member states by 2010. Many countries have adopted various
policy initiatives. Specific legislation to promote and regulate the use of biodiesel is in force in Germany, Italy,
France, Austria and Sweden (Canakci, et al., 2009). By 2010, the United States is expected to become the
world’s largest single biodiesel market, accounting for roughly 18% of world biodiesel consumption, followed
by Germany. New and large single markets for biodiesel are expected to emerge in China, India and Brazil
(Yusuf, et al., 2011).
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Adv. in Nat. Appl. Sci., 6(6): 802-818, 2012
World BiodieselProduction
a/
T
M
r
o
r
Y
/
e
n
o
t
n
o
ill
i
M
40
30
20
10
0
2002 2003 2004 2005 2006 2007 2008
Fig. 6: Biodiesel 2020: A Global Market Survey
Malaysia’s biodiesel production is mainly palm oil based and since 1980s, Malaysian Palm Oil Board
(MPOB) in collaboration with the local oil company ‘‘Petronas’’ has begun Trans esterification of crude palm
oil into palm biodiesel (also known as palm diesel) (Kalam & Masjuki, 2008). It is currently the world’s second
largest producer, accounting for 42.3% of worldwide production and 48.3% of the world’s total exports of palm
oil. The Malaysian Government has been researching the use of a B5 (5% processed palm oil and 95% diesel)
blend for vehicles and industrial sectors (Zhou & Thomson, 2009). According to Malaysian Biodiesel
Association (MBA), there are 10 active biodiesel plants in the country with a total annual biodiesel installed
capacity of 1.2 million tons. An additional four biodiesel plants with combined annual capacity of 190,000 tons
are on the way to be completed soon (Lim & Teong, 2010).
Alongside the rapid pace of biodiesel development and commercialization, there are several key challenges
emerging and one of them is the inevitable low value production of glycerol as by-product of biodiesel from
transesterification and esterification of vegetable oil. Stoichiometrically, glycerol is produced by 10 wt.% of
total biodiesel production. The mass production of biodiesel would bring about surplus glycerol production
which has 80–88% purity (Pramanik and Tripathi, 2005). The conventional application and current market of
glycerol could not cope the excess production that need further costly purification step to meet the purity of
crude glycerin of industrial grade (98% purity) (Kenkel and Holcomb, 2008). A report from Tyson (2003)
indicated that by taking into account the value of glycerol, the biodiesel cost production could be reduced from
US$ 0.63 per L to US$ 0.38 per L. Figure 7 represent the biodiesel production over the years and its impact on
the price trend of glycerol (Yazdani and Gonzalez, 2007).
Fig. 7: Increasing production of biodiesel and crude glycerol leading to drop in glycerol price (Yazdani and
Gonzalez, 2007).
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Glycerol, or its chemical name propa-1,2,3-triol is the simplest trihydric alcohol The glycerol molecule
has two primary and a secondary hydroxyl groups. Glycerol is a water soluble, colorless, odorless, viscous, and
hygroscopic liquid with a specific gravity of 1.261 g/mL, melting temperature of 18.2°C, and a boiling
temperature of 290°C (accompanied by decomposition). Chemically, glycerol is available for reacting with a
stable alcohol under most operation conditions, and it is basically non-toxic to human health and to the
environment. The key feature of its usefulness is the particular combination among its physicochemical
properties, compatibility with other substances, and easy handling. Due to these particular properties glycerol
has found more than 1500 end-uses or large volume applications. Figure 8 shows traditional glycerin
applications (Van Loo, 2006).
In general, about 10 kg crude glycerol is produced for every 100 kg of biodiesel. Crude glycerol generated
by homogeneous base-catalyzed transesterification contains approximately 50 to 60% of glycerol, 12 to 16% of
alkalis, especially in the form of alkali soaps and hydroxides, 15 to 18% of methyl esters, 8 to 12% of methanol,
and 2 to 3% of water. In addition to that, crude glycerol also contains a variety of elements such as Ca, Mg, P, or
S (Thompson & He, 2006). The wide range of the purity values can be attributed to different glycerol
purification methods used by biodiesel producers and the different feedstocks used in biodiesel production.
Despite the wide applications of pure glycerol in pharmaceutical, food, and cosmetic industries, the refining of
crude glycerol to a high purity is very expensive, especially for small and medium biodiesel producers (Pachauri
and He, 2006). To improve the economic feasibility of the biodiesel industry, alternate ways of using the crude
glycerol phase have recently been studied. One of many promising applications of the glycerol surplus is its
bioconversion to high value compounds through microbial fermentation. Bioconversion is a cheap way to obtain
reduced chemicals (e.g., succinate, ethanol, xylitol, propionate, hydrogen, etc.), at higher yields than those
obtained from sugars (Dharmadi, et al., 2006).
Fig. 8: Traditional glycerin applications (Van Loo, 2006).
Glycerol as a new type of feedstock:
The amount of raw glycerol increases constantly and this triol has become an attractive feedstock material.
The substantial increase in crude glycerol has created a need for a quick conversion of large quantities of
glycerol into useful products. Although combustion of glycerol for heat is one solution that can consume large
quantities, it is not ideal from an economics perspective. To help make biodiesel plants more profitable, it is
desirable to convert glycerol into a commodity chemical that brings a higher price and has (or could have) a
large market (Anand & Saxena, 2011). Clearly, the development of processes to convert crude glycerol into
higher-value products is a dire need. Table 6 shows contamination in raw glycerol obtained as a by-product of
biodiesel production (Papanikolaou, et al., 2000).
Table 6: Residues in raw glycerol obtained as a by-product of biodiesel production (Papanikolaou, et al., 2000).
Contamination
%
Na and K salts
Methanol
Heavy metals and lignin
Other organic materials
Water
4-5
3
1
0.5
26
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Adv. in Nat. Appl. Sci., 6(6): 802-818, 2012
Glycerol from biodiesel is considered as a cheap reagent in bio-chemistry and biotechnology for synthesis
of value-added products. As shown in figure 9, glycerol can be fermented to syngas that can be used for the
Fisher-Tropsch or methanol synthesis, or reformed to H2 or CH4 (Simonetti, et al., 2007). Dihydroxyacetone, a
chemical which is used as a tanning agent, can be obtained both biochemically using bacteria and by oxidation
over Pd, Pt, Au catalysts (Demirel, et al., 2007). Succinic acid obtained mainly via fermentation of glycerol is
used as an intermediate in organic synthesis and as a monomer in polymer production (Song and Lee, 2006).
1,3-Propanediol, propylene glycol, acrolein and glycerol carbonate are used for synthesis of biodegradable
polymers (Kurian, 2005). Many useful products like ethanol, butanol, lactic, propionic, acetic and butyric acids
can be obtained via biological fermentation of glycerol (Silva, et al., 2009). One can say that the increased
production of glycerol as a by-product has provoked a huge interest in its utilization for the synthesis of useful
value-added products, gases and fuels. However, only processes with low cost are commercially viable.
6.1 Bacterial fermentation of glycerol:
Fermentation is one of the most convenient methods of conversion of glycerol. In the last few years
glycerol conversion to 1,3-propanediol by anaerobic bacteria (Figure 10) was studied intensively due to the
potential use of the product diol in the production of polymers (Kurian, 2005). Although many microorganisms
are able to metabolize glycerol in the presence of external electron acceptors (respiratory metabolism), few are
able to do so fermentatively (i.e., absence of electron acceptors). Fermentative metabolism of glycerol has been
reported in species of the genera Klebsiella, Citrobacter, Enterobacter, Clostridium, Lactobacillus, Bacillus,
Propionibacterium, and Anaerobiospirillum (Yazdani & Gonzalez, 2007). Enterobacteria of the genera
Klebsiella, Enterobacter, Citrobacter and Clostridium are capable to ferment glycerol producing 1,3propanediol as major product. The fermentative transformation of glycerol to 1,3-propanediol by C. butyricum
bacteria attracts much attention compared to other strains (Klebsiella pneumoniae, Citrobacter freundii,
Enterobacter agglomerans), because it allows to obtain high yield of the product. It is also more stable, since
the key enzyme, glycerol dehydratase, is vitamin B12-independent, not deactivated by glycerol, and the toxic
intermediate - 3-hydroxypropionaldehyde - does not accumulate in this process (Barbirato, et al., 1998).
Fermentations of glycerol by Enterobacteria results in the accumulation of two main products, 1,3-propanediol
and acetate, whereas byproducts, such as lactate, formate, succinate and ethanol, are differentially produced
according to culture conditions (Fig. 11).
Fig. 9: Possible useful products from glycerol (Kurian, 2005; Simonetti, et al., 2007; Silva, et al., 2009).
Fig. 10: Anaerobic biotransformation of glycerol.
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Fig. 11: Metabolic pathways of glycerol fermentation by Entrobacteria (Biebl, et al., 1999).
Fermentation of glycerol by Clostridium leads to 1,3-propanediol as a major component, two acids
(butyrate and acetate) as byproducts and gaseous products, CO2 and H2. The theoretical yield of 1,3-propanediol
is 64 or 50 %, if acetic or butyric acid is formed as the main by-product. Usually microorganisms use all the
pathways simultaneously, therefore the total yield of 1,3-propanediol can vary (Biebl, et al., 1999; Saxena, et
al., 2009). Industrial glycerol available directly from biodiesel production is frequently contaminated with salts
and other residues. Despite the opinion that this glycerol is not suitable for biochemical processes, an example
of using industrial glycerol for 1,3-propanediol production was demonstrated by Papanikolaou, et al., (2000). It
is still unclear whether high concentration of 1,3-propanediol inhibits the consumption of glycerol or not, but
undoubtedly pH plays a significant role, leading to the decrease in 1,3- propanediol production at low pH values
due to the formation of acetic and butyric acids (Biebl, et al., 1999). High concentrations of glycerol up to 130
g/ L can be fermented in batch cultures with Clostridium bacteria, however, it was shown in batch cultures that
the initial glycerol concentration higher than 150 g/L can cause prolonged lag phase and inhibits bacterial
growth (Colin, et al., 2000). High concentrations of 1,3-propanediol up to 80 g/ L are likely to be non-inhibitory
(Papanikolaou, et al., 2000).
To date, most research has focused on fermentation methods using pure microbial cultures (bacterial strains
in the genera Citrobacter, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, Pelobacter, and Clostridium)
(Pagliaro, et al., 2007). However, this involves expensive equipment and complicated protocols, and is costly.
The use of microbes such as Escherichia coli, an organism very amenable to industrial applications, can easily
help overcome the aforementioned problems (Murarka, et al., 2008). The non-native producers, Escherichia
coli and Saccharomyces cerevisiae, have also been engineered for 1, 3-propenediol production. In S. cerevisiae,
due to ineffective transport of vitamin B12 needed for 1, 3-proponediol synthesis, only low levels of the product
has been obtained. On the other hand, E. coli has been metabolically engineered by DuPont and Genencor
International, Inc., to produce 1, 3-propenediol at a concentration of 135 g/l, (Maervoet, et al., 2011) the highest
reported so far in the industry. A major concern with the existing 1, 3-propenediol non-native producers is that a
majority of them are opportunistic pathogens. The potentials for using these organisms at the industrial level
could be limited due to issues that include pathogenicity, requirement of strict anaerobic conditions, need of
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supplementation with rich nutrients, and unavailability of the genetic tools and physiological knowledge
necessary for their effective manipulation. Besides the usual production of 1,3-propenediol from glycerol,
Jarvis, et al. (1997) demonstrated that formate and ethanol are the major products of glycerol fermentation by
Klebsiella planticola isolated from the rumen of red deer. Ito, et al. (2005) stated that Enterobacter aerogenes
mutant can be used for the high-yield production of ethanol from biodiesel wastes glycerol. The task for the
future is to find effective non-pathogenic microorganisms capable of producing 1,3-propenediol from glycerol
(Saxena, et al., 2009).
By contrast, methods involving mixed cultures are simple to undertake, incur lower costs, and are not
affected by contamination problems. The drawback is that productivity is lower than that with pure cultures.
Because of the recent increases in glycerol production from biodiesel refining superior options for the biological
conversion of glycerol into value added products are extremely important. There are various ways to improve
the productivity of methods using mixed cultures. These include controlling substrate concentrations, light and
temperature as well as using fermentation promoters. Studies have been done to develop a novel bioprocessing
system using mixed bacterial cultures for the anaerobic conversion of high-loading glycerol. A recent
development in the microbial fermentation of glycerol is that Escherichia coli can ferment glycerol
anaerobically (Dharmadi, et al., 2006; Tokumoto and Tanaka, 2012). A study by Tokumato and Tanaka (2012),
showed that the bacteria Escherichia coli and particularly Schizosaccharomyces pombe (found within sewage
sludge) and glucose (found within bacterial components (i.e., cell walls)) acted as efficient promoters of
fermentation. A native, nonpathogenic strain of E. coli, able to ferment glycerol to useful products under
anaerobic condition without the need of genetic engineering is the main objective of current research by many
researches. The key factor is not the type of strain, but rather on the appropriate environment including an acidic
pH, avoiding accumulation of fermentation gas hydrogen and appropriate medium composition.
6.2
Downstream processing of biotechnologically produced 1,3-Propenediol:
The recovery of 1,3-Propenediol from complex fermentation broth represents a true bottleneck in the
development of a commercially viable bioprocess. This could be mainly attributed to its high boiling point and
presence of two hydroxyl groups which make it strongly hydrophilic and therefore complicate its extraction.
Therefore, its recovery from complex aqueous fermentation broth solutions containing macromolecules, salts,
remaining substrates and by-products, is a difficult task. First, microbial cells are removed by filtration or
centrifugation. Then, 1,3-propanediol and by-products are distilled. This step requires a substantial energy input
which accounts for high cost of the final product. The application of relatively simple approaches of evaporation
and vacuum distillation for the recovery of 1,3-PD has been attempted but appeared unattractive and
uneconomical due to the requirement of large amounts of energy, desalination and low product yield (Hao, et
al., 2006). Li, et al (2001) attempted to recover 1,3-propanediol by pervaporation through a X-type zeolite
membrane from a model 1,3-propanediol/glycerol/water solution and broth solutions containing glucose.
Extraction is the most promising and studied method with the ethanol/inorganic salts solution being the most
effective (Hao, et al., 2006). The use of ion exclusion for extraction of 1,3-PD has been reported. The method
consisted of desalination of the fermented broth by the aid of strongly cationic and weakly basic anionic resins
with subsequent passage over a cationic exchange resin for purification of 1,3-PD (Hao, et al., 2006). Probably
the easiest and the most effective method of 1,3-propanediol recovery is a two-phase aqueous extraction with
ethanol (or methanol)/inorganic salts solution. This approach showed the highest recovery of 93.7 % of 1,3propanediol from fermentation broth by a mixture of ethanol and saturated (NH4)2SO4 solution. Moreover, this
method allowed the removal of cells and proteins from the fermentation broth. As one can see, the problem of
the down-stream processing of 1,3-propanediol obtained via microbial fermentation has not been solved
effectively so far. Extraction is the most promising and studied method with the ethanol/inorganic salts solution
being the most effective.
7.0 Enzymatic process in cascade reactions:
There is an increasing demand for environmentally friendly technologies and processes in the chemical
industry due to the growing concern about the irrevocable harmful anthropologic impact on the environment. In
some cases traditional organic/inorganic catalysts or stoichiometric reactions could be replaced by biocatalytic
(or enzymatic) routes, which offer environmental benefits. Biocatalysts seem to satisfy the criteria of green
chemistry in terms of biodegradability, high selectivity, mild reaction conditions and low amounts of waste and
by-products. A concept of combining bio-catalysts within one system emerged as a possible way to develop a
chain of feedstocks transformations to value-added products through multi-step reactions. Such systems also
allow to reduce waste and to intensify chemical processes by avoiding extraction and purification of the
intermediates. Enzymes can also be immobilized by: 1) ionic binding to ion-exchange supports (e.g. cellulose
and carboxylmethyl cellulose), 2) adsorption through van der Waals interactions to hydrophobic supports (e.g.
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Adv. in Nat. Appl. Sci., 6(6): 802-818, 2012
polypropylene and teflon), 3) covalent binding between the amino or carboxyl groups of amino acids and the
support membrane (Giorno and Drioli, 2000). Despite some mass-transfer problems, immobilized systems offer
considerable advantages in comparison with conventional free biocatalysts: a) prolonged stability of the
biocatalyst, increased tolerance to high substrate concentrations and reduced end-product inhibition; b) high
biocatalyst density per unit of a bioreactor volume, which leads to high volumetric productivity and short
reaction times; c) easy product recovery without separation and filtration, thus reduction in the cost of
equipment and energy; d) regeneration and reuse of the biocatalysts for extended periods of time.
The concept of developing a chain of feedstocks transformations to value-added products through multistep reactions allow to reduce waste and to intensify chemical processes by avoiding extraction and purification
of the intermediates. It also reduces time and labor required to effect a given transformation. A tandem or
cascade reaction is a reaction in which several bonds are formed in sequence without isolating intermediates,
changing reaction conditions, or adding reagents. A sequential transformation of 1,3-propanediol formed via a
biocatalytic fermentation by a catalyst integrated within the same reactor system. Several approaches can be
proposed to carry out multistep bio-chemical transformation within a single reaction system. This is usually
based on the principles of spatial separation by using immiscible solvents or by supporting the catalyst or cell on
a support (immobilization).
Conclusion:
Ethanol has experienced unseen levels of attention due to its value as fuel alternative to gasoline, the
increase of oil prices, and the climatic changes, besides being a renewable and sustainable energy source,
efficient and safe to the environment. Currently, worldwide ethanol production is in high levels, and corn is the
main raw material used for this purpose, but this scenario may change due to technological improvements that
are being developed for production of low cost cellulosic ethanol, as well as for ethanol production from
microalgae. Is important to emphasize that, to be a viable alternative, bio-ethanol must present a high net energy
gain, have ecological benefits, be economically competitive and able to be produced in large scales without
affecting the food provision. The use of various wastes (such as crude glycerol from biodiesel production, wood,
agricultural wastes) and unconventional raw materials (such as microalgae) can solve the problem without
sacrificing food demands. Under anaerobic fermentation conditions, facultative anaerobes such as Escherichia
coli are able to convert glycerol into soluble metabolite products and gaseous products, including 1,3propanediol,2,3-butanediol, ethanol, acetic acid, succinic acid, H2, and CO2. Glycerol the most effective carbon
substrate for Escherichia coli, which can form1, 3-PDO and 2,3-BDO, gaseous (H2) and liquid (ethanol)
biofuels. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by using
Escherichia coli and Enhance the enzyme by immobilization process generate a large amount of bioethanol and
decrease the cost process. The usage of low-grade quality of glycerol obtained from biodiesel production is a big
challenge as this glycerol cannot be used for direct food and cosmetic uses. An effective usage or conversion of
crude glycerol to specific products will cut down the biodiesel production costs. Glycerol can cover possible
conversion into useful products such as 1,3-propanediol, 1,2-propanediol, dihydroxyacetones, hydrogen,
polyglycerols, succinic acid, polyesters, and ethanol.
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