Saranraj & Stella
ISSN-2277-6079
TECHNOLOGIES
AND
MODERN
TRENDS
FOR
BIOETHANOL
PRODUCTION
USING
CELLULOSIC
AGRICULTURAL WASTES.
P Saranraj*, D Stella
Department of Microbiology, Annamalik University,Annamalik Nagar,Chidambaram- 608 002.
Science Instinct Publications
Abstract
Global warming alerts and threats are on the rise due to the utilization of fossil fuels. Alternative
fuel sources like bioethanol and biodiesel are being produced to combat against these threats.
Bioethanol can be produced from a range of substrates. Cellulose rich substances which are
generated in tons in agricultural countries like India can be used for the production of bioenergy
in the form of bioethanol with the help of microbial catalytic enzyme cellulose. Celluloses and
hemicelluloses, when hydrolyzed into their component sugars, can be converted into ethanol
through well established fermentation technologies. In this review, technologies and modern
trends for bioethanol production using cellulosic agricultural wastes is discussed. This review
assesses the following topics: cellulose in agriculture waste, raw materials for bioethanol
production, microorganisms for bioethanol production, ethanol production technologies,
cellulosic ethanol, current bioethanol production processes and trends in bioethanol production
development. Indeed, the world's strongest economies are deeply committed to the development
of technologies aiming at the use of renewable sources of energy. In this view, the substitution
with fuel, bioethanol is of foremost importance.
Keywords: Agricultural wastes, Bioethanol, Cellulose and Microorganisms.
*
Corresponding Author:
P Saranraj,
Department of Microbiology,
Annamalik University,
Annamalik Nagar,
Chidambaram- 608 002,
Email-ID: microsaranraj@gmail.com
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Introduction
thanol is a clear, colourless, flammable, oxygenated hydrocarbon with the chemical
formula C2H5OH. Ethanol has been made since ancient times by fermenting sugars. All
the ethanol used for fuel and alcoholic drinks including most of the industrial ethanol, is
made by this process. Fuel ethanol is also known as bioethanol, since it is produced from
plant materials by biological processes. Fuel ethanol has the largest market by far, accounting
for 60% of total ethanol production worldwide. The term biofuel is attributed to any
alternative fuel that is derived from organic material, such as energy crops (e.g. corn, wheat,
sugar cane, sugar beet, cassava, etc.), crop residues (e.g. rice straw, rice husk, corn stover,
corn cobs, etc.) or waste biomass (food waste, livestock waste, paper waste, constructionderived wood residues, etc).
Industrial ethanol accounts for 20% of the market and beverages for about 15%; both these
markets are growing comparatively slow. Ethanol can be used as a transport fuel in at least
four forms: anhydrous ethanol (100% ethanol), hydrous ethanol (95% ethanol and 5% water),
anhydrous ethanol-gasoline blends (10–20% ethanol in gasoline) and as raw material for
ethyl tert-butyl ether (ETBE) [1]. The term biofuel is referred as the liquid or gaseous fuels
produced from biomass, for the transport sector. Bioethanol is one of the most famous
biofuels. Ethanol fermented from renewable sources for fuel or fuel additives are known as
bioethanol. Additionally, the ethanol from biomass-based waste materials is considered as
bioethanol. The use of bioethanol as an alternative motor fuel has been steadily increasing
around the world for a number of reasons. The production of ethanol from biomass is
progressing in many countries worldwide however the production costs are still relatively
high when compared to petrol. The environmental benefits coupled with the social benefits
and economic benefits can be seen to have a dollar value offsetting the higher relative cost
compared to petrol.
E
Cellulose in agricultural wastes
Agricultural wastes contain a high proportion of cellulosic matter which is easily decomposed
by a combination of physical, chemical and biological processes. The bunch consists of 70%
moisture and 30% solid, of which holocellulose accounts for 65.5, lignin 21.2, ash 3.5, hot
water-soluble substances 5.6 and alcohol-benzene soluble 4%. Lignin is an integral cell wall
constituent, which provides plant strength and resistance to microbial degradation [2]. The
recognition that environmental pollution is a worldwide threat to public health has given rise
to a new massive industry for environmental restoration. Biological degradation, for both
economic and ecological reasons, has become an increasingly popular alternative for the
treatment of agricultural, industrial, organic as well as toxic waste.
Plant lignocellulosics, as organic substances, are often subjected to attacks by biological
agents such as fungi, bacteria and insects. Acids can breakdown the long chains in cellulose
to release the sugars through hydrolysis reaction, but because of the higher specificity of
cellulase higher yield of glucose from cellulose can be achieved. A portion of pretreated
biomass can be used to feed a fungus or other organism to produce cellulase that can then be
added to pretreated solids to release glucose from cellulose. Filamentous fungi which use
cellulose as carbon source possess the unique ability to degrade cellulose molecules in plant
lignocellulose. Although, a large number of microorganisms are capable of degrading
cellulose, only a few of these produce significant quantities of cell-free enzymes capable of
completely hydrolyzing crystalline cellulose in vitro [3].
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Raw materials for ethanol production
A variety of sources can provide sugars for bioethanol production, including crops and
lignocellulose, as discussed below. The relevance of certain crops as raw material for ethanol
production is indicated by the fact that over 90% of the world’s bioethanol is derived from
crops [4]. Crops such as sugar cane and sugar beet contain sucrose, which can be converted
into its monomeric components; other crops, such as corn and cereals, contain starch, which
can be converted into glucose. Sugar cane is the preferred raw material for ethanol production
in Brazil, India, and South Africa, whereas corn is used in USA and sugar beet in France.
In contrast to sugar-containing crops, the utilization of lignocellulose as a substrate for
ethanol production has a barrier in its complex structure, which resists degradation.
Lignocellulose is composed of three main fractions: cellulose (~45% of dry weight),
hemicelluloses (~30% of dry weight), and lignin (~25% of dry weight) [5]. Cellulose, the
most abundant polymer on earth, is composed of thousands of molecules of anhydroglucose
linked by β-(1,4)-glycosidic bonds. The basic repeating unit is the dissacharide cellobiose.
The secondary and tertiary conformation of cellulose, as well as its close association with
lignin, hemicellulose, starch, protein and mineral elements, makes cellulose a hydrolysisresistant molecule. Cellulose can be hydrolyzed chemically by diluted or concentrated acid,
or enzymatically.
Hemicellulose is a highly branched heteropolymer containing sugar residues such as hexoses
(D-galactose, L-galactose, D-mannose, L-rhamnose, L-fucose), pentoses (D-xylose, Larabinose), and uronic acids (D-glucuronic acid). Hemicellulose is more easily hydrolyzed
than cellulose [6]. The composition of hemicellulose will depend on the source of the raw
material. Lignin, the most abundant aromatic polymer in nature, is a macromolecule of
phenolic character, being the dehydration product of three monomeric alcohols (lignols),
trans-p-coumaryl alcohol, trans-p-coniferyl alcohol, and trans-p-sinapyl alcohol, derived
from p-cinnamic acid.
Microorganisms for bioethanol production
Microorganisms found to be efficient ethanol producers from pure xylose in well-defined
growth media will not necessarily ferment the lignocellulosic hydrolysate efficiently. The
pre-treated hydrolysates contain various inhibitors of microbial fermentation such as weak
organic acids like acetate, furan derivatives and phenolic monomers from sugar and lignin
degradation respectively. Sometimes the inaccessibility of the carbohydrates in the
hydrolysates to the microorganisms is responsible for the incomplete fermentation and
subsequently lower ethanol yields. Furthermore, the microorganisms have to be able to grow
and produce ethanol in a pre-treated hemicellulose hydrolysate to make them usable in an
industrial bioconversion process of lignocellulosic biomass. Only a few studies have been
conducted with thermophilic anaerobic ethanol producers on pretreated lignocellulosic
hydrolysate [7].
Only enteric bacteria and some yeast are able to ferment pentoses but with low yields.
Natural xylose fermenting yeast (Pichia stipitis, Candida shehatae and Pachysolen
tannophilus) are not tolerant to high ethanol concentrations, require microaerophilic
conditions and are very sensitive to inhibitors and pH changes [8]. With the introduction of
ethanol genes in enteric bacteria, hard efforts are to incorporate pentose conversion pathways
in natural ethanol producers such as Saccharomyces cerevisiae or ethanologenic bacterium
Zymomonas mobilis.
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The most common way of bioethanol production today is by fermentation using the yeast
Saccharomyces cerevisiae with high ethanol yields from starch based substrates. In the past
decades, thermophilic bacteria have gained more attention because of fast growth rates and
their ability to degrade a broad variety of both hexoses and pentoses [9]. Although, ethanol
tolerance of thermophiles is generally less than those of Saccharomyces cerevisiae and the
well known mesophilic bacterium Zymomonas mobilis, they have several advantages like
lower risk of contamination, increased bioconversion rates and product recovery.
Role of Thermophilic bacteria in ethanol production
Thermophilic anaerobic strains can accomplish almost the theoretical ethanol yield of 1.67
mol of ethanol/mol of xylose (0.51 g of ethanol/g of xylose). However, these yields have only
been obtained at concentrations upto 10 g/l for D-xylose. When the concentration of
Dxylose exceeded 10 g/l, the ethanol yield was decreased, probably because of low substrate
and product tolerance. This would be disadvantageous for application of thermophilic
anaerobic ethanol-producing bacteria in industrial ethanol production.
A variety of ethanol producing thermophilic microorganisms have been isolated and
characterized in the past two decades from different environments, including farm soils,
sewage plants, riverbanks, thermal springs, sediments, as well as waste composts often, with
the intention to evaluate and develop them for large-scale ethanol production. These bacteria
include Thermoanaerobacter ethanolicus, Thermoanaerobacter thermohydrosulfuricus,
Thermoanaerobacter
mathranii,
Thermoanaerobacter
brockii,
Clostridium
thermosaccharolyticum and Clostridium thermocellum [10].
Economical analyses have shown that the hemicellulose fraction also needs to be converted
to ethanol in order to obtain an economically feasible lignocellulosic bioconversion process.
Thermophilic anaerobic bacteria have been examined for their potential as ethanol producers.
The investigated species were Thermoanaerobacter ethanolicus, Clostridium thermocellum,
Clostridium
thermohydrosulfuricum
(reclassified
as
Thermoanaerobacter
thermohydrosulfuricus), Thermoanaerobium brockii (reclassified as Thermoanaerobacter
brockii), Clostridium thermosaccharolyticum (reclassified as Thermoanaerobacterium
thermosaccharolyticum and Thermoanaerobacterium saccharolyticum. The major advantage
of using these microorganisms for ethanol production is their ability to degrade a variety of
carbohydrates found in lignocellulosic biomass. However, none of these strains can compete
with Saccharomyces cerevisiae or Zymomonas mobilis when it comes to conversion of
hexoses and ethanol tolerance. Thermophilic anaerobic ethanol-producing bacteria could,
however, be promising candidates for conversion of the hemicellulose fraction (xylose,
arabinose, mannose and galactose).
Maney Sveinsdottir et al. [11] isolated seven strains of thermophilic bacteria from several
Icelandic geothermal areas on various carbohydrates (glucose, xylose, xylan, pectin,
cellulose). Phylogenetic studies (16S rRNA) revealed that four of the isolates belong to the
genus Thermoanaerobacterium, two to Thermoanaerobacter and one to Paenibacillus. The
Thermoanaerobacterium strains had pH optima at low pH (pH 5.0 - 6.0), the
Thermoanarobacter at slightly acidic to neutral pH’s (pH 6.0 - 7.0) and the Paenibacillus
strain at pH 8.0. Similarly there was a clear distinction of temperature optima between the
various genera; Thermoanerobacterium strains had temperature optima close to 60°C,
Thermoanaerobacter at 70°C and the Paenibacillus at 50°C. Ethanol tolerance was from low
(MIC = 1.6% v/v) for Thermoanaerobacter to moderately high (MIC = 3.2% v/v) for the
Thermoanaerobacterium and Paenibacillus strains. Ethanol production capacity on 20 mM of
glucose and xylose showed that six of the strains produced between 1.0 to 1.5 mol-EtOH
mol-l glucose and 0.4 to 1.3 mol- EtOH mol-l xylose, respectively. One strain showed much
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lower yields. Strain AK17 gave the best yields on glucose and xylose with 1.5 mol-EtOH
mol-l glucose and 1.1 mol-EtOH mol-l xylose, respectively. Other end products analyzed in
the culture broth were acetate and hydrogen but in lower amounts. Growth on 0.75 % (w/v)
hydrolysates made from cellulose (Whatman paper), non inked paper, inked paper, glossy
paper, saw dust and grass (Phleum pratense) resulted in good ethanol production yields for
most of the strains.
Henstra and Stams [12] examined the use of thermophilic bacteria to produce pure hydrogen,
acetate, butyrate, ethanol or butanol from hot syngas. Here, metabolic engineering might be
required to produce other products. The advantage of thermophilic fermentation of syngas is
the faster conversion rate compared with mesophilic fermentation.
Role of Genetically engineered bacteria in ethanol production
Some ethanologenic bacteria (Escherichia coli, Klebsiella oxytoca and Zymomonas mobilis)
have shown promising alternatives for industrial exploitation. Escherichia coli was the first
successful bacterium genetically modified for ethanol production [13]. It can grow on a wide
range of carbon sources, can sustain high anaerobic and aerobic glycolytic fluxes and
presents quite good ethanol tolerance [14]. Wild type Escherichia coli shows low ethanol
yield because it converts sugar most efficiently to organic acids (acetic or lactic acid) instead
of ethanol. Due to that, several approaches have been performed with the aim of redirecting
fluxes to ethanol. Most successful approach has been transformation of Escherichia coli with
a plasmid containing genes from Zymomonas mobilis encoding for PDC (pyruvate
decarboxylase) and ADH in an artificial operon (PET) [15].
In recombinant strain Escherichia coli, both enzymes are over expressed to high level [16].
Strain KO11 has been evaluated at laboratory scale for producing ethanol from many types of
Luria Bertani including barley hull, water energy crops, orange peel, corn cobs or even waste
house wood [17]. Escherichia coli, derived from KO11, showed higher tolerance to inhibitors
present in lignocellulosic hydrolysates [18]. Major disadvantage in using Escherichia coli is
public acceptance because of the existence of some pathogens strains as well as neutral pH.
Thus, it is not widely employed in Solid State Fermentation approaches because its optimum
pH (6.5) is not compatible with optimum pH for cellulolytic enzymes [19].
Apart from Escherichia coli, xylose is employed by engineered bacterium Zymomonas
mobilis. Glucose can easily cross cell membrane by facilitated diffusion and can efficiently
convert it into ethanol by an overactive PDC- ADH system-1. Wild strain cannot produce
ethanol from xylose but it has been successfully engineered by introducing a xylosemetabolizing pathway from Escherichia coli. It presents high ethanol yields, selectivity and
specific productivity as well as low pH and high ethanol tolerance [20]. Zymomonas mobilis
has been mainly used in processes for ethanol production from starch108 or sugar biomass
but new strain AX101 can use xylose after glucose consumption in lignocellulosic
hydrolysates showing high sensitivity to inhibitors present in the broth [21].
Klebsiella oxytoca has been also transformed with PET operon diverting carbon flow to
ethanol production. It can grow on sugars including hexoses and pentoses as well as
cellobiose and cellotriose [22]. This latter trait makes strain attractive for processes for
ethanol production from LB presenting chance of adding lower amount of extracellular ßglucosidase for cellobiose hydrolysis. Klebsiella oxytoca has been employed successfully on
corn fiber or sugar beet pulp. However, bacterial processes for ethanol production are not still
commercial [23].
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Role of Genetically engineered yeast in ethanol production
Saccharomyces cerevisiae shows high ethanol productivity, high tolerance to ethanol and
tolerance to inhibitory compounds present in hydrolysate of LB. However, wild type
Saccharomyces cerevisiae has limitation being unable to ferment pentoses and hard efforts
have been made to design a suitable engineered Saccharomyces cerevisiae [24]. Main
strategies have been the construction of recombinant strains by introduction of genes XYL1
and XYL2 encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) respectively or
by introduction of gene coding for xylose isomerase (XI) due to its ability to ferment xylulose
to ethanol [25]. Former strategy also need an over expression of endogenous xylulokinase
(XK) for efficient xylose metabolism. Another hurdle to overcome is that when xylose
fermenting Saccharomyces cerevisiae is used xylose uptake competes with glucose uptake,
because of the sharing of membrane transporters [26].
Saccharomyces cerevisiae takes up xylose by both low and high affinity glucose transport
systems, however, xylose uptake through these transporters is significantly less efficient
compared to glucose. Therefore, various metabolic engineering efforts involving recombinant
Saccharomyces cerevisiae have led to improvements in the initial rate of xylose consumption,
with improvement of xylose transport in Saccharomyces cerevisiae, being a great challenge
to optimize xylose metabolic pathway [27].
Ethanol production technologies
Ethanol can be obtained by chemical synthesis or by ethanol fermentation. Fermentation is a
reaction induced by catalysts - enzymes produced by living cells. There are a number of
advanced technologies of ethyl alcohol production in the world presently, depending on the
raw material subjected to fermentation. The raw materials containing simple sugars and
suitable for direct processing through fermentation are white beet and its processing products,
sugar cane, domestic and citrus fruits, some tropical plants, juices of certain trees and honey.
The group of raw materials containing starch and polysaccharides, such as cellulose and
inulin used for the production of ethanol should comprise cereals in the form of food grain of
rye, barley, corn, oat, wheat, sorghum, besides vegetable bulbs of potato vegetable roots,
seeds of bifoliate plants, fruits, timber, grass, moss, etc.
Using current production technology the cheapest bioethanol produced in world comes from
sugarcane in Brazil and from starch crops in Europe [28]. Presently, the production of ethanol
(for fuel) largely depends on waste materials: lignocellulosic biomass such as crop residues,
wasted and energy crops (switch grass), fast-growing trees such as poplar and willow, waste
paper and package material, cereals in the form of grain unsuitable for consumption,
domestic and agricultural waste (maize and wheat stalks). However ethanol production from
lignocellulosic biomass is not yet used at commercial scale, even though many technologies
are mooted. The total potential bioethanol production from crop residues and wasted crops is
about 16 times higher than the current world ethanol production.
Production costs of bioethanol vary and are dependent on the prices of raw materials, the
method of production, the extent of refining undertaken and the supplementary utilization of
bio-products and waste. Depending on the degree of processing the raw materials for the
production of ethanol, the energy output of the process defined as the ratio of energy contents
and energy supplied for production is different. The energy output in case of ethanol
production ranges from 1.7 to 3.8. The more processed the materials subjected to
fermentation, the lower the energy gain of the entire process. Hence, the current vast interest
is in biofuel production technologies using waste materials e.g., agricultural and forest waste
such as straw or shavings [29].
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Among other examples, the continuous production process composed of thermo-pressure
hydrolysis, enzymatic hydrolysis, fermentation and ethanol dewatering is proposed, which
are characterized by a high level of heat recovery and recuperation (2.95) and low production
price (0.24 EUR/kg EtOH). Another alternative for the future are biorefinery - multisystems
producing fuels, solvents, plastics and food from waste biomass as well as involving ethanol
and lactic acid fermentation [30].
Cellulosic ethanol
Lignocellulosic materials offer a fuel source to supplement fossil fuels. Conversion of
naturally occurring lignocellulosic materials to ethanol currently requires pretreatment to
enhance the accessibility of reactive agents and to improve conversion rates and yields.
According to one patent, agricultural biomass was prepared to approximately 1–6 mm by a
disc refiner for ethanol production. Nowadays, there is an increasing interest in many
countries in the use of fuel ethanol which is produced from renewable biomass as a
replacement of fossil fuels for the consideration of environment and energy security [31].
Lignocellulosic biomass is considered one of most promising feedstocks for production of
fuel ethanol due to its global availability and environmental benefits of its use.
Consequently wide varieties of processes for the production of ethanol from lignocellulosic
biomass are studied and are currently under development [32]. One of the main challenges
for cost effective production from lignocellulosic biomass is high energy consumption. So,
process design integration is needed and more efficient use of energy is necessary.
Furthermore, to reduce operating costs, energy integration is very important to meet the heat
and electricity consumption for the whole process.
Lignocellulosic materials consist primarily of three components, namely cellulose (40-50%),
hemicelluloses (20-30%) and lignin (20-30%) [33]. The potential conversion of these
components into bioethanol was performed by several researchers. The soluble sugar
products are primarily xylose, and further mannose, arabinose and galactose. A small portion
of the cellulose may already be converted to glucose. However, the cellulose bulk will be
converted in a separate step. The product is filtered and pressed, solids (cellulose + lignin) go
to cellulose hydrolysis, and liquids (containing the sugars) go to the fermentation step. The
choice of a pretreatment technology heavily influences cost and performance in subsequent
hydrolysis and fermentation.
An alternative to the use of energy crops as feedstock for ethanol production is to utilize
rougher and woodier parts of plants for producing ethanol, the so-called “cellulosic ethanol”.
This field has gained attention in the latest decades, as lignocellulosic biomass is a potential
source for ethanol that is not directly linked to food production [34]. The conversion of
lignocellulosic material to ethanol is generally more complex, compared to starch hydrolysis
and fermentation. In case of cellulose, more drastic hydrolysis steps are necessary for
achievement of high conversion yields, due to the presence of various amounts of other
sugars, such as xylose and arabinose.
Lignocellulosic materials are an abundant and renewable source of sugar substrate that can be
fermented to ethanol. Using lignocellulosic materials such as agricultural residues, forestry
and municipal wastes and other low-cost biomasses, can significantly reduce the cost of raw
materials for ethanol production and it has been estimated that this accounts for about 50% of
the biomass in the world. In the conversion of lignocellulosic into ethanol, a pre-treatment
step is therefore included because of the high crystalline nature of the cellulose and the
presence of lignin, which makes the cellulose recalcitrant to degradation. The pretreatment
step should improve the accessibility of the cellulose component to hydrolytic enzymes while
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avoiding degradation of solublized hemicelluloses and cellulose. Sugar degradation not only
decreases the final ethanol yield but also results in degradation products that are inhibitory to
the yeast used in the subsequent fermentation [35].
Processing of lignocellulosics to ethanol consists of four major unit operations: pretreatment,
hydrolysis, fermentation, and product separation/purification. Pretreatment is required to alter
the biomass macroscopic and microscopic size and structure as well as its submicroscopic
chemical composition and structure so that hydrolysis of the carbohydrate fraction to
monomeric sugars can be achieved more rapidly and with greater yields. Hydrolysis includes
the processing steps that convert the carbohydrate polymers into monomeric sugars. Although
a variety of process configurations have been studied for conversion of cellulosic biomass
into ethanol, enzymatic hydrolysis of cellulose provides opportunities to improve the
technology so that biomass ethanol is competitive when compared to other liquid fuels on a
large scale [36].
Corn fiber is a potential raw material for the production of various products, including fuel
ethanol, because it is available in countries in which corn grains are processed [37]. It is
obtained in the process of wet milling of corn. Corn fiber, similar to other lignocellulosic
materials is the complex of polysaccharides (35% hemicelluloses, 20% cellulose, up to 20%
starch) and lignin. The main component of corn fiber is the outer corn grain layer pericarp
and residual part of starchy endosperm.
Current bioethanol production processes
Ethanol has been produced by anaerobic yeast fermentation of simple sugars since early
recorded history. These fermentations used the natural yeast found on fruits and the sugars of
these fruits to produce wines. Beer fermentations made use of the amylases of germinating
grain to hydrolyze the grain starches to ferment sugars. Current practices utilize bacterial and
fungal amylases to efficiently hydrolyze grain or tuber starch to glucose for fermentation to
ethanol.
Ethanol can be produced by biologically catalyzed reactions. In much the same way that
sugars are fermented into beverage ethanol by various organisms including yeast and
bacteria, sugars can be extracted from sugar crops, such as sugar cane, and fermented into
ethanol. For starch crops such as corn, starch is first broken down to simple glucose sugars by
acids or enzymes, known as amylases. Acids or cellulase enzymes similarly catalyze the
breakdown of cellulose into glucose, which can be then fermented to ethanol. The
hemicellulose fraction of biomass is broken down into various sugars, e.g. xylose, in the
presence of acids or enzymes known as xylanases; conventional organisms cannot ferment
many of the sugars derived from hemicelluloses into ethanol with reasonable yields.
However, recently new technologies capable of efficiently converting hemicelluloses into
ethanol are under development.
Innumerous reports related to biomass conversion into ethanol have published recently, for
instance, using starch crops such as wheat for bioethanol production resulted in considerable
high ethanol concentration in reduced fermentation time [38]. In that case, slurries containing
300 g/L of raw wheat flour were initially liquefied using 0.02 g α-amylase/g starch at 95°C
for 2 hours, followed by saccharification using two different levels of amyloglucosidase
activity (270 U/kg starch and 540 U/kg starch) and simultaneous fermentation by
Saccharomyces cerevisiae at 35°C for 21 hours, reaching a final ethanol concentration of 67
g/L. As for the hydrolysis of lignocellulosic biomass, various levels of enzyme load have
been reported in the various literatures.
Basically, two different processes can be used to produce ethanol from starch crops: dry grind
and wet milling. In dry grind, the feed material is ground mechanically and cooked in water
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to gelatinize the starch. Enzymes are then added to break down the starch to form glucose,
which are fermented by yeasts to ethanol. In that case, a fixed amount of ethanol is produced,
along with other feed products and carbon dioxide, and has almost no process flexibility. In
wet milling, the insoluble protein, oil, fiber, and some solids are removed initially with only
the starch slurry fed remaining upto the ethanol production step. This process has the
capability to produce various end products and considerable higher process flexibility,
compared to the dry milling. Currently, about 65% of the ethanol is produced from dry grind
corn processing plants [39].
Biological processing offers a number of advantages for converting biomass into biofuels.
First, the enzymes used in bio-processing are typically capable of catalyzing only one
reaction, and so formation of unwanted degradation products and by-products is avoided [40].
Additionally, material not targeted for conversion can pass through the process unchanged
and be used for other applications. Although the individual steps for converting biomass into
ethanol can be conveniently isolated, these can otherwise be combined in various ways in
order to minimize the production cost.
Trends in bioethanol production development
In the last couple of years, technological breakthrough has been enormously necessitated due
to the lack of alternative feedstock and considerable shortage of agricultural land. In this
sense, advances in metabolic pathway engineering and genetic engineering have led to the
development of microorganisms capable of efficiently converting biomass sugars into
ethanol. Generally, such development relies on broadening the substrate range to include
other biomass sugars such as arabinose or xylose in strains that cannot ferment sugars other
than glucose. Examples of such microorganisms include Escherichia coli, Saccharomyces sp.
and Zymomonas mobilis [41].
As for the cellulosic ethanol industry, aside from Pichia stipitis, a natural xylose-fermenting
yeast, most efforts have concentrated on obtaining recombinant strains of bacteria and yeast
which are able to ferment pentose sugars, such as xylose and arabinose. Basically the tail end,
as in Escherichia coli and Klebsiella oxytoca, or the front end of metabolism, as for
Saccharomyces cerevisiae and Zymomonas mobilis can be recombined. Future directions for
the development of lignocellulose to ethanol processes should necessarily include the
efficient depolymerization of cellulose and hemicellulose to soluble sugars. For instance,
considering the SSF process, if pentose sugars present in the lignocellulosic material could be
fermented at the same time as glucose, the ethanol concentration in the slurry after
fermentation could be increased significantly, leading to considerable reduction in the energy
demand during the distillation afterwards [42].
The genetic engineering of plants is another promising research field, which will most likely
play a major role on the biofuel industry. The latest developments on hybrid varieties have
enabled considerable increases in starch yield from energetic crops. In the near future, that
same bushel may contain as much as 17 kg of starch through improved hybrid corn. This
would result in a gain of nearly $2 million annual revenue from processing the same bushel
of corn in a 120 million litres per year facility [43].
Protein engineering using the “informational” approach offers powerful opportunities to
enhance the efficiency of enzymatic hydrolysis. Simple modifications to the amino acid
sequence of a protein can have dramatic impacts on performance. Though ethanol is the
major biofuel actually produced on commercial scale, its usage as transport fuel poses a few
obstacles, such as: the tendency of ethyl alcohol to pick up water endurances its transport,
particularly in pipeline; in addition, it is corrosive, considerably volatile and its energy
density is low compared to regular petrol. In order to overcome such disadvantages, another
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form of alcohol has been thought to replace ethanol, such as butanol, an alcohol with four
carbon atoms in its molecule, which has been produced experimentally in the U.S. using
sugar beet and cellulosic feedstock. The stability of e-diesel, a new biofuel under
development has been investigated recently [44]. This biofuel is produced by direct blending
of bioethanol and diesel fuel, and has a considerable potential to reduce particulate emissions,
with low production cost. One drawback in this process is the fact that ethanol is ordinarily
immiscible with diesel fuel, thus requiring in many cases the presence of surfactants.
Future works
Extensive research has been done in the development of advanced technologies to prepare
more digestible biomass to ease bioconversion of biomass into cellulosic ethanol. An ideal
cost-effective pretreatment method might have several characteristics. (a) maximum
fermentable carbohydrate recovery; (b) minimum inhibitors produced from carbohydrate
degradation during pretreatment; (c) low environmental impact; (d) low demand of postpretreatment processes such as washing, neutralization, and detoxification; (e) minimum
water and chemical use; (f) low capital cost for reactor; (g) moderately low energy input; (h)
relatively high treatment rate; and (i) production of high value-added by-products. Therefore,
the future research on pretreatment would be focused on the following areas, (i) reduction of
water and chemical use; (ii) recovery of carbohydrates and value-added by-products to
improve the economic feasibility; (iii) development of clean delignification yielding benefits
of co-fermentation of hexose and pentose sugars with improved economics of pretreatment.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Prasad S, Singh A, Joshi HC. Ethanol as an alternative fuel from agricultural, industrial and urban
residues, Resources Conservation and Recycling 2007; 50(1):1.
Shibata MG, Singhal RS, Kulkarni PR. Resistant starch: A review. Comprehensive Reviews in Food
Science and Food Safety 2006; 5: 1-17.
Immanuel LO, Gomez PF, Moniruzzaman M, York SW. Metabolic engineering of bacteria for ethanol
production. Biotechnology and Bioengineering, 2006; 58: 205-214.
Berg, C. World ethanol production 2001. The distillery and Bioethanol Network. Available at
http://www.distill.com/world ethanol production.htm.
Wiselogel A, Tyson J, Johnsson D. Biomass feedstock resources and composition. In: Wyman CE (ed)
Handbook on bioethanol: production and utilization. Taylor and Francis, Washington, DC 1996: 05–
118.
Brigham JS, Adney WS, Himmel ME. Hemicelluloses: diversity and applications. In: Wyman CE (ed)
Handbook on bioethanol: production and utilization. Taylor and Francis, Washington, DC 1996: pp
119–142.
Ahring A, D Atkin, Rigsby L. Corn fiber: Structure and response to enzymes for fermentable sugars
and co-products. Applied Biochemistry and Biotechnology 1999; 144: 59-68.
Hahn-Hagerdal B, Jeppsson H, Prior BA. Biochemistry and physiology of xylose fermentation by
yeast. Enzyme Microbial Technology 1998; 16: 933-943.
Koskinen EP, Beck SR, Orlygsson JO, Puhakka J. Ethanol and hydrogen production by two
thermophilic, anaerobic bacteria isolated from Icelandic geothermal areas. Biotechnology and
Bioengineering 2008; 101: 679-690.
Larsen L, Nielsen P, Ahring BK. Thermoanaerobacter mathranii an ethanol-producing, extremely
thermophilic anaerobic bacterium from a hot spring in Iceland. Arch Microbiol 1997; 168: 114-119.
Maney Sveinsdottir, Liming Xiaa, Peijian Xueb. Enzymatic hydrolysis of corncob and ethanol
production from cellulosic hydrolysate. International Biodeterioration and Biodegradation, 2009; 59:
85–89.
Henstra, Stams. Biomass recalcitrance: engineering plants and enzymes for biofuels production.
Science 2009; 315: 804-807.
Ingram LO, Gomez PF, Moniruzzaman M et al. Metabolic engineering of bacteria for ethanol
production. Biotechnology and Bioengineering1998; 58: 205-214.
Dien BS, Cotta MA, Jeffries TW. Bacteria engineered for fuel ethanol production: Current status.
International Journal of Applied Microbiology Science 2012; 1(2): 1-12
10
Saranraj & Stella
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
Applied Biochemistry and Biotechnology 2003; 63: 258-266.
Wang Z, Chen M, Xu Y et al. An ethanol-tolerant recombinant Escherichia coli expressing
Zymomonas mobilis pdc and adh B genes for enhanced ethanol production from xylose, Biotechnology
Letters 2008; 30: 657-663.
Doran-Peterson J, Cook DM, Brandon SK. Microbial conversion of sugars from plant biomass to
lactic acid or ethanol. Plant J 2008; 54: 582-592.
Kim TH, Taylor F, Hicks KB. Bioethanol production from barley hull using SAA (soaking in aqueous
ammonia) pretreatment, Bioresource Technology 2008; 99: 5694 -5702.
Mishima D, Kuniki M, Sei K et al. Ethanol production from candidate energy crops: Water hyacinth
(Eichhornia crassipes) and water lettuce (Pistia stratiotes L.), Bioresource Technology 2008; 99:
2495-2500.
Brandon SK., Eiteman MA, Patel K et al. Hydrolysis of Tifton 85 bermuda grass in a pressurized batch
hot water reactor. Journal of Chemical Technology and Biotechnology 2008; 83: 505-512.
Patle S, Lal B. Investigation of the potential of agro-industrial material as low cost substrate for
ethanol production by using Candida tropicalis and Zymomonas mobilis. Biomass Bioenergy 2008; 32:
596-602.
Mohagheghi A, Evans K, Zhang M. Co-fermentation of glucose, xylose, and arabinose by genomic
DNA-integrated xylose/arabinose fermenting strain of Zymomonas mobilis AX101. Applied
Biochemistry and Biotechnology 2002; 98: 885-898.
Golias H, Dumsday GJ, Stanley GA et al. Evaluation of a recombinant Klebsiella oxytoca strain for
ethanol production from cellulose by simultaneous saccharification and fermentation: comparison with
native cellobiose-utilizing yeast strains and performance in co-culture with thermotolerant yeast and
Zymomonas mobilis. J Biotechnol 2002; 96: 155-168.
Doran JB., Cripe J, Sutton M, Foster B. Fermentations of pectin-rich biomass with recombinant
bacteria to produce fuel ethanol. Applied Biochemistry and Biotechnology 2000; 84-86: 141-152.
Chandrakant P, Bisaria VS. Simultaneous bioconversion of glucose and xylose to ethanol of
Saccharomyces cerevisiae in the presence of xylose isomerise. Applied Biochemistry and
Biotechnology 2000; 53: 301-309.
Zaldivar J, Borges M, Johansson Bet al. Fermentation performance and intracellular metabolite
patterns in laboratory and industrial xylose-fermenting Saccharomyces cerevisiae. Applied
Biochemistry and Biotechnology 2002; 59: 436-442.
Chu BCH and Lee H. Genetic improvement of Saccharomyces cerevisiae for xylose fermentation,
Biotechnol Adv 2007; 25: 425-441.
Hamacher T, Becker J, Gardonyi M et al. Characterization of the xylose-transporting properties of
yeast hexose transporters and their influence on xylose utilization. Microbiology 2002; 148: 27832788.
Ryan L, Convery F, Ferreira S. Stimulating the use of biofuels in the European Union: Implications for
climate change policy. Energy Policy 2006; 34: 3184-3194.
Murphy JD, McCarthy. Ethanol production from energy crops and wastes for use as a transport fuel in
Ireland. Applied Energy 2005; 82:148-166.
Ohara H. Biorefinery. Journal of Applied Microbiology and Biotechnology 2003; 62: 474-477.
Yu SR, Tao J. Life Cycle Simulation-based Economic and Risk Assessment of Biomass-based Fuel
Ethanol (BFE) Projects in Different Feedstock Planting Areas. Energy 2008; 33: 375−384.
Zhang SP, Yan YJ, Ren Z. Fuel Ethanol Production from Lignocellulosic Biomass [J]. Progress in
Chemistry 2007; 7: 1129−1133.
Ehara K, Saka S. A comparative study on chemical conversion of cellulose between the batch-type and
flow-type systems in supercritical water. Cellulose 2002; 9: 301-311.
Chum HL, Overend RP. Biomass and renewable fuels. Fuel Processing Technology 2001; 73: 187-195.
Ohgren K, Rudolf A, Galbe M et al. Fuel ethanol production from stream-pretreated corn stover using
SSF at higher dry matter content. Biomass and Bioenergy 2007; 30: 863-869.
Wyman CE. Biomass ethanol: technical progress, opportunities, and commercial challenges. Annual
Review of Energy and the Environment 1999; 24: 189–226.
Noureddini H, Byun J, Yu T. Stage wise dilute-acid pretreatment and enzyme hydrolysis of distillers'
grains and corn fiber. Applied Biochemistry and Biotechnology, (in press). 2009.
Montesinos T, Navarro JM. Production of alcohol from raw wheat flour by Amyloglucosidase and
Saccharomyces cerevisiae. Enzyme and Microbial Technology 2000; 27: 362-370.
DOE, Department of Energy. Office of Energy Efficiency and Renewable Energy, Washington DC,
US, http://www.doe.gov. 2007.
International Journal of Applied Microbiology Science 2012; 1(2): 1-12
11
Saranraj & Stella
[40]
[41]
[42]
[43]
[44]
Schmidt AS, Mallon S, Thomsen AB et al. Comparison of the chemical properties of wheat straw and
beech fibers following alkaline wet oxidation and laccase treatments. Journal of Wood Chemistry and
Technology 2002; 22: 39-53.
Neves MA, Kimura T, Shimizu N et al. Production of alcohol by simultaneous saccharification and
fermentation of low-grade wheat flour. Brazilian Archives of Biology and Technology 2006; 49: 481490.
Xu, H., Sun L, Zhao D, et al. Production of α-amylase by Aspergillus oryzae. As 3951 in Solid state
fermentation using spent brewing grain as substrate. Journal of Science and Food Agriculture, 2008,
88: 529-535.
Rani P, Garg FC, Sharma SS et al.. Ethanol production from potato starch. Indian Food Packer, 2009,
63(4): 63-68.
Lapuerta M, Armas O, Contreras RG. Stability of diesel-bioethanol blends for use in diesel engines.
Fuel, 2007, 86: 1351-1357.
International Journal of Applied Microbiology Science 2012; 1(2): 1-12
12