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Bioethanol production: Feedstock and current technologies

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Journal of Environmental Chemical Engineering 2 (2014) 573–584

Contents lists available at ScienceDirect

Journal of Environmental Chemical Engineering

journal homepage: www.elsevier.com/locate/jece

Review

Bioethanol production: Feedstock and current technologies

Mustafa Vohra a, Jagdish Manwar b,*, Rahul Manmode c, Satish Padgilwar b, Sanjay Patil a
a
Department of Alcohol Technology, Vasantdada Sugar Institute, Manjari (Bk), Pune 412 307, India
b
Department of Quality Assurance, SGSPS Institute of Pharmacy, Hingna Road, Kaulkhed, Akola 444 004, India
c
Department of Chemistry, University of Massachusetts, Lowell 01854, USA

A R T I C L E I N F O A B S T R A C T

Article history: Fossil fuels such as oil, coal and natural gases have become the prime sources of energy in the current era.
Received 11 April 2013 However, it is anticipated that these sources will deplete within the next 40–50 years. The expected
Received in revised form 11 October 2013 environmental damages like global warming, acid rain and urban smog have tempted us to reduce the
Accepted 22 October 2013
carbon emissions by 80% (v/v) and shift toward utilizing a variety of renewable energy resources such as
solar, wind, biofuel, etc. that are less environmentally harmful in a sustainable way. Ethanol is one of the
Keywords: most promising alternative biofuel. Although the energy equivalent of ethanol is 68% lower than that of
Ethanol
petroleum fuel, the combustion of ethanol is cleaner (because it contains oxygen) and thus it recognize as
Lignocellulosic waste
Sugarcane
a potential biofuel alternative to gasoline. Ethanol has been frequently used for the blended gasoline in
Starch the concentration range 10–85% (v/v). More recently, ethanol is identified as a fuel for the direct ethanol
Syngas fuel cells (DEFC) and biofuel cells. Sugarcane and corn feedstock, are the main source of ethanol.
Nevertheless, it is barely sufficient to meet the current demand. Lignocellulosic biomass is an alternative
source but its availability is poorly documented. This review discusses the current status of ethanol
production from different feedstocks and the state of technologies involved in ethanol production from
such different feedstock.
ß 2013 Elsevier Ltd All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
Overall process of bioethanol production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
Bioethanol production from various sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
Production of ethanol using sucrose based feedstocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
Bioethanol production using starch based feedstocks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
Corn dry-milling process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
Corn wet-milling process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
Bioethanol production using lignocellulosic based feedstocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
Lignocellulosic feedstock composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
Conversion paths for ethanol from lignocellulosic feedstock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Integrated technologies based on hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
Simultaneous saccharification and fermentation, SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
Simultaneous saccharification and co-fermentation, SSCF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Consolidated bioprocessing, CBP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Syngas platform: thermochemical conversion processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Gasification: syngas catalytic conversion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Biological path is gasification: syngas fermentation route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
Preferred technology route. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583

* Corresponding author at: Department of Quality Assurance, SGSPS Institute of Pharmacy, Hingna Road, Kaulkhed, Akola, Maharashtra 444 003, India.
Tel.: +91 9811805653.
E-mail addresses: mustafa_vohra1@yahoo.co.in (M. Vohra), jvmanwar@gmail.com (J. Manwar).

2213-3437/$ – see front matter ß 2013 Elsevier Ltd All rights reserved.
http://dx.doi.org/10.1016/j.jece.2013.10.013
 Author's personal copy
574 M. Vohra et al. / Journal of Environmental Chemical Engineering 2 (2014) 573–584
Introduction
Ethanol (CH3CH2OH) is the most popular alcoholic biofuel
available in the current world market. Henry Ford has used the
term ‘‘fuel of the future’’ for the ethanol. There are several reasons
for being its use as alternative fuel such as (i) it is produced from
the renewable agricultural products like corn, sugar, molasses
including other products rather than nonrenewable petroleum
products, (ii) it is less toxic than other alcoholic fuels, and (iii) by-
products of incomplete oxidation of ethanol (e.g. acetic acid and
acetaldehyde) are less toxic than the by-products formed from
other fuel alcohols [1].
The world total ethanol production during 2009–2010 was
almost 100 billion liters. The world ethanol consumption was 68%
for fuel, 21% for industrial and 11% for potable purpose [2]. It is
used to partially replace gasoline to make gasoline-ethanol
mixtures, E15 (15% ethanol and 85% gasoline) and E85 (85%
ethanol and 15% gasoline). Bioethanol is a liquid biofuel which can
be produced from several different biomass feedstocks and
conversion technologies. The feedstock used for fuel ethanol
production is mainly sugarcane in tropical areas such as India,
Brazil and Colombia, while it is dominantly corn in other areas such
as the United States, European Union, and China [3]. Ethanol
production from sugar crops such as sugarcane and sugar beet
account for about 40% of the total bioethanol produced and nearly
60% corresponding to starch crops [4]. Due to increasing demand
for ethanol in the last few years and due to shortage of molasses
and corn, the prices of ethanol are increasing day by day. Also due
to its increase in demand as a food source and its rising price, the
availability and feasibility of using corn as a feedstock is in stake.
The further expansion of ethanol production from many of these
feedstock’s, thus triggers debate on food/feed versus fuel, limiting
the use of first generation feed stock for ethanol production. Thus
for sustainable fuel grade ethanol production, non-food feedstock
should be used. There is an urgent need for development of second
generation bioethanol [5].
Second-generation fuels are generally those made from non-
edible lignocellulosic (LC) biomass, either residues of forest
management or food crop production (e.g. corn stalks or rice
husks) or whole plant biomass (e.g. grasses or trees grown
specifically for biofuel purposes). LC biomass, also called cellulosic
biomass, is a complex composite material consisting primarily of
cellulose, hemicellulose and lignin bonded to each other in the
plant cell wall. This Lignocellulosic biomass such as agri-residues
(e.g., corn stover, wheat and barley straws), agri-processing
byproducts (e.g., corn fiber, sugarcane bagasse, seed cake, etc.),
woody biomass (hardwood and softwood) and energy crops (e.g.,
switch grass, poplar, banagrass, miscanthus, etc.) do not compete
with food and feed, and are considered to be renewable feedstocks
for ethanol production [6].
This review examines what is currently known regarding
different feedstock’s and technologies that are used in bioethanol
production with respect to its overall conversion technology. This
review also provides a brief summary of the current challenges and
barriers that interfere with each substrate based-ethanol pathway
and places the emphasis on potential issues challenging biotech-
nological conversion and performance of the bioethanol produc-
tion process.
Overall process of bioethanol production
The process of ethanol production depends on the raw
materials used. Ethanol production commonly carried out into
the major three steps: (1) to obtain the solution containing
fermentable sugars, (2) conversion of sugars into ethanol by
fermentation and (3) ethanol separation and purification, usually
by distillation–rectification–dehydration. The fermentation pro-
cess can use any sugar-containing material to produce ethanol
(Fig. 1) [7].
Based on Fig. 1, one or more steps can be combined depending
on the feedstock and the conversion technology. Once the biomass
reaches the ethanol plant, it is stored in a warehouse where it is
conditioned to prevent early fermentation and bacterial contami-
nation. Through the pretreatment, carbohydrates are extracted or
made more accessible to the further extraction. During this step,
the amounts of available sugars depend on biomass and pre-
treatment used. A large portion of fibers may remain for conversion
into simple sugars through hydrolysis reactions or other techni-
ques. The hydrolysate, yeasts, nutrients and other ingredients are
added to the fermentation at the beginning of the batch operation.
In a fed batch process, one or more inputs are added as
fermentation progresses. Continuous processes in which ingre-
dients are constantly input and products removed from the
fermentation vessels are also used. In efficient processes, the cell
densities may be made high by recycling or immobilizing the
yeasts in order to improve their activity and increase the
fermentation productivity [8]. The fermentation reactions occurs
at temperatures between 25 8C and 30 8C and it last between 6 h
and 72 h depending on the composition of the hydrolysate, cell
density, physiological activity and yeast species. The broth
typically contains 8–14% of ethanol on a volume basis. Above this
latter concentration, inhibition of yeasts may occur that reduces
their activity. The distillation step yields an azeotropic mixture
made up of 95.5% alcohol and 4.5% water that is the ‘‘hydrous’’ or
‘‘hydrated’’ ethanol which is then dehydrated to obtain an
‘‘anhydrous’’ ethanol containing up to 99.6% alcohol and 0.4%
water. The remaining flow from the distillation column, known as
[(Fig._1)TD$IG]
vinasse, or stillage can be volatilized to produce co-products,
Corn wet mill Enzymes/ Cellulosic ethanol
acid
Corn Cellulosic
Pretreatment
biomass
Hydrolysis
Oil Starch Heat/
CGM Wet chemicals
milling
CGF
Grinding Corn
Canesugar ethanol Sugar
Fermentation
Cane juice Fermentation Corn
organism dry
mill
Sugar
extraction
DDGS
Product
Sugarcane recovery
Lignin and other
Vinasse residuals
Sugarcane
Ethanol
bagasse
Fig. 1. Schematic representation of production of ethanol from cane sugar, corn, and
cellulosic biomass. All have similar fermentation and ethanol recovery operations
but use different approaches to release sugars and generate differentcoproducts.
Sugar can be directly extracted from sugarcane, and the residual bagasse is used as a
boiler fuel to provide much of the energy for the extraction and ethanol production
and recovery operations. In a corn dry mill, corn is ground, and enzymes and heat
are added to hydrolyze starch to sugars for conversion to ethanol, while the oil,
protein, and fiber in corn are recovered after fermentation as an animal feed known
as DDGS. Wet mills first fractionate corn to separate corn oil, corn gluten meal
(CGM), and corn gluten feed (CGF) to capture value for food and animal feed, and the
starch can then be hydrolyzed to sugars for fermentation to ethanol. For cellulosic
biomass, heat and acids or enzymes hydrolyze the hemicellulose and cellulose
portions to release sugars that can be fermented to ethanol, and the lignin and other
remaining fractions can be burned to provide all the process heat and electricity for
the conversion step with excess electricity left to export [8].
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M. Vohra et al. / Journal of Environmental Chemical Engineering 2 (2014) 573–584
which may include process steam and electricity, products for
feeding animals, stillage of moderate concentration used as
fertilizer, and other valuable by-products [9].
Bioethanol production from various sources
Bioethanol is commonly produced from the agricultural raw
materials containing sugar. This material can be classified as first
generation material (includes sugar and starch) and second
generation material (includes lignocellulosic sugar). Sugars (e.g.,
from sugarcanes, molasses, sugar beet, and fruits) can be directly
fermented using yeast to produce ethanol. It should be noted that if
sugar materials such as molasses and sugarcane juice are used for
fermentation, then processes like milling, pretreatment, hydroly-
sis, and detoxification are not necessary. For the production of
fermentable sugar from starchy materials, processes like milling,
liquefaction, and saccharification are used; but in case of
lignocellulosic material, milling, pretreatment, and hydrolysis
are required. Further, a detoxification unit is not always consid-
ered, unless a toxic substrate is fed to the bioreactors. The step that
precedes the fermentation processes to obtain fermentable sugars,
is the main difference between the ethanol production processes
from simple sugar, starch or lignocellulosic material (Fig. 1) [4].
Production of ethanol using sucrose based feedstocks
Sugar cane, sugar beets, and sweet sorghum are sugar crops
used as feedstocks for ethanol production. Their chief advantages
are high yield of sugar per acre and low conversion costs. The main
disadvantage of using these crops is their natural seasonal
availability. Since the fibers in the stalks and leaves (bagasse)
are used to generate process steam and electricity for the
biorefinery, particularly the sugarcane became a cost effective
source of biofuel. In addition, the liquid effluent (vinasse) is used as
a fertilizer and irrigation supply to the cane fields, thereby
eliminating costs for wastewater treatment [10].
Sugar cane must be processed within the period of 24–72 h
after harvesting. Sugar is first extracted by crushing the stalks with
specialized rollers to release the juice (Fig. 2). Lime (calcium
hydroxide) is then added to precipitate the fiber and sludge, and
mixture is then filtered. The filtrate solution is evaporated to
concentrate and crystallize the sugar, prior to its removal by
centrifugation. The noncrystallized sugar and accompanying salts
are concentrated to form syrup called ‘blackstrap molasses (BSM)’
that is used as a raw material, which is subsequently converted
[(Fig._2)TD$IG]into ethanol [10]. After extraction, which is different according to
Sugarcane
Milling
Juice extraction
Sugar juice Bagasse
Juice treatment Juice treatment Energy
generation
Sugar production Fermentation
Steam
Sugar Distillation CO2 Electricity
Bioethanol
...to process....
Fig. 2. Main processes for bioethanol production from sugar cane [11].
575
the type of distillery (producing ethanol only, or ethanol and
sugar), the sugar content has to be adjusted in the range of 14–18%
to achieve optimum fermentation efficiency of yeast Saccharomy-
ces cerevisiae, the most commonly used microorganism at a
temperature around 33–35 8C and cell density of 8–17% (v/v). Cell
recycle system are used in which yeast is typically concentrated
and separated from the fermentation effluent, washed with
sulfuric acid to reduce bacterial contamination, and recycled to
fermenters, resulting in high cell densities that shorten fermenta-
tions to 6–10 h at temperatures of about 33–35 8C with high cell
ethanol yield [8]. Fermentation is interrupted at concentration of
approximately 10% (v/v) ethanol: then, the broth is send to the
distillation and rectification phase, which product is an azeotropic
solution of 95% (v/v) ethanol. Further concentration to absolute
ethanol (high grade or anhydrous ethanol) is finally achieved by
molecular sieves or distillation using benzene or cyclohexane
(azeotropic distillation) [11].
Sugar beets represent a major source of sugar in Europe and
North America, and used for biofuel production in France. Sugar
beets generate good yields (25–50 tons per acre) and grow in
temperate climate with a lower rainfall than is needed for sugar
cane. On average, the ethanol yield is 25 gallons per ton of sugar
beets; however, ethanol production from sugar beet requires a
greater chemical and energy input and consequently is more
expensive than the process using sugar cane [10].
Sweet sorghum varieties are few in number and not widely
grown, although some have a significant sucrose content (500
gallons syrup per hectare). In sweet sorghum, the sugar is stored in
the main stalk, and is recovered by pressing the stalks with rollers
(similar to the process used for sugar cane). Yields, on average, are
20 gallons of ethanol per ton of stalks. China is currently seeking to
switch from corn to sweet sorghum as a feedstock for ethanol,
since sorghum is more drought-tolerant and can grow in the arid
regions of China where corn does not perform well. An additional
benefit is that the farmers can use the sorghum grain, since the
ethanol is produced from the sweet juice in the stalk [10].
It is generally accepted that alcoholic fermentation from
sucrose containing feedstock is a well-known and mastered
process. There is increase in overall yield of Brazilian fuel ethanol
industry due to several agricultural improvements, including
selection of new sugarcane varieties with increased amounts of
sugarcane biomass per hectare. While the amount of total sugar
per ton of sugarcane also increased significantly during the initial
years, in the last 15 years it seems to have reached a plateau around
140 kg of sugar per ton of sugarcane. Thus, there is a need for
improvement in sucrose production and accumulation by the
sugarcane plant, a feature that is under current research in Brazil
[12]. High ethanol concentration, high temperature, osmotic stress
due to sugar and salts, acidity, sulfite and bacterial contamination
are recognized stress conditions faced by yeast during the
industrial processes, some of them acting synergistically and
particularly with cell recycling. It is very difficult to understand
how the yeast cells respond to the stressful conditions of industrial
fermentation without precisely reproducing these conditions [13].
Main challenges of bioethanol production from sucrose containing
feedstock are: (a) Introduction of new feedstocks, (b) production of
second generation bioethanol from bagasse, (c) selection of new
yeast strains more adapted to stressing conditions of industrial
fermentations, (d) bacterial and yeast contamination in the
fermentation process with cell recycling, and (e) reduction of
vinasse volume. The challenges in using sweet sorghum as a
feedstock includes the breeding of new sorghum varieties, planting
techniques, harvesting time (especially during the rainy season),
crushing and fermentation, as well as reduction of compounds that
affect industrial processes such as aconitic acid, phenolic
compounds, and starch [14].
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576 M. Vohra et al. / Journal of Environmental Chemical Engineering 2 (2014) 573–584
Bioethanol production using starch based feedstocks
Grains (corn, wheat or barley) mainly provide starch. For
example, corn contains 60–70% starch. Starch stored in grains is
made up of long chains of glucose units, containing 1000
monomeric units or more per amylose structure and 1000–6000
units or more monomers per amylopectin structure. To produce
ethanol from starch it is necessary to break down the chains of this
carbohydrate for obtaining glucose syrup, which can be converted
into ethanol by yeasts. This type of feedstock is the most utilized
for ethanol production in North America and Europe. Corn and
wheat are mainly employed with the purpose of producing
ethanol. In tropical countries, other starchy crops as tubers (e.g.
cassava) can be used for commercial production of fuel ethanol
[15]. In starch, polymers of glucose are broken into glucose through
a hydrolytic reaction catalyzed by gluco-amylase enzyme. The
resulting sugar is known as dextrose or D-glucose that is an isomer
of glucose. The enzymatic hydrolysis is then followed by
fermentation, distillation and dehydration to yield anhydrous
ethanol [16].
There are two distinct methods for processing corn, wet milling
and dry milling, and each method generates unique co-products.
Dry mills are usually smaller in size (capacity) and are built
primarily to produce only ethanol. Wet mill facilities are called
corn refineries, that also produce a list of high-valued co-products
such as high-fructose corn syrup (HFCS), dextrose, and glucose
syrup. Both wet and dry milling operations convert corn into
ethanol [17].
Corn dry-milling process
Corn dry-milling process is carried out in five steps viz: (i)
biomass handling (milling), (ii) liquefaction, (iii) hydrolysis
(saccharification), (iv) fermentation, and (v) distillation and
recovery. In dry-grind process, the corns are passes through the
hammer mills that grind it into the fine particles. This process
facilitates the entry of water and enzymes in the next steps [10]. In
a typical dry mill process, the grains are milled to a powder and
heated with water at 85 8C. While still hot, powder of alpha-
amylase enzymes are added and the mixture is heated at 110–
150 8C for an hour. This causes the liquefaction of starch and
reduces the level of bacteria. It is again cool to 85 8C, and held at
[(Fig._3)TD$IG]
Corn - amylase
Grinding Mashing Cooking Liquefaction
Fresh
water
Treated Thin stillage recycle
Water for
reuse CH4
Condensate
Methanator Evaporation
Solubles
Excess
Blending
biosolids
Distillers dried grains
With solubles (DDGS)
Fig. 3. Corn dry milling process flow diagram.
this temperature for 1 h after adding of with more quantity of
alpha-amylase enzymes. It is cooled to room temperature and
gluco-amylase enzymes are added to ensure the conversion of corn
starch to dextrose. Thus, the overall process is catalyzed by the
enzymatic hydrolysis [8]. In most of the dry-grind milling plants,
the gluco-amylase enzymes are directly added into the fermentor
using the process known as ‘simultaneous saccharification and
fermentation’ (SSF). This process reduces the cost of requirement
of saccharification vessels and minimizes the risk of contamination
(Fig. 3) [10].
In the fermentation process, yeasts convert glucose into ethanol
and carbon dioxide. Fermentation process can be operated in batch
held until the process is completed within 48 h or can be operated
continuously with ongoing addition of sugar and taking out of
fermented broth know as beer. Usually, for the completion of
fermentation process it takes about 40–50 h. During the fermen-
tation, the mash is agitated continuously to distribute of yeast
uniformly and kept it cool and highly active. After fermentation,
the resulting beer is transferred to distillation columns where
ethanol is separated from the remaining stillage [18]. The stillage
containing the remaining protein, oil, and fiber are dried to a 27%
protein product known as distillers dried grains with solubles
(DDGS) or just distillers dried grains (DDG), depending on whether
process syrup is combined with the solids or not, and used in
animal feeds.
Corn wet-milling process
In wet milling process, the corn kernel is separated into three
parts in an aqueous medium prior to fermentation: (1) the hull, (2)
the germ, and (3) the endosperm. The primary products of wet
milling include starch and starch-derived products (e.g. high
fructose corn syrup and ethanol), corn oil, and corn gluten.
As schematically shown in Fig. 4, the process consists of several
steps. A wet mill generally receives shelled corns, which pass
through mechanical cleaners designed to remove unwanted
material, such as pieces of cobs, sticks, husks, meal and stones.
The cleaned corns are next fed into ‘‘steep’’ tanks, where these are
soaked in dilute sulfuric acid from 24 to 48 h at a temperature of
52 8C. Steeping softens the kernel, helps to break down the protein
holding the starch particles, and removes various soluble
constituents. A number of tanks are used in series. Corn that
Glucoamylase
Saccharification Simultaneous
saccharification
And fermentation
Yeast Fermentation
Distillation Ethanol
stillage
Thin stillage
Centrifugation
Wet cake
Distillers
dried grains Drying
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Corn
Kernels Steeping
Starch,
Degermination Gluten,
(grinding, centrifugation) fiber
Fiber
Seed germ
Gluten
Corn oil
Corn gluten meal
Corn gluten feed
Fig. 4. Corn wet milling process flow diagram.
has steeped for the desired length of time is discharged from the
tank for further processing, and the tank is filled with fresh corn.
Generally, water drained from the first steep tank is discharged to
evaporators, called ‘‘light steepwater’’ that contains about 6% of the
original dry weight of the grains. The solids from steep water are
rich in protein and are concentrated to 30–55% solids in multiple-
effect evaporators. The resulting steeping liquor can be sold as
animal feeds [19].
The germ is removed from the steeped corn in the degerminat-
ing mills, which break the kernel apart to free both the germ and
about half of the starch and gluten. The germ is separated in liquid
cyclones from the mixture of fiber, starch, and gluten. It is
subsequently washed, dewatered, and dried, and further processed
to extract corn oil [20].
The starch and gluten from the product slurry are removed from
the rest of the fibrous material by further washing, grinding, and
screening operations. The discarded hulls are dried for use in
animal feed. The starch is separated from the gluten by
centrifugation. A number of stages may be necessary [19,20].
When the purified starch slurry is obtained, the wet-mill
process is very similar to that of dry milling. First, the pH of the
starch slurry is adjusted to 5.8–6.2 with lime, after which alpha-
amylase is added to convert the starch polymer into soluble short-
chain dextrins (liquefaction). Calcium is often added (20–
100 ppm) to enhance enzyme stability. As the starch stream is
relatively free of fiber or other components, it is well suited to the
high temperature and short time of jet cooking and subsequent
enzyme liquefaction. Hence, solid slurries of 30–40% starch are
common [10].
The slurry from the liquefaction stage is mixed with heat-
sterilized steep water and sent for saccharification. The steep water
provides both the fermentation nutrients and pH adjustment for
saccharification, in which the added glucoamylase converts the
dextrins to glucose at a pH of 4.5 and a temperature of 65 8C. After
saccharification, S. cerevisiae is added to ferment the sugars to
ethanol and CO2. The total fermentation time varies from 20 to
60 h, depending mainly on the degree of saccharification prior to
fermentation. Most wet mills practice continuous-cascade fer-
mentation. Very few insoluble solids are found in these fermenta-
tion systems, which facilitate yeast recycling and improves the
overall fermentation rates. The final product from a continuous
process will have an ethanol content of 8–10% by volume [10,20].
The starch-based bioethanol industry has been commercially
viable for about 30 years; in that time, tremendous improvements
have been made in developing high fermentable hybrid strains,
577
Steepwater Corn steep liquor
(45% Solids)
Starch
Liquefaction,
saccharification
Fermentation Distillation
Solids
Aqueous ethanol (95%)
Anhydrous ethanol
separation technology, fermentation process, enzyme efficiency,
reducing process costs and time, and increasing bioethanol yields.
New tendencies in corn-to-ethanol industry are aimed at dry-
milling processes. Research efforts are oriented to the develop-
ment of corn hybrids with higher extractable starch or higher
fermentable starch content. Another area of industrial interest is
illustrated in the efforts of Syngenta Biotechnology (Syngenta
Biotechnology, Inc., SBI) to direct the accumulations of starch-
hydrolyzing enzymes in the endosperm of transgenic corn kernels.
Stable accumulations of enzymes, without detriment to grain
viability and composition, allows ‘‘processing capability’’ to be
built into the grain itself. Self-processing grains can be designed to
meet specific and novel process constraints due to flexibilities in
engineering enzymes with distinct biophysical properties and
enzymatic specificities [20]. Modifications of the dry-grind facility
have made the recovery of corn germ possible in dry milling. Dry
fractionation technology separates the corn kernel into its
components without the soaking step. Advancement in fermenta-
tion has led to development of improved quality of yeast and
reactor configuration. The SSF performed at a temperature above
34 8C employing a thermotolerant yeast enables the reduction of
cooling requirements and the improvement of the conversion
process. Very-high-gravity fermentation accomplishes saving in
energy by using a highly concentrated mash with more than 30%
solids. Experiments have resulted in 23%-alcohol fermentation,
much higher than with the conventional process. Potential savings
would come from the reduced cost of water and wastewater
cleanup, as well as from reduced energy use. In last years, the
possibility of hydrolyzing starch at low temperatures for achieving
energy savings is also being investigated. Costs have fallen 70%
over the last 25 years. Improved enzyme use also results in reduced
soak time, higher starch and gluten yield, better protein quality,
and reduced water and energy use [21].
Bioethanol production using lignocellulosic based feedstocks
Bioethanol can also be produced from lignocellulosic materi-
als, which is commonly known as second generation bioethanol.
The feedstocks for the second generation bioethanol include
agricultural residues, grasses, and forestry and wood residues.
There have been tremendous research efforts in developing cost-
effective second generation or advanced technologies for fuel
ethanol production in the literature. However, there are some
challenges for the commercial applications of the advanced
technologies [3].
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578 M. Vohra et al. / Journal of Environmental Chemical Engineering 2 (2014) 573–584
Lignocellulosic feedstock composition
Cellulosic feedstock is composed of cellulose, hemi-cellulose,
lignin, and solvent extractives. Lignin acts as a cementing material
that binds all other constituents together. It is also responsible for
providing structural rigidity to a cellulosic feedstock. Cellulose is a
polymer of repeating b-D-glucopyranose units and is a chief
constituent of the lignocellulosic feedstock. Hemi-cellulose, like
cellulose, is a polysaccharide but it is less complex and of easy
hydrolysis. In the feedstock, soluble materials or extractives
consist of those components that are soluble in neutral organic
solvents. Sugars present in the cellulose are mostly glucose.
However, hemi-cellulose is a mixture of different types of sugars. It
contains both C6 (glucose, mannose, and galactose) and C5 (xylose,
arabinose, and rhamnose) sugars [22].
Chemical composition of lignocellulosic materials is a key factor
affecting efficiency of biofuel production during conversion
processes. The structural and chemical composition of lignocellu-
losic materials is highly variable because of genetic and
environmental influences and their interactions. A typical chemi-
cal composition of lignocellulosic materials is 48 wt.% C, 6 wt.% H,
and 45 wt.% O, the inorganic matter being a minor component.
Cellulose + hemicellulose contents are more in hardwoods (78.8%)
than softwoods (70.3%), but lignin is more in softwoods (29.2%)
than hardwoods (21.7%) [23].
Conversion paths for ethanol from lignocellulosic feedstock
At present, several technologies are in use for converting
cellulosic feedstocks into ethanol. However, technologies for the
conversion of lignocellulosic feedstocks to ethanol have been
grouped in to two broad platforms, which can be referred to as the
sugar platform (Biochemical conversion) and the syngas platform
(Thermochemical conversion). The basic steps of these platforms
are shown in Fig. 5.
The sugar platform uses enzymes to convert pretreated
lignocellulosic biomass materials into sugars, which can then be
fermented into ethanol. In the syngas platform, a biomass
feedstock is gasified to produce syngas (carbon monoxide,
hydrogen and carbon dioxide) which is then converted into
ethanol by a chemical reaction utilizing chemical catalysis or
biological reaction using microorganisms [24].
Sugar platform: biochemical conversion. Lignocellulosic materials
are abundant almost all over the world and they can be used for
bioethanol production because they have a high content of
cellulose and hemicelluloses. However, the conversion of ligno-
cellulosic materials into ethanol is much more difficult than that of
sugar-rich or starch-rich materials [25]. The biochemical platform
consists of three main process elements – pretreatment, enzymatic
[(Fig._5)TD$IG]
Lignocellulosic Pretreatment
Milling
biomass
Hydrolysis
Sugar
Plat Form
Fermentation Ethanol
Gasification Fermentation Ethanol
Syngas
Plat Form Catalytic
conversion
Fig. 5. Basic pathway for ethanol production from lignocellulosic biomass.
hydrolysis, and fermentation. Process steps also include feedstock
harvesting, handling, recovery and transport, milling of the
biomass to give small and homogeneous particles, fractionation
of the polymers, separation of the solid lignin component, and end
product recovery. Furthermore, detoxification and fermentation of
pentoses released during the pretreatment step can be carried out.
The cellulose undergoes enzymatic hydrolysis to produce hexoses
such as glucose [26].
Pretreatment. The pre-treatment process to expose the cellulose
and hemicellulose for subsequent enzymatic hydrolysis is a critical
process step. Pretreatment options can be classified as biological,
physical, chemical or a combination of them, where temperatures
and reaction times are the variables. The pre-treatment process is a
major cost component of the overall process. No ‘‘best’’ option
exists and research and development (R&D) continues to improve
cost and performance goals, though steam explosion and dilute
acid are probably closest to commercialization [27]. Pre-treatment
can be carried out in a number of ways e.g. using dilute acids (such
as sulphuric or hydrochloric acid), alkalis (such as calcium
hydroxide), liquid ammonia (the ammonia fiber explosion pre-
treatment) or steam explosion [25]. Pre-treatment with dilute acid
and intermediate temperatures is generally considered the most
cost-effective and acts by loosening the cell wall matrix through
degradation of hemicelluloses. Lignin is unaffected by this process.
Accessibility to cellulose microfibrils is sufficiently increased to
provide a higher yield of monomeric sugars for fermentation. Acid
treatment will result in other high value products like furfural,
hydroxyl-methyl furfural (HMF), phenolics, aldehydes, and ali-
phatic compounds. These products have to be removed before
subjecting the residues for further biochemical treatments. Acid
pretreatment processes using mineral acids requires corrosion free
reactors and more over neutralization and detoxification process
are also necessary [28]. Steam explosion, compared to other
pretreatment methods, offers potential for lower capital invest-
ment, significantly lower environmental impact, more potential
for energy efficiency, less hazardous process conditions and
complete sugar recovery. In these treatments, the substrate will
be subjected to high pressures and temperatures for short duration
of retention times followed by the rapid release of the elevated
pressures to atmospheric pressure, which will break the polymeric
bonds in the substrate. The conventional mechanical methods
require 70% more energy than steam explosion to achieve the same
size reduction. Steam explosion is considered the most cost
effective option for hardwood and agriculture residues, but is less
effective for softwood. Most important factors affecting effective-
ness of steam explosion are particle size, temperature and
residence time [4,23]. Acid catalysis also studied within the steam
explosion treatment and is found to reduce the temperature and
retention time with decrease in formation of unwanted products
and complete hydrolysis of hemicellulose [29]. Lime has emerged
as a promising chemical for the pre-treatment step due to its low
cost and wide use in agricultural processing [30]. Ammonia
treatment at high temperatures reduces crystallinity of cellulose
microfibrils and depolymerizes lignin. Recent advances in ammo-
nia pretreatment using liquid ammonia result in production of a
cellulose III polymorph, which significantly enhances the effec-
tiveness of enzymatic depolymerisation [31]. Sulfite pretreatment
to overcome recalcitrance of lignocellulose (SPORL) is a recently
developed pretreatment method mainly used for the pretreatment
of woody biomass. In the process, wood chips first react with a
solution of sodium bisulfite (or calcium or magnesium or other
bisulfite) at 160–190 8C and pH 2–5 (for about 10–30 min in batch
operations). The pretreated wood chips are then fiberized through
mechanical milling (using a disk refiner) to generate fibrous
substrate for subsequent saccharification and fermentation. The
removal of the strong recalcitrance of woody biomass by SPORL is
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M. Vohra et al. / Journal of Environmental Chemical Engineering 2 (2014) 573–584
achieved by the combined effects of dissolution of hemicelluloses,
depolymerization of cellulose, partial delignification, partial
sulfonation of lignin, and increasing surface area by fiberization
through disk milling. SPORL pretreatment was shown to increase
sugar yields (from 57% to 88%) and reduce inhibitors by up to 65%
compared to dilute sulfuric acid pretreatment of softwood [32,33].
Many pretreatment processes have been developed in the last
decades and are being continuously improved through research
studies. The pretreatment process has significant effects on all
downstream processes and ultimately influence the overall biofuel
yield and cost [28].
Hydrolysis. Hydrolysis involves breakdown of the polysaccharides
to their simple sugar. Three methods are commonly used for
hydrolyzing the cellulose into glucose (C6 sugar for fermentation):
(1) dilute acid hydrolysis (H2SO4 <1%, 215 8C and 3 min to achieve
satisfactory glucose yields) which is no longer a viable candidate;
(2) concentrated acid (H2SO4 30–70%, 40 8C, a few hours to achieve
>90% glucose yields) which has been used in Japan; and (3)
enzymatic hydrolysis (cellulase mixture, 50 8C several days to
reach 75–95% glucose yields) [34]. The current trend is toward
enzymatic hydrolysis to avoid costly recovery and wastewater
treatment requirements resulting from the use of acid. Enzymatic
hydrolysis is attractive because it produces better yields than acid-
catalyzed hydrolysis, while enzyme manufacturers have recently
reduced costs substantially using biotechnology [25]. Acid
hydrolysis has been considered as a more expensive option [35].
Enzymatic hydrolysis of pretreated lignocellulosic materials
involves enzymatic reactions that convert cellulose into glucose
and hemicellulose into pentoses (xylose and arabinose) and
hexoses (glucose, galactose, and mannose). The conversion of
cellulose and hemicellulose is catalyzed by cellulase and hemi-
cellulase enzymes, respectively. The enzymes are highly specific.
The enzymatic hydrolysis is usually carried out at mild conditions
(pH 4.8 and temperature 45–50 8C) [3]. At least three major groups
of cellulases are involved in the hydrolysis process: (a) endoglu-
canase (EG, endo-1,4-D-glucanohydrolase, or EC 3.2.1.4) which
attacks regions of low crystallinity in the cellulose fiber, creating
free chain-ends; (b) exoglucanase or cellobiohydrolase (CBH, 1,4-
b-D-glucancellobiohydrolase, or EC 3.2.1.91.) which degrades the
molecule further by removing cellobiose units from the free chain-
ends; and (c) b-glucosidase (EC 3.2.1.21) which hydrolyzes
cellobiose to produce glucose [36].
The economic production of cellulolytic enzymes and reducing
the enzyme-to-biomass ratio required for hydrolysis remains a key
determinant for commercialization of the fuels derived from
biomasses. Trichoderma reesei remains the most effective com-
mercial host for cellulase production, although other fungal hosts
may be attractive. Protein concentrations in excess of 100 g/L are
reported with T. reesei [37]. Another problem is that large amounts
of cellulase are required to hydrolyze cellulose [38].
Fermentation. Fermentation of the sugars generated from enzy-
matic hydrolysis of biomass is another important step, where a lot
of technical advances are needed to make lignocellulosic ethanol
technology feasible. What is desired for an ideal organism for
biomass-ethanol technology would be a high yield of ethanol,
broad substrate utilization range, resistance to inhibitory com-
pounds generated during the course of lignocellulose hydrolysis
and ethanol fermentation, ability to withstand high sugar and
alcohol concentrations, higher temperatures, lower pH and
minimal by-product formation [6]. Unfortunately, all these
features seldom exist together in any wild organism and the need
of the industry would be to develop an organism which will at least
partially satisfy these requirements [39]. The ability to use the
hemicellulose component in biomass feedstock is critical for any
bio-ethanol project. Saccharomyces cereviseae and Zymomonas
mobilis, the commonly employed organisms used in alcohol
579
fermentation lacks the ability to ferment hemicellulose derived
pentose (C5) sugars. While there are organisms that can ferment
C5 sugars (e.g. Pichiastipitis, Pachysolentannophilus, Candida she-
hatae) the efficiencies are lower. They also need microaerophilic
conditions and are sensitive to inhibitors, higher concentrations of
ethanol and lower pH [40].
The conversion of glucose into ethanol during fermentation of
the enzymatic hydrolysate is not difficult, since there is an absence
of certain inhibitory substances such as furfural, hydroxyl methyl
furfural, or natural wood- derived inhibitors such as resin acids
containing fatty acids, rosin acids, and sterols. For more than 20
years, research activities have been directed toward the develop-
ment of improved micro-organisms for the fermentation of the
pentose sugars [40]. For cost effective processing, such organisms
must be able to co-ferment the feed streams containing both
glucose and xylose together. Although C5 pentoses are generally
more difficult to ferment, new yeast strains are being developed
that can effectively use these sugars [39]. Currently, there are no
known natural organisms that have the ability to convert both
these C6 and C5 sugars at high yields although there have been
some claims toward this goal using genetically modified (GM)
microorganisms [41]. Significant progress has been made in
engineering microorganisms for the co-fermentation of glucose
and pentose sugars, but their sensitivity to inhibitors and the
production of unwanted by-products remain serious problems, yet
to be overcome to allow a viable commercialization [42].
One promising GM strategy has been to take ethanologen, a
natural hexose, and add the pathways needed to convert other
sugars. This has been achieved by adding pentose conversion to
Saccharomyces cerevisiae, Zymomonas mobilis, Klebsiella and E. coli
strains. The need to understand and manipulate ethanol and sugar
tolerance and resistance to potential inhibitors generated in pre-
saccharification treatments remains a scientific goal [43]. The most
frequently used microorganism for fermenting bio-ethanol in
industrial processes is S. cerevisiae, which has proved to be very
robust and well suited to the fermentation of lignocellulosic
hydrolysates. S. cerevisiae strains have been metabolically engi-
neered by introducing heterologous xylose utilization pathways
for conversion of xylose to ethanol. However, neither metabolic
engineering nor evolutionary engineering has been successful in
generating a single S. cerevisiae capable for fermenting both xylose
and arabinose efficiently. Engineered S. cerevisiae strain capable of
fermenting mixtures of glucose and xylose with high ethanol yields
without the formation of side products such as xylitol or arabinitol
was also developed. Further improvements in the kinetics of
fermentation of the sugar mixture (glucose, xylose, arabinose) was
achieved by evolutionary engineering strategy. This strategy
involves repetitive cultivation of recombinant strain in three
different sugar mixtures which resulted in rapid anaerobic
fermentation of mixtures of glucose, xylose and arabinose to
ethanol [44]. A xylose fermenting Z. mobilis was generated by
introducing a xylose-metabolizing pathway from E. coli having
advantages of requiring a minimum of nutrients, growing at low
pH and high temperatures, and it is considered generally
recognized as safe (GRAS) [45]. PDC (encoding for pyruvate
decarboxylate) and adhB (encoding for alcohol dehydrogenase)
genes from Z. mobilis were over-expressed in E. coli. This was an
outstanding contribution which made E. coli an ethanol producer
yielding 40 g/L of ethanol with 90–100% of the theoretical yields
from hemicellulose hydrolysates of bagasse, corn hulls and corn
stover [44,45]. E. coli, as a biocatalyst for bioethanol production,
has ability to ferment a wide spectrum of sugars, no requirements
for complex growth factors, and prior industrial use (e.g., for
production of recombinant protein). The major disadvantages
associated with using E. coli cultures are a narrow and neutral pH
growth range (6.0–8.0), less hardy cultures compared to yeast, and
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580 M. Vohra et al. / Journal of Environmental Chemical Engineering 2 (2014) 573–584

Lignocellulosicwaste Enzyme fermentation process produces a nutrient-rich microbial cell mass

Enzymatic
Pretreatment Sugars
hydrolysis
Separator lignin
Combustion or
gasification
Process heat
electricity mineral
Fig. 6. Biochemical process for conversion of lignocellulosic waste to ethanol.
public perceptions regarding the danger of E. coli strains [23].
Solutions to these issues will also need to accommodate the
variability in biomass resources. While pentose fermentation has
been achieved on ideal substrates, (i.e. laboratory preparations of
sugars designed to imitate a perfectly-pretreated feedstock),
significant work remains to apply this to actual lignocellulosic
feedstocks [35].
Overall fuel ethanol production from lignocellulosic biomass is
shown in Fig. 6. There are no large-scale cellulosic ethanol facilities
in commercial production as yet, although several companies have
announced plans to build facilities with capacities of more than 30
million gallons per year. A typical pilot-scale process for making
cellulosic ethanol involves treatment of pulverized biomass with
hot acid, which partially hydrolyzes hemicellulose and other
polysaccharides and disrupts the association of lignin with the
polysaccharides [46]. The hydrolysate is neutralized, separated
from the insolubles, and fermented to produce ethanol. The
insoluble fraction is then treated with cellulase and glycosidases
to release glucose which is also fermented to produce ethanol.
The residual insoluble material, mostly lignin, is burned to
generate energy for the overall process [47]. The future
development of plants with modified lignin that can be readily
hydrolyzed, or improved enzymes or chemical catalysts for lignin
hydrolysis may allow the use of lignin as a component of plastics or
[(Fig._7)TD$IG] a fermentation feedstock for liquid fuel production. The
as
which could be inactivated and used as fertilizer so as to recycle the
mineral nutrients to the land [38]. This process is called separate
Fermentation
hydrolysis and fermentation (SHF).
SHF is one of the configurations that have been tested more
Distillation
dehydration
extensively. Pentose fermentation, when it is carried out, is
accomplished in an independent unit. In the SHF configuration the
joint liquid flow from both hydrolysis reactors first enters the
Ethanol glucose fermentation reactor. The mixture is then distilled to
remove the bioethanol leaving the unconverted xylose behind. In a
second reactor, xylose is fermented to bioethanol, and the
Waste water
bioethanol is again distilled. The advantage of SHF is the ability
treatment to carry out each step under optimal conditions, i.e. enzymatic
hydrolysis at 45–50 8C and fermentation at about 308K [15,23]. But
this approach proves to be very costly. So based on the different
combinations of technologies adopted at the pretreatment,
hydrolysis, and fermentation stages of ethanol synthesis, several
integrated technologies have been developed. In the section below,
existing integrated conversion technologies are discussed [48].
Integrated technologies based on hydrolysis
Simultaneous saccharification and fermentation, SSF
Saccharification and fermentation are carried out simulta-
neously in a single reactor, thus allowing for cost saving and
reduction of inhibitors, increasing hydrolysis rate (Fig. 7) [49].
Obviously, the optimization of process conditions concerning both
enzymes and microorganisms at the same time is the critical issue
of this solution [11]. The key of the SSF process from biomass is its
ability for rapidly converting the sugars into ethanol as soon as
they are formed diminishing their accumulation in the medium.
Bearing in mind that the sugars are much more inhibitory for
conversion process than ethanol is, SSF can reach higher rates,
yields and ethanol concentrations compared to the SHF process
[50]. SSF offers an easier operation and a lower equipment
requirement than the sequential process since no hydrolysis
reactors are needed; moreover, the presence of ethanol in the broth
makes the reaction mixture less vulnerable to the action of
undesired microorganisms. Nevertheless, SSF has the inconvenient
that the optimal conditions for hydrolysis and fermentation are
different, which implies a difficult control and optimization of

(C+H+L)
Pretreatment
Biomass

Solid fraction Liquid fraction Liquid fraction

(C+L) (P+I)
Detoxification

(Cel) SSF SSFC

Production Cellulose
of cellulose hydrolysis
(G) (P+I)

Hexose Pentose
fermentation fermentation
CF

CBP (EtOH+L) (EtOH)

Conventional Ethanol
distillation dehydration
Anhydrous
Waste Effluent ethanol
streams treatment

Fig. 7. Generic block diagram of fuel ethanol production from lignocellulosic biomass. Possibilities for reaction–reaction integration are shown inside the shaded boxes: CF,
co-fermentation; SSF, simultaneous saccharification and fermentation; SSCF, simultaneous saccharification and co-fermentation; CBP, consolidated bioprocessing. Main
stream components: C, cellulose; H, hemicellulose; L, lignin; Cel, cellulases;G, glucose; P, pentoses; I, inhibitors; EtOH, ethanol [15].
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M. Vohra et al. / Journal of Environmental Chemical Engineering 2 (2014) 573–584 581

process parameters; in addition, larger amounts of exogenous marxianus strain codisplaying endoglucanase and b-glucosidase
enzymes are required [51]. Saccharification with cellulolytic on the cell surface grew well at temperature as high as 48 8C, at
enzymes is best done at around 50 8C, while most fermenting which ethanol was produced from the cellulosic material b-glucan
microbes have an optimum temperature for ethanol fermentation with a yield of 0.47 g ethanol per gram of consumed carbohydrate.
between 28 8C and 37 8C. In practice, it would be difficult to lower This study gives supports the development of CBP yeast for
the optimum temperature of cellulases through protein engineer- effective bioethanol production [54]. Other proposed approach is
ing. Accordingly, high-temperature fermentation is in high the utilization of mixed cultures in such a way that the hydrolysis
demand for simultaneous saccharification and fermentation, and and fermentation of lignocellulosic biomass be carried out
thermotolerant yeast strains have been screened for the ability to simultaneously [15].
ferment ethanol [52]. Kluyveromyces marxianus appears to be
particularly promising. Many strains of K. marxianus grow well at
Syngas platform: thermochemical conversion processes
temperatures as high as 45–52 8C and can efficiently produce
ethanol at temperatures between 38 8C and 45 8C. Moreover, K.
The production of bioethanol from syngas is an emerging
marxianus offers additional benefits including a high growth rate
technology that can utilize a wide variety of biomass. This route of
and the ability to utilize a wide variety of sugar substrates (e.g.,
ethanol production has the advantage of utilizing the entire
arabinose, galactose, mannose, xylose) at elevated temperatures
biomass including the lignin content, which is usually difficult to
[15,52].
break down. Biomass is converted to syngas via a process called
gasification, a process in which solid or liquid carbonaceous
Simultaneous saccharification and co-fermentation, SSCF
material, such as biomass, coal, or oil, that react with air, oxygen,
and/or steam to produce a gas product called syngas or producer
This kind of integration is oriented to the complete assimilation
gas that contain CO, H2, CO2, CH4, and N2 in various proportions
by the microorganisms of all the sugars previously released during
[47]. Biomass-syngas can then be converted into biofuels such as
the pretreatment and hydrolysis of lignocellulosic biomass (Fig. 7).
methanol, ethanol and hydrogen via the metal-catalytic or
The use of mixed cultures of yeasts that assimilate both hexoses
biocatalytic methods [15].
and pentoses has been proposed, but problems related to the fact
that hexose-utilizing microorganisms grow faster than pentose-
Gasification: syngas catalytic conversion
utilizing microorganisms and that the conversion of hexoses to
ethanol is consequently more elevated, are arisen [39]. Other
Gasification of lignocellulosic biomass at a high temperature
variant of co-fermentation consists in the utilization of a single
(750–800 8C) produces a gas mixture containing carbon monoxide
microorganism capable of assimilating both hexoses and pentoses
(CO), hydrogen (H2), methane (CH4), nitrogen (N2), carbon dioxide
in an optimal way allowing high conversion and ethanol yield [6].
(CO2) and some higher hydrocarbons commonly known as
Although in the nature these microorganisms exist, a high
producer gas [15]. The overall gasification process is endothermic,
efficiency in the conversion to ethanol can be reached through
that is, it requires heat-energy input to drive the process. The
the genetic modification of yeasts or bacteria already adapted to
composition of producer gas depends on the types of gasifier and
the ethanolic fermentation [15].
biomass, and the gasification conditions among others. The
synthesis gas predominantly contains H2 and CO, and is commonly
Consolidated bioprocessing, CBP
known as syngas in for short. After gasification, the syngas mixture
passes through a series of filters to remove undesirable pollutants
All enzymes and bioethanol are produced in a single reactor by a
such as tar and solid particles [55]. The syngas is than passed
single microorganisms community [53]. The logic culmination of
through the Fischer–Tropsch (FT) process to create a range of liquid
reaction–reaction integration for the transformation of biomass
fuels suitable for aviation and marine applications as well as
into ethanol is the consolidated bioprocessing (CBP), known also as
chemicals including ethanol (Fig. 8).
direct microbial conversion (DMC) (Fig. 7). The key difference
Before entering the chamber, gas is heated to 300 8C at a
between CBP and the other strategies of biomass processing is that
pressure of 69 bar. Gas is also mixed with water and methanol to
only one microbial community carries out both the production of
improve yield of higher alcohols. The mixture is passed through the
cellulases and fermentation, i.e., cellulase production, cellulose
synthetic catalyst to obtain methanol, ethanol, and higher linear
hydrolysis, and fermentation are carried out in a single step. This
alcohols such as pentanol, water, methane, and minor amounts of
difference has an important advantage as no capital or operation
other hydrocarbon byproducts [48]. The reaction rate is high, and it
expenditures are required for enzyme production within the
is over within periods ranging from seconds to minutes, with up to
process [43]. Similarly, part of the substrate is not deviated for the
60% CO (carbon monoxide) conversion into ethanol. Next, the gas is
production of cellulases. Moreover, the enzymatic and fermenta-
cooled, this allows the alcohols to condense and separate from the
tion systems are entirely compatible [15]. Thermophilic cellulo-
unconverted syngas. The liquid alcohols are then further refined by
lytic anaerobic bacteria have also been extensively examined for
alcohol separation and purification steps [56]. Some of the
their potential as bioethanol producers. These bacteria include
catalysts that have been historically used contain rhodium (Rh),
Thermoanaerobacter ethanolicus, Clostridium thermohydrosulfuri-
cobalt, molybdenum and others including multi-component
cum, Thermoanaerobacter mathranii, Thermoanaerobium brockii,
catalysts. Rh seems to be the best catalytic metal for a natural
Clostridium thermosaccharolyticum strain, etc. including others.
gas conversion to ethanol due to its ability to perform all of the four
Thermophilic cellulolytic anaerobic bacteria have a distinct
specific functions that a catalyst should perform. This include: the
advantage over conventional yeasts for bioethanol production in
adsorption and dissociation properties of carbon monoxide (CO)
their ability to directly use variety of inexpensive biomass
molecule and oxygen on the catalyst, hydrogenation of the
feedstocks and their ability to withstand temperature extremes.
adsorbed carbon to methyl species, insertion of non-dissociated
The low bioethanol tolerance of thermophilic anaerobic bacteria
CO into the methyl species to form an adsorbed acyl species, and
(<2%, v/v) is a major obstacle for their industrial exploitation for
hydrogenation of the acyl species to form the ethanol product [57].
bioethanol production [23,53]. Cell surface engineering has been
applied to a thermotolerant strain of the yeast K. marxianus for the catalyst
display of cellulolytic enzymes on the cell surface. Recombinant K. 2CO þ 4H2 ! CH3 CH2 OH þ H2 O
 Author's personal copy
[(Fig._8)TD$IG]
582 M. Vohra et al. / Journal of Environmental Chemical Engineering 2 (2014) 573–584
Fig. 8. Production of ethanol by catalytic conversion of biomass-derived syngas.
Biological path is gasification: syngas fermentation route
Another possible option for bioethanol production via mixed
thermo-chemical-biological path is represented by syngas fer-
mentation. Syngas conversion using microbial catalysts offer
several advantages, since they require significantly lower temper-
ature and pressure conditions (usually atmospheric conditions)
and are less susceptible to varying feed gas compositions. Chemical
catalysts are more susceptible to poisoning, and their specificity is
lower compared to microbial processes, although faster conversion
times are often possible [58].
Similarly to the above reported syngas catalyst route, the initial
stage is again biomass gasification. Cleaned, gas is cooled to the
normal ambient temperature and stored at a high pressure. Cooled
and cleaned gas is fed into an ethanol conversion chamber where
microbes ferment the gas into ethanol and acetic acid. After
fermentation is complete, the liquid is distilled to separate ethanol
from other products. The ethanol produced is then dehydrated to
produce fuel quality ethanol [48] (Fig. 9). The cell mass can be
recycled to the gasifier, while it is not approved as animal feed [15].
A large number of bacterial strains have been isolated that have
the ability to ferment producer gas (the CO, CO2, and H2
components) to ethanol, acetic acid, and other useful liquid
[(Fig._9)TD$IG]
Syngas/waste gas
fermentation
CO2 rich Gas cleaning
waste gas & conditioning
Gasification
Pretreatment
• Sizing
• Drying
Biomass • Carbonization
feedback • Leaching
Fig. 9. Production of ethanol by microbial fermentation of biomass-derived syngas [60].
products. Clostridium ljungdahlii was the first organism recognized
as able to form ethanol from components of producer gas [59]. The
organism favors the production of acetate at a higher pH (5–7), but
ethanol is the dominant product at pH between 4 and 4.5. Recently,
an additional clostridial acetogen was isolated and was shown to
produce ethanol from producer gas generated from biomass. Other
organisms that can produce ethanol from producer gas, although
not as the major product, include Butyribacterium methylotrophi-
cum and Clostridium autoethanogenum [60].
Commercialization of producer gas fermentations is currently
hindered by low productivity in the bioreactor. Several factors,
such as low cell density, lack of regulation of metabolic pathways
to yield only the desired product, inhibition of the biological
catalysts by products and substrates, and low gas–liquid mass
transfer, need to be addressed to establish the economic feasibility
of producer gas fermentations [61]. The transport of gases to the
bulk liquid through the liquid film around gas bubbles is the rate-
limiting step in most fermentation processes that involve sparingly
soluble gaseous substrates. Producer gas fermentations are
primary examples of such mass transfer limited fermentations.
At mild temperatures, CO and H2 have aqueous solubilities of 60%
and 4%, respectively, compared to oxygen on a mass basis. These
low solubilities result in low concentration gradients and, hence,
Ethanol recovery
• Distillation
• Molecular sieve
• Pervaporation
• Dephlegmation
• Cyclone
• Scrubbing
Fuel graded
• Adsorption
• Filtration ethanol
• Cooling
Heat recovery
to generate
power for the
process
 Author's personal copy
M. Vohra et al. / Journal of Environmental Chemical Engineering 2 (2014) 573–584
low mass transfer rates. Higher mass transfer rates can be achieved
by the use of an agitator system. Increasing the operating pressure
can also increase the gas mass transfer rate in producer gas
fermentations. Microbubble dispersions, bubbles with diameters
of about 50–100 mm, have been used to provide a large gas
transport area at low power consumption [62].
Preferred technology route
There is currently no clear commercial or technical advantage
between the biochemical and thermochemical pathways, even
after many years of R&D and the development of near-commercial
demonstrations. Both sets of technologies remain unproven at the
fully commercial scale, are under continual development and
evaluation, and have significant technical and environmental
barriers yet to be overcome.
For the biochemical route, much remains to be done in terms of
improving feedstock characteristics; reducing the costs by
perfecting pretreatment; improving the efficacy of enzymes and
lowering their production costs; and improving overall process
integration [63]. The potential advantage of the biochemical route
is that cost reductions have proved reasonably successful to date,
so it could possibly provide cheaper biofuels than via the
thermochemical route. Conversely, as a broad generalization,
there are less technical hurdles to the thermochemical route since
much of the technology is already proven [49].
One key difference between the biochemical and thermochem-
ical routes is that the lignin component is a residue of the
enzymatic hydrolysis process and hence can be used for heat and
power generation. In the thermochemical process it is converted
into synthesis gas along with the cellulose and hemicellulose
biomass components [64,65]. A second major difference is that
biochemical routes produce ethanol whereas the thermo-chemical
routes can also be used to produce a range of longer-chain
hydrocarbons from the synthesis gas. These include biofuels better
suited for aviation and marine purposes.
Among all the issues that need to be considered when selecting
lignocellulosic ethanol production technologies, net environmen-
tal impact is one of the most important decision factors. One of the
most commonly used methods for evaluating the environmental
profile of engineered products or processes is life cycle assessment
(LCA), which takes a holistic approach and provides a comprehen-
sive view of the environmental impacts over entire life cycle of a
process or product, thus presenting a more accurate picture of the
true environmental trade-offs [66]. According to LCA study done by
Mu et al., environmental impacts associated with process inputs
for thermochemical conversion though can be neglected, this is not
the case for biochemical conversion where sulfuric acid, lime, and
nutrients consumed contribute significantly to life cycle fossil fuel
consumption, GHG emission, and water consumption. In the near
short term, biochemical conversion has better performance on
GHG emissions and fossil fuel consumption, mainly due to credits
from electricity exported. In all the scenarios analyzed thermo-
chemical conversion has significantly less fresh water consump-
tion both direct and indirect. For thermochemical plant, if the
higher molecular mixed alcohols are separated as co-products, the
environmental performance can be significantly improved. This
further supports the concept of a thermochemical biorefinery [66].
As described in literature [49], a comparison between biochemical
and thermochemical ethanol conversion approaches concerning
operational and capital cost was performed. This study addition-
ally investigated yields, thermal efficiencies, and important
sustainability metrics, such as water usage for both the processes.
It was shown that the overall economics of the two processes are
very similar. On an energy efficiency basis, these processes are
essentially the same when the energy credits for the co-products
583
are taken in to account. This is also true from an environmental
perspective with very similar emission profiles for both conversion
processes [49].
Only time will tell which conversion route will be preferred, but
whereas there may be alternative drives becoming available for
light vehicles in future (including hybrids, electric plug-ins and
fuel cells), such alternatives for aeroplanes, boats and heavy trucks
are less likely and liquid fuels will continue to dominate [35].
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