FEMS Microbiology Letters, 363, 2016, fnw033

doi: 10.1093/femsle/fnw033
Advance Access Publication Date: 11 February 2016
Minireview

M I N I R E V I E W Biotechnology & Synthetic Biology

Acetone-butanol-ethanol fermentation of corn stover:

current production methods, economic viability and
commercial use
Nawa R. Baral1,2 , Lauren Slutzky1 , Ajay Shah1, , Thaddeus C. Ezeji3 ,
Katrina Cornish1,4 and Ann Christy1
1
Department of Food, Agricultural and Biological Engineering, The Ohio State University, Room 110 FABE
Building, 1680 Madison Avenue, Wooster, OH 44691, USA, 2 Department of Mechanical Engineering, Institute
of Engineering, Tribhuvan University, Kathmandu, 44600, Nepal, 3 Department of Animal Sciences, The Ohio
State University, Room 305 Gerlaugh Hall, 1680 Madison Avenue, Wooster, OH 44691, USA and 4 Department
of Horticulture and Crop Science, The Ohio State University, OH 44691, USA

Corresponding author: Department of Food, Agricultural and Biological Engineering, The Ohio State University, Room 110 FABE Building, 1680 Madison
Avenue, Wooster, OH 44691, USA. Tel: 330-263-3858; Email: shah.971@osu.edu
One sentence summary: Corn stover is a viable substrate for microbial butanol production.
Editor: Michael Sauer

ABSTRACT
Biobutanol is a next-generation liquid biofuel with properties akin to those of gasoline. There is a widespread effort to
commercialize biobutanol production from agricultural residues, such as corn stover, which do not compete with human
and animal foods. This pursuit is backed by extensive government mandates to expand alternative energy sources. This
review provides an overview of research on biobutanol production using corn stover feedstock. Structural composition,
pretreatment, sugar yield (following pretreatment and hydrolysis) and generation of lignocellulose-derived microbial
inhibitory compounds (LDMICs) from corn stover are discussed. The review also discusses different Clostridium species and
strains employed for biobutanol production from corn stover-derived sugars with respect to solvent yields, tolerance to
LDMICs and in situ solvent recovery (integrated fermentation). Further, the economics of cellulosic biobutanol production
are highlighted and compared to corn starch-derived ethanol and gasoline. As discussed herein, the economic
competitiveness of biobutanol production from corn stover largely depends on feedstock processing and fermentation
process design.

Keywords: ABE recovery; butanol production cost; corn stover; fermentation techniques; LDMIC; solventogenic Clostridium
species

INTRODUCTION folio. In 2012, 13 billion gallons of ethanol were produced in

the US compared to the 134 billion gallons of gasoline used an-
Worldwide research in alternatives to gasoline fuels, such as
nually (Tao and Aden 2009; Kumar and Murthy 2011; Qureshi
ethanol and butanol, is rapidly increasing due to volatility in
et al. 2014). Commercial bioethanol is currently being produced
crude oil prices and climatic concerns. However, as of now, al-
from the fermentation of corn grain starch and cane sugar by
ternative fuels contribute only about 10% of the US energy port-

Received: 14 December 2015; Accepted: 8 February 2016


C FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

1
 2 FEMS Microbiology Letters, 2016, Vol. 363, No. 6
Saccharomyces cerevisiae (Kumar et al. 2012). However, ethanol has
a range of problems impeding its larger scale implementation
including its miscibility with water, difficulty in transportation
and inability of industrial S. cerevisiae strains to ferment xylose
(Baeyens et al. 2015). Compared to ethanol, butanol has superior
fuel properties, such as high calorific value, high hydrophobic-
ity, low flammability and corrosiveness, ability to be mixed with
gasoline and diesel in high proportions, ability to be transported
by pipes, and can be produced from both glucose and xylose sug-
ars (Pfromm et al. 2010; Green 2011; Mariano et al. 2011; Kumar
et al. 2012; Baral, Li and Jha 2014). While biobutanol production
from lignocelluloses on a commercial scale is currently not oc-
curring, the US, Brazil and China all plan to develop large-scale
butanol production plants (Green 2011; Kumar et al. 2012; Tao
et al. 2014a).
Industrial biobutanol production also has support from gov-
ernment mandates, such as the Energy Independence and Se-
curity Act of 2007, which require 36 billion gallons of renewable
fuels be produced by 2022, including 16 billion gallons derived
from cellulosic biomass (DOE 2011; Tao et al. 2014a). Converting
cellulosic biomass to butanol will significantly help fulfill fed-
eral biofuels production mandates; however, improvements to
the cellulosic fermentation process are needed to improve the
production efficiency before commercialization is possible.
Pretreatment of cellulosic feedstocks for optimal extraction
of hexoses (C6 ) and pentoses (C5 ) provides a sugar-rich feed-
stock, but the yeast, S. cerevisiae, cannot use C5 sugars in conven-
tional ethanol fermentation systems. However, solventogenic
Clostridium species metabolize both C6 and C5 sugars during ABE
fermentation (Saha et al. 2005; Ezeji and Blaschek 2008; Green
2011). Some major problems of butanol production through cur-
rent ABE fermentation include low butanol yield, product inhibi-
tion, multiple end products, low product concentration and high
downstream processing cost (Pfromm et al. 2010; Green 2011; Ju-
rgens et al. 2012; Kumar et al. 2012; Kurkijarvi, Lehtonen and Lin-
nekoski 2014). Currently, researchers are trying to improve the
efficiency of pretreating lignocellulosic feedstock, as well as sub-
sequent fermentation and recovery processes.
The viability of biobutanol commercialization depends on
feedstock prices as well as design and process parameters for
feedstock processing. Corn stover, despite being an agricultural
waste, is susceptible to price inflation that would increase the
price of biobutanol. For example, the cost of biobutanol produc-
tion from corn grain is estimated as $1.56 per gal at $1.80 per
bushel of corn, but this increases to $2.10 per gal if the corn
price is $3.35 per bushel (Tao and Aden 2009). Additionally, vari-
ations in design and process parameters, including fermenter
size, plant capacity, microbial strain(s), production yield, recov-
ery methods and waste disposal and utilization, significantly in-
fluence butanol yield and production cost (Tao and Aden 2009;
Pfromm et al. 2010; Kumar et al. 2012). The scope of this review
is to discuss the appropriateness of corn stover as a substrate
for butanol production, the current state of ABE fermentation
methods, challenges and possible mitigation strategies. This re-
view is divided into four major aspects of ABE fermentation: (i)
feedstock for ABE fermentation, (ii) pretreatment and enzymatic
hydrolysis of lignocellulose, (iii) ABE fermentation and recovery
and (iv) economic viability and potential commercialization.
FEEDSTOCK FOR ABE FERMENTATION
Butanol can be produced from different types of biomass, such
as forest residues, agricultural residues, energy crops and mu-
Figure 1. Available agricultural residues, price and yield in the US. The US De-
partment of Energy projected average values, 201230, for available agricultural
residues and corresponding price, and variations are represented by error lines
(DOE 2011).
nicipal solid wastes. Agricultural residues have great potential
as reliable feedstocks due to widespread availability. Corn stover
dominates the other agricultural residues (Fig. 1, DOE 2011) in
terms of yield per hectare, availability and price, but also has
by far the greatest price variability. Cellulose is the largest con-
stituent of agricultural residues, followed by hemicelluloses and
lignin. Whereas cellulose and hemicellulose are desirable con-
stituents of lignocellulosic biomass for butanol production, the
lignin component cannot be metabolized by most fermenting
microorganisms. The specific amount of constituents of agricul-
tural residues depends on crop origin, climate, maturity and har-
vesting season (Pordesimo et al. 2005).
Corn stover is an abundant feedstock because of its ready
availability, high residue yield rate and ease of cultivation (DOE
2011). The US Department of Energy projects that annual corn
stover supply will increase from 19 to 65 million dry tons be-
tween 2012 and 2030 at a farm-gate price of $40 per dry ton (DOE
2011). If the farm-gate price increased to $60 per dry ton, the es-
timated annual corn stover supply increases from 85 to 140 mil-
lion dry tons between 2012 and 2030 (DOE 2011) as higher prices
motivate more producers to engage in stover collection for biore-
fineries. The DOE (2011) also estimated the corn stover supply
based on yield rate. With 1% increase in current yield, the avail-
able corn stover increases 2-fold. Thus, maximum annual corn
stover of 271 million dry tons can be supplied by 2030, which is
about 85% of total primary agricultural residues in the US (DOE
2011). The major agricultural residues in the US are corn stover,
wheat and barley straw. Corn stover also has similar structural
composition and ABE yield to other lignocellulosic feedstocks
such as wheat straw, switchgrass, miscanthus and poplar (DOE
2011; Jurgens et al. 2012; Qureshi et al. 2013, 2014).
Corn stover is a promising alternative feedstock for butanol
production, as discussed in a recent review (Baral and Shah
2014). A single 25 million gallon per year cellulosic ethanol
biorefinery requires delivery of around 18 500 semi-trailer truck
loads of 36 rectangular bales (1.2 1.2 2.4 m) per truck (Darr
and Shah 2012). Currently, the semi-trailer truck is the most
common means of feedstock transportation. Supplying enough
biomass to meet direct demand of large scale biorefineries is
limited by the low biomass bulk density of corn stover bales of
130200 kg m3 (Darr and Shah 2012). The transportation cost for
lignocellulosic biomass feedstock will be greater than corn grain
feedstock because the grain bulk density is 720 kg m3 (CAES
2015) and the concentration of convertible biomass (starch ver-
sus biomass) is also greater. This results in an increase in the

number of trucks required to supply dry tonnage of lignocellu-
losic biomass than corn to the biorefinery with the same biofuel
production capacity. Thus, feedstock transportation has been
identified as a major cost contributor to the cellulosic feedstock
logistics (Shah 2013).
PRETREATMENT AND ENZYMATIC
HYDROLYSIS
Figure 2 illustrates structural compositions of corn stover and
various products and byproducts from pretreatment and hydrol-
ysis (Pordesimo et al. 2005; Wyman et al. 2005; Yu et al. 2007; Ku-
mar et al. 2009; Ezeji et al. 2010; Bondesson, Galbe and Zacchi
2013; Liu et al. 2013; Saha et al. 2013). Cellulose (glucan), hemi-
cellulose (xylan) and lignin are the major constituents of corn
stover, whose morphology is characterized by cellulose fibers
surrounded in a hemicellulose and lignin matrix (Jurgens et al.
2012; Baral, Li and Jha 2014). The lignin component of corn stover
(21.4% by weight) cannot be fermented by Clostridia (Fig. 2). How-
ever, sugars from cellulose (37.45% of corn stover by weight) and
hemicellulose (24.8% of corn stover by weight) can be utilized
to produce biobutanol (Li, Baral and Jha 2014). Pretreatment and
enzymatic hydrolysis are essential steps before fermentation to
break lignin constituents and liberate fermentable sugars, such
as glucose, xylose and galactose (Fig. 2; Jurgens et al. 2012; Zhang
et al. 2013; Baral, Li and Jha 2014).
Various pretreatment techniques can be used to disrupt the
lignin matrix of corn stover, including hydrothermal, dilute acid
hydrolysis, steam explosion, acid-catalyzed steam explosion,
wet oxidation, wet explosion, alkaline hydrolysis, ammonia
fiber explosion, ionic and biological pretreatment with actino-
mycetes, bacteria, fungi or ligninolytic enzymes such as lignin-
degrading peroxidase, manganese peroxidase and laccase
(Table 1) (Jurgens et al. 2012; Kumar et al. 2012; Baral, Li and
Jha 2014; Yao and Nokes 2014). Different pretreatment methods
have different processing conditions, such as temperature, pH,
pressure, water activity and nutrients. For instance, biological
pretreatments yield fermentable sugars that range from 42 to 66
g per 100 g dry corn stover (Kim et al. 2003; Varga, Reczey and
Zacchi 2004; Kim and Lee 2005; Lloyd and Wyman 2005; Mosier
et al. 2005; Teymouri et al. 2005; Li et al. 2011; Shen and Wyman
2011; Qin et al. 2012; Avci et al. 2013; Bondesson, Galbe and Zac-
chi 2013; Liu et al. 2013; Feng et al. 2014; Uppugundla et al. 2014).
These operating parameters and corresponding sugar yield are
summarized in a recent review (Baral and Shah 2014).
The sugar components of corn stover can be fermented us-
ing solventogenic Clostridia into butanol via separate enzymatic
hydrolysis and fermentation and recovery or through simul-
taneous saccharification, fermentation and recovery (Qureshi
et al. 2014). However, a major problem for ABE fermentation us-
ing lignocellulosic biomass hydrolysate (LBH) is lignocellulose-
derived microbial inhibitory compounds (LDMICs) (Fig. 2), which
are formed during pretreatment processes (Ezeji, Qureshi and
Blaschek 2007b; Jurgens et al. 2012; Kumar et al. 2012; Qureshi
et al. 2013; Baral and Shah 2014; Rasmussen, Srensen and Meyer
2014). The LDMICs, such as 5-hydroxymethylfurfural (HMF), fur-
fural, acetic acid, ferulic acid, glucuronic acid and phenolic com-
pounds at a concentration of 13 g L1 (even less than 1 g L1 for
phenolic compounds) can inhibit the fermentation (Baral and
Shah 2014; Zhang and Ezeji 2014; Ujor et al. 2015). Generally,
alkali salts, such as Ca(OH)2 , are used to remove such LDMICs
(Qureshi et al. 2010b; Wang and Chen 2011). Detailed discussions
Baral et al. 3
on different LDMICs and possible ways of their reduction are in-
cluded in a recent review (Baral and Shah 2014).
ACETONE-BUTANOL-ETHANOL
FERMENTATION AND RECOVERY
BY DISTILLATION
The main products of pretreatment and hydrolysis are glucose
and xylose, which can be metabolized by Clostridium species
during growth and acetone-butanol-ethanol (ABE) fermentation
(Green 2011; Kumar et al. 2012; Mariano et al. 2013). Clostridium
acetobutylicum ATCC 824 and C. beijerinckii NCIMB 805212 are two
major microbes frequently used in ABE fermentation (Pfromm
et al. 2010; Kumar et al. 2012; Li, Baral and Jha 2014). In recent
years, researchers have screened butanol tolerant and high bu-
tanol yield strain(s) using genetic engineering, metabolic engi-
neering techniques and chemical mutation methods such as
N-methyl-N-nitro-N-nitrosoguandine (Table 2) (Li, Baral and Jha
2014). Various strain(s), their parent microbes, cultivation condi-
tions and growth media specifically designed for corn stover as
a substrate, have been used in batch, fed batch and continuous
fermenters to produce ABE (Table 2).
Clostridium species can metabolize a wide range of sugars
such as hexoses, several pentoses and polysaccharides (Qureshi
et al. 2006; Green 2011). While glucose is a common hexose car-
bon source for butanol production using solventogenic Clostrid-
ium species, other substrates such as lactose, xylose, arabinose,
saccharose and starch have been used as carbon sources, al-
though ABE yield (g/g) from C. beijerinckii P260 fermentation var-
ied with substrate (Qureshi et al. 2006). Indeed, ABE production
by C. beijerinckii P260 with glucose, arabinose, xylose and galac-
tose as substrates were 21.37, 15.18, 11.12 and 10.09 g L1 , respec-
tively, which suggests glucose is superior to other sugar types.
While the butanol-producing solventogenic Clostridium
species, C. beijerinckii BA101, converts xylose and glucose to
ABE, the microorganism performed poorly in the presence of
LDMICs (Ezeji, Qureshi and Blaschek 2007b). Additionally, C.
acetobutylicum ATCC 824 can utilize both xylose and glucose
during ABE fermentation, although xylose was fermented more
slowly than glucose. While C. acetobutylicum ATCC 824 and C.
beijerinckii NCIMB 8052 are known to produce acetone butanol
and ethanol, other strains such as C. beijerinckii DSM 6423, C.
isopropylicum IAM 19239, C. tetanomorphum and C. thermosaccha-
rolyticum can ferment substrates into butanol and ethanol with
little to no acetone production (Patakova et al. 2013). Of the best
available bacterial strains for ABE fermentation with corn stover
hydrolysate, the maximum ABE of 26.27 g L1 ,with yield of
0.44 g g1 and productivity 0.31 g L1 h1 , were achieved in
batch fermentation of dilute sulfuric acid pretreated corn stover
using C. beijerinckii P260 (Qureshi et al. 2010b). In addition to
C. beijerinckii P260, other solventogenic Clostridium species and
strains, including C. acetobutylicum P262, C. beijerinckii BA101,
C. acetobutylicum 206 and C. beijerinckii NCIMB 8052, have been
reported to produce maximum ABE concentrations of 25.8,
24.8, 24.7, 23.5 and 19.2 g L1 , respectively (Parekh, Parekh
and Wayman 1988; Hespell 1998; Parekh and Blaschek 1999;
Campos, Qureshi and Blaschek 2002; Gao et al. 2012). These data
suggest there is room for improvement and that using a single
strain may not be the most feasible approach for commercial
scale butanol production from corn stover. Consequently,
utilization of mixed culture strain(s) of efficient strain(s) such as
C. beijerinckii P260, C. acetobutylicum P262, C. beijerinckii BA101, C.
acetobutylicum 206 and C. beijerinckii NCIMB 8052 may enhance
 4 FEMS Microbiology Letters, 2016, Vol. 363, No. 6

Figure 2. Breakdown of corn stover into sugars and LDMICs. The average sugar yield from 100 g corn stover was calculated based on best available results of different
pretreatment methods and range, and is given in a bar diagram. More variation in LDMICs than sugar concentrations was observed in pretreatment technologies
evaluated. Thus, concentration range is presented in the case of inhibitors. (Modified from Ibraheem and Ndimba 2013, Baral and Shah 2014).
Table 1. Comparison of different types of pretreatment processes (Mosier et al. 2005; Jurgens et al. 2012; Mood et al. 2013; Baral, Li and Jha 2014).

Pretreatment Particle Energy Accessible surface Decrease in Hemicellulose Lignin Change lignin LDMICs Appropr- Reagent
method size needed area enhancement cellulose crystallization removal removal structure formation iateness recovery Cost

Mechanical comminution # #

Hydrothermal

Steam explosion

Acidic thermal

Alkali thermal

Ammonia fiber explosion

CO2 explosion #

Irradiation #

Oxidative

Ionic liquids

Biphasic

Biological

Note: not known; # not applicable/none; low/small; medium; high/large.

Table 2. Cultivation conditions and growth media of common strain(s) for ABE fermentation.

Strain(s) Parent species/strain Cultivated conditions Germination medium References

C. beijerinckii IB4, C. beijerinckii RT66 C. beijerinckii NCIMB 8052 37 C for 14 h Yeast extract/ peptone / starch (YPS) Guo et al. (2012); Du et al. (2013)
medium
C. beijerinckii NCIMB 8052 C. beijerinckii 37 C for 12 h. TGY (tryptone/glucose/yeast extract) Parekh and Blaschek (1999)
medium
C. beijerinckii BA101 C. beijerinckii NCIMB 8052 37 C for 12 h. TGY Parekh and Blaschek (1999)
C. beijerinckii BA101 C. beijerinckii NCIMB 8052 35 C for 1214 h and 35 C for Cooked meat medium and Ezeji, Qureshi and Blaschek (2007)
34 h tryptoneglucosemannose
arabinosexyloseyeast extract
(TGMAXY) medium
C. acetobutylicum 206 C. acetobutylicum ATCC 824 37 C for 18 h Seed medium Gao et al. (2012)
C. acetobutylicum ATCC 824 C. acetobutylicum 37 C for 2036 h Corn mash Wang and Chen (2011)
C. acetobutylicum CICC 8008 C. acetobutylicum 37 C for 1416 h Basal medium Lin et al. (2011)
C. beijerinckii ATCC 55025 C. beijerinckii 37 C for 12 h Reinforced clostridial media (RCM, Zhang et al. (2009); Mu et al. (2011)
Oxoid CM 149)
C. acetobutylicum P262 C. acetobutylicum 30 C for 48 h Cooked meat medium (CMM) Ennis and Maddox (1985)
C. beijerinckii P260 C. beijerinckii 35 C for 1618 h CMM Qureshi et al. (2007, 2014)
Baral et al.

C. beijerinckii P260 C. beijerinckii 35 C for 18 h P2 medium Qureshi et al. (2010b)

5
 6 FEMS Microbiology Letters, 2016, Vol. 363, No. 6
butanol yield and productivity with suitable fermentation
parameters (Li, Baral and Jha 2014).
In addition to obtaining/developing high butanol yielding
strains, contamination and the failure to switch from acido-
genic to the solventogenic phase, are major problems in ABE fer-
mentation of lignocellulosic biomass substrates (Pfromm et al.
2010). Continuous fermentation processes may be used to im-
prove product yield, concentration and productivity (Tao et al.
2014a). In parallel, optimization of culture conditions, such as
temperature, pH, agitation and manipulation of gases (hydrogen
partial pressure, or gassing with carbon monoxide, pure CO2 or
N2 or the ratio of CO2 and N2 ) during fermentation, have been
shown to enhance product concentration and productivity (Li,
Baral and Jha 2014). Apart from microbes and process parame-
ters, cost and efficiency of components ascribed to recovery and
stillage utilization sections, also impact the economic viability
of the production of butanol from corn stover.
Prior research (Ezeji et al. 2010; Qureshi et al. 2010a,b, 2013,
2014; Cheng et al. 2012; Jurgens et al. 2012; Tashiro et al. 2013) used
batch, fed-batch, continuous and cell-recycle fermentations for
butanol production. Results of those studies offer insight into
strain physiology and behavior. While batch fermentation has
several advantages, such as simplicity, easy manipulation, low
risk of contamination and versatility in types of feedstock used
(Ezeji, Qureshi and Blaschek 2007a, Ezeji et al. 2010; Jurgens et
al. 2012), fed-batch and continuous fermentation processes of-
fer advantages like higher cell density, which has a positive im-
pact on butanol productivity and selectivity (Jiang et al. 2009;
Cheng et al. 2012; Qureshi et al. 2013, 2014). Whereas higher bu-
tanol concentrations may be obtained in fed-batch cultures than
in continuous cultures (Jiang et al. 2009), higher butanol pro-
ductivity may be achieved in continuous fermentation cultures
(Qureshi et al. 2014). Another advantage of continuous culture
over fed-batch system is a longer fermentation period made pos-
sible because toxic products are continually removed from the
bioreactor while nutrients are continually supplied to support
the health of fermenting microorganisms (Ezeji, Qureshi and
Blaschek 2007b; Cheng et al. 2012; Jurgens et al. 2012).
When using a continuous bioreactor, only one batch of in-
oculum culture is needed over the course of the ABE produc-
tion period. Therefore, the volume of the bioreactor can be re-
duced without decreasing productivity, and the time necessary
for cleaning and sterilization of the equipment is drastically re-
duced when compared to batch culture. In addition, the continu-
ous culture system is an important research tool for the determi-
nation of parameters that influence the physiology and activity
of solventogenic Clostridium species (Ezeji, Qureshi and Blaschek
2007a). Currently, simultaneous saccharification and fermenta-
tion (SSF) is a popular alternative to separate enzymatic hydrol-
ysis and fermentation (He and Chen 2013; Qureshi et al. 2013),
and SSF may be integrated with in situ product removal (to al-
leviate butanol toxicity to solventogenic Clostridia; Qureshi et al.
2013; Baral and Shah 2014). A mass balance model and a com-
parative analysis of different ABE fermentation approaches are
illustrated in Fig. 3.
End products of ABE fermentation using corn stover as sub-
strate are grouped into two phases, gas and liquid. In the efflu-
ent or liquid phase, the primary end products are butanol (25%
yield by weight from glucose) and acetone (8% yield by weight
from glucose) (Tao et al. 2014a), and ethanol (3% yield by weight
from glucose). While intermediate fermentation products such
as butyric and acetic acid (each with approximately a 3% yield
by weight from glucose) may end up in effluents, they are typi-
cally reabsorbed by solventogenic Clostridium species during sol-
ventogenesis. However, if the ABE fermentation process is sub-
optimal or compromised, end products are predominantly bu-
tyric and acetic acid due to poor acids re-assimilation and con-
version to acetone, butanol and ethanol. Notably, acids formed
via the acidogenic pathway of solventogenic Clostridium species
include acetic and butyric acid, and relatively small amounts of
formic and lactic acid. Gas products associated with ABE fer-
mentation are CO2 and H2 , which help to maintain anaerobic
conditions during ABE fermentation. Previous studies (Qureshi
and Blaschek 2001; Ezeji, Quresi and Blaschek 2003) reported in-
creased ABE production when sparging the bioreactor with CO2
and H2 during ABE fermentation due to increased utilization of
acids in the bioreactor system and reduction of inhibitory ef-
fects of butanol. It should be noted that end product concen-
trations of ABE fermentation are dependent on the species and
strain of solventogenic Clostridium used as well as feedstock, pre-
treatment and hydrolysis of the feedstock (Ezeji, Qureshi and
Blaschek 2007a; Qureshi et al. 2010a,b, 2014; Jurgens et al. 2012;
Baral and Shah 2014).
While commercial ABE fermentation from lignocellulosic
biomass is yet to be established, distillation is an established
commercial recovery method for ethanol production facilities.
Similarly, distillation methods can be used to separate ABE from
the fermentation broth and purify butanol from the ABE mix-
ture. The ABE separation and purification through traditional
distillation may require similar level of energy as ethanol or even
more due to relatively higher boiling point than ethanol. For
instance, traditional distillation is an energy-intensive process,
which requires more energy (up to 42 MJ L1 of ethanol) than is
available in the recovered ethanol (23.5 MJ L1 of ethanol) (Pitt,
Haag and Lee 1983) or butanol (36 MJ L1 of butanol). On the other
hand, traditional distillation process requires 12 tons of steam
to recover 1 ton of ABE in addition to the azeotrope formation
with water (i.e. a mixture of two liquids whose vapor has the
same portions of constituents as the mixture and which cannot
be separated by distillation) (Green 2011). Because of azeotrope
formation, ethanol fuel or butanol (more than 99% pure) cannot
be produced by traditional distillation. As a result, commercial
ethanol plants employ distillation process along with molecular
sieve adsorption to produce fuel-grade ethanol.
ABE FERMENTATION AND IN SITU RECOVERY
Apart from conventional distillation, several studies have fo-
cused on techniques for real-time ABE recovery during fer-
mentation due to the butanol toxicity to the culture. These
techniques include vacuum recovery, adsorption, gas strip-
ping, steam stripping, freeze crystallization, liquidliquid ex-
traction, perstraction (membrane extraction), reverse osmosis,
membrane distillation, sweeping gas pervaporation, thermo-
pervaporation, vacuum pervaporation and hybrid processes in
which distillation-pervaporation and distillation-vapor perme-
ation are used (Ezeji, Quresi and Blaschek 2003; Vane, 2005, 2008;
Mariano et al. 2012; Hecke, Hofmann and De Wever 2013; Lin
et al. 2013; Abdehagh et al. 2015; Kujawska et al. 2015). These
non-conventional methods for the recovery of ABE solvents are
being developed in order to increase butanol concentration and
productivity, reduce energy and butanol production costs. Some
in situ recovery methods, including membrane-based systems,
such as pervaporation, perstraction, reverse osmosis and ad-
sorption, might not be good recovery options for fibrous LBHs
due to potential membrane fouling. However, gas stripping,
liquidliquid extraction and vacuum assisted stripping appear

Figure 3. Comparison of fermentation methods. (Mass balance for each fermentation method was summarized from several previous studies (Qureshi et al. 2010b,
2013; Amiri, Karimi and Zilouei 2014; Xue et al. 2014).
to be compatible with LBH. Nonetheless, membrane based sys-
tems, molecular sieve processes included, may be used to dehy-
drate ABE after recovery using vacuum, gas stripping or liquid
liquid extraction. These different single and hybrid recovery
methods require different levels of energy input and summa-
rized in Table 3. The energy requirement to recover butanol (and
ethanol) from fermentation broth is influenced by their initial
concentration in the broth, expected concentration in the dis-
tillate, and the type of the recovery systems (Table 3). Detail re-
views of ABE fermentation and in situ recovery methods can be
found elsewhere (Groot, van der Lans and Luyben 1992; Durre
1998; Ezeji, Qureshi and Blaschek 2004; Vane 2005, 2008; Ku-
jawska et al. 2015).
ECONOMIC VIABILITY
AND COMMERCIALIZATION
Owing to increasing interest in biobased butanol, various com-
panies including Gevo, Inc., Butamax Advanced Biofuels LLC,
DuPont, Chevron Oronite and Cobalt Technologies, are actively
involved in the promotion and development of ABE fermen-
tation plants (Tao et al. 2014a,b). The success of the biofuels
industry depends not only on economic viability but also on en-
vironmental sustainability. However, commercial production of
butanol from biobased sources needs to be economically com-
Baral et al. 7
petitive with the butanol produced from petroleum (Ranjan and
Moholkar 2012). Economic and sustainable ABE fermentation
depends on several factors, such as cost of substrate, butanol
concentration in the fermentation broth and product recovery
(Mu et al. 2011; Ranjan and Moholkar 2012). The present fermen-
tative production of butanol is not cost effective because of (i) the
spore-forming life cycle of solventogenic Clostridium species, (ii)
butanol toxicity, (iii) instability of the butanol producing strains
and (iv) production of other unwanted byproducts such as bu-
tyrate, acetate, acetone and ethanol (Ranjan and Moholkar 2012).
Techno-economic analysis (TEA) and life cycle assessment
(LCA) are essential for process development and identification
of technical, economic and environmental barriers to ABE fer-
mentation from lignocellulose. Underscoring the significance of
feedstock and design costs, several TEA and LCA studies of com-
mercial scale ABE production from corn stover feedstock have
been reported (Kumar et al. 2012; Tao et al. 2014a,b). Raw ma-
terial cost accounted for 65% of annual biobutanol production
costs (Kumar et al. 2012). Although corn stover is an agricultural
waste, it may be susceptible to price inflation, like corn grain,
thereby causing volatility in the butanol production cost. Addi-
tionally, design and process parameters that have significant im-
pact on economic viability of biobutanol include fermenter size,
plant capacity and production yield (Kumar et al. 2012). The bu-
tanol yield and productivity may also vary due to the particular
 8 FEMS Microbiology Letters, 2016, Vol. 363, No. 6

Table 3. Energy requirement to recover ethanol and butanol or ABE (summarized data for some potential recovery methods from Vane 2008).

Recovery Initial concentration Energy requirement: MJ fuel/kg-EtOH; Final concentration (wt%):

method(s) (wt%): ethanol; (butanol or ABE) (MJ fuel/kg-BuOH or ABE) ethanol; (butanol)

Distillation without heat 4.5 9.4 94

recovery
Distillation with heat 5 4.5 94
recovery
Gas stripping 5 9 94
Distillation with 8.8 5.02 99.8
pervaporation
Distillation with molecular 10 4.47.6 99.5
sieve
Distillation with vapor 10 5.17 99.5
permeation
Distillation with (Butanol: 0.5) (8.2 MJ fuel/kg-BuOH) (99.9)
pervaporation
Distillation-decanter (Butanol:0.5) (88.3 MJ fuel/kg ABE) (99.9)
system
Distillation with silicalite (Butanol: 0.5) (9 MJ fuel/kg-BuOH) 810 g L1
adsorption
Multicolumn distillation (Butanol: 0.5) (26.9 MJ fuel/kg-BuOH) 810 g L1
Four-column distillation (Acetone- 1.25, butanol-3.35, ethanol- (9.7 MJ fuel/kg ABE)
0.4: ABE-5)
Distillation with silicalite (ABE-1: (A:B:E- 6:3:1)) (37 MJ fuel/kg ABE)
adsorption
Liquidliquid extraction 10 2.8 70
with fatty acid

strain of the solventogenic Clostridium species used and whether
traditional fermentation, or fermentation with in situ product re-
covery, was employed.
Lignocellulosic feedstock seems suitable for economically vi-
able butanol production with an average production cost of $0.48
per liter butanol from a plant capacity of 12.34 million liters (3.26
million gallons) per year (Kumar et al. 2012). Butanol produc-
tion cost was highly sensitive to plant capacity, ABE yield and
feedstock cost (Kumar et al. 2012). Their estimated butanol pro-
duction cost varied from $0.45 to $0.54 per liter with a change
in plant capacity from 6.17 to 30.86 million liter butanol per
year, probably most influenced by feedstock transportation cost.
Similarly, the estimated butanol production cost decreased from
$0.77 to $0.54 per liter when the ABE yield increased from 20% to
40%, and was $0.40$0.55 per liter when the price of corn stover
varied by 50% of average price of $33 per metric ton. A decrease
in sugar yield from corn stover ($58.5 per dry ton) from 85% to
60%, would increase the minimum n-butanol selling price from
$0.88 per liter to $1.38 per liter in a butanol plant with a pro-
duction capacity of 94.63 million liters per year (25 million gals
per year) (Tao et al. 2014a). However, when the increase in ABE
molar ratio from 3:6:1 to 0.5:9:0.5 (increased butanol concentra-
tion from 60% to 90%) was considered, the n-butanol selling price
decreased from $0.88 per liter to $0.81 per liter. In addition to n-
butanol, TEA and LCA of iso-butanol production from corn stover
was estimated to have competitive selling price with n-butanol
(Tao et al. 2014b). It was estimated that the selling price of iso-
butanol increases from $0.78 per liter to $1.47 per liter when
sugar yield decreased from 85% to 45% (Tao et al. 2014b). Remark-
ably, n-butanol and iso-butanol production processes emit more
greenhouse gases than ethanol production (Tao et al. 2014b),
generating 4.3, 26.42 and 21.91 kgCO2 -eq per gallon gasoline
equivalent, during ethanol, n-butanol and iso-butanol produc-
tion, respectively. In parallel, a recent economic study on the
production of butanol from wheat straw hydrolysate (Qureshi
et al. 2013) identified utility cost to be one of the most significant
factors affecting price of butanol. The estimated production cost
of butanol with recovery through distillation was $1.11 per liter
for a grass rooted/green field plant (i.e. new plant requires the
costs for the building, yard improvement and auxiliary facilities)
while the production cost of butanol from an annexed plant (i.e.
plant annexed to an existing distillery and does not require site
development cost) would decrease to $0.87 per liter. Recovery of
butanol using a pervaporation membrane should further reduce
the cost to $0.66 per liter (Qureshi et al. 2013).
Feedstock, design and process costs are major factors that in-
fluence the production cost of biobutanol (Kumar et al. 2012; Tao
et al. 2014a,b). While corn stover feedstock accounts for 54%
of total butanol production cost, a significant amount of the re-
maining 46% was contributed to facilities and utilities (Tao et al.
2014a). Thus, to make butanol production commercially attrac-
tive, it is essential that the right solventogenic Clostridium strain,
feedstock, and plant capacity are used, as well as compatible in
situ butanol recovery method.
CONCLUSIONS
Biobutanol has the potential to be a viable liquid fuel for the
transportation industry. Corn stover appears to be an ideal fer-
mentation substrate for biobutanol production due to its sugar
content and widespread availability. Although much progress on
the use of nonfood substrates for the production of fuels and
chemicals has been made, substantial research efforts are still
being expended on the development of technologies that im-
prove sugar yields from corn stover and similar lignocellulosic
biomass without generating significant levels of LDMICs. Since
the LDMICs content of corn stover hydrolysates is the most im-
portant cause of poor fermentation of corn stover hydrolysates

to butanol, and other biofuels and chemicals, over time, it should
be possible to develop a process that produces high sugar yields
without the generation of inhibitory levels of LDMICs. Nonethe-
less, to develop commercializable technology for the production
of corn stover based biobutanol, the primary obstacles to com-
mercial biobutanol production such as feedstock cost, butanol
yield and productivity, and downstream processing cost, must
be addressed. Thus, developments of microbial strains with im-
proved tolerance to butanol and LDMICs, in conjunction with
compatible integrated fermentation with in situ product recov-
ery technology, are expected to significantly increase the bu-
tanol concentration in the fermentation broth, increase the bu-
tanol yield and productivity, and ultimately, reduce the produc-
tion cost of biobutanol to a level that is competitive with the
petroleum-derived butanol.
ACKNOWLEDGEMENTS
The authors thank Ms. Mary Wicks (Food, Agricultural and Bio-
logical Engineering Department, The Ohio State University) and
Dr Victor Ujor (Renewable Energy Program, Agricultural Tech-
nical Institute, The Ohio State University, 1328 Dover Road,
Wooster OH 44691, USA) for critical review of the manuscript.
FUNDING
This work is supported in part by State funds appropriated to
The Ohio State University, Ohio Agricultural Research and De-
velopment Center (OARDC), and the USDA NIFA Hatch projects
1005665 and OHO01333.
Conflict of interest. None declared.
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