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Applied Energy 88 (2011) 1999–2012

Contents lists available at ScienceDirect

Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Developments in biobutanol production: New insights


Manish Kumar, Kalyan Gayen ⇑
Department of Chemical Engineering, Indian Institute of Technology Gandhinagar, VGEC Complex, Chandkheda, Ahmedabad 382 424, Gujarat, India

a r t i c l e i n f o a b s t r a c t

Article history: Biobutanol will become an attractive, economic and sustainable fuel as petroleum oil leads towards
Received 2 October 2010 expensive fuel due to diminishing oil reserves and an increase of green house gases in the atmosphere.
Received in revised form 16 December 2010 The major challenges in biobutanol production are low butanol titer, availability of compatible feed-
Accepted 18 December 2010
stocks, and product inhibition. These hurdles are being resolved using several genetic engineering tech-
Available online 13 January 2011
niques, metabolic engineering strategies, and promising integrated continuous fermentation processes
with efficient product recovery techniques (like gas stripping). Adequate success in utilizing renewable
Keywords:
and cost-effective cellulosic materials as feedstocks has opened up novel grounds for the advancement
Biobutanol
Lignocellulosic material
in economic biobutanol production. In this direction, Clostridium beijerinckii is being explored as promis-
Process development ing strain to produce biobutanol from cellulosic materials. Moreover, high biobutanol titer is being
Metabolic engineering focused through genetic modifications of Clostridia and non-Clostridia organisms (e.g., Escherichia coli,
Economic feasibility Saccharomyces cerevisiae, Pseudomonas putida, and Bacillus subtilis) in both aerobic and anaerobic fermen-
tation. Further, application of various novel genetic tools and genome sequencing of hyper-butanol-
producing Clostridial organism will enhance the scope of genetic engineering for biobutanol production.
Therefore, consolidation of academic and industrial research towards economic synthesis of biobutanol
illustrates the possibility of substantial breakthrough in future. In this review, we focus on (i) selection
of suitable bacterial strain (ii) availability of cheaper biomass to produce butanol (iii) metabolic engineer-
ing strategies of various microorganisms (iv) attempts at process development and (v) biobutanol recov-
ery techniques that provide future direction of economical biobutanol fermentation.
Ó 2010 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2000
2. History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2000
3. Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2001
4. Biomass selection for butanol production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2001
5. Development in fermentation processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2002
5.1. Batch and fed-batch fermentation processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2002
5.2. Continuous fermentation processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2003
5.2.1. Free cell continuous fermentation processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2003
5.2.2. Immobilized cell fermentation processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2003
5.2.3. Improved free cell fermentation processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2004
5.2.3.1. Cell recycling and bleeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2004
5.2.3.2. Continuous flash fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2004
6. Butanol inhibition and recovery techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2004
7. Importance of agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2005
8. Metabolic pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2006
8.1. Acid producing pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2006
8.2. Solvent producing pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2006
9. Strain improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2006
9.1. Strain improvement by mutation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2006
9.2. Strain improvement by genetic engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2008

⇑ Corresponding author. Tel.: +91 79 23972324; fax: +91 79 23972622.


E-mail address: gkalyan@iitgn.ac.in (K. Gayen).
URL: http://www.iitgn.ac.in/faculty/chemical/gkalyan.htm (K. Gayen).

0306-2619/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apenergy.2010.12.055
2000 M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012

10. Economics of biobutanol fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2009


11. Future prospectives . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2010
12. Conclusion . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2010
Acknowledgements . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2010
References . . . . . . . . . . . . ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2010

1. Introduction introduction of ABE fermentation, cost and availability of raw


materials have always played an important role in the economical
Continuous depletion of petroleum fuel-reserves is one of the production of biofuel. During World War I and II, sugar and starchy
prime concerns in this fuel dependent era. Despite of oil reserve grains (cereal grains) were the main substrates for ABE fermenta-
availability, current estimates show that accessing them will be- tion. Unfortunately, these raw materials turned cost-intensive
come extremely difficult in a few decades [1]. In addition to the due to increased food demand worldwide, with an exception of
depletion of petroleum fuels, environmental issues like greenhouse sugarcane-rich countries [31]. Hence, there is a constant demad
effect, global warming and climate change, are also the issues to be to search cost-effective raw materials for economic biobutanol
resolved worldwide [2]. Specifically, consumption of petroleum production. In such a scenario, cheaper and readily available ligno-
fuels in generating energy raises the atmospheric concentration cellulosic materials such as agriculture waste (corn stover [32],
of harmful carbon dioxide (CO2) and other greenhouse gases [e.g. wheat straw [33,34], corn fiber [35], barley straw [36], Carob pod
methane (CH4), nitrous oxide (N2O) etc.] [3]. Thus, the world re- [37], and switch grass [32]), and wood residues, can be used for
quires alternative, renewable fuels to substitute fossil fuels [4–6]. economical production of biobutanol [4,38,39].
In this scenario, economic production of biofuels has the potential Various modes of fermentation processes for the production of
to compensate the current needs of petroleum fuels. It addresses biobutanol have been examined, e.g., batch, fed-batch, and contin-
the issue of energy production with zero/near zero emission of uous processes [32–36,40–49]. Despite of a few demerits such as
greenhouse gases due to its nobility of counterbalance capacity high capital cost and contamination, continuous processes (free
[2,7–9]. In other words, biomass is utilized as raw material to pro- cells, immobilized cells and cell recycling) tend to be more eco-
duce biofuel and the emitted CO2 during the consumption of this nomical over batch and fed-batch processes. Continuous processes
biofuel, is recycled in synthesizing biomass. Therefore, utilization offer various advantages such as reduction in sterilization and re-
of the biofuel acts as a counterbalance leading to reduction in inoculation time, superior productivity, and reduction in butanol
atmospheric greenhouse gas emissions. Additionally, benefits of inhibition [50]. The extensive problems in butanol fermentation
biofuels include energy security, foreign exchange savings, and can be attributed to (i) selection of sustainable biomass (ii) low
socioeconomic issues that are directly associated with the develop- production (iii) constraints executed on butanol inhibition and
ment of rural areas [8,10]. Liquid biofuels (biobutanol, bioethanol (iv) high product recovery costs [51–56]. Recent research efforts
[11,12], and biodiesel [13]) can compensate the stimulus demand increasingly are focused towards investigating the economic feasi-
of petroleum based liquid fuels [8,14–18]. bility of low cost lignocellulosic materials as feedstocks [57], novel
Brazil and USA [19] have set up their trade mark on economic processes compensating product inhibition, and efficient recovery
production of bioethanol due to rich availability of raw materials techniques. Genetic engineers also play a significant role towards
and bioethanol is being consumed in automobiles as gasoline developing tools to improve the capability of microbial strains aid-
blends [20]. This has triggered the production and subsequent ing the increments in fermentation yield. This review focuses on
use of bioethanol blended gasoline in developing countries like In- the challenges in biosynthesis of biobutanol at metabolic and pro-
dia [19,21]. Similarly, biobutanol, a recently introduced organic cess level along with the future perspectives of economic ABE fer-
alcohol, can also be utilized as biofuel or fossil fuel extender [3]. mentation. It also emphasizes on utilization of novel lignocellulosic
Biobutanol owing to significant properties such as high energy materials as feedstocks, improvement in fermentation processes,
content, hydrophobicity, blending ability, compatibility to com- strain improvement efforts, attempts on depreciating the butanol
bustion engines, corrosion and octane rating, is increasingly re- inhibition and factors affecting economics of ABE fermentation.
garded as a renewable source of energy [22,23]. Moreover, the
properties of biobutanol such as density (810 kg/m3) [23], energy
content (27.08 MJ/l) [24] and synthetic yields of biobutanol 2. History
(0.42 wt.% from corn stover having 90% cellulose content) [24])
have promise for its use as biofuel. Historically, ABE fermentation was largely used to synthesize
Industrial synthesis of biobutanol was first achieved during acetone that was solvent for preparation of smokeless nitrocellu-
1912–1914 by Acetone–Butanol–Ethanol (ABE) fermentation of lose explosive (cordite). Meanwhile, butanol was increasingly
molasses and cereal grains using Clostridium acetobutylicum being used as a solvent in rubber production and quick-drying lac-
(Weizmann’s organism) [25]. However, activity of biobutanol syn- quer to render a good finish on car bodies. Butanol was also used as
thesis was first elucidated at laboratory scale by Louise Pasteur in precursor for production of acryl esters, glycol ethers, butyl ace-
1861 [26]. Later, a few promising strains (Clostridium beijerinckii, tate, butyl amines, and amino resins. Some other applications are
Clostridium saccharoperbutylacetonicum and Clostridium saccha- listed below [22]:
robutylicum) were also identified which are capable of producing
biobutanol with higher yield [27]. Prior to 2005, butanol was pri-  Solvent for dyes, e.g. in printing, inks.
marily used as a solvent or a precursor for production of other  Feedstock for the production of flotation aids, e.g. butyl
chemicals. However, since 2005, after recognition of its novelty xanthate.
as biofuel, North American based company DuPont and UK based  Extractant in the production of drugs and natural substrates
company BP have declared their intension to restart the industrial such as antibiotic, hormones, vitamins, alkaloids and camphor.
scale ABE fermentation. Its importance is exemplified by large  Additive in polishes and cleaners, e.g. floor cleaners and stain
number of filed patent and patent applications [28–30]. Since the removers.
M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012 2001

 Solubilizer in the textile industry, e.g. additive in de-icing fluids. acetobutylicum (10–16 g l1) [25,52]. Further, studies on C.
 Gasoline additive (anti-icing). aurantibutyricum, revealed its ability for production of acetone
 Mobile phase in paper and thin layer chromatography. and isopropanol in addition to butanol [64]. Its efficacy was
 Humectants for cellulose nitrate. probed in ABE fermentation utilizing palm oil mill effluents as
substrates. Instead of using only anaerobic organism, co-culturing
After recognizing increased demand of butanol, a group of of Bacillus subtilis TISTR 1032 (aerobic) with C. butylicum WD 161
American businessmen (Commercial Solvent Corporation) ob- (anaerobic) led to substantial enhancement (5.4 and 6.5 folds) of
tained a license on the microbial fermentation process developed ABE fermentation production utilizing soluble cassava starch as
and patented by Weizmann (US Patent 1315585, 1919) and started substrate. This higher yield was due to higher amylase activity
butanol production at Terre Haute, Indiana, USA in 1920. Another during desaccharification process in the presence of aerobic B.
extended plant (a distillery in Peoria, Illinois) with 32 fermentors subtilis. Interestingly, co-culture system of aerobic and anaerobic
(capacity of 189,250 l) was acquired by the same group and its organisms favored to omit costly reducing agents, specifically
capacity was enhanced to 96 fermentors (capacity of 567,750 l) used for maintaining anaerobic conditions in fermentation [65].
during 1924–1927. Subsequently, many other plants were estab- Unfortunately, possibility of infection of these species by a bacte-
lished in countries like Japan, India, Australia, and South Africa riophage on increasing transfer/sub-culturing steps provided a
within 1936 [25]. However, microbial production of butanol and drawback [25]. To overcome this inviability of the organisms from
acetone declined rapidly due to availability of cheaper crude oil bacteriophage infection, control strategies such as sterilization,
based butanol and high cost of fermentative raw material (molas- decontamination, disinfection and immunization of resistant
ses). In 1982, solvent production plants came to a standstill strains were implemented [66].
throughout the world on critical shortage of molasses. The selection of bacterial strains in producing biobutanol is
Recently a greater emphasis is placed on biobutanol production primarily based on type of feedstocks, targeted productivity, addi-
after recognizing its potentiality as biofuel. This use of biobutanol tional nutrient requirement, butanol tolerance, and bacteriophage
as a biofuel has received a huge impact in 2005, when David Ramey resistance. More number of endeavors should be made on
drove his unmodified vehicle across the USA fuelled solely on buta- isolation of novel organisms having desired characteristics and
nol [22]. Subsequently, BP and DuPont have announced a joint ven- on enhancement of novel strains using different genetic engineer-
ture, approved by European Commission, towards economic ing strategies.
synthesis of butanol in 2007 and targeted the market induction
in a short period of time. This joint venture also focused on devel-
oping the cost-effective technologies to synthesize biobutanol 4. Biomass selection for butanol production
from biomass [28,29,58].
Feedstock for fermentation is most accountable parameter on
rationale of its economic feasibility [67,63,68,69]. On the basis of
3. Microorganisms utilization of feedstocks, biofuels including biobutanol were classi-
fied into first generation and second generation biofuels. In the first
Genus Clostridia embrace a vast variety of biobutanol producing generation biofuels, raw materials were sugarcane and cereal
bacteria (e.g. C. acetobutylicum, C. beijerinckii, Clostridium saccaro- grains while in the second generation biofuel, lignocellulosic mate-
perbutylacetonicum, Clostridium saccharoacetobutylicum, Clostridium rials (e.g. agriculture and forest wastes) were used as feedstocks. It
aurantibutyricum, Clostridium pasteurianum, Clostridium sporogenes, should be noted that the raw materials used for first generation
Clostridium cadaveris, and Clostridium tetanomorphum). Fortunately, biofuels is food competitive while second generation biofuels are
C. acetobutylicum, C. beijerinckii, C. saccaroperbutylacetonicum, and non-edible biomass [14,15,38]. Currently, there is an increased fo-
C. saccharoacetobutylicum exhibited significant activity for synthe- cus on second generation biofuels due to huge availability of
sis of butanol with higher yields [5]. C. acetobutylicum (a spore cheaper raw materials [2].
forming, rod shaped, obligate anaerobe and gram-positive bacte- Traditional attempts, utilizing cereal grains and sugar as feed-
ria) was first microorganism that was employed in industrial fer- stocks in ABE fermentation for industrial scale production, were
mentation from sugar and starchy grains for production of encouraged by availability of these raw materials and vast require-
acetone and butanol during the first half of last century [59]. Ear- ment of fermentation products [26]. However, utilization of these
lier, it was believed that this was the only species that could be substrates was criticized as ‘price hikers’ as well as contributing
used for ABE fermentation. However, later on (during 1990s), three to food shortages except in sugarcane and cereal grains rich
other species were identified in this mixed culture, namely C. countries like Brazil [22,31,70]. This concern forced to explore
beijerinckii, C. saccharoperbutylacetonicum and C. saccharobutylicum inexpensive and non- food competitive raw materials for ABE fer-
along with C. acetobutylicum. Subsequently, various strains of these mentation. Fortunately, it is found that various carbohydrates such
four species have been compared and reclassified on the basis of as glucose, fructose, mannose, sucrose, lactose, starch, and dextrin
16S rRNA gene sequencing and DNA fingerprinting [22,59–61]. were completely consumed by Clostridial organisms, while galact-
Moreover, Gutierrez et al. [62] investigated several strains of ose, xylose, arabinose, raffinose, melezitose, inulin and minnitol
C. acetobutylicum and C. beijerinckii (C. acetobutylicum DSM 1731, were partially utilized. Studies have revealed that xylose and arab-
C. acetobutylicum ATCC 824, C. acetobutylicum NCIMB 8052, C. acet- inose could also be completely fermented by most Clostridial bac-
obutylicum NCP 260, C. acetobutylicum NCP 262, and C. beijerinckii teria. However, these organisms were unable to consume
NRRL 592), which were capable of producing biobutanol utilizing trehalose, rhamnose, melibiose, and glycerol [25]. It was clear that
potato as a substrate. In this study, it was reported that C. acetobu- raw materials containing above mentioned simple sugars because
tylicum DSM 1731 was most proficient strain (0.24 g l1 h1). of their low cost, and enough availability can maintain the eco-
Apart from the C. acetobutylicum, the efficacy of other butanol- nomics of ABE fermentation.
producing Clostridia species was examined based on utilization of The usefulness of several starch (sago, defibrated-sweet-potato-
raw materials and ratio of solvent production. In this direction, slurry, degermed corn, extruded corn, liquefied corn starch, cas-
Qureshi et al. [32–36,63] cultivated C. beijerinckii using different sava, etc.) [46,50,56,71] and lactose (whey permeate) [49,72] were
waste cellulosic materials and reported approximately same examined as energy and carbon sources for microorganisms in-
ratio of solvents with higher butanol titer (18–25 g l1) than C. volved in ABE fermentation (Table 1). Ezeji et al. [71] used a novel
2002 M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012

Table 1
Various raw materials utilized in ABE fermentation.

Raw materials Composition Hydrolysis/gelatinization Bacterial strain Refs.


required
Barley straw 42% Cellulose, 28% hemicellulose, 7% lignin, 11% ash Yes C. beijerinckii [36]
Wheat straw 38% Cellulose, 29% hemicellulose, 24% lignin, 6% ash Yes C. beijerinckii [33,75,117]
Corn fiber 20% Starch, 50–60% non-starch polysaccharides Yes C. beijerinckii [35]
Corn stover 38% Cellulose, 26% hemicellulose, 23% lignin. 6% Ash Yes C. beijerinckii [32]
Switchgrass (Panicumvirgatum) 37% Cellulose, 29% hemicellulose, 19% lignin Yes C. beijerinckii [32]
Domestic organic waste 59% Sugars, 13% lignin, 17% ash Yes C. acetobutylocum [118]
Sago 86% Starch, small amounts of mineral Yes C. saccharobutylicum [45]
and nitrogenous matters
Defibrated-sweet-potato-slurry Starch No C. acetobutylocum [41]
(DSPS)
Degermed corn 73% Starch, 3% ash, 13% proteins Yes C. beijerinckii [50]
Extruded corn 61% Starch, 3.8% corn oil, 8.0% Protein, 11.2% fiber
Yes C. acetobutylocum [56]
Liquefied corn starch 39% Starch, 45% moisture Yes C. beijerinckii [71]
Cassava 70% Starch, 2.7% protein, 2.4% fiber, 0.2% ash No Co-culture of B. Subtilis and [65]
C. butylicum
Whey permeate 5% Lactose, 0.36% fat, 0.86% protein No C. acetobutylocum [49,53,72]

and inexpensive substrate viz. liquefied corn starch (a product of pretreatment of macro-algal biomass are the focus of current re-
corn processing industry) in producing biobutanol. However, accu- search efforts with enormous scope for further development. Re-
mulated sodium metabisulfite (Na2S2O5, part of liquefied corn search efforts are required to optimize the processes including
starch [LCS]) was found toxic above the concentration of 0.6 g l1 pretreatment (or hydrolysis) of substrate, and removal of inhibitors
for fermentative bacteria. Therefore, authors postulated that LCS especially for utilization of lignocellulosic raw materials in synthe-
could be cost-effective raw material in producing biobutanol after sizing biobutanol.
removal of Na2S2O5 during pretreatment of raw material and on
line removal of product (by gas stripping). Apart starchy sub-
5. Development in fermentation processes
strates, the suitability of lactose containing substrates (wastes of
dairy industry) like cheese whey were investigated in batch fer-
ABE fermentation was the second largest industrial fermenta-
mentation using C. acetobutylicum DSM 792 and C. acetobutylicum
tion process after ethanol (utilizing yeast fermentation) due to
AS 1.224. Results demonstrated that cheese whey had better per-
the economic importance of acetone and butanol prior to introduc-
formance than lactose solution or synthetic media [72].
tion of petrochemical solvents. Typically, the total concentration of
Recently, it is known that Clostridial bacteria can also ferment
solvents (acetone, butanol, and ethanol) in ABE fermentation broth
other cheaper alternative biomass such as, lignocellulosic materi-
was 20 g l1(ratio of butanol, acetone, and ethanol is 6:3:1) with
als due to their saccharolytic ability [73]. However, acidic or enzy-
butanol around 13 g l1 [78,79]. In this review, wide range of raw
matic hydrolysis of lignocellulosic materials is essential to convert
materials and various Clostridia species used in different fermenta-
into monosaccharides before using them as substrates in ABE fer-
tion processes for butanol production were discussed in the follow-
mentation. The lignocellulosic biomass is most abundant renew-
ing sections (Table 2) [33,34,36,41,42,46,49,51,52,80].
able resource on the earth for biofuel production [3,74]. For
instance, developing countries like India generates over 370 mil-
lion tonnes of biomass every year directly from plants, rice husk 5.1. Batch and fed-batch fermentation processes
from rice mills, saw dust from saw mills, bagasse from sugar mills
etc. [39]. It is known that up to 30% of fertile land of world is still One stage and two stage fermentation studies were performed
being used to cultivate the crops primarily as feed for horses and to enumerate the performance of batch and fed-batch process in
oxen used in transportation. This critically reveals availability of producing biobutanol from numerous feedstocks (like lignocellu-
lignocellulosic raw materials [3]. Experimentally, the potential of losic and starchy materials) and various microbes. Wheat straw
numerous lignocellulosic feedstocks like wood forestry residues, was examined by five combinations of pretreatments (acidic and
corn stover, wheat straw, corn fibers, barley straw, and switch enzymatic hydrolysis) of raw material and fermentation processes
grass has been tested for ABE fermentation (Table 1) [4,32– using C. beijerinckii P260 [33]. These five combinations were: (i)
34,36,75,76]. The wheat straw, pretreated with alkaline peroxide fermentation with pretreated wheat straw, (ii) separate hydrolysis
and hydrolytic enzymes, has been employed for butanol produc- and fermentation of wheat straw without removing sediments, (iii)
tion using C. beijerinckii P260 by Qureshi et al. [75]. Regrettably, simultaneous hydrolysis and fermentation of wheat straw without
dramatic reduction in productivity was detected due to the inhibi- agitation, (iv) simultaneous hydrolysis and fermentation with
tion caused by high concentration of salts. Significant increase of addition of sugar supplementation, and (v) simultaneous hydroly-
butanol titer (22.17 g l1) has been achieved on electrolytic reduc- sis and fermentation with agitation by gas stripping. The results re-
tion of salt concentration than without reduced salt concentration vealed the maximum productivity of butanol (0.31 g l1 h1) using
hydrolysate (2.59 g l1) even higher than glucose fermentation a combination process (v) i.e.; simultaneous hydrolysis of wheat
(21.37 g l1). straw and fermentation with agitation from gas stripping. Here,
Another non-food biomass, namely macro-algal biomass, con- monosaccharides, viz. glucose, xylose, arabinose, galactose, and
taining significant quantity of carbohydrates, with low or no lignin mannose, were identified in hydrolysate. Authors claimed that
and fermentation inhibitors, has been acquired as alternative sub- the use of simultaneous hydrolysis of wheat straw towards simple
strate [77]. Interestingly, environmental carbon dioxide is being sugar and fermentation to butanol was an attractive method than
consumed for growth of autotrophic macro-algae leading to reduc- expensive glucose in ABE fermentation processes. Subsequently,
tion in global warming. Challenges of optimized growth and hydrolysis of wheat and fermentation were attempted in a single
M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012 2003

Table 2
Comparison of fermentation processes for butanol production based on raw materials, and yield/productivity.

Fermentation process Strain used Substrate Yield (g/g)/productivity (g Maximum titer of Refs.
l1 h1) ABE (g/l)
Batch fermentation C. beijerinckii P260 Barley straw 0.43/0.39 26.64 [36]
C. beijerinckii P260 Wheat straw 0.41/0.31 21.42 [33]
C. beijerinckii BA101 Corn fibers 0.36–0.39/0.10 9.3 [35]
C. beijerinckii P260 Corn stover and switchgrass 0.43/0.21 21.06 [32]
(1:1)
C. beijerinckii P260 Switchgrass 0.37/0.09 14.61 [32]
Fed-batch fermentation C. beijerinckii P260 Wheat straw –/0.36 16.59 [34]
C. Synthetic medium with 0.49/0.42 16.0 [43]
saccharoperbutylacetonicumN1– butyric acid
4

Continuous fermentation
(i) Free cell continuous fermentation C. saccharobutylicum DSM Sago starch 0.29/0.85 9.1 [45]
13864
C. beijerinckii BA101 Degermed corn –/0.29–0.30 14.28 [50]
C. beijerinckii BA101 Starch and glucose –/0.42 9.9 [46]
(ii) Immobilized cells continuous C. acetobutylicum P262 Whey permeate 3.5–3.6/0.36–1.10 8.6 [49]
fermentation
C. acetobutylicum 824A Lactose and yeast extract –/0.78 1.43 [81]
C. acetobutylicum ATCC 55025 Corn 0.42/4.6 12.50 (butanol) [40]
C. acetobutylicum P262 Defidered-sweet-potato- 0.20/1.0 7.73 [41]
slurry
C. beijerinckii BA101 Synthetic medium 0.36/12.43 8.8 [44]
(iii) Cells recycling and bleeding C. saccharoperbutylacetonicum Synthetic medium –/11.0 8.58 [119]
N1-4

reactor with semi batch mode aiming 100% hydrolysis of wheat consumption in sterilization of bioreactors, re-inoculation, solvent
straw and its fermentation towards butanol. In this case, produc- inhibition and low productivity. These limitations were compen-
tivity was doubled (0.77 g l1 h1) in batch fermentation. Further, sated by using continuous fermentation processes.
supplement of sugar feed to the reactor helped in improving the
productivity of the fed-batch fermentation [34]. In another study, 5.2. Continuous fermentation processes
the effect of feeding butyric acid in pH-stat fed-batch fermentation
process using C. saccharoperbutylacetonicum was examined [43]. Continuous fermentation processes have several advantages
Results demonstrated enhanced productivity of butanol by feeding over batch fermentation processes as fermentation can be operated
butyric acid in the broth containing glucose. The maximum buta- in a continuous mode [47]. Only one inoculum culture is sufficient
nol concentration (of 16 g l1) was achieved with a 72% higher pro- in fermentation for long time. Moreover, drastic reduction of ster-
ductivity than conventional batch process. Therefore, the pH-stat ilization and inoculation time leads to enhanced productivity. The
fed-batch process was carried out by maintaining a constant pH most common strategies of continuous fermentation including free
through controlled addition of butyric acid. cell, immobilized cell, and cell recycles, have been attempted
Prior to batch fermentation process, pretreatment of the raw [42,44].
materials was crucial step to control the concentration of inhibitors
in hydrolysate (product from hydrolysis of raw materials). Two 5.2.1. Free cell continuous fermentation processes
pretreatment processes (acidic and enzymatic) were analyzed with In the free cell fermentation process, cells are free to move
corn fiber hydrolysate in batch fermentation. Fermentation using within fermentation broth due to the agitation by mechanical agi-
corn fiber hydrolysate, treated with enzymatic method, resulted tator or by air lifting. This maintains the microbial cells and nutri-
a higher yield (0.35 g/g) than acidic method as fermentation inhib- ents in the suspension and helps in promoting mass transfer. The
itors were eliminated in the case of enzymatic method [35]. Like- sustainability of the continuous cultivation of C. beijerinckii
wise, the presence of inhibitors in hydrolysates of other cellulosic BA101 in degermed corn based P2 medium for biobutanol fermen-
substrates (barley straw, corn stover, and switch grass hydrolysate) tation was investigated by Ezeji and Blaschek [47]. Authors
was detected in several studies of biobutanol production using reported that butanol production was merely possible in sacchari-
batch fermentation. Fortunately, these inhibitors could be success- fied degermed corn or in combination of saccharified and normal
fully removed by treatment of hydrolysate with calcium hydroxide degermed corn, while normal degermed corn showed negative re-
[Ca(OH)2] [32,36]. sults. Further, stability of physical and chemical properties of gel-
In addition to utilization of cellulosic substrates, biobutanol atinized corn starch as raw material was also examined
synthesis was attempted using carbon dioxide as a substrate ob- thoroughly in free cell mode and noted that the degree of hydroly-
tained from coal-derived synthesis gas. In this fermentation study, sis of gelatinized corn starch decreased with time. It was also no-
suitable organism was Butyribacterium methylotrophicum. Experi- ticed that the physical and chemical properties change adversely
ments were carried out in two stage process: in the first stage, bu- during storage time due to retro-gradation (reaction takes place
tyric acid and acetic acid formation (acidogenesis) was carried out in gelatinized starch to make less digestive structures of starch),
using B. methylotrophicum, and in the second stage, butanol syn- resulting low productivity in ABE fermentation [45,46].
thesis (solventogenesis) was achieved from acids using C. acetobu-
tylicum. Interestingly, it was found that the composition of final 5.2.2. Immobilized cell fermentation processes
products could be dependent on pH [48]. Batch and fed-batch fer- Immobilized cell fermentation process is advantageous over
mentation processes were limited by several factors such as time free cell continuous fermentation process. For example, cell
2004 M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012

immobilization allows long survival time of cells (due to lack of butanol from broth). Simulation analysis (dynamic behavior of
mechanical agitation) in solventogenesis phase without frequent the process) revealed that the flash fermentation process could
cell regeneration. This process was applied in a fibrous bed biore- be promising for high productivity of butanol. Moreover, final con-
actor with C. acetobutylicum cells using corn as substrate. Results centration of butanol was expected to be more than 20 g l1 and
revealed a significant enhancement in butanol yield (20% higher this process could also be helpful in reduction of distillation costs
over conventional continuous fermentation techniques). Moreover, and benefit environment due to lower quantity of waste water
shorter acidogenesis phase was elucidated using butyric acid as co- generation [82]. Another mathematical model was formulated base
substrate in feed stream which improved the duration of solvento- on experimental results to optimize productivity, energy require-
genesis phase [40]. Continuous fermentation process at stable bio- ment and product purity [83]. The Model showed that the
film using C. acetobutylicum on lactose and yeast extract as sub- performance of the fermentation process can be improved using
strates was also performed in packed bed reactor, containing high feed concentration. Also, high product purity could be
hydrophobic plastic carriers [81]. Results showed bare improve- achieved using two-vessel partial flash system (first vessel of two
ment in the performance of fermentation by increasing pH and to three plates and second as a flash vessel) over two-vessel flash
dilution rate. system.
Immobilized cell fermentation technique was also employed to
investigate the a-amylase activity with eight strains of C. acetobu-
tylicum (B-527, B-528, B-529, B-530, B-591, B-594, ATCC 824, 6. Butanol inhibition and recovery techniques
P262) using defibered-sweet-potato-slurry (DSPS). Two strains,
namely B-591 and P262 of the anaerobic bacteria showed highest It is well known that during repeated batch sub-culturing or
activity in that scenario. However, C. acetobutylicum P262 (active continuous culturing of Clostridia, butanol formation declines as
amylase producer), using DSPS, showed 12-fold more productivity butanol is toxic for the microorganisms. It is one of the most crucial
over batch processes [41]. In a different study, fluidized bed biore- hurdles for butanol fermentation at commercial scale as bacterial
actor containing immobilized cells, was examined to study the uti- strains rarely tolerate more than 2% butanol [84]. High butanol
lization of lactose from whey permeate using C. acetobutylicum concentration induces an adverse change in phospholipid and fatty
P262 organism. Lactose utilization was enhanced significantly with acid composition in cell membrane of C. acetobutylicum. This ad-
feeble reduction in productivity as fermentation was operated in verse change diminishes the unsaturated and the saturated fatty
integrated system with liquid–liquid extraction [49]. On the basis acids ratio (U/S) and also decreases the specific interaction of alco-
of intensive observations of cell immobilized technique in ABE fer- hol with individual component of lipids [85]. Specifically, butanol
mentation, scale up became a crucial factor to meet the demands (a noxious organic solvent) enters into cytoplasmic membrane
for commercial production. In this direction, Qureshi et al. [44] and alters the membrane structure. It also disrupts a number of
scaled up an immobilized cell reactor utilizing C. beijerinckii physicochemical characteristics such as preferential solute trans-
BA101 strain and achieved productivity closer to that obtained port, membrane permeability, maintenance of the proton motive
using a laboratory scale reactor. However, due to excessive cell force (or maintenance of intracellular pH), intracellular ATP level,
growth in packed bed, blockage problem was recognized after glucose uptake, and conformation and activity of intrinsic mem-
2302 h of continuous operation. Nutrient constraints were applied brane proteins. Approximately, increment of 20–30% of fluidity in
to reduce the cell growth, but this approach was unsuccessful due membrane is observed with only 1% butanol exposure [86,87]. It
to inactivation (spore forming) of a significant number of cells or should be noted that butanol-induced degeneration is more severe
presence of a fewer number of cells in solventogenesis phase. than acetone [55,79].
Authors postulated that nutrient limitation should be used only Various endeavors were implemented in organism level as well
to avoid the blockage from excessive cell growth in packed bed. as in process level to facilitate the butanol tolerance of Clostridial
Still, research efforts are necessary in proper scaling up of contin- organisms. At organism level, several butanol tolerated strains
uous immobilization cell bioreactor in economical production of were developed from classical butanol producer organism (C.
butanol. acetobutylicum) using mutagenesis and genetic manipulation. A
few butanol tolerant strains of C. acetobutylicum ATCC 824 (SA-1,
5.2.3. Improved free cell fermentation processes SA-2, and G1) showed butanol tolerance limit up to 1.8% [84]. Fur-
5.2.3.1. Cell recycling and bleeding. Another novel technique, a mod- ther, consequences of butanol on cell membrane of SA-2 mutant
ified and improved version of free cell continuous fermentation were scrutinized; however, butanol tolerance could not be en-
(cell recycling and bleeding) process was examined using high cell hanced significantly because of vicissitudes in inherent properties
density of C. saccharoperbutylacetonicum. In this technique, mem- of cell membrane. In addition, the destructive consequences on sol-
brane module (filtration) was used to recycle the cells into the bio- vent producing ability of microorganisms were caused by muta-
reactor for improvement of cell concentration leading to better tions [88]. One efficacious achievement in the direction of strain
butanol productivity. On the other hand, optimization of dilution improvement was carried out by Lin et al. and a butanol tolerant
rate facilitated cell bleeding (i.e., removal of excess concentration derivative (called SA-1) of C. acetobutylicum ATCC 824 was ob-
of cells) from bioreactor to maintain optimum density of fermenta- tained by serial enrichment procedure. The mutant propagated
tion broth. In this study, 10-fold higher cell concentration has been (with 66% growth rate of uninhibited controlled strain) at higher
recorded and six fold higher yield of butanol (11.0 g l1 h1) was concentration of butanol (15 g l1) than native organism. Authors
observed than conventional continuous fermentation without cell stated that the strain SA-1 was superior over other mutants in
recycling (1.85 g l1 h1). This process included advantages like terms of growth rate, butanol tolerance, butanol production, pH
homogeneity of broth that facilitated diffusion over fermentation resistance, carbohydrate utilization, conservation efficiency to
process in cell-immobilized bioreactor [43]. butanol, and solvent ratio [56]. Interestingly, Assobhai et al. stud-
ied that repeated sub-culturing of C. acetobutylicum ATCC 824 re-
5.2.3.2. Continuous flash fermentation. A flash fermentation technol- sulted spore formation, and ceased butanol and acid production
ogy was proposed by Mariano et al. to overcome the low produc- after 23–25 sub-culturing. Feeding of sufficiently high concentra-
tivity hurdle in synthesizing butanol. This innovative technology tion of acids (acetic acid or butyric acid) was suggested in fermen-
consisted of three interconnected units, viz. fermentor, cell reten- tation broth to maintain the solventogenesis phase longer with
tion system, and vacuum flash vessel (for continuous recovery of reduction of butanol inhibition [54].
M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012 2005

Table 3
Advances in product recovery to enhance the production of ABE.

Recovery Raw material Bacterial strain Type of reactor Max. titer of ABE (without online Max. titer of ABE (with online Refs.
method recovery) in g/l recovery) in g/l
Gas stripping Whey permeate C. acetobutylicum Batch 8.7 70.0 [51]
P262
Sugar solution C. beijerinckii Batch – 69.7 [52]
BA101
Sugar solution C. beijerinckii Fed-batch – 120 [52]
BA101
Glucose C. beijerinckii Fed-batch 17.7 232.8 [78,120]
BA101
Liquefied corn starch C. beijerinckii Batch 18.4 23.9 [71]
BA101
Saccharified liquefied C. beijerinckii Batch 18.2 26.5 [71]
corn starch BA101
Saccharified liquefied C. beijerinckii Fed-batch – 81.3 [71]
corn starch BA101
Glucose C. beijerinckii Batch 17.7 75.9 [120]
BA101
Pervaporation Glucose C. beijerinckii Fed-batch 25.3 165.1 [121]
BA101
Glucose C. acetobutylicum Fed-batch – 154.97 [122]
ATCC 824
Perstraction Whey permeate C. acetobutylicum Batch 7.72 136.58 [53]
(lactose) P262
Whey permeate C. acetobutylicum Fed-batch 7.72 57.8 [53]
(lactose P262
Potato wastes C. acetobutylicum Fed-batch 19 33 [123]
DSM 1731
Adsorption C. acetobutylicum Batch 13.5 23.2 [124]
C. acetobutylicum Fed-batch 13.5 59.8 [124]
C. acetobutylicum Fed-batch 13.5 387.3 [124]
(repeated cycles)

One attractive achievement in process development, namely using C. acetobutylicum P262. This process was simple and eco-
extractive fermentation was also being examined to reduce the nomical (no need of expensive equipments); and was advanta-
butanol-induced inhibition effect. In this approach, production as geous over others as it reduces the butanol inhibition without
well as selective removal of butanol was carried out simulta- affecting the culture, concentration of nutrients and reaction inter-
neously to maintain low concentration of butanol in fermentation mediates. It was also successful to improve the productivities and
broth. The common online butanol removal techniques, are to reduce the process volume (reactor size) efficiently [51,52].
adsorption, liquid–liquid extraction, perstraction, reverse osmosis, Nitrogen [52] and a combination of CO2 and H2 (own gases of fer-
pervaporation, and gas stripping, which could be integrated with mentation) [51,71,78] or the mixture of all three were employed
ABE fermentation (Table 3) [89]. on stripping in fermentation broth. However, use of nitrogen gas
In the direction of liquid–liquid extraction, the use of a mixed provided better results than any other gas or combination of gases.
extractant containing 20% decanol in oleyl alcohol enhanced buta- Nowadays, fermentation integrated with gas stripping is attracting
nol formation using C. acetobutylicum ATCC 4259 in controlled pH the attention of researchers in scaling up of biobutanol synthesis.
conditions while decanol (extractant) alone was toxic for organ- Apart from product inhibition, other inhibitory factors were also
ism. It was interesting to note that decanol held larger butanol dis- detected in ABE fermentation. These were substrate inhibition, salt
tribution coefficient over either oleyl alcohol or the mixture of concentration inhibition, presence of dead cells, low water activity,
both. Authors optimized these two opposite consequences (toxic- macromolecule (like polysaccharides) accumulation, nutrient defi-
ity and distribution coefficient) of extractants and exceeded buta- ciency, and O2 diffusion through connecting tubes during addition
nol yield of 0.47 [53,55]. Ishii et al. preferred a mixture of oleyl of nutrients into the fermentor [51,78]. It is interesting to note that
alcohol and guerbet alcohol as extractant for butanol among 29 or- high concentrations (about 200 g l1) of whey permeates (lactose)
ganic compounds, based on their non-toxicity towards bacterial as substrate did not exhibit inhibitory effect [51], while glucose
cells (C. acetobutylicum IAM 19012). In their study, biosynthesis concentration greater than 161 g l1 showed inhibitory effects
of butanol was 2.5-fold higher than conventional fermentation [78]. Fed-batch fermentation was proposed to overcome this inhib-
processes (without extraction of product) [90]. However, the main itory effect of substrates [78]. High salt concentration (30–45 g l1)
concern of this extractive technique was to optimize the combina- also exhibited inhibitory effect towards growth of C. acetobutyli-
tion of non-toxicity and distribution coefficient, as extractant hav- cum. Interestingly, growth occurred with unfavorable shifting of
ing significant distribution coefficient was toxic to the bacterial fermentation from solventogenesis phase to acidogenesis phase
cells and vice versa. Consequently, identification and/or synthesis at lower salt concentrations [51].
of any compound or mixture of compounds having non-toxic activ-
ity and high distribution coefficient will be advantageous for 7. Importance of agitation
liquid–liquid extraction of butanol from fermentation broth.
Another viable recovery technique, namely gas stripping, was It is always beneficial to reduce the non-uniformities in fluid by
integrated with batch [51,52,71], fed-batch [71,78], and continu- removing concentration gradients of nutrients and temperature
ous fermentation processes [52]. Butanol fermentation integrated gradient (promoting heat transfer). Other notable functions of
with gas stripping was investigated in concentrated sugar broth the process are to maintain the cell suspension and gas dispersion
2006 M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012

in broth. Agitation serves the uniformity of aeration, suspension of 8.2. Solvent producing pathways
microbes and nutrients in aerobic fermentation process. However,
in anaerobic fermentation, aim of agitation is limited in maintain- At the final stage of acidogenesis phase, acid production
ing homogeneity of nutrient and microbes. Homogeneity is neces- slows down due to effect of low pH. To compensate the unfavor-
sary for high mass transfer between nutrients and microbes to able effect of low pH, organism shifts its metabolic activity from
enhance the rate of fermentation by increased interfacial area. Yer- acidogenesis phase to solventogenesis phase. In this phase, ace-
ushalmi and Volesskhy [91] studied the effect of agitation with C. tate and butyrate are consumed as substrates for biosynthesis of
acetobutylicum ATCC 824 using semi-solid reinforced clostridium acetone and butanol while no growth is observed [22]. However,
medium (RCM). Authors demonstrated that the specific rate of metabolic activity of this complex phase shifting differs from
anaerobic solvent production enhanced on increasing impeller organism to organism. For example, conversion of butyrate to
speed from 190–340 rpm with working volume of 10 l. Maximum butanol is hardly observed in the case of C. saccharoperbutylace-
specific rates of butanol, acetone, and ethanol production were tonicum N1-4 in absence of glucose in the media. Interestingly,
5.54, 3.85, and 0.8 mmole h1 g1 respectively. However, higher highest yield of butanol (0.671 mol-butanol/mol-butyrate)
impeller speed (higher than 340 rpm) showed low specific rates from butyrate was observed in presence of glucose in the media
of production. This phenomenon can be attributed due to compe- [98].
tition between solvent production and biomass biosynthesis, and The acetyl-CoA and butyryl-CoA are the key intermediates in
mechanical damage of cells at high shear rates. synthesizing ethanol and butanol (Fig. 1). These pathways produce
acetaldehyde and butyraldehyde respectively, as intermediate in
the presence of two sets of dehydrogenases. The reduction of bu-
8. Metabolic pathways tyryl-CoA to butanol is mediated by butyraldehyde dehydrogenase
and butanol dehydrogenase [96,99]. Six enzymes (thiolase,
Fermentation process inherently relies on the level of metabolic 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA
activities of the organism. Therefore, understanding of metabolic dehydrogenase, butyraldehyde dehydrogenase, and butanol dehy-
network of an organism is essential to engineer the strain leading drogenase and corresponding seven genes thiL, hbd, crt, bcd, etfA,
better productivity. Insights of the metabolic pathway and the etfB, and adhe) were identified for the conversion of acetyl-CoA
metabolic network analysis are key steps for metabolic engineer- to butanol (Fig. 1). This multi-gene expression was investigated
ing. Fortunately, the metabolic pathways of clostridium species in many organisms (e.g. E. coli, S. cerevisiae, and Pseudomonas
(C. acetobutylicum and C. beijerinckii) are alike [92]. Major end putida) aiming improvement of butanol productivity (discussed
products from metabolic pathway activities are butanol, acetone, in subsequent section) [5,99–101]. In both C. acetobutylicum and
ethanol, acetic acid, butyric acid, CO2, and H2 [92]. Recently, in C. beijerinckii, the activity of butanol dehydrogenase was NADPH
the investigation of cellulosic and hemicellulosic biomass as feed- dependent rather than NADH dependent [26]. Ethanol could be
stocks, it has been reported that Clostridia secrete extracellular en- produced independently under certain adverse media conditions.
zymes like amylase, glucosidase, glucoamylase, pullulanase, and However, ethanol formation was observed in traceable amount in
amylopullulanase to digest complex polysaccharides into simple comparison to acetone and butanol. The uptake of acids (butyric
monosaccharides like glucose, xylose and arabinose (Fig. 1) [73]. and acetic acids) was coupled with formation of acetone for buta-
However, cellulosic Clostridia were known to be extremely nol synthesis resulting from complex mechanism of metabolic
sensitive to oxygen and have low aerotolerance power. Peroxide pathways (Fig. 1). However, mechanism of complex regulatory
repressor (PerR) gene, responsible to regulate genes encoding aspects of butanol synthesis associated with acetone formation is
detoxification component, was identified as the reason of low aero- still an open question [25,93,97].
tolerance of C. acetobutylicum. Further investigations will provide
the information about aerotolerance as well as cellulosic activity
9. Strain improvement
of microorganisms [93–95]. Metabolic activities of the organism
broadly observed in acid producing and solvent producing phases
The prime challenge of industrialization of ABE fermentation
are briefly discussed in subsequent sections.
lies on low butanol tolerance capability of the organism as it de-
stroys the cell membrane. To overcome this challenge, strain
improvement programs were applied to engineer the high butanol
8.1. Acid producing pathways
producing strains [5,22,73].
Bacteria grows exponentially in the first phase of fermentation
(acidogenesis phase) along the formation of acids (mostly acetate 9.1. Strain improvement by mutation
and butyrate), leading to decrease of pH to 4.5 [96]. In this
phase, glycolysis pathway is active to produce pyruvate consum- Mutagenesis was executed randomly to metamorphose the
ing glucose, which is converted to Acetyl-CoA (Fig. 1). In this DNA sequence of genes responsible for ABE production. One suc-
organism, synthesis of lactate was observed, while inhibition of cessful endeavor of mutant SA-1 strain was developed by serial
hydrogenase was occurred by carbon monoxide or depletion of enrichment of diluted n-butanol from C. acetobutylicum ATCC 824
iron [22,96]. in 1983. The strain was found facultative to cultivate higher buta-
Acetyl-CoA is the prime precursor for synthesis of acetate, buty- nol titer and lower acetone enhancing butanol tolerance signifi-
rate, ethanol, butanol and acetone anaerobically (see Fig. 1). Ace- cantly (121% higher) over native strain (Table 4). Additional
tate and butyrate are produced in acid producing phase through advantages of mutant included higher carbohydrate utilization
two analogous steps from acetyl-CoA and butyryl-CoA respec- and higher a-amylase activity promoting potential utilization of
tively. Phosphate acetyltransferase and acetate kinase enzymes waste cellulosic materials as feedstocks [56]. Another novel mu-
are involved for acetate formation, while phosphate butyltransfer- tated strain (MEMS-7) was developed from C. acetobutylicum hav-
ase and butyrate kinase are active for butyrate synthesis. Four en- ing better potency in molasses. This strain was obtained by
zymes, namely thiolase, b-hydroxybutyryl-CoA dehydrogenase, treating the parent organism with N-methyl-N-nitro-N-nitroso-
crotonase, and butyryl-CoA dehydrogenase catalyze the synthesis guanidine and ethyl methane sulphonate and under UV exposure
of butyryl-CoA from acetyl-CoA [25,97]. [102]. MEMS-7 was recognized as effective mutant having 20%
M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012 2007

Fig. 1. Metabolic pathways in C. acetobutylicum for the acidogenic and solventogenic phase. Enzymes are indicated by numbers as follows: (1) enzymes including in glycolysis
process (2) pyruvate–ferredoxinoxidoreductase; (3) acetaldehyde dehydrogenase; (4) ethanol dehydrogenase; (5) phosphate acetyltransferase (phosphotransacettylase); (6)
acetate kinase; (7) thiolase (acetyl-CoA acetyltransferase); (8) 3-hydroxybutyryl-CoA dehydrogenase; (9) acetoacetyl-CoA: acetate/butyrate:CoA-transferase; (10)
acetoacetate decarboxylase; (11) crotonase; (12) butyryl-CoA dehydrogenase; (13) phosphate butyltransferase (phosphotransbutyrylase); (14) butyrate kinase; (15)
butyraldehyde dehydrogenase; (16) butanol dehydrogenase; (17) hydrogenase [5,25,73].

more butanol yield than the parent strain. The most worthwhile maintenance of anaerobic conditions. One successful mutant of
achievement was the development of the C. beijerinckii E. coli (which can grow aerobically) was recognized for the synthe-
BA101[52] strain having highest butanol producing capability sis 3-methyl-1-butanol (9.5 g l1). This strain was mutated through
(19–20 g l1) [35,52,103,104]. Another focus in strain improve- treatment of 4-aza-D,L-leucine, structural analogue to L-leucine
ment program was the elimination of tedious and expensive [105].
2008 M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012

Table 4
Various mutagenesis attempts in butanol producing organisms.

Parent organism Mutagens Mutants Remarks Refs.


C. acetobutylicum ATCC 824 Butanol SA-1 Enhanced butanol tolerance (121% higher than parent [56]
organism)
C. acetobutylicum PTCC-23 UV exposure, N-methyl-N-nitro-N-nitrosoguanidine MEMS- Yield enhanced by 20% [102]
and ethyl methane sulphonate 7
C. acetobutylicum ATCC 824 N-methyl-N0 -nitro-N-nitrosoguanidine together with BA101, Amylolytic activity increased by 82% (BA-101), 25% (BA- [103,104]
(or C. beijerinckii NCIMB selectiveenrichment on the glucoseanalog 2- BA-105 105), and increase to nearly 2% final concentration of
8052) deoxyglucose. butanol
E. coli JCL16 4-aza-D,L-leucine, a structural analogue to L-leucine AL-1, Yield reached 33% of theoretical maximum (0.33 g/g) [105]
AL-2

9.2. Strain improvement by genetic engineering unable to produce acetone and butanol due to destruction (called
as ‘Degeneration process’) of solvent producing genes (ctfA, ctfB,
Genetic modification in butanol producing bacteria was at- adc, and aad) after serial sub-culturing [108]. Plasmid pSOLI con-
tempted after identifying the butanol and acetone producing genes taining these four genes was inserted in degenerated mutants for
(Table 5). Prior to genetic engineering, metabolic engineering pro- expressing appropriate enzymes involved in butanol and acetone
vided the targeted gene for manipulation and it came in light that synthesis. Results demonstrated that production of butanol and
organisms were incapable to produce significant amounts of fer- acetone using engineered organisms was no longer due to the
mentative products [106]. One investigation was attempted to destruction of plasmid pSOLI. Similar results were observed in Sil-
eliminate the consequences of acetone in ABE fermentation. The lers’s study aiming over-expression of gene aad towards improving
TargeTron technology [107] was utilized to disrupt acetoacetate the butanol production in non-sporulating and non-solventogenic
decarboxylase gene (adc), which was responsible for acetone pro- strain C. acetobutylicum M5 using Plasmid pSOLI [99]. Therefore,
duction in hyper-butanol producing industrial stain EA 2018. As due to the genetic complexity of Clostridial strains and lack of
a result, butanol ratio enhanced from 70% to 80.05%, while acetone the suitable genetic tools, none of the attempts using Clostridial
production was reduced to 0.21 g l1 [97]. Recently, whole genome organism as a host was successful. This hurdle encouraged the
of two butanol producing microorganisms has been sequenced. In researchers in investigating other organisms as host for butanol
future, the sequencing of genomes of more hyper-butanol produc- producing genes.
ing bacteria will provide the scope of genetic engineering for Heterologous organisms (e.g. E. coli and some other organisms)
enhancement of butanol production [93]. as host were also employed to introduce butanol producing genes
Recombinant DNA technology (RDT) was another attractive ge- of Clostridia [92]. In this context, expression of butanol synthesis
netic engineering tool in facilitating the solvent producing ability genes of C. acetobutylicum ATCC 824 was investigated in E. coli.
of microbial strains. A few attempts of RDT were performed using During these investigations, genes thiL, hbd, crt, bcd-etfB-etfA,
C. acetobutylicum ATCC 824 as a host. Unfortunately, the strain was adhE1, and adhE2 were introduced into E. coli, which code

Table 5
Genes involved in genetic engineering of Clostridia for ABE fermentation.

Genes Encoding enzymes Products Mode of the Host Expression results Refs.
genetic
modification
adc Acetoacetate decaboxylase Acetone Deletion C. Yield has been improved [97]
acetobutylicum 57–70.8%
thiL, hbd, crt, bcd-etfB-etfA, Acetyl-CoA acetyltransferase, b-hydroxybutyryl- Butanol Insertion E. coli Expression of all genes were [100]
and adhe1 (adhe) CoA dehydrogenas, crotonase, butyryl-CoA detected except bcd-etfB-
dehydrogenas, butyraldehydedehydrogenas, and etfA
butanol dehydrogenase
thiL, hbd, crt, bcd-etfB-etfA, Acetyl-CoA acetyltransferase, b-hydroxybutyryl- Butanol Insertion E. coli, P. Pathway was constructed [101]
adhe1, and adhe2 CoA dehydrogenas, crotonase, butyryl-CoA putida, and B. successfully in three
dehydrogenas, butyraldehydedehydrogenas, and subtilis organism, butanol titer was
butanol dehydrogenase greatest in E. coli
thiL, hbd, crt, bcd-etfB-etfA, Acetyl-CoA acetyltransferase, b-hydroxybutyryl- Butanol Deletion and E. coli Expression of all genes were [109]
and adhe2 (deletion of CoA dehydrogenas, crotonase, butyryl-CoA insertion detected except bcd-etfB-
adhE, ldhA, frdBC, fnr, dehydrogenas, butyraldehydedehydrogenas, and etfA
pflB from E. coli) butanol dehydrogenase
ctfA, ctfB, adc, and aad Acetyl-CoA acetyltransferase, b-hydroxybutyryl- Butanol Insertion C. No longer expression of [108]
CoA dehydrogenas, crotonase, butyryl-CoA and acetobutylicum genes due to plasmid loss
dehydrogenas, butyraldehydedehydrogenas, acetone
butanol dehydrogenase, and acetoacetate
decaboxylase
aldA, ctfA, bdhA, bdhB, hbdA, Crotonase, butyryl-CoA dehydrogenase, electron- Butanol, Insertion E. coli Expression of all genes was [110]
crt A, bcd-etfB-etfA, adhA, transport protein subunits A and B, 3- acetone, detected except adhA
adh-1, adcA, and aad hydroxybutyryl-CoA dehydrogenase, alcohol and
dehydrogenase, CoA-transferase (subunits A and ethanol
B), aldehyde dehydrogenase, and acetoacetate
decaboxylase
hbd, crt, bcd, etfA, etfB, and 3-Hydroxybutyryl-CoA dehydrogenase, crotonase, Butanol Insertion S. cerevisiae Expression of all gens was [111]
adhe2 butyryl-CoA, electron-transport protein subunits A found properly
and B dehydrogenase
M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012 2009

Fig. 2. Schematic presentation of 1-butanol synthesis in engineered E. coli. The six enzymes and corresponding genes of C. acetobutylicum pathway (in brackets) are involved
in 1-butanol synthesis: (1) thiolase (acetyl-CoA acetyltransferase) (thl); (2) 3-hydroxybutyryl-CoA dehydrogenase (hbd); (3) crotonase (crt); (4) butyryl-CoA dehydrogenase
(bcd); (5) electron transfer flavoprotein (etf) (6) phosphate butyltransferase(phosphotransbutyrylase), butyrate kinase (adhE2) [92,100,109].

acetyl-CoA acetyltransferase (THL), b-hydroxybutyryl-CoA dehy- and genetic manipulations, none of the efforts were successful.
drogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyr- Simultaneously, recombinant DNA technology in non-Clostridial
yl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase organisms was unable to improve the yields over native Clostridial
(BYDH), and butanol dehydrogenase (BDH) enzymes (Fig. 2). Re- bacteria [5]. However, all the above studies provided crucial infor-
sults reported that expression of butanol producing gene was pos- mation for future research in the field of genetic engineering to-
sible in E. coli in aerobic environment. However, E. coli was able to wards producing hyper-butanol synthesizing strain. Focus should
tolerate up to 1.5% concentration of 1-butanol approximately sim- now be concentrated on developing more suitable genetic tools
ilar to native strain (C. acetobutylicum) [100,109]. Nielsen et al. for the better polycistronic expression in host microorganism.
[101] also examined the insertion of butanol producing genes in
E. coli, S. cerevisiae, P. putida, and B. subtilis. In this case, maximum
butanol titer of 5.8 g l1 and highest solvent-tolerant ability was 10. Economics of biobutanol fermentation
reported in E. coli and P. putida respectively. One novel butanol pro-
ducing Clostridium strain, C. saccharobutylicum, having high level of Fermentation processes are exothermic and hence, the products
hemicellulosic activity, was isolated and its genes, coding of cro- will have less energy than substrates. The theoretical mass and en-
tonase, butyryl-CoA dehydrogenase (bcd), electron-transport pro- ergy yield of ABE fermentation are 37% and 94% respectively, calcu-
tein subunits A and B, 3-hydroxybutyryl-CoA dehydrogenase, lated on the basis of energy combustions and products ratio
alcohol dehydrogenase, CoA-transferase, acetoacetate decarboxyl- obtained in fermentation [68]. During a study, it was reported that
ase, and aldehyde dehydrogenase, were cloned in E. coli. Except economic feasibility of ABE fermentation might not be possible on
bcd, expression of the all genes were detected in host organism meeting 100% yield. However, substrate costs, accounting to
[110]. In another study, Gene bcd was successfully expressed in approximately 60% of the total production costs, play a major role
S. cerevisiae with other butanol producing genes but insignificant in the economics of fermentation [8,114]. None of the starch and
improvement in butanol production was reported [111]. sugar containing crops can make fermentation economically feasi-
With modification, production of butanol and propanol, from ble on the basis of the current market needs and scenario. Conse-
glucose through keto-acid intermediates of amino acid pathways quently, cheaper agriculture wastes (lignocellulosic materials)
in E. coli, was also examined [112]. Applying appropriate genetic and other industrial wastes may be suitable for the economical
tools, the deletion of genes namely metA, tdh, ilvB, ilvl, and adhE ABE fermentation [67,115]. Ethanol distilleries have already pro-
and insertion of luc, tdcB, leuABCD, thrABC, ilvA, and kivd were per- ven to be more economically productive when utilizing low cost
formed. Reconstructed microorganism illustrated butanol and pro- raw materials. However, the economy of ABE fermentation is more
panol titer of approximately 2 g l1 with 1:1 ratio [113]. In spite of sensitive to substrate than yield; but it is estimated that butanol
several attempts in improving the industrial strains by mutation fermentation process will not be feasible while yield becomes less
2010 M. Kumar, K. Gayen / Applied Energy 88 (2011) 1999–2012

than 25% [68]. Strain improvement is effective technique in techniques. Additionally, efforts are needed in identifying better
improving the yield; however it has lower influence in economics butanol tolerance strains. Development of novel strains may be
than other factors (mainly cost of substrate and product recovery). more challenging as butanol has direct effect on cell membrane
Interestingly, capital costs of butanol fermentation was higher and ceasing molecular activity of the cell. In other words, butanol
production costs were lesser than petrochemical production of inhibits number of biochemical reactions, which are essential in
butanol [116]. Batch fermentation processes have been found less surviving the organism. However, development of process technol-
economical than continuous processes comprising extra burden of ogies with efficient butanol recovery techniques may be easily
sterilization equipment, piping and valves. However, the contami- achievable and crucial in economics. New recovery processes such
nation problem in continuous fermentation is a key challenge. as gas stripping, advanced membrane separation with super criti-
Product market is also a vital point in economics of butanol fer- cal extraction may contribute to the economic feasibility in future.
mentation. Still, the market is good enough in absorbing the prod- Multidirectional endeavors are constrained in achieving the eco-
ucts of ABE fermentation. It is hoped that after adapting biobutanol nomical usefulness of microbiological butanol synthesis, which
as a liquid fuel, demands will be huge. Byproducts including ace- will be competitive with petrochemical based butanol. In this
tone, ethanol, H2, and CO2 can also contribute substantially to eco- direction, co-culture fermentation processes with efficient technol-
nomics of butanol production [63]. Another cost-intensive process ogy may be a novelty for future.
in butanol production is recovery of products from diluted fermen-
tation broth. It should be noted that butanol production itself is a 12. Conclusion
cost-intensive process, and recovery technology of this fermenta-
tion is far away from feasibility. The following endeavors can mate- Biobutanol production can aid in extending the life of petro-
rialize in economical feasibility of ABE fermentation. Firstly, leum oil reserves and diminish environmental concerns. Problems
improvement in butanol tolerance of strain can enhance the fer- associated with feedstock availability and economic feasibility of
mentation time, productivity and economic feasibility of the pro- fermentation, are the challenges in current age. The adequate pos-
cess. Estimated fermentation time of 40–60 h was significant in sibility of using waste cellulosic materials as feedstocks ushers a
commercialization of biobutanol production. Secondly, reduction new direction in the production of biobutanol at industrial scale.
in product inhibition and recovery cost can be achieved by online Genetic engineering methods, for strain improvement, with inser-
selective removal of product [69]. Till date, the gas stripping pro- tion of butanol producing gene of Clostridia in high butanol toler-
cess was found the most effective process for online recovery of ant organisms leading to significant production of butanol is a
butanol [92]. Combination of fluidized bed reactor using immobi- great opportunity. Researchers are also attempting aerobic produc-
lized cells and removal of product by membrane was also sug- tion of biobutanol using genetically engineered organisms like
gested to ensure the economic sustainability of ABE fermentation E. coli, S. cerevisiae etc. However, the emerging scope of process
[114]. Therefore, it is concluded that raw materials being used development can be explored in developing novel continuous sys-
and product recovery are the most crucial factors for cost-effective tem, feedstock pretreatment, and efficient integrated butanol
butanol fermentation process. It is a considerable challenge in recovery to improve the productivity of biobutanol fermentation.
identifying cheaper substrates and developing suitable recovery Moreover, it is needed at commercial level by optimized utilization
processes for cost effective ABE fermentation. of waste materials and suitable organism in achieving the break-
through in economics. It will be the milestone to attract the atten-
tion of government, commercial, and research organizations for
11. Future prospectives further support in implementing the innovative fermentation and
extraction technology.
Utilization of foodstuff (like sugar and cereal grains) as raw
materials for energy production (for example, ethanol from maize, Acknowledgements
barley grain and sugar cane) enhances the price of food materials,
meat and milk. Therefore, these factors increase the risk of food Kalyan Gayen acknowledges financial support for the research
scarcity. Similarly, the drought and soil infertility increase the risk from Department of Science and Technology, India. The authors
of human and animal nutrition. To reduce these risks, application are also thankful to Sriram Kanvah Gundimeda and Sameer Dalvi
of food operating certificate should be initiated. For this purpose, for their assistance in improving the manuscript.
a food using protocol (similar to Kyoto Protocol on green house
emissions) must be organized internationally with broader partic- References
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