Accepted Manuscript

Title: Fermentative production of butanol: Perspectives on

synthetic biology

Authors: Sonil Nanda, Dasantila Golemi-Kotra, John C.

McDermott, Ajay K. Dalai, Iskender Gokalp, Janusz A.
Kozinski

PII: S1871-6784(16)32429-3
DOI: http://dx.doi.org/doi:10.1016/j.nbt.2017.02.006
Reference: NBT 959

To appear in:

Received date: 23-9-2016

Revised date: 20-2-2017
Accepted date: 22-2-2017

Please cite this article as: Nanda, Sonil, Golemi-Kotra, Dasantila, McDermott,
John C., Dalai, Ajay K., Gokalp, Iskender, Kozinski, Janusz A., Fermentative
production of butanol: Perspectives on synthetic biology.New Biotechnology
http://dx.doi.org/10.1016/j.nbt.2017.02.006

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 Fermentative production of butanol: Perspectives on synthetic biology

Sonil Nanda1, Dasantila Golemi-Kotra2, John C. McDermott2, Ajay K. Dalai3, Iskender Gkalp4,
Janusz A. Kozinski1,*

1
Department of Earth and Space Science and Engineering, York University, Ontario, Canada
2
Department of Biology, York University, Ontario, Canada
3
Department of Chemical and Biological Engineering, University of Saskatchewan,
Saskatchewan, Canada
4
Institut de Combustion, Arothermique, Ractivit et Environnement (ICARE), Centre National
de la Recherche Scientifique (CNRS), Orlans, France

* Corresponding author:
Dr. Janusz A. Kozinski
Founding Dean and Professor
Lassonde School of Engineering
York University, Toronto, Ontario, M3J 1P3, Canada
E-mail: janusz.kozinski@lassonde.yorku.ca
Tel: (416) 736-5484, Fax: (416) 736-5360

37
Graphical abstract

38
Highlights

1. Butanol is an advanced drop-in biofuel for vehicles without engine modification.

2. Butanol is produced by acetone-butanol-ethanol fermentation using Clostridium spp.

3. Acetone-butanol-ethanol fermentation consists of acidogenic and solventogenic phases.

4. Butanol fermentation is hampered by phage contamination, solvent stress and toxicity.

5. Genetic and metabolic engineering can generate hyper-butanol producing bacteria.

39
Abstract:
Apprehensions relating to global warming, climate change, pollution, rising energy demands as
well as fluctuating crude oil prices and supply are leading to a shift in global interest to find
suitable alternatives to fossil fuels. This review aims to highlight the many different facets of
biobutanol as an advanced next-generation transportation biofuel. Butanol has fuel properties
almost on a par with gasoline, such as high energy content, low vapour pressure, non-
hygroscopic nature, less volatility, flexible fuel blends and high octane number. The paper
reviews some recent advances in acetone-butanol-ethanol fermentation with special emphasis on
the primary challenges encountered in butanol fermentation, including butanol toxicity, solvent
intolerance and bacteriophage contamination. The mechanisms for butanol recovery techniques
have been covered along with their benefits and limitations. A comprehensive discussion of
genetic and metabolic engineering of butanol-producing microorganisms is made for the
prospective development of industrially-relevant strains that can overcome the technical
challenges involved in efficient butanol production.

Keywords: Acetone-butanol-ethanol fermentation; Clostridium; butanol; butanol toxicity;

genetic engineering; metabolic engineering

40
Introduction
Awareness concerning the production of renewable transportation fuels has been
growing rapidly. Waste biomasses that have tremendous potential to supplement the
production of alternative biofuels include lignocellulosic feedstocks (e.g. agricultural and
forestry residues), algae, municipal solid wastes, sewage sludge, industrial effluents and
cattle manure [1-3]. Biobutanol, especially n-butanol (C4H9OH) is a four carbon primary
alcohol that is gaining attention as a gasoline substitute. Butanol is often considered as a
next-generation fuel over ethanol (C2H5OH) that can be derived from lignocellulosic
feedstocks through bacterial fermentation. The many advantages of biobutanol are illustrated
in Fig. 1. In contrast to ethanol, which is usually blended up to 85% with gasoline, butanol
can be used in its pure form or blended at higher concentrations [4]. Biobutanol is often
referred to as a drop-in fuel that can be used in existing motor engines and vehicular
infrastructures without mechanical tailoring.
Butanol is considered to have superior fuel properties to ethanol that make it a
better replacement for gasoline. Table 1 summarizes some significant fuel properties of
butanol, ethanol and gasoline. The energy value of butanol (29.2 MJ/L) is 30% greater than
that of ethanol (21.2 MJ/L) and 10% less than that of gasoline (32.5 MJ/L). On the other
hand, the octane ratings (research octane number, RON and motor octane number, MON) of
butanol are much closer to gasoline, indicating similar fuel properties. The air-to-fuel ratio of
butanol (11.2) is much closer to that of gasoline (14.7) [7]. Ethanol is highly oxygenated
(35% oxygen content) compared to butanol (22% oxygen content) [8]. Compared to ethanol,
the restorative qualities of butanol as a fuel are non-sensitivity to water, less corrosiveness,
lower volatility, less flammability and reduced toxicity to physical exposure. Moreover,
butanols low vapor pressure enables its use in existing gasoline transport pipelines and
filling stations without considerable retrofitting. Furthermore, the rising crude oil prices and
aggregating concerns over pollution and global warming have renewed a common interest in
the biological production of butanol as an alternative fuel.
Approximately 10-12 billion pounds of butanol is generated every year through
petrochemical routes [9], which currently accounts for a nearly $7-8.4 billion market [5].
Moreover, butanol is expected to achieve about 20% of the global gasoline and diesel
requirements in the near future. The current worldwide demand for butanol exceeds 1.2

41
 billion gallons per annum, which is valued at over $6 billion [10]. The market for butanol is
expanding by 3% per year [5], which is predicted to reach $9.9 billion by 2020 [11].
Recently, GranBio (Brazilian Biotech Company in Alagoas) and Rhodia (Solvay
International Chemical Group in Belgium) announced their partnership to produce biobutanol
from sugarcane bagasse in an impending refinery in Brazil [12]. The biorefinery is envisaged
to produce 100 kilotons of solvents every year. Butamax Advanced Biofuels, a joint
endeavor of British Petroleum (London, UK) and DuPont (Delaware, USA), is developing
sustainable technologies to make butanol an inexpensive, high-value and drop-in biofuel for
the future transportation market. Butamax technology is intended for converting
polysaccharides from different feedstocks including corn and sugarcane into butanol using
some existing biorefining facilities [13]. Furthermore, some global enterprises that have
lately announced their endeavors in commercializing butanol as a next-generation biofuel
include Butamax (a joint venture of British Petroleum and DuPont), GreenBiologics
(Oxon, UK), Cobalt Biofuels (California, USA), Tetravitae Bioscience Inc. (Illinois, USA),
Gevo (Colorado, USA), METabolic EXplorer (Clermont-Ferrand, France), Butalco (Frigen,
Switzerland) and Cathay Industrial Biotech (Shanghai, China).
Although promising, the butanol biorefining process suffers from several
drawbacks such as solvent toxicity towards microorganisms, lesser yields, expensive
recovery technologies, and metabolic intolerance of butanol-producing bacteria. This review
aims to highlight the advantages of butanol as a drop-in biofuel, its fermentative production
and associated challenges. Finally, some recent approaches to advance butanol production
through manipulation at the genetic, enzymatic and metabolic levels are discussed.

Acetone-butanol-ethanol fermentation
The steps involved in butanol fermentation and recovery are represented in Figure
2. While the upstream processing includes the feedstock treatment, the downstream
processing is related to the butanol recovery and purification. The starch-based biomass can
readily undergo acetone-butanol-ethanol (ABE) fermentation due to the amylolytic activity
of Clostridia whereas lignocellulosic feedstocks are pretreated using dilute acids and
hydrolytic enzymes as discussed above. Clostridium secretes many enzymes to facilitate the
breakdown of carbohydrate (i.e. polysaccharides) into fermentable sugars (i.e.

42
monosaccharides). The enzymes secreted by Clostridium to degrade carbohydrates include -
amylase, -amylase, glucoamylase, -glucosidase, -glucosidase, pullulanase and
amylopullulanase [14]. The monosaccharide sugars are transported into the bacterial cell by
specific membrane-bound transport systems that are further metabolized through glycolysis
or the pentose phosphate pathway. After butanol is produced, its partial solubility in water
creates impediments in its downstream processing and extraction from the fermentation
broth. Some butanol recovery techniques include adsorption, distillation, gas stripping,
liquid-liquid extraction, perstraction, pervaporation and supercritical fluid extraction. These
butanol recovering techniques and their associated challenges are discussed in the subsequent
sections.
The biological production of butanol is achieved through acetone-butanol-ethanol
or ABE fermentation which is one of the oldest fermentation processes. Butanol production
by ABE fermentation is mostly carried out using Clostridium spp., particularly C.
acetobutylicum and C. beijerinckii that can convert broad-ranging carbon sources (e.g.
arabinose, cellobiose, galactose, glucose, mannose and xylose) to butanol, acetone and
ethanol [15]. Clostridium spp. known to carry out ABE fermentation include C.
acetobutylicum, C. beijerinckii, C. aurantibutyricum, C. butylicum, C. saccharobutylicum, C.
saccharoperbutylacetonicum, etc.
Table 2 lists a few Clostridium spp. used in ABE fermentation with a wide variety of organic
substrates. Some traditional feedstocks for butanol production include corn, millet, molasses,
potatoes, rice, wheat, whey permeate and tapioca. Since these substrates are starch-based, the
intense amylolytic activity of Clostridium restricts the requirement of biomass pretreatment
and hydrolysis [38]. The high cost of starch-based substrates and concerns of food versus
fuel have diverted the attention towards using lignocellulosic feedstocks for butanol
production. Some lignocellulosic biomass used in ABE fermentation are barley straw, corn
cobs, corn fiber, corn stover, hemp waste, pinewood, rice straw, sunflower shells,
switchgrass, timothy grass and wheat straw [24, 39, 40].
The ABE fermentation in a typical batch system is initiated with carbohydrate
substrates (usually glucose or equivalent sugars). The fermentation medium containing the
substrate and nutrients are prepared under sterile conditions followed by creation of an
anaerobic environment inside the reactor using N2 or CO2. The medium is then inoculated

43
with a suitable butanol-producing Clostridium culture followed by incubation at 35C. The
fermentation typically lasts for 36-72 hours producing total ABE up to 20-25 g/L [39]. Cell
growth inhibition can be observed at butanol concentrations of 5-10 g/L leading to the
accumulation of unutilized residual sugars in the medium.
Clostridium acetobutylicum is the most widely studied butanol-producing
bacterium. The metabolic pathways involved in ABE fermentation are illustrated in Figure 3.
The ABE fermentation performed by anaerobic Clostridium is biphasic involving
acidogenesis and solventogenesis. While the acidogenic phase implicates the generation of
acids (e.g. acetic acid and butyric acid), the solventogenic phase encompasses the production
of acetone, butanol and ethanol. The ABE biosynthesis involves key enzymes such as
acetoacetyl-CoA thiolase, aldehyde/alcohol dehydrogenase, butyryl-CoA dehydrogenase, 3-
hydroxybutyryl-CoA dehydrogenase and crotonase. These enzymes, respectively encoded by
thl, adhE/adhE2, bcd, hbd and crt genes, are required to complete the conversion of acetyl-
CoA to butanol [41]. Conversion of acetyl-CoA to butyryl-CoA demonstrates enhanced
thermodynamic stability; however, the condensation of acetyl-CoA to acetoacetyl-CoA is the
rate-limiting step [42].
Clostridium produces butanol along with acetic acid, butyric acid, acetone and
ethanol as the liquid products as well as CO2 and H2 as the gaseous products. The production
of H2 is considered as a waste of electrons (or energy) that could otherwise be utilized to
prime the solventogenic phase for generating acetone, butanol and ethanol. Purging CO into
the fermentation medium is known to shrink H2 formation by retarding hydrogenase activity,
and to escalate the electron flux from the reduced ferredoxin (FdH2) to nicotinamide-adenine
dinucleotide (NAD+) in the electron transport chain [43].
The acidogenic phase typically occurs in the log (exponential) phase of bacterial
growth. However, Clostridium undergoes a major shift in its metabolism during the end of
log phase [42]. The exponential growth stage of Clostridium is favorable for the acidogenic
phase because the acid formation is complemented by the synthesis of adenosine triphosphate
(ATP) required for cell growth. As Clostridium is an obligate anaerobe, the acidogenic phase
plays a vital role in its energy metabolism. Due to the formation of acetic acid and butyric
acid, the pH of the fermentation medium falls, which inhibits the bacterial growth [44]. As a
result, the bacterium slows down acid production and utilizes the excreted acetic acid and

44
butyric acid by converting them to acetone and butanol. During the acidogenic phase, more
butyrate is generated than acetate because the former has a better balance of the redox
equilibrium [42]. Nicotinamide-adenine dinucleotide (NADH) formed through glycolysis is
only consumed in butyrate formation rather than in acetate formation. This is because more
butanol is produced than ethanol in ABE fermentation indicating more conversion of butyrate
than acetate in the solventogenetic phase. Additionally, almost twice as much butyrate is
produced compared to acetate [4]. Butanol is a chief product of ABE fermentation as the
typical proportions of acetone, butanol and ethanol are 3:6:1 [45].
Clostridium metabolism involves the phase shift from acidogenesis to
solventogenesis followed by sporulation (70-80% of the viable bacterial cells) and cessation
of growth [43]. At around pH 5.5, the solvent (acetone, butanol and ethanol) production starts
while the acid concentration remains nearly constant or slightly lower due to its dissociation.
The non-dissociated butyric acid usually triggers the phase shift from acidogenesis to
solventogenesis. Solventogenic phase can also be induced even at pH 6 or higher at a greater
butyric acid concentration [43]. Acetic acid and butyric acid act as inducers for the respective
enzymes such as acetate kinase and butyrate kinase necessary for the butanol biosynthesis
[46]. The intracellular metabolic precursors, namely butyryl-CoA and butyrylphosphate also
contribute to the phase shift [47].
The pH level of the fermentation medium is crucial to biphasic ABE
fermentation. The formation of acetic acid and butyric acid lowers the pH in the acidogenic
phase. The solventogenic phase starts after the pH level reaches a steady critical point after
which the acids are re-assimilated thereby generating acetone and butanol [5]. Although low
pH is preferable for solvent production, a pH below 4.5 results in enough acid formation,
thereby lowering the duration and effectiveness of the solventogenic phase. Increasing the
buffering capacity of the fermentation medium is a potential solution for enhancing the
Clostridial growth, carbohydrate utilization and butanol production [48].
Considerable amounts of acetic and butyric acid remain non-dissociated, which
start diffusing into the cytoplasm [49]. An acid crash occurs as the concentrations of non-
dissociated acids exceed a threshold value (57-60 mmol/L) [42]. This causes the transcellular
proton gradient to collapse causing cell death. Under normal conditions for cell survival, the
proton gradient is higher outside the cell than inside. In addition to the negative effects that

45
proton gradient has on the cells, the produced butanol also has adverse effects on Clostridium
spp. [50]. The accumulated butanol damages the cell membranes and disintegrates the
membrane proteins [51]. Hence, the bacterium prevents it from utilizing all of the produced
acids and remaining sugar substrates to acetone, butanol and ethanol. As a response to higher
butanol concentration, Clostridium synthesizes endospores for survival [49]. However, this
does not salvage their ability to produce acetone and butanol from acetic acid and butyric
acid, respectively. This is a major disadvantage in the biological butanol production process
which results in very low final product yields and high residual substrate levels.
During the anaerobic ABE fermentation, which lasts for 36-72 hours, total ABE
up to 20-25 g/L can be obtained [39]. Butanol inhibition can be detected at a concentration
range of 5-10 g/L as Clostridia can rarely withstand more than 2% butanol [39, 52]. Recently,
the advance in synthetic biology has created metabolic engineering approaches for
developing hyper butanol-producing strains such as C. beijerinckii P260 [53] and C.
beijerinckii BA101 [14]. Moreover, a process engineering approach such as simultaneous
removal of produced butanol from the fermentation medium through solvent recovery
techniques (e.g. membrane separation and gas stripping) could prevent product inhibition [5].
However, the amendment of such technologies contributes to the overall process economics
[54]. Typically, in a batch fermentation, the final concentration of total solvents (i.e. acetone,
butanol and ethanol) can range from 12 to 20 g/L [5]. Although the yield can be enhanced
using fed-batch and continuous fermentation, they do not overcome the butanol toxicity
problems. As a solution to reduce butanol toxicity, fed-batch fermentations systems have
been annexed with an in-situ recovery process such as gas stripping [55].

Challenges in butanol production

Butanol toxicity
There are several challenges associated with butanol at upstream (i.e. biomass
selection and pretreatment), downstream (i.e. solvent recovery) and production (i.e.
fermentation) stages. The decline in butanol formation as a result of its toxicity towards
microorganisms is a major hurdle for industrial scale production as Clostridium spp. rarely
tolerate more than 2% butanol. Butanol toxicity is a limiting factor for the final butanol yield
in ABE fermentation which is more severe than acetone and ethanol. In batch fermentation,

46
Saccharomyces cerevisiae is completely inhibited by ethanol at a concentration of 90 g/L,
although inhibition can start at 24 g/L ethanol [56]. High concentrations of ethanol can alter
cell membrane composition, structure and functionality, thereby inhibiting cell division and
metabolic activity [52]. However, yeast demonstrates ethanol tolerance by cell organelles
(e.g. mitochondria and vacuoles) and cellular metabolism (e.g. sugar transport systems). In
the conventional ABE fermentation, a butanol level up to 12-13 g/L is usually reported as
the upper limit tolerated by wild-type Clostridium strains [43, 57]. A maximum butanol
concentration of 19.6 g/L is reportedly produced from genetically modified C. beijerinckii
BA101 [58].
The toxicity imparted by butanol is related to its hydrophobic nature and other
long-chain aliphatic alcohols that disrupt the phospholipid component of bacterial cell
membranes. High butanol levels induce an adverse alteration in the cell membrane of C.
acetobutylicum, especially in the phospholipid and fatty acid composition. Short-chain
aliphatic alcohols e.g. ethanol can decrease membrane fluidity while long-chain aliphatic
alcohols e.g. butanol can increase membrane fluidity [45]. Butanol enters the cell membrane
of Clostridium and modifies its structural framework resulting in membrane fluidity. Nearly
a 20-30% rise in membrane fluidity has been observed for C. acetobutylicum in response to
1% butanol exposure [59]. During the mid-exponential growth phase, the presence of 1%
butanol results in an increase in the levels of saturated acyl chains and a decrease in the
concentration of unsaturated chains. The accumulation of saturated fatty acids in the cell
membrane is a result of the physiological response of Clostridium to counter the effect of
increased membrane fluidity.
Enhancement in butanol tolerance by Clostridium could result in higher yields of
butanol. Studies have shown that by externally supplementing saturated fatty acids to the
growth medium, the indigenous saturated fatty acid content in the bacterial cell membrane
can be enriched, thus increasing cell growth, ATPase activity and butanol tolerance by up to
two-fold [45]. Additionally, membrane fluidity is found to increase with rising temperature
suggesting that a decrease in temperature during late-lag phase (i.e. butanol-producing
phase) could promote butanol tolerance. Butanol toxicity is triggered by the generation of
cell-free autolysin during solventogenesis causing autolytic degradation of C.
acetobutylicum P262 [45]. A pleiotropic autolysis-deficient mutant (lyt-1) of C.

47
acetobutylicum P262 was isolated by Allcock et al. [60] which not only produced less
autolysin than its parent strain but also showed resistance to it. The vegetative cells of the
mutant lyt-1 demonstrated enhanced butanol tolerance and produced higher concentrations
of butanol (i.e. 14.2 g/L compared to 13.3 g/L).
Other impacts of butanol on the bacterial cells as a result of membrane fluidity are
destabilization of cell membranes and interruption of many physiochemical features such as
membrane permeability, glucose uptake, solute transport, intracellular ATP level, proton
motive force (or chemiosmosis), as well as conformation and activity of intrinsic membrane
proteins [61]. Apart from butanol toxicity, other factors inhibiting ABE fermentation include
substrate type, salt and nutrient concentration, low water activity, macromolecular
accumulation, oxygen diffusion, presence of dead cells and bacteriophage contamination
[45, 55, 62].

Bacteriophage contamination
Bacteriophage infection during ABE fermentation is a common technical
challenge, but there are very few reports documented in the literature on this issue.
Lysogenic phages belonging to Siphoviridae and Podoviridae have been reported to infect
Clostridium madisonii and C. beijerinckii P260, respectively leading to slower growth and
reduced solvent production [62]. The symptoms of bacteriophage infection include: (i)
lethargic fermentation rate, (ii) longer fermentation time, (iii) change in bacterial cell
population and morphology, (iv) reduced butanol yields, (v) lower evolution of fermentation
gases (e.g. H2 and CO2), (vi) high organic acid levels (acetic acid and butyric acid), and (vii)
high levels of unutilized sugars in the medium. An initial assumption of bacteriophage
infection can be made by visual observation of the fermentation medium, which turns
abnormally dark in color, unlike normal fermentation where the substrate color is lighter.
Bacteriophage contamination causes a decrease in Clostridial cell population, loss of
motility and appearance of etched and elongated cells. Degenerative cellular changes and
premature cell lysis appear to be common symptoms of Clostridium infected with
bacteriophage [45].
The difficulties can mount up in the diagnosis of ABE fermentation with low
levels of bacteriophage contamination. Contamination by virulent bacteriophages with a

48
short latent period and large burst size can lead to very rapid and severe infection. Viral
burst size is related to lytic infections and refers to the amount of virus released from the
infected bacterial cells at the burst time. Less obvious and prolonged symptoms are caused
by lysogenic and pseudo-lysogenic phages that can be extremely difficult to eliminate from
the infected fermentation medium. The infections occurring during the initial fermentation
stages are likely to become pervasive and difficult to control whereas those occurring at later
stages of ABE fermentation can have relatively fewer invasive effects [62]. The
bacteriophage infection can be checked through chemical treatment methods such as: (i)
addition of chelating agents to remove divalent cations essential for phage infection, and (ii)
use of non-ionic detergents and antibiotics.

Limitations in butanol recovery

The partial miscibility of butanol in water is a progressive quality in preventing
engine corrosion and use in higher concentrations. However, this feature also leads to
several technical issues during its recovery from the fermentation broth. The solubility of
biobutanol (n-butanol or 1-butanol) in water is 7.7% (77 g/L) at 20C, 7.3% (73 g/L) at 25C
and 7.1% (71 g/L) at 30C. While isobutanol and 1-butanol are sparingly water-soluble, sec-
butanol (2-butanol) is largely water-soluble and tert-butanol is completely miscible in water.
The hydroxyl group in alcohols makes them polar and tends to enhance aqueous solubility.
As the alcohols carbon chain increases, its solubility in water decreases. Methanol, ethanol
and propanol are completely miscible in water, but butanol is moderately soluble because of
the opposing solubility trend rendered by its longer carbon chain.
Butanol can be recovered from the fermentation broth through several chemi-
mechanical technologies such as adsorption, distillation, gas stripping, liquid-liquid
extraction, perstraction, pervaporation and supercritical fluid extraction. In adsorption,
butanol is conveyed from the fermentation broth to a solid adsorbent (e.g. packed column)
and subsequently desorbed using a displacer or by heat treatment to attain a concentrated
solution. The selectivity of adsorption is reliant on the relative binding of the adsorbent with
butanol versus water [63]. Amberlite (e.g. XAD2, XAD4, XAD7, XAD8 and XAD16),
bonopore, polyvinyl pyridine, resins and silicalite have been scrupulously tested for butanol
separation.

49
 During butanol recovery by adsorption, there are chances that ethanol or acetone
could compete with butanol for adsorption sites on the solid adsorbent material [64]. In the
same way, acetic acid and butyric acid, as well as nutrients from the fermentation broth may
also compete and block the adsorption sites thereby lowering butanol recovery efficiency.
Silicalite is reported to concentrate butanol up to 790-810 g/L from dilute solutions [65].
Adsorption of ~40 mg water per g of silicalite-1 at room temperature has been reported in
the absence of alcohol [66]. Upon addition of alcohol into the water, it is favorably adsorbed
causing a reduction in water adsorption. The regeneration of adsorbent also needs more
research to make the recovery process cost-effective and efficient.
Distillation is a traditional solvent recovery method that involves an azeotrope to
separate alcohol from a dilute solution. Distillation has high energy requirements due to
butanols boiling point (i.e. 117.7C) being greater than that of water. Although a mature
technology, distillation struggles in being commercially viable because of its greater energy
requirement and high cost due to lower butanol concentration in the fermentation medium
[63]. Typically, acetone, butanol and ethanol together comprise about 18-33 g/L in the
fermentation broth of which butanol accounts for only 13-18 g/L [67]. The energy savings in
distillation can be achieved if butanol yield is augmented from 10 to 40 g/L [68].
Gas stripping is a promising technology that selectively removes alcohols from the
vapor phase through condensation in a cold trap or separation using a molecular sieve. In
this process, a gas or mixture of gases (e.g. N2, CO2 and H2) is purged into the fermentation
medium using a rotating shaft and the volatiles (including butanol) are condensed and
recovered [55]. The gases create bubbles upon purging in the broth that capture the solvents
including butanol. The gas-solvent vapor mixture passes through a condenser before being
collected in the liquid state. After condensation, the gases can be recycled back into the
fermenter to recover more alcohol in a continuous mode. Since CO2 and H2 are released
during ABE fermentation, they do not interfere with the Clostridial growth cycle.
Additionally, the inert gas N2 creates an anaerobic environment optimal for ABE
fermentation.
Owing to cell toxicity and low final titer levels of butanol, the condensate obtained
after gas stripping usually contains less than 70 g/L of butanol, which is much lower than its
critical concentration for phase separation [69]. With a low butanol selectivity of 4-22, gas

50
stripping often removes considerable amounts of water necessitating high energy input for
its vaporization and condensation [63]. The energy requirement by gas stripping and
consequent concentration techniques such as distillation or steam evaporation is estimated to
be around 14-31 MJ/kg [69], which is quite high to make the downstream separation process
energy-intensive. The energy requirement is directly influenced by butanol concentration
and contributes pointedly to the operating cost of the recovery system [63, 70].
Liquid-liquid extraction relies on the differences between the distribution
coefficients of the solvents and involves mixing an extraction solvent with the fermentation
medium. Acetone, butanol and ethanol are recovered using an extracting solvent and further
separated through distillation or back-extraction by another extracting solvent [67]. Some
extractants that have been studied for butanol recovery include aromatic hydrocarbons,
alkanes, esters, ketones [71], corn oil [72], crude palm oil [73], dibutyl phthalate [74],
decanol and oleyl alcohol [75], hexanol [76], polyoxyalkylene ethers [77], vegetable oil [78]
and biodiesel [79]. In addition, a few imidazolium-based ionic liquids (e.g. [Tf2N] and
[Omim][Tf2N]) have been studied for butanol recovery [80].
Some common drawbacks with liquid-liquid extraction include cell toxicity by the
extractant and subsequent emulsion formation [64]. Using a membrane to separate the
fermentation broth from the extractant could help overcome these issues. This could also
provide larger surface area for the transfer of butanol from one immiscible phase to another.
Certain considerations for an ideal extraction solvent are: (i) cost-effectiveness, (ii) non-
toxicity to Clostridia, (iii) immiscibility in the fermentation broth, (iv) no emulsion
formation, (v) high partition coefficient for fermentation products, and (vi) thermal stability
during sterilization and product recovery.
Perstraction is an improved version of liquid-liquid extraction. It uses butanol-
permeable membranes between the fermentation broth and the extraction solvents to
overcome problems such as: (i) inactivation of bacterial cells at the two-phase interface, (ii)
mass loss of extractant by incomplete phase separation, (iii) extraction of other intermediates
along with butanol, and (iv) emulsion formation [81, 82]. The diffusion of fermentation
products through the permeable membrane in perstraction is a rate-controlling process
because of the differences in vapor pressure of the diffusing components at feed phase and

51
extractant phase. It is highly desirable that the diffusion rates of fermentation products
exceed their production rate in the fermenter to make the process energy efficient [83].
Pervaporation is a butanol recovery technique that combines membrane
permeation with evaporation. While one side of the membrane is in contact with the
fermentation medium, the other side is connected with vacuum or gas purge to create
permeate vapor stream. Butanol diffuses through the membrane, desorbs as permeate vapor
under vacuum or low pressure and finally condenses at lower temperatures [64]. Some
hydrophobic polymeric membranes investigated in pervaporation of butanol include
polysiloxane, polyurethane, polyether block amide, poly[1-(trimethylsilyl)-1-propyne],
polydimethylsiloxane (PDMS), silicalite-filled PDMS, zeolite-filled PDMS, polypropylene,
polytetrafluoroethylene (PTFE), polyvinylidene fluoride, silicone, silicalite-silicone, zeolite
membrane Ge-ZSM-5 and oleyl alcohol liquid membrane [63, 64, 84].
Pervaporation usually requires relatively higher temperatures to subordinate the
operating cost [66]. The membrane area required for butanol separation decreases as
temperature increases inferring that the optimal temperature for pervaporation could be
inhibitory to bacterial cells, enzymes, proteins and essential metabolites. The energy
efficiency of pervaporation can be increased with an improved alcohol-water separation
factor and reuse of the process heat. A higher membrane thickness in pervaporation tends to
decrease the product transport flux [63]. The operational costs can be lowered by increasing
the membrane flux to reduce the area, which consequently minimizes the membrane/module
cost per unit area.
Supercritical fluids are new candidate extractants for liquid-liquid extraction. The
most commonly explored supercritical fluids include supercritical water and supercritical
carbon dioxide. Water transforms into supercritical water when its critical temperature and
critical pressure are higher than 374C and 22 MPa, respectively [85, 86]. Similarly,
supercritical-CO2 is formed when the critical temperature and critical pressure of CO2 are
above 30.95C and 7.38 MPa, respectively [87]. The recovery of butanol using supercritical
fluids is a function of the partition coefficient of butanol and water as well as the solubility
of organic solvents in the aqueous phase [63]. The extracted compound is recovered by
depressurizing the system and reducing the temperature thereby allowing the supercritical
fluid to return to gaseous state for recycling. There is a lack of accessible information on the

52
use of supercritical fluids for the downstream recovery of butanol; therefore future research
could only address their potential use and commercial applications.

Genetic and metabolic engineering in butanol biosynthesis

The recent approach towards the genetic and metabolic engineering of butanol-
producing microorganisms has been underway to overcome several limitations such as low
butanol titer, yield and productivity. This could remarkably help to improve the performance
of solventogenic Clostridium and make it competitive for ABE fermentation on a
commercial scale. Recombinant DNA technology [14], transcriptomics [88], proteomics
[89] and mutagenesis [90] have been used to tailor the targeted metabolic pathways in
butanol-producing Clostridia. Metabolic engineering has been performed on both natural
butanol-producing Clostridium and potential microorganisms other than Clostridium capable
of fabricating butanol synthesis pathways. The metabolic studies of Clostridia have been
targeted towards [91]: (i) growth at high cell density, (ii) persistent cell viability, (iii) no
spore formation, (iv) high butanol selectivity, (v) tolerance to aerobic conditions, (vi) high
tolerance to acetone, butanol and ethanol, as well as (vii) direct utilization of lignocellulosic
biomass. Table 3 summarizes a few ideal traits of genetically engineered microorganisms
and their possible roles in improving butanol fermentation.
Inducing mutagenesis in Clostridium and other microorganisms and isolating the
ideal mutants is a key factor in improving and shaping an industrial scale butanol refinery.
Ethyl methanesulfonate, metronidazole, N-methyl-N-nitro-N-nitrosoguanidine, nalidixic
acid, hydrogen peroxide and UV irradiation are some of the mutagens studied to induce
mutations in Clostridia [14]. Through chemical mutagenesis i.e. treatment with N-methyl-N-
nitro-N-nitrosoguanidine, the hyper-amylolytic and hyper-solventogenic C. beijerinckii
BA101 was produced from C. beijerinckii NCIMB 8052 (formerly C. acetobutylicum) [67].
This mutant C. beijerinckii BA101 can produce and tolerate up to 23 g/L of butanol and 33
g/L of total ABE in a batch fermentation under optimized conditions [54]. This is a major
yield improvement of 69% over the parental strain C. beijerinckii NCIMB 8052 which
produced only 15-20 g/L of total solvents. The mutant strain should be developed to tolerate
and produce higher concentrations of butanol for making the overall biorefining process

53
economical. The mutant C. beijerinckii BA101 also grows well in simple and inexpensive
media which is highly desirable at industrial scales.
Antisense RNA technology has also been implemented in producing mutants for
improved ABE fermentation. Antisense RNA (asRNA) is a single strand of RNA that is
complementary to a transcribed messenger RNA (mRNA) strand. Introduction of antisense
RNA into a cell can inhibit translation of a complementary mRNA through base pairing and
hindering the translation machinery, thereby making the effects stoichiometric. In C.
acetobutylicum 824, antisense RNA was used to downregulate the enzymes (i.e. acetoacetate
decarboxylase [AADC] and coenzyme A-transferase [CoAT]) involved in the acetone
formation pathway [92]. Although the mutant showed a downregulation of CoAT gene
leading to suppressed acetone yield, there was no rerouting of carbon flux towards butanol
production. Furthermore, butanol yields were radically lowered in the mutants. The plasmid
pAADB1 was used to overexpress alcohol-aldehyde dehydrogenase (aad) gene and
downregulate CoAT using antisense RNA against ctfB in C. acetobutylicum ATCC 824 to
escalate butanol-to-acetone ratio [93]. ctfB is the second CoAT gene on the polycistronic
aad-ctfA-ctfB operon. While butanol yields significantly decreased in the mutant, ethanol
concentration increased by 23 times (~200 mM) over that of the control, making it the
highest ever ethanol production by C. acetobutylicum.
Attempts have been made to avoid the formation of acetone to develop a non-
sporulating C. acetobutylicum M5 strain with increased butanol productivity [94]. From an
industrial perspective, a non-spore forming strain is most desirable for butanol production
because Clostridium produces solvents only during a short period of sporulation regime with
characteristic cigar-shaped cell structures [45]. As the remaining mixed-cell population does
not contribute to solvent production, the specific cell productivity is restricted. The
megaplasmid pSOL1 has the sol operon genes for alcohol-aldehyde dehydrogenase (aad),
acetoacetate decarboxylase (adc) and CoA transferase (ctfA and ctfB). Sillers et al. [94] used
the aad gene to complement C. acetobutylicum M5 which had lost the megaplasmid. The
strategy successfully restored butanol biosynthesis, but its titer level (~9 g/L) was moderate
with high concentrations of organic acids and no acetone production. The genome of C.
acetobutylicum ATCC 824 comprises a 3,940,880 base pair chromosome and a 192,000
base pair megaplasmid pSOL1 [5]. In C. acetobutylicum ATCC 824, nearly 3,740 and 178

54
open reading frames (ORFs) have been identified on the chromosome and megaplasmid,
respectively.
Jiang et al. [95] enhanced butanol yield in C. acetobutylicum EA 2018 by
deactivating the gene encoding acetoacetate decarboxylase (adc) using TargeTron
technology. The TargeTron plasmid pSY6adc was constructed for disruption of adc in C.
acetobutylicum EA 2018 in order to overcome a leaky expression problem during antisense
RNA downregulation. The study resulted in the rise in butanol ratio from 70% to 80.1% and
a decline in acetone yield to 0.21 g/L by the adc-disrupted mutant (2018adc). The addition
of methyl viologen altered the regulation of electron flow by shifting the carbon flux from
acetic acid synthesis to butanol production in 2018adc. This caused the overall butanol yield
to increase from 57% to 70.8%. However, pH played a significant role in cell growth of
2018adc and its solvent production. Metabolic engineering has impacted in Clostridium due
to its lack of an efficient gene knock-out system. The genes adhE, buk, pta, solR and spo0A
have been reported to be knocked-out in C. acetobutylicum with low success rate [5].
Nair et al. [96] did not observe any increase in the solvent production by
Clostridium after overexpressing the adhE gene encoding aldehyde dehydrogenase. Although
the strain lacked the pSOL1 megaplasmid, it expressed adhE and produced butanol and
ethanol [97]. AdhE is involved in synthesizing both acetaldehyde and butyraldehyde, but its
overexpression for high solvent yields appears to be influenced by Clostridial cell physiology
[5]. The adhE overexpression led to improved butanol production in the scenario of butanol
yield less than 10 g/L by the control strain [96, 97]. However, adhE overexpression resulted
in more ethanol production in the case where the control strain had a butanol yield of more
than 10 g/L.
A few studies have employed integrational plasmid technology to interrupt the
metabolic pathways for acetate and butyrate formation in C. acetobutylicum. The genes buk
and pta encoding butyrate kinase and phosphotransacetylase respectively were inactivated to
redirect the carbon flow from acid production to solvent formation. This was executed by
constructing a non-replicative plasmid containing either buk or pta gene fragments
integrated into the chromosomes homologous regions [98]. Acetate production was
suppressed by the inactivation of pta, which in turn compromised phosphotransacetylase and
acetate kinase activities. On the other hand, butanol production was promoted by the

55
inactivation of buk, which hampered butyrate kinase activity leading to reduced butyrate
formation. The overall solvent ratios produced by the mutant PJC4BK were twice as high as
in the parental strain. The mutant also produced 50% less acetone and 10% more butanol
compared to the wild-type strain. The combined effects of buk inactivation and aad over-
expression were tested using PJC4BK (pTAAD) [47]. In contrast to the wild-type strain, the
PJC4BK produced higher concentrations of ABE, notably 4.4 g/L acetone, 16.7 g/L butanol
and 2.6 g/L ethanol. Acetone and butanol production by PJC4BK (pTAAD) was similar to
that of PJC4BK. However, a relatively higher ethanol yield (4.5 g/L) was found for PJC4BK
(pTAAD).
Atsumi et al. [99] expressed several butanol pathway genes coding for
alcohol/aldehyde dehydrogenase (adhE2), 3-OH-butyryl-CoA dehydrogenase (hbd), butyryl-
CoA dehydrogenase (bcd), crotonase (crt), electron transferring flavoprotein (etf) and
thiolase (thl) in Escherichia coli using two plasmids (i.e. pJCL50 and pJCL60). Several
native host genes (e.g. adhE, ldhA, frdBC, fnr and pflB) from E. coli were deleted that would
otherwise compete for acetyl-CoA and NADH required in butanol synthesis. This resulted in
approximately a three-fold rise in butanol yield compared to the wild-type strain (373 mg/L).
The use of Terrific broth as the fermentation medium also enhanced butanol yield up to 552
mg/L. The butanol-producing E. coli mutant also exhibited the ability to grow semi-
anaerobically, unlike the anaerobic Clostridium.
E. coli could be a potential host for metabolic engineering by cloning and
expressing essential genes to produce fuel alcohols via biosynthetic pathways for amino
acids (e.g. isoleucine, leucine, phenylalanine, threonine and valine) [100]. Two crucial steps
are required to produce alcohol-based fuels using engineered E. coli by shunting the
intermediates formation from amino acid biosynthesis pathways. In the first step, a group of
intermediate compounds in amino acid biosynthesis, i.e. 2-ketoacids, are converted to
aldehydes by 2-ketoacid decarboxylases (KDC). In the second step, alcohol dehydrogenases
(ADH) work to reduce the resulting aldehydes to alcohols. The overexpression of 2-ketoacid
decarboxylases and alcohol dehydrogenases in E. coli can produce long-chain alcohols such
as isobutanol, n-butanol, n-propanol, active amyl alcohol, isoamyl alcohol and phenethyl
alcohol [101]. Thus, increasing the metabolic flux to the 2-ketoacid pathway in E. coli can
considerably improve the productivity of butanol and other higher alcohols.

56
 Ralstonia eutropha, S. cerevisiae, E. coli and C. beijerinckii have been engineered
for butanol biosynthesis by substituting their isozymes for Clostridial enzymes [102]. The
most prolific strains contained 3-hydroxybutyryl-CoA dehydrogenase from C. beijerinckii,
which used the co-factor NADH in contrast to the R. eutropha isozyme that instead used
nicotinamide adenine dinucleotide phosphate (NADPH). The expression of genes (bcd and
etfAB) encoding butyryl-CoA dehydrogenase did not advance butanol production
remarkably as evidenced in the case of E. coli. By choosing the appropriate isozymes,
butanol production increased by up to ten-fold i.e. 2.5 mg/L.
Lactic acid bacteria such as Lactobacillus delbruckii and Lactococcus lactis can
sustain growth in the presence of 3 wt% butanol [43]. One of the initial steps in the butanol
biosynthesis pathway implicates acetyl-CoA acetyltransferase (ACoAAT) which regulates
the diversion from acetyl-CoA to butanol. To catalyze the ligation of two acetyl-CoA into
acetoacetyl-CoA, thl encodes acetyl-CoA acetyltransferase or thiolase. Using gene-specific
primers, Liu et al. [103] amplified the thl gene from C. beijerinckii P260 by genomic
polymerase chain reaction (PCR). Upon subsequent introduction of thl into Lactobacillus
buchneri and L. lactis, respective butanol yields of 66 mg/L and 28 mg/L were obtained.
Bacillus subtilis and Pseudomonas putida can tolerate butanol levels of up to
4.5% w/v and 6% v/v, respectively [103]. The polycistronic expression of butanol-producing
genes in P. putida and B. subtilis led to respective butanol yields of 120 mg/L and 24 mg/L
[104]. However, butanol biosynthesis in P. putida was dependent on the expression of gene
bcd/etfAB. Previous reports suggest that butyryl-CoA dehydrogenase (bcd) and electron-
transfer proteins, i.e. electron-transfer flavoprotein alpha and beta-subunit (etfAB) contain
flavin adenine dinucleotide (FAD) and require strict anaerobic conditions [105].
Nevertheless, P. putida KT2440 is reported to have acyl-CoA dehydrogenase (acd) with
catalytic activity on crotonyl-CoA/butyryl-CoA under aerobic conditions [106].
In a biological system, cellulolytic enzymes are arranged in a multi-enzyme
complex called the cellulosome, with biocatalytic components organized around a scaffold
protein [43]. The occurrence and arrangement of the cellulosome in Clostridia are influenced
by: (i) gene silencing of a function-limiting protein, (ii) mutation in such an essential protein,
or (iii) failure to transport and assemble the cellulosome [91]. Despite the presence of a
complete cellulosome (a set of 11 genes and proteins), C. acetobutylicum ATCC 824 is

57
incapable of degrading crystalline cellulose [107]. This creates some concerns in its
industrial utilization and bioconversion of lignocellulosic materials. For example, compared
to C. beijerinckii NCIMB 8052, which that lacks cellulosome genes and is incompetent in
hydrolyzing xylan [14], C. acetobutylicum ATCC 824 successfully degrades xylan although
at a slower rate [108].
Metabolic engineering has also been used to reconstruct recombinant
cellulosomes with improved functioning. The cipA gene encodes the scaffolding protein
CipA in C. acetobutylicum. CipA consists of six hydrophilic domains, five type I cohesin
domains, a family 3a cellulose-binding domain and an N-terminal signal peptide.
Minicellulosomes were synthesized in C. acetobutylicum with the overexpression of a mini-
CipA polypeptide comprising of two cohesin domains and a family 3a cellulose-binding
domain [109].
Similar studies also aimed to construct heterologous minicellulosomes containing
different cellulase enzymes bound to mini-scaffoldin protein. With cellular components from
C. thermocellum and C. cellulolyticum, these studies attempted to create heterologous and
chimeric scaffoldin proteins as well as to assemble and secrete minicellulosomes in C.
acetobutylicum ATCC 824 [110, 111]. Initially, functional scaffoldins were produced that
comprised Scaf 3 (hybrid scaffoldin from C. thermocellum) and miniCipC1 (truncated CipC
from C. cellulolyticum). Expression and folding of cellulases were performed to determine
their binding with the scaffold after the proteins were synthesized and became functional.
The man5K gene encoding mannanase (Man5K protein) from C. cellulolyticum was cloned
alone and as an operon with cipC1 gene encoding the truncated scaffoldin miniCipC1 in C.
acetobutylicum [111]. The recombinant strains produced active Man5K protein but it was
truncated when cloned alone. A full-length mannanase bound to miniCipC1 heterocomplex
was secreted by the recombinant strain when man5K was co-expressed with cipC1,
indicating the candidacy of C. acetobutylicum as an appropriate host for expression of
minicellulosomes. Owing to the fact that C. acetobutylicum has a unique family of proteins
for anaerobic metabolism, sporulation, energy and carbon flux, as well as carbohydrate
degradation, it could serve as a model host for genetic and metabolic engineering.
Perret et al. [112] have cloned and expressed the man5K gene of C. cellulolyticum
in E. coli resulting in the purification of two forms of protein. One corresponded to the full-

58
 length enzyme (45 kDa) while the other was a truncated form (39 kDa) that lacked the N-
terminal dockerin module. Compared to the full-length enzyme, the truncated form displayed
a 4-fold higher activity in the presence of galactomannans. The engineered cellulosomes
exhibited 20-fold greater specific activity with galactomannan than the control species, but
the specific activity on crystalline cellulose was reduced by 20%. The composition of
cellulosomes is regulated by the produced enzymes but their composition can be altered in
Clostridia through the structural gene cloning approach.
Synthetic biology has also been applied to develop better microbial strains
tolerant to high butanol concentration and other solvent stress. The stress response and
solvent tolerance by C. acetobutylicum involve overexpression of several significant genes
for sporulation, fatty acid synthesis, molecular pumps, transcriptional regulators and
chaperones (e.g. dnaKJ, groES, hsp18 and hsp90) [5]. An increase in butanol concentration
in the fermentation media provokes a response that is similar to heat shock. The role of heat
shock proteins (or stress proteins) in solvent tolerance is not well understood. However, it is
clear that they assist in protein folding under heat shock and high solvent stress conditions.
The plasmid pGROE1 was created for the overexpression of the groESL operon, under the
control of thiolase promoter with pSOS95del as the control plasmid [113]. The genes in the
groESL operon comprise groES and groEL, which are responsible for the production of heat
shock proteins GroES and GroEL. By stabilizing solventogenic enzymes, these heat shock
proteins increase the final ABE concentration compared to the wild-type strain (~40%
higher) and the plasmid 824(pSOS95del) control strain (~33% higher). The transcriptional
regulators are the most effective targets to identify complex phenotypes that require
exhaustive alteration of metabolic pathways.

Conclusions and future prospective

Butanol is an advanced biofuel with fuel properties superior to ethanol and almost
comparable with gasoline. C. acetobutylicum is the most widely studied microorganism
involved in ABE fermentation, which produces acetone, butanol and ethanol in the ratio of
3:6:1. The ABE fermentation has two metabolic phases, namely acidogenesis and
solventogenesis. In the acidogenic phase, acetic acid and butyric acid are produced whereas
in the solventogenic phase acetone, butanol and ethanol are generated. Along with acids and

59
 solvents, Clostridial metabolism also generates CO2 and H2 as the gaseous products of ABE
fermentation. During ABE fermentation, Clostridium is prone to butanol toxicity, spore
formation, opportunistic bacteriophage infection and incomplete sugar conversion.
The synthetic biology approach has recently been applied to genetically modify
Clostridium in order to knock-out several genes and induce certain desirable traits for
efficient butanol production. Metabolic engineering of butanol-producing microorganisms
can induce oxygen and butanol tolerance, high cell density, prolonged viability,
asporogenesis and high butanol selectivity. Despite much potential, the use of recombinant
DNA technology struggles to develop a hyper butanol-producing strain due to the unique
physiology of Clostridium and lack of understanding of its induced genomic regulation.
Nevertheless, the development of simultaneous fermentation and concomitant butanol
recovery processes can relieve butanol toxicity and help progress ABE fermentation.
Progressive developments in butanol production could make it a drop-in fuel replacement
for gasoline.

Acknowledgements
The funding support by Natural Sciences and Engineering Research Council of Canada
(NSERC) for this bioenergy research is hugely acknowledged.

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List of figures
Fig. 1: Advantages of butanol as an advanced next-generation fuel
Fig. 2: Different phases in the biological production of butanol and its recovery
Fig. 3: Metabolic pathways in ABE fermentation by Clostridium

71
Fig. 1: Advantages of butanol as an advanced next-generation fuel. Note: The properties of
butanol mentioned here are comparative to those of ethanol.

72
Fig. 2: Different phases in the biological production of butanol and its recovery

73
Fig. 3: Metabolic pathways in ABE fermentation by Clostridium

74
Tables

Table 1: Fuel properties of ethanol, butanol and gasoline

Property Gasoline Ethanol Butanol

Chemical formula H, C4 to C12 C2H5OH C4H9OH
Molecular weight (g/mol) 114.23a 46.07 74.12
Density at 20C (g/m3) 0.7 0.789 0.81
Acidity (pKa) - 15.9 16.1
Viscosity at 25C (mPas) 0.6 1.074 2.573
Flash point (C) 43 17.2 35
Auto-ignition temperature (C) 280 365 343
Calorific value (MJ/L) 32.5 21.2 29.2
Air-fuel ratio 14.7 9 11.2
Research octane number 91-99 129 96
Motor octane number 81-89 102 78
Heat of vaporization (MJ/kg) 0.36 0.92 0.43
Specific gravity at 15.6C 0.72-0.78 0.79 0.81
Boiling point (C) 125a 78.4 117.4
Melting point (C) 56.6a 114 89.8
Lower heating value (MJ/kg) 43.45 26.95 34.37
Higher heating value (MJ/kg) 46.54 29.85 37.33
Reid vapor pressure (psi) 8-15 2.3 0.3
Solubility in water (%) at 25C - 100 7.3
Oxygen content (%) 0 35 22
a
The values represent octane as a major component of gasoline.
References: Drre [4]; Lee et al. [5]; Surisetty et al. [6]; MacLean and Lave [7]; Szulczyk [8]

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Table 2: Summary of a few microorganisms used in acetone-butanol-ethanol (ABE) fermentation
Culture Feedstock Total ABE ABE Total Reference
concentration productivity ABE
(g/L) (g/L/h) yield
(g/g)
C. acetobutylicum 824A Lactose 1.43 0.77 0.15 Napoli et al. [16]
C. acetobutylicum ATCC Jerusalem artichokes juice 19.8 - - Marchal et al. [17]
824 (IFP 904)
C. acetobutylicum IFP 921 Wheat straw 17.7 0.47 0.18 Marchal et al. [18]
C. acetobutylicum DSM Potato 33 0.63 0.13 Grobben et al. [19]
1731
C. acetobutylicum P262 Defibrated sweet potato slurry 7.73 1.0 0.2 Badr et al. [20]
C. acetobutylicum P262 Aspen wood 20.1-24.6 - 0.31- Parekh et al. [21]
0.34
C. acetobutylicum P262 Corn stover 25.8 - 0.34 Parekh et al. [21]
C. acetobutylicum P262 Pinewood 17.6 - 0.36 Parekh et al. [21]
C. acetobutylocum P262 Whey permeate 8.6 3.5 0.36 Qureshi and
Maddox [22]
C. aurantibutyricum Model palm oil mill effluent 12.6 - - Sombrutai et al.
[23]
C. beijerinckii B-592 Pinewood 18.5 0.31 0.27 Nanda et al. [24]
C. beijerinckii B-592 Timothy grass 17.4 0.29 0.3 Nanda et al. [24]
C. beijerinckii B-592 Wheat straw 17.9 0.3 0.28 Nanda et al. [24]
C. beijerinckii BA101 Corn fiber 9.3 0.1 0.39 Qureshi et al. [25]
C. beijerinckii BA101 Degermed corn 14.3 0.3 - Ezeji et al. [26]
C. beijerinckii BA101 Liquefied corn starch 26.5 0.4 0.41 Ezeji et al. [27]
C. beijerinckii BA101 Starch and glucose 9.9 0.42 - Ezeji et al. [28]
C. beijerinckii P260 Barley straw 26.6 0.39 0.43 Qureshi et al. [29]
C. beijerinckii P260 Wheat straw 21.4 0.31 0.41 Qureshi et al. [30]
C. beijerinckii P260 Wheat straw 16.6 0.36 - Qureshi et al. [31]
C. beijerinckii P260 Corn stover and switchgrass (1:1 ratio) 21.1 0.21 0.43 Qureshi et al. [32]
C. beijerinckii P260 Switchgrass 14.6 0.09 0.37 Qureshi et al. [32]

76
C. butylicum NRRL B592 Apple pomace 1.05 - - Voget et al. [33]
C. butylicum TISTR 1032 Cassava 7.4 0.1 - Tran et al. [34]
and B. subtilis WD 161
C. saccharobutylicum Sago 9.1 0.85 0.29 Liew et al. [35]
DSM 13864
C. Bagasse 18.1 0.3 0.33 Soni et al. [36]
saccharoperbutylacetonic
um ATCC 27022
C. Rice straw 13.0 0.15 0.28 Soni et al. [36]
saccharoperbutylacetonic
um ATCC 27022
C. Butyric acid and glucose 16.0 0.42 0.49 Tashiro et al. [37]
saccharoperbutylacetonic
um N1-4

77
Table 3: Summary of salient phenotypic features of metabolically engineered microorganisms
and their biorefining attributes
Phenotypic trait
Oxygen tolerance and aerobic metabolism
Higher cell densities
Prolonged cell viability
Utilization of lignocellulosic biomass
Non-spore forming strains
Butanol tolerance
Improved butanol selectivity
Biorefinery attributes
(i) Simplify bioprocessing requiring less
infrastructural requirements for anaerobic
fermentation.
(ii) Aerobic fermentation could enable higher
cell densities without higher acid levels.
(i) Better volumetric productivity and faster
completion of fermentation.
(ii) Less vulnerability to inhibitory
compounds.
(i) Improved volumetric productivity.
(ii) Prolonged productive fermentation.
(iii) Possibility of culture recycling.
(iv) Less degeneration of vegetative cells.
(i) Reduces substrate costs.
(ii) No competition towards food-based
feedstocks.
(iii) Bioconversion of both pentose and hexose
sugars.
(i) Improved specific cell growth and
volumetric productivity.
(ii) Ability to use continuous or semi-
continuous bioprocessing.
(iii) Enhanced bioconversion of sugars.
(i) Higher final butanol levels.
(ii) Improved cell growth and volumetric
productivity.
(i) Improved butanol yield.
(ii) Efficient downstream processing.
(iii) Less undesirable byproducts formation.
78