Biomass and Bioenergy 94 (2016) 187e200

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Biomass and Bioenergy

journal homepage: http://www.elsevier.com/locate/biombioe

Review

A re-look at the biochemical strategies to enhance butanol production

Sampa Maiti a, Gorka Gallastegui a, b, Saurabh Jyoti Sarma a, Satinder Kaur Brar a, *,
Yann Le Bihan c, Patrick Drogui a, Gerardo Buelna c, Mausam Verma d
a
Institut National de la Recherche Scientifique, Centre-Eau Terre Environnement, 490, Rue de la Couronne, Qu

ebec (QC) G1K 9A9, Canada
b
University of the Basque Country (UPV/EHU), Department of Chemical and Environmental Engineering, Faculty of Engineering, Alameda Urkijo s/n, 48013
Bilbao, Spain
c
Centre de Recherche Industrielle du Qu
ebec (CRIQ), Qu
ebec (QC), Canada
d
CO2 Solutions Inc., 2300, rue Jean-Perrin, Qu

ebec (QC) G2C 1T9, Canada

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

Article history: Butanol produced from renewable feedstock is defined as an emerging biofuel and biochemical. Research
Received 16 June 2015 efforts made during the last three decades on biochemical production of butanol via conventional ABE
Received in revised form (acetone-ethanol-butanol) fermentation has tried to bring biobutanol close to competition with petro-
16 August 2016
butanol. However, each new effort of development has been often countered by new challenges,
Accepted 2 September 2016
Available online 14 September 2016
confining biobutanol production mostly to the laboratory scale. This review provides a systematic,
comparative analysis of different steps in biochemical production of butanol and identifies the coun-
teractive aspects and challenges to overcome. A special emphasis is given on process inhibitors, applied
Keywords:
ABE fermentation
detoxification techniques, chemical supplements and research & development in industry in order to
Biobutanol enhance and update ABE fermentation and make it cost effective. Biobutanol future lies in utilization of
Clostridia inexpensive cellulose enriched lignocellulosic hydrolysates and hyper-butanol producing bacteria,
Inhibitors combined with specific detoxification techniques and followed by efficient continuous fermentation
Detoxification technologies together with in situ product recovery.
Chemical supplements © 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
2. Biochemical production of butanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
2.1. Selection of feedstock for ABE production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
2.2. Pretreatment of feedstock for ABE production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
2.3. Inhibitory compounds in feedstock hydrolysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3. pH adjustment of feedstock hydrolysate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
3.1. Effect of soluble salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
3.2. Effect of metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
3.3. Selection of microorganism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
3.4. Fermentation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Abbreviations: ABE, acetone-butanol-ethanol; AQDS, anthraquinone-2, 6-

disulphonate; ASF, absorbed substrate fermentation; ATP, adenosine triphos-
phate; BSG, brewery spent grain; CaCO3, calcium carbonate; Ca(OH)2, calcium hy-
droxide; CO2, carbon dioxide; DMSO, dimethyl sulfoxide; FSC, fermentable sugar
concentration; GHG, greenhouse gases; H2, hydrogen; H2SO4, sulfuric acid; HBA, 4-
hydroxybenzoic acid; HCl, hydrochloric acid; HMF, 5-hydroxymethyl furfural; log
Kow, log of partition coefficients in a standard octanol/water system; MW, molecular
weight; NaCl, sodium chloride; NaOH, sodium hydroxide; NaOAc, sodium acetate;
Na2SO4, sodium sulphate; TRS, total reduced sugar.
* Corresponding author.
E-mail address: satinder.brar@ete.inrs.ca (S.K. Brar).

http://dx.doi.org/10.1016/j.biombioe.2016.09.001
0961-9534/© 2016 Elsevier Ltd. All rights reserved.
188 S. Maiti et al. / Biomass and Bioenergy 94 (2016) 187e200

3.5. Optimization of fermentation medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

3.6. Recovery processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
3.7. Hybrid technology of separation-fermentation process integrated with more than one recovery process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
4. Commercial biochemical production of butanol and R&D in industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
5. Future outlook and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

1. Introduction 2. Biochemical production of butanol

Gradual depletion of non-renewable energy sources, crude oil 2.1. Selection of feedstock for ABE production
price rise, overpopulation and increasing concentrations of green-
house gases (GHG) in the atmosphere has accelerated the urge for Feedstock selection and conditioning is one of the key factors for
searching green alternative solutions for gasoline. Liquid biofuels butanol biochemical production as it accounts for approximately
from biomass biorefining have garnered significant attention for 60% of the total production cost [9]. Substrate selection for ABE
their environmental friendly production method. Butanol, four fermentation is determined by its abundance, annual supply, pro-
carbon containing aliphatic saturated alcohol (C4H9OH, MW 74.12), cessing cost, type of fermentable sugar monomers and the amount
is considered a promising renewable liquid transportation biofuel of inhibitory compounds generated during the process.
in comparison with ethanol, which is widely established in North Complex polymers, such as lignocellulose (composed of cellu-
America and Europe, due to its higher heat of combustion, higher lose (40e80%), hemicellulose (10e40%) and lignin (1e20%)), starch
octane number, miscibility, less volatility and its ability to blend or affordable agro-industrial residues enriched with directly
with gasoline in higher percentage without any modification of fermentable sugar and micronutrients have been traditionally used
conventional Otto-cycle engine [1]. for anaerobic ABE fermentation by means of solventogenic Clos-
From 1945 to 1960, about two thirds of the butanol production tridia [10e12]. Lignocellulose based feedstock containing a higher
in North America was based on the conventional ABE (acetone- proportion of lignin, non-carbohydrate biopolymers, was not
butanol-ethanol) fermentation [2]. However, several downsides considered as a promising substrate due to significant inhibitory
including unforeseeable feedstock prices, inconsistent substrate effect caused by the release of crosslinking phenolic compounds as
supply, high processing cost, low volumetric productivity, low well as the residual soluble lignin obtained after hydrolysis [13,14].
yield, less explored hyper-butanol-producing bacterial strains, un- Currently, alternative carbon sources, such as algae, carbon dioxide,
availability of sophisticated infrastructure and inhibitory effect of syngas, glycerol, mannitol, galactose, protein derived amino acids,
degradation products and metabolites made biochemical butanol amongst other have begun to be used either by genetic engineered
production economically unattractive [3,4]. With the emergence of butanol production microorganisms or by wild varieties of Clos-
petrochemical industry in 1950s, attention shifted towards butanol tridia [15e22].
synthesis from fossil-oil-derivatives, mainly prop-1-ene via oxo- Unlike ethanol producing microorganisms, butanol producing
process. Biochemical production of butanol globally lost its bacteria utilize both aldohexose and pentose as sources instead of
competitiveness, except in Russia and South Africa due to low la- only aldohexose [23e26]. In any case, utilization of aldohexoses is
bour cost [5]. preferred over aldopentoses, and only when aldohexose is limiting,
The 1973e74 oil crisis put an end to years of inexpensive energy the pentose sugars are used [27]. Raganati et al. (2012), showed that
and exacerbated the economic difficulties faced by many indus- butanol producing Clostridium acetobutylicum was able to convert
trialized nations, forcing them to regain the interest in ABE both hexose and pentose sugars into ABE, while the conversion
fermentation in order to develop a least oil-dependent economy. degree decreased in the following order:
According to the most recent process report published by DOE glucose > mannose > arabinose > xylose [28]. Therefore, for large-
(2011) [6] more than 1 billion metric ton of butanol could be pro- scale production purposes, cellulose, a polysaccharide of b-D
duced using cellulose based feedstock by 2030. Thus, the current glucose, and starch, a polysaccharide of a-D glucose, have been
prospects of biomass-based butanol production, particularly from preferred for ABE fermentation over hemicellulose, a hetero-
lignocellulosic material as substrate, look positive and it could even polysaccharide primarily composed of D-pentose sugars (xylose in
compete with synthetic butanol in the chemical market [7]. Pres- the largest amount).
ently, ABE fermentation process for biobutanol production based In conclusion, lignocellulosic feedstocks containing large per-
on corn starch transformation is already economically viable when centage of cellulose, followed by hemicellulose and a minimum
n-biobutanol is sold as a premium product in the market for amount of lignin would have positive effects on ABE fermentation.
chemicals [8]. Bearing this in mind, agricultural residues as well as partly pro-
Several attempts have been made in the last decades to address cessed agro-industrial wastes enriched with nutrients and
the hurdles in biochemical butanol production. However, most of fermentable reducing sugars have been the preferred choices for
the efforts are often counteracted by new challenges. In this pro- butanol biochemical process. It has to be taken into account that
spective, this review presents a systematic and comparative study efficient reuse of these residues becomes a major logistical, finan-
of various counteractive aspects in the updates of biochemical cial and environmental issue. North America, one of the largest
butanol production based on authoritative reports and thus iden- agro-industrial waste producers (Canada is in fact the second
tifies the challenges to enhance ABE fermentation and make it cost overall supplier of wood lignocellulosic biomass), retrieves only
effective again. 20% of agro-industrial food wastes for animal feed, while the rest is
used for landfilling, incineration or composting, which causes GHG
emissions [29].
 S. Maiti et al. / Biomass and Bioenergy 94 (2016) 187e200
Corn stover has been reported as the best substrate for ABE
production (DOE (2011)) [30]. However, most of the agro-industrial
feedstock, such as apple pomace [31], apple pomace ultrafiltration
sludge, maple industry waste [32], juice processing industry wastes
[33] or brewery industry liquid wastes, contains relatively lower
amounts of fermentable reducing sugars to promote a profitable
biobutanol production. As butanol is the end product in Clostridial
metabolism, more than 15e20 g L
1 of initial reducing sugar con-
centration is required to promote the acidogenic phase to sol-
ventogenic phase [34e36]. The pretreatment of complex
agricultural residues and agro-industrial wastes is thus a promising
avenue for generation and enhancement of water-soluble,
fermentable sugars to produce biobutanol via ABE fermentation.
2.2. Pretreatment of feedstock for ABE production
Pretreatment and hydrolysis of feedstock implies the unravel-
ling of lignocellulosic biomass complex polymeric structure and the
release of simple reducing sugar monomers along with other cross-
linked units and the remaining non-hydrolyzed raw material [37].
Process conditions during pretreatment of feedstock are considered
to be the “heart” of the hydrolysis step, as slight changes in oper-
ation parameters may result in sensible differences in total reduced
sugar (TRS) and inhibitory compound concentrations in the
solution.
Different chemical, physical or biological techniques have been
explored to enhance the release of fermentable sugars from agri-
cultural and agro-industrial wastes. Physical techniques, such as
grinding [38], hydrothermal pretreatment [39], steam explosion or
auto-hydrolysis [40], microwave irradiation [41,42] and ultra-
sonication [43,44] have been evaluated. Steam explosion at high
temperature (121e260  C) for short time (5e60 min) has been
reported to be more efficient for higher cellulose based corn stover
hydrolysis [10].
Chemical techniques, such as dilute or concentrated acid cata-
lysed hydrolysis [37], acid or base catalysed microwave digestion
[45], solid acid (H-form zeolites, transition-metal oxides, cation-
exchange resins, supported solid acids) catalysed hydrolysis [12],
ammonia fiber explosion [46], alkali treatment (sodium, potassium
or calcium hydroxide), hydrogen peroxide catalysed hydrolysis [47]
and nano-particle catalysed [48] lignocellulose based biomass hy-
drolysis have been well reported in literature. Agro-industrial res-
idues, such as brewery industry spent grains (BSG) released highest
amount of reducing sugar via acid catalysed microwave digestion
[45]. Higher total phenolic compounds concentration was observed
when acid hydrolysis was applied to lignin in comparison with
hydrogen peroxide or alkali catalysed pretreatment. Together with
phenolic compounds, acid-soluble lignin also remained in solution
inhibiting ABE fermentation [13,14]. According to Carvalheiro et al.
(2008), low pH pretreatments facilitate hemicellulose solubilisa-
tion and lignin precipitation. In contrast, alkaline processing
selectively solubilized lignin but the impact was minimized on
hemicellulose [37]. Alkaline pretreatments induced slightly lower
overall sugar yields, but resulting hydrolysate was less inhibitory to
the microorganisms [49].
Biological techniques are mainly either in situ (simultaneous
saccharification and fermentation process) or ex situ enzymatic
hydrolysis by means of different enzymes, such as cellulase, xyla-
nase and amylase under relatively mild operation conditions. In situ
application hampers overall process optimal performance, since
fermentation and hydrolysis usually have different process condi-
tions. On the contrary, it ensures lower contamination risk and
equipment cost [50e52]. Enzymatic hydrolysis has been proven to
be superior to other common treatments, such as acid catalysed
hydrolysis, due to its selectiveness and efficiency (it is possible to
189
obtain cellulose hydrolysis of close to 100%). It is indeed the most
common method of producing bioethanol from lignocellulosic
biomass [47]. Nevertheless, enzyme cost, estimated running time
and product inhibition during hydrolysis are the main barriers to
take the bioprocess to industrial scale using agro-industrial and
agricultural wastes [52].
The formation of fermentable compounds and unintended
process inhibitors is substrate and pretreatment dependent and
they must be looked into for achieving full exploitation from each
lignocellulose based feedstock [53]. As production of inhibitory
compounds plays a vital role in ABE fermentation, attention needs
to be drawn towards their formation before selecting a hydrolysis
process for large scale production. Every pretreatment method has
its own advantages from sugar release and inhibitory byproduct
generation. Regarding industrial application, dilute sulfuric acid
process (0.5e1.5%, 121e160  C) has been traditionally most fav-
oured even if it is hampered by non-selectivity and by-product
formation [54]. However, exploration of a moderate, highly selec-
tive, less inhibitory product forming, and economically viable
process would be the way forward. In this regard, application of
metallic nanoparticles would not only catalyse substrate hydrolysis,
but also act as a nutrient supplement source for ABE fermentation
process [12].
2.3. Inhibitory compounds in feedstock hydrolysate
During hydrolysis of lignocellulosic materials, a wide range of
compounds which are inhibitory to bacteria are formed. Inhibitory
compound formation pathways are represented in Fig. 1. Based on
their origin (cellulose, hemicellulose and lignin), inhibitors are
classified in three main groups: furan derivatives, aliphatic acids
and polyphenolic aromatic compounds. When cellulose and
hemicellulose are ideally degraded, glucose and other mono-
saccharide sugars, such as mannose, galactose, xylose and arabi-
nose are liberated (Fig. 1). However, the application of non-selective
pretreatment methods (e.g. acid catalysed hydrolysis) enables the
further conversion of previously cited fermentable sugars into
heterocyclic organic compounds, such as furfural and 5-
hydroxymethyl furfural (HMF). According to the extremity of
working conditions, these by-products may likewise break down
and several aliphatic acids, such as formic acid, acetic acid or lev-
ulinic acid would emerge. Polyphenolic aromatic compounds
(ferulic acid, syrindic aldehyde, vanilline) are mainly generated
from partial decomposition of lignin [55,56]. A summary of in-
hibitors present in main lignocellulosic materials (sugarcane
bagasse, corn stover, spruce) was briefly reported by Chandel et al.
(2011) and Carvalheiro et al., 2008 [11,37].
The sensitivity to the inhibitors varies with the fermenting mi-
croorganisms and within different strains of bacteria. Effects of
inhibitory compounds and detoxification mode applied have
already been thoroughly explored in bioethanol production
(Table 1). Although key aspects could be drawn for butanol pro-
duction, yet the specific effects of inhibitory compounds must be
investigated in-depth for this particular fermentation. Different
inhibitory compounds produced during hydrolysis and their cor-
responding effects in microorganisms responsible for ABE
fermentation have been summarized in Table 2. Furfural and HMF
concentrations over 2 g L
1 are known to damage DNA, inhibit
glycolytic enzymes, unsettle redox balance and disrupt cell mem-
branes [57]. Among all the weak organic acids produced during
hydrolysis, inhibitory effect of formic acid was found to be signifi-
cant, since 0.04 g L
1 of the compound was enough to affect mi-
crobial growth due to its high permeability through the cell
membrane [10,33]. Formic acid inactivation was also previously
verified for ethanol bioproduction [58]. Two mechanisms were
190 S. Maiti et al. / Biomass and Bioenergy 94 (2016) 187e200

O
OH HO
O HO O
Formic acid
Furfural O O
O OH
HO Acetic acid
HO O OH
Cellulose, OH Process inhibitor
Hemicellulose Aldohexose O
HO Levulinic acid
O
O 5-Hydroxymethyl furfural
Hydrolysis
HO
HO HO OH
HO
HO OHHO
Aldopentose OHOH
HO
Agro-industrialwastes HO O HO OH
/agriculturalresidues Mannonic acid
O HO OH
(Lignocellulos,reducing
sugars etc.) Gluconic acid
O
HO O
Hydrolysis

Lignin HO
OH
O
Phenolic compounds
Ferulic acid O O
HO
Syringaldehyde

O OH O HO
HO
p- Coumaric acid OH O
O
O Vanillic acid Vanillin

Fig. 1. Different inhibitory compounds produced during hydrolysis.

proposed by Ref. [59] to understand the damage caused by weak
acids: uncoupling (ATP unavailability for biomass formation) and
intracellular anion accumulation (undissociated acid diffusion into
the cell). Mills et al. (2009), observed in tests with Escherichia coli
that phenolic compounds were more toxic than aliphatics acids or
furans with the same functional group [60]. Inhibition mechanism
of phenolic compounds has been based on their partition into
biological membranes with the subsequent loss of integrity [61].
Removal of inhibitory compounds is an unavoidable necessity to
ensure ABE fermentation success [57,62e64]. ABE solvent produc-
tion was increased from 3.8 g L
1 to 16.0 g L
1 and from 18.0 g L
1 to
26.3 g L
1 when substrate was treated with Ca(OH)2 as detoxifi-
cation agent in corn cob and in corn stover hydrolysate, respectively
[57,63,65]. Several detoxification methods, such as physical
(membrane mediated detoxification, evaporation), chemical (acti-
vated charcoal treatment, calcium hydroxide over liming, ion ex-
change resins, neutralization and extraction with ethyl acetate) and
biological detoxification (microbial detoxification using genetically
modified strains, enzymatic mediated catalysis using lignin
peroxidase, laccase, etc.) have been described in the literature
(Table 2). Glycerol supplement to reduce furfural to furfural alcohol
and excess lime treatment to oxidize the inhibitory compounds to
carbon dioxide have been widely used in butanol production by
solventogenic Clostridia from lignocellulose feedstock [66,67].
Mechmech et al. (2015), proposed a novel detoxification method
for wood hydrolysate by flocculation with ferric sulphate. These
authors achieved removal efficiencies of 80%, 56%, 20% and 6% for
acetic acid, phenolic compounds, HMF and furfural, respectively,
with a total sugar loss of 3 g L
1 (z10%) [68]. Apart from the re-
ported techniques (Table 2), the addition of an extractive water-
immiscible phase of high affinity towards the inhibitory byprod-
ucts, but less toxic to microorganism (compounds with log Kow in
the range 1.5e4 have been found generally harmful to microor-
ganisms [69]), might be explored for detoxification in feedstock
hydrolysate to enhance ABE solvent yield. The adequate selection of
the non-aqueous phase should fulfil certain requirements, such as
non-biodegradability, inexpensive, available in bulk quantities, low
emulsion-forming tendency and good hydrodynamic characteris-
tics. Two phase bioreactor equipped with the proper organic sol-
vent could be promising alternative, especially for phenolic
compounds elimination. However, neutralization of hydrolysate
after removal of inhibitory compounds is compulsory for growth of
solventogenic Clostridia.
3. pH adjustment of feedstock hydrolysate
3.1. Effect of soluble salt
TRS enriched hydrolysate is not directly applied for subsequent
fermentation process. A neutralization step is essential before
starting fermentation. Initial hydrolysate pH should be around
6.5e6.7 to allow solventogenic Clostridia adaptation. Common al-
kalis, such as sodium hydroxide (NaOH) and calcium hydroxide
(Ca(OH)2) have been traditionally used for neutralizing the pH after
sulfuric acid (H2SO4) or hydrochloric acid (HCl) pretreatment step.
Nevertheless, formation of neutralization products, such as sodium
sulphate (Na2SO4), sodium chloride (NaCl) along with sodium ac-
etate (NaOAc) (e.g. acetate from hydrolysate of complex biomass)
has been observed. Certain concentrations of aforementioned sol-
uble salts, which are difficult to separate from the hydrolysate, were
found to be inhibitory (Table 2), both to cell growth and ABE
fermentation [24,70].
3.2. Effect of metals
Inhibitory effect of certain heavy metals on anaerobic digestion
and ABE fermentation has long been known [71e73]. Biosorption
by plants from soil and unintended release from the walls of the
pretreatment system reactor are the main two sources of metal
content found in the hydrolysate derived from agriculture based
 S. Maiti et al. / Biomass and Bioenergy 94 (2016) 187e200 191

Table 1
Detoxification modes applied for typical inhibitory compounds in bioethanol production.

Inhibitor Detoxification mode Removal efficiency (RE) FSC Microorganism Substrate Reference

Furan derivatives
Furfural, Electro-dialysis Furfural: 0.03 g L
1 (RE z 7%), 5-HMF: FSC unaffected e Mixed [156]
Hydroxymethylfurfural z0.15 g L
1 (RE z 7%). softwood
(5-HMF) Absorption with treated wood Furfural: 260 mg L
1 (RE 100%), 5-HMF: FSC unaffected Saccharomyces Spruce chips [157]
charcoal (600  C) 490 mg L-1 (RE 100%). cerevisiae
20% (w/v) Ca(OH)2 up to pH 10 for Furfural: 0.20 g L
1 (RE 20%), 5-HMF: FSC unaffected Saccharomyces Spruce chips [158]
1h 1.30 g L
1 (RE 22%). cerevisiae
NH4OH (pH 10 and 80  C for 3 h) Furfural: 0.75 g L
1 (RE 93%); 5-HMF: 25% and 16% Saccharomyces Spruce chips [159]
2.76 g L
1 (RE 89%). reduction in cerevisiae
glucose and
mannose
concentration
(Na2SO3) 1% (w/v) at pH 10 under Furfural: 0.53 g L
1 (RE 53%), 5-HMF: FSC unaffected Saccharomyces Spruce chips [158]
an atmosphere of helium for 1 h 3.07 g L
1 (RE 52%). cerevisiae
Neutralization with CaO (pH 6; (Furfural þ 5-HMF): 327 mg L
1 (RE 42%). FSC reduced 9% Pichia stipites Sunflower [160]
60  C for 30 min) seed hull
Over liming with CaO (pH 10) (Furfural þ 5-HMF): 319 mg L
1 (RE 41%). FSC reduced 12% Pichia stipites Sunflower [160]
seed hull

1
Over liming with CaO (pH (Furfural þ 5-HMF): 529 mg L (RE 68%). FSC reduced 11% Pichia stipites Sunflower [160]
10) þ charcoal (1 g/200 mL) seed hull
stirred for 24 h at 30  C
Evaporation of 90% of the initial Furfural: 1.0 g L
1 (RE 100%), 5-HMF: FSC unaffected Saccharomyces Spruce chips [158]
volume and water addition to 0.24 g L
1 (RE 4%). cerevisiae
100% of initial volume
Biological detoxification with Furfural: 0.25 g L
1 (RE 50%); 5-HMF: FSC reduced less Saccharomyces Waste house- [161]
Ureibacillus thermosphaericus 0.18 g L
1 (RE 20%). than 5% cerevisiae wood
(2.4 g L
1) (50  C for 24 h)
Ethyl acetate extractions (1:1), Furfural: 0.28 g L
1 (RE 100%). 6% and 19% Pichia stipitis Aspen wood [162]
followed by roto-evaporation reduction in CBS 5776 chips
glucose and
xylose
concentration
Surfactant-based cloud point Furfural: z0.15 g L
1 (RE z 30%), 5-HMF: FSC unaffected e Corn Stover [163]
extraction (CPE) with non-toxic z0.1 g L
1 (RE z 10%).
thermo-separating copolymer
(L62D 5%)
Aliphatic acids
Acetic acid, Formic acid, Electro-dialysis Acetic acid: 1.84 g L
1 (RE 100%). FSC unaffected e Mixed [156]
Levulinic acid softwood

1
Electrodialysis Formic acid: 6.9 g L (RE 100%); Levulinic FSC reduced 17% K. marxianus; e [152]
acid: 6.1 g L
1 (RE 100%). Pterocladiella
capillacea
Evaporation of 90% of the initial Acetic acid: 1.56 g L
1 (RE 65%), Formic acid: FSC unaffected Saccharomyces Spruce chips [158]
volume and water addition to 1.18 g L-1 (RE 74%) cerevisiae
100%
Over liming with CaO (pH 7.0 and Acetic acid: 3.0 g L
1 (RE 38%). 18%, 28% and 9% C. shehatae Corn cob [164]
100  C for reduction in ACCC 20335 hemicellulose
15 min) þ filtration þ activated glucose, xylose
charcoal (3% at 40  C for 1 h and and arabinose
200 rpm)
Over liming with Ca(OH)2 to pH 10 Formic acid: 3.59 g L
1 (RE 52%), Levulinic FSC reduced 42% K. marxianus; e [152]
e11 at 60  C for 30 min acid: 2.93 g L
1 (RE 48%). Pterocladiella
capillacea
Neutralization to pH 6 Levulinic acid: 0.73 g L
1 (RE 12%). FSC reduced 25% K. marxianus; [152]
Pterocladiella
capillacea
Poly-phenolic aromatic acids
Vanillic acid, Vanillin, 4- Absorption with treated wood Vanillic acid: 33 mg L
1 (RE 100%), Vanillin: FSC unaffected Saccharomyces Spruce chips [157]
Hydroxybenzoic acid charcoal (600  C) 36 mg L
1 (RE 100%). cerevisiae
(HBA), p-Coumeric acid Ethyl acetate extractions (1:1) HBA: 1.07 g L
1 (RE 100%), Vanillin: 6% and 19% Pichia stipitis Aspen wood [162]
followed by rotoevaporation 0.21 g L
1 (RE 100%). reduction in CBS 5776 chips
glucose and
xylose
concentration
Over liming with Ca(OH)2 (pH 12 p-Coumeric acid: 0.35 g L
1 (RE 34%) e e Olive stones [165]
and 60  C for 60 min) as feedstock
Surfactant-based cloud point p-Coumeric acid: z0.45 g L
1 (RE z 90%), FSC unaffected e Corn Stover [163]
extraction (CPE) with non-toxic Vanillin: z0.5 g L
1 (RE z 100%), Ferulic acid
thermo-separating copolymer z0.5 g L
1 (RE z 100%), Syringaldehyde:
(L62D 5%) z0.5 g L
1 (RE z 100%).
Electro-dialysis Total phenolic compounds: z2 g L
1 (RE FSC unaffected e Mixed [156]
70%). softwood
(continued on next page)
192 S. Maiti et al. / Biomass and Bioenergy 94 (2016) 187e200
Table 1 (continued )
Inhibitor Detoxification mode Removal efficiency (RE)
Over liming with CaO (pH 7.0 and Total phenolic compounds: 0.95 g L
1 (RE
100  C for 97%).
15 min) þ filtration þ activated
charcoal (3% at 40  C for 1 h and
200 rpm)
FSC: Fermentable sugar concentration.
waste biomass [74,75]. Moreover, metal concentration in the media
might increase when hydrolysate pre-concentration step is carried
out to increase reducing sugar concentration for butanol
production.
Multiple essential metals are required for ABE fermentation
reaction as they are involved in Clostridial metabolism [76]. How-
ever, they are micronutrients (e.g. Zn, Cu, Fe, Co) with a relatively
narrow optimum concentration range favourable to microorgan-
isms, and at higher concentrations they produce toxic effects.
Several heavy metal ions, such as Zn2þ and Cu2þ produce hydro-
peroxide radicals which may interfere with electron transport
pathways of cell metabolism. Interaction of Cu2þ with cysteine
residues present in spore membranes and coat proteins adversely
affects sporulation and germination of microorganism. Chiu-Yue
Lin (1993), showed that Cu2þ and Zn2þ were more toxic to acid-
ogens than to methanogens [71]. Drop in alcohol production in
presence of Cd3þ and Cr3þ was studied by Ref. [77]. Effects of Al3þ
and Fe3þ were also reported by Ref. [73]. Heavy metal ions pre-
cipitate as metal hydroxides within the hydrolysate by increasing
the pH of the medium above 10 and subsequently removed by
centrifugation at 10000 c g.
3.3. Selection of microorganism
Availability of microbial catalysts to achieve robust and high
product yields, titer and productivity is another bottleneck in
biochemical production of butanol [78]. Solventogenic microbial
activity is certainly butanol production limited as 2 mol of carbon
dioxide (CO2) are evolved per mole of glucose together with other
side products, such as acetone, ethanol, acetic acid, butyric acid and
hydrogen during ABE fermentation. Compared to 10% (w/v) of
ethanol obtained in yeast fermentation, the butanol level at 2% (w/
v) is low and uneconomical because of intense energy consumption
in its recovery [79,80].
Several solventogenic Clostridia strains (C. acetobutylicum, C.
beijerinckii, C. saccharobutylicum, and
C. saccharoperbutylacetonicum) have been studied for butanol pro-
duction [2]. Typical production of butanol by solventogenic Clos-
tridia is around 11e13 g L
1, which is not economically feasible for
large scale production [2]. The intolerance of butanol-producing
Clostridia to metabolites accumulated in the fermentation broth
(i.e. acetone, butanol and ethanol) is an additional constraint to
overcome. Total cell growth inhibition was set at 70 g L
1,
12e16 g L
1 and 50e60 g L
1 for acetone, butanol and ethanol,
respectively (Jones and Woods, 1986). Butanol toxicity weakens
ATPase activity and cell membrane fluidity, causing membrane
leaking and disruption of the lipid structure [2,81,82].
One approach to maximize butanol recovery, reduce side
product levels, make use of alternative substrates or find alterna-
tive microbial hosts was proposed through mutagenesis and
metabolic engineering [79,83,84]. Higashide et al. (2011), devel-
oped a metabolically engineered Clostridia cellulolyticum able to
directly convert cellulose to produce isobutanol [21]. To the best
knowledge of the authors [85], found the highest production of
butanol by means of solventogenic Clostridia. These authors
FSC Microorganism Substrate Reference
18%, 28% and 9% C. shehatae Corn cob [164]
reduction in ACCC 20335 hemicellulose
glucose, xylose
and arabinose
concentration
developed a solvent hyper-producing mutant bacterium (Clos-
tridium beijerinckii BA101) capable of producing 20.9 g L
1 of
butanol using glucose as substrate [86]. noticed a high organic-
solvent concentration tolerance by Pseudomonas putida and ob-
tained a genetically engineered Pseudomonas putida DOT-T1E strain
which tolerated up to 6% (v/v) (48.6 g L
1) butanol content. Kataoka
et al. (2011), isolated a bacterium, Bacillus subtilis GRSW2-B1, with a
remarkable tolerance ability to butanol 5% (v/v) [87]. This bacte-
rium showed higher butanol tolerance than other Bacillus sp.
strains (B. subtilis 168 and B. subtilis KS438) previously used as in-
dustrial heterologous hosts. It is expected that a more solvent
tolerant strain can reduce overall cost of butanol production by
lowering 50% of purification process. Exploration of hyper-butanol
producing, higher solvent tolerant microorganisms able to
consume different monosaccharides equally efficiently can result in
a profitable ABE fermentation. However, nowadays, with existing
tools for genetic manipulation, it seems still difficult to develop a
genetically engineered microorganism with combined ability of
lignocellulose feedstock utilization and solvent production without
losing its activity after long-term use.
3.4. Fermentation techniques
Biphasic ABE fermentation is composed of acidogenesis fol-
lowed by solventogenesis. Substrate is converted into acids during
the acidogenesis (acetic acid and butyric acid are produced by
exponentially growing cells). Under certain conditions (minimum
glucose concentration of 15e20 g L
1 (1), minimal intracellular ATP
concentration (2), high NADH: NAD ratio (3), and accumulation of
undissociated fatty acids (pH below 5.5) (4)), the bacterial culture
stops growing and solventogenic phase is stimulated [36,79,88,89].
In fermentation broth, when the butyric acid concentration reaches
around 13 mM and the total acid concentration (butyric acid and
acetic acid) becomes at least 40e45 mM, the acidogenesis phase is
completed and it enters solventogenic phase to produce butanol,
acetone and ethanol [90]. Unfortunately, higher butyrate concen-
tration (overproduction of acids) leads to inactivation of the mi-
crobial culture and absence of solvent formation [89].
ABE fermentation can be accomplished under strictly anaerobic
conditions in batch [85], fed-batch [91], semi-continuous [92], free
cell continuous [93], immobilized cell continuous [94], cell recycle
continuous [95], cell recycling and bleeding [96], continuous flash
fermentation [96,97], biofilm bioreactor, fed-batch extractive
fermentation [98] and fed batch with in situ product recovery,
among others modes [91,99e101]. The most reported is the batch
process due to its simplicity and reduced risk of contamination [2].
In butanol batch fermentation, a maximum total ABE solvent con-
centration of 25e30 g L
1 is rarely attained. 26.64 g L
1 of ABE
using C. beijerinckii P260 were recorded by Qureshi et al. (2010)
from fermentation of dilute sulfuric acid barley straw hydrolysate
treated with lime.
Reduced bacterial cell density and resulting low bio-butanol
productivity may be attributed to longer cell lag-phase, substrate
inhibition and solvent toxicity. However, increase in cell concen-
tration and solvent productivity has been achieved by fixing cells
 S. Maiti et al. / Biomass and Bioenergy 94 (2016) 187e200 193

Table 2
List of inhibitory compounds produced during hydrolysis and their corresponding effect in subsequent ABE fermentation.

Types of substrates Inhibitory Microorganism Effect of inhibitory in ABE Effect in cell Inhibitors removal strategies References
compounds in for ABE production
hydrolysate fermentation

Aromatic hetero HMF; Furfural C. Beijerinckii 1. <0.5 g L
1 enhances 1. Adverse effect on enzymes Evaporation; Sodium sulphite [63
cyclic P260; production and required for metabolism addition; Anion exchange resin e65,114,166,167]
C. acetobutylicum productivity due to and long lag phase during (70%) recovery; XAD-4 resin
ATCC 824; increase in enzyme cell growth recovery; Over liming and
C. beijerinckii activity as well as used 2. Strong inhibition on ADH common alkaline treatment.
BA101 as precursor (anti-diuretic hormone)
2. z2e3 g L
1 converted to leads to accumulation of
other less toxic acids by acetaldehyde (>0.5 mM)
specific microorganism which inhibits DNA and

1
3. >3 g L is deleterious protein synthesis
for ABE fermentation 3. Membrane permeability
decrease and cell
replication deactivation
Phenolic Vanillic acid; C. beijerinckii IB4; 1. >1 g L
1 stopped 1. Disrupt electrochemical Peroxidase and laccase (80% [66,114,166,168
compounds HBA C. beijerinckii completely butanol gradient by transporting recovery) based enzymatic e170]
RT66 production protons back across the treatment; Flocculation; Ion
2. Total soluble phenolic mitochondrial membranes exchange resins; Activated
compound 2. Deterioration of cell charcoal; Common alkalis, such

1
>2.1 g L has strong membranes ability to serve as Ca(OH)2, NaOH, KOH are
negative effect in as selective barriers and employed at industrial level
production enzyme matrices, causing
Ferulic acid C. beijerinckii >0.3 g L
1 no butanol leads adverse effect in cell
BA101 production growth and sugar

1
p-Coumaric C. beijerinckii >0.5 g L no butanol assimilation
acid BA101 production

1
Syringaldehyde C. beijerinckii 1. 0.05 g L strong Reduction of cell growth and
NCIMB 8052 inhibition on cellulase sugar assimilation due to
enzyme activity partition and loss of integrity
2. 0.3e1.0 g L
1 complete of biological membranes
stop ABE production
Vanillin C. beijerinckii z1 g L
1 complete butanol
NCIMB 8052 production inhibition
Organic acids Acetic acid; C. acetobutylicum 0.04 g L
1 of formic acid 1. Decrease cell pH, causing Evaporation; Flocculation; Over [65,114,166]
Formic acid; inhibit the conversion inhibition of cell activity liming; Common alkaline
Levulinic acid process from acidogenic and cell lysis treatment.
phase to solventogenic 2. At low pH, acetic acid
phase accumulation in cytoplasm
via plasma membrane
diffusion
Non-carbohydrate Soluble Clostridium 1. Drop in ABE production Destruction of enzymes by Hydrogen peroxide and activated [10,13,14]
Biopolymer unhydrolyzed acetobutylicum by 30% compared to adsorption during metabolism charcoal; Polyethylene oxide
lignin ATCC 824 control sample without flocculation (>99% removal);
soluble lignin Amberlite XAD-4 resin by 90%
2. Reduced sugar
consumption
Inorganic soluble Sodium C. beijerinckii z25% drop in production Increase cell membrane Electrolysis; Extraction; Reverse [70,114,171]
salts sulphate BA101 due to cell inhibition in permeability which leads to osmosis
presence of 13.3 g L
1 cell growth inhibition
Sodium z36% reduction in ABE
sulphate þ production in presence of
Sodium acetate 13.3 g L
1 sodium
sulphate þ 8.9 g L
1 sodium
acetate
Sodium C. beijerinckii 1. 1.98 g L
1 inhibit cell
chloride P260; growth
C. beijerinckii 2. 5 g L
1 reduce ABE
BA101 production

onto a support or gel in absorbed substrate fermentation (ASF)
[102] and bio-film reactors [103,104] or by applying membrane
technology (i.e. perstraction) [105]. Besides, performance of batch
fermenters has been improved by developing simultaneous
saccharification and fermentation of the substrate [50], extractive
fermentation [7,106], multistage fermentation [107], co-culture
[108] and electrochemical production [19].
Substrate inhibition has been overcome by fed batch process
[109]. Fed-batch fermentation keeps substrate concentration below
detrimental toxic level by gradual addition of substrate to the
reactor at the same time as fermentation culture consumes it. The
dilution effect created during the addition of the substrate solution
may also partially solve the problem of accumulated solvent
products. Nevertheless, fed-batch process must be associated with
one or more simultaneous product removal techniques to subside
product inhibition [91,99,101,110]. Production of butanol and ABE
using fed batch integrated with in situ product recovery by gas
trapping were 151.7 g L
1 and 232.8 g L
1 using 500 g of glucose
[99] and 76.4 g L
1 and 108.5 g L
1 using 584.4 g L
1 glucose from
concentrated cassava bagasse hydrolysate, respectively [91].
Continuous operation has several advantages over batch and
fed-batch operations, including minimizing downtime and the lag-
194 S. Maiti et al. / Biomass and Bioenergy 94 (2016) 187e200

Table 3
Optimization of fermentation broth by addition of supplements.

Supplement Substrate Bacterial strain Effect in ABE production References

Glucose Wheat straw hydrolysate C. beijerinckii P260 Butanol production increased from 9.36 to 17.92 g/ [66]
L
Glucose Non-nitrogen containing butyrate (10 g L
1) C. saccharoperbutylacetonicum Butanol production increased from 0.2 to 7.5 g L
1 [119]
media N1-4 (ATCC 13564)
Butyric acid (B) þ Glucose Triptoneeyeast extracteacetate (TYA) C. saccharoperbutylacetonicum 1. Butanol production rate increased from 0.10 [96]
(G) medium N1-4 gbutanol g
1
cell h

1
to 0.42 gbutanol g
1
cell h

1

2. Maximum butanol production rate: 16 g L
1

Butyrate Modified CAB medium C. beijerinckii NCIMB 8052 Butanol product increased from 0.45 g L
1 to [172]
11.2 g L
1
Butyric acid P2 media C. beijerinckii ATCC 55025 Butanol: total ABE ratio was increased by addition [173]
of 4 g L
1 butyric acid.
Butanol production was stopped due to addition of
8 g L
1 butyric acid.
Lactic acid Tryptone-yeast (TY) media. C. saccharoperbutylacetonicum Butanol production increased from 5 g L
1 to [174]
N1-4 15.5 g L
1. No increase when 10 g L
1 lactic acid
were added.
Acetate Modified P2 media C. bezrinckii BA101 Production of 20.9 g L
1 butanol. [85]
Yeast Cassava meal medium C. acetobutylicum ATCC824 15% increase in butanol concentration. [175]
Hydrogen at head space Semi-solid reinforced Clostridium medium C. acetobutylicum ATCC824 Ethanol and butanol yields were improved by 13% [176]
(RCM) and 18%, respectively
Glycerol P2 media C. beijerinckii NCIMB 8052 2-3 times enhancement in ABE production [67]
Methyl viologen; Neutral Non-nitrogen containing butyrate (10 g L
1) C. saccharoperbutylacetonicum Methyl viologen and neutral red increased butanol [119]
red media N1-4 yield from 0.577 mol mol
1 to 0.671 mol mol
1 and
0.645 mol mol
1 respectively.
Methelene blue; Dimethyl Negative effect in ABE production
sulfoxide
Ca(OH)2 Corn stover and switchgrass hydrolysate C. beijerinckii P260 Production increased from 18 g L
1 to 26.27 g/L [62]
CaCO3 Yeast þ P2 media supplemented with single C. acetobutylicum DSM 792 ABE and butanol concentrations were increased. [28]
sugars (glucose, mannose, arabinose, and C. acetobutylicum ATCC 824; ABE production was enhanced by 31% and 46%. [126]
xylose investigated separately) C. beijerinckii 260 Butanol tolerance was enhanced by more than 40%
Fe(3þ) Synthetic media C. beijerinckii NICMB8052 Butanol yield was increased by 5 times. [125]
Yeast, glucose and other 9 P2 and TYA medium C. beijerinckii YM1 The compounds with the greatest to least positive [127]
chemicals effect on biobutanol production: glucose, MgSO4,
K2HPO4, peptone, FeSO4, K2HPO4.
The compounds with the greatest to least negative
on biobutanol production: Yeast, Na2CO3,
Tryptone, NaCl, NH4Ac.

phase on the microbial culture, and it has been preferred over batch
and fed-batch mode in terms of both butanol yield and productivity
[109,111]. Continuous mode increased volumetric productivity up
to 1.74 g L
1 h
1 using 6% maltodextrin, but product concentration
was reduced because of the fluctuating solvent level [112]. Con-
centration of the product has been increased by using two or
multistage continuous fermenter [113], free cell continuous reactor
[114] immobilized cell continuous reactor [103,115], cell recycling
and bleeding [96,116] and continuous flash fermentation [97,117].
Compared to free cell mode, cell recycling reactor and immobilized
cell fermenters ensure higher yield. Controlled cell bleeding in a
continuous membrane cell-recycle bioreactor using Clostridium
acetobutylicum BKM19 produced 17.6 g L
1 bio-butanol with a
maximum butanol productivity of 9.6 g L
1 h
1 [116]. Continuous
adsorbed fermentation would be a promising option in this context.
3.5. Optimization of fermentation medium
Different attempts have been made to optimize microbial cul-
ture conditions to increase bio-butanol production. Optimization of
fermentation medium by means of different chemicals has been
summarized in Table 3.
Addition of substrate supplements, such as glucose, butyrates
and acetates, butyric acid and hydrogen sparging in headspace has
been explored by several researchers [27,85,96,118,119]. Supple-
mentation of these co-substrates in optimum amount drives
metabolism in the desired direction. The inclusion of allopurinol
has been demonstrated to increase the ability of C. beijerinckii to
tolerate and detoxify furfural as it inhibited xanthine oxidoreduc-
tase activity, an enzyme involved in dissimilation of furfural and
furfuryl alcohol [120]. Addition of artificial electrons carriers, such
as methyl viologen, neutral-red, methylene blue and dimethyl
sulfoxide (DMSO) have been reported to increase bio-butanol
production, possibly due to electron flow regulation, favouring
solventogenesis step [119,121e124]. Addition of 20 mM ferric cit-
rate to batch culture of Clostridium beizerinckii represented an in-
crease of bio-butanol yield up to 5.5-fold. Fe3þ acted as an electron
sink and increased metabolism by readily recycling anthraquinone-
2, 6-disulphonate (AQDS) to transfer more electrons [73,125].
Besides, utilization of different chemical compounds (calcium
hydroxide, calcium carbonate, sodium carbonate, potassium
hydrogen phosphate) has allowed the removal of accumulated toxic
substances and buffering of fermentation media to maintain the
appropriate pH [28,62,126]. Calcium carbonate (CaCO3) supply
(5 g L
1) to several sugar solutions (glucose, mannose, arabinose,
and xylose) in standard culture medium of Clostridium acetobuty-
licum DSM reported remarkable increase in the degree and rate of
sugar consumption and higher concentration of both bio-butanol
and ABE final solution [28]. Al-Shorgani et al. (2013), screened
eleven nutrient factors affecting biobutanol production using the
Placket Burman Design [127]. Glucose, magnesium sulphate,
dipotassium phosphate, peptone, ferrous sulphate and monop-
otassium phosphate showed, in this order, the most positive effect
on biobutanol production. Several low cost, nutrient rich, waste
biomass, such as brewery industry liquid waste or apple pomace
ultrafiltration sludge could be supplemented to enhance
 S. Maiti et al. / Biomass and Bioenergy 94 (2016) 187e200 195

Table 4
Key factors limiting butanol production.

Achievements Limitations Solution(s) proposed

Biomass 1. Easy fermentation of food crops with higher 1. Food scarcity and high feedstock cost 1. Use of agroindustrial waste and agricultural residues
selection productivity 2. Water management crisis, nitrogen and 2. Microbial strains capable of one pot conversion of
2. Use of low cost cellulosic biomass phosphorus pollution. cellulose to biobutanol
3. Use of renewable low cost lingocellulosic 3. Higher cost to remove lignin layer and 3. Simultaneous microbial saccharification and
biomass inhibitors fermentation process
4. High water usage due to low butanol 4. Use of wastes generated after algal biodiesel
concentration in the fermenter extraction
Microbial strain 1. Clostridia strains have higher sugar intake and 1. High butanol toxicity leads to low Metabolic modification and genetic manipulation of
selection higher butanol productivity butanol titer strains and their co-cultures to increase butanol
2. Non-Clostridia strains have higher butanol 2. Low ABE productivity production
tolerance (microbial hosts)
Optimization of Addition of methyl viologen to increase biobutanol Slower fermentation rate due to cell Addition of viologen with non-toxic metal ion electron
fermenter yield growth inhibition donor
feed
Fermentation 1. Continuous fermentation mode leads to higher 1. Low butanol titer due to product 1. Integration of novel on-line product removal
technique productivity inhibition 2. Search for novel high-capacity absorbents
2. Higher ABE titer and productivity in absorbent 2. Decreased solvent yield on increased
fermentation (solid-state fermentation sugar concentration (>100 g L
1)
evolution)
Recovery 1. Multiple column distiller decanter reduces 1. Installation of supplementary columns 1. Integration of liquid-liquid-extraction or pervapora-
methods energy required increase cost of separation tion with distillation will reduce separation cost.
2.1. Use of extractant alcohols with high 2.1. Low selectivity and toxicity of 2. Mixture of decanol and oleyl alcohol used for high
distribution coefficient alcohol countered separation. distribution coefficient and low toxicity
2.2. Utilization of alkanes with high biobutanol 2.2. Alkanes have low distribution 3. Alternative ionic liquids of higher selectivity and
selectivity coefficient lower synthesis cost
3. Use of non-toxic ionic liquids with high bio- 3. Higher initial cost required for 4. Manufacture of stable membranes with high
butanol selectivity have increased solvent synthesis of ionic liquids selectivity and high butanol flux
separation 4. Membrane selectivity counter balances 5. Manufacture of mixed matrix membranes having
4. Use of low-energy demand pervaporation to permeate flux thin and stable hydrophobic layer on porous support
remove efficiently ABE from the broth 5. Decrease of membrane stability and 6. Design of supported ionic liquid membranes with
5. Thinner hydrophobic membrane for higher ABE selectivity optimized support liquid interface
permeate flux 6. Low mechanical stability due to loss of 7. Adsorption viability can be explored by integrating it
6. Utilization of supported ionic liquid membranes liquid membrane from the support with ionic liquid extraction or gas stripping.
for high selectivity and promising flux matrix 8. Multi stage gas stripping coupled with adsorption
7. Use of bio-compatible adsorption method 7. Scarce possibility in industrial use due would be a more efficient method
8. Gas stripping with self-producing gases (CO2, H2) to low capacity and low selectivity
for operational simplicity 8. Low selectivity, high water and energy
requirements

production and make biochemical production of butanol cost
competitive.
3.6. Recovery processes
Several obstacles limit conventional ABE fermentation: low
yield (0.28e0.33 g g
1) due to conversion of approximately 53% of
substrate into CO2 and H2; low sugar consumption (<60 g L
1); and
low productivity (0.30e0.50 g L
1 h
1) resulting from severe in-
hibition caused by accumulated butanol [3,4,128]. These factors
lead to high downstream processing cost and large quantities of
disposal water for recovering butanol by conventional distillation
method, the main separation process used in industrial plants
[99,129]. As an alternative to distillation, several on-line product
removal techniques, such as liquideliquid extraction [130], perva-
poration [131], perstraction [132], gas stripping [91,101], reverse
osmosis [133], aqueous two-phase separation [134], ionic liquid
extraction [135] and adsorption [136] have been investigated to
eliminate the effect of butanol inhibition and enhance ABE pro-
ductivity and sugar utilization [137].
These techniques had been operated both in situ and ex situ. In
situ product recovery is simple and energy saving, but microor-
ganisms could be damaged and fouling and clogging episodes could
happen. A filtration step of the product broth prior to the separation
will eliminate clogging possibility. However, higher capital invest-
ment is required for these kinds of changes. Ex-situ mode of sepa-
ration is defined as a more bio-compatible system, since it is carried
out in a separate column or chamber. Most of these processes were
developed at laboratory-scale but they have not been economically
viable as yet for industrial scale butanol recovery. Hybrid separa-
tion processes using suitable combinations of two or more sepa-
ration processes seem to be the future trend that industry will
follow.
3.7. Hybrid technology of separation-fermentation process
integrated with more than one recovery process
As no process or unit operation is 100% efficient, attention has
been brought to hybrid technology of separation [129,138e143].
Energy-related footprint data for the recovery of butanol from ABE
mixtures using combined alternatives have been extensively
described in the literature. Thus, Groot et al. (1992), estimated that
3.67 MJfuel kg
1
ABE would be required to recover butanol and an
ethanol/acetone mixture from an aqueous feed containing a total of
5% of ABE solvents using a combination of a beer stripper and a
three-phase distillation system [144,145]. proved that combination
of pervaporation and distillation was more effective (7.4 MJfuel
kg
1
BuOH of energy consumption) to recover 99% pure butanol from
0.5% (w/v) feed stream in comparison to energy intensive simple
distillation (79.5 MJfuel kg
1
1
BuOH). Similarly, 9.0 MJfuel kgBuOH was
required when a combination of silicate adsorption and distillation
was used to recover butanol from 0.5 wt% ABE solution [146].
Compared to bench distillation/decantation process, about 63%
energy was saved using membrane assisted vapour stripping
hybrid system (7.81 MJ Kg
1BuOH) [129,138]. developed an innovative
two-stage gas stripping process for in situ butanol recovery from
196 S. Maiti et al. / Biomass and Bioenergy 94 (2016) 187e200
Biomass
Lignocellulose
cellulose>hemicellulose> lignin
Microorganism
pH adjustment
GMO :
Hyper producing Ca(OH)2 and
dilute HCl
+ High solvent
tolerant
Supplementation
in fermentation broth
Brewery Industry
Liquid waste +
Crude Glycerol
Spent fermented broth
Biogasification/
Phosphate solubilizer
Fig. 2. Suggested process diagram to enhance biochemical production of butanol.
ABE fermentation with a fibrous bed bioreactor that was able to
significantly reduce at least 50% of the energy consumption
(7e15 MJ kg
1) in final product recovery. In the first-stage, gas
stripping was used to remove ABE solvent from the fermentation
broth. The second-stage gas stripping was operated to further
concentrate butanol from the aqueous phase formed with the
condensate collected from the first-stage gas stripping. The final
concentrated mixture purified by two-stage gas stripping con-
tained more than 400 g L
1 butanol.
Kraemer et al. (2011), observed that extraction of butanol using
mesityl alcohol or oleyl alcohol followed by distillation was 71% and
23% respectively less energy consuming in comparison to conven-
tional distillation (19.4 MJ kg
1) [147]. Moreover, the butanol pro-
cessing industry Butamax™, patented the method of butanol
recovery by extraction with oleyl alcohol followed by gas stripping,
a hybridized method for efficient in situ product recovery [148].
Proper selection of combination of lower cost and more efficient
separation processes based on fermentation techniques and prod-
uct composition is essential for cost effective production of butanol.
Green, (2011) showed that 2% increase in volumetric productivity
could reduce approximately 20% of capital expenditure and sig-
nificant operation cost reduction. Future research will be directed
towards a rigorous optimization of the hybrid process, bringing
down the energy demand and reducing the capital costs.
4. Commercial biochemical production of butanol and R&D
in industry
Biochemical production of butanol as a liquid transportation
second generation biofuel is, globally, second largest anaerobic
fermentation industrial process after bioethanol production by
yeast [149]. Biobutanol has closer resemblance to gasoline in the
Pretreatment
More reducing sugar release together
with less inhibitory generation
Removal of inhibitors
Two phase bioreactor
Recovery and
purification
Fermentation
In-situ hybridge
technology
Continuous Liquid liquid
Batch followed by fed batch extraction
with in-situ product recovery followedd by
pervoporation
Pure
Butanol
airefuel ratio than ethanol, which enables the usage of a greater
percentage of butanol in its blend with gasoline (85% Butanol/
gasoline) without impacting the fuel storage and fuel economy.
Besides, butanol can be blended with gasoline without phase sep-
aration and without modification to the vehicle engine, whereas
modifications are required for automobiles to use E85 (85% ethanol,
15% gasoline) [150,151]. Additionally, Wu et al. (2014), observed
that, as compared to gasoline, corn-based n-butanol could save
39e56% fossil fuel while reducing GHG emissions by up to 48% on a
lifecycle basis [152].
Lignocellulosic biomass based butanol production could be a
promising renewable energy source for a country with abundant
biomass resources, such as Canada. However, in developed coun-
tries with ample fossil fuel provisions, commitment with these
kinds of gasoline alternatives is still not sufficient. In Canadian case,
less than 5% of the internal energy demand was met through
biomass consumption (International Energy Association, 2004).
Nevertheless, the multiple advantages of butanol have attracted the
attention of a number of oil companies [151]. Thus, more than 470
patent families have been issued in the last decade (2004e2014),
with claims covering all the aspects potentially improvable in
biobutanol full-scale production: biomass pretreatment process,
fermentation, genetic improvement, separation and purification
methods, reactor engineering, compounds concentration control in
the medium culture and genetic vectors construction to increase
yield [153]. Companies already involved in bioethanol production
have taken advantage of the fact that all necessary sophisticated
infrastructures for the separation and purification of ABE fermen-
tation products generated are already in place [154].
In 2006, Dupont and BP enterprises led the way to produce
butanol using sugar beets with the target of 30000 tons of pro-
duction per year. In 2008, Green Biologics signed an agreement
 S. Maiti et al. / Biomass and Bioenergy 94 (2016) 187e200
with Laxmi Bio Industries to build a biobutanol production com-
mercial plant in India. Further, in 2009, a joint venture of Dupont
and BP resulted in Butamax™, which has been actively involved in
butanol biochemical production [129]. Since 2010, Cobalt Tech-
nologies (California) operates a pilot plant producing up to 20000 L
of biobutanol per year. In May 2012, Gevo, a USA biofuel producer
backed by French oil company Total SA and specialty-chemicals
maker Lanxess AG, started the production of butanol with an
estimated annual capacity of z68 million litres. It was reported as
world's first commercial scale production, developed by conversion
of a former corn ethanol plant in Luverne (Minnesota). After
overcoming challenges during initial stages (including temporal
shutdown due to microbial contamination), the company was on
target to produce 189000e379000 L per month of isobutanol by the
end of 2014 [155].
Apart from these petroleum refineries, biotech companies, such
as Cathay Industrial Biotech (China), Butalco GmBH (Switzerland),
Optinol (US), Metabolic explorer (France), Abengoa (Spain) are now
investigating novel alternatives to traditional ABE fermentation,
which would enable biobutanol to be produced on an industrial
scale, either by exploring with modified Clostridium strains,
fermentation technology or recovery techniques.
5. Future outlook and conclusions
Re-establishment of sustainable biochemical production of
butanol is essential to minimize environmental and social re-
percussions caused by the forthcoming depletion of fossil fuels.
Butanol profitable production via ABE fermentation requires a
multidisciplinary approach, coordinating the most recent advances
in environmental science, biochemistry, microbiology, genetic en-
gineering, chemical engineering, process technology and market
economics. Biobutanol production involves many different steps,
such as biomass selection, procurement, hydrolysis and detoxifi-
cation, optimization of fermenting solution, bacterial strain selec-
tion, acclimation and development, solvent separation and
purification, storage, transportation and marketing. Any change in
one or more steps resulting from scientific research has been
proven to create new challenges to meet sustainable production of
butanol. In Table 4, different counteractive factors and their corre-
sponding workable solutions have been summarized. Additionally,
a process diagram has been proposed to enhance biochemical
production of butanol via ABE fermentation (Fig. 2). In conclusion,
the success of biobutanol re-commercialization after so many de-
cades should be linked to the utilization of low-cost cellulose
enriched lignocellulose feedstock by hyper-butanol producing and
high solvent tolerant microbial strains. Industrial standardization of
integrated hybrid separation techniques in order to achieve effi-
cient continuous substrate fermentation and solvent in situ recov-
ery could be the best option in the next future. Utilization of huge
amount of spent fermented broth as phosphate solubilizer instead
of landfilling could have positive effect to make biobutanol cost
effective again.
Acknowledgements
Financial supports from the Natural Sciences and Engineering
Research Council of Canada (NSERC, Discovery Grant), MAPAQ (No.
809051) 90330229, Ministe re des Relations Internationales du

bec (coope
ration Parana

-Que
bec 2010e2012; Quebec-Vietnam
Que
2012e2015) 23525 and the Centre de Recherche Industrielle du

bec (CRIQ) 122629 are sincerely acknowledged. We thank
Que
PRQNT for the postdoctoral scholarship of short duration to Dr.
Gorka Gallastegui for working on biobutanol production.
197
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