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A Review on Fermentative Production of Biobutanol From Biomass

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Current Biochemical Engineering, 2016, 3, 37-46 37

A Review on Fermentative Production of Biobutanol From Biomass

Anand Prakash1, Ravi Dhabhai1 and Vinay Sharma1,*

1
Department of Bioscience and Biotechnology, Banasthali University, P.O. Banasthali Vidyapith, Ra-
jasthan, 304022, India

Abstract: Fossil fuels are the primary source of energy production. However, owing to their negative
effects and ever increasing demand of energy, biofuels, especially, biobutanol is gaining increased in-
terest. At present, the biofuel market is dominated by biodiesel, bioethanol, biobutanol, and biogas,
much relying on the substrates such as- sugars, starch, oil crops, agricultural and animal residue, and
lignocellulosic biomass. Butanol (C4H9OH) has superior fuel properties over traditional fuel ethanol in
terms of energy density and hygroscopicity. There are some bottlenecks that need to be overcome in
the production of butanol in order to get a sustainable alternative for fossil fuels. Lignocellulosic bio-
mass due to its low cost and yearlong availability, is a suitable raw material for butanol production. In this mini review,
feedstock, microorganism, process modifications, and past and present trends in biobutanol production have been dis-
cussed. In this review, an attempt has been made to develop a better understanding about acetone-butanol-ethanol fermen-
tation process.
Keywords: Energy, biofuel, butanol, ABE fermentation, lignocellulosic biomass.

1. INTRODUCTION such as - higher energy content (29.2 MJ/L), lower miscibil-

ity with water, less corrosiveness, higher octane number, and
Fosil fuels, such as petroleum, are the most utilized en-
higher flash point than traditional biofuel like ethanol and
ergy resources which are not renewable and fast depleating.
has similar characteristics with gasoline. Due to these prop-
The continuous and abundant use of petroleum fuels has
erties, it can be seen as a superior fuel candidate as well as
increased the greenhouse gases (GHG) emissions including can directly be used as an alternative to gasoline or fuel addi-
CO, CO2, NOx, and other gases. By reducing the use of pe-
tive in current internal combustion engine without any modi-
troleum fuels, these emissions could be reduced which can
fication [6, 7]. Chemical or biochemical conversion of buta-
lead to a cleaner environment [1]. Considerable interest has
nol to other valuable chemical compounds such as ethers,
been evoked in alternative energy resources due to several
esters and acetates can also be carried out [8]. Every year 4
environmental concerns [2]. All over the world, the focus of
to 6 billion kilograms of butanol are produced with the ap-
research is to develop novel processes for producing fuel and proximate cost of US$ 7.0–8.4 billion. The use of butanol as
related products from renewable resources like biomass, to
fuel or fuel additive is not very encouraging as nearly half of
find efficient and sustainable energy sources that are eco-
the produced butanol utilized for ester and acrylate produc-
nomically competitive, technically feasible, easily available
tion, this clearly indicates the high requirement of butanol
and eco-friendly. In Indian context, there is an immense need
production [6].
to develop a sustainable technology for alternative fuel pro-
duction [3]. Microbial production of acetone-butanol-ethanol (ABE)
has been considered as one of the pioneer industrial biotech-
Biofuels have been considered as a relevant technology
nological processes for solvent production [9]. The historical
for energy production and possibly to replace fossil fuels.
development of ABE fermentation can be divided into dif-
Biofuels have several advantages which include energy secu-
ferent time periods [10] and is described in (Table 2) [11-
rity, cleaner environment, less dependence on foreign cur-
28]. There are some challenges in the fermentative produc-
rency, and socio-economic issues [4]. Biobutanol (C4H9OH) tion of butanol and their possible solutions. These are pre-
is one such superior biofuel and could be seen as a potential
sented in (Table 3) [5, 29].
alternative to conventional fuels. The comparative analysis
of different fuels with respect to their fuel properties is In the present review paper, the technology such as feed-
summarized in (Table 1) [5]. stock, microorganism, process modifications has been dis-
cussed and recent advancements in biobutanol production
Butanol is a colorless liquid and is miscible with other have been critically reviewed.
organic solvents [6]. There are various advantages of butanol
2. MICROORGANISMS EMPLOYED FOR BUTANOL
*Address correspondence to this author at the Department of Bioscience and PRODUCTION
Biotechnology, Banasthali University, P. O. Banasthali Vidyapith, Ra-
jasthan, 304022, India; Tel: +91-1438-228302; The selection of bacterial strains for butanol production
E-mail: vinaysharma30@yahoo.co.uk is dependent upon feedstock, nutritional requirements,

2212-7127/16 $58.00+.00 © 2016 Bentham Science Publishers

38 Current Biochemical Engineering, 2016, Vol. 3, No. 1 Prakash et al.

Table 1. Comparison of properties of different fuels (modified from [5]).

Fuel Property Diesel Rapeseed Oil Methanol Ethanol n-butanol

Cetane number 40-55 50 3 8 12

Density (g/mL) 0.86 0.92 0.80 0.79 0.81

Auto-ignition temperature (°C) 200-220 >300 470 434 385

Lower heating value (MJ/Kg) 42.5 37.6 19.9 26.8 35.1

Boiling point (°C) 180-230 >350 64.5 78.4 117

Saturation temperature (°C) - <0.1 20 20 20

Saturation pressure (kPa) - - 11.83 5.93 0.6

Table 2. History and development of ABE fermentation.

Years Scientists Contributions and Developments References

1861 Louis pasteur First reported the butanol production from microbial source and termed as ‘Vibron Butyrique’. [11]

1905 Schardinger Production of acetone by fermentation [12]

1910 Perkins and Weiz- Reported the best metabolic pathway for isoprene or butadiene production from isoamyl alcohol
[13, 14]
mann or butanol

1911 Fernbach Isolated microbial population that could ferment potatoes to butanol, but not maize starch. [14, 15]

1912 Weizmann Butanol or isoamyl alcohol formation through fermentation found to be important for the success
[16]
of synthetic rubber process.

1912-1914 Weizmann During this, multiple numbers of cultures reported, one from these BY. Later this was named as
Clostridium acetobutylicum. This strain possessed ability to convert various starchy biomass into [14]
acetone and butanol.

1913 Strange and Gra- Strange and Graham Ltd. Applied for a patent of a process using Fernbach’s bacillus strain. By
[15]
ham this year they started production of acetone and butanol at Rainham, using potatoes as substrate.

1916 Weizmann British Admiralty took over the Strange and Graham Ltd. plant at King's Lynn under "The De-
fence of the Realm Act". They made changes to Weizmann process and started utilizing maize as [17]
basic material for the process.

1918 Weizmann During this time acetone production for cordite and airplane dope started and in Canada butanol
manufacturing and storage in vats also started. Conversion of butanol to methyl ethyl ketone [15]
begins in small quantity.

1919-1920 Weizmann and In 1920 U.S. Weizmann patent allotted them a license to start butanol manufacturing at the Terre
EloiRicard Haute plant. They started operation according to U.S license and later got worldwide process [18]
rights of this patent.

1923 Terre Haute plant, This was the time when bacteriophase contamination affected the yield badly. To rectify this
[17-19]
with Weizmann problem a research team developed at the Terre Haute plant under the supervision of Weizmann.

1935 J. Hastings During this time the Commercial Solvents Corp and the Distiller's Co. in a joint venture project
developed plant that could produce industrial level of alcohol, acetone and buanol with distillation
[19]
units. The plant was completely operated by trained British personnel under guidance of
J.Hastings. The phase immunized C.saccharoacetobutylicum strain used for the process.

1936-1941 New Acetone Butanol fermentation plants constructed in Philadelphia, Pa., Baltimore, Md., and
Puerto Rico and became functional with the expire of Weizmann patent in 1936. After this year
many new isolated strains reported with better fermentation capability from molasses. During this
[20, 21]
period about 18 patents issued covering process using different strains, adapted by big production
houses like Publiker Industrial Inc., Commercial Solvents Corp., Western Condensing Co., and
U.S. Industrial Chemicals Co.
A Review on Fermentative Production of Biobutanol From Biomass Current Biochemical Engineering, 2016, Vol. 3, No. 1 39

(Table 2) contd….

Years Scientists Contributions and Developments References

1936-1960 Plants construction for the solvent production also started in different part of world such as South
Africa, Australia, Japan, and India. In between 1950 and 1960 various researches about AB fer-
[22-24]
mentation process published and implemented for acetone and butanol production by continuous
process. With these modification operation started in Dokshukino in the USSR in 1960.

1950-1960 After the last phase of second world war acetone-butanol fermentation rapidly retarded in differ-
ent part of the world, may be due to rise in substrate cost and increased competition to the indus- [19, 25]
try for cheap raw substrate material.

1970-1982 By 1970 Organic chemical Factories of Egyptian Sugar and Distillation Company was partially
[26, 27]
functional. South African plant, the only plant working until 1982.

1980-1990 During this period intensive research focused in a direction of process improvement, process
optimization, strain improvement, improvement of culture cultivation, using various alternative [28]
fermentation substrates and improvement of product removal technologies.

1990 onwards With the development of recombinant DNA technology, genetic manipulation and strain im-
provement of solvent-producing clostridia successfully completed in laboratories. These ap-
[28]
proaches are proper documented in various research articles, patents and reviews. These are well
implemented at industrial scale production.

Table 3. Biobutanol production: Challenges and solutions (Adapted from [29-30]).

Challenges Solutions

Increased operating cost due to high raw material cost Switching to cheaper and sustainable raw material such as lignocellulosic
materials

High cost of recovery due to low yield, limited sugar loading Development of improved microoraganisms with improved yield and/or
butanol selectivity; Development of novel reactive separation processes

Capital and operating cost increases due to low volumetric productivity Development of continuous fermentation processes for reduction in reaction
time

Product recovery with conventional distillation is expensive and energy Development of low cost energy efficient methods for solvent purification
intensive and recovery.

High water usage is not sustainable and caused higher cost of effluent treat- Recycling the process water back through the fermentation.
ment.

Formation of undesirable or low value by-products Engineering of respective knock-out or deletion strains

Culture degeneration Avoid the excessive acidification of the culture.

Phage contamination Good culture practices, proper sterilization, use of phage resistant strains

substrate and product tolerance, high productivity, and phage
resistance. In order to achieve maximum growth of microor-
ganisms, various strategies have been used such as steriliza-
tion, decontamination, and disinfection [30].
The gram positive, sporulating, rod shaped, anaerobic
genus Clostridium is one of the best organisms for butanol
production. For the biobutanol production, the most com-
monly used strains are Clostridium acetobutylicum, C. sac-
charobutylicum, C. beijerinckii, C. aurantibutyricum, C.
saccharoperbutylacetonicum, and C. carboxidivorans [30,
62]. Strain C. beijerinckii P260 and BA101 are the best
available strains till date that produce high concentration of
butanol [31, 32]. Other species have also been reported to
produce acetone and butanol than acetobutylicum group of
solventogenic Clostridia. Clostridium ljungdahlii is capable
of utilizing hydrogen and carbon monoxide from synthesis
gas as a substrate [34].
ABE-producing Clostridia have large substrate utilization
range as a multitude of carbon sources such as xylose, glu-
cose, lactose, sucrose, xylan, glycerol, and starch [33]. The
metabolism for butanol production is presented in (Fig. 1).
The process starts with glucose and after production of 2
moles of pyruvate from 1 mole of glucose, undergoes the
process of acidogenesis followed by solventogenesis to pro-
duce acetone, butanol and ethanol.
40 Current Biochemical Engineering, 2016, Vol. 3, No. 1 Prakash et al.

2NADH, 2ATP
Glucose 2Pyruvate

Oxidized ferredoxin Hydrogenase

2COA
2NADPH 2NADH
2Co2 2NADP 2NAD H2
Reduced ferredoxin
2NAD(P)H 2 Co-A Acetate kinase
2Pi
2 Acetaldehyde 2Acetyl-CoA 2 Acetyl-P 2 Acetate
2Co-A 2ATP
2NAD(P)H Phosphotrans-acetylase
Thiolase

2 Ethanol Co-A Transferase

CoA Co2
Acetoacetyl Co-A Acetoacetate Acetone

3-OH-butyryl-CoA dehydrogenase NADH

-hydroxybutyryl-CoA

Crotonase
H2O
Crotonyl-CoA

Butyryl-CoA dehydrogenase NADH

CoA
ATP
Butyraldehyde Pi
Butyryl-CoA Butyryl-P Butyrate
CoA NADH
NADH Butyrate kinase
Butyraldehyde dehydrogenase Phosphotrans butyrylase

Fig. (1). Metabolic reactions for butanol production.

3. LIGNOCELLULOSIC RAW MATERIALS FOR BU-
TANOL FERMENTATION
The major factor in determining the economy of ABE
fermentation is the cost of substrate. Initially starch or car-
bohydrate sources such as potatoes, rice, sorghum, pearl mil-
let, apple pomace, cheese whey, and Jerusalem artichokes
were used as substrate. But these substrates are used as food
source. In this context, lignocellulosic biomass can be used
as a cheaper substrate for biobutanol production due to its
several advantages such as availability in abundance and no
competetion with the food chain. Lignocellulosic biobutranol
production consists of major three steps: pretreatment, en-
zymatic hydrolysis and fermentation. After pretreatment,
there is a need of hydrolysis step for fermentabe sugar pro-
duction. The use of enzymes for hydrolysis could be expen-
sive and also causes end point inhibition due to the incom-
plete hydrolysis [64].
Lignocellulosic materials contain lignin, hemicellulosic,
and cellulose with their varying proportions. Climate and
associated environmental factors govern the availability of
lignocellulosic materials in different parts of the world [35].
As they are abundant and renewable resources, lignocellu-
losic materials have the potential to be the largest source of
biomass for the production of biofuels [36]. Agricultural
residues like wheat straw [31], rice straw [37], barley straw
[38], wheat bran [39], switch grass, corn stover [40], bagasse
[41], etc. have been extensively studied. Apart from agricul-
tural residues, other feedstocks, such as municipal waste,
used paper, soil organic carbon, wheat flour, and other type
of lignocellulosic materials have also been studied exten-
sively [42]. Lignocellulosic biomass with higher content of
cellulose and hemicellulose can be hydrolysed readily into
pentoses and hexoses, which are potential feedstock for
biobutanol production [31].
Rice straw can be seen as easily available and abundant
lignocellulosic biomass, which is a larger component of
agro-residue, could be used as a potential source of feedstock
for biobutanol and other biofuels production. Total annual
rice straw production in India is 97 MMTPA (million metric
tonnes per annum). Out of this 22 MMTPA is unused yearly,
could be seen as potential feedstock for biobutanol produc-
tion [37]. The typical composition of milled rice straw pow-
der is; cellulose 37 wt.%, hemicelluloses 24 wt.%, and lignin
14 wt.%, with particle size of ~4 mm [43]. Rice straw cannot
be used directly for bioconversion due to the presence of the
high lignin content. Therefore, pre-treatment of rice straw is
required which releases simpler sugars (glucose, mannose,
xylose, etc.), for the production of alcoholic biofuels through
a fermentation process [44]. Dilute sulfuric acid pre-
treatment method is widely used to open up crystalline and
rigid complex of lignin, hemicellulose and cellulose. This
pretreatment allows the hydrolytic enzyme to act upon hemi-
cellulose and thus fermentable sugars (arabinose, glucose,
xylose, etc.) are generated [45].
The fractionation of biomass by dilute acid pretreatment
is a very promising approach, but it often produces degrada-
tive side products, which tend to inhibit efficient fermenta-
tion process of such biomass [46]. Inhibitors like acetic acid,
A Review on Fermentative Production of Biobutanol From Biomass
formic acid, furfurals, and hydroxymethylfurfural (HMF) are
produced in hydrolysate. These inhibitors can be removed by
detoxification method using seralite SRA400 anionic resin.
The hydrolysate can be analysed before detoxification and
the efficiency of detoxification can be further determined by
analyzing the remaining acids and furfurals present in resin
treated samples. Yeast extract and calcium carbonate me-
dium with detoxified pre-treated rice straw hydrolysate
yields biobutanol 4.46 g l-1 and productivity of 0.05 g l-1h-1
using Clostridium sporogenes [47]. The inhibitors can also
be minimized by using milder acidic condition, followed by
enzymatic hydrolysis, but high cost and comparatively low
efficiency of enzymes may add to the operating cost and
increase the duration of fermentation [48].
In a recent study, batch fermentation of acidic pre-treated
rice straw under anaerobic condition was carried out. Clos-
tridium acetobutylicum NCIM 2337, an obligate anaerobic
and mesophilic strain used for this process, capable of utiliz-
ing a range of carbohydrates. During the process of conven-
tional hydrolysis of lignocellulosic biomass at higher tem-
perature, an additional high pressure and shear stress can
also be applied. There was a rapid release of fermentable
sugar mainly glucose and other sugars such as xylose, arabi-
nose, etc. in minor concentrations as a result of such pre-
treatment. This stress assisted with acid hydrolysis of rice
straw resulted in maximum sugar yield with the concentra-
tion of ~3.9% (w/v) from aqueous solution of the 5% (w/v)
rice straw. Total fermentable sugar utilized during fermenta-
tion found to be 61.4%, with reducing sugars 57.8% and
glucose with 55.1%. This study manifests that rice straw as
an economical and easily available substrate for butanol pro-
duction. Further improvement of this process can be done by
nutrient supplementation of rice straw hydrolyzates, using
genetically modified strains and optimization of physical
parameters [37]. Lignocellulosic biomass hydrolysis by en-
zymes is carried out at 45ºC [49] or 50ºC [50]. Another ad-
vantage of pretreatment at high temperature is the inhibition
of the growth of other mesophilic anaerobic microbes
thereby minimizing of contamination in ABE fermentation
process by these organisms [51].
Another potential substrate for butanol production is cas-
tor. India is the major castor seed producer followed by Bra-
zil and China. Ricinus communis is castor oil yielding herba-
ceous plant from Euphorbiaceae family, containing a potent
cytotoxin. A huge quantity of this is either burnt or buried
because it is not fit for animal fodder [67]. However, the
stem and leaf of this plant can be used as the source of fer-
mentable sugars. There is need of an efficient pretreatment
mechanism of such lignocellulosic biomass that could make
the cellulose and hemicellulose more accessible for hydroly-
sis and reduce the cost of fermentable sugar production from
cellulosic biomass [68, 69]. Among various hydrolysis
methods, enzymatic hydrolysis stands out as it does not re-
sult in byproducts such as HMF, furfural and acetic acid,
which act as inhibitors during the fermentation process. Fur-
thermore, enzymatic hydrolysis may produce higher reduc-
ing sugar yield. Application of laccases and celluloses for the
biodeploymerization R. communis has been reported. Lac-
cases (EC 1.10.3.2) oxidize the phenolic groups of lignin,
which is commonly produced by white rot fungi and cellu-
lases (EC 3.2.1.4) catalyze the hydrolysis of cellulose, which
Current Biochemical Engineering, 2016, Vol. 3, No. 1 41
is produced by fungi, bacteria and protozoans. A very few
reports have been published on the biodepolymerization of
lignocellulosic materials using whole cell and enzymatic
process. Potential of R. communis as a substrate for biofuel
production is manifested by its biochemical composition,
lignin 19.8% and cellulose 42%. Biodelignification up to
85.69% has been reported with laccase and a low concentra-
tion of cellulase in 4 h. The yield of reducing sugar is re-
ported to be enhanced by about 2.68 times from the deligni-
fied R. communis [70]. Optimization of such processes can
have a great impact on the development of more economical
processes for the production of biobutanol.
There are various advantages of using extremophiles in
biobutanol production. Extremophiles produce pH and tem-
perature stable enzymes. Moreover, fermantation process
using extremphiles, are less prone to contamination and re-
quire lower energy input. Thermophilic clostridia are the
fermentative anarobes, which are reported an optimal growth
at 60-65°C . To this regard, Clostridium thermocellum has
been used in mixed fermentations with Thermoanaerobacte-
rium thermosaccharolyticum, Thermoanaerobacter thermo-
hydrosulphuricum, Thermoanaerobacter ethanolicus,
Geobacillus stearothermophilus, Thermoanaerobacter
brockii and Thermoanaerobacterium saccharolyticum. As,
this mixed culture can uptake both hexoses and petoses as
fermentable sugars, can be used for biofuel production [71].
4. FERMENTATIVE PRODUCTION OF BUTANOL
The production of biobutanol can be more successful
when it achieves certain targets, such as- higher butanol po-
ductivity, higher butanol titer and purity, process simplifica-
tion, to overcome the problem of coupling between sporula-
tion and solvent production, minimize the energy consump-
tion at down stream processing, and enhanced rate of fer-
mentation [52]. During the process, some frequent problems
are encountered while achieving above mentioned targets
such as selection of sustainable raw material, substrate and
product inhibition, low product yield, high cost of purifica-
tion and process complexity [30].
The presence of carbon, nitrogen, iron, and factors like
temperature, pH of medium is very important aspect of the
fermentation process. For higher productivity of butanol,
excess of carbon under limited nitrogen is needed. Iron is
another important supplement, because the conversion of
pyruvate to acetyl-CoA includes a ferredoxin oxidoreductase
iron-sulfur protein. The pH of fermentation broth plays an
important role in the solventogenesis phase of the process
[54].
(i) Batch Process
Determination of various parameters of fermentation
process such as suitable biomass application, standardization
of pretreatment method, and saccharification through differ-
ent experiments, all these can be done with the batch reactor
process initially. This process gives lower butanol concentra-
tion compared to other types of reactor processes [6]. The
initial studies manifested the productivity of a traditional
batch ABE fermentation process in the range of 0.1 to 0.3 g
l-1 h-1 and needed larger fermentors for batch production as
well as long periods of operation time with 0.5 g l-1 h-1 aver-
42 Current Biochemical Engineering, 2016, Vol. 3, No. 1
age volumetric productivity by Clostridia [52, 54]. The
maximum concentration of solvents, acetone, butanol and
ethanol by using C. acetobutylicum ATCC 824 was found to
be 20 g/L with the ratio 6:3:1 [30, 53].
In batch fermentation, it is projected that in order to pro-
duce 80 giga gram of butanol annually, at least 25,000 m3 of
the fermentation capacity would be needed [52]. In a study
employing C. beijerinckii BA101 in batch mode, need of
reactor size ranging from 1 to 10 L by applying a variety of
carbon sources, where production achieved up to 18-33 g L-1
in 72 h with productivity in the range of 0.25–0.46 gL-1h-1
[54]. The reasons behind low productivity in batch reactor
process can be cited due to long lag phase, downtime dura-
tion between batches, product inhibition, and low cell con-
centration in the reactor [54]. Improvement in butanol yield
can be achieved if the butanol products are removed along
with the fermentation or by strain improvement for example,
to make C. beijerinckii amenable to high butanol concentra-
tion, it needs to be mutated or genetically modified [6].
pH is an important factor in ABE fermentation. Several
reports suggest that solvents (butanol, acetone, and ethanol)
being produced at pH values approaching to neutral, i.e. fer-
mentation performed at relatively higher pH values, solvents
are produced rather than acids, whereas at relatively low pH,
acids are produced rather than solvents [55]. Higher initial
sugar concentration promotes solvent production at pH near
to neutral. Interaction between pH, initial sugar concentra-
tion, and the fermenting strain has been studied by several
scientists. It has been reported that relatively lower lactose
concentration and low pH favor solventogenesis with poor
utilization of carbon source. With the same carbon source, at
higher pH value, both growth and carbon source utilization
have been improved [56]. Furthermore, pH should be con-
trolled during the process rather than simple pH main-
tainance for a given substrate and bacterial strain [53].
The size of the reactor for ABE fermentation is not lim-
ited by the oxygen transfer rate as it is an anaerobic process.
Therefore, improvement in volumetric productivity can be
made by increasing biomass concentration in the system
[52]. It can simply be done by fixing the cells onto support
medium or gel particle, i.e. cell immobilizing, by cell recy-
cling using a membrane, or by reactive separation techniques
[54].
(ii) Fed-batch Process
The Fed-batch is extremely useful for alleviating sub-
strate inhibition. In such type of process, the bioreactor is
started in batch mode with a low initial feed concentration
and low medium volume [57]. This process can be trans-
formed into a fed-batch process by adding fresh concentrated
culture medium slowly as the replacement of used substrate
by the culture. When the process volume is nearly 75% of
the reactor volume, the process is stopped [54]. The produc-
tion of butanol can be enhanced by using substrates in the
media that can favor butanol production in ABE fermenta-
tion processes. Various organic acids like acetate, lactate,
butyrate, and propionate can be utilized for this purpose. The
acids produced in the process, viz. acetic acid and butyric
acid during acidogenesis phase, can be reutilized in solven-
Prakash et al.
togenesis, which manifest a great potential as substrates for
butanol production [33].
The fed-batch process can be more successful for butanol
production when product removal strategy is carried out
along with the butanol production. The butanol in higher
concentration is reported to be toxic to the butanol producing
strains such as C. acetobutylicum and C. beijerinckii. This
approach has been applied for the production of biobutanol
using C. beijerinckii where a feed containing 500 gL1 and
product recovery carried out by membrane separation (per-
vaporation) or gas stripping [56]. In a study, 15 gL1 of buta-
nol recovered with volumetric productivity of 0.6 g l1 h1 in
a fed batch process, when 50% of feed glucose was utilized
along with a mixture of butyric acid and glucose were sup-
plied [52]. It was found that no butyrate used in the process,
the production of butanol found to be very low and volumet-
ric productivity was found to be 0.4 g l1 h1 with a dilution
rate of 0.04 h1 [52]. In another study of feeding butyric acid
in the broth along with glucose in pH-stat condition, the fed-
batch fermentation process preformed using C. accharoper-
butylacetonicum. The maximum of 16 gL-1 butanol concen-
tration was achieved with productivity higher than routine
batch process [30]. The effect of lactic acid on fermentation
and butanol yield was also studied using C. saccharoperbu-
tylacetonicum N1-4 strain [33]. Along with the modified
technology of pH-stat lactic acid and glucose feeding system
in fed-batch process, the butanol concentration found to be
elevated up to 15.5 gL-1 [33].
(iii) Continuous Process
There are various factors that limit the fermentation proc-
ess, which can be compensated by using continuous fermen-
tation process. Demerits of batch and fed-batch processes
such as time consuming sterilization of bioreactors, substrate
and product inhibition, and low productivity (in case of batch
process) can be overcome with this mode of operation [30].
The continuous culture process technology can be used for
the culture physiology study at steady state. The culture can
be initiated in batch mode and biomass is allowed to grow
till exponential phase. At this stage of the process, the reac-
tor is continuously fed with medium. The primary advantage
of continuous process is that the steady state can be main-
tained by optimizing the process for as long as required.
There are several limitations associated with single-stage
continuous reactor. High productivity can only be obtained,
when the product concentration is maintained low during the
process. Therefore, two or more multistage systems are in-
troduced [54]. The most common strategies can be practiced
under continuous fermentation processes are free and immo-
bilized cell, and biomass recycle processes [30].
Free Cell Continuous Fermentation Processes
It is a type of continuous process, where cells freely
move within fermentation broth. The cells and the nutrients
are maintained in the suspension form. The free cell suspen-
sion condition is being maintained by providing proper agita-
tion either by a mechanical agitator or by air lifting method,
to promote the mass transfer [30]. It is one of the most poten-
tial strategies that can alleviate product inhibition by feeding
fresh medium at a constant rate. In a chemostat culture study,
A Review on Fermentative Production of Biobutanol From Biomass
where glucose used as substrate and cell wash-out of C. sac-
charoperbutylacetonicum N1-4 occurred at a dilution rate of
0.26 h-1. This resulted a ABE productivity of 1.85 gL-1h-1,
similar productivities are observed in case of C. acetobutyli-
cum and C. beijerinckii [33].
Immobilized Cell Continuous Fermentation Process
There are various advantages of Immobilized cell fer-
mentation over free cell continuous fermentation. Some of
these have been stated below:
1) Increased survival time of cells: The immobilized cells
are in a condition where mechanical agitation is lacking,
there is long survival time of the cells present in solven-
togenesis phase. This process was applied in a fibrous-
bed process bioreactor with C. acetobutylicum cells us-
ing corn as substrate, showed 20% higher butanol yield
over conventional continuous fermentation techniques
[58].
2) Higher cells concentration: In immobilized cell process,
higher cell concentrations can be maintained, which re-
sult in high reactor productivity.
3) Stabilization of metabolic and operational activity: In-
creased cell density can be achieved by cell immobiliza-
tion that provides longer operational stability and stabi-
lized metabolic activity in ABE-producing clostridia, as
culture degeneration is a problem associated with con-
tinuous process [33].
The immobilized cell technique in ABE fermentation has
been successful at laboratory level, facing the challenge to
meet the demands for commercial production [30]. An im-
mobilized cell continuous fermentation process by using C.
beijerinckii BA101 strain has been scaled up and productiv-
ity achieved closer to that of a laboratory scale reactor [30].
Some other state of the art techniques for butanol fermenta-
tion is as follows:
Cell-recycle Fermentation Process
This is a new technique giving the opportunity to im-
prove the butanol productivity, where cell density has been
maintained by recycling of cells using ultrafiltration or mi-
crofiltration membrane modules. This process manifested
advantages such as homogeneity of broth that facilitates dif-
fusion and total recycling of microorganisms over fermenta-
tion process in a cell-immobilized bioreactor [30, 33]. In
such systems, the process is started as batch mode and cells
are allowed to grow with process proceeds. As the culture
reaches stationary phase, the fermentation broth is passed
through ultrafiltration or microfiltration membrane. Selection
of the membrane is such so as to allow product as permeate
[54]. If this system of cell-recycle is compared with continu-
ous fermentation method without cell recycling, using high
cell density of C. saccharoperbutylacetonicum it allowed
tenfold higher cell concentration with six fold higher product
yield (11 gL–1h–1), compared to conventional continuous
fermentation without cell recycling (1.85 gL–1h–1) [30].
Continuous Flash Fermentation Process
The Continuous flash fermentation process came forward
with potential that can overcome the problem of low produc-
Current Biochemical Engineering, 2016, Vol. 3, No. 1 43
tivity, which was a biggest hurdle in biobutanol production.
This new process consists of three different processes: fer-
mentation process, cell retention system, and a vacuum flash
vessel for continuous separation of butanol from fermenta-
tion broth. This technology can be useful and more economi-
cal during distillation process, butanol production is ex-
pected to be more than 20 gL–1 butanol and also generates
lower quantity of waste water that means more eco-friendly
technology [30].
5. TECHNICAL AND ECONOMIC FEASIBILITY
Continuous mode of operations comprising of free/im-
mobilized cells and/or biomass recycling processes are pre-
ferred over conventional batch or fed batch due to the cost
effectiveness, higher productivity, and lower inhibition
percentage [30]. However, it is an established fact that
continuous processes are prone to contamination. Separation
of product by distillation or pervaporation in all the three
modes have been reported [53]. Entrapment of Clostridium
spp. cells into lens shaped polyvinyl alcohol (PVA) hydrogel
is one of the methods used for immobilization. Due to the
several advantages, viz. lower cost of the matrix, simpler
method of gel preparation, simple separation from medium,
excellent mechanical stability, nearly non-degradable and
low diffusion limits, increases the application of PVA for
immobilization in repeated batch, fed-batch and continuous
fermentation processes. Immobilization with PVA does not
inhibit the growth, but allows accumulation of the biomass
within the matrix and thus increases the efficiency of fer-
mentation [63]. Using this method, significant time reduction
of 3.3 h in repeated batch fermentation process has been re-
ported while maintaining equal yields. With repeated batch
and fed-batch fermentations, the highest butanol productivity
1.21 g/L/h and solvent productivities 1.91 g/L/h have been
reported after 6.5 h with a final butanol yield of 0.15 g/g
[62]. The various types of processes for butanol production
have been described here.
Development of economic process with product recov-
ery, scale-up of the process with mathematical models as a
tool, and optimization and control of feasible technologies
are important areas of research. Process can be developed
with the batch, fed-batch, and continuous modes of bioreac-
tor operation. Time reduction in sterilization and inoculation,
high productivity and low butanol inhibition are the advan-
tages of continuous process whereas high product recovery
cost is the disadvantage caused by low concentration of the
product [59]. The process which starts with low substrate
concentration and substrate is periodically added to maintain
the fermentation process is the fed-batch process. Another
alternative process is the continuous packed bed reactor
(PBR) where immobilized microorganisms are used in the
fermentation process. This technology makes the product
recovery easier. Batch process is the mostly used and pre-
ferred over other processes because it can be handled easily
and maintenance of anaerobic condition, temperature con-
trol, pH control, sampling can easily be carried out. Moreo-
ver, a separation unit can also be added with lesser difficul-
ties with this mode of operation [60].
Mathematical modeling in ABE fermentation is a useful
tool to short-list the best combinations of process parameter
44 Current Biochemical Engineering, 2016, Vol. 3, No. 1
out of different permutation and combinations of the experi-
mental parameter, which is much impractical. Model of Pa-
pautsakis (1984) provides ABE fermentation equation and
stoichiometric balance equation. Stoichiometric model with
nonlinear constraint proposed by Desai et al., 1999 proposed
new method based on uptake of butyric acid and same en-
zyme catalyzes acetic acid. Intracellular concentration of
acid was related to extracellular concentration of acid.
Yerushalmi et al., 1986 proposed physiological model,
which is based on both intra and extracellular culture condi-
tions and transport parameters for solvents. The concept of
general framework for extractive fermentation developed at
Nagoya University (Japan) by Honda and his colleuges
which could be applied to batch, repeated-batch and repeated
fed-batch modes. Fermentation model with cell retention is
based on inhibition of specific growth rate due to butyric
acid. Models with immobilized cultures in trickle bed reactor
employ the concept of an equilibrium stage with steady state
and isothermal operation and surface reaction with no diffu-
sion limitation in an immobilization matrix [61].
There are three main factors which govern the economics
of butanol fermentation: mode of fermentation, recovery of
solvent, and feedstock used. The first two factors decide total
fixed capital investment and third factor is responsible for
total production cost. According to analysis of Roeffler et al.
[65], capital cost of extractive fed-bath fermentation is 20%
lower than normal batch process because of removal of buta-
nol during the process and treatment of broth with concen-
trated sugar in extractive fermentation [64]. Some of the fac-
tors which influence the economic viability of ABE fermen-
tation such as batch process is always economical than con-
tinuous process but maintaining sterility is a crucial issue,
continuous process operation is very advantageous but there
is huge investment in fermentation plant, absolute sterile
operation being very expensive than non-sterile which is
susceptible to contamination. Plant automation can reduce
the cost of production, the sale of byproduct or utilizing the
byproduct in plant itself can be economical production and
process plant with substrate flexibility is highly economical
one [66].
6. CONCLUSION
Compared to other alcoholic fuels, butanol has its own
advantages, but these are overshadowed by the shortcomings
or challenges encountered for the successful industrial adap-
tation of the technology. The cost of fermentation substrate
is the key factor in the microbial production of biobutanol.
Different low cost lignocellulosic substrates have been inves-
tigated for ABE fermentation process, however, there is need
of intensive research with regard to the efficient utilization of
these materials. Out of the different pretreatment methods,
biodepolymerization of lignocellulosic materials using whole
cell and enzymatic process is proven to be more efficient.
Extremophiles are robust organisms producing stable en-
zymes, and are often able to tolerate changes in environ-
mental conditions. Strain improvement, growth condition
optimization, and suitable substrate utilization can give bet-
ter results for biobutanol production in future. Over the
years, there have been several technological advancements in
the ABE fermentation which would certainly help to conquer
these shortcomings of the process.
Prakash et al.
CONFLICT OF INTEREST
The author(s) confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
Declared none.
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