A Review On Fermentative Production of Biobutanol From Biomass

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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 produce acetone and butanol than acetobutylicum group of
resistance. In order to achieve maximum growth of microor- solventogenic Clostridia. Clostridium ljungdahlii is capable
ganisms, various strategies have been used such as steriliza- of utilizing hydrogen and carbon monoxide from synthesis
tion, decontamination, and disinfection [30]. gas as a substrate [34].
The gram positive, sporulating, rod shaped, anaerobic ABE-producing Clostridia have large substrate utilization
genus Clostridium is one of the best organisms for butanol range as a multitude of carbon sources such as xylose, glu-
production. For the biobutanol production, the most com- cose, lactose, sucrose, xylan, glycerol, and starch [33]. The
monly used strains are Clostridium acetobutylicum, C. sac- metabolism for butanol production is presented in (Fig. 1).
charobutylicum, C. beijerinckii, C. aurantibutyricum, C. The process starts with glucose and after production of 2
saccharoperbutylacetonicum, and C. carboxidivorans [30, moles of pyruvate from 1 mole of glucose, undergoes the
62]. Strain C. beijerinckii P260 and BA101 are the best process of acidogenesis followed by solventogenesis to pro-
available strains till date that produce high concentration of duce acetone, butanol and ethanol.
butanol [31, 32]. Other species have also been reported to
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- of lignocellulosic materials have also been studied exten-
TANOL FERMENTATION sively [42]. Lignocellulosic biomass with higher content of
cellulose and hemicellulose can be hydrolysed readily into
The major factor in determining the economy of ABE
pentoses and hexoses, which are potential feedstock for
fermentation is the cost of substrate. Initially starch or car-
biobutanol production [31].
bohydrate sources such as potatoes, rice, sorghum, pearl mil-
let, apple pomace, cheese whey, and Jerusalem artichokes Rice straw can be seen as easily available and abundant
were used as substrate. But these substrates are used as food lignocellulosic biomass, which is a larger component of
source. In this context, lignocellulosic biomass can be used agro-residue, could be used as a potential source of feedstock
as a cheaper substrate for biobutanol production due to its for biobutanol and other biofuels production. Total annual
several advantages such as availability in abundance and no rice straw production in India is 97 MMTPA (million metric
competetion with the food chain. Lignocellulosic biobutranol tonnes per annum). Out of this 22 MMTPA is unused yearly,
production consists of major three steps: pretreatment, en- could be seen as potential feedstock for biobutanol produc-
zymatic hydrolysis and fermentation. After pretreatment, tion [37]. The typical composition of milled rice straw pow-
there is a need of hydrolysis step for fermentabe sugar pro- der is; cellulose 37 wt.%, hemicelluloses 24 wt.%, and lignin
duction. The use of enzymes for hydrolysis could be expen- 14 wt.%, with particle size of ~4 mm [43]. Rice straw cannot
sive and also causes end point inhibition due to the incom- be used directly for bioconversion due to the presence of the
plete hydrolysis [64]. high lignin content. Therefore, pre-treatment of rice straw is
required which releases simpler sugars (glucose, mannose,
Lignocellulosic materials contain lignin, hemicellulosic,
xylose, etc.), for the production of alcoholic biofuels through
and cellulose with their varying proportions. Climate and a fermentation process [44]. Dilute sulfuric acid pre-
associated environmental factors govern the availability of
treatment method is widely used to open up crystalline and
lignocellulosic materials in different parts of the world [35].
rigid complex of lignin, hemicellulose and cellulose. This
As they are abundant and renewable resources, lignocellu-
pretreatment allows the hydrolytic enzyme to act upon hemi-
losic materials have the potential to be the largest source of
cellulose and thus fermentable sugars (arabinose, glucose,
biomass for the production of biofuels [36]. Agricultural
xylose, etc.) are generated [45].
residues like wheat straw [31], rice straw [37], barley straw
[38], wheat bran [39], switch grass, corn stover [40], bagasse The fractionation of biomass by dilute acid pretreatment
[41], etc. have been extensively studied. Apart from agricul- is a very promising approach, but it often produces degrada-
tural residues, other feedstocks, such as municipal waste, tive side products, which tend to inhibit efficient fermenta-
used paper, soil organic carbon, wheat flour, and other type tion process of such biomass [46]. Inhibitors like acetic acid,
A Review on Fermentative Production of Biobutanol From Biomass Current Biochemical Engineering, 2016, Vol. 3, No. 1 41

formic acid, furfurals, and hydroxymethylfurfural (HMF) are is produced by fungi, bacteria and protozoans. A very few
produced in hydrolysate. These inhibitors can be removed by reports have been published on the biodepolymerization of
detoxification method using seralite SRA400 anionic resin. lignocellulosic materials using whole cell and enzymatic
The hydrolysate can be analysed before detoxification and process. Potential of R. communis as a substrate for biofuel
the efficiency of detoxification can be further determined by production is manifested by its biochemical composition,
analyzing the remaining acids and furfurals present in resin lignin 19.8% and cellulose 42%. Biodelignification up to
treated samples. Yeast extract and calcium carbonate me- 85.69% has been reported with laccase and a low concentra-
dium with detoxified pre-treated rice straw hydrolysate tion of cellulase in 4 h. The yield of reducing sugar is re-
yields biobutanol 4.46 g l-1 and productivity of 0.05 g l-1h-1 ported to be enhanced by about 2.68 times from the deligni-
using Clostridium sporogenes [47]. The inhibitors can also fied R. communis [70]. Optimization of such processes can
be minimized by using milder acidic condition, followed by have a great impact on the development of more economical
enzymatic hydrolysis, but high cost and comparatively low processes for the production of biobutanol.
efficiency of enzymes may add to the operating cost and
There are various advantages of using extremophiles in
increase the duration of fermentation [48].
biobutanol production. Extremophiles produce pH and tem-
In a recent study, batch fermentation of acidic pre-treated perature stable enzymes. Moreover, fermantation process
rice straw under anaerobic condition was carried out. Clos- using extremphiles, are less prone to contamination and re-
tridium acetobutylicum NCIM 2337, an obligate anaerobic quire lower energy input. Thermophilic clostridia are the
and mesophilic strain used for this process, capable of utiliz- fermentative anarobes, which are reported an optimal growth
ing a range of carbohydrates. During the process of conven- at 60-65°C . To this regard, Clostridium thermocellum has
tional hydrolysis of lignocellulosic biomass at higher tem- been used in mixed fermentations with Thermoanaerobacte-
perature, an additional high pressure and shear stress can rium thermosaccharolyticum, Thermoanaerobacter thermo-
also be applied. There was a rapid release of fermentable hydrosulphuricum, Thermoanaerobacter ethanolicus,
sugar mainly glucose and other sugars such as xylose, arabi- Geobacillus stearothermophilus, Thermoanaerobacter
nose, etc. in minor concentrations as a result of such pre- brockii and Thermoanaerobacterium saccharolyticum. As,
treatment. This stress assisted with acid hydrolysis of rice this mixed culture can uptake both hexoses and petoses as
straw resulted in maximum sugar yield with the concentra- fermentable sugars, can be used for biofuel production [71].
tion of ~3.9% (w/v) from aqueous solution of the 5% (w/v)
rice straw. Total fermentable sugar utilized during fermenta- 4. FERMENTATIVE PRODUCTION OF BUTANOL
tion found to be 61.4%, with reducing sugars 57.8% and
The production of biobutanol can be more successful
glucose with 55.1%. This study manifests that rice straw as
when it achieves certain targets, such as- higher butanol po-
an economical and easily available substrate for butanol pro-
ductivity, higher butanol titer and purity, process simplifica-
duction. Further improvement of this process can be done by
tion, to overcome the problem of coupling between sporula-
nutrient supplementation of rice straw hydrolyzates, using
tion and solvent production, minimize the energy consump-
genetically modified strains and optimization of physical
parameters [37]. Lignocellulosic biomass hydrolysis by en- tion at down stream processing, and enhanced rate of fer-
mentation [52]. During the process, some frequent problems
zymes is carried out at 45ºC [49] or 50ºC [50]. Another ad-
are encountered while achieving above mentioned targets
vantage of pretreatment at high temperature is the inhibition
such as selection of sustainable raw material, substrate and
of the growth of other mesophilic anaerobic microbes
product inhibition, low product yield, high cost of purifica-
thereby minimizing of contamination in ABE fermentation
tion and process complexity [30].
process by these organisms [51].
The presence of carbon, nitrogen, iron, and factors like
Another potential substrate for butanol production is cas-
temperature, pH of medium is very important aspect of the
tor. India is the major castor seed producer followed by Bra-
fermentation process. For higher productivity of butanol,
zil and China. Ricinus communis is castor oil yielding herba-
excess of carbon under limited nitrogen is needed. Iron is
ceous plant from Euphorbiaceae family, containing a potent
another important supplement, because the conversion of
cytotoxin. A huge quantity of this is either burnt or buried
because it is not fit for animal fodder [67]. However, the pyruvate to acetyl-CoA includes a ferredoxin oxidoreductase
iron-sulfur protein. The pH of fermentation broth plays an
stem and leaf of this plant can be used as the source of fer-
important role in the solventogenesis phase of the process
mentable sugars. There is need of an efficient pretreatment
[54].
mechanism of such lignocellulosic biomass that could make
the cellulose and hemicellulose more accessible for hydroly-
(i) Batch Process
sis and reduce the cost of fermentable sugar production from
cellulosic biomass [68, 69]. Among various hydrolysis Determination of various parameters of fermentation
methods, enzymatic hydrolysis stands out as it does not re- process such as suitable biomass application, standardization
sult in byproducts such as HMF, furfural and acetic acid, of pretreatment method, and saccharification through differ-
which act as inhibitors during the fermentation process. Fur- ent experiments, all these can be done with the batch reactor
thermore, enzymatic hydrolysis may produce higher reduc- process initially. This process gives lower butanol concentra-
ing sugar yield. Application of laccases and celluloses for the tion compared to other types of reactor processes [6]. The
biodeploymerization R. communis has been reported. Lac- initial studies manifested the productivity of a traditional
cases (EC 1.10.3.2) oxidize the phenolic groups of lignin, batch ABE fermentation process in the range of 0.1 to 0.3 g
which is commonly produced by white rot fungi and cellu- l-1 h-1 and needed larger fermentors for batch production as
lases (EC 3.2.1.4) catalyze the hydrolysis of cellulose, which 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 Prakash et al.

age volumetric productivity by Clostridia [52, 54]. The togenesis, which manifest a great potential as substrates for
maximum concentration of solvents, acetone, butanol and butanol production [33].
ethanol by using C. acetobutylicum ATCC 824 was found to
The fed-batch process can be more successful for butanol
be 20 g/L with the ratio 6:3:1 [30, 53].
production when product removal strategy is carried out
In batch fermentation, it is projected that in order to pro- along with the butanol production. The butanol in higher
duce 80 giga gram of butanol annually, at least 25,000 m3 of concentration is reported to be toxic to the butanol producing
the fermentation capacity would be needed [52]. In a study strains such as C. acetobutylicum and C. beijerinckii. This
employing C. beijerinckii BA101 in batch mode, need of approach has been applied for the production of biobutanol
reactor size ranging from 1 to 10 L by applying a variety of using C. beijerinckii where a feed containing 500 gL1 and
carbon sources, where production achieved up to 18-33 g L-1 product recovery carried out by membrane separation (per-
in 72 h with productivity in the range of 0.25–0.46 gL-1h-1 vaporation) or gas stripping [56]. In a study, 15 gL1 of buta-
[54]. The reasons behind low productivity in batch reactor nol recovered with volumetric productivity of 0.6 g l1 h1 in
process can be cited due to long lag phase, downtime dura- a fed batch process, when 50% of feed glucose was utilized
tion between batches, product inhibition, and low cell con- along with a mixture of butyric acid and glucose were sup-
centration in the reactor [54]. Improvement in butanol yield plied [52]. It was found that no butyrate used in the process,
can be achieved if the butanol products are removed along the production of butanol found to be very low and volumet-
with the fermentation or by strain improvement for example, ric productivity was found to be 0.4 g l1 h1 with a dilution
to make C. beijerinckii amenable to high butanol concentra- rate of 0.04 h1 [52]. In another study of feeding butyric acid
tion, it needs to be mutated or genetically modified [6]. in the broth along with glucose in pH-stat condition, the fed-
batch fermentation process preformed using C. accharoper-
pH is an important factor in ABE fermentation. Several
butylacetonicum. The maximum of 16 gL-1 butanol concen-
reports suggest that solvents (butanol, acetone, and ethanol)
being produced at pH values approaching to neutral, i.e. fer- tration was achieved with productivity higher than routine
batch process [30]. The effect of lactic acid on fermentation
mentation performed at relatively higher pH values, solvents
and butanol yield was also studied using C. saccharoperbu-
are produced rather than acids, whereas at relatively low pH,
tylacetonicum N1-4 strain [33]. Along with the modified
acids are produced rather than solvents [55]. Higher initial
technology of pH-stat lactic acid and glucose feeding system
sugar concentration promotes solvent production at pH near
in fed-batch process, the butanol concentration found to be
to neutral. Interaction between pH, initial sugar concentra-
tion, and the fermenting strain has been studied by several elevated up to 15.5 gL-1 [33].
scientists. It has been reported that relatively lower lactose
(iii) Continuous Process
concentration and low pH favor solventogenesis with poor
utilization of carbon source. With the same carbon source, at There are various factors that limit the fermentation proc-
higher pH value, both growth and carbon source utilization ess, which can be compensated by using continuous fermen-
have been improved [56]. Furthermore, pH should be con- tation process. Demerits of batch and fed-batch processes
trolled during the process rather than simple pH main- such as time consuming sterilization of bioreactors, substrate
tainance for a given substrate and bacterial strain [53]. and product inhibition, and low productivity (in case of batch
The size of the reactor for ABE fermentation is not lim- process) can be overcome with this mode of operation [30].
ited by the oxygen transfer rate as it is an anaerobic process. The continuous culture process technology can be used for
Therefore, improvement in volumetric productivity can be the culture physiology study at steady state. The culture can
made by increasing biomass concentration in the system be initiated in batch mode and biomass is allowed to grow
[52]. It can simply be done by fixing the cells onto support till exponential phase. At this stage of the process, the reac-
medium or gel particle, i.e. cell immobilizing, by cell recy- tor is continuously fed with medium. The primary advantage
cling using a membrane, or by reactive separation techniques of continuous process is that the steady state can be main-
[54]. tained by optimizing the process for as long as required.
There are several limitations associated with single-stage
(ii) Fed-batch Process continuous reactor. High productivity can only be obtained,
when the product concentration is maintained low during the
The Fed-batch is extremely useful for alleviating sub-
process. Therefore, two or more multistage systems are in-
strate inhibition. In such type of process, the bioreactor is
troduced [54]. The most common strategies can be practiced
started in batch mode with a low initial feed concentration
under continuous fermentation processes are free and immo-
and low medium volume [57]. This process can be trans-
bilized cell, and biomass recycle processes [30].
formed into a fed-batch process by adding fresh concentrated
culture medium slowly as the replacement of used substrate
Free Cell Continuous Fermentation Processes
by the culture. When the process volume is nearly 75% of
the reactor volume, the process is stopped [54]. The produc- It is a type of continuous process, where cells freely
tion of butanol can be enhanced by using substrates in the move within fermentation broth. The cells and the nutrients
media that can favor butanol production in ABE fermenta- are maintained in the suspension form. The free cell suspen-
tion processes. Various organic acids like acetate, lactate, sion condition is being maintained by providing proper agita-
butyrate, and propionate can be utilized for this purpose. The tion either by a mechanical agitator or by air lifting method,
acids produced in the process, viz. acetic acid and butyric to promote the mass transfer [30]. It is one of the most poten-
acid during acidogenesis phase, can be reutilized in solven- 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 Current Biochemical Engineering, 2016, Vol. 3, No. 1 43

where glucose used as substrate and cell wash-out of C. sac- tivity, which was a biggest hurdle in biobutanol production.
charoperbutylacetonicum N1-4 occurred at a dilution rate of This new process consists of three different processes: fer-
0.26 h-1. This resulted a ABE productivity of 1.85 gL-1h-1, mentation process, cell retention system, and a vacuum flash
similar productivities are observed in case of C. acetobutyli- vessel for continuous separation of butanol from fermenta-
cum and C. beijerinckii [33]. tion broth. This technology can be useful and more economi-
cal during distillation process, butanol production is ex-
Immobilized Cell Continuous Fermentation Process pected to be more than 20 gL–1 butanol and also generates
lower quantity of waste water that means more eco-friendly
There are various advantages of Immobilized cell fer-
technology [30].
mentation over free cell continuous fermentation. Some of
these have been stated below:
5. TECHNICAL AND ECONOMIC FEASIBILITY
1) Increased survival time of cells: The immobilized cells
Continuous mode of operations comprising of free/im-
are in a condition where mechanical agitation is lacking,
mobilized cells and/or biomass recycling processes are pre-
there is long survival time of the cells present in solven-
ferred over conventional batch or fed batch due to the cost
togenesis phase. This process was applied in a fibrous-
effectiveness, higher productivity, and lower inhibition
bed process bioreactor with C. acetobutylicum cells us-
percentage [30]. However, it is an established fact that
ing corn as substrate, showed 20% higher butanol yield
over conventional continuous fermentation techniques continuous processes are prone to contamination. Separation
of product by distillation or pervaporation in all the three
[58].
modes have been reported [53]. Entrapment of Clostridium
2) Higher cells concentration: In immobilized cell process, spp. cells into lens shaped polyvinyl alcohol (PVA) hydrogel
higher cell concentrations can be maintained, which re- is one of the methods used for immobilization. Due to the
sult in high reactor productivity. several advantages, viz. lower cost of the matrix, simpler
3) Stabilization of metabolic and operational activity: In- method of gel preparation, simple separation from medium,
creased cell density can be achieved by cell immobiliza- excellent mechanical stability, nearly non-degradable and
tion that provides longer operational stability and stabi- low diffusion limits, increases the application of PVA for
lized metabolic activity in ABE-producing clostridia, as immobilization in repeated batch, fed-batch and continuous
culture degeneration is a problem associated with con- fermentation processes. Immobilization with PVA does not
tinuous process [33]. inhibit the growth, but allows accumulation of the biomass
within the matrix and thus increases the efficiency of fer-
The immobilized cell technique in ABE fermentation has mentation [63]. Using this method, significant time reduction
been successful at laboratory level, facing the challenge to of 3.3 h in repeated batch fermentation process has been re-
meet the demands for commercial production [30]. An im- ported while maintaining equal yields. With repeated batch
mobilized cell continuous fermentation process by using C. and fed-batch fermentations, the highest butanol productivity
beijerinckii BA101 strain has been scaled up and productiv- 1.21 g/L/h and solvent productivities 1.91 g/L/h have been
ity achieved closer to that of a laboratory scale reactor [30]. reported after 6.5 h with a final butanol yield of 0.15 g/g
Some other state of the art techniques for butanol fermenta- [62]. The various types of processes for butanol production
tion is as follows: have been described here.

Cell-recycle Fermentation Process Development of economic process with product recov-


ery, scale-up of the process with mathematical models as a
This is a new technique giving the opportunity to im- tool, and optimization and control of feasible technologies
prove the butanol productivity, where cell density has been are important areas of research. Process can be developed
maintained by recycling of cells using ultrafiltration or mi- with the batch, fed-batch, and continuous modes of bioreac-
crofiltration membrane modules. This process manifested tor operation. Time reduction in sterilization and inoculation,
advantages such as homogeneity of broth that facilitates dif- high productivity and low butanol inhibition are the advan-
fusion and total recycling of microorganisms over fermenta- tages of continuous process whereas high product recovery
tion process in a cell-immobilized bioreactor [30, 33]. In cost is the disadvantage caused by low concentration of the
such systems, the process is started as batch mode and cells product [59]. The process which starts with low substrate
are allowed to grow with process proceeds. As the culture concentration and substrate is periodically added to maintain
reaches stationary phase, the fermentation broth is passed the fermentation process is the fed-batch process. Another
through ultrafiltration or microfiltration membrane. Selection alternative process is the continuous packed bed reactor
of the membrane is such so as to allow product as permeate (PBR) where immobilized microorganisms are used in the
[54]. If this system of cell-recycle is compared with continu- fermentation process. This technology makes the product
ous fermentation method without cell recycling, using high recovery easier. Batch process is the mostly used and pre-
cell density of C. saccharoperbutylacetonicum it allowed ferred over other processes because it can be handled easily
tenfold higher cell concentration with six fold higher product and maintenance of anaerobic condition, temperature con-
yield (11 gL–1h–1), compared to conventional continuous trol, pH control, sampling can easily be carried out. Moreo-
fermentation without cell recycling (1.85 gL–1h–1) [30]. ver, a separation unit can also be added with lesser difficul-
ties with this mode of operation [60].
Continuous Flash Fermentation Process
Mathematical modeling in ABE fermentation is a useful
The Continuous flash fermentation process came forward tool to short-list the best combinations of process parameter
with potential that can overcome the problem of low produc-
44 Current Biochemical Engineering, 2016, Vol. 3, No. 1 Prakash et al.

out of different permutation and combinations of the experi- CONFLICT OF INTEREST


mental parameter, which is much impractical. Model of Pa-
The author(s) confirm that this article content has no con-
pautsakis (1984) provides ABE fermentation equation and
flict of interest.
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- ACKNOWLEDGEMENTS
zyme catalyzes acetic acid. Intracellular concentration of Declared none.
acid was related to extracellular concentration of acid.
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Received: February 20, 2015 Revised: July 03, 2015 Accepted: July 31, 2015

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