Journal ofFlora

Chemical Technology
Tsvetanova, andPetrova,
Penka Metallurgy, 53, 4,
Kaloyan 2018, 683-696
Petrov

MICROBIAL PRODUCTION OF 1-BUTANOL – RECENT ADVANCES

AND FUTURE PROSPECTS
(Review)
Flora Tsvetanova , Penka Petrova2, Kaloyan Petrov1
1

1
Institute of Chemical Engineering, Bulgarian Academy of Sciences Received 02 November 2017
“Georgi Bonchev” str., bl. 103, Sofia, Bulgaria Accepted 05 April 2018
2
Institute of Microbiology, Bulgarian Academy of Sciences
“Georgi Bonchev” str., bl. 26, Sofia, Bulgaria
E-mail: florablue@abv.bg

ABSTRACT

Butanol is an alcohol that can be obtained by fermentation of organic feedstocks sugars. Biobutanol is soon
expected to surpass ethanol as a renewable fuel due to the intensive scientific efforts aimed at substrates broadening,
process parameters improvement and genetic manipulations of butanol producing bacteria. This review focuses on
the biotechnological synthesis of 1-butanol by natural isolates (Clostridium acetobutylicum, C. beijerinkii) and re-
combinant producers (E. coli, C. cellulovorans, C. cellulolyticum, C. thermocellum, S. cerevisiae, etc.). Concerning
the economical butanol fermentation, the contemporary utilization of cheap renewable and non-food substrates
(lignocellulosic materials, glycerol, syngas, and algae) is decribed. Different strategies for advanced 1-butanol pro-
duction are considered. Along with media optimizations, immobilization, and co-culturing, the exploration of the
strains’ tolerance to butanol, and their genetic improvement is widely applied. The recombinants obtained in case
of naturally producing strains application as hosts possess enhanced metabolic flux, shifted towards higher butanol
titer, yield, and productivity. Another approach refers to the insertion of “butanol pathway” genes in a non-solvent
producing host, which is resistant to high butanol concentrations. The present review analyses and summarizes the
problems and the future prospects of the development of economically feasible bio-processes for butanol production.
Keywords: butanol, ABE fermentation, Clostridium, fuel.

INTRODUCTION Significant efforts are made to achieve an eco-

The increasing demand for alternative fuels employ-
ment due to natural resources depletion has forced sci-
entists to explore different strategies in this direction. To
date, ethanol is classified as the most proper biofuel due
to its simple production through existing fermentation
technologies. However, ethanol is not the best choice
as a biofuel because of its high vapor pressure and its
hygroscopicity.
Butanol is probably the most promising among the
possible conventional fuel alternatives because of its
undoubtable advantages over the other known biofuels
and the fact that it can completely replace gasoline with
no engine modifications required (Table 1).
nomical microbial production of butanol in view of its
advantages over other fuels. It is superior to ethanol in
many regards including a high energy content and low
vapor pressure as it is less volatile [1]. Butanol’s low
hygroscopicity defines its low degree of corrosivity to
engines. It can be directly applied with no engines modi-
fications; it blends with gasoline at any ratios and can
be easily transported. Table 1 shows the fuel properties
of butanol compared to those of ethanol and gasoline.
Combined with gasoline, higher alcohols like butanol
can contribute to minimization of the global warming
effect as smoke gases formation is thus decreased. The
alcohols OH-group increases their oxygen content which
leads to decreased smoke gases release [2, 3].

683
 Journal of Chemical Technology and Metallurgy, 53, 4, 2018
Table 1. Comparison of the fuel properties of butanol, ethanol and gasoline.
Property Butanol
Energy density (MJ L-1) 29.2
Water solubility (mL 100 mL-1) 9.1
Vapor pressure 6.4
Oxygen content (% mass) 21.6
Octane rate 96
Butanol (butyl alcohol) is a five carbon alcohol with
four isomeric forms: 1–butanol (n–butanol), 2–butanol,
tert–butanol and isobutanol. It is considered that 1–
butanol and isobutanol have the properties providing
their use as biofuels. Butanol is an important precursor
in plastic and polymers production [4]. It is used as a
solvent in the production of antibiotics, hormones, vita-
mins, in the textile industry [5], and as a parfume base
in the cosmetics [6].
Butanol can be obtained from fossil fuels by chemi-
cal synthesis (as petro – butanol) or from biomass by
microbial fermentation (as biobutanol). The chemical
synthesis is unprofitable and unfounded because of the
over–consumption of petroleum–based products.
As a fermentation metabolite, butanol is mentioned
by Pasteur in 1861. Its industrial production on the
ground of starch and sugars starts a century ago [7].
Its biological production follows the acetone–butanol–
ethanol (ABE) fermentation pathway. The products
ratio usually is 3:6:1 [8]. A major disadvantage of ABE
fermentation is butanol’s low final concentration (< 20
g L-1) and its low yield (0.28 - 0.33 g g-1) as the other
fermentation products are obtained in higher concentra-
tions. The high cost of the substrate is another problem
as feed-stocks are usually used. The cost for its separa-
tion from the rest of the reaction products is high. Also,
butanol is toxic to the microorganism–producers [9].
MICROORGANISMS - PRODUCERS OF 1-BU-
TANOL
Butanol production in nature is extensively per-
formed with the participation of Clostridium genus
members. In 1916 C. acetobutylicum is isolated by
Chaim Weizmann who discovers that it produces butanol
and ethanol from starch. During WWII the strain is used
684
Ethanol Gasoline
21.2 32.5
miscible <0.01
18 - 22 6.4 – 11.9
34.8
112 - 129 91 - 99
for acetone synthesis, which is a precursor for cordite
production [10]. In 1980, the strain C. acetobutylicum
that performs ABE fermentation in ratio 7 (acetone) :
2 (butanol) : 1 (ethanol) is isolated [11]. However, C.
acetobutylicum is Gram-positive spore–forming anaero-
bic bacterium that can survive just a couple of hours in
aerobic atmosphere. This strictly anaerobic physiology
impedes its application [10].
C. acetobutylicum conducts a two–phase fermen-
tation process. Acetic and butyric acids, as well as H2
and CO2 are formed during the first phase. Usually this
phase coincides with the bacteria exponential growth,
when ATP is formed. Then a solvent formation phase
follows when the acids are consumed. Thus acetone, bu-
tanol and ethanol are synthesized [12]. Fig. 1 illustrates
the metabolic pathway of C. acetobutylicum and the
metabolites formed. Clostridia secrete enzymes which
facilitate carbohydrates breaking down to monomeric
units. α–amylase, α–glucosidase, β-amylase, β–glucosi-
dase, glucoamylase, pullulanase and amylopullulanase
are among these enzymes. The monosaccharides ob-
tained are transported into the cell throughout membrane
bounded transport systems. The carbohydrates are
subsequently metabolized following the glycolysis or
pentosophosphate pathway [13].
As C. acetobutylicum produces the highest concen-
tration of butanol among the known microorganisms,
it is the most studied bacterial species in relation to
optimizing the fermentation process and the butanol
final concentration. Tsai et al. study the effect of the
media components and pH on butanol fermentation
by C. acetobutylicum. They add ammonium acetate,
acetate buffer and calcium carbonate in order to control
the media pH. They find out that the addition of 6 g L-1
of ammonium acetate increases butanol concentration
 Flora Tsvetanova, Penka Petrova, Kaloyan Petrov

from 2 g L-1 to 3.6 g L-1. The role of the acetate buffer
is similar as it increases butanol concentration from 2 g
L-1 to 9.8 g L-1. Calcium carbonate affects pH control.
It sustains pH above 4.8 when added in a concentration
higher than 8 g L-1. According to the authors, the optimal
pH for the process is 4.5. The solvent synthesis begins
when pH reaches a certain value above which acids are
re-assimilated and solvents are produced [14]. On the
other hand, the way of pH control conductance is also
of importance [15].
Table 2 lists the natural and engineered microbial
producers of butanol.

Table 2. The natural and recombinant strains used for 1-butanol production.
Organism Substrate Gene Strain engineering Titer Yield Fermentation reactor Reference
overexpressed
C. acetobutylicum Cassava - Genome shuffling 20.1 g L-1 0.23 g g-1 Bioreactor, batch [16]
Glucose pfkA, pykA - 19.12 g L-1 - Bioreactor, fed-batch [17]
Glucose adhE1D485G Δbuk, Δpta 18.9 g L-1 0.29 g g-1 Bioreactor, batch [18]
Glucose groESL operon - 17.1 g L-1 - Bioreactor, fed-batch [19]
C. tyrobutyricum Sugarcane scrB, scrA, scrK - 16 g L-1 0.24 g g-1 Bioreactor [20]
juice
C. pasteurianum Glycerol - - 12 g L-1 0.34 mol mol-1 Bioreactor, batch [21]
C. carboxidovorans CO - - 2.66 g L-1 - Bioreactor, batch [22]
C. cellulovorans Cellulose adhE2 - 1.42 g L-1 0.39 g g-1 Bottle [23]
C. beijerinckii BA101 Glucose - - 18.6 g L-1 0.32 g g-1 Bioreactor, batch [24]
E. coli Glucose atoB, hbd, crt, ΔadhE2, ΔldhA, 15 g L-1 0.36 g g-1 Tube [25]
adhE2, fdh ΔfrdBC, Δpta
E. coli Glucose cimA3.7, leuABCD, Δilvl, ΔilvB 0.524 g L-1 - Flask [26]
kivid, ADH2
E. coli Glucose thrAfbrBC, ilvA, Δilvl, ΔilvB, 1 g L-1 - Flask [27]
leuABCD, kivD, ΔmetA, Δtdh,
ADH2 ΔadhE
E. coli BuT-8 Glucose Δfdh, Δfdh1, 6.1 g L-1 0.31 g g-1 Bioreactor, batch [28]
Δhbd, Δcrt,
ΔadhE2, ΔphaA,
ΔadhE, ΔldhA,
Δpta, ΔfrdA
Glucose yqeF, fucO ΔarcA, ΔadhE, 2.2 g L-1 0.28 g g-1 Flask [29]
Δpta, ΔfrdA,
ΔyqhD, Δeut,
ΔfadR, ato(Con),
crp
TB+glycer atoB, hbd, crt, bcd, ΔadhE, ΔldhA, 0.552 g L-1 - Flask [30]
ol etfAB, adhE ΔfrdBC, Δfnr,
Δpta
S. cerevisiae Glucose CoaA, ΔADH1-5, Δ 130 mg L-1 0.071 g g-1 Batch [31]
adhEA267T/E568K adhEA267T/E568K
Glucose Δgpd1, Δgpd2, 14.1 g L-1 - Flask [32]
Δter, ΔbcdΔatfA,
ΔetfB
Galactose thl,hbd, crt, - 2.5 mg L-1 - Vial [33]
bcd,etfAB, adhE2
T. saccharolyticum Xylose thl, hbd, crt, bcd, Δldh, ermR 1.05 g L-1 0.10 g g-1 Tube [34]
bcd,etfAB, adhE2
P. putida Glycerol thl, hbd, crt, bcd, - 0.122 g L-1 - Flask [35]
bcd,etfAB, adhE1
L. brevis Glucose thl, hbd, crt, bcd, 0.300 g L-1 - Vial [36]
bcd,etfAB
B. subtilis Glycerol thl, hbd, crt, bcd, - 24 mg L-1 - Flask [35]
bcd,etfAB, adhE2
S. elongatus PCC7942 CO2 atoB, hbd, crt, ter, - 15 mg L-1 - Tube [37]
adhE2
C. acetobutylicum/B. Glucose - - 11.2 g L-1 - Bioreactor, batch [38]
cereus
C. beijerinckii/C. Cassava - - 13.26 g L-1 0.42 g g-1 Batch [39]
tyrobutyricum starch

685
 Journal of Chemical Technology and Metallurgy, 53, 4, 2018
SUBSTRATES FOR BUTANOL PRODUCTION
The price of raw materials for fermentations con-
stitutes about ¾ of the total fermentation cost [40]. It is
important to use cheap renewable non-food raw materi-
als aiming to decrease the substrate expenses. However,
mostly glucose and starch-based substrates are used to
date for ABE fermentation [2].
Different raw materials can be used for butanol pro-
duction. Butanol producing clostridia are able to utilize a
wide spectrum of substrates - hexoses, pentoses, syngas,
and glycerol [7]. The ability of engineered cellulitic C.
cellulovorans, C. cellulolyticum and C. thermocellum
to convert directly cellulose to butanol is reported [41].
These species have cellulosomes for degrading and
hydrolyzing crystalline cellulose and hemicelluloses.
Higashide et al. introduce C. cellulolyticum to the isobu-
tanol metabolic pathway obtaining 0.66 g L-1 isobutanol
from cellulose in 7 - 9 days. C. cellulovorans produces
butyric acid as a main product on the ground of different
carbon sources like glucose, xylose, cellulose, hemicel-
luloses, cellobiose. Small amounts of acetic, lactic and
formic acids are formed as by-products. Thus, this bac-
terium is a promising host for butanol production from
cellulosic biomass [42]. According to Tamaru et al. the
high butyrate/acetate ratio (5:1 - 6:1) suggests the exist-
ence of a favorable metabolic pathway from cellulose
to butyryl–CoA. Therefore, only one heterologous gene
(aad/adhE) is required to obtain butanol from cellulose
as a native metabolic pathway exists from cellulose to
butyryl-CoA using C. cellulovorans (Fig. 2) [43].
Glycerol is an attractive opportunity for obtaining
valuable metabolites as 1,3-propanediol, ethanol and
biopolymers. It is synthesized as a by-product in the bio-
diesel industry. However, the naturally butanol produc-
ing microorganisms (C. acetobutylicum, C. beijerinckii,
C. saccharobutylicum) can not utilize glycerol as a sole
carbon source. This is possible if only a glycerol–glucose
mixture can be used [45]. C. pasteurianum is the most
studied microorganism capable of utilizing glycerol as a
carbon source. CO2, H2, butanol, 1,3-propanediol, acetic
acid, lactic acid, and butyric acid are its fermentation
products. Acetone is not formed. Sarchami et al. optimize
the fermentation process using C. pasteurianum DSM
525. They study the effects of the innoculum age, the cel-
lular density, the initial pH and the temperature. Optimal
results are obtained in case of an inocullum age of 16 h,
a cellular density 0.4 g L-1CDW, an initial pH value of 6.8
686
and temperature of 30°C. Thus 0.34 mol butanol/mol
glycerol (starting with 50 g L-1 glycerol) are produced
[21] with the application of this optimal model. Recently,
another strain able to convert glycerol to butanol has
been isolated from sediments. C. tetanomorphum GT6
produces 11.5 g L-1 butanol for 72 h [46].
The lignocellulosic biomass is another possible
substrate for butanol production. It consists mainly of
cellulose, hemicelluloses and lignin. It contains a major
part of the pentoses (mainly D-xylose and L-arabinose),
which is about 30 % of the total sugar content of the
lignocellulosic hydrolysate [47]. Butanol producing
clostridia are able to convert pentoses to butanol as
a sole carbon source. On the other hand, pentose me-
tabolism is repressed in presence of glucose as it is the
preferred carbon source when compared with xylose
and arabinose. The metabolic regulation of pentose
utilization in clostridia is extensively explored [48].
An over-expression of some of the genes related to the
carbon metabolism (ccpA, talA) and the pentose–phos-
phate pathway (tal, tkl, rpe, rpi) is provoked [49] for
pentose utilization acceleration. Thus, a strain capable
of simultaneous fermentation of hexoses and pentoses
as substrates for solvents production is constructed.
Syngas is also a promising substrate for butanol fer-
mentation. It represents a cheap gas mixture of H2, CO
and CO2. It can be obtained from different sources like
natural gas, coal, and biomass [50]. C. glycolicum [51], C.
ljungdahlii [52], C. autoethanogenum [53], and Acetobac-
terium woodii [54] are acetogenic organisms that are able
to utilize it as a carbon and energy source. These bacteria
produce solvents and organic acids via Wood–Ljungdahl
pathway, where CO or CO2 are converted to acetic acid.
Bruant et al. [55] report the strain C. carboxidovorans that
synthesizes 8.91 mmol butanol per CO mol consumed.
Although syngas has the advantages to be used as an
eventual substrate for solvents production, the fermentation
process with the acetogenic bacteria participation has still
to be optimized to provide a controlled culture media [56].
The scientists are lately focusing their attention on
algae utilization. It is an attractive raw material due to
its low cost. It is not a feedstock; it does not require
agricultural lands, fertilizers and fresh water for cultiva-
tion [57]. Marine algae are brown, red and green and are
characterized by their high carbohydrate and low lignin
content. The experimental data show that butanol can be
produced by algal hydrolysates [58] although the yields
 Flora Tsvetanova, Penka Petrova, Kaloyan Petrov
achieved are low. The main disadvantage of the microalgal
employment is that the major part of the microorganisms
is not able to utilize the alginates. The latter can be rapidly
assimilated by Sphingomonas sp. A1 using alginate–lyase
enzymes [59]. Wargacki et al. [57] construct an engineered
strain E. coli by cloning a 36 kb DNA fragment from
the brown microalgae Vibrio splendidus, responsible for
alginate metabolism. Wang et al. [60] use Chlorella vul-
garis JSC-6 pretreated with 1 % NaOH and 3 % H2SO4.
The high level of glucose utilization (97.5 %) evidences
that the hydrolysate does not contain inhibitors of ABE
fermentation. 13.1 g L-1 butanol with a yield of 0.58 mol
mol-1 sugar and productivity of 0.66 g Lh-1 is reached.
STRATEGIES FOR BUTANOL PRODUCTION
ENHANCEMENT
Metal ions impact
The impact of Zn2+ on fructose and xylose assimi-
lation as well as on butanol synthesis is investigated.
Wu et al. [61] report that the addition of 0.001 gL-1
ZnSO4x7H2O to the media including fructose as a sole
carbon source results in 12.8 g L-1 butanol with pro-
ductivity 0.089 g L h-1, compared to the fermentation
in absence of Zn2+ (4.5 g L-1 butanol and productivity
0.028 g L h-1). Zn2+ ions have also a positive impact in
experiments with xylose as a carbon source - 8.3 g L-1
butanol is produced with a productivity increased by
31.7 %. The fermentations with fructose/glucose (4:1)
and xylose/glucose (1:2) mixtures result in an increase of
butanol final concentration and productivity - by 130.2
% and 8.5 % in the first case, while by 203.4 % and
18.4 % in the second one. Zn2+ ions affect probably the
regulatory mechanisms responsible for sugar transport
and metabolism of Clostridium acetobutylicum.
Nitrogen and carbon sources impact
The importance of glucose concentration, butyric
acid addition, carbon and nitrogen ratios for ABE fer-
mentation is reported. Al–Shorgani et al. [62] conduct
batch–processes with C. acetobutylicum YM1. They reach
13.87 g L-1 butanol at optimal conditions. Glucose initial
concentration is 50 g L-1, while the nitrogen sources ratio
(tryptone, yeast extract, ammonium acetate) refers to
6:2:3. They find also that the increase of the initial glucose
concentration above 50 g L-1 leads to diminished butanol
production. Different concentrations of butyric acid
(which is a precursor of ABE fermentation) are added to
the medium. The highest butanol concentration is obtained
in case of butyric acid addition of 87 g L-1.
Immobilization
Aiming to increase the butanol yield, Börner et al.
[63] apply a new immobilization technique. Cells of C.
acetobutylicum DSM 792 form a macroporous aggregate
through cryogelation and simultaneous crosslinking with
activated polyethyleneimin (PEI) and polyvinyl alcohol
(PVA). The cryogel is a highly porous elastic structure
with walls consisting of densely packed crosslinked cells.
The immobilization increases the cell’s vitality. Unlike
the free cells, the immobilized one demonstrate 2.7 fold
increase of butanol concentration and yield - 18.2 g L-1
and 0.41 g g-1, respectively. The immobilized cells have
another advantage - they can be used from 3 to 5 times.
Chen et al. [64] fix cells of C. acetobutylicum CG-
MCC 5234 on pretreated cotton towel carriers. The sub-
strates used refer to glucose and xylose. The immobilized
bacterial cells synthesize 10 g L-1 butanol with a yield of
0.141 g g-1. The butanol obtained is 11.1 g L-1, while its
yield is 0.190 g g-1 in case of using a mixed sugar media
(30 g L-1 glucose + 30 g L-1 xylose). These values are by
28.3 % higher than those found in free cells processes.
Co-culturing
Another approach to the increase of the concentration
of the synthesized butanol refers to the co-culturing of two
different butanol producing strains. Free and immobilized
cells of C. beijerinckii and C. tyrobutyricum are cultivated
[65] in media containing glucose, cassava starch and cane
molasses. The highest values are reached in experiments
with cassava starch, i.e. 6.66 g L-1 butanol, a yield of
0.18 g g-1, and a productivity of 0.96 g Lh-1. According
to the authors, this type of co-culturing can be applied in
industry. The same strains are used by Zhang et al. [39].
They combine immobilized cells of C. beijerinckii with
C. tyrobutyricum. Sugar cane wastes pretreated with
diluted H2SO4 are the substrates used. Thus, 6.78 g L-1
isopropanol and 12.33 g L-1 butanol are obtained.
The main obstacle in the course of the work with C.
acetobutylicum refers to their high sensibility to oxygen.
In this regard Wu et al. [66] co-culture C. acetobutylicum
and Bacillus cereus strain at microaerophilic conditions.
The role of B. cereus is to deplete oxygen and to provide
favorable conditions for C. acetobutylicum. It is found
that the ratio between the inoculated cultures is of im-
687
 Journal of Chemical Technology and Metallurgy, 53, 4, 2018

portance. Butanol concentration of 11 g L-1 is reached
at the optimal ratio of 5 % C. acetobutylicum: 0.5 % B.
cereus. The total solvents concentration is 18.1 g L-1.
Another disadvantage of working with C. acetobu-
tylicum is connected with the bacteria low tolerance to
organic solvents. It is considered that the limitation of
16 g L-1 - 20 g L-1 of total solvents concentration is due
to butanol toxicity [67]. It is known [68] that butanol
penetrates the cell membrane and destructs the hydrogen
bonds between the lipids. This leads to disruption of the
cell membrane and hence to cellular death [68].
Genetic improvement of strains naturally producing
1-butanol
Genetic engineering is widely applied to strains ma-
nipulated for obtaining the desired products. In case of C.
acetobutylicum, genetic manipulations are focused mainly
on increasing butanol titer, yield and productivity. Experi-
ments are concentrated at the genes responsible for acids
forming (pta, ack, ptb, buk) and those concerning solvents
production (adc, adhE, ctfAB) (Fig 1). This aims at the
direct metabolic flow to synthese butanol and to eliminate
acetone and ethanol [18, 69, 70]. High butanol titer (16.7
g L-1) is achieved by inactivation of the butyrate–kinase
gene (buk) [71]. The inactivation of pta and buk with con-
comitant over-expression of adhE (encoding aldehyde/
alcohol dehydrogenase) contributes to an increase by 60
% of the final butanol titer (18.9 g L-1) and 154 % of the
yield (0.29 g g-1) [18]. The over-expression of the genes
aad and thl (encoding acetaldehyde dehydrogenase and
thiolase, respectively) leads to a decrease of the accumu-
lation of acetone, acetic and butyric acid and hence, to
a considerable increase of alcohols production (mainly
ethanol) [72]. Some functional transcription regulators
like solR, spo0A, groESL, grpE and htpG are examined
aiming butanol titer improvement. The inactivation of
putative transcriptional repressor gene (solR) results in
17.8 g L-1 butanol concentration [73].

Fig. 1. Metabolic pathway of Clostridium acetobutylicum for ABE fermentation from glucose [5]. Genes and enzymes:
ldh - lactate dehydrogenase, pfor - pyruvate - ferredoxinoxidoreductase, ack - acetate kinase, pta - phosphotransacetylase,
thl - thiolase, adc - acetoacetate decarboxylase, ctfAB - acetoacetyl CoA:acetate/butyrate:CoA transferase, hbd - 3-hy-
droxybutyryl-CoA, crt - crotonase, bcd - butyryl-CoA dehydrogenase, buk - butyrate kinase, ptb - phosphotransbutyrylase,
1 - acetaldehyde dehydrogenase, 2 - ethanol dehydrogenase, 3 - butyraldehyde dehydrogenase, 4 - butanol dehydrogenase.

688
 Flora Tsvetanova, Penka Petrova, Kaloyan Petrov

Fig. 2. Engineered butanol pathway in cellulolytic Clostridium with over-expressed heterologous adhE2 or aad from
C. acetobutylicum with acetate, lactate and butyrate pathways knocked out to produce butanol and ethanol from
cellulose [44]. Wild cellulolytic Clostridium can produce acids and ethanol, but not butanol. Genes and enzymes:
ack - acetate kinase, adhE2 - aldehyde-alcohol dehydrogenase, buk - butyrate kinase, ldh - lactate dehydrogenase,
pta - phosphotransacetylase, ptb - phosphotransbutyrylase.

Under certain conditions of ABE fermentation Genetic manipulations of non–solvents producing

the cells do not switch from acids production to
solvents accumulation. This constitutes a common
problem. In this regard, Lu [74] achieves an increased
re-assimilation of acetic and butyric acid and there-
fore, higher butanol concentration in presence of C.
beijerinckii by over-expression of CoA-transferase
(ctfAB) and aldehyde/alcohol dehydrogenase (aad).
The over–expression of the genes encoding the
enzymes phosphofructokinase (pfkA) and pyruvate
kinase (pykA) in C. acetobutylicum 824 contributes
to the higher butanol titer. This is due to the increased
levels of ATP and NADH in bacterial cells. In contrast
to the wild type, higher concentrations of butanol and
ethanol are reached - 29.4 % and 85 %, respectively.
In a fed–batch process with a glucose substrate 28.02
g L-1 of butanol are produced [17].
microbial species
Experiments concerning strains which do not syn-
thesize butanol naturally, but are capable of growing in
presence of higher concentrations of solvents are carried
out. Genetic engineering approaches manipulating such
strains find application as they lend them the ability to
produce butanol. Usually genes responsible for solvents
accumulation are cloned and genes responsible for acids
formation are eliminated. Modified organisms should
possess three important qualities: tolerance to butanol, a
limited ability to assimilate it (not to metabolize it) and
ability to utilize substrates used in the industry (glucose,
lactic acid, glycerol) [75]. It is presumed that the toler-
ance depends on physico–chemical factors like pH and
cultivation temperature. However, there are not enough
experiments concerning these effects [76].

689
 Journal of Chemical Technology and Metallurgy, 53, 4, 2018

Knoshaug and Zhang [77] screen 24 different
microorganisms for their tolerance to butanol. Some
of the yeast strains (Pichia methanolica, Pachysolen
tannophilus, P. guillermondii) are found sensitive to
butanol concentrations higher than 2 %. Candida so-
norensis, Saccharomyces cerevisiae, and Zymomonas
mobilis withstand 2 % butanol. The representatives of
Lactobacillus genus are more sustainable than yeasts.
At 2 % butanol strains of L. delbrüeckii and L. brevis
demonstrate a relative growth rate of 55 % and 58 %,
respectively. These two strains also withstand 2.5 %
butanol, but the growth rate is diminished to 30 % and
40 %, correspondingly. The authors explore the tempera-
ture effect on E. coli sensitivity. They find out that the
culture grows faster at 37°C, but is more sensitive to 1 %
butanol when compared to that cultivated at 30°C [77].
It is established that Pseudomonas putida has the
potential to be used for industrial butanol production due
to its relatively high tolerance and ability to metabolize
cheap substrates. According to Molina-Santiago et al.
[78] the strain possesses pump systems used for detoxi-
cation. That is to say these systems are the reason for
the tolerance to large number of antibiotics and organic
solvents of some organisms. Cuenca et al. [75] investi-
gate the tolerance of Ps. putida BIRD-1 and find that the
strain shows no significant decrease in its vitality up to
2 % butanol presence. Analyzing a mutant library, the
authors find 16 genes responsible for butanol tolerance.

Fig. 3. The convertion of CO, CO2 and H2 to butyric acid, acetic acid and ethanol via Wood-Ljungdahl pathway
[55]. Genes and enzymes: acs - acetyl-CoA synthase, cod - CO dehydrogenase, fd - formate dehydrogenase, ftc -
formyl-THF cyclohydrolase, fts - formyl-THF synthase, hyda - hydrogenase, mtd - methylene-THF dehydrogenase,
mtf - methyl transferase, mtr - methylene-THF reductase.A

690
 Flora Tsvetanova, Penka Petrova, Kaloyan Petrov
An aerobic butanol producing bacteria from soil in
Singapore, identified as Bacillus sp. is isolated [79]. It
produces 10.38 g L-1 butanol in a batch process. The op-
timization of the fermentation conditions by minimizing
the by-products quantities results in 12.3 g L-1 of butanol.
Butanol-tolerant Enterococcus faecalis CM4A is
isolated from soil polluted with grease [76]. The strain
sustains 3.5 % butanol without assimilating or degrading
it. A strain Staphylococcus aureus is reported [39] to with-
stand 3 % butanol. It is determined that the addition of 10
g L-1 - 20 g L-1 glucose increases the bacterial tolerance.
Another microorganisms found to be tolerant to
butanol belong to genus Saccharomyces-yeast. They
utilize sugar compounds for biomass accumulation
and conversion to ethanol. Saccharomyces cerevisiae,
among them, is widely spread in bread preparation, wine
and beer production [80]. The species was reported to
withstand high ethanol concentration [81] and it turns
out lately that it is tolerant to butanol too. Zaki et al.
[83] examine 90 different Saccharomyces strains and
detect that some of them (S. cerevisiae DBVPG 1788,
S. cerevisiae DVBPG 6044, S. cerevisiae YPS 128) are
tolerant to 4 % butanol. It is found out that S. uvarum
and S. castelli do not tolerate more than 3 % butanol.
The authors suppose that the tolerance is related to the
over-expression of HOR2 gene [82]. Hansson [83] iso-
lates S. cerevisiae strains from different types of beer
and wine. The most tolerant strain is isolated from beer
and is capable to grow in 3 % butanol.
In regard to its tolerance to butanol, S. cerevisiae is
used as a host where the synthetic metabolic pathway
for butanol production from C. acetobutylicum is cloned.
The authors eliminate the glycerol production pathway
and achieve improved butanol production. In addition,
the gene encoding trans-enoil-CoA reductase is cloned,
and after 48 h of fermentation 14.1 mg L-1 butanol is
reached [32]. The priority of S. cerevisiae as a host for
genetic manipulations is that it is a well-characterized
organism which is genetically tractable [84].
The ability of carboxydotrophic bacteria to tolerate
1 % - 3 % butanol is reported [85]. These bacteria utilize
syngas compounds (CO, H2 and CO2) for butanol pro-
duction. B. licheniformis YP1A, Pediococcus acidilacti
IMUA 20068 and Enterococcus faecalis IMUAU 60169
are found among 11 different strains to be less sensi-
tive to butanol. The advantage of the carboxydotrophic
bacteria is that they are capable to tolerate up to 3 %
butanol and besides, they synthesize it in small quanti-
ties from CO. These properties make them exceptionally
appropriate for genetic investigation and manipulations.
Fig. 3 shows the Wood-Ljungdahl pathway of CO, CO2
and H2 conversion.
E. coli, another bacterium used intensively by the
genetic engineering, is also manipulated in order to pro-
duce butanol. Saini et al. [28] construct a recombinant
strain on the ground of clostridia by introducing CoA-
dependent synthetic pathway. This strain demonstrates
enhanced NADH levels and produces 6.1 g L-1 butanol
with a yield of 0.33 g g-1 and a productivity of 0.21 g
Lh-1 [28].
L. brevis is modified by Berezina et al. [36]. They
use plasmid shuttle vector pHYc to clone genes encoding
enzymes of the lower part of butanol metabolic pathway
of C. acetobutylicum - crt, bcd, etfA, etfB, hbd (included
in bcs – operon), and the thl gene. However, butanol is
found to be produced at very low levels - just 300 mg L-1.
MAIN PROBLEMS AND FUTURE PERSPECTIVES
The preferred use of biobutanol refers to the produc-
tion of motor fuels for spark ignition engines by mixing
with conventional gasoline. The biobutanol concentra-
tion in the fuel can reach up to 30 % v/v, and since the
butanol fuel contains oxygen atoms, the stoichiometric
air/fuel ratio is smaller than that of gasoline and more
fuel could be injected to increase the engine power for
the same amount of air induced. The oxygen content is
supposed to improve the combustion, therefore lower
CO emissions can be expected. That is why biobutanol is
currently an exceptionally desired fermentation product,
superior to ethanol because of its physical properties and
the fact that it can completely replace gasoline.
However, there are several obstacles impeding fer-
mentative butanol production. The work with natural
butanol producers is very challenging. Clostridia are
not very suitable because of their intolerance to oxygen;
they suffer from butanol toxicity, they grow slowly.
Moreover, butanol is not synthesized in economically
desirable quantities. Thus, there is an interest in produc-
ing butanol by using more appropriate organisms. A
possible approach to butanol levels improvement refers
to the application of methods of genetic engineering and
manipulation of the genes providing acid formation and
solvents accumulation. Thus, the by-products concen-
trations can be minimized. Another strategy is finding
691
 Journal of Chemical Technology and Metallurgy, 53, 4, 2018
organisms, which do not naturally produce butanol,
but are tolerant to it. They can be included in a butanol
metabolic pathway.
Considering the global demand of n-butanol, it is
expected that it will be worth 9.9 billion USD by 2020.
The Asia-Pacific region is the world’s largest market in
2016 with a share of 51.3 % in terms of volume. China
is the key consumer there. It is also the biggest import
market of n-butanol. Almost 15.0 % demand is met
through import from other regions including Russia,
Taiwan, Malaysia, the U.S., and Germany although vari-
ous projects for construction of n-butanol manufacturing
facilities are approved . Since the production deficit is
still expected to exist in the near future, the production of
biobutanol from waste biomass is particularly promising.
For instance, the EU funded ButaNexT project (costing
€4.6 million) unites the efforts of scientists from 15 Eu-
ropean countries in search of alternative lignocellulosic
substrates for the production of biobutanol. The use
of inexpensive renewable non-food carbon substrates
would solve some of the problems worldwide, like
global warming and the over-consumption of petroleum
- based products.
Acknowledgements
This work is financially supported by Bulgar-
ian Academy of Sciences through Grant DFNP-17-
46/26.07.2017.
REFERENCES
1. S. Atsumi, A. Cann, M. Connor, C. Shen, K.
Smith, M. Brynildsen, Metabolic engineering of
Escherichia coli for 1 - butanol production, Metab.
Eng., 10, 2008, 305-311.
2. C. Xue, X. Zhao, C. Liu, L. Chen, F. Bai, Prospective
and development of butanol as an advanced biofuel,
Biotechnol. Adv., 31, 2013, 1575-1584.
3. J. Zhao, C. Lu, C. Chen, S. Yang, Biological
production of butanol and higher alcohols,
Bioprocess. Technol. Biorefin. Sustainable Prod.
Fuels, Chem., Polym., 2013, 235-261.
4. E. Papoutsakis, Engineering solventogenic clostridia,
Curr. Opin. Biotech., 19, 2008, 420-429.
5. J. Zheng, Y. Tashiro, Q. Wang, K. Sonomoto,
Recent advances to improve fermentative butanol
production: Genetic engineering and fermentation
692
technology, J. Biosci. Bioeng., 119, 2015, 1-9.
6. O. Harris, S. Wilbor, J. George, C. Eisenmann,
Toxicological profile for 2 – butoxyethanol and
2 – butoxyethanol acetate, US Dept. of Health and
Human Services, 1998.
7. D. Jones and D. Woods, Acetone - butanol
fermentation revisited, Microbiol. Rev., 50, 1986,
484-524.
8. V. Garcia, J. Păkkilă, H. Ohamo, E. Muurinen, R.
Keiski, Challenges in biobutanol production: How
to improve the efficiency, Renew. Sust. Energ. Rev.,
15, 2011, 964-980.
9. N. Qureshi and H. Blaschek, Recent advances in
ABE fermentation: hyper – butanol producing
Clostridium beijerinckii BA 101, J. Ind. Microbiol.
Biotechnol., 27, 2001, 287-291.
10. P. Dürre, Biobutanol: An attractive biofuel,
Biotechnol. J., 2, 2007, 1525-1534.
11. S. Hu, H. Zheng, Y. Gu, J. Zhao, W. Zhang, Y. Yang,
Comparative genomic and transcriptomic analysis
revealed genetic characteristics related to solvent
formation and xylose utilization in Clostridium
acetobutylicum EA2018, BMC Genomics, 2011,
12:93.
12. M. Harmanis, T. Klason, S. Gatenbeck, Uptake and
activation of acetate and butyrate in Clostridium
acetobutylicum, Appl. Microbiol. Biotechnol., 20,
1984, 66-71.
13. N. Qureshi, H. Blaschek, Economics of butanol
fermentation using hyper – butanol producing
Clostridium beijerinckii BA101, Trans. IchemE.,
78 (part C), 2000, 139-144.
14. T. Tsai, Y. Lo, J. Chang, Effect of medium
composition and pH control strategies on butanol
fermentation with Clostridium acetobutylicum,
Energy Procedia, 61, 2014, 1691-1694.
15. B. Kim, P. Bellows, Z. Data, J. Zeikus, Control
of carbon and electron flow in Clostridium
acetobutylicum fermentations: utilization of carbon
monoxide to inhibit hydrogen production and to
enhance butanol yields, Appl. Environ. Microbiol.,
48, 1984, 767-770.
16. S. Li, Y. Qian, Z. Liang, Y. Guo, M. Zhao, Z. Pang,
Enhanced butanol production from cassava with
Clostrodium acetobutylicum by genome shuffling,
World. J. Microbiol. Biotechnol., 2016, 32-53.
17. J. Ventura, H. Hu, D. Jang, Enhanced butanol
 Flora Tsvetanova, Penka Petrova, Kaloyan Petrov
production in Clostridium acetobutylicum ATCC 824
by double overexpression of 6-phosphofructokinase
and pyruvate kinase genes, Appl. Microbiol.
Biotechnol., 97, 2013, 7505-7515.
18. Y. Jang, Y. Lee, J. Lee, J. Park, J. Im, M. Eom, J. Lee,
S. Lee, H. Song, J. Cho, D. Seung, S. Lee, Enhanced
butanol production obtained by reinforcing the
direct butanol-forming route in Clostridium
acetobutylicum, MBio, 3, 2012, (5):e00314-12.
19. C. Tomas, N. Welker, E. Papoutsakis, Overexpression
of groESL in Clostridium acetobutylicum results
in increased solvent production and tolerance,
prolonged metabolism, and changes in the cell’s
transcriptional program, Appl. Environ. Microb.,
69, 2003, 4951-4965.
20. S. Zhang, C. Qu, X. Huang, Y. Suo, Z. Liao, J. Wang,
Enhanced isopropanol and n-butanol production by
supplying exogenous acetic acid via co-culturing
two Clostridium strains from cassava bagasse
hydrolysate, J. Ind. Microbiol. Biotechnol., 43,
2016, 915-925.
21. T . S a r c h a m i , E . J o h n s o n , L . R e h m a n n ,
Optimization of fermentation condition favoring
butanol production from glycerol by Clostridium
pasteurianum DSM525, Biores. Technol., 208,
2016, 73-80.
22. A. Naveira, H. Abubackar, M. Veiga, C. Kennes,
Efficient butanol-ethanol (BE) production from
carbon monoxide fermentation by Clostridium
carboxidovorans, Appl. Microbiol. Biotechnol.,
100, 2016, 3361-3370.
23. X. Yang, M. Xu, S. Yang, Metabolic and process
engineering of Clostridium cellulovorans for biofuel
production of cellulose, Metab. Eng., 32, 2015,
39-48.
24. J. Formanek, R. Mackie, H. Blaschek, Enhanced
butanol production by Clostridium beijerinckii
BA101 grown in semidefined P2 medium containing
6 percent maltodextrin or glucose, Appl. Environ.
Microb., 63, 1997, 2306-2310.
25. C. Shen, E. Lan, Y. Dekishima, A. Baez, K. Cho,
J. Liao, Driving forces enable high titer anaerobic
1-butanol synthesis in Escherichia coli, Appl.
Environ. Microb., 77, 2011, 2904-2915.
26. S. Atsumi, J. Liao, Directed evolution of
Methanococcus janaschii citramalate synthase
for biosynthesis of 1-propanol and 1-butanol by
Escherichia coli, Appl. Environ. Microb., 74, 2008,
7802-7808.
27. C. Shen, J. Liao, Metabolic engineering of
Escherichia coli for 1-butanol and 1-propanol
production via the keto-acid pathways, Metab. Eng.,
10, 2008, 312-320.
28. M. Saini, S. Li, Z. Wang, C. Chiang, Y. Chao,
Systematic engineering of the central metabolism
in Escherichia coli for effective production of
n-butanol, Biotechnol. Biofuels, 9, 2016, 69-79.
29. C. Dellomonaco, J. Clomburg, E. Miller, R.
Gonzalez, Engineered reversal of the β-oxidation
cycle for the synthesis of fuels and chemicals,
Nature, 476, 2011, 355-359.
30. S. Atsumi, A. Cann, M. Connor, C. Shen, K. Smith,
M. Brynlidsen, K. Chon, T. Hanai, J. Liao, Metabolic
engineering of Escherichia coli for 1-butanol
production, Metabol. Eng., 10, 6, 2008, 305-311.
31. V. Schadeweg, E. Boles, n-butanol production
in Saccharomyces cerevisiae is limited by the
availability of coenzyme A and acetyl Co A,
Biotechnol. Biofuels, 9, 2016, 44-56.
32. H. Sakuragi, Studies on applications of Clostridium
species for biorefinery, Kyoto University, 2014.
33. E. Steen, R. Chan, N. Prasad, S. Myers, C. Petzold,
A. Redding, M. Ouellet, J. Keasling, Metabolic
engineering of Saccharomyces cerevisiae for the
production of n-butanol, Microb. Cell. Fact., 7,
2008, 36-42.
34. A. Bhandiwad, A. Shaw, A. Guss, A. Guseva,
H. Bahl, L. Lynd, Metabolic engineering of
Thermoanaerobacterium saccharolyticum for
n-butanol production, Metab. Eng., 21, 2014, 17-25.
35. D. Nielsen, E. Leonard, S. Yoon, H. Tseng, C.
Yuan, K. Prather, Engineering alternative butanol
production platforms in heterologous bacteria,
Metabol. Eng., 11, 2009, 262-273.
36. O. Berezina, N. Zakharova, A. Brandt, S. Yarotski, W.
Scwartz, V. Zverlov, Reconstructing the clostridial
n-butanol metabolic pathway in Lactobacillus
brevis, Appl. Microbiol. Biotechnol., 87, 2010,
635-646.
37. E. Lan, J. Liao, ATP drives direct photosynthetic
production of 1-butanol in cyanobacteria, Proc. Natl.
Acad. Sci. USA, 109, 2012, 6018-6023.
38. P. Wu, G. Wang, G. Wang, B. Borresen, H. Liu,
J. Zhang, Butanol production under microaerobic
693
 Journal of Chemical Technology and Metallurgy, 53, 4, 2018
conditions with a symbiotic system of Clostridium
acetobutylicum and Bacillus cereus, Microb. Cell.
Fact., 15, 2016, 8-19.
39. J. Zhang, S. Huang, Y. Ma, M. Zhang, S. Zou,
Isolation and characterization of butanol tolerant
strain Staphylococcus faecalis, Biotechnol. Lett.,
2016.
40. Y. Jiang, J. Liu, W. Jiang, Y. Yang, S. Yang, Current
status and prospects of industrial bio-production of
n-butanol in China, Biotechnol. Adv., 33, 7, 2014,
1493-1501.
41. B. Tracy, S. Jones, A. Fast, D. Indurthi, E.
Papoutsakis, Clostridia: the importance of their
exceptional substrate and metabolite diversity for
biofuel and biorefinery applications, Curr. Opin.
Biotechnol., 23, 2012, 364-381.
42. W. Higashide, Y. Li, Y. Yang, J. Liao, Metabolic
engineering of Clostridium cellulolyticum for
production of isobutanol from cellulose, Appl.
Environ. Microbiol. 77, 2011, 2727–2733.
43. Y. Tamaru, H. Miyake, K. Kuroda, M. Ueda, R.
Doi, Comparative genomics of the mesophilic
– producing Clostridium cellulovorans and its
application to biofuel production via consolidated
bioprocessing, Environ. Technol., 3, 2010, 889-903.
44. Z. Wang, G. Gao, J. Zheng, D. Fu, J. Song, J. Zhang,
L. Zhao, Q. Yang, Developing a mesophilic co-
culture for direct conversion of cellulose to butanol
in consolidated bioprocess, Biotechnol. Biofuels, 8,
1, 2014. 84-92.
45. I. Vasconcelos, L. Girbal, P. Soucaille, Regulation
of carbon and electron flow in Clostridium
acetobutylicum grown in chemostat culture at
neutral pH on mixtures of glucose and glycerol, J.
Bacteriol., 176, 5, 1994, 1443-1450.
46. J. Panitz, V. Zverlov, V. Pham, S. Stürzl, D.
Schieder, W. Schwarz, Isolation of solventogenic
Clostridium sp. strain: fermentation of glycerol
to n-butanol, analysis of the bcs operon region
and its potential regulatory elements, Syst. Appl.
Microbiol., 37, 2014, 1-9.
47. S. Ha, J. Galazka, S. Kim, J. Choi, X. Yang, J. Seo,
N. Glass, J. Cate, Y. Jin, Engineered Saccharomyces
cerevisiae capable of simultaneous cellobiose and
xylose fermentation, Proc. Natl. Acad. Sci. USA,
108, 2, 2011, 504-509.
48. C. Grimmler, C. Held, W. Liebl, A. Ehrenreich,
694
Transcriptional analysis of catabolyte repression in
Clostridium acetobutylicum growing on mixtures
of D-glucose and D-xylose, J. Biotechnol., 150, 3,
2010, 315-323.
49. L. Jin, H. Zhang, L. Chen, Yang, C. Yang, S Jiang
, W. Jiang, Combined overexpression of genes
involved in pentose phosphate pathway enables
enhanced D-xylose utilization by Clostridium
acetobutilycum, J. Biotechnol., 173, 2014, 7-9.
50. K. Weissermel and H. Arpe, Industrial Organic
Chemistry, Wiley, Hoboken, 2008.
51. K. Küsel, A. Karnholz, T. Trinkwalter, R. Devereux,
G. Acker, H. Drake, Physiological ecology of
Clostridium glycolicum RD-1, an aerotolerant
acetogen isolated from sea grass toots, Appl.
Environ. Microbiol., 67, 10, 2001, 4734-4741.
52. M. Köpke, C. Held, S. Hujer, H. Liesegang, A.
Wiezer, A. Wollherr, A. Ehrenreich, W. Liebl,
G. Gottschalk, P. Dürre, Clostridium ljungdahlii
represents a microbial production platform based
on syngas, Proc. Natl. Acad. Sci. USA, 107, 2010,
13087-13092.
53. Y. Guo, J. Hu, Y. Zhang, H. Hu, Z. Yuan, D.
Li, Medium optimization of ethanol production
with Clostridium autoethanogenum with carbon
monoxide as sole carbon source, Bioresour.
Technol., 101, 22, 2010, 8784-8789.
54. J. Bertsch, V. Muller, Bioenergetic constraints
for conversion of syngas to biofuels in acetogenic
bacteria, Biotechnol. Biofuels, 8, 2015, 210-222.
55. G. Bruant, M. Levesque, C. Peter, S. Guiot, L.
Masson, Genomic analysis of carbon monoxide
utilization and butanol production by Clostridium
carboxidovorans strain P7, PLoS ONE, 5, 9, 2010,
e13033.
56. Y. Jang, A. Malaviya, C. Cho, J. Lee, S. Lee, Butanol
production from renewable biomass by clostridia,
Bioresour. Technol., 123, 2012b, 653-663.
57. A. Wargacki, E. Leonard, M. Win, D. Regitsky, C.
Santos, P. Kim, S. Cooper, R. Raisner, A. Herman,
A. Sivitz, A. Lakshmanaswamy, Y. Kashiyama,
D. Baker, Y. Yoshikuni, An engineered microbial
platform for direct biofuel production from brown
microalgae, Science, 335, 2012, 308-313.
58. Y. Kim, D. Kim, T. Kim, M. Shin, Y. Kim, J. Yoon,
I. Chang, Use of red algae, Ceylon moss (Gelidium
amansii), hydrolysate for clostridial fermentation.
 Flora Tsvetanova, Penka Petrova, Kaloyan Petrov
Biomass Bioenergy, 56, 2013, 38-42.
59. K. Murata, S. Kawai, B. Mikami, W. Hashimoto,
Superchannel of bacteria: biological significance
and new horizons, Biosci. Biotechnol. Biochem.
72, 2, 2008, 265-277.
60. Y. Wang, W. Guo, C. Cheng, S. Ho, J. Chang,
N. Ren, Enhanced bio-butanol production from
biomass of Chlorella vulgaris JSC-6 with sequential
alkali pretreatment and acid hydrolysis, Bioresour.
Technol., 200, 2016, 557-564.
61. Y. Wu, C. Xue, L. Chen, F. Bai, Impact of zinc
supplementation on the improved fructose/xylose
utilization and butanol production during acetone-
butanol-ethanol fermentation, J. Biosci and Bioeng.,
1, 2016, 66-72.
62. N. Al-Shorgani, H. Shukor, P. Abdeshahian, M.
Kalil, W. Yussof, A. Hamid, Enhanced butanol
production by optimizing medium parameters using
Clostridium acetobutylicum YM1, Saudi J. Biol.
Sci., 2016, 1-14.
63. R. Börner, O. Zaushitsyna, D. Berillo, N. Scaccia,
B. Mattiasson, H. Kirsebom, Immobilization of
Clostridium acetobutylicum 792 as macroporous
aggregates through cryogelation for butanol
production, Process. Biochem., 49, 2014, 10-18.
64. Y. Chen, T. Zhou, D. Liu, A. Li, S. Xu, Q. Liu, B.
Li, H. Ying, Production of butanol from glucose
and xylose with immobilized cells of Clostridium
acetobutylicum, Biotechnol. Bioprocess Eng., 18,
2013, 234-241.
65. L. Li, H. Ai, S. Zhang, S. Li, Z. Liang, Z. Wu,
S. Yang, J. Wang, Enhanced butanol production
by coculture of Clostridium beijerinckii and
Clostridium tyrobutyricum, Bioresour. Technol.,
143, 2013, 397-404.
66. P. Wu, G. Wang, G. Wang, B. Borresen, H. Liu,
J. Zhang, Butanol production under microaerobic
conditions with a symbiotic system of Clostridium
acetobutylicum and Bacillus cereus, Microb. Cell.
Fact., 15, 2016, 8-19.
67. A. Moreira, D. Ulmer, J. Linden, Butanol toxicity in
the butylic fermentation, Biotechnol. Bioeng. Sym.,
Proceedings of the 3th symposium on biotechnology
in energy production and conservation, Gatlinburg,
TN, USA, 1981, 11.
68. M. Jain & N. Wu, Effect of small molecules on
the dipalmitoyl lecithin liposomal bilayer. Phase
transition in lipid bilayer. J. Membr. Biol., 34, 1977,
157-20.
69. C. Cooksley, Y. Zheng, H. Wang, S. Redl, K.
Winzer, N. Minton, Targeted mutagenesis of the
Clostridium acetobutylicum acetone butanol ethanol
fermentation pathway, Metab. Eng., 14, 2012, 630-
641.
70. M. Mann, T. Lutke-Eversioh, Thiolase engineering
for enhanced butanol production in Clostridium
acetobutylicum, Biotechnol. Bioeng., 110, 2013,
887-897.
71. L. Harris, R. Desai, N. Welker, E. Papoutsakis,
Characterisation of recombinant strains of the
Clostridium acetobutylicum butyrate kinase
inactivation mutant: need for new phenomenological
models for solventogenesis and butanol inhibition?
Biotechnol. Bioeng., 67, 2000, 1-11.
72. R. Sillers, M. A-Hanai, E. Papoutsakis,
Aldehyde-alcohol dehydrogenase and/or thiolase
overexpression coupled with CoA transferase
downregulation lead to higher alcohol titers
and selectivity in Clostridium acetobutylicum
fermentations, Biotechnol. Bioeng., 102, 2009,
38-49.
73. R. Nair, E. Green, D. Watson, G. Bennett,
E. Papoutsakis, Regulation of the sol locus
genes for butanol and acetone formation in
Clostridium acetobutylicum ATCC 824 by a putative
transcriptional repressor, J. Bacteriol., 181, 1999,
319-330.
74. C. Lu, Butanol production from lignocellulosic
feedstock by acetone-butanol-ethanol fermentation
with integrated product recovery, Ohio State
University, Colombus, OH, 2011.
75. M. Cuenca, A. Roca, C. Molina-Santiago, E.
Duque, J. Armengaud, M. Gómez-Garcia, J. Ramos,
Understanding butanol tolerance and assimilation in
Pseudomonas putida BIRD-1: an integrated omics
approach, Microb. Biotechnol., 9, 2015, 100-115.
76. M. Kanno, H. Tamaki, Y. Mitani, N. Kimura, S.
Hanada, Y. Kamagata, pH-induced change in cell
susceptability to butanol in a high butanol-tolerant
bacterium, Enterococcus faecalis strain CM4A,
Biotechnol. Biof., 8, 2015, 69-75.
77. E. Knoshaug, M. Zhang, Butanol tolerance in
a selection of microorganisms, Appl. Biochem.
Biotechnol., 153, 2008, 13-20.
695
 Journal of Chemical Technology and Metallurgy, 53, 4, 2018
78. C. Molina-Santiago, A. Daddaoua, S. Fillet, E.
Duque, and J. Ramos, Interspecies signalling:
Pseudomonas putida efflux pump TtgGHI is
activated by indole to increase antibiotic resistance,
Environ. Microbiol., 16, 2014, 1267–1281.
79. C. Ng, K. Takahashi, Z. Liu, Isolation,
characterization, and optimization of an aerobic
butanol-producing bacterium from Singapore,
Biotechnol. Appl. Biochem., 2014, 86-91.
80. G. Naumov, E. Naumova, I. Masneuf-Pomarėde,
Genetic identification of new biological species
Saccharomyces arboricolus, Antonie van
Leeuwenhoek, 98, 1, 2010, 1-7.
81. C. Belloch, S. Orlic, E. Barrio, A. Querol,
Fermentative stress adaptation of hybrids within
the Saccharomyces sensu strict complex, Int. J. Food
696
Microbiol., 122, 1-2, 2008. 188-195.
82. A. Zaki, T. Wimalasena, D. Greetham, Phenotypic
characterization of Saccharomyces spp. for tolerance
to 1-butanol, J. Ind. Microbiol. Biotechnol., 41,
2014, 1627-1636.
83. C. Hansson, Identification of butanol tolerant
Saccharomyces cerevisiae strain and of a gene
associated with enhanced butanol tolerance,
Linköping University, 2016.
84. D. Ro, E. Paradise, M. Ouellet, R. Fisher, Production
of the antimalarial drug precursor artenisinic acid in
engineered yeast, Nature, 440, 7086, 2006, 940-943.
85. E. Pomaranski and S. Tiquia-Arashiro, Butanol
tolerance of carboxydotrophic bacteria isolated
from manure composts, Environ. Technol., 37, 15,
2016, 1970-1982.