A green process for the production of butanol from butyraldehyde using alcohol dehydrogenase: Process details

Article abstract of DOI:10.1039/c3ra47821a

Depletion of energy sources has drawn attention towards production of bio-butanol by fermentation. However, the process is constrained by product inhibition which results in low product yield. Hence, a new strategy wherein butanol was produced from butyraldehyde using alcohol dehydrogenase and NADH as a cofactor was developed. Butyraldehyde can be synthesized chemically or through fermentation. The problem of cofactor regeneration during the reaction for butanol production was solved using substrate coupled and enzyme coupled reactions. The conventional reaction produced 35% of butanol without regeneration of cofactor using 300 μM NADH. The process of substrate coupled reaction was optimized to get maximum conversion. NADH (30 μM) and 100 μg per ml of alcohol dehydrogenase (320 U mg^-1) could convert 17.39 mM of butyraldehyde to butanol using ethanol (ratio of butyraldehye to ethanol 1:4) giving a maximum conversion of 75%. The enzyme coupled reaction under the same conditions showed only 24% conversion of butyraldehyde to butanol using the glutamate dehydrogenase-l-glutamate enzyme system for the regeneration of cofactor. Hence, substrate coupled reaction is suggested as a better method over the enzyme coupled reaction for the cost effective production of butanol. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3ra47821a

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A green process for the production of butanol from
butyraldehyde using alcohol dehydrogenase:
process details†
Cite this: RSC Adv., 2014, 4, 14597
ab
ab
a
a
Swati B. Jadhav, Shirish Harde,b Sandip B. Bankar, Tom Granstrom,
¨
a
a
Heikki Ojamo, Rekha S. Singhal and Shrikant A. Survase*
Depletion of energy sources has drawn attention towards production of bio-butanol by fermentation.
However, the process is constrained by product inhibition which results in low product yield. Hence, a
new strategy wherein butanol was produced from butyraldehyde using alcohol dehydrogenase and
NADH as
a cofactor was developed. Butyraldehyde can be synthesized chemically or through
fermentation. The problem of cofactor regeneration during the reaction for butanol production was
solved using substrate coupled and enzyme coupled reactions. The conventional reaction produced 35%
of butanol without regeneration of cofactor using 300 mM NADH. The process of substrate coupled
reaction was optimized to get maximum conversion. NADH (30 mM) and 100 mg per ml of alcohol
À1
dehydrogenase (320 U mg ) could convert 17.39 mM of butyraldehyde to butanol using ethanol (ratio
of butyraldehye to ethanol 1 : 4) giving a maximum conversion of 75%. The enzyme coupled reaction
under the same conditions showed only 24% conversion of butyraldehyde to butanol using the
glutamate dehydrogenase-L-glutamate enzyme system for the regeneration of cofactor. Hence,
substrate coupled reaction is suggested as a better method over the enzyme coupled reaction for the
cost effective production of butanol.
Received 20th December 2013
Accepted 5th March 2014
DOI: 10.1039/c3ra47821a
www.rsc.org/advances
needs high temperature with very low yield of 15–21%. The
1
. Introduction
ꢀ
temperature required for liquid phase reaction is 120 C and for
ꢀ
4,5
High prices of conventional oil in the global market coupled gas phase reaction 300–450 C. Moreover, the process involves
with depletion of resources have been the driving force for multiple steps and many byproducts. Hence attempts were
researchers all over the world to work on renewable energy made successfully to selectively synthesize butanol from
6
sources. This has made bio-fuels commercially important and ethanol over strontium phosphate hydroxyapatite catalysts.
efforts to produce it economically are being made worldwide.
Butanol is an alternative source of energy and is better than Clostridium spp. It can be obtained from renewable sources by
Biological synthesis of butanol is by the fermentation using
7,8
ethanol. Butanol is less volatile, less hydroscopic and less acetone–butanol–ethanol (ABE) fermentation. The traditional
1
corrosive. Production of bio-butanol by fermentation process is ABE fermentation gives low yield which is due to an inhibitory
9
well reported by several researchers. Chemical method of effect of the produced butanol on the growth of the organism.
synthesis of butanol has been reported using either the oxo Recently, many molecular and fermentation techniques have
process starting from propylene or the aldol process starting been used to increase the production of butanol. The develop-
2
from acetaldehyde. Ethanol is a renewable source which can be ment of hyper-butanol producing strain and integrated ABE
derived from biomass. It can be converted to valuable chem- fermentation for the simultaneous production and removal of
10
icals. Chemical conversion of ethanol to butanol has been butanol from the fermentation broth has been reported. The
previously reported. The yield of butanol production using need for inexpensive feedstocks, improved fermentation
3
ethanol with MgO as catalyst was 18.39%. This conversion performance, sustainable solvent recovery, and water recycle are
11
the major challenges for ABE fermentation. ABE fermentation
has a drawback of low yield due to butanol toxicity for the
Aalto University, School of Chemical Technology, Department of Biotechnology and organism which makes it necessary to explore new strategies for
a
Chemical Technology, POB 16100, 00076 Aalto, Finland. E-mail: shrikantraje1@
the production of butanol. In Clostridium spp., the last step for
butanol production is the conversion of butyraldehyde to
butanol using butanol dehydrogenase. Based on this in vivo
reaction occurring in the cell, we report a new strategy where
butyraldehyde (which itself can be obtained from variety of
gmail.com; Fax: +358 9 462 373; Tel: +358 400368375
b
Food Engineering and Technology Department, Institute of Chemical Technology,
Nathalal Parekh Marg, Matunga, Mumbai 400 019, India
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c3ra47821a
1
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chemical and microbial sources) can be biocatalytically con- 2.2 Production of butanol from butyraldehyde by
verted to butanol in vitro.
conventional reaction
Butyraldehyde has been produced from 1,2-butanediol by
The reaction mixture was prepared by adding butyraldehyde
1
2
catalytic dehydration in supercritical water. It can also be
À1
(
(
17.39 mM), alcohol dehydrogenase (100 mg ml ) and NADH
50–300 mM). Control was prepared similarly but with the
1
3
synthesized by hydroformylation of propylene. Butyralde-
hyde can also be obtained by fermentation to overcome the
limitations of chemical processes and raw materials. A spon-
taneous mutant of Clostridium acetobutylicum has been shown
enzyme being replaced by water. The reaction mixture was
continuously mixed by keeping it on the shaker for 3 h. The
À1
reaction was stopped by adding oxidized pectin (0.5 mg ml
)
1
4
to produce signicant amounts of butyraldehyde. Strepto-
myces cinnamonensis has been shown to excrete butyraldehyde,
which inhibits the alcohol dehydrogenase. In our previous
work, oxidized pectin had shown complete inhibition of alcohol
dehydrogenase (data not shown). An aliquot of the mixture was
then checked for the production of butanol using gas chroma-
tography (GC) (Hewlett Packard series 6890) equipped with a
1
5
isobutyraldehyde
and
valeraldehyde.
Butyraldehyde
obtained by the above processes can be used for the butanol
production.
Recent concerns on the environmental quality and energy
resources have made biocatalysis attractive for industrial
applications. Biocatalytic reactions are environmentally

ame ionization detector and DB-WAXetr capillary column
ꢀ
(
30 m  0.32 mm  1 mm). The injector temperature was 200 C
ꢀ
and the detector temperature was 250 C. The heating prole
1
6
friendly, selective and energy efficient. Butanol dehydro-
genase can be used to obtain butanol from butyraldehyde.
However, this enzyme is not easily available and literature on
the production and purication of butanol dehydrogenase is
ꢀ
À1
ꢀ
was 10 C min starting from the initial value of 40 C. Internal
standard (1-propanol) was used for all samples. The retention
times for acetaldehyde, butyraldehyde, ethanol and butanol
were 1.2, 2.2, 2.8 and 5.6 minutes, respectively. The injector
volume was 10 ml.
1
7,18
scant.
Here, we have used alcohol dehydrogenase for the
production of butanol from butyraldehyde using NADH as a
cofactor. Alcohol dehydrogenase is easily available from
many sources including Saccharomyces cerevisiae. It is an
important catalyst for organic synthesis especially for chiral
2.3 Production of butanol from butyraldehyde by substrate
coupled reaction
1
9
alcohols.
2.3.1 Optimization of concentration of NADH for the
The cofactors are usually expensive. This necessitates production of butanol. Reaction mixture was prepared by add-
2
0

nding a way to regenerate and reuse them efficiently.
ing butyraldehyde (17.39 mM), ethanol (69.56 mM), alcohol
À1
Different cofactor regeneration strategies including biological, dehydrogenase (100 mg ml ) and NADH (2.5–50 mM). The
enzymatic (substrate coupled and enzyme coupled), electro- reaction mixture was continuously mixed by keeping it on the
chemical, chemical and photochemical have evolved over a shaker for 3 h. Control was prepared similarly but with enzyme
period of time. Substrate coupled reaction contains one replaced by water. The reaction was stopped by adding oxidized
enzyme which use both oxidized and reduced form of cofactor. pectin (0.5 mg ml ) which inhibited the alcohol dehydroge-
2
1
À1
It synthesizes the product in one reaction using one substrate nase. An aliquot of mixture was then checked for the production
and regenerates the cofactor in another reaction using second of butanol using GC.
substrate. Enzyme coupled reaction uses two enzymes to carry
2.3.2 Optimization of ratio of butyraldehyde to ethanol for
out two different reactions. Each enzyme needs separate the production of butanol. The reaction mixture was prepared
substrate to convert into product and regenerate the as explained in Section 2.3.1. The ratio of butyr-
1
6
cofactor.
aldehyde : ethanol was varied from 4 : 1–1 : 8 using 17.39 mM
Here, we have attempted a biocatalytic conversion of butyr- butyraldehyde and ethanol varying from 4.34–139.13 mM.
À1
aldehyde to butanol using alcohol dehydrogenase as the catalyst NADH (30 mM) and alcohol dehydrogenase (100 mg ml ) were
and NADH as the cofactor. The cofactor regeneration was kept constant. All other conditions were maintained as
attempted using ethanol as substrate for the substrate coupled explained above and checked for the production of butanol aer
reaction and glutamate dehydrogenase-L-glutamate system for 3 h.
the enzyme coupled reaction. We compared two methods of
2.3.3 Optimization of concentration of enzyme for the
cofactor regeneration to suggest a possible cost effective production of butanol. The reaction mixture was prepared as
method for the butanol production from butyraldehyde.
explained in Section 2.3.1. Enzyme concentration was varied
from 10–120 mg ml . Ratio of butyraldehyde : ethanol (1 : 4)
À1
and NADH (30 mM) were kept constant. Other conditions were
maintained as explained above and checked for the production
of butanol aer 3 h.
2
. Experimental
2.1 Materials
À1
Alcohol dehydrogenase (320 U mg ) from Saccharomyces cer-
2.3.4 Optimization of concentration of butyraldehyde for
evisiae, butyraldehyde, NADH and butanol was purchased from the production of butanol. The reaction mixture was prepared
Sigma Aldrich, USA. Ethanol, L-glutamic acid and glutamate as explained in Section 2.3.1. The concentration of butyralde-
dehydrogenase were purchased from Altia Oy, Finland, M.P. hyde was varied from 4.34–43.47 mM, while keeping the ratio of
Biomedicals Inc., Germany and Roche Diagnostics, Germany, butyraldehyde : ethanol at 1 : 4. Enzyme concentration (100 mg
À1
respectively.
ml ) and NADH (30 mM) were kept constant. Other conditions
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were maintained as explained above and checked for produc-
tion of butanol aer 3 h.
2.3.5 Optimization of time for the production of butanol
using 17.39 mM butyraldehyde. The reaction mixture was
prepared similarly as explained in Section 2.3.1. Butyraldehyde
(
(
17.39 mM), ethanol (69.56 mM), alcohol dehydrogenase
100 mg ml ) and NADH (30 mM) were used in the reaction. The
À1
reaction mixture was continuously mixed by keeping it on the
shaker till 3 h. Samples were withdrawn every 30 min and
checked for production of butanol.
2.3.6 Optimization of time for the production of butanol
using 34.78 mM butyraldehyde. The reaction was carried out as
explained in Section 2.3.1. Butyraldehyde (34.78 mM), ethanol
À1
(
(
155.78 mM), alcohol dehydrogenase (100 mg ml ) and NADH
30 mM) were used in the reaction. The reaction mixture was
Fig. 1 The production of butanol from butyraldehyde by conventional
reaction.
continuously mixed by keeping it on the shaker till 9 h. Samples
were withdrawn every 60 min and checked for production of
butanol.
3
.2 Production of butanol from butyraldehyde by substrate
coupled reaction
.2.1 Optimization of concentration of NADH for the
2
.4 Production of butanol from butyraldehyde using enzyme
coupled reaction
3
Reaction mixture was prepared by adding butyraldehyde
production of butanol. Substrate coupled reaction overcome
the drawbacks of conventional reaction by continuous supply of
NADH by its regeneration through another reaction occurring
in the process. Butyraldehyde and ethanol were used as
substrates for the enzyme alcohol dehydrogenase. Lower level of
NADH was used for the substrate coupled reaction as there is
continuous supply of NADH in the reaction. Butanol production
was seen to increase with an increase in concentration of NADH
À1
(17.39 mM), alcohol dehydrogenase (100 mg ml ), glutamate
À1
dehydrogenase (100 mg ml ), L-glutamate (69.56 mM) and
NADH (30 mM). The reaction mixture was continuously mixed by
keeping it on the shaker for 3 h. Control was prepared by using
same reaction mixture except that the enzyme was replaced with
water. The reaction was stopped by adding oxidized pectin
À1
(0.5 mg ml ) which inhibits the alcohol dehydrogenase. An
aliquot of the mixture was then checked for the production of
butanol using GC.
(2.5 to 30 mM). Maximum conversion of butyraldehyde to
butanol (75%) was obtained at 30 mM of NADH (Fig. 2). The
butanol production remained unaltered above 30 mM of NADH.
Butyraldehyde was converted to butanol with simultaneous
conversion of NADH to NAD. The NAD so produced was used by
the alcohol dehydrogenase to convert the ethanol to acetalde-
hyde and which regenerated the NADH. There was a continuous
3
3
. Results and discussion
.1 Production of butanol from butyraldehyde by
conventional reaction
Clostridium spp. produces butanol by the fermentation supply of NADH due to which the low initial concentration of
process in which the last step is the conversion of butyral-
2
2
dehyde to butanol by butanol dehydrogenase. The same
reaction can be mimicked in vitro using alcohol dehydroge-
nase. Alcohol dehydrogenase is easily available, less expen-
sive as compared to butanol dehydrogenase and it catalyses
2
3
the reduction of aldehyde to alcohol. Conventionally,
butyraldehyde can be enzymatically converted to butanol
using NADH, where NADH is the limiting factor in the reac-
tion. The reaction stops aer all the NADH is consumed.
Hence there is a need to provide large amount of NADH in the
reaction to convert all the available butyraldehyde to butanol.
An increase in the NADH supply from 50 to 300 mM increased
the butanol production from 0.4 to 1.2 mM (Fig. 1). The
conversion of butanol production varied from 25 to 35%. The
conventional reaction is not cost effective as there is a
continuous need to supply large amount of NADH. This is a
drawback of the reaction to proceed further. Hence the
cofactor should be regenerated to make it continuously
available in the reaction.
Fig. 2 Optimization of concentration of NADH for the production of
butanol by substrate coupled reaction.
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NADH could convert all the available butyraldehyde to butanol
giving a high conversion. NADH at 30 mM was sufficient to
convert 17.39 mM of butyraldehyde to butanol in 3 h.
3.2.2 Optimization of ratio of butyraldehyde : ethanol for
the production of butanol. Concentration of both the substrates
is an important parameter for the substrate coupled reaction
because it affects the reaction equilibrium. Concentration of
ethanol should be sufficiently high to carry out the forward
reaction of conversion of ethanol to acetaldehyde and simul-
taneously the conversion of NAD to NADH. Different ratios of
butyraldehyde : ethanol were used in the reaction. When the
ethanol concentration was lower or equal to butyraldehyde
(butyraldehyde to ethanol ratio 4 : 1–1 : 1), the production of
butanol was 3 to 8 mM (Fig. 3). The production of butanol was
found to be maximum (10.63 mM) at butyraldehyde : ethanol Fig. 4 Optimization of concentration of enzyme for the production of
ratio of 1 : 4, where the conversion was 75%. The butanol butanol by substrate coupled reaction.
production was found to decrease with increasing amount of
ethanol. At ratios of 1 : 6 and 1 : 8, the butanol production was
6
9% and 68%, respectively. Hence, a 1 : 4 ratio of butyr-
determined using butyraldehyde concentration ranging
between 4.34 and 43.47 mM. The production of butanol were
aldehyde : ethanol was selected for the reaction. Higher
concentration of ethanol inhibited alcohol dehydrogenase and
thereby decreased the production of butanol.
.2.3 Optimization of concentration of enzyme for the
production of butanol. The use of minimum amount of enzyme
is important in the enzymatic reaction to obtain the product
cost effectively. Various concentrations (10–120 mg ml ) of
alcohol dehydrogenase were used for the reaction of butanol
production. At lowest concentration (10 mg ml ) of alcohol
10.63 mM and 15.16 mM from 17.39 mM and 34.78 mM of
24
butyraldehyde, respectively (Fig. 5). The conversion of butyral-
dehyde to butanol was 74% at butyraldehyde concentration of
3
17.39 mM. Whereas it was 65% at the butyraldehyde concen-
tration of 34.78 mM. Higher concentration of enzyme would be
needed to convert 34.78 mM of butyraldehyde with better
conversion.
À1
À1
3.2.5 Optimization of time for the production of butanol
dehydrogenase, the butanol production was 46% (Fig. 4). The
highest butanol production (10.63 mM) and conversion effi-
ciency (75%) were obtained at 100 mg per ml of alcohol dehy-
drogenase, beyond which there was no further increase in the
yield. Hence the optimum concentration of alcohol dehydro-
using 17.39 mM and 34.78 mM butyraldehyde. Time needed for
the completion of reaction was determined for 17.39 mM and
34.78 mM of butyraldehyde. The production of butanol was
found to increase with progress of time. The maximum
production of butanol from 17.39 mM of butyraldehyde was
obtained aer 150 min (Fig. 6). Initially, the rate of butanol
production increased until 60 min and decreased thereaer.
The butanol production from 34.78 mM of butyraldehyde was
monitored every 60 min (Fig. 7). It was seen that the rate of
butanol production increased until 240 min and decreased
genase for the conversion of 17.39 mM of butyraldehyde to
À1
butanol was 100 mg ml
.
3.2.4 Optimization of concentration of butyraldehyde for
the production of butanol. The maximum conversion of
substrate to the product that an enzyme could carry out was
Fig. 3 Optimization of ratio of butyraldehyde to ethanol for the Fig.
5
Optimization of concentration of butyraldehyde for the
production of butanol by substrate coupled reaction.
production of butanol by substrate coupled reaction.
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Fig. 8 Production of butanol using enzyme coupled reaction. AD:
alcohol dehydrogenase and GD: glutamate dehydrogenase.
Fig. 6 Optimization of time for the production of butanol using
1
7.39 mM butyraldehyde by substrate coupled reaction.
of 4 : 1–1 : 1) showed lower butanol production. Glutamate
dehydrogenase was needed at higher amount (ratio of 1 : 2–
1
: 6) as compared to alcohol dehydrogenase. Higher amount of
glutamate dehydrogenase could initiate the conversion of NAD
to NADH even at lower concentration of available NAD and
make it possible to supply NADH continuously in the reaction.
The substrate coupled reaction where ethanol was used as a
second substrate for continuous supply of NADH showed a
conversion of 75%. However the enzyme coupled reaction
showed only 24% conversion of butyraldehyde to butanol. A
comparison between two above mentioned methods suggested
substrate coupled reaction to be a better approach than the
enzyme coupled reaction. Enzyme coupled reaction is costly as
it needs two enzymes and two substrates for the reaction to
proceed, whereas substrate coupled reaction needs only one
enzyme with two substrates. Besides, ethanol is an inexpensive
substrate needed for the substrate coupled reaction and
conversion obtained was higher as compared to enzyme
coupled reaction. Hence it can be suggested that conversion of
butyraldehyde to butanol using alcohol dehydrogenase with
substrate coupled reaction for cofactor regeneration can be
used as a green, cost effective and rapid method for the
production of butanol.
Fig. 7 Optimization of time for the production of butanol using
4.78 mM butyraldehyde by substrate coupled reaction.
3
thereaer. The production of butanol aer 240 min was
14.62 mM and increased to 18.86 mM aer 540 min.
3.3 Production of butanol from butyraldehyde using enzyme
coupled reaction
Two enzymes can be coupled together for continuous regener-
ation of NADH. An enzyme coupled reaction is an alternative to
4. Conclusions
substrate coupled reaction. Two enzymes, viz. alcohol dehy- To overcome the limitation of product inhibition in butanol
drogenase and glutamate dehydrogenase were used in the production, a new strategy was developed for the production of
reaction with butyraldehyde and L-glutamate as substrates. butanol using butyraldehyde as the substrate and NADH as the
Glutamate dehydrogenase could convert NAD to NADH by the cofactor. The regeneration of cofactor NADH was comparatively
using L-glutamate as substrate and generating NADH continu- evaluated using substrate coupled and enzyme coupled reac-
ously in the reaction. Different ratios of alcohol dehydrogen- tions. The optimized process of butyraldehyde to butanol
ase : glutamate dehydrogenase (4 : 1–1 : 6) were used for the conversion with substrate coupled cofactor regeneration
reaction to determine the effect on the production of butanol. showed conversion of 75%. However, the enzyme coupled
The maximum butanol production was obtained at 1 : 4 ratio of reaction showed 24% conversion of butyraldehyde to butanol.
alcohol dehydrogenase : glutamate dehydrogenase 1 : 4 (Fig. 8). Substrate coupled reaction would also be more cost effective
The maximum production of butanol was 1.39 mM than enzyme coupled reaction with respect to butanol produc-
and conversion of butyraldehyde to butanol was 24% using tion. The process could be further scaled up and made
30 mM of NADH. Lower level of glutamate dehydrogenase (ratio continuous by packing a column with beads of enzyme and
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regeneration of cofactor.
343.
12 L. Ott, S. Kohl, M. Bicker and H. Vogel, Chem. Eng. Technol.,
2
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3 M. M. T. Khan, N. S. Rao and S. B. Halligudi, J. Mol. Catal.,
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