Chemo-enzymatic conversion of glucose into 5-hydroxymethylfurfural in seawater

Article abstract of DOI:10.1002/cssc.201200065

Do you sea water? Water consumption will be a challenge in biorefineries, and the use of non-drinkable sources of water will be preferred. Herein, glucose is converted into 5-hydroxymethylfurfural (HMF) in a chemo-enzymatic one-pot, two-step procedure, involving immobilized glucose isomerase to produce fructose and oxalic acid to dehydrate it to HMF. Copyright

Full text of DOI:10.1002/cssc.201200065

DOI: 10.1002/cssc.201200065
Chemo-Enzymatic Conversion of Glucose into 5-Hydroxymethylfurfural in
Seawater
[
a]
Philipp M. Grande, Christian Bergs, and Pablo Domꢀnguez de Marꢀa*
Interest in biorefineries is increasing because they represent an
alternative to tackle the challenges created by the depletion of
raw (fossil) materials as well as the environmental concerns
the on-spec standards for economics and efficiency must be
kept. Following these considerations, this Communication dis-
closes a combined chemo-enzymatic concept for the conver-
sion of glucose into HMF in seawater as reaction medium
(Scheme 1).
[
1]
created by chemical processes. Taking lignocellulose as raw
material, pretreatment steps lead to separation of the main
[
2]
components,
subsequently
producing a broad number of in-
teresting platform chemicals de-
rived thereof, to finally process-
ing them to generate fuels and
[
1,3–5]
commodities.
In this area, 5-
hydroxymethylfurfural (HMF), de-
rived from the dehydration of C6
sugars (e.g., glucose), represents
a versatile building block known
[
1,3–5]
for centuries
that still has
not found an efficient and eco-
nomic industrial process at large
[
5]
scale. Recent strategies to form
HMF (and other furans such as
furfural) typically include acid
[
6]
catalysis in aqueous media as
well as in solvents such as alco-
hols, ionic liquids, or deep-eutec-
Scheme 1. Conceptual approach for the chemo-enzymatic conversion of glucose into HMF in seawater.
[
7]
[8]
tic-solvents, or sub- and supercritical fluids. Likewise, the
set-up of biphasic systems has gained importance in the last
years, since the in situ extraction of HMF suppresses the by-
product formation caused by the inherent reactivity of these
The use of seawater as medium for biomass processing re-
presents an opportunity for using non-drinkable water sources
at large scale, especially at coastal cities, and for the valoriza-
[14]
tion of existing resources (e.g., algae). Likewise, it would pro-
vide the source of NaCl needed for efficient sugar dehydra-
[
9]
aldehydes. Higher conversions in furans are obtained when
inorganic salts (e.g., NaCl) are added, an effect that has been
[9b,10,11]
tion.
Several articles dealing with the use of seawater in
[
10]
related to the salting-out effect. Actually, this salt effect was
first reported in the 1930s, when acid-catalyzed dehydration of
biomass processing have been recently reported, focusing on
chemical and biocatalytic approaches for cellulose depolymeri-
[
11]
[9b,15]
xylose was studied. Apart from this salting-out effect, other
roles of the salts in the reaction mechanism cannot be exclud-
ed (e.g., decrease of the pH of an acidic solution by adding in-
zation and fermentations.
For the HMF production, the
first step comprised the use of commercially available, immobi-
lized d-glucose/xylose isomerase (EC 5.3.1.5) for the isomeriza-
tion of glucose to fructose until equilibrium (ca. 50:50 glucose/
[
11,12]
organic neutral salts).
Other recent examples to produce
[16]
HMF include the combination of glucose isomerization with
fructose). After enzyme filtration, a biphasic system is set-up
by the addition of a second phase of 2-methyltetrahydrofuran
(2-MTHF), as biobased solvent for the in situ extraction of
[
13]
fructose dehydration.
Sustainable biomass processes may be envisaged when raw
materials, reaction media, and catalysts can be derived from bi-
obased resources, thus being readily available worldwide and
in large and regenerable amounts. Furthermore, reactions
must be conducted in relatively mild and ecological conditions
with diminished waste formation. Obviously, at the same time
[17]
formed HMF. 2-MTHF has been recently used as a useful sol-
[2,9b,10b]
vent in the field of biomass processing.
For fructose de-
hydration oxalic acid was chosen, being an efficient, biobased,
and easily biodegradable catalyst with promising use in ligno-
[2,3a,15c]
cellulose (pre)treatments.
After HMF formation (out of
[2]
fructose), oxalic acid can be recovered by crystallization, and
a solution of seawater with glucose (50%) and 2-MTHF (ca. 5%
v/v) would remain. This aqueous solution might be directly
transferred to a fermentation plant, making use of the remnant
of glucose as carbon source for microorganism growth. Exam-
ples of fermentative processes in seawater have already been
[
a] P. M. Grande, C. Bergs, Dr. P. Domꢀnguez de Marꢀa
Institut fꢁr Technische und Makromolekulare Chemie (ITMC)
RWTH Aachen University
Worringerweg 1. 52074 Aachen (Germany)
Fax: (+49)241-8022177
E-mail: dominguez@itmc.rwth-aachen.de
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[
15b,d]
reported.
Alternatively, the effluent can be recycled back
water medium combined with organic acids may represent
a promising approach for an integrated HMF production, com-
bined with algae and desalination plants (e.g., using 2ꢂ con-
centrated seawater, Table 1, entry 4).
in the enzymatic step to isomerize glucose again into fructose.
First of all, the performance of oxalic acid (compared to HCl)
in fructose dehydration to HMF at different temperatures in bi-
phasic systems (2-MTHF-water and NaCl) was studied
Once HMF is formed and extracted in 2-MTHF and oxalic
acid is recovered, the seawater containing glucose (ca. 50%
from initial loading) might be directly used as fermentative
broth to produce biofuels and
(
Figure 1). As observed, biobased organic acids lead to analo-
gous results in the dehydration of fructose to HMF as mineral
other chemicals, without the
need for downstream processing
[15b]
of glucose.
In addition, to
evaluate whether that seawater
effluent could still be recycled
and treated again with the im-
mobilized d-glucose/xylose iso-
merase step (Scheme 1), the en-
zymatic performance in seawater
(
containing 2-MTHF 5% v/v) was
studied. In a first step, a compari-
son between the glucose iso-
merization in buffer and in sea-
water was conducted (Figure 2).
Although the initial rate of
glucose isomerase is lower in
À1
Figure 1. HMF yield at different temperatures (100–1608C) in the 2-MTHF phase. Conditions: Fructose 36 gL
2
,
0 wt% NaCl, water, pH 1. Oxalic acid (black) or HCl (grey) (0.1m); 1 h reaction time. Yield determined by HPLC
(see Experimental).
ones, providing high HMF yields (under such non-optimized
process conditions) in the range of 140–1608C. Glucose dehy-
dration by using oxalic acid under these conditions was not
observed (<1% HMF formed from glucose) when different
fructose/glucose mixtures (1:1 or 3:7) were used (data not
shown). This finding is in agreement with our previous studies
on oxalic acid as a selective catalyst for cellulose depolymeriza-
tion at those conditions, leading to glucose formation without
[
15c]
significant degradation to HMF.
Subsequently, fructose de-
hydration was assessed in different concentrated seawater sys-
tems, using oxalic acid as catalyst (Table 1).
Figure 2. Glucose isomerization catalyzed by d-glucose/xylose isomerase in
phosphate buffer (^) and seawater saturated with 2-MTHF (&). Conditions:
Table 1. Oxalic-acid-catalyzed dehydration of fructose in seawater at dif-
ferent concentrations.
[
a]
À1
À1
Glucose 140 gl ; 3 wt% immobilized glucose isomerase (ꢁ300 Ug );
phosphate buffer pH 8; seawater pH 8.2 (without extra buffering); 608C;
Entry
Solvent
NaCl [wt%]
HMF yield [%]
600 rpm. Glucose determined by UV/Vis glucose (HK) assay kit (see
Experimental).
1
2
3
4
5
water
water
–
20
ꢀ4
ꢀ8
ꢀ16
27
52
45
54
57
seawater
concentrated seawater (2ꢂ)
concentrated seawater (4ꢂ)
seawater (saturated with 2-MTHF) than in phosphate buffer,
under these non-optimized conditions equilibrium in seawater
was reached in ca. 5 h (data not shown). This is consistent with
previous literature describing glucose isomerase as a robust
enzyme, also able to catalyze the glucose isomerization in
À1
[a] Conditions: 36 gL fructose; 1408C; oxalic acid (0.1m), pH 1; 1 h reac-
tion time. HMF yield determined in the 2-MTHF phase (HPLC).
[18]
ionic liquids or in ethanol-aqueous systems. To further study
the activity and stability of the immobilized enzyme, several re-
cycling experiments were conducted by filtering the enzyme
and testing it again. Gratifyingly, the enzyme displayed a high
stability along the different cycles (Figure 3).
The direct use of seawater provided an efficient medium for
the fructose dehydration with oxalic acid. As seawater compris-
[
15a,b]
es several salts,
an effect of all of them in the performance
[
12]
might be assumed. Since the dehydration of algae-derived
saccharides to afford HMF has been reported as straightfor-
Since both steps (enzymatic and chemical) proceeded well
in the seawater media, a combination of them was performed.
[
14]
ward (compared to that of glucose), the herein reported sea-
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À1
(
36 gL ) were prepared, in which
different amounts of acids and
NaCl were aggregated. In a closed
glass reactor a mixture of 2.5 mL
reaction phase and 2.5 mL MTHF
phase was magnetically stirred at
different reaction temperatures.
Standard reaction conditions (glu-
cose isomerization). Solutions of
À1
glucose (140 gL ) were prepared
in phosphate buffer and in 2-
MTHF-saturated
seawater.
In
a closed glass reactor 5 mL reac-
tion phase and 3% (w/w) of immo-
bilized glucose isomerase were
added. The reaction mixture was
shaken in an Eppendorf Thermo-
À1
Figure 3. Enzymatic glucose isomerization with recycled enzymes. Conditions: glucose 140 gL ; seawater saturat- mixer Comfort at 600 rpm and
À1
ed with 2-MTHF; pH 8.2; 3 wt% glucose isomerase (ꢁ300 Ug ); kinetics up to 40 min reaction time; 608C;
608C. Phosphate buffer: In 1 L dis-
tilled water 7.4897 gK HPO ,
.9526 g KH PO , and 0.300 mg
6
00 rpm; glucose concentration determined by UV/Vis glucose (HK) assay kit (see Experimental).
2
4
0
2
4
MgSO4 were dissolved under stir-
ring at room temperature. The solution was stored at 48C until
use.
À1
Glucose isomerase was added to a glucose solution (140 gL )
À1
in 5 mL seawater. After 2 h ca. 90 gL of fructose were formed
(
calculated as difference from measured glucose concentration
À1
of 52 gL ). After enzyme filtration, oxalic acid was added, to-
Analysis (HPLC). After stopping the reaction by cooling the reaction
mixture to room temperature, the 2-MTHF phase was removed
completely, and the water phase was extracted with 2.5 mL 2-
MTHF. The 2-MTHF fractions were collected, filtered, and analyzed
by using HPLC, 250ꢂ4.0 mm Kromasil 100C18 5 mm (eluent: 90:10,
gether with 5 mL of 2-MTHF. After 1 h reaction at 1408C, analy-
sis of the 2-MTHF phase afforded ca. 40 gL of HMF, equiva-
À1
lent to 64% yield. The organic phase was removed, the pH
À1
was adjusted to 8 (with NaOH), and 90 gL glucose was
H O/MeOH), Jasco UV detector, 210 nm.
2
added. After addition of new enzyme, the reactor was stirred
À1
again for 2 h at 608C, leading to 60 gL of fructose (measured
Analysis (Glucose (HK) Assay Kit). 10 mL of the reaction solution was
diluted with 990 mL distilled water. After mixing for 30 s, 50 mL of
the diluted solution was mixed with 200 mL of the glucose (HK)
assay kit (GAHK20) in a microtiter plate, and the absorption at
À1
glucose concentration of 77 gL ).
In summary, we disclose a chemo-enzymatic concept for the
conversion of glucose into HMF in seawater. In a first step, glu-
cose isomerase produces fructose up to equilibrium condi-
tions. Later on, the set-up of a biphasic system with bio-based
3
60 nm was measured. With glucose as standard substrate a cali-
bration curve was recorded. Equation for linear region: Absorb-
2
ance=4.77[Glucose]+0.12 (R =0.9999).
2
-MTHF and after the addition of oxalic acid as catalyst, fruc-
tose is dehydrated to HMF, which is extracted in situ into the
organic phase. The remnant seawater with glucose (ca. 50%)
can be either used further for fermentative processes, or con- Acknowledgements
versely may be recycled back in the enzymatic tank, since glu-
cose isomerase proves to be very stable under the applied re-
action conditions. After concept optimization, the combination
of enzymes and bio-based acids may provide useful and sus-
tainable synergies for future biorefineries, making use of sea-
water as readily available and non-drinkable water (and salt)
source.
This work was performed as part of the Cluster of Excellence
“Tailor-Made Fuels from Biomass”, which is funded by the Excel-
lence Initiative of the German Research Foundation to promote
science and research at German universities. We thank Prof. Dr.
Walter Leitner, Zaira Maugeri, Henning Kayser, and Thorsten vom
Stein for fruitful and stimulating discussions. Hannelore Es-
chmann and Julia Wurlitzer are acknowledged for support with
analytical measurements (HPLC).
Experimental Section
Keywords: acid catalysis · biomass · enzyme catalysis ·
renewable resources · seawater
Chemicals. Fructose, glucose, oxalic acid, hydrochloric acid, sodium
chloride, sodium hydroxide, dipotassium hydrogen phosphate, po-
tassium dihydrogen phosphate, glucose isomerase Sweetzyme IT
À1
Extra (5.1.3.5; ꢁ300 mg ), glucose (HK) assay kit (GAHK20), and 2-
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Received: January 26, 2012
Published online on && &&, 0000
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These are not the final page numbers!

COMMUNICATIONS
P. M. Grande, C. Bergs,
P. Domꢀnguez de Marꢀa*
&& – &&
Chemo-Enzymatic Conversion of
Glucose into 5-Hydroxymethylfurfural
in Seawater
Do you sea water? Water consumption
will be a challenge in biorefineries, and
the use of non-drinkable sources of
water will be preferred. Herein, glucose
is converted into 5-hydroxymethylfurfu-
ral (HMF) in a chemo-enzymatic one-
pot, two-step procedure, involving im-
mobilized glucose isomerase to produce
fructose and oxalic acid to dehydrate it
to HMF.
ChemSusChem 0000, 00, 1 – 4
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemsuschem.org
&
5
&
ÞÞ
These are not the final page numbers!