Biocatalytic Reduction of HMF to 2,5-Bis(hydroxymethyl)furan by HMF-Tolerant Whole Cells

Article abstract of DOI:10.1002/cssc.201601426

Catalytic upgrading of 5-hydroxymethylfurfural (HMF), an important biobased platform chemical for high-value products, is currently of great interest. In this work, a new highly HMFtolerant yeast strain—Meyerozyma guilliermondii SC1103 was isolated, and biocatalytic reduction of HMF to 2,5-bis(hydroxymethyl)furan (BHMF) using its resting cells was reported. Cosubstrates exerted a significant effect on the catalytic activity and selectivity of microbial cells as well as their HMF-tolerant levels whereas the nitrogen source and mineral salts had no effects. In addition, M. guilliermondii SC1103 cells exhibited good catalytic performances within the range of pH 4.0–10.0. The yeast was highly tolerant to both HMF (up to 110 mm) and BHMF (up to 200 mm). In addition, 100 mm HMF could be selectively reduced to BHMF within 12 h by its resting cells in the presence of 100 mm glucose (as cosubstrate), with a yield of 86 % and selectivity of >99 %. The production of 191 mm of BHMF was realized within 24.5 h by using a fed-batch strategy, with a productivity of approximately 24 g L^?1per day. In addition, this new biocatalytic approach was applied for the reduction of furfural and 5-methylfurfural, affording the corresponding furfuryl alcohols with yields of 83 and 89 %, respectively.

Full text of DOI:10.1002/cssc.201601426

DOI: 10.1002/cssc.201601426
Full Papers
Biocatalytic Reduction of HMF to 2,5-
Bis(hydroxymethyl)furan by HMF-Tolerant Whole Cells
Yan-Mei Li, Xue-Ying Zhang, Ning Li,* Pei Xu, Wen-Yong Lou, and Min-Hua Zong*[a]
Catalytic upgrading of 5-hydroxymethylfurfural (HMF), an im-
portant biobased platform chemical for high-value products, is
currently of great interest. In this work, a new highly HMF-
tolerant yeast strain—Meyerozyma guilliermondii SC1103 was
isolated, and biocatalytic reduction of HMF to 2,5-bis(hydroxy-
methyl)furan (BHMF) using its resting cells was reported. Co-
substrates exerted a significant effect on the catalytic activity
and selectivity of microbial cells as well as their HMF-tolerant
levels whereas the nitrogen source and mineral salts had no ef-
fects. In addition, M. guilliermondii SC1103 cells exhibited good
catalytic performances within the range of pH 4.0–10.0. The
yeast was highly tolerant to both HMF (up to 110 mm) and
BHMF (up to 200 mm). In addition, 100 mm HMF could be se-
lectively reduced to BHMF within 12 h by its resting cells in the
presence of 100 mm glucose (as cosubstrate), with a yield of
86% and selectivity of >99%. The production of 191 mm of
BHMF was realized within 24.5 h by using a fed-batch strategy,
with a productivity of approximately 24 gLÀ1 per day. In addi-
tion, this new biocatalytic approach was applied for the reduc-
tion of furfural and 5-methylfurfural, affording the correspond-
ing furfuryl alcohols with yields of 83 and 89%, respectively.
Introduction
Recently, utilization of renewable and carbon-neutral biomass
has attracted a lot of interest for the production of biobased
fuels and platform chemicals.[1] 5-Hydroxymethylfurfural (HMF)
obtained through the dehydration of hexoses was recognized
by the U.S. Department of Energy as one of “Top 10+4” bio-
based chemicals.[2] HMF could be converted into various useful
chemicals owing to the presence of functional groups such as
primary hydroxyl and formyl. For example, HMF could undergo
oxidation and esterification as well as etherification.[3] In addi-
tion, HMF could be transformed into 2,5-bis(hydroxymethyl)-
furan (BHMF), 2,5-dihydroxymethyltetrahydrofuran (DHMTHF),
and 2,5-dimethylfuran (DMF) through selective reduction.[3b]
BHMF is the hydrogenation product of the formyl group in
HMF and is a versatile building block for the synthesis of poly-
mers,[4] drugs,[5] macrocycle polyether compounds,[6] and crown
ethers.[7]
because of the formation of an equimolar byproduct
(5-hydroxymethyl-2-furancarboxylic acid, HMFCA).[10] In addi-
tion, several groups reported environmentally benign (photo)-
electrochemical routes for the reduction of HMF to BHMF.[11]
Biocatalysis represents an attractive and promising option to
supplement or replace chemical methods for organic transfor-
mations because of a number of advantages such as excellent
selectivity, mild reaction conditions, high efficiency, and envi-
ronmental friendliness.[12] However, biocatalytic valorization of
HMF received little attention[13] compared to chemical routes.
The reason may be that the costly enzymes have been used
generally for the synthesis of enantiopure and high-value
building blocks or drugs.[14] Driven by environmental concerns,
biocatalytic production of bulk commodities such as biodiesel
and biobased polyesters has received growing attention.[15] Re-
cently, our group has reported enzyme-catalyzed oxidation
and esterification of HMF.[16]
To date, BHMF is synthesized mainly by chemical reduction
of HMF. Cottier et al. described BHMF synthesis by stoichio-
metric reduction of HMF using sodium borohydride.[6] Catalytic
hydrogenation of HMF to BHMF was reported on noble metal
catalysts[8] as well as non-noble metals.[9] The Cannizzaro
reaction was used for the synthesis of BHMF from HMF, but
the theoretic selectivity toward the desired product was 50%
Biocatalytic reduction of HMF to BHMF can be conducted
theoretically using isolated oxidoreductases as well as whole
cells. Compared to isolated enzymes, whole cells were prefera-
ble for HMF reduction because they are not only inexpensive
and more stable but also do not require complex cofactor re-
generation systems that are necessary for isolated enzymes.[17]
However, efficient synthesis of BHMF from HMF using whole
cells is still a great challenge because the substrate HMF is
a well-known potent inhibitor to microorganisms.[18] To our
knowledge, there are only two reports on whole cell-catalyzed
transformation of HMF in the literature,[13f,19] in which 2,5-
furandicarboxylic acid (FDCA) was synthesized by oxidation of
HMF. Some microorganisms were reported to be capable of
transforming HMF in biological detoxification of the inhibitors
present in lignocellulosic hydrolysates.[20] Nonetheless, these
[a] Y.-M. Li, Dr. X.-Y. Zhang, Dr. N. Li, Dr. P. Xu, Prof. W.-Y. Lou, Prof. M.-H. Zong
School of Food Science and Engineering
South China University of Technology
381 Wushan Road, Guangzhou 510640 (P.R. China)
E-mail: lining@scut.edu.cn
btmhzong@scut.edu.cn
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cssc.201601426.
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microorganisms were not appropriate biocatalysts for efficient
synthesis of BHMF from HMF because of the following reasons:
i) Their biodetoxification efficiencies remained low,[21] suggest-
ing that HMF reduction rates were low; for instance, the high-
est HMF transformation rate was reported to be less than
2 mmolLÀ1 hÀ1.[20c,21a] ii) Their tolerance to HMF, especially in
high concentrations, was poor; for example, HMF of more than
30 mm would exert a significantly deleterious effect on bio-
transformation, leading to long reaction periods (>48 h).[20c,d]
iii) The selectivities were not satisfactory; in addition to BHMF,
the oxidation products of HMF were also formed.[21b,22] In this
work, we report a new HMF-tolerant yeast strain—Meyerozyma
guilliermondii SC1103 isolated from soil samples for efficient
synthesis of BHMF from HMF (Scheme 1). The effects of some
key conditions on whole-cell catalytic synthesis of BHMF were
studied to obtain an optimized biocatalytic process. In addi-
tion, the tolerance of this strain to the substrate (HMF) and
vs. 1). A yield of 87% was achieved after 7 h of reaction using
glucose as cosubstrate (Table 1, entry 2); in addition, the yield
changed slightly with the prolongation of the reaction time,
suggesting that the product is very stable and cannot be de-
graded further by the yeast. In addition, the selectivity was
higher for glucose compared to the one obtained for glycerol.
In the case of glycerol, the lower selectivity (93.1%) was owed
to the formation of more HMFCA, an HMF oxidation product.
The results indicate that glucose is the preferred cosubstrate
for this reduction reaction possibly because, compared to glyc-
erol, glucose is a better carbon source that can sufficiently pro-
vide the reduced form of nicotinamide cofactor (NAD(P)H) for
HMF reduction.[23] This could be confirmed by the results on
glucose concentration effect on BHMF synthesis (Table 1,
entries 2–4). The catalytic performances of microbial cells de-
creased significantly with decreasing glucose concentrations.
Lower glucose concentrations led to longer reaction times as
well as lower yields and selectivities. The poor results (55%
yield and 61% selectivity) were obtained after 24 h in the ab-
sence of cosubstrate, suggesting that the cosubstrate plays
a key role in whole cell-catalyzed reduction of HMF. In addition
to using oxidized cosubstrates (glucose and glycerol) for bio-
catalytic reduction, we attempted to use acetone as reduced
cosubstrate for biocatalytic oxidation of HMF (Figure S1 in the
Supporting Information). Unfortunately, BHMF remained to be
the major product with a low yield (21%). Both nitrogen
source and mineral salts had no significant effects on the re-
duction reaction (Table 1, entries 5–7). Good yield (89%) and
excellent selectivity (99%) were achieved in the absence of
both nitrogen source and mineral salts (Table 1, entry 7), which
would significantly simplify the reaction mixture and facilitate
the downstream product isolation and purification.
Scheme 1. Biotransformation of HMF to BHMF in buffer.
product (BHMF) was evaluated, and a substrate feeding strat-
egy was used for the production of a high concentration of
BHMF. Besides, the established biocatalytic process was used
for the synthesis of other furfuryl alcohols.
Results and Discussion
Effects of cosubstrates, nitrogen source, and mineral salts
on BHMF synthesis
Effects of key reaction conditions on BHMF synthesis
Table 1 shows the effects of cosubstrates, nitrogen source, and
mineral salts on BHMF synthesis. It was found that cosub-
strates exerted a significant effect on the catalytic performance
of M. guilliermondii SC1103 cells, particularly on the reaction
rate and selectivity. The reaction time was much lower using
glucose as cosubstrate than using glycerol, although compara-
ble BHMF yields were obtained in both cases (Table 1, entries 2
The influences of some key conditions on whole cell-catalyzed
reduction of HMF were studied (Figure 1). Figure 1a shows the
effect of pH value on the reduction of HMF when pH value
varies from 4.0 to 10.0. M. guilliermondii SC1103 cells exhibited
good catalytic performances within the pH range examined.
The reaction rates were comparable at pH 4.0–10.0, and the
maximal yields of 86–91% were achieved after a reaction time
Table 1. Comparison of the catalytic performances of resting and culturing cells.[a]
Entry
Cosubstrate
[mmolLÀ1
Nitrogen source
[gLÀ1
Mineral salts[b]
t [h]
BHMF yield
[%]
Selectivity
[%]
]
]
1
2
3
4
5
6
7
glycerol, 30
glucose, 30
glucose, 15
none
glucose, 30
glucose, 30
glucose, 30
(NH4)2SO4, 2
(NH4)2SO4, 2
(NH4)2SO4, 2
(NH4)2SO4, 2
none
yes
yes
yes
yes
yes
none
none
24
7
12
24
7
88.0Æ0.3
87.1Æ0.2
88.7Æ0.8
55.2Æ1.5
90.5Æ3.5
85.8Æ1.5
89.3Æ0.9
93.1Æ0.1
98.9Æ0.0
97.6Æ0.2
61.4Æ0.4
99.0Æ0.1
98.9Æ0.0
99.0Æ0.0
(NH4)2SO4, 2
none
7
7
[a] Reaction conditions: 40 mm HMF, 20 mgmLÀ1 microbial cells, 4 mL phosphate buffer (100 mm, pH 7.2), 200 rpm, 308C. [b] MgCl2·6H2O (0.1 gLÀ1), EDTA
(10 mgLÀ1), ZnSO4·7H2O (2 mgLÀ1), CaCl2·2H2O (1 mgLÀ1), FeSO4·7H2O (5 mgLÀ1), Na2MoO4·2H2O (0.2 mgLÀ1), CuSO4·5H2O (0.2 mgLÀ1), CoCl2·6H2O
(0.4 mgLÀ1), and MnCl2·2H2O (1 mgLÀ1).
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Figure 1. Effects of a) pH, b) biocatalyst dosage, c) temperature, and d) reaction time on BHMF synthesis. The following general conditions applied unless
otherwise stated: 40 mm HMF, 30 mm glucose, 20 mgmLÀ1 microbial cells, 4 mL phosphate buffer (100 mm, pH 7.2), 308C, 200 rpm; a) buffers used:
pH 4.0–6.0 citrate buffer, pH 6.0–8.0 phosphate buffer, pH 8.0–9.0 Tris-HCl buffer, pH 9.0–10.0 glycine-NaOH buffer, 7 h; b) gradual increase of cell dosage
from 10 to 30 mgmLÀ1 with corresponding periods of 9, 7, 7, 5, and 5 h; c) gradual increase of temperature from 20 to 408C with corresponding periods of
24, 9, 7, 7, and 5 h; d) 358C.
of 7 h. In addition, excellent selectivities (ca. 99%) were ob-
served. Figure 1b shows the impact of the cell dosage on
BHMF synthesis. Although the cell dosage exerted no signifi-
cant effect on the maximal yields (88–91%) and selectivities
(ca. 99%), its effect on the reaction rates was substantial. For
example, a reaction period of 9 h was needed for reaching
equilibrium with 10 mgmLÀ1 of cell dosage whereas only 5 h
were needed for more than 25 mgmLÀ1 microbial cells. The
effect of the reaction temperature on BHMF synthesis is shown
in Figure 1c. Similarly, the reaction rates were also affected sig-
nificantly by the temperature, but its effect on yield and selec-
tivity was slight. The reaction time was reduced significantly by
increasing the temperature (e.g., 24 h at 208C vs. 5–7 h at
>308C). These results suggest that M. guilliermondii SC1103
cells can exhibit good catalytic performances over a wide
range of conditions, which demonstrates its great application
potential for HMF reduction. Figure 1d shows the time course
of whole-cell catalytic reduction of HMF under optimized con-
ditions. BHMF was synthesized quickly, and a maximal yield of
89% was obtained within 7 h. Moreover, excellent selectivities
were retained during the reaction.
presence of 30 mm glucose, and further increasing the sub-
strate concentrations resulted in significant decrease of the ini-
tial reaction rates. This effect suggests that significant substrate
inhibition occurs when HMF concentrations are more than
80 mm. In addition, the yield was recorded at various HMF con-
centrations in the presence of 30 mm glucose. As shown in
Figure 2a, high yields (92–97%) were obtained within 5–7 h,
with excellent selectivities (99%) when HMF concentrations
were less than 50 mm. Although the selectivity remained high
at higher substrate concentrations, the yield decreased signifi-
cantly to 37–84%.
A yield of only 69% was obtained when the HMF concentra-
tion was 70 mm, which could not be explained rationally by
Substrate inhibition and toxicity toward microbial cells
As described above, HMF is an inhibitory and toxic compound
toward microbes because it may result in damage of cell walls
and membranes as well as inhibition of the activities of various
dehydrogenases and of ribonucleic acid (RNA) synthesis.[20b,24]
Therefore, the HMF-tolerant level of microbial cells is critical
for efficient synthesis of BHMF from HMF. The tolerance of
M. guilliermondii SC1103 cells toward HMF was evaluated
(Figure 2). As shown in Figure 2a, the highest initial reaction
rate (14.4 mmolLÀ1 hÀ1) was observed at 70 mm HMF in the
Figure 2. Effects of substrate concentrations on a) BHMF synthesis and
b) cell viability. Reaction conditions: 30 mm glucose, 20 mgmLÀ1 microbial
cells, 4 mL phosphate buffer (100 mm, pH 7.2), 200 rpm, 358C; a) reaction
periods: 5, 7, 7, 12, 24, 36, 12 and 7 h, with gradually increasing HMF con-
centrations from 20 to 110 mm, respectively; b) incubation period of 12 h.
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the results on substrate inhibition, because no substrate inhibi-
tion occurs at this substrate concentration. Besides inhibiting
enzyme activities, HMF might also be toxic to microbial cells.
Therefore, cell viability was assayed after the yeast cells were
incubated in the presence of various HMF concentrations (Fig-
ure 2b). It was found that more than 89% of the cells re-
mained alive after an incubation period of 12 h in the presence
of less than 70 mm HMF. However, the cell viability decreased
significantly to 52–73% for more than 80 mm HMF, indicating
that these concentrations of HMF may be highly toxic to the
yeast cells. However, the total number of cells including dead
and alive cells did not change considerably before and after in-
cubation in HMF solutions, which suggested that no cell lysis
occurred. The above results on HMF inhibition and toxicity
suggest that M. guilliermondii SC1103 cells may be tolerant to
substrate concentrations up to 70 mm in the presence of
30 mm glucose. Nonetheless, the yields (69–84%) were not sat-
isfactory when substrate concentrations were 60–70 mm. It is
well known that efficient regeneration of the reduced cofactors
(e.g., NAD(P)H) is critical for biocatalytic reduction reactions,
which is closely related to the cosubstrates and their concen-
trations.[17] Accordingly, the changes in glucose concentrations
were monitored during the reaction when the HMF concentra-
tion was 50 mm (data not shown). It was found that glucose
was used up within 3 h. Therefore, we reasoned that the rela-
tively low yields might be owed to insufficient regeneration of
the reduced cofactors for the reduction of HMF in the pres-
ence of 30 mm glucose, when HMF concentrations were more
than 60 mm.
tions were 80–100 mm. Even microbial cells were capable of
smoothly reducing HMF in the concentration up to 110 mm to
the desired product, resulting in a yield of 87% after 36 h. To
our knowledge, this is the highest HMF-tolerant concentration
of microbial cells ever reported. In general, the effects of furan
on microbial cells could be explained by a redirection of micro-
bial energy for repairing the damage, relieving enzyme inhibi-
tion, and regenerating the cofactors.[25] Therefore, supplemen-
tation of excess cosubstrate might favor the regeneration of
NAD(P)H as well as fixing the damage, thus significantly en-
hancing HMF-tolerance of microbial cells.[23] However, low
yields (42–51%) were obtained when HMF concentrations
were higher than 150 mm (Figure 3). In addition, no significant
improvements in the yields were observed when higher con-
centrations of glucose (150–200 mm) were supplemented (Fig-
ure S2, available in the Supporting Information), suggesting
significant substrate inhibition and toxicity at substrate con-
centrations of above 150 mm. It is worth noting that the excel-
lent selectivities (>99%) remained in the range of the sub-
strate concentrations tested.
Product inhibition and toxicity toward microbial cells
In addition to substrate inhibition and toxicity, the products
also may exert such negative effects on biocatalytic reduction
reactions.[17] Product inhibition and toxicity toward microbial
cells were studied. As shown in Figure 4, no significant product
inhibition was observed when BHMF concentrations were less
than 150 mm because the initial reaction rates (13.4 and
14.4 mmolLÀ1 hÀ1) were comparable. The initial reaction rate
slightly decreased to 10.6 mmolLÀ1 hÀ1 when the BHMF con-
centration was increased to 300 mm. In addition, BHMF had
almost no toxicity toward microbial cells when its concentra-
tions were less than 100 mm and slight toxicity was found at
150 mm BHMF (82% of cell viability). Cell viability (63%) re-
mained moderate at BHMF concentration up to 300 mm. Simi-
To verify our assumption, the reduction of HMF was con-
ducted in the presence of 100 mm glucose (Figure 3). The bio-
catalytic reduction of HMF was improved significantly when
a high concentration of glucose was supplemented. For exam-
ple, the yield of 87% was obtained in the case of 70 mm HMF
in the presence of 100 mm glucose, which is much higher than
that in the presence of 30 mm glucose (87% vs. 69%). More
importantly, the HMF-tolerant level of microbial cells became
higher with 100 mm glucose as cosubstrate (Figure 3): good
yields (ca. 85%) were achieved in 12 h when HMF concentra-
Figure 4. Effect of the product concentrations of BHMF synthesis and cell
viability. Conditions: 50 mm HMF, 30 mm glucose, BHMF of the designated
concentration, 20 mgmLÀ1 microbial cells, 4 mL phosphate buffer (100 mm,
pH 7.2), 200 rpm, 358C; after microbial cells were incubated for 12 h under
the above conditions without HMF, cell viability was measured.
Figure 3. Whole cell-catalyzed reduction of HMF in the presence of 100 mm
glucose. Conditions: HMF of the designated concentration, 20 mgmLÀ1
microbial cells, 4 mL phosphate buffer (100 mm, pH 7.2), 200 rpm, 358C.
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larly, no significant changes in the total cell populations were
observed before and after incubation in BHMF solutions. The
results indicate that the product is less inhibitory and toxic to
the yeast cells than the substrate, which is in good agreement
with a previous study.[20a]
Synthesis of BHMF by a fed-batch strategy
Continuous accumulation of a high concentration of the prod-
uct in the reactor is highly desirable because it can not only
significantly improve the reactor productivity but also facilitate
the product purification. Inspired by the above results on in-
hibition and toxicity, biocatalytic synthesis of BHMF was con-
ducted by fed-batch feeding of the substrate (Figure 5). BHMF
Figure 6. Biocatalytic reduction of furfurals to furfuryl alcohols. Reaction
conditions: 50 mm furfurals, 30 mm glucose, 20 mgmLÀ1 microbial cells,
4 mL phosphate buffer (100 mm, pH 7.2), 200 rpm, 358C.
The results obtained in this work were compared with se-
lected literature results. As shown in Table 2, HMF was effi-
ciently hydrogenated to BHMF with an excellent yield and se-
lectivity using Pt/MCM-41 as catalyst under mild conditions.[8d]
Compared to noble metals, non-noble metal catalysts usually
required relatively harsh reaction conditions (e.g., higher tem-
perature) for BHMF synthesis owing to lower reactivity.[9a,c] The
efficiency of electrocatalytic hydrogenation of HMF to BHMF
remained low[11c] although the method appeared to be envi-
ronmentally friendly and selective. Biocatalytic reduction of
HMF is of considerable interest owing to its many advantages
such as not needing to use metal catalysts, H2, and organic sol-
vents; being highly selective; and requiring mild reaction con-
ditions. Although culturing cells of some microorganisms were
reported to be HMF-tolerant, their transformation efficacies
were very poor (>48 h required for complete conversion of
HMF).[20c,d,21b] And the quantitative results on the selectivity
remained unknown in most of the previous publications al-
though qualitative results were described. For example, Feld-
man et al. reported that both BHMF and FDCA were formed
during detoxification by culturing cells of Pleurotus ostrea-
tus.[21b] M. guilliermondii SC1103 was proven to be a good bio-
catalyst for the reduction of HMF to BHMF because it is highly
tolerant to both HMF and BHMF. Moreover, a good yield as
well as an excellent selectivity was obtained in 12 h when the
HMF concentration was up to 100 mm. The biocatalytic ap-
proach established in this work appeared to be more eco-
friendly than metal-mediated chemical methods, in spite of
a lower productivity, because the biocatalyst was environmen-
tally benign, and no volatile organic solvent and H2 were used.
In addition, this biocatalytic process was energy efficient and
highly selective. Its productivity and substrate concentrations
may be improved significantly by increasing cell dosage and
using a biphasic system, and the work is currently in progress.
Figure 5. Synthesis of BHMF by a fed-batch strategy. Reaction conditions:
50 mm HMF, 30 mm glucose, 20 mgmLÀ1 microbial cells, 4 mL phosphate
buffer (100 mm, pH 7.2), 200 rpm, 358C. After HMF was almost used up,
0.2 mmol HMF and 0.12 mmol glucose were supplemented.
up to 191 mm was produced within 24.5 h after three-batch
feeding of HMF, and the total yield was approximately 88%.
The productivity of approximately 24 gLÀ1 per day was ob-
tained. In addition, only traces of HMFCA (about 1 mm) were
formed as the sole byproduct. The selectivity toward the de-
sired product reached >99%.
Reduction of furfurals to furfuryl alcohols
In addition to HMF reduction, the biocatalytic process estab-
lished in this work was applied for the reduction of other plat-
form chemicals—furfural and 5-methylfurfural (Figure 6).
Furfuryl alcohol is an important intermediate for the produc-
tion of thermostatic resins, synthetic fibers, farm chemicals,
and plasticizers.[26] It is well known that furfural is a much
stronger inhibitory compound to microorganisms than HMF.[27]
Interestingly, it was found that this compound was also a good
substrate of M. guilliermondii SC1103 and furfuryl alcohol was
obtained with a yield of 83% in 5 h, along with 2-furoic acid as
the byproduct (Figure 6). The selectivity was approximately
96%. In addition, M. guilliermondii SC1103 could efficiently
catalyze the reduction of 5-methylfurfural, affording 5-methyl-
furfuryl alcohol with a yield of 89% and selectivity of >99%.
Conclusions
We developed an efficient and selective biocatalytic approach
for the synthesis of 2,5-bis(hydroxymethyl)furan (BHMF) from
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Table 2. HMF reduction catalyzed by various catalysts.
Catalysts
Reaction conditions
t [h]
C/Y [%][a]
BHMF
Ref.
selectivity [%]
Pt/MCM-41
~2m HMF, 50 mgmLÀ1 catalyst, 0.8 MPa H2, 358C,
in H2O
~0.2m HMF, 25 mgmLÀ1 catalyst, 1508C, in ethanol
(as both hydrogen donor and solvent)
0.02m HMF, E=À1.3 V vs Ag/AgCl, 60 C charge passed,
pH 9.2, in borate buffer
2
98.9 (Y)
94.1 (C)
100 (C)
98.9
88.9
[8d]
[9c]
ZrO(OH)2
2.5
Ag catalytic electrode
n.a.[b]
>99
[11c]
Pleurotus ostreatus
Saccharomyces cerevisiae 307-12H60
0.03m HMF, 308C, 180 rpm, in a GP medium[c]
0.06m HMF, 308C, 250 rpm, 1% of the inoculate
culture, in a complete synthetic medium
0.06m HMF, 308C, pH 5.5, 220 rpm, in seaweed
hydrolysates of 10% (w/v) slurry
0.1m HMF, 20 mgmLÀ1 cells, 100 mm glucose as cosubstrate,
pH 7.2, 358C, 200 rpm, in phosphate buffer
48
48
100 (C)
100 (C)
n.a.
n.a.
[21b]
[20c]
Scheffersomyces stipitis KCTC 7228
M. guilliermondii SC1103
60
12
100 (C)
86.0 (Y)
n.a.
[20d]
99.2
This work
[a] Y: Yield, C: conversion. [b] n.a.: Not available. [c] GP: glucose peptone.
medium containing 1% yeast extract, 2% peptone, and 2% glu-
cose. Then, the 2% seed culture was inoculated to the fresh YPD
medium. After incubation at 308C and 200 rpm for 12 h, the cells
were harvested by centrifugation (3500 rpm, 15 min, 48C) and
washed twice with distilled water, followed by dispersing in phos-
phate buffer to yield cell concentrations of 10–30 mg (cell wet
weight) per mL.
5-hydroxymethylfurfural (HMF) using resting cells of Meyerozy-
ma guilliermondii SC1103. The new isolated M. guilliermondii
SC1103 was highly tolerant to HMF as well as BHMF and
proved to be an excellent biocatalyst for the reduction of HMF.
The resting cells of M. guilliermondii SC1103 still retained good
catalytic performances over a wide range of conditions.
BHMF up to 191 mm was synthesized in 24.5 h by a fed-batch
strategy, which is promising for the development of an indus-
trially sound biocatalytic process for HMF reduction. Moreover,
the yeast cells were able to efficiently transform furfural and 5-
methyfurfural into target furfuryl alcohols with good selectivi-
ties. In addition to the reduction of furfurals, M. guilliermondii
SC1103 may have promising application potential in biological
detoxification because of its high detoxification efficiency.
General procedure for biocatalytic reduction of HMF
Typically, phosphate buffer (4 mL, 100 mm, pH 7.2) containing
50 mm HMF, 30 mm glucose and 20 mg (cell wet weight) per mL
microbial cells was incubated at 358C and 200 rpm. Aliquots were
withdrawn from the reaction mixtures at specified time intervals
and diluted with the corresponding mobile phase prior to HPLC
analysis. The initial reaction rate (V0) was calculated based on the
decrease in HMF concentrations at the initial reaction stage. The
yield was defined as the ratio of the measured product amount to
the theoretical product amount based on the initial amount of
HMF. The selectivity was defined as the ratio of BHMF amount (in
mmol) to the sum of all the products. All experiments were con-
ducted at least in duplicate, and the values were expressed as the
means Æstandard deviations.
Experimental Section
Biological and chemical materials
M. guilliermondii SC1103 was isolated from soil samples obtained
from the grounds of an industrial plant. Based on the analysis of
the D1/D2 domain of nuclear large subunit (26S) ribosomal DNA
(available in the Supporting Information) as well as physiological
and biochemical characteristics, the strain was identified as M. guil-
liermondii. The phylogenetic tree of this train is shown in Figure S3
in the Supporting Information. M. guilliermondii SC1103 (CCTCC No.
M2016144) was maintained in the China Center for Type Culture
Collection (CCTCC, Wuhan, P.R. China).
HMF (98%) was purchased from J&K Scientific Ltd. (Guangzhou,
P.R. China). BHMF (98%) and furfural (99%) were obtained from
Macklin Biochemical Co., Ltd. (Shanghai, P.R. China). HMFCA (98%)
was purchased from Adamas Reagent Ltd. (Shanghai, P.R. China).
Furfuryl alcohol (98%) was obtained from Alfa Aesar (Tianjin, P.R.
China). 2-Furoic acid (98%) and 5-methylfurfural (97%) were pur-
chased from TCI (Japan). 5-Methyl-2-furoic acid (97%) was ob-
tained from Sigma–Aldrich (USA). 5-Methylfurfuryl alcohol (98%)
was purchased from Apollo Scientific Ltd. (UK).
HPLC analysis
The reaction mixtures were analyzed using an Eclipse XDB-C18
column (4.6 mmꢁ250 mm, 5 mm, Agilent, USA) by reversed-phase
HPLC equipped with a Waters 996 photodiode array detector
(Waters, USA). The mobile phase was the mixture of acetonitrile
and 4.0% (NH4)2SO4 solution (10:90, v/v) with a flow rate of
0.6 mLminÀ1. The retention times of HMFCA, BHMF, and HMF were
6.1, 8.3, and 9.9 min, respectively.
Cell viability assay
Yeast cell viability was measured using the methylene-blue staining
method.[28] Briefly, the cell suspension (100 mL) was withdrawn and
diluted 40 times with phosphate buffer (100 mm, pH 7.2) after in-
cubation for 12 h under the designated conditions. Then, the dilut-
ed cell suspension (50 mL) was added into 0.1% methylene blue
dissolved in physiological saline (2 mL). After staining for 5 min,
blue dead and colorless viable cells were counted using a blood
Cultivation of M. guilliermondii SC1103 cells
M. guilliermondii SC1103 cells were precultivated at 308C and
200 rpm for 12 h in the yeast extract peptone dextrose (YPD)
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Full Papers
haddam, M. T. M. Koper, ChemSusChem 2015, 8, 1745–1751; c) J. J. Roy-
lance, T. W. Kim, K.-S. Choi, ACS Catal. 2016, 6, 1840–1847.
counting chamber. The cell viability was expressed as the percent-
age of viable cells in the total cells, and the values were expressed
as the means Æstandard deviations (n=3).
[12] a) R. Wohlgemuth, Curr. Opin. Biotechnol. 2010, 21, 713–724; b) B.
Schulze, M. G. Wubbolts, Curr. Opin. Biotechnol. 1999, 10, 609–615.
[13] a) M. van Deurzen, F. van Rantwijk, R. Sheldon, J. Carbohydr. Chem.
1997, 16, 299–309; b) M. Krystof, M. Pꢂrez-Sꢃnchez, P. Domꢄnguez de
Marꢄa, ChemSusChem 2013, 6, 630–634; c) M. Krystof, M. Pꢂrez-Sꢃnchez,
P. Domꢄnguez de Marꢄa, ChemSusChem 2013, 6, 826–830; d) J. Carro, P.
Ferreira, L. Rodrꢄguez, A. Prieto, A. Serrano, B. Balcells, A. Ardꢃ, J. Jimꢂ-
nez-Barbero, A. Gutiꢂrrez, R. Ullrich, FEBS J. 2015, 282, 3218–3229;
e) W. P. Dijkman, D. E. Groothuis, M. W. Fraaije, Angew. Chem. Int. Ed.
2014, 53, 6515–6518; Angew. Chem. 2014, 126, 6633–6636; f) F. Koop-
man, N. Wierckx, J. H. de Winde, H. J. Ruijssenaars, Bioresour. Technol.
2010, 101, 6291–6296; g) J. Donnelly, C. R. Mꢅller, L. Wiermans, C. J.
Chuck, P. Domꢄnguez de Marꢄa, Green Chem. 2015, 17, 2714–2718;
h) S. M. McKenna, S. Leimkuhler, S. Herter, N. J. Turner, A. J. Carnell,
Green Chem. 2015, 17, 3271–3275; i) N. Wierckx, T. D. Elink Schuurman,
L. M. Blank, H. J. Ruijssenaars in Microorganisms in Biorefineries (Ed.: B.
Kamm), Springer, Berlin, 2015, pp. 207–223.
Optimized synthesis of BHMF by fed-batch feeding of
substrate
The reaction mixture containing 4 mL phosphate buffer (100 mm,
pH 7.2), 50 mm HMF, 30 mm glucose and 20 mgmLÀ1 microbial
cells was incubated at 358C and 200 rpm. After HMF was almost
used up, 0.2 mmol HMF and 0.12 mmol glucose were repeatedly
supplemented into the reaction mixture. The changes in the con-
centrations of various compounds were monitored by HPLC.
Acknowledgements
[14] J. M. Woodley, Trends Biotechnol. 2008, 26, 321–327.
This research was financially supported by the National Natural
Science Foundation of China (21676103) and the Natural Science
Foundation of Guangdong Province (2014A030313263).
[15] a) S. Hama, A. Kondo, Bioresour. Technol. 2013, 135, 386–395; b) A.
Pellis, E. Herrero Acero, V. Ferrario, D. Ribitsch, G. M. Guebitz, L. Gardos-
si, Trends Biotechnol. 2016, 34, 316–328.
[16] a) Y. Z. Qin, Y. M. Li, M. H. Zong, H. Wu, N. Li, Green Chem. 2015, 17,
3718–3722; b) Y. Z. Qin, M. H. Zong, W. Y. Lou, N. Li, ACS Sustainable
Chem. Eng. 2016, 4, 4050–4054.
Keywords: biocatalysis · furan · furfural · platform chemicals ·
reduction
[17] Y. Ni, J.-H. Xu, Biotechnol. Adv. 2012, 30, 1279–1288.
[18] a) E. Palmqvist, B. Hahn-Hꢆgerdal, Bioresour. Technol. 2000, 74, 25–33;
b) S. I. Mussatto, I. C. Roberto, Bioresour. Technol. 2004, 93, 1–10.
[19] C.-F. Yang, C.-R. Huang, Bioresour. Technol. 2016, 214, 311–318.
[20] a) Z. L. Liu, P. J. Slininger, B. S. Dien, M. A. Berhow, C. P. Kurtzman, S. W.
Gorsich, J. Ind. Microbiol. Biotechnol. 2004, 31, 345–352; b) Y. Zhang, B.
Han, T. C. Ezeji, New Biotechnol. 2012, 29, 345–351; c) Z. L. Liu, P. J. Sli-
ninger, S. W. Gorsich, Appl. Biochem. Biotechnol. 2005, 121–124, 0451–
0460; d) C. H. Ra, G.-T. Jeong, M. K. Shin, S.-K. Kim, Bioresour. Technol.
2013, 140, 421–425.
[21] a) D. Zhang, Y. L. Ong, Z. Li, J. C. Wu, Biochem. Eng. J. 2013, 72, 77–82;
b) D. Feldman, D. Kowbel, N. L. Glass, O. Yarden, Y. Hadar, Biotechnol. Bi-
ofuels 2015, 8, 63.
[22] H. Ran, J. Zhang, Q. Gao, Z. Lin, J. Bao, Biotechnol. Biofuels 2014, 7, 51.
[23] V. Ujor, C. V. Agu, V. Gopalan, T. C. Ezeji, Appl. Microbiol. Biotechnol.
2014, 98, 6511–6521.
[1] a) C. O. Tuck, E. Pꢂrez, I. T. Horvꢃth, R. A. Sheldon, M. Poliakoff, Science
2012, 337, 695–699; b) R. A. Sheldon, Catal. Today 2011, 167, 3–13;
c) R. A. Sheldon, Green Chem. 2014, 16, 950–963.
[2] J. J. Bozell, G. R. Petersen, Green Chem. 2010, 12, 539–554.
[3] a) R.-J. van Putten, J. C. van der Waal, E. de Jong, C. B. Rasrendra, H. J.
Heeres, J. G. de Vries, Chem. Rev. 2013, 113, 1499–1597; b) A. A. Rosatel-
la, S. P. Simeonov, R. F. Frade, C. A. Afonso, Green Chem. 2011, 13, 754–
793.
[4] a) C. Zeng, H. Seino, J. Ren, K. Hatanaka, N. Yoshie, Macromolecules
2013, 46, 1794–1802; b) C. Zeng, H. Seino, J. Ren, N. Yoshie, ACS Appl.
Mater. Interfaces 2014, 6, 2753–2758; c) A. Gandini in Agricultural and
Synthetic Polymers, Vol. 433, American Chemical Society, 1990, pp. 195–
208.
[5] I. Matsumoto, K. Nakagawa, K. Horiuchi, Jpn. Kokai 1973, 73, 763.
[6] L. Cottier, G. Descotes, Y. Soro, Synth. Commun. 2003, 33, 4285–4295.
[7] J. M. Timko, D. J. Cram, J. Am. Chem. Soc. 1974, 96, 7159–7160.
[8] a) J. Ohyama, A. Esaki, Y. Yamamoto, S. Arai, A. Satsuma, RSC Adv. 2013,
3, 1033–1036; b) M. Tamura, K. Tokonami, Y. Nakagawa, K. Tomishige,
Chem. Commun. 2013, 49, 7034–7036; c) T. Pasini, G. Solinas, V. Zanotti,
S. Albonetti, F. Cavani, A. Vaccari, A. Mazzanti, S. Ranieri, R. Mazzoni,
Dalton Trans. 2014, 43, 10224–10234; d) M. Chatterjee, T. Ishizaka, H.
Kawanami, Green Chem. 2014, 16, 4734–4739.
[9] a) Y. Zhu, X. Kong, H. Zheng, G. Ding, Y. Zhu, Y.-W. Li, Catal. Sci. Technol.
2015, 5, 4208–4217; b) A. J. Kumalaputri, G. Bottari, P. M. Erne, H. J.
Heeres, K. Barta, ChemSusChem 2014, 7, 2266–2275; c) W. Hao, W. Li, X.
Tang, X. Zeng, Y. Sun, S. Liu, L. Lin, Green Chem. 2016, 18, 1080–1088.
[10] a) E.-S. Kang, D. W. Chae, B. Kim, Y. G. Kim, J. Ind. Eng. Chem. 2012, 18,
174–177; b) S. Subbiah, S. P. Simeonov, J. M. S. S. EsperanÅa, L. P. N.
Rebelo, C. A. M. Afonso, Green Chem. 2013, 15, 2849–2853.
[24] a) T. Modig, G. Liden, M. J. Taherzadeh, Biochem. J. 2002, 363, 769–776;
b) J. Zaldivar, A. Martinez, L. O. Ingram, Biotechnol. Bioeng. 1999, 65, 24–
33.
[25] J. R. M. Almeida, T. Modig, A. Petersson, B. Hꢆhn-Hꢆgerdal, G. Lidꢂn,
M. F. Gorwa-Grauslund, J. Chem. Technol. Biotechnol. 2007, 82, 340–349.
[26] a) B. M. Nagaraja, A. H. Padmasri, B. David Raju, K. S. Rama Rao, J. Mol.
´
Catal. A 2007, 265, 90–97; b) J. Kijenski, P. Winiarek, T. Paryjczak, A. Lew-
icki, A. Mikołajska, Appl. Catal. A 2002, 233, 171–182.
[27] B. Sanchez, J. Bautista, Enzyme Microb. Technol. 1988, 10, 315–318.
[28] a) S. S. Lee, F. M. Robinson, H. Y. Wang, Biotechnol. Bioeng.. 1981, 11,
641–649; b) K. Qi, X.-X. Xia, J.-J. Zhong, Bioresour. Technol. 2015, 189,
36–43.
Manuscript received: October 9, 2016
Revised: October 22, 2016
[11] a) Y. Kwon, E. de Jong, S. Raoufmoghaddam, M. T. M. Koper,
ChemSusChem 2013, 6, 1659–1667; b) Y. Kwon, Y. Y. Birdja, S. Raoufmog-
Accepted Article published: && &&, 0000
Final Article published: && &&, 0000
ChemSusChem 2016, 9, 1 – 8
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These are not the final page numbers! ÞÞ

FULL PAPERS
Y.-M. Li, X.-Y. Zhang, N. Li,* P. Xu,
W.-Y. Lou, M.-H. Zong*
&& – &&
Biocatalytic Reduction of HMF to 2,5-
Bis(hydroxymethyl)furan by HMF-
Tolerant Whole Cells
High HMF tolerance and selectivity!
An efficient and selective biocatalytic
approach for the synthesis of 2,5-bis
(hydroxymethyl)furan (BHMF) from
5-hydroxymethylfurfural (HMF) is suc-
cessfully developed by using highly
HMF-tolerant Meyerozyma guilliermondii
SC1103 cells for the first time. A fed-
bath strategy was used for the synthesis
of BHMF, obtaining a good yield and
excellent selectivity.
&
ChemSusChem 2016, 9, 1 – 8
www.chemsuschem.org
8
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!