A. Rodríguez M et al.
Catalysis Today 372 (2021) 220–225
2
.2. Microorganism
The Laboratory of Vegetal Physiology at Universidad Pedag o´ gica y
Tecnol o´ gica de Colombia provided a B. cereus strain isolated from soils
not treated with furanic aldehydes. This strain was cultured in NA solid
ꢀ
1
medium containing (g L ): peptone (5.0), beef extract (3.0), agar (15),
◦
and 30 mM of FAL as a carbon source, and incubated at 30 C [29].
Single colonies were cultured in modified NB liquid medium con-
Fig. 1. Scheme of the biotransformation of furfural using whole cells.
ꢀ 1
taining (g L ): yeast extract (2.0), meat extract (1.0), and sodium
◦
chloride (5.0), and incubated at 30 C overnight with agitation. The
biotransformation and fermentation of FAL into FOL using microor-
ganisms [2,12–15]. Furan toxicity can be overcome by increasing the
biomass density. Thus, higher initial biomass concentration implies that
more enzymes are present initially converting into the less toxic com-
pound, resulting in apparently increased furanic aldehyde tolerance
purity was verified by BBL Crystal GP identification system and micro-
scopy observation previous to Gram staining. The strain was conserved
◦
in 30% glycerol at -80 . To verify the identity, single colonies were
inoculated in NB and grown overnight; 1 mL of bacterial culture was
used to extract genomic DNA and to amplify the partial sequence of the
[
16]. Other strategies such as the regeneration of cofactors have allowed
1
6S rDNA gene. The similarity tree was generated using the Neighbor-
the increase of FAL tolerance in microorganisms. Thus whole cells of
B. coagulans NLO1 present a 96% yield from 42 mM of FAL using glucose
as co-substrate [12]. Glucose serves to regenerate the NADH cofactor,
and at the same time is converted to lactic acid using a biphasic system
leading to 86% FOL yield from 208 mm of FAL [17]. In fact, glucose has
also been used as co-substrate to improve the bioconversion of FAL and
hydroxymethylfurfural in yeasts [2,18]. Meyerozyma guilliermondii
SC1103 cells tolerate up to 200 mM of FAL with a yield to FOL of 96%
Joining method (Fig. S1) [30].
Samples of the NB were taken every 2 h for 24 h to quantify the total
number of viable cells and construct the growth curve (Fig S2). Serial
ꢀ 8
-9
dilutions in sterile water of each sample and dilutions of 10 , 10 , and
-10
◦
1
0
were plated on NA, and the plates were incubated at 30 C. The
results were expressed in log of colony forming units (CFU log) per mL.
2
.3. FAL bioconversion using growing cells
[
2], but with immobilized cells the yield to FOL decreases slightly to
1%. Therefore NAD(P)H dependent alcohol dehydrogenases catalyze
8
FAL concentration effect (0–75 mM) on growth rate (
μ
) of B. cereus
the reduction of FAL, while glucose as a co-substrate is enzymatically
oxidized for generating NAD(P)H catalysts [2].
◦
was studied on cultures growing in modified NB medium, pH 7.2, 30 C,
and 250 rpm, using an initial optical density at 600 nm (OD600) of 0.05
On the other hand, B. cereus has been reported previously as a strain
that degrades FAL up to a concentration of 40 mmol/L; the best FAL
degradation ability (35%) occurred in long periods (7 days). However,
the authors did not establish the products formed or the possible FAL
adsorption in this sporulating microorganism [19]. B. cereus is a
gram-positive, facultative anaerobic rod-shaped endospore-forming
bacterium [20–22]. The mechanism of sporulation of B. cereus has been
described by various authors [20,21]. In all cases, it has been observed
that its sporulation begins at the end of the late stationary phase [21].
Although B. cereus has been tested in distinct organic transformations
9
or 1.9 × 10 CFU/mL. A Whittaker ELX808 spectrometer with flat-
bottom 96-well cell culture plates (Nest) was used to measure OD600
.
In each well, 200
μ
L of growing bacteria was added. The specific growth
rate ( max) was calculated from the steepest part of the ln(OD600) curve.
μ
The concentration of 30 mM was chosen to analyze the conversion and
yield to FOL and FA until 24 h. The viable cells were determined by
plating in medium containing 30 mM of FAL. The presence of spores was
determined by staining with 5% malachite green aqueous solution.
2
.4. Biocatalysis using resting cells
[
23–25], none of these studies correlate spore formation with the yields
obtained. Besides, B. cereus is a typical bacterium that expresses glucose
dehydrogenase even after exponential growth is completed and sporu-
lation has started. [26] Glucose dehydrogenase from B. cereus has been
used to exploit its ability to reduce prochiral ketones stereoselectively to
chiral alcohols [27,28].
The assays using resting cells involved a culture of this strain in NA
overnight. The growing cells were resuspended in 5 mL of buffer phos-
10
phate at pH 7.2 until obtaining an OD600 of 1.0 or 200 ± 10 × 10
CFU/mL. The cells in this OD600 remained in stationary phase (Fig. S3).
Four treatments were assayed to analyze the bioconversion of FAL.
In this work, a strain of B. cereus, isolated from soils without previous
treatment with furanic aldehydes, was grown in the presence of FAL.
The ability of the bacterium to convert this compound was evaluated.
Later, resting cells of B. cereus in different conditions were assayed until
optimizing FOL production. The effect of glucose to regenerate NAD(P)H
in this bacterium was studied, thus as the addition of Mo6 as a cofactor
of oxidoreductases involved in the transformation of furfural.
First, concentrations from 30 to 200 mM of FAL were assayed at pH 7.2,
◦
3
0 C, and 250 rpm. The FAL concentration with the highest conversion
and yields was used in the next experiments. Second, different concen-
trations of glucose (0ꢀ 150 mM) as co-substrate were assayed, using
3
0 mM of FAL and keeping the pH, temperature, and rpm constant.
+
6+
Third, an assay involving the effect of Mo using Mo(SO
4
)
3
on the
conversion of FAL was performed. In the experiment the concentration
of Mo6 was varied from 0.1 to 0.8 mM using 30 mM of FAL, pH 7.2, and
+
2
. Materials and methods
◦
3
0 C. The last treatment evaluated was the effect of glucose (100 mM)
6
+
with Mo (0.1 mM). In this treatment temperatures ranging from 25 to
2
.1. Materials
◦
3
5 C, and pH ranging from 5.2–9.2 were studied using phosphate
buffer. Each treatment was performed in triplicate. The percentage of
viable cells for each treatment was expressed as the ratio between CFU/
mL after 12 or 24 h over the initial CFU/mL.
The following reagents and culture media were purchased from
commercial sources and used without further purification: Nutrient agar
NA, HiMedia ref. M561), nutrient broth (NB, Scharlau ref. 02,140,500),
furfural (Aldrich, 99%), furfuryl alcohol (Aldrich, 99%), furoic acid
Aldrich, 99%), malachite green oxalate (Merck, 98%), sodium mono-
(
2
.5. Analytical methods
(
hydrogen phosphate (Merck, 99%), sodium dihydrogen phosphate (J.T.
Baker, 99%).
FAL, FOL, and FA were quantitatively determined by high-
performance liquid chromatography using an apparatus Knauer Azura
equipped with a Waters C-18 column. The column temperature was kept
◦
constant at 35 C, and as mobile phase water/acetonitrile (80:20) with a
flow rate of 0.4 mL/min was used. FAL, FOL, and FA concentrations
2
21