Bioresource Technology 291 (2019) 121931

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Bioresource Technology
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Improved upstream processing for detoxification and recovery of xylitol T

produced from corncob
Vinod Kumar, Pankaj Preet Sandhu, Vivek Ahluwalia, Bhuwan Bushan Mishra,
Sudesh Kumar Yadav
⁎

Center of Innovative and Applied Bioprocessing (CIAB), Sector-81 (Knowledge City), Mohali 140306, Punjab, India

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Keywords: This work deals with the development of an improved process for xylitol production from corn cob hydrolysate
Corn cob by biotechnological routes emphasizing the detoxification of corncob acid hydrolysate. The acid hydrolysate
Acid hydrolysis obtained by acid hydrolysis of corn cob was concentrated and detoxified by activated charcoal, membrane
Membrane filtration process and ion exchange resin process. The resultant partially purified corncob hydrolysate was used in fer-
Fermentation
mentation. The fermentation of acid hydrolysate containing 56.5 g/L xylose was carried out in a 14 L fermenter
Carbonation
at pH 4.5 for 48 h with 150 rpm stirring rate at 30 °C. A xylitol yield of 62% was achieved from the partially
purified acid hydrolysate medium during fermentation using Candida tropicalis MTCC 6192. The purity of xylitol
was increased to 92–94% upon downstream processing of carbonation, subsequently ion exchange process and
activated charcoal.

1. Introduction
Xylitol, a five-carbon sugar alcohol is widely used as a sugar sub-
stitute in food industry because of its properties, such as similarity in
sweetness to sucrose, no insulin requirement, prevent dental caries and
low in calories (Makinen, 2000; Rafiqul and Mimi, 2012; López-Linares
et al., 2018). Production of the xylitol is mainly reported through
chemical reduction of xylose obtained from wood hydrolysate.
Catalysts like nickel, palladium and ruthenium are used in the xylitol
conversion process. Such processes are energy consuming and cost in-
tensive (Parajó et al., 1995; Cortez et al., 2016). Biotechnological
conversion of xylose to xylitol can be an economical and efficient
process than chemical route. Xylitol is produced from xylose through
fermentation. Xylose, a pentose sugar mainly found in lignocellulosesic
biomass. Currently many processes available for extraction of xylose
from biomass. However, during pretreatment process of biomass many

⁎
Corresponding author.
E-mail address: sudesh@ciab.res.in (S.K. Yadav).

https://doi.org/10.1016/j.biortech.2019.121931
Received 6 June 2019; Received in revised form 26 July 2019; Accepted 27 July 2019
Available online 30 July 2019
0960-8524/ © 2019 Published by Elsevier Ltd.
V. Kumar, et al.
inhibitors and salts are produced. These inhibitors drastically inhibit
the fermentation process and therefore yield is very low (López-Linares
et al., 2018; Cortez et al., 2016; Mussatto, 2012). Xylitol bioproduction
also depends upon different parameters like feedstock types, pretreat-
ment methods, microorganism used and cultivation conditions. High
concentration of xylose rich hemicellulose in corncob make it an effi-
cient raw material for production of xylitol (Hasan et al. 2011; Kumar
et al., 2018). Sugar oligomers, monosaccharides, and acetic acid are the
main autohydrolysis reaction products reported from corn cob (Yang
and Wyman, 2004; Agbor et al., 2011; Rabemanolontsoa and Saka,
2016).
The mild acidic reaction conditions generate liquors with reduced
contents of undesired byproducts, particularly those from sugar de-
composition and acid-soluble lignin, that may still cause inhibition in
subsequent fermentation steps (Marzialetti et al., 2008; Xu et al., 2008;
Lee et al., 2011). The major challenge in the production of xylitol from
biomass is the detoxification of acid hydrolysate and recovery of pure
xylitol. In view of this, present study was planned to develop a new and
improved process involving activated carbon and membrane separation
technology for detoxification of acid hydrolysate as upstream proces-
sing. Further, better quality xylitol recovery from fermentation broth of
corn cob hydrolysate was optimized through innovative sequential
downstream processing using carbonation, ion-exchange and activated
charcoal.
2. Materials and method
2.1. Materials
Ultrafiltration and nanofiltration was performed using poly-
ethersulfone polymer-based membranes and were procured from
CleaNsep system Pvt. Ltd, Mumbai. Corncobs were locally collected and
air dried followed by fine milling to get particles. All the chemicals and
medium components were purchased form Sigma Aldrich (analytical/
reagent grade) and used as such unless otherwise indicated. All the
experiments were performed in duplicate.
2.2. Acid hydrolysate preparation from corncob
Acid hydrolysate was prepared from corncob as described earlier
(Kumar et al., 2018). Different concentration of corn cob powder was
hydrolyzed with 0.5% HNO3 in a glass batch flask with solid and liquid
ratio (1:10). The reaction mixture was autoclaved (121 °C, 15 psi) for
30 min followed by filtration and pH adjustment with 5 M NaOH. The
concentration of xylose and other contents were analyzed using high
performance liquid chromatography (HPLC).
2.3. Detoxification of acid hydrolysate using activated charcoal
The detoxification of corncob hydrolysate was through activated
charcoal and membrane filtration separation techniques.
2.3.1. Decolorization of corncob acid hydrolysate using activated charcoal
Activated charcoal was added to the acid hydrolysate at different
concentrations from 0.5 to 10% (w/v). The charcoal suspension was
agitated at 150 rpm, 45 °C for 60 min and then activated charcoal was
removed by filtration system.
2.3.2. Concentration and detoxifications of decolorized acid hydrolysate of
corn cob
After charcoal treatment, decolor acid hydrolysate was further
concentrated and detoxified by passing through membrane filtration
system. Ultrafiltration was used to separate high molecular weight
compounds (xylan etc.) from solutions. The treated acid hydrolysate
broth was filtered using a cross-flow membrane unit with a MWCO
1000 Da poly ether sulfone membrane with a surface area of 2.5 m2 and
2
Bioresource Technology 291 (2019) 121931
treated acid hydrolysate broth was pumped using a booster pump. The
following conditions were used for the cross-flow separation; flow rate
(3 L/min), pressure (7.25 kPa), pH (4.0), and ambient temperature. The
permeate of acid hydrolysate obtained after ultrafiltration was con-
centrated further using nanofiltration (MWCO 150 Da) with a surface
area of 2.5 m2. The following conditions were used for the cross-flow
nanofiltration separation: flowrate (2 L/min), pressure (56.05 kPa), pH
(4.0), and ambient temperature. Finally, the concentrated (retentate
fraction) acid hydrolysate was collected in tank for further process
through ion exchange to remove salts. Similarly, xylose rich acid hy-
drolysate has been passed through sequential cationic and anion resins
(Kumar et al., 2018).
2.4. Yeast fermentation for xylitol production
Xylitol production through yeast fermentation was conducted in a
14 L fermenter using 5 L of optimized and detoxified concentrated
corncob acid hydrolysate medium (New Brunswick Sci. Inc., Fermentor
Bioflow 415, USA). The optimized and purified corncob acid hydro-
lysate medium contained 56.5 g xylose/L and 0.5% yeast extract. The
appropriate medium was sterilized in situ at 110 °C for 20 min and in-
oculated with 5.0% of the optimized inoculum of Candida tropicalis
adapted yeast strain. Fermentation was carried out at 30 °C with agi-
tation of 150 rpm and aeration rate of 0.5 vvm. Samples were with-
drawn for 60 h at regular time intervals of 6 h, centrifuged and were
analyzed for xylitol production as well as leftover xylose.
2.5. Recovery and purification of xylitol from fermentation broth
The obtained broth after fermentation was used for recovery and
purification of xylitol through sequential processing through carbona-
tion, ion exchange and activated charcoal as described below.
2.5.1. Carbonation process
The carbonation process for xylitol purification was followed as
described earlier with some modifications (Moodley et al., 2002). The
required amount of lime was added, in xylitol rich fermentation broth
(w/v) and stirred continuously at 40 °C. After the sample was mixed for
5 min, the carbonatation reaction was initiated by introducing carbon
dioxide into the reaction vessel. As soon as the pH of the liquor reached
to 7.0, the gas supply was stopped. The carbonated broth was stirred for
a period of one minute. The carbonated liquor was then filtered, cooled
and stored in a freezer for analysis.
2.5.2. Ion exchange process
Post carbonation process, fermentation broth was treated with A-
IRN78 strong anionic exchange resin. Fabricated steel columns of 32 cm
long and 2.5 cm diameter were loaded with these resins and used for
the purification process. Peristaltic pump was used for feed inlet to the
subsequent columns that were tightly packed with the resins at 4 mL/
min flow rate at room temperature (Kumar et al., 2018). The effluents
of the final column were collected and filtered through 0.4 μm filter and
stored at 4 °C. The concentration levels of salt and xylitol was de-
termined in the inlet and outlets samples obtained from high perfor-
mance liquid chromatography (HPLC). Following filtration, the filtrate
was concentrated using rotary evaporator under vacuum.
2.5.3. Activated charcoal treatments
In this study, activated carbon was mainly applied to discolor and to
remove other impurities like protein, phenolics, etc. as described earlier
under Section 2.3.1.
2.6. Quantitation method:
Quantitative determination of carbohydrate sugars (5- and 6-carbon
sugars), furan compounds (furfural and 5-hydroxymethyl furfural
V. Kumar, et al.
(HMF)), acetic acid and xylitol collected intermittently during the
conversion processes were performed using high performance liquid
chromatography (HPLC; Agilent, model 1200, Palo Alto, CA) equipped
with refractive index detector (55 °C). The analytical column used was
Agilent HiPlex H (300 mm × 7.7 mm, 8 μm) and operated at 60 °C with
5 mM H2SO4 mobile phase at the flow rate of 0.7 mL/min. The mobile
phase was filtered through a 0.22 μm nylon membrane (Millipore
Corporation, MA) and degassed. Each data point was the average of two
replicates.
3. Results and discussion
3.1. Extraction of xylose using acid hydrolysis
The effect of particle size on acid hydrolysis of corncob biomass was
evaluated for the optimum extraction of xylose. The yield of xylose
production was compared for three different sized particles of corncob
obtained by sieving through mesh of 2 mm, 4 mm, and 6 mm. However,
xylose yield was found highest in the corncob powder obtained by di-
rect grinding without sieve. The physical structure of the feed such as
surface area plays an important role in the yield of xylose. Hydrolysis of
plant and wood cell walls could be affected by its chemical composition
as well as structural and morphological features. The idea behind
choosing three particle sizes was to see the effect of increased surface
area on lignocellulosic hydrolysis of corn cob. As particle size decreases,
the ratio of surface area to mass grows enormously. This may provide
many more reaction sites or places where molecules can collide and
interact. Therefore, the xylose yield was lesser from the minimum
particle size (2.0 mm) of corncob. It is evident that the maximum ex-
traction of 16.6 mg/ml xylose was obtained from the acid hydrolysis
(0.5% HNO3) of corncob without sieve at 121 °C temperature for 30 min
treatment. A decline in extraction of xylose to 14.2 mg/ml was recorded
from 6 mm particle size of corn cob biomass and was further drastically
declined to 11.2 mg/ml and 9.0 mg/ml from 4 mm and 2 mm particle
size of biomass, respectively. The concentration of released sugars
during pretreatment is directly dependent upon the type of lig-
nocellulosic material, composition of substrates, temperature, time,
acid concentration, and solid to liquid ratio employed in the process
(Karimi et al., 2006; Taherzadeh and Karimi, 2007; Akpinar et al.,
2009; Lenihan et al., 2011; Kim et al., 2011). Kim et al. (2011) reported
that the maximum hemicellulose hydrolysis can be achieved from
1.16% of acid hydrolysis of barley straw at 150 °C for 16.9 min. A
combined dilute acid-catalyzed hydrolysis of oil palm empty fruit
bunch has been reported for the optimum production of xylose (Zhang
et al., 2012). Xylose yield of 91.3% was obtained after hydrolysis cat-
alyzed by 0.5% (w/v) of H2SO4 and 0.2% (w/v) of H3PO4 at 160 °C at a
liquid to solid ratio of 20 mL/g of oil palm empty fruit bunch for
10 min.
3.2. Decolorization of corn cob acid hydrolysate using activated charcoal
Four set of experiments were carried out with 10 L of acid hydro-
lysate of corncob. Wherein the activated charcoal concentration was
optimized by varying from 1 to 10 g/L. Each set was kept at the opti-
mized treatment temperature of 30 °C for 1 h on magnetic stirrer
equipped with temperature controller. After the desired incubation, the
acid hydrolysate was filtered using ceramic filter (prototype design).
The filtrate thus obtained in each set after charcoal treatment was
analyzed for xylose concentration (Table 1). Use of 1 g/L activated
charcoal concentration was found to be optimum for maximum deco-
lorization of corncob acid hydrolysate. At this concentration, there was
minimum binding of xylose (3.4%) to activated charcoal and maximum
color was removed. Further, an increase in activated charcoal con-
centration beyond an optimal concentration of 1 g/L was resulted in
higher binding of xylose and thereby decreasing their yield. Activated
charcoal has been reported as a cost-effective detoxification method for
3
Bioresource Technology 291 (2019) 121931
Table 1
Effect of charcoal concentration on treatment of corncob acid hydrolysate.
S. No. Charcoal Recover of Loss of xylose Decolorization of
concentration xylose during (%) acid hydrolysate
(% w/v) process (g)
1 Initial 187.0 ± 8.7 – –
2. 0.5 ± 0.02 185.6 ± 8.7 0.75 +
3. 1.0 ± 0.05 180.7 ± 7.7 3.4 ± 0.15 ++++
4. 2.5 ± 0.15 172.0 ± 6.21 8.0 ± 0.35 ++++
5. 5.0 ± 0.21 171.1 ± 7.15 8.5 ± 0.45 ++++
6. 10.0 ± 0.50 158.0 ± 6.50 15.5 ± 0.65 ++++
Initial xylose concentration: 187.0 ± 8.7 mg/ml and was considered 100%;
decolorization of acid hydrolysate visible indication (+: not decolorization, +
+++, approx. 95% decolorization of acid hydrolysate.
the treatment of lignocellulose hydrolysates with good capacity to ab-
sorb inhibitory compounds without sugars loss (Mussatto et al., 2014;
Canilha et al., 2008; Sene et al., 2011). Inhibitory compounds like
phenolics, furfurals, 5-HMF from the lignocellulose hemicellulosic hy-
drolysates have been removed by charcoal treatment to achieve the
desired xylitol production (Grzenia et al., 2010). The efficiency of the
activated charcoal for decolorization depends upon some factors like
concentration, temperature, contact time and pH of the reaction mix-
ture (Mussatto et al., 2014).
3.2.1. Concentration and detoxification of corn cob acid hydrolysate using
membrane process and ion exchange resin
Membrane filtration was used to detoxify and concentrate the acid
hydrolysate of corncob obtained after treatment with activated char-
coal. In present study, ultrafiltration (1000 Da) and nanofiltration
(150 Da) was used for removal of inhibitors from acid hydrolysis and
successfully achieved the removal of inhibitors like acetic acid (82.4%)
and salts of acid (57.8%), respectively. Xylose concentration was in-
creased from 16.2 g/L to 56.1 g/L (3.4-fold) using ultrafiltration and
nanofiltration with minimum loss of xylose. Ultrafiltration was used for
removal of high molecular weight of compounds produced during acid
hydrolysis of corn cob. Increase in xylose concentration to the level of
90.6% has been reported using nanofiltration (Murthy et al., 2005).
Monosaccharides recovery from dilute acid hydrolysis with the removal
of 90% inhibitors has also been documented by nanofiltration Jiang
et al., 2018). In addition, used diananofiltration mode to detoxification
of hemicellulose hydrolysate from olive pomace has been detoxified
through nanofiltration with 99% removal of inhibitors and a loss of
40% mono sugars Bras et al. (2014). The recovery of xylose, salt of
acids, and acetic acid obtained through membrane process are shown in
Table 2. The concentration of xylose in the tank was increased from
13.6 to 38.4 g/L. Also, 150 Da polymeric membrane was found to be
effective in removal of inhibitors from corn cob acid hydrolysate with
simultaneous concentration of the xylose. Similarly, 150 Da polymeric
membrane has been used for the removal of inhibitors and concentra-
tion of sugars from acid hydrolysate of rice straw (Maitai et al., 2012).
3.3. Xylitol production in a 14 L fermenter
Xylitol production from detoxified and concentrated acid hydro-
lysate of corncob was used for xylitol production through fermentation.
Process of xylitol production was performed in a 14 L fermenter using 5
L of detoxified and concentrated acid hydrolysate medium (New
Brunswick Sci. Inc., Fermenter Bioflow 415, USA) contained 56.5 g/L of
xylose, 5.0 g/L yeast extract pH 4.5 was used for xylitol production.
With the increase in production of xylitol from xylose by yeast Candida
tropicalis in fermentation, the pH of the medium was increased to 5–7
(Fig. 1). This increase in pH had transformed dissociated acetic acid
into undissociated form that delayed the xylitol production in fermen-
tation. Therefore, the pH of the fermentation broth was maintained at 5
V. Kumar, et al. Bioresource Technology 291 (2019) 121931

Table 2
Process for concentration and detoxification of acid hydrolysate using membrane filtration process and ion exchange resin:
Step Acid hydrolysate Total volume (L) Salt of acid (g) Xylose (g) Acetic acid (g)

1 Acid hydrolysate initial 45.0 ± 3.1 321.2 ± 24.5 729 ± 45 84.5 ± 4.6
2 Ultrafiltration P 40.0 ± 0.15 266.4 ± 9.15 626.9 ± 24.8 69.6 ± 3.2
R 5.0 ± 0.25 49.2 ± 2.6 65.8 ± 3.5 11.7 ± 0.4
3 Nano filtration P 30.0 ± 1.1 39.9 ± 1.5 45.7 ± 2.1 36.0 ± 1.8
R 10.0 ± 0.5 226.4 ± 11.3 561.2 ± 22.1 23.5 ± 1.2
4 First ion exchanger (H+) 9.9 ± 0.7 200.8 ± 9.8 552.8 ± 19.4 23.5 ± 1.8
5 Second ion exchanger (OH−) 9.7 ± 0.8 35.5 ± 1.8 518.8 ± 19.2 16.16 ± 1.5

Note: P: Permeate; R: Retentate.

to overcome the inhibitory effect of acetic acid in xylitol production.
The C. tropicalis strain produced good titres of xylitol in a wide pH range
of 4–6 with maximum (62%) at pH 4.5. In an earlier optimized process
at 1.4 L, a yield of 0.6 g xylitol/g xylose and a productivity of 0.26 g/L/
h has been documented in the fermentation of model corn fiber hemi-
cellulose hydrolysate using Candida tropicalis ATCC 96745. However,
the process yielded by-products like furfural, and 5-hydro-
xymethylfurfural. Importantly, the yield of xylitol obtained in our study
was higher compared to the previous reported study on corncob bio-
mass (Canettieri et al., 2007). However, the use of synthetic medium for
xylitol production in fermenter by C. guilliermondii yielded 0.79 g/g
xylitol (Faria et al., 2002). But such processes will be costly because of
the synthetic medium. While the use of sugarcane bagasse and barley
bran hydrolysate in fermentation produced lesser xylitol (Um and Bae,
2011; Cruz et al., 2000). Hence, the fermentation process optimized for
xylitol production in sale up fermenter from corncob hydrolysate and C.
tropicalis produced higher concentration.
exchange chromatography that removed salt impurities. The xylitol
obtained was 89.7% (from initial concentration of xylitol) after anion
exchange chromatography. Final purification of the broth containing
xylitol was performed using activated charcoal for removal of remain
colours or phenolic content. After three step purification, highly pure
xylitol broth of 78.06% was recover from initial concentration of xylitol
obtained (Table 3). Carbonation has been reported to be effective in the
removal of the colours (40–50%) turbidity (95%), starch (93%), sul-
phates (86%) and phosphates (100%), gums (29%) and magnesium
(67%) from solutions (Moodley et al. 2002). The anionic (Amberlite
94S) and cationic (Amberlite 200C) resins have been used for xylitol
purification and found that there was a loss of about 40–55% in yield
due to xylitol due to adhesion to the resin surface (Gurgel et al., 1995).
Another study carried out by Misra et al. (2011) used activated charcoal
for purification process and then used vacuum concentrated and crys-
tallization of xylitol. They have obtained a yield of 44% xylitol by using
15.0 g/L charcoal at 30 °C for 1 h and crystallization temperature of
−20 °C as purification mechanism.

3.4. Purification of xylitol from fermentation broth

Carbonatation has been used for over 100 years for the refining of
raw sugar. In view of this, carbonation process was used for the first
time in purification of xylitol obtained from fermentation of detoxified
acid hydrolysate of corn cob using Candida tropicalis. In the process,
calcium hydroxide was added after diluting the obtained xylitol from
fermenter broth. Thereafter, carbon dioxide gas was bubbled into the
xylitol rich fermentation broth in saturation, under controlled condi-
tions of pH and temperature. The impurities of the broth were absorbed
and enmeshed in the conglomerated particles of the calcium carbonate
formed by precipitation of carbon dioxide and calcium hydroxide. Clear
liquor was separated from calcium carbonate by centrifugation. The
recovery of xylitol was as high as 94.6%. The obtained clear liquor
containing xylitol was further purified by passing through anion
4. Conclusion
For the maximum production and recovery of xylitol, a detoxifica-
tion process for the removal of salts, acids and phenolic compounds
from lignocellulosic acid hydrolysate of corn cob has been developed.
Optimized process could successfully remove 60% salts and 90% phe-
nolic compounds from corn cob acid hydrolysate. Along with fermen-
tation performance of Candida tropicalis was significantly improved
with a productivity of 1.12 g/l/h. A recovery and purification of xylitol
to the extent of more than 78% with 92–94% (purity) was achieved
from corn cob hydrolysate through downstream processing of carbo-
nation, ion exchange and activated charcoal.

Fig. 1. Fermentation profile of xylitol production using partial purified acid hydrolysate.

4
V. Kumar, et al.
Table 3
Downstream recovery of xylitol from fermentation broth using carbonation and ion exchange chromatography.
S. No. Purification step Total volume (ml)
1 Initial 1000
2 Carbonation 980 ± 35.1
3 Ion exchanger (OH−) 950 ± 30.3
4 Activated charcoal (0.5%) 930 ± 30.3
Acknowledgments
Authors are thankful to CEO, CIAB for continuous support and en-
couragement. The authors express their sincere thanks to the
Department of Biotechnology (DBT), Government of India, New Delhi
for financial support.
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