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Bioconversion of Lignocellulosic biomass to xylitol: An overview

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DOI: 10.1016/j.biortech.2016.04.092

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 Bioresource Technology xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Bioconversion of lignocellulosic biomass to xylitol: An overview

Linga Venkateswar Rao ⇑, Jyosthna Khanna Goli, Jahnavi Gentela, Sravanthi Koti
Dept. of Microbiology, Osmania University, Hyderabad, Telangana State 500 007, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Effective pre treatment strategies

were discussed. Lignocellulosic Biomass Hydrolysis

Recent detoxification methodologies
have been elaborated. Acid Based Enzyme Based

Emphasis on the overall processes Processing Inhibitors

involved in the bioconversion of

biomass to xylitol. Hexoses Hexoses
Detoxification Pentoses

Focus on the upcoming fermentation
Pretreatment
strategies.
Xylose
Reduction in crystallinity of HC Fermentation
Concentration,
Lignin removal
Purification &
Xylitol Crystallization
Hydrolysis

a r t i c l e i n f o a b s t r a c t

Article history: Lignocellulosic wastes include agricultural and forest residues which are most promising alternative
Received 31 December 2015 energy sources and serve as potential low cost raw materials that can be exploited to produce xylitol.
Received in revised form 16 April 2016 The strong physical and chemical construction of lignocelluloses is a major constraint for the recovery
Accepted 19 April 2016
of xylose. The large scale production of xylitol is attained by nickel catalyzed chemical process that is
Available online xxxx
based on xylose hydrogenation, that requires purified xylose as raw substrate and the process requires
high temperature and pressure that remains to be cost intensive and energy consuming. Therefore, there
Keywords:
is a necessity to develop an integrated process for biotechnological conversion of lignocelluloses to xylitol
Xylitol
Pretreatment
and make the process economical. The present review confers about the pretreatment strategies that
Lignocellulose facilitate cellulose and hemicellulose acquiescent for hydrolysis. There is also an emphasis on various
Detoxification detoxification and fermentation methodologies including genetic engineering strategies for the efficient
Fermentation conversion of xylose to xylitol.
Ó 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Pretreatment of lignocellulosic materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Physical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. Chemical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3. Physico-chemical pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.4. Biological pretreatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Hydrolysis of biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

⇑ Corresponding author.
E-mail address: vrlinga@gmail.com (L. Venkateswar Rao).

http://dx.doi.org/10.1016/j.biortech.2016.04.092
0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Venkateswar Rao, L., et al. Bioconversion of lignocellulosic biomass to xylitol: An overview. Bioresour. Technol. (2016),
http://dx.doi.org/10.1016/j.biortech.2016.04.092
2 L. Venkateswar Rao et al. / Bioresource Technology xxx (2016) xxx–xxx

4. Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Detoxification of acid hydrolysate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.1. Chemical detoxification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.2. Nanofiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.3. Vacuum membrane distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.4. Electrochemical detoxification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5.5. Biological detoxification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. Enzymatic hydrolysis by hemicellulases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7. Biotechnological production of xylitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7.1. Fed batch fermentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
7.2. Cell recycling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8. Fermentation parameters influencing xylitol production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8.1. Carbon source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8.2. Nitrogen source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8.3. Aeration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8.4. Effect of temperature and pH on xylitol production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
8.5. Co-substrate and oxygen transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
9. Continuous fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
10. Molecular approaches for xylitol production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
11. Strain Improvement strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
12. Enzymatic approach for xylitol production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
13. Purification and recovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
14. Cost economics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
15. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction
Xylitol (five carbon sugar alcohol), an artificial sweetener, could
be produced from second most abundant polysaccharide, xylan
rich hemicellulose which on hydrolysis produces xylose. It is a
sugar substitute used in dietary, food and pharmaceutical indus-
tries due to its properties like low energy content, anticariogenic-
ity, tooth rehardening, preventive against otitis, ear and upper
respiratory infections etc. Though most of xylitol is produced
chemically based on catalytic reduction of pure xylose under high
pressure and temperature using expensive catalyst (usually
Ni-catalyst), biotechnological production of xylose to xylitol offers
a better alternative in terms of energy intensiveness of the produc-
tion process of xylitol and overall process cost.
As the consumers are inclining towards sugar free and low calo-
rie food products due to health and weight consciousness, the
demand for xylitol is increasing. The global consumption of xylitol
was approximately 160 thousand metric tons in 2013 which is
equivalent to market value of US$ 670 million and is expected to
reach 242 thousand metric tons by 2020 equivalent to US $ 1bn
with a CAGRS of over 6% by value and volume (http://www.
prnewswire.com). Globally Asia alone produces 50% of the world’s
total xylitol and rest of the production is by Europe, United States
and Australia (Rao et al., 2012). Asia pacific is the largest consumer
of xylitol. In Asia Pacific, China has the largest market for xylitol
and next India is also projected to have fastest growing market
(http://www.marketresearch.com/product/sample-8164119.pdf)
(Fig. 1). Chewing gum industry has the largest market for xylitol
consumption compared to other industries and was found to be
80% in 2010 and estimated to consume about 163 thousand metric
tons by 2020 with 67% of global xylitol consumption (Fig. 2).
(http://www.marketresearch.com/product/sample-8164119.pdf).
Chewing gum brands like Gross and Wrigley use only xylitol for
the manufacture of sugar-free chewing gums. DuPont, Cargill, CSPC
Shengxue Glucose, Ingredion, Mitsubishi Shoji Food tech were
found to be the leading vendors of xylitol in the market, other
important vendors include ZuChem, Hangzhou Shouxin Biological
Technology, Novagreen, Tata Chemicals, Roquette Frres and Thom-
son Biotech. http://www.prnewswire.com.
Lignocellulosic wastes are abundantly available, renewable and
are inexpensive energy sources generally found in nature (Misra
et al., 2013). Lignocelluloses comprise of cellulose, hemicellulose
and lignin. Hemicellulose content in lignocellulosic biomass is
usually in the range of 20–35%. Some of the lignocellulosic raw

Fig. 1. Asia-pacific xylitol market overview (2009–2020) in metric tons.

Please cite this article in press as: Venkateswar Rao, L., et al. Bioconversion of lignocellulosic biomass to xylitol: An overview. Bioresour. Technol. (2016),
http://dx.doi.org/10.1016/j.biortech.2016.04.092
 L. Venkateswar Rao et al. / Bioresource Technology xxx (2016) xxx–xxx
Fig. 2. Global xylitol chewing gum (2009-2020) market by geographic region –in metric tons.
materials used for the production of xylitol mainly include
corncobs, wheat straw, corn stover, wheat bran, miscanthus etc.
A prerequisite step in the production of xylitol is the pretreat-
ment of the substrate which is essential for removal of lignin and
to reduce the crystallinity of the substrate. Hydrolysis of lignocel-
lulosic biomass using acids is most widely used, during which
optimization of various process parameters is required with a focus
on xylose recovery. During the process of acid hydrolysis, various
inhibitors are released which show negative impact on fermenta-
tion. Detoxification process reduces the concentration of inhibitors.
Later the sugars especially xylose will be converted to xylitol
during fermentation and the xylitol thus produced is further
concentrated, purified and crystallized.
In the earlier reviews there was focus on the conversion of lig-
nocellulosic wastes, the effect of process parameters and evalua-
tion on biotechnological strategies involved in xylitol production.
This review mainly focuses on the recent technologies employed
in the exploitation of lignocellulosic wastes with respect to pre-
treatment, detoxification strategies for removal of inhibitors and
the use of various genetic engineering aspects that necessitates
xylitol production.
2. Pretreatment of lignocellulosic materials
Pretreatment is the prerequisite step which plays a key role in
subsequent saccharification of biomass and its conversion to xyli-
tol. The methods of pretreatment can be divided into physical,
chemical, physico-chemical and biological methods as there are
many reports available on pretreatment, in the current review
we tried to put forth in brief about the recent pretreatment
technologies.
2.1. Physical pretreatment
Physical pretreatment methods disrupt the structural integrity
of lignocellulosic substrates and thereby increase their accessibil-
ity to acids or enzymes. Various types of physical pretreatment
methods which can improve the efficiency of lignocellulosic sac-
charification include reducing size through chipping, grinding, or
milling and irradiation by gamma rays, electron beam or micro-
wave (Taherzadeh and Karimi, 2008).
2.2. Chemical pretreatment
Chemical pretreatment methods involve the use of alkali, dilute
acid, ammonia, organic solvents, oxidative agents and other chem-
icals. These methods are effortless and give good conversion yields
in less span of time. However, the alkali used in the treatment has
to be recovered after the process which needs additional energy
Please cite this article in press as: Venkateswar Rao, L., et al. Bioconversion of lignocellulosic biomass to xylitol: An overview. Bioresour. Technol. (2016),
http://dx.doi.org/10.1016/j.biortech.2016.04.092
3
input. Alkaline pretreatment digests lignin and make holocellulose
accessible for acid or enzymatic degradation. Generally hydroxides
of sodium, potassium, calcium and ammonium are employed in
this process. Xu et al. (2007) studied the influence of equal concen-
trations of sodium and potassium hydroxides in the recovery of
hemicellulose from perennial ryegrass leaves, and found sodium
hydroxide was effective for the removal of hemicelluloses whereas
use of potassium hydroxide resulted in higher purity of hemicellu-
lose. Manas and Krishnan (2015) studied the pretreatment of rice
straw using 15% aqueous ammonia at 120 °C, which removed sig-
nificant amount of lignin i.e., 65.84% and enhanced subsequent
enzymatic digestibility by four fold.
In acid pretreatment method, acids like sulfuric, hydrochloric,
hydrofluoric, nitric and phosphoric acid etc. are used as catalysts.
Dilute sulfuric acid hydrolysis is a well-known process for depoly-
merization of hemicelluloses in lignocellulosic biomass into mono-
meric sugars (Garcia Martin et al., 2013). In the presence of
concentrated acids, hemicellulose and cellulose get hydrolyzed
completely and high yield of monomeric sugars are obtained but
the major drawback is their corrosive nature and the need to recy-
cle acids to lower the cost of pretreatment. In recent times, organic
acids were also recommended as alternatives to inorganic acids to
avoid reactor corrosions and to reduce energy requirement for the
recovery of acid. Acids like maleic, succinic, oxalic, fumaric and
acetic acid have been extensively studied. Qin et al. (2012)
observed that, when corn stover was pretreated with sulfuric, oxa-
lic, citric, tartaric and acetic acid, it was found that 91.7–96.8% glu-
can as well as higher xylan and less furfural was obtained using
acetic acid. Pretreating lignocellulosic biomass using oxidative
reagents involves the reduction of crystallinity in hemicellulose
and cellulose and also disrupts the association between carbohy-
drates and lignin. Nevertheless the cost of oxidative pretreatment
is more than conventional alkali and acid pretreatments hence it
is generally used as a support for other pretreatments to remove
residual lignin from lignocellulosic substrates (Qi et al., 2009).
The oxidative methods comprise of alkaline peroxide pretreat-
ment, ozonolysis, and wet oxidation. In wet oxidation, the biomass
is treated with water and air or oxygen at high temperatures for
comparatively short residence time. Recent studies have stated
that hydrogen peroxide is the most frequently used oxidizing agent
that disintegrate about 50% of lignin and most of the hemicellulose
with 2% H2O2 at 30 °C. As ozone has the electron deficiency in the
terminal oxygen, it is used as the strongest oxidizing agent. It
attacks lignin, an electron-rich substrate selectively than carbohy-
drates. In a study conducted by Garcia Cubero et al. (2009), O3
appreciably reduced lignin content with meagre loss in hemicellu-
lose during oxidative delignification process.
Organic solvent pretreatment is an additional probable method
in the extraction of hemicelluloses. As compared to other chemical
pretreatments, this process has many advantages such as easy
4 L. Venkateswar Rao et al. / Bioresource Technology xxx (2016) xxx–xxx
recovery of solvent by distillation, low environmental impact, and
recovery of high quality lignin as by-product (Pan et al., 2006).
Dimethyl sulfoxide (DMSO) is the most common neutral solvent
that has been used to recover hemicelluloses from holocellulose
without disturbing the acetyl ester compounds and the glycosidic
bonds. Though most of the solvents used in the organosolv pre-
treatment can be recycled from the reactor to decrease the cost,
the high price and potential hazards of handling large volumes of
organic solvents limit the utilization of organosolv process. Process
using ionic liquids (IL) is another alternative for pretreatment of
lignocellulosic materials. Ionic liquids disturb the non-covalent
interactions in the lignocellulosic matrix without any significant
degradation. Hemicellulose liberated from IL solutions show
enhanced enzymatic digestibility. Although the inhibitor formation
is minimal, a small amounts of ILs remained in the residual
pretreated materials are toxic to fermentative microorganisms
(Yang and Wyman, 2008) significantly. On the other hand, ILs are
mostly expensive and thus further research is essential to
standardize the solubility of ILs for lignocellulosic substrates.
2.3. Physico-chemical pretreatment
This category includes the majority of pretreatment technolo-
gies such as ultrasonication, microwave irradiation, steam
explosion, twin screw extruder and so on. These forms of pretreat-
ment exploit the use of conditions and compounds that affect the
physical and chemical properties of biomass. Twin-screw extruder
or extrusion reactor is a thermo-mechanic and chemical fractiona-
tion system that allows the integration of extrusion, cooking,
liquid–solid extraction, and filtration in a single step. The twin-
screw reactor extracts up to 90% of the original hemicellulose
depending upon the content of xylan. The yield after extraction
depends significantly on temperature whereas reduction in screw
rotation speed and solid flow rate influenced the average residence
time of the liquid phase which increased the extraction yield of
hemicellulose (N0 diaye and Rigal, 2000).
Hydrothermal pretreatment includes steam explosion and
liquid hot water hydrolysis. In the process of steam explosion, bio-
mass is generally treated with high pressure and temperature for a
certain amount of time after which the sample is rapidly decom-
pressed resulting in the breakdown of the lignin carbohydrate
complex. The principal fraction of the biomass digested in the liq-
uid phase during pretreatment is hemicellulose whereas the lignin
is transformed as result of the high temperature (Kumar et al.,
2012). In hot-compressed water or liquid hot water pretreatment,
temperature above 200 °C at various pressures below the critical
point is used. This method can recover comparatively high hemi-
cellulose with low levels of inhibitory compounds (Abdullah
et al., 2014). Moretti et al. (2014) reported that microwave-
assisted extraction can be a novel eco-friendly way for the recovery
of hemicelluloses. The difficulty in microwave-assisted extraction
is to get a good yield of sugars without extensive degradation of
the hemicelluloses and contamination with dissolved lignin. This
method is efficient than the traditional chemical pretreatment.
2.4. Biological pretreatment
Biological pretreatment using either organism directly or its
metabolite for pretreatment of biomass is a promising eco-
friendly technology as it has various advantages and is economi-
cally viable strategy for enhancing the rate of enzymatic sacchari-
fication. Microorganisms like brown, white and soft rot fungi were
used for degradation of lignin in biological pretreatment. Biological
pretreatment of Eucalyptus grandis, saw dust degradation patterns
and saccharification kinetics with white rot fungi was reported by
Castoldi et al. (2014). The treatment produced structural changes
Please cite this article in press as: Venkateswar Rao, L., et al. Bioconversion of lignocellulosic biomass to xylitol: An overview. Bioresour. Technol. (2016),
http://dx.doi.org/10.1016/j.biortech.2016.04.092
in the saw dust fibers and after pretreatment there was a twenty
fold increase in reducing sugars. Fungal laccases are the most
widely used lignolytic enzymes in biotechnology but pretreatment
with a single enzyme cannot completely remove lignin in either
wood or herbaceous species.
3. Hydrolysis of biomass
Hydrolysis of lignocellulosic biomass can be exploited using
acids/enzymes. In chemical hydrolysis, sulfuric acid is the most
investigated and predominantly used acid since it is less volatile,
do not corrode the equipment and less expensive. Although other
acids such as hydrochloric acid, phosphoric acid, nitric acid have
also been reported. Dilute acid hydrolysis is preceded by enzymatic
hydrolysis and is more favored over enzymatic hydrolysis due to
its lower cost, simplicity, effectiveness, economic feasibility and
faster kinetics (Garcia Martin et al., 2013).
Varying the concentration of acid, temperature, residence time,
and solid to liquid ratio play a crucial role during acid hydrolysis in
sugar recovery. In a study conducted by Misra et al. (2013), hydrol-
ysis of corn cob at optimized condition of 121 °C for 30 min. using
1.0% H2SO4, with solid: liquid ratio of 1:8 resulted in the (g/L):
xylose 21.98, glucose 3.56, arabinose 2.17, acetic acid 1.31, furfural
0.21. Recently, Zahed et al. (2015) conducted dilute acid hydrolysis
of rice straw by varying the concentrations of sulfuric acid (2%,
3.5%, and 5%) at fixed temperature of 100 °C. At an optimum con-
centration of 3.5% sulfuric acid, 2.3 and 15.05 g/L of glucose and
xylose was obtained.
Meranti wood sawdust, is the most promising and economic
lignocellulosic biomass used for manufacturing xylitol. The opti-
mum temperature, acid concentration and time was found to be
124 °C, 3.26%, and 80 min. respectively to attain maximum saccha-
rification. Under these conditions xylose 18.8 g/L, glucose 4.64 g/L,
arabinose 2.55 g/L, acetic acid 4.14 g/L, furfural 0.55 g/L, HMF
0.08 g/L, and lignin degradation products of 1.55 g/L with a xylose
yield and selectivity attained of 90.6% and 4.05 g/g, respectively
(Rafiqul et al., 2014).
Saleh et al. (2014) conducted acid hydrolysis of olive stones
with 0.025 M sulfuric acid at a temperature of 195 °C for 5 min.
in a 2 L Parr reactor, series 4522 (Moline, IL, USA). The hydrolysis
resulted in the recovery of 89.7% of the total D-xylose (20.5 g
extracted from 100 g raw material), which was further concen-
trated and detoxified under vacuum at 40 °C. The hydrolysate con-
tained D-xylose 20.0 g/L, L-arabinose 0.88 g/L, D-galactose 0.55 g/L,
D-glucose 0.83 g/L, and acetic acid 2.86 g/L.
The use of phosphoric acid has been advantageous since the
neutralization of acid hydrolysate is performed using NaOH, and
the salt, sodium phosphate thus formed is used as nutrient by
microorganisms during xylitol fermentation. Using phosphoric
acid Martínez et al., 2012) performed hydrolysis in a stirred batch
reactor of 1 L capacity, where sunflower stalk was used as sub-
strate and hydrolysis was performed at 95 °C. At phosphoric acid
concentration of 2.67 mol/L, the maximum hemicellulose of
82.7% was obtained. The polyphenols like hydroxytyrosol
(12.98 mg/L), vanillin (1.25 mg/L), veratric acid (1.23 mg/L), syrin-
gic acid (0.88 mg/L), vanillic acid (0.52 mg/L), protocatechuic acid
(0.42 mg/L) and caffeic acid (0.17 mg/L) were detected in the
hydrolysate.
The use of potential catalysts such as amorphous carbon mate-
rial bearing SO3H and OH groups, micro porous crystalline material
(zeolites), and sulfated zirconia were found to be promising for the
hydrolysis of lignocelluloses. In a recent study by Zhong et al.
(2015), a nanoscale catalyst, solid acid SO2
4 / Fe2O3 with both Bron-
sted and Lewis acidity was used for hydrolysis of wheat straw,
which led to the hemicellulose recovery of 63.5%. This catalyst
 L. Venkateswar Rao et al. / Bioresource Technology xxx (2016) xxx–xxx
was found to be more promising as green catalyst and could be
recycled six times with high activity.
The use of plasma acid is more beneficial over sulfuric acid in
cost as it can be obtained and separated more easily. Large num-
bers of protons are produced by the dielectric barrier discharge
of water vapor and these protons make plasma acid that could
hydrolyze hemicellulose. At optimum conditions of 100 °C,
50 min. and pH 2.81, 38.67% xylose; 9.28% glucose and 3.09% galac-
tose was obtained. It was observed and finally concluded that the
use of plasma acid over sulfuric acid is environmental-friendly
and likely to bring about efficient hydrolysis of hemicellulose
(Wang et al., 2013). However, the major bottleneck with acid
hydrolysis is the degradation of sugars into inhibitory components
that affects yeast and there by consequently impinge the process of
xylitol fermentation.
4. Inhibitors
The hydrolysis of lignocellulosic biomass (LB) using acids
results in the generation of various inhibitors in addition to sugars.
The inhibitors released affect the growth of microorganism with an
impact on fermentation process. The tolerable degree of inhibitors
is reliant on the microorganism used and its level of adaptation, the
fermentation process employed, and the presence of other inhibi-
tors (Kumar et al., 2009).
Phenolics are the major inhibitors obtained as a result of lignin
breakdown which lead to the loss in integrity of biological mem-
branes. Usually 0.1 g/L concentration of phenolics compounds
affects the xylose consumption, cell growth and xylitol production.
Phenolics include vanillic acid, syringic acid, vanillone, and
syringaldehyde. The most commonly observed phenolic deriva-
tives in the lignocellulosic acid hydrolysate include 4-hydroxy ben-
zoic acid, ferulic acid, and guaiacol (Mussatto and Roberto, 2004).
Furfural and HMF: The higher temperature and pressure during
acid hydrolysis result in the degradation of xylose that leads to the
formation of furfural while 5-HMF is generated during hexose
degradation. Furfural and HMF not only affect the enzymatic and
biological activities of yeast but damage DNA and further inhibit
protein and RNA synthesis (Mussatto and Roberto, 2004).
Aliphatic acids such as acetic, formic and levulinic acid are also
formed by de-acetylation of hemicellulose or HMF breakdown. The
lower concentration i.e., 1.0 g/L in the fermentation medium ema-
nates the bioconversion of xylose to xylitol. The presence of acetic
acid at a concentration of 2.6 g/L compared to the control has
reduced the consumption of xylose, xylitol production, and cell
growth by 13%, 18%, and 30%, respectively (Kumar et al., 2009).
The concentration of inhibitors obtained by hydrolysis of different
lignocellulosic wastes and their respective detoxifying agents used
were given in Table 1.
Table 1
Concentration of inhibitors from different lignocellulosic wastes and their respective detoxifiying agents.
Lignocellulosic Inhibitors (g/L)
biomass
Mixed hard wood Acetic acid, 11.25; HMF, 0.19; furfural, 1.75; Total
phenolic compound, 4.52
Yellow poplar Acetic acid, 11.33; HMF, 0.18; Furfural, 1.4; Total
phenolic compound, 4.53
Sugarcane bagasse Acetic acid, 2.31; HMF, 0.02; Furfural, 0.19; Total
phenolic compound, 5.48
Brewery’s spent grain Acetic acid, 1.31; HMF, 0.05; Furfural, 0.64; Total
phenolic compound, 1.32; Formic acid, 0.81; Levulinic
acid, 0.16
ED-Electrodialysis.
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5
5. Detoxification of acid hydrolysate
Several detoxification strategies were used to remove toxic
compounds from the hydrolysates as the minute amounts may
interfere and prevent the microbial growth. The efficiency of
detoxification process depends on the raw material, type of hydrol-
ysis process, and microorganism employed (Rao et al., 2006).
5.1. Chemical detoxification
Adsorption with activated charcoal is a well-established and
low cost detoxification method. The detoxification of sago trunk
hydrolysate with 2.5% activated charcoal at the reaction time of
60 min facilitated the reduction of 53% and 78% of furfural and
phenolic compounds. It is evident that the maximum removal of
phenolic compounds is attained by activated charcoal with a xyli-
tol yield of 0.78 g/g when compared with the untreated hydroly-
sate (0.307 g/g). This study proposes that detoxification method
using activated charcoal has a significant impact on xylitol produc-
tion (Siti et al., 2011).
Arruda et al. (2011) elaborated the process involved in using
ion-exchange resins. Sugarcane hemicellulosic hydrolysate was
treated with the resins at the ratio of 1:2 v/v. The hydrolysate
was vacuum evaporated and then subjected to ion-exchange resins
like A-860S (macroporous strong base anion exchanger), A-500PS
(macroporous type I strong base anion exchanger) and C-150
(macro porous strong acid cation exchanger), which led to the
removal of 93% phenolics and 64.9% acetic acid.
Zhu et al. (2011) performed trialkylamine extraction to remove
the inhibitors from corn stover prehydrolysate. The advantages in
using trialkylamine is that it is highly efficient in removing inhibi-
tors, requires low temperature, low energy, low cost. The trialky-
lamine contained 50% n-octanol-20% kerosene. It was able to
remove 73.3% of acetic acid, 45.7% of 5-HMF and 100% of furfural.
5.2. Nanofiltration
Nanofiltration (NF) is a potential and cost-competitive mem-
brane separation technology. The molecular weight cut-off ranges
from 150 to 1000 g/mol, thus facilitating the retention of com-
pounds with molecular weight up to 150–250 g/mol as well as
charged molecules. NF finds its applications in detoxification,
fermentation broth separation, fractionation of sugars and sugar
concentration (Weng et al., 2009).
Recently Nguyen et al. (2015) worked on NF and reverse osmo-
sis (RO). The low molecular weight cut-off (150–400 g/mol) of the
membranes of NF and RO were monitored on a flat-sheet plant for
their capability in separating C5 and C6 sugars from acetic acid,
furfural, 5-HMF and vanillin in a model solution. RO led to the
Detoxifying agents employed for inhibitor References
removal
Resin XAD, XAD-ED, ED, ED-XAD Kundu et al. (2015)
XAD, XAD-ED, ED, ED-XAD Kundu et al. (2015)
Ion exchange resin (A-860S; A500PS and C- Arruda et al. (2011)
150-Purolite
pH adjustment to 5.5, overliming, adsorption Carvalheiro et al. (2005)
with activated charcoal, Anion exchanger pH
5.5
Anion exchanger pH 0.77
Cation exchanger pH 5.5
Cation exchanger pH 0
6 L. Venkateswar Rao et al. / Bioresource Technology xxx (2016) xxx–xxx
sugar rejection of >97% but with the low transmission in inhibitors.
The membranes of NF chiefly NF270, NF- and NF245 (Dow) and DK
(GE Osmonics) were suitable with >94% glucose rejection and >80%
inhibitors transmission.
Brás et al. (2014) used nanofiltration under diafiltration mode
for the removal of inhibitory compounds from diluted acid hydro-
lysate of olive pomace substrate to improve its xylose fermentabil-
ity. They also used the membranes NF270 and NF90 to explore the
best membrane suitable for detoxification. NF270 was chosen to be
used in the diananofiltration experiment as it showed the lowest
rejection for toxic compounds and highest permeate flux and can
deplete 99.7% of acetic acid and 100% of formic acid and furfural.
5.3. Vacuum membrane distillation
Chen et al. (2013) studied the efficiency of vacuum membrane
distillation (VMD), a membrane separation technology which is
an increasingly popular and cost-effective in recovering inhibitors.
It works on the principle of liquid–vapor phase equilibrium that
controls the process selectivity, facilitating the removal of volatile
components. VMD was used to eliminate two inhibitors acetic acid
and furfural from lignocellulosic hydrolysates. More than 98% of
furfural could be removed by VMD under optimal conditions but,
the removal of acetic acid was considerably lower when compared
with furfural.
5.4. Electrochemical detoxification
Electrochemical detoxification is used for the removal of pheno-
lic compounds, p-coumaric acid, ferulic acid, syringaldehyde, and
vanillin. The phenolic compounds were converted electrochemi-
cally to a radical form at oxidation potential and are then removed
after radical polymerization as polymeric compounds. Nearly 71%
of total phenolics were removed from rice straw hydrolysate with-
out any sugar-loss (Lee et al., 2015).
5.5. Biological detoxification
There is increasing interest on biological mode of detoxification
because of the formation of low side reaction products, requires
less energy without the use of chemicals (Parawira and Tekere,
2011). For the proficient microbial detoxification to occur, there
should be a focus on the process parameters like optimal nutrient
addition, pH 4–6, temperature 25–50 °C, incubation time
12–144 h, size of inoculum 1–10% v/v or 0.5–10 g/L (dry weight))
and the strain employed in carrying out detoxification. The use of
an efficient Coniochaeta ligniaria NRRL30616 metabolizes furfural,
HMF, and other inhibitory compounds present in the dilute acid
hydrolysate (Nichols et al., 2008).
Table 2
List of saccharification of different lignocellulosic substrates by xylanase Zhang and Sang (2015).
Enzymes sources Pretreatment conditions
Penicillium chrysogenum Aqueous ammonia solution (15%),
QML-2 60 °C
Aspergillus foetidus MTCC Ammonia solution (15%), 121 °C,
4898 60 min
Penicillium sp. ECU0913 Steam-pretreated at 200 °C, 2.0
MPa, 4 min
Trichoderma reesei Sodium hydroxide solution (1.0%),
Aspergillus candidus Ammonium solution (15%)
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Zhang et al. (2013) performed detoxification of oil palm empty
fruit bunch hydrolysate with a newly isolated Enterobacter sp. FDS8
and reported the depletion of furfural and HMF at a rate of up to
0.54 g/L/h and 0.12 g/L/h with total sugar loss of <5%. It is even pos-
sible to recover the cells and used again for at least 5 times without
losing their ability of detoxification. Biological detoxification is
performed in single step without any additional manipulation,
and without volume loss. It also offers numerous other benefits
like complete reduction of furfural, acetic acid, and guaiacol over
chemical detoxification.
The enzyme mediated detoxification involves the enzymes lac-
cases and peroxidises. Lee et al. (2012) was the first to report a
novel laccase from the yeast, Yarrowia lipolytica, which demon-
strates a higher efficiency in the reduction of non-phenolic and
phenolic components than any previously reported enzyme from
bacteria or fungi.
6. Enzymatic hydrolysis by hemicellulases
Bioconversion of hemicellulose into monomeric sugars by the
action of xylanases is promising, economical but time consuming.
The complex structure of xylan needs different enzymes for its
complete hydrolysis. Endo-1, 4-b-xylanases (1, 4-b-D-
xylanxylanohydrolase, E.C.3.2.1.8) depolymerise xylan by the
random hydrolysis of xylan backbone and 1,4-b-D-xylosidases (1,4,
b-D-xylan xylohydrolase E.C.3.2.1.37) split off small oligosaccha-
rides. List of different pretreated lignocellulosic materials
hydrolyzed by xylanases is depicted in Table 2. Though these organ-
isms produce high yields of enzyme, there is still a need to improve
the production of xylanases to make the enzymatic conversion of
hemicellulose to xylitol more economic.
Microbial hemicellulases for biomass hydrolysis are cost inten-
sive. Mostly xylanases need to be either modified genetically or
immobilized to meet the requirements like activity, substrate
specificity, enantioselectivity, sterospecificity, stability and toler-
ance to toxic reagents and extreme conditions. Overexpression of
these enzymes has been attained in many studies. Some of the
recombinant xylanases with heterologous expression are summa-
rized in Table 3. The over expressed enzyme in majority of the
reports obtained increased thermal stability up to 80–90 °C. When
the recombinant C. thermocellum GH43 hemicellulase genes
expressed in E. coli the enzyme production has been enhanced
from 1.6 to 2.9 fold in repetitive batch mode of fermentation
(Das et al., 2013).
Hydrogels comprising polyanion xylan and polycation chitosan
were used as a carrier for immobilization of the endo-xylanase
from Trichoderma viridae. Because of the induced electrostatic
interaction between the enzyme and gel, the immobilized endo-
xylanase was active at 98 °C while the free enzyme was active only
Saccharification conditions Substrate Reducing sugar Xylose (mg/g dry
(mg/g dry substrate)
substrate)
Substrate loading (2.0%, w/v), Corn cob powder 553.94 236.63
50 °C, pH 4.8, 24 h
Substrate loading (2.5%, w/v), Wheat straw 196 –
45 °C, pH 5.3, 20–24 h Rice straw 188 –
Corn cob 184 –
Substrate loading (10%, w/v) Corn stover powder – 139.33
Room temperature, 60 min. Corn Stover powder 467 156.91
Substrate loading (1.0%, w/v), pH 4.8,
50 °C, 120 h
121 °C, 60 min. Substrate loading Corn cob powder 438.47 190.66
(0.5%, w/v), pH 5.0, 43 °C, 7.12 h
 L. Venkateswar Rao et al. / Bioresource Technology xxx (2016) xxx–xxx
Table 3
Patents on expression of xylanases in different hosts (Juturu and Wu, 2012).
Source of gene Enzyme Expression host
Bacillus sp. NG-27 Endo Chloroplasts of tobacco
xylanase A plant
T. reesei Endo H. insolens
xylanase
N. frontalis Endo E. coli, P. Methanolica
N. patriciarum xylanase
Mixed population of rumen Endo- E. coli BL21(DE3)
microorganism xylanase
Trichoderma reesei Endo H. polymorpha
xylanase
Alicyclobacillus acidocaldarius b xylosidase E. coli, P. pastoris
Selenomonas ruminantium GA192 b xylosidase E. coli
Aspergillus niger b xylosidase H. polymorpha
up to 45 °C. b-Xylosidase was extracted from Aspergillus niger and
30% of the enzyme was immobilized on a polyamide membrane
by adsorption. The immobilized enzyme showed only 6.8% of the
maximal activity of the free enzyme but has a better thermal sta-
bility than the free enzyme. The immobilized enzyme was still
active after 20 cycles of usage for xylan hydrolysis releasing xylose
at 1.7 g/L enzyme (Delcheva et al., 2008).
7. Biotechnological production of xylitol
The microbial xylitol production has been extensively studied
as an alternative to the chemical process as it does not need exten-
sive steps for purification of xylose and operated at low cost.
Microorganisms like bacteria, yeasts and fungi possess the ability
of fermenting commercial xylose or xylose present in the hydroly-
sate derived from lignocellulosic residues to xylitol.
Xylose metabolism occurs in two steps. Xylose is reduced to
xylitol in the presence of an enzyme xylose reductase (XR), which
is either secreted from the cell or oxidized to xylulose by xylitol
dehydrogenase (XDH). The above two reactions are considered to
be rate-limiting steps in xylitol production. Further by the enzyme
xylulokinase (XKS), xylulose is phosphorylated to xylulose
5-phosphate (EC 2.7.1.17) and catabolized by entering to pentose
phosphate, glycolytic, or phosphoketolase pathways. Xylitol pro-
duction is extensively carried out using free cells or immobilized
cells in batch, continuous or fed-batch process using synthetic
media or lignocellulosic substrates (Arruda et al., 2011).
7.1. Fed batch fermentation
Francisco et al. (2015) carried out simultaneous production of
ethanol and xylitol under different operation bioreactor modes
using co-cultures of Candida tropicalis IEC5-ITV and Saccharomyces
cerevisiae ITV01-RD in sugarcane bagasse hydrolysates. Sequential
cultivation using simultaneous co-culture of S. erevisiae and
C. tropicalis, the productivity and yield for xylitol were found to
be xylitol, QXylOH = 0.10 g/L/h and YXylOH/S = 0.31 g/g, respectively.
Whereas by using fed-batch culture, the productivity and yield of
xylitol were found to be QxytOH = 0.27 g/L/h and YxytOH/s = 0.57 g/g
respectively. Continuous multistep production showed a 50%
increase than in fed-batch culture, where the productivity and
yield of xylitol were found to be QxytOH = 0.26 g/L/h and YxytOH/s
= 0.62 g/g, respectively. Himabindu and Gummadi (2015) carried
out fed batch fermentation to increase the yield, productivity and
to determine the effect of volumetric oxygen transfer coefficient
(KLa) on xylitol production by Debaromyces nepalensis. By varying
oxygen transfer coefficients in the range of 12–39.6 h1and provid-
ing xylose and nitrogen source to the medium intermittently,
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7
maximal xylitol yield of 0.64 g/g and productivity of 0.43 g/l/h
were obtained at 13.68 KLa.
7.2. Cell recycling
Cunha et al. (2007) investigated the conversion of xylose to xyl-
itol by Candida guillermondii cells entrapped within poly vinyl alco-
hol hydrogel by repeated batch method with cell recycling. The
yeast cells were immobilized on to the support using freeze thaw
method, and fermentation was carried out in a reactor at 30 °C,
400 rpm at air flow rate of 1.04 vvm for 6 cycles. It was found that
the biocatalytic activity of cells retained till the 6th cycle but the
conversion performance was slightly decreased in the 5th cycle.
The xylitol production, volumetric productivity and yield increased
with continuous recycling of cells and reached to a maximum of
39.7 g/L, 0.53 g/L/h and 0.77 g/g after third cell cycling.
Kwon et al. (2006) reported a maximal volumetric productivity
of 3.5 g/L/h from an initial xylose concentration of 200 g/L by an
osmophilic C. tropicalis KCTC 10457. In a fed-batch fermentation
of xylose and glucose, xylitol production of 234 g/L was obtained
in 48 h, with a production rate of 4.88 g/L/h utilizing 260 g/L
xylose. This strain produced moderate xylitol of 2.07 g/L/h even
at a very high xylose concentration of 350 g/L due to its high osmo-
tolerance. To increase the productivity of xylitol, cell recycling was
carried out for 10 cycles during which an average xylitol concen-
tration of 180 g/L was produced with a productivity and yield of
8.5 g/l/h and 0.84 g/g respectively for each recycle. When recycling
was carried out using two fold concentrated cell mass, average xyl-
itol concentration, productivity and yield for each recycle was
found to be 182 g/L, 12 g/L/h and 0.85 g/g respectively.
8. Fermentation parameters influencing xylitol production
Microbial production of xylitol is influenced by various factors.
Hence optimization of these factors is necessary to obtain efficient
xylitol production and it depends upon the specificity of the
microorganism used.
8.1. Carbon source
Lignocellulosic materials are cheap, renewable and abundantly
available promising carbon sources. Upon hydrolysis of hemicellu-
losic fraction of lignocellulosic biomass xylose is released, which is
further fermented to xylitol. Corncobs, rice straw, rice husk, sugar-
cane bagasse, sorghum straw, sawdust and oat hulls are generally
used as lignocellulosic substrates.
Misra et al. (2013) used corncobs as potential substrate for xyl-
itol production by C. tropicalis. A maximum of 20.92 g/L xylose was
obtained by acid hydrolysis. The hydrolysate was further concen-
trated by rotavapor that increased the amount of xylose to
40.16 g/L that resulted in the xylitol production of 22.63 g/L and
yield of 0.57 g/g. But, by microwave assisted concentration of the
hydrolysate, the amount of xylose increased to 52.71 g/L resulted
in the xylitol production of 15.19 g/L and yield of 0.31 g/g.
8.2. Nitrogen source
Several organic nitrogen sources such as yeast, peptone, urea,
casamino acid and inorganic sources like ammonium sulfate,
ammonium nitrate, and ammonium chloride are generally used
in the production of xylitol. Organic nitrogen sources are more
beneficial than inorganic nitrogen sources and show a greater
impact on the cell growth and xylitol production. Zhang et al.
(2012) investigated the effect of nitrogen sources on xylitol pro-
duction from horticultural waste hemicellulosic hydrolysate using
8 L. Venkateswar Rao et al. / Bioresource Technology xxx (2016) xxx–xxx
Fig. 3. Effect of different nitrogen sources on xylitol yield.
the strain Candida athensensis SB18. The YNB-YE synthetic medium
was supplemented with different nitrogen sources like ammonium
sulfate, ammonium nitrate, urea and casaminoacid. Among which,
the addition of urea enhanced the conversion of xylose with a
higher xylitol yield of 0.75 g g1. These results were similar and
consistent when compared with Rao et al. (2006) where urea
was found to be beneficial for maximum xylitol yield of 0.87 g/g
when compared with other different nitrogen sources like casein
hydrolysate, yeast extract, urea, sodium nitrate, ammonium chlo-
ride and ammonium sulfate (Fig. 3).
8.3. Aeration
The availability of oxygen, which is often expressed in terms of
volumetric oxygen transfer coefficient (KLa) and oxygen transfer
rate (OTR), is an important process parameter that plays a major
role in regulating cell growth and formation of xylitol by affecting
the activity of xylose reductase and xylitol dehydrogenase (Branco
et al., 2008). To study the influence of KLa on XR and XDH, batch
fermentation under different initial KLa values was carried out.
Generally to maintain micro-aerobic conditions, low oxygen trans-
fer volumetric coefficient (KLa) values are used. However Branco
et al. (2008) reported that high KLa values resulted in high xylitol
production. By evaluating the effect of aeration on XR and XDH
activities, it was found that at lowest KLa value of 12 h1, maxi-
mum XR specific activity of 1.45 ± 0.21 U/mg protein was obtained,
whereas the maximum XD specific activity of 0.19 ± 0.03 U/mg
protein was obtained at a high KLa value of 25 h1. Further when
KLa was increased from 12 to 50 h1, there was a greater increase
in xylitol production with a volumetric productivity of
1.50 ± 0.08 g/L/h and efficiency of 71%.
To evaluate the influence of different KLa values on xylitol pro-
duction by C. tropicalis W103, Cheng et al. (2010) carried out batch
fermentation and observed that at lower KLa of 10.8 h1, the bio-
mass was less with low xylitol productivity but, as KLa increased
from 10.8 h1 to 16.5 h1, biomass is increased with increased xyl-
itol yield. Further increase of KLa to 18.3 h1 led to the increase of
biomass but decrease in xylitol yield, may be due to increased XRD
activity which converts xylitol to xylulose. The maximum xylitol
yield of 0.73 g g1 and productivity of 1.43 g L1 h1 were obtained
at KLa 16.5 h1 and 18.3 h1 respectively.
8.4. Effect of temperature and pH on xylitol production
Most of the yeasts grow at 30 °C to 37 °C for biotechnological
xylitol production. However, some thermotolerant yeasts possess
Please cite this article in press as: Venkateswar Rao, L., et al. Bioconversion of lignocellulosic biomass to xylitol: An overview. Bioresour. Technol. (2016),
http://dx.doi.org/10.1016/j.biortech.2016.04.092
a higher temperature optimum (40–45 °C) because of acceleration
of their enzymes at high temperature. It was observed that, conver-
sion of xylose to xylitol by Candida sp. B-22 was found to be con-
stant in the temperature range of 35–40 °C. Whereas at 45 °C and
higher, there was a great decline in the xylitol yield, may be due
to the inactivation of NADPH and NADH dependent XR
(Ur-Rehman et al., 2012).
Sirisansaneeyakul et al. (2013) evaluated the xylitol production
by Candida magnolia TISTR 5663 under oxygen limited conditions.
Xylitol yield of 0.72 g/g was obtained utilizing the biomass of
50 g/L at pH 7.0.
8.5. Co-substrate and oxygen transfer
Hernández-Pérez et al. (2016) demonstrated the effect of
various sugars as co-substrate for xylitol production by Candida
guilliermondii FTI20037, by supplementing sugar cane bagasse
hydrolysate containing 57 g/L xylose with sugars such as maltose,
sucrose and glycerol. It was observed that the addition of 10 g/L
sucrose and 0.7 g/L glycerol as co-substrate led to the significant
increase in the xylose uptake by 8.8% and 6.8% respectively. But
addition of sucrose alone increased the xylitol concentration to
36 g/L. This study makes the process economical by using sucrose
derived from lignocellulosic substrates.
To avoid the requirement of co-substrate for co-factor regener-
ation and cell growth, Ko et al. (2011) followed a new method of
decreasing the XDH activity by engineering the attenuation of
XDH of C. tropicalis strain, by reducing its gene copy number. Stud-
ies on xylitol production by mutants, strain XDH-5 (with only one
copy of the XDH gene), and ARSdR-16 (with a mutated XDH gene)
showed 70% and 40% of wild type (WT) XDH activity respectively.
Xylose to xylitol conversion yield by wild type and mutant strains
XDH-5 and ARSdR-16 were 62%, 64%, and 75%, respectively, with
productivity of 0.52, 0.54, and 0.62 g/L/h respectively. ARSdR-16
mutant showed high xylitol yield and productivity without using
any co-substrate.
9. Continuous fermentation
Continuous fermentation maintains high productivities for
longer periods of time by eliminating the idle time for cleaning
and sterilization. It shows greater steadiness in product synthesis,
hence several bioreactors have been used for continuous produc-
tion of xylitol.
However, immobilized cells are generally used during continu-
ous fermentation as they possess high cell density, improved
 L. Venkateswar Rao et al. / Bioresource Technology xxx (2016) xxx–xxx
stability with higher productivity and can be re-used when com-
pared to free cells.
A three-phase fluidized-bed bioreactor using immobilized Can-
dida sp. ZU04 cells was built by Ding (2011) for efficient continu-
ous production of xylitol with high yield from corn cob
hemicellulosic hydrolysate. It was found that at high aeration rate
of 1.00 vvm, within first 24 h, the glucose was rapidly consumed
and biomass in alginate beads was quickly increased. But when
the aeration rate was decreased to 0.30 vvm in the second phase,
xylitol yield increased remarkably. The immobilized cells were
reused for further batches leading to the xylitol yield of 0.73 g/g
and productivity of 0.84 g/L/h.
Sarrouh et al. (2010) determined hydrodynamic characteristics
and fermentation parameters of a bench-scale three-phase flu-
idized bed reactor (FBR) for xylitol production using calcium algi-
nate immobilized C. guilliermondii. When the affect of total
particle density, terminal velocity, particles drag force, minimum
fluidization velocity and bed porosity (n) on cell immobilization
was evaluated, it was found that the reactor operates similar to a
fixed bed reactor at porosity <0.5 and as fluidized bed reactor at
>0.5 porosity. It is observed that maximum bed fluidization in
the reactor can be obtained when the flow rate is equal to the ter-
minal velocity of immobilized cells. When the effects of the aera-
tion rate and fermentation time on the process productivity and
yield were analyzed, a high aeration rate of 600 ml/min favors
better oxygen transfer into the immobilized cells and lead to the
maximum xylitol productivity, yield and xylitol concentration of
0.41 g/L/h, 0.582 g /g and 28.9 g/L respectively at 70 h of
fermentation.
Comparison of regeneration and bioconversion efficiencies of
free and immobilized cells of C. guilliermondii FTI 20037 on xylitol
production from oat hull hemicellulosic hydrolysate was evaluated
and found that the aeration rate or oxygen mass transfer coeffi-
cient KLa plays a crucial role on xylitol production. When fermen-
tation of hydrolysate containing 74.5 g/L xylose was carried out
using free cells of Candida at an aeration rate of 1.25 vvm or KLa
of 15.8 h1, a maximum xylitol yield of 0.87 g/g and production
of 55 g/L was obtained which is almost similar to the fermentation
carried out by immobilized cells with aeration rate of 1.25–1.5 vvm
(Soleimani and Tabil, 2014).
10. Molecular approaches for xylitol production
Xylose reductase (XR) is considered as a key enzyme in biotech-
nological xylitol production. In recent decades, various metabolic
engineering strategies have been explored to modify the XR and
XDH in microbes to increase the production of xylitol.
In a study conducted by Jo et al. (2015), NADPH-dependent XR
and NADH-preferring mutant XR along with co expression of ZWF1
for NADPH regeneration and ACS1 for NADH regeneration was
engineered into S. cerevisiae that resulted in the xylitol productivity
of 4.27 g/l/h and a yield of 1.0 g/g. The XR gene from N. crassa was
codon optimized and expressed in XYL2 deficient C. tropicalis
mutant. This engineered C. tropicalis was able to metabolize both
glucose and xylose that resulted in higher xylitol yield of 0.96 g/g
than without the expression of XR gene from N. crassa.
The disruption of Glucose specific phospho transferase system
(ptsG) which is phosphoenol dependent resulted in the elimination
of catabolite repression and allowed immediate uptake of glucose
and xylose. Deleting the genes xylA (xylose isomerase) and xylB
(xylulokinase) resulted in the blockage of xylose catabolic pathway
in E. coli W3110 to avoid xylose consumption. The phospho enol
pyruvate-dependent fructose phospho transferase system (ptsF)
was disrupted that consequently blocked the xylitol phosphoryla-
tion pathway with a xylitol yield of 0.95 g/g (Su et al., 2015).
Please cite this article in press as: Venkateswar Rao, L., et al. Bioconversion of lignocellulosic biomass to xylitol: An overview. Bioresour. Technol. (2016),
http://dx.doi.org/10.1016/j.biortech.2016.04.092
9
Qi et al. (2016) conducted a study in which xdh gene of Glu-
conobacter oxydans CGMCC 1.49 was cloned and expressed in
E. coli BL21 successfully, and recombinant XDH was characterized.
The results showed that, in the mixed culture of resting cells, the
mixed strains (G. oxydans and recombinant strain BL21-xdh)
improved the yield of xylitol from 0.297 to 0.960 g/g using 30 g/L
D-xylulose as substrate.
Xylose from mother liquor was converted to xylitol by using a
novel strategy of combining Bacillus subtilis and Candida maltosa.
In this process C. maltosa was used to detoxify the inhibitors like
furfural and 5-HMF present in the mother liquor by utilizing the
glucose that causes catabolite repression in B. subtilis. Further
xylose isomerase gene disrupted B. subtilis was constructed for uti-
lizing the arabinose, thereby increasing the concentration of xylose
in the media. Finally C. maltosa is reused for the reduction of xylose
to xylitol with a yield and productivity of 0.85 g/g and 4.25 g/L/h
respectively (Cheng et al., 2011).
11. Strain Improvement strategies
Strain improvement methods such as mutation, adaptation and
recombination are generally employed to enhance the perfor-
mance of microorganisms used in fermentation process. To evalu-
ate xylitol production capabilities of K. marxianus, Kim et al. (2015)
carried out random mutagenesis and selected seven K. marxianus
strains (ATCC 124224, ATCC 46537, ATCC 200963, ATCC 36907,
ATCC 26548, CBS 1555, and CBS 607). Among seven strains,
K. marxianus ATCC 36907 produced maximum amount of xylitol
24 g/L with a yield and productivity of 0.48 g/g and 0.17 g/L/h
respectively from 50 g/L xylose at 30 °C for 144 h, indicating that
XR from ATCC 36907 may possess higher activity than
other strains. Further mutagenesis of K. marxianus 36907 lead to
K. marxianus 36907-FMEL1 strain which exhibited more than two
fold improvement in xylitol production which corresponds to
0.67 g/g xylitol yield and productivity of 0.36 g/L/h. Xylitol concen-
tration and yield from K. marxianus 36907-FMEL1 were 120% and
39% more than the parental strain, K. marxianus ATCC 36907,
respectively. The reason being, XR activity of the mutant is twofold
higher than its parent and genes sequencing of its KmXYL1 gene
show that cysteine was substituted to tyrosine at position 36 after
strain development which might cause enhanced XR activity in
K. marxianus 36907-FMEL1.
Mahmud et al., 2013 disrupted the xdhA and ladA genes of
Aspergillus oryzae which encode XDH and LAD respectively to
increase xylitol production. The xdhA- and ladA-disrupted mutants
were constructed by homologous transformation into A. oryzae P5
(DpyrG) and pyrG was used as a selectable marker. The mutants
were grown on different carbohydrate-containing media, colony
diameters of mutants were measured, and gene disruption was
confirmed by PCR. The xylitol productivity of the mutants was
studied using D-xylose and oat spelt xylan as carbon sources. The
xdhA-disrupted mutant (xdhA2-1) produced 16.6 g/L xylitol with
a yield of 0.43 g/g D-xylose and productivity of 0.248 g/L/h from
D-xylose, while 10.2 g/L xylitol was produced at a yield of
0.204 g/g from oat spelt xylan.
Adaptation is an inexpensive approach used as an alternative to
the detoxification methods which adapts the microbes to inhibi-
tory compounds. Fermentative efficiency for xylitol production
can be improved by adapting the cells to the fermentation med-
ium. Misra et al. (2013) reported xylitol production of 7.46 g/l with
a yield of 0.37 g/g by adapted C. tropicalis from 21.98 g/L of xylose
present in untreated corn cob hydrolysate. Further adaptation of C.
tropicalis in optimized and treated corncob hydrolysate containing
20.92 g/l xylose, found to produce 12.23 g/L of xylitol with a yield
of 0.61 g/g which is 1.22-fold higher compared to parent strain. In
10 L. Venkateswar Rao et al. / Bioresource Technology xxx (2016) xxx–xxx
comparison with the above study, Rao et al. (2006) reported an
increase in xylitol yield from of 0.43 g/g to 0.58 g/g and 0.45 g/g
to 0.65 g/g by adaptation of C. tropicalis cells for 20 cycles in neu-
tralized, activated charcoal and ion exchange resin treated corn
fiber and sugarcane bagasse hydrolysates respectively.
12. Enzymatic approach for xylitol production
Enzymatic xylitol production technology is an alternative and
promising approach for conversion of xylose to xylitol. During
enzymatic process, xylose was stoichiometrically converted to xyl-
itol with almost equivalent consumption of NADPH using NADP+
to xylose ratio of over 1:30, and the coenzyme was regenerated
and retained successfully using a membrane reactor. About 90%
conversion of xylose to xylitol could be achieved at 35 °C and pH
7.5 after a 24 h reaction period (Rafiqul and Sakinah, 2012).
To interpret the xylitol production by enzymatic process,
Branco et al. (2011) used RSM, a statistical design which resulted
in quadratic model that could predict 98.6% volumetric productiv-
ity of xylitol with respect to xylose and NADPH concentration.
A maximum xylitol volumetric productivity of 1.5 g/L/h was
obtained using the predicted experimental conditions of pH 7.0,
temp 25 °C, 1.2 mM NADPH, 0.34 M xylose and glucose, 0.2 U/mg
xylose reductase and 0.2 U/mg of xylitol dehydrogenase. The xyli-
tol productivity of 1.58 g/L/h obtained by the statistical model was
higher and significant compared to the traditional mode.
Zhoua et al. (2012) investigated the biotransformation of
D-arabitol into xylitol with focus on the conversion of xylulose into
xylitol by co expression of XDH gene from G. oxydans and co-factor
regenerating enzyme gene glucose dehydrogenase (GDH) from
B. subtilis in E. coli (system 1) and co-expression of alcohol dehy-
drogenase gene ADH from G. oxydans and co-factor regenerating
enzyme gene GDH from B. subtilis in E. coli (system 2). Both the
above systems successfully converted xylulose to xylitol without
addition of NADH. Approximately 92% conversion yield i.e.,
26.91 g/L xylitol was obtained from 30 g/L xylulose by system
1 and 24.9 g/L xylitol with 85.2% conversion yield by system 2.
The xylitol yields of both systems are 3 fold higher than G. oxydans
NH-10 cells (7.32 g/L).
13. Purification and recovery
Depending upon the type of lignocellulosic substrate used for
fermentation, its acid hydrolysis process varies and generates dif-
ferent type of sugars and inhibitors making the purification and
recovery process complicated. Crystallization is an energetically
advantageous process generally used to separate a single com-
pound from a mixture of raw materials or reaction by-products
(Misra et al., 2013).
Economical and environmental-friendly method for the purifi-
cation and crystallization of xylitol from corn cob hydrolysate
was reported by Wei et al. (2010). Initially by using 1% activated
carbon at a temperature of 60 °C and 165 rpm, the xylitol fermen-
tation broth was decolourised and then desalted with a two ion
exchange resins 732 and D301. Later the left over sugars in the
broth were separated with the resin UBK-555(Ca2+). Further the
broth was vacuum-concentrated up to xylitol super saturation of
750 g/L. Finally to the clarified fermentation broth 1% xylitol crys-
tal seeds, were added and then the supersaturated solution was
cooled to 20 °C for 48 h to obtain crystalline xylitol of tetrahedral
shape with 95% purity and crystallization yield of 60.2%.
To purify and crystallize xylitol present in wheat straw hemicel-
lulosic hydrolysate, Canilha et al. (2008), initially used two anion
exchange resins A-860S and A-500PS to clarify the hemicellulosic
hydrolysate by sequential adsorption. By adsorption 95% of
Please cite this article in press as: Venkateswar Rao, L., et al. Bioconversion of lignocellulosic biomass to xylitol: An overview. Bioresour. Technol. (2016),
http://dx.doi.org/10.1016/j.biortech.2016.04.092
hydrolysate was decolourised and by-products such as acetic acid,
lignin derivatives were removed. The clarified fermentation broth
was concentrated 40 fold, and then the xylitol content in concen-
trated medium was 726.5 g dm3, a density of 1.3443 g cm3 and
viscosity of 15cP. Further to promote xylitol crystallization, the
concentrated medium was cooled in the presence of alcohol as
the solubility of xylitol is decreased in the presence of ethanol.
Finally to favor the nucleation pure xylitol crystal were seeded in
to crystallizer at a temperature below the saturation so that small
amount of crystals 3.3 g with a crystalline yield of 43.5% is
obtained which is close to the 51.4% obtained for commercial xyl-
itol solution.
14. Cost economics
Xylitol production cost varies and depends mainly on cost of
raw material used and its transport cost which depends on feed-
stock mass and location of the manufacturing plant. The estimated
cost for production of xylose from rye straw hemicellulose was
observed to be €59.2 per tonne. This can be further broken down
in to 30% for raw materials, utilities 24.4%, enzyme cost 6.5%,
10.2% maintenance, 22.9% depreciation and 6.0% labor cost. This
has to be further added up with fermentation and downstream
processing cost (Franceschin et al., 2011).
15. Conclusions
The production of xylitol is receiving greater interest because of
its increasing demand in food and pharmacy industries. Efforts are
being made by several researchers to improve xylitol yield to make
the process economical and competitive. The focus should be more
on novel strategies like genetic engineering for complete utiliza-
tion of lignocellulosic substrates and also making organisms more
stress tolerant. Understanding the technology involved in the
hydrolysis, detoxification and the conversion of wastes to xylitol
plays vital role in commercial production of xylitol. Economic fea-
sibility of the process involved in the conversion of xylose to xylitol
has to be elucidated.
Acknowledgements
Authors wish to acknowledge the financial support from UGC
DRS, SAP-I and UGC BSR Faculty fellowship.
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Please cite this article in press as: Venkateswar Rao, L., et al. Bioconversion of lignocellulosic biomass to xylitol: An overview. Bioresour. Technol. (2016),
http://dx.doi.org/10.1016/j.biortech.2016.04.092
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