Bioresource Technology 273 (2019) 641–653

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

Review

Biotechnological applications of inulin-rich feedstocks T

a,⁎ a b
R.S. Singh , Taranjeet Singh , Christian Larroche
a
Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala 147 002, Punjab, India
b
Université Clermont Auvergne, Institut Pascal, UMR, CNRS 6602, and Labex, IMobS3, 4 Avenue Blaise Pascal, TSA 60026, CS 60026, F-63178 Aubiere Cedex, France

ARTICLE INFO ABSTRACT

Keywords: Inulin is a naturally occurring second largest storage polysaccharide with a wide range of applications in
Inulin-rich feedstocks pharmaceutical and food industries. It is a robust polysaccharide which consists of a linear chain of β-2, 1-linked-
Fructose D-fructofuranose molecules terminated with α-D-glucose moiety at the reducing end. It is present in tubers, bulbs
Fructooligosaccharides and tuberous roots of more than 36,000 plants belonging to both monocotyledonous and dicotyledonous fa-
Biofuels
milies. Jerusalem artichoke, chicory, dahlia, asparagus, etc. are important inulin-rich plants. Inulin is a potent
Organic acids
substrate and inducer for the production of inulinases. Inulin/inulin-rich feedstocks can be used for the pro-
duction of fructooligosaccharides and high-fructose syrup. Additionally, inulin-rich feedstocks can also be
exploited for the production of other industrially important products like acetone, butanol, bioethanol, single
cell proteins, single cell oils, 2, 3-butanediol, sorbitol, mannitol, etc. Current review highlights the biotechno-
logical potential of inulin-rich feedstocks for the production of various industrially important products.

1. Introduction many health-promoting properties due to which it is considered as a

Inulin, a naturally occurring polysaccharide belongs to a class of
dietary fibres known as fructans. The term fructans is generally used for
those compounds in which fructosyl moieties constitute the molecule.
Inulin, a peculiar substance was first isolated from Inula helenium by a
German scientist, Rose (1804) and later on it was named inulin by
Thomson (1817). In nature it is the second most abundant storage
polysaccharide. Structurally inulin [α-D-glucopyranosyl-β-D-fructofur-
anosyl-(n-1)-D-fructofuranoside] is composed of β-D (2 → 1) linked
fructosyl oligomer with a glucose moiety at the reducing end. Glucose
unit joined by α-D (1 → 2) glycosidic bond is present in pyranose form
(4C1 conformation), whereas fructose unit is in the furanose form.
Generally, the inulin has a glucose moiety at the reducing end, but in
some cases it has only fructose molecules lacking the glucose at the
terminal end (Hebette et al., 1998). The unique aspect of the inulin
structure is that no bond of its fructose ring is the part of macro-
molecular backbone (Andre et al., 1996).
Inulin was designated a GRAS (Generally Recognised As Safe) status
since 2002, because of its use in food industries including meat and
poultry products and also in the baby products. In many countries,
inulin-rich plants are used as an essential part of regular diet. An
average daily intake of inulin in Western and American diet has been
estimated to 1–10 g and 2.5 g, respectively. However in European diet,
inulin consumption is relatively higher (3–11 g) per day. Inulin has
functional food. Owing to its high degree of polymerization (DP), it acts
as a potential prebiotic in food processing industries (Singh and Singh,
2010; Singh et al., 2016a). Inulin in combination with FOSs (fructoo-
ligosaccharides) is used as a non-digestible dietary fibre which pro-
motes the human gut microbiota by stimulating the growth of bifido-
bacteria in human intestine that beneficially affects the host’s body.
Apart from its prebiotics effect, it is also used in the lipid metabolism,
absorption of mineral ions from gut, control of blood sugar level and
prevention of obesity, chemically induced aberrant crypts, colon
cancer, etc. (Singh and Singh, 2010; Singh et al., 2017a).
A lot of inulin-rich plants are reported from both monocots and
dicotyledonous families. Inulin is present in a considerable amount in
bulbs, tubers and tuberous roots of many plants like Dahlia pinnata
(Dahlia), Taraxacum officinale (Dandelion), Asparagus officinalis
(Shatwaar), Helianthus tuberosus (Jerusalem artichoke), Asparagus ra-
cemosus (Safed musli), Cichorium intybus (chicory), etc. (Singh and
Singh, 2010). Therefore, such inulin-rich feedstocks are of great con-
sideration as they are inexpensive, renewable and abundant substrate
for the production of various bioproducts. Inulin-rich plant materials
and mixed substrates can be used as potent substrates for various bio-
processes. Inulin-rich feedstocks has been extensively used for the
production of high fructose syrup, inulinases, inulooligosaccharides,
biofuels, organic acids, single cell oil, single cell proteins, mannitol,
sorbitol, 2,3-butanediol, pullulan, etc. The current review highlights the

⁎
Corresponding author.
E-mail address: rssbt@pbi.ac.in (R.S. Singh).

https://doi.org/10.1016/j.biortech.2018.11.031
Received 3 October 2018; Received in revised form 5 November 2018; Accepted 8 November 2018
Available online 09 November 2018
0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
R.S. Singh et al. Bioresource Technology 273 (2019) 641–653

Table 1
Inulin content of various inulin containing plants.
Common name Botanical name Plant part Inulin content (%)* References

Agave Agave americana Lobes 7–10 Partida et al. (1998)

Artichoke Cynara cardunculus Leaves-heart 3–10 Van Loo et al. (1995), De Leenheer (1996)
Banana Musa acuminata Fruit 0.3–0.7 Van Loo et al. (1995)
Barley Hordeum vulgare Grains NS Van Loo et al. (1995)
Buckwheat Fagopyrum esculentum Grains NS Bonciu et al. (2012)
Burdock Arctium sp. Roots 3.5–4.0 Van Loo et al. (1995), De Leenheer (1996)
Camas Camassia sp. Bulbs 12–22 Van Loo et al. (1995), De Leenheer (1996)
Chicory Cichorium intybus Roots 15–20 Van Loo et al. (1995), Gupta and Kaur (1997), De Leenheer (1996)
Dahlia Dahlia sp. Root tubers 15–20 Gupta and Kaur (1997)
Dandelion Taraxacum officinale Leaves 12–15 Van Loo et al. (1995)
Garlic Allium sativum Bulbs 9–16 Van Loo et al. (1995), De Leenheer (1996)
Globe thistle Echinops ritro Roots NS Vergauwen et al. (2003)
Jerusalem artichoke Helianthus tuberosus Roots 12–19 Bach et al. (2015)
Kuth Saussurea lappa Roots 18–20 Kuniyal et al. (2005)
Leek Allium ampeloprasum var. porrum Bulbs 3–10 Van Loo et al. (1995), De Leenheer (1996)
Lettuce Lactuca sativa Roots NS Hendry and Wallace (1993)
Murnong Microseris lanceolata Roots 8–13 Van Loo et al. (1995)
Onion Allium cepa Bulbs 2–6 Van Loo et al. (1995), De Leenheer (1996)
Rye Secale cereale Grains 0.5–1.0 Van Loo et al. (1995)
Safed musli/shatwaar Asparagus racemosus Root tubers 10–15 Gupta and Kaur (1997)
Salsify Tragopogon sp. Roots 15–20 Gupta and Kaur (1997)
Shatwaar Asparagus officinalis Root tubers 10–15 Gupta and Kaur (1997)
Spanish salsify Scorzonera hispanica Roots 8.15–10.75 Dolota and Dąbrowska (2004)
Suma Pfalia glomerate Roots 11.45a Caleffi et al. (2015)
Sunflower Helianthus annuus. Root tubers NS Chi et al. (2011)
Sweet leaf Stevia rebaudiana Roots 18-23a Lopes et al. (2015)
Yacon Smallanthus sonchifolius Roots 3–19 Van Loo et al. (1995)
Yam Dioscorea esculenta Root tubers 7.5 Handayani et al. (2016)

NS: Not specified.

* Percentage of fresh mass.
a
Percentage of dried mass.

utilization of inulin-rich feedstocks for the production of high fructose inulinases on inulin and this phenomenon occurs when the chicory
syrup, inulinases, fructooligosaccharides, biofuels, organic acids, etc. roots are fully developed. Thus, chicory roots are rich source of both
inulin and oligofructose. Dahlia, a tuberous perennial flowering plant
belonging to family Asteraceae is another important inulin-rich feed-
2. Inulin-rich feedstocks and mechanism of inulin synthesis
stock. It is cultivated worldwide for ornamental purposes and is char-
acterized by a variety of colors and inflorescence forms. Its tuberous
Inulin is present as a storage carbohydrate in more than 36,000
roots are very rich in inulin (15–20%). Due to the absence of highly
plants belonging to the families Amaryllidaceae, Asteraceae,
active enzymes and caramel-producing fructans, the inulin from dahlia
Boraginaceae, Campanulaceae, Liliaceae, Malpighiaceae, Poaceae,
tubers can be easily purified, which saves the downstream processing
Primulaceae, Styracaceae, Violaceae, etc. Usually, inulin is stored in
cost. Asparagus is another perennial herb and its tuberous roots contain
tubers, bulbs and tuberous roots of plants (Table 1). In these plant parts,
10–15% inulin. Additionally, its roots also contain short-chain fruc-
inulin can be easily extracted and processed to a purified product due to
tooligosaccharides, minerals (manganese, zinc and selenium), vitamin
the absence of interfering components. Commercially, inulin is pro-
(C and E), glutathione, etc. Due to all these nutrients, asparagus roots
duced from tubers of Dahlia pinnata (Dahlia), Helianthus tuberosus
are potent feedstock for inulinase production. Belgium, Netherland,
(Jerusalem artichoke) and roots of Cichorium intybus (chicory). The
France and Chile are major producers of inulin. The yield of inulin
purification of inulin is done on the basis of solubility difference of the
depends upon the harvesting time of crop, climate conditions, cultiva-
DP fractions present in the extract prepared from plant material.
tion strategies, etc. Higher content of inulin can be achieved, if the
Heating or cooling in connection with filtration, decantation and ul-
crops are harvested annually.
tracentrifugation are used for the purification of different molecular
Many inulin-rich plants like chicory, Jerusalem artichoke, aspar-
weight fractions of inulin. Additionally, organic solvents can also be
agus, leek, garlic, onion, yacon are commonly used for human nutrition.
used to precipitate long-chain inulin (DP 25-40). The left over inulin
Inulin has been recognized as a natural food ingredient by European
that has not been precipitated by organic solvents can be further turned
Union and it also has GRAS status in the United States. The DP plays a
into solid by spray drying. Currently, the worldwide production of in-
predominant role in the functionalities of inulin. The average chain
ulin is estimated to be about 37514.659 metric tons. Jerusalem arti-
length (DP) of the inulin ranges from 20 to 60 fructose units. The DP of
choke is an important inulin-rich feedstock which accumulates about
inulin from plants is low (DP < 200) than bacterial inulin
50–70 g/kg of its fresh weight fructan as inulin. Inulin content accounts
(DP < 10,000). These variations in the DP of inulin are due to the
for 80% of its total carbohydrate present in the tuberous roots. Apart
different plant species, physiological age of the plant, climatic condi-
from inulin, Jerusalem artichoke tubers also comprises of fat (1.77%),
tions, maturity at harvest and storage time, etc. (Singh et al., 2017a).
proteins (1.58%), minerals, vitamins and other microelements. The
The storage of inulin-rich plant materials leads to lower DP due to the
cultivation of Jerusalem artichoke is easier due to its strong resistance
depolymerization of high molecular weight carbohydrate moiety. The
towards plants diseases and pests. It is cultivated in Israel and its tu-
DP of inulin also influences its caloric value, prebiotic activity, digest-
berous roots are consumed as vegetables. Chicory is a inulin-rich plant
ibility, water binding capacity, sweetening power, etc. Moreover, DP
and its fleshy roots contain 15–20% inulin. Depending upon the de-
also determines the solubility of inulin in water. It is well established
velopmental stage of chicory crop, either inulin or oligofructose can be
that short-chain oligomers are much more soluble than long-chain
obtained from it. Oligofructose is formed by the hydrolytic action of

642
R.S. Singh et al.
polymers. Depending upon the inulin solubility or insolubility, its
polymers of different chain length are utilized in different industries.
For example in food processing industries, generally short-chain inulin
dissolves properly in order to make gels that are important for main-
taining the bulk and texture of products. Whereas, long-chain inulin in
crystalline form is utilized in pharmaceutical industries with lesser so-
lubility in water as a vaccine adjuvant.
Inulin also functions as a reserve energy resource and regulator of
cold resistance in plants like onion, garlic, wheat, banana, leek, as-
paragus, chicory, etc. Additionally, it allows the growth of plants under
water scarcity conditions generated either by drought or by low-tem-
perature conditions. Inulin is synthesized enzymatically in inulin-rich
plants. It is represented by a gross molecular formula GFn, where ‘G’, ‘F’
and ‘n’ stands for glucosyl, fructosyl and number of fructosyl units,
respectively. The natural biosynthesis of inulin in plant cells begins by
sucrose-sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) which cat-
alyzes the production of one glucose moiety and trisaccharides (kes-
tose) from two sucrose molecules. This enzyme is active at a high
concentration of sucrose (6–15%). Therefore, it can be concluded that
fructan production is an extension of sucrose metabolism. Another en-
zyme, fructan-fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) uti-
lizes the trisaccharide formed by the SST and transfers fructose from
one sucrose moiety to the primary hydroxyl group which is linked to
anomeric carbon through methylene group at C1 of fructose molecule of
trisaccharide or another oligosaccharide (nystose and 1-fructofur-
anosylfructose). This leads to chain elongation of fructan polymer and
releases inulin as the main end product. Whereas, in case of bacterial
and fungal spores, inulin synthesis takes place by transfer of an addi-
tional fructose molecule of sucrose to the terminal fructose unit of 1-
kestose or higher inulin-type fructans by sucrases (inulosucrase or le-
vansucrase). Inulin from these sources usually has very high DP. Inulin
biosynthesis in plants can be improved by metabolic engineering or
enzyme engineering to improve the pathway efficiency.
3. Bioprocessing strategies for the conversion of inulin-rich
feedstocks into microbial products
Inulin-rich feedstocks are the abundant renewable resources which
can be exploited for the production of various microbial products like
inulinases, high fructose syrup, fructooligosaccharides, biofuels, or-
ganic acids, single cell oil, single cell proteins, etc. Inulin, a naturally
occurring fructan of plant-origin can be used in its native form for the
production of inulinases, high fructose syrup and fructooligosacchar-
ides. It is a very costly substrate, so inulin-rich feedstocks can be used as
cost-effective substitute of inulin. Raw inulin extracted from various
inulin-rich feedstocks has been used for the production of inulinases,
high fructose syrup and fructooligosaccharides (Singh and Chauhan,
2018a; Singh et al., 2016a, 2017a, 2018a). Raw inulin extraction pro-
cess involves, firstly milling of wet or dry inulin-rich feedstocks and
then extraction of raw inulin either by boiling or at high temperature in
moist-steam under pressure using a autoclave. Inulin easily diffuses out
from inulin-rich feedstocks in hot water at higher temperature. The
increase in temperature upto a certain extent during the extraction
process increases the quantity of raw inulin from inulin-rich feedstocks.
Most of the research work on the utilization of raw inulin is at la-
boratory-scale. Raw inulin extraction from inulin-rich feedstocks is a
simple process, so the utilization of raw inulin for the production of
these products can be exploited at industrial scale.
Inulin-rich feedstocks cannot be utilized directly for the production
of biofuels, organic acids, single cell oil, single cell proteins, 2, 3-bu-
tanediol, sorbitol, mannitol, etc. Three approaches i.e. separate hy-
drolysis and fermentation (SHF), simultaneous saccharification and
fermentation (SSF) and consolidated bioprocessing (CBP) are used for
the utilization of inulin-rich feedstocks for the production of these
products. SHF involves hydrolysis and fermentation of inulin-rich
feedstocks separately in two reactors. During hydrolysis, inulin from
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Bioresource Technology 273 (2019) 641–653
inulin-rich feedstocks is hydrolysed into fermentable sugars (fructose
and glucose) by inulinases. These sugars are separated from the solid
residues and used as carbon source for the production of above men-
tioned products by fermentation using different microbial sources. The
freedom of independent operation during both the processes (hydro-
lysis and fermentation) is the major advantage of this strategy. The risk
of contamination during hydrolysis due to longer residual time is its
main drawback. SSF is a good alternative to conventional SHF. In this
process, hydrolysis and fermentation of inulin-rich feedstocks is carried
out in a single-step. The principal benefits for performing hydrolysis
and fermentation in a single-step are reduced end-product inhibition of
enzymatic hydrolysis and the reduced investment costs. Improved hy-
drolysis efficiency, reduced contamination and immediate utilization of
released sugars by the hydrolysis are the other major advantages of SSF.
The principal drawbacks of SSF are to find suitable conditions (e.g.
temperature and pH) for both hydrolysis and fermentation. Recycling
the enzyme and fermenting microorganism is the another limitation of
this process. CBP is a promising strategy in which all the processes
including enzyme production, saccharification and fermentation of the
resulting sugars for the production of desired products proceed si-
multaneously. It is an excellent technology which reduces the number
of unit operations and overall capital cost of the process. Furthermore,
CBP enhances processing efficiencies, eliminates the need of addition of
exogenous hydrolytic enzymes and also reduces the sugar inhibition of
inulinases. The major limitation of this technology is the requirement of
a suitable microorganism or engineered microbial strains capable of
hydrolysing the inulin-rich feedstocks by inulinases produced by them
and also producing the desired product from the saccharified sugars.
Literature survey reveals not even a single trial for the production of
biofuels, organic acids, single cell oil, single cell proteins, etc. from
inulin-rich feedstocks at industrial scale. Saccharification of inulin-rich
feedstocks is a major hindrance for the production of these products.
SHF is not cost-effective at industrial scale. So, SSF or CBP can be opted
at industrial scale. Operational conditions are problematic for SSF. So,
CBP is a good alternative to overcome this problem. Native strains
(having both inulinolytic activity and desired product producing
ability), microbial co-cultures (co-culture of inulinolytic and desired
product producing microorganism) and genetically engineered micro-
organism can be used in CBP for the production of these products. CBP
have a bright future for the production of these products at industrial
scale. Overall, to achieve successful commercialization of these pro-
ducts from inulin-rich feedstocks, it is necessary to bring all the barriers
into focus and combined a multitude of individual successes into high
yield and low cost processes.
4. Biotechnological applications of inulin-rich feedstocks
Various inulin-rich feedstocks have been used for the production of
a wide range of bioproducts which are discussed in the following sec-
tions:
4.1. Inulinases production
Inulin hydrolyzing enzymes belong to glycoside hydrolases (GH)
family 32 and 91. Exoinulinase (E.C. 3.2.1.80), endoinulinase (E.C.
3.2.1.7), 1, 2-β-fructan 1F-fructosyltransferase (E.C. 2.4.1.100), 1-exo-
hydrolase (E.C. 3.2.1.153), and sucrose 1F-fructosyltransferase (E.C.
2.4.1.99) are the inulinolytic enzymes belonging to family GH32.
Inulinases of family GH32 have plenty of applications in food and
pharmaceutical industry. Exoinulinase carries out the sequential de-
gradation of inulin from its terminal end releasing fructose with a
molecule of glucose, whereas endoinulinase acts on the internal β-2,1-
glycosidic linkages of inulin to produce FOSs of varied chain length.
The action pattern of 1-exohydrolase is same as that of exoinulinase as
it acts on inulin type fructans to produce fructose. By the hydrolytic
action, 1, 2-β-fructan 1F-fructosyltransferase produces
R.S. Singh et al.
fructooligosaccharides from inulin, whereas 1F-fructosyltransferase
produces short-chain fructooligosaccharides from sucrose. Family
GH91 includes inulin transferases (E.C. 4.2.2.18 and E.C. 4.2.2.17)
which hydrolyzes the inulin from the D-fructosyl-D-fructosyl dis-
accharide terminal releasing α-D-fructofuranose-β-D-fructofuranose
1,2′:2,3′dianhydride (DFA-III) and α-D-fructofuranose-β-D-fructofur-
anose 1,2′:2,1′dianhydride (DFA-I), respectively. These biocatalysts are
also known as inulin lyases.
Inulinases have been reported from microbes, plants and animals.
Inulinases from plants and animals are insufficient for industrial ap-
plications. Microorganisms are the potent source of inulinases as they
possess several advantages like rapid multiplication, easy genetic ma-
nipulation, high production yield and considerable variability in their
biochemical and biophysical characteristics (Singh and Chauhan,
2018a; Singh et al., 2017a). Inulinases from fungal strains are preferred
over other microbial strains due to their low substrate requirement and
ability to tolerate low pH and high-temperature conditions. Amongst
Aspergilli, A. ficuum, A. terreus, A. niger, A. tamarii, A. niveus, A. tu-
bingensis and A. tritici are reported as efficient inulinase producers
(Singh and Singh, 2017). Whereas, Penicillium trzebinski, P. purpur-
ogenum, P. subrubescens, P. rugulosum and P. expansum are potent in-
ulinases producing Penicilli. Recently, Penicillium oxalicum (Singh and
Chauhan, 2017, 2018b; Singh et al., 2018b,c) and Mucor circinelloides
(Singh et al., 2018d) has been reported as new inulinase producer.
Bacterial strains like Clostridium sp., Bacillus sp., Xanthomonas sp.,
Streptomyces sp., etc. have been used for inulinase production due to
their ability to survive under acidic, alkaline and high-temperature
conditions. Recently, Acinetobacter baumannii (Muslim et al., 2015), B.
safensis (Singh et al., 2013; Singh and Singh, 2014) and Bacillus cen-
trosporus (Zhang et al., 2015) are reported as new inulinase producers.
Amongst yeasts, Cryptococcus aureus (Kango and Jain, 2011), Kluyver-
omyces marxianus (Singh and Bhermi, 2008) and Zygosaccharomyces
cerevisiae (Liu et al., 2014) are efficient inulinase producers. Ad-
ditionally, few yeast strains like Hanseniaspora, Saccharomyces,
Metschnikowia, Torulosopra, Lachancea, Gordonia and Zygosaccharomyces
have also been reported as inulinase producers (Paixao et al., 2013).
Inulin is used as a carbon source and an inducer for the production
of inulinases. The major limiting factor of an enzyme for industrial
application is its cost. A significant reduction in the cost of inulinases
can be achieved by using a cheap inulin-rich feedstock for its produc-
tion. Various inulin-rich feedstocks like Jerusalem artichoke tubers,
asparagus roots, dandelion roots, chicory roots and dahlia tubers have
been used for inulinase production in both solid-state and submerged
fermentation (Table 2). Numerous fungal strains including Aspergillus
awamori (Rawat et al., 2015a), A. tritici (Singh et al., 2016b), Peni-
cillium citrinum (Rawat et al., 2015a) and yeast strains like Kluyver-
omyces marxianus (Singh et al., 2006; Singh and Bhermi, 2008), etc.
have been used for the production of inulinases from asparagus root
tubers. Chicory roots have been utilized on laboratory scale for the
production of inulinases by Acinetobacter baumannii (Muslim et al.,
2015), Aspergillus tamarii (Saber and El-Naggar, 2009), Thielavia ter-
restris (Fawzi, 2011), Xanthomonas sp. (Park and Yun, 2001), etc. Dahlia
tubers are promising inulin-rich feedstock which has been explored for
inulinase production from Aspergillus niger (Cruz et al., 1998), Asper-
gillus tamarii (Saber and El-Naggar, 2009), Cryptococcus aureus (Jain
et al., 2012), Kluyveromyces marxianus (Singh et al., 2007a; Singh and
Saini, 2013), etc. Jerusalem artichoke tubers have been utilized for
inulinases production by a diverse group of microorganisms like As-
pergillus tamarii (Saber and El-Naggar, 2009), Geotrichum candidum
(Erdal et al., 2011), Streptomyces sp. (Laowklom et al., 2012), Asper-
gillius wentii (Karatop and Sanal, 2013), etc. Many more inulin-rich
feedstocks like garlic extract (Muslim et al., 2015), dandelion roots
extract (Kango, 2008), yacon tubers extract (Cazetta et al., 2005), yacon
meal (Pivetta et al., 2016), etc. have also being explored for inulinase
production by a variety of microorganisms like Aspergillus niger, Aci-
netobacter baumannii, Penicillium sp., Stenotrophomonas maltophila, K.
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Bioresource Technology 273 (2019) 641–653
marxianus, etc. Recently, Tithonia rotundifolia extract has been explored
for inulinase production in shake-flask fermentations (Kamble et al.,
2018). Most of the microbial inulinases produced from inulin-rich
substrates have been purified using conventional chromatographic
techniques like ion-exchange, gel-exclusion, Ni2+-affinity chromato-
graphy, etc. (Singh and Chauhan, 2018a; Singh et al., 2017a).
4.2. High fructose syrup production
Fructose is a ketonic monosaccharide present in various vegetables
and fruits including raw carrots, apples, raisins, etc. It is a naturally
occurring low-calorie sweetener which is about 1.2–2.0 times sweeter
than conventional sugar sucrose. Due to its high sweetening index, it is
used in many countries as a sugar substitute. Fructose plays a pivotal
role in maintaining the human health. Some of its physiological roles
include bypassing glucose metabolic pathway, high intestinal iron ab-
sorption by forming iron-chelates complex, low glycemic index, in-
creases ethanol metabolism, free from problems causing athero-
sclerosis, corpulence, cariogenicity, etc. (Singh et al., 2018a).
Additionally, fructose also possesses several functional and technical
properties which are superior to other disaccharide sugars like high
osmotic pressure, high solubility at low temperature, low molecular
weight, flavour enhancer, stable in acidic foods, excellent humectant,
antioxidant and hygroscopic properties, etc. (Singh et al., 2017a; Singh
et al., 2018a). Due to these technical superiorities, it is used as an im-
portant ingredient in various food and beverage industries. In food
industries, it is used as a sweetening agent in various products like
yoghurt, chocolate, ice-cream, etc. In fruit-caning industries fructose
gives a glossy appearance to fruits due to its similarity with fruit flavors.
Moreover, fructose is also resistant to microbial spoilage which en-
hances its usage in carbonated drinks in beverage industries. In phar-
maceutical industries, fructose is used in the formulation of capsules
and solutions for infusions and injections.
High fructose syrup (HFS) is a nutritive and idyllic sweetener which
is used in numerous food and beverages due to its high-quality char-
acteristics. It has GRAS status and is in great demand around the globe.
Apart from these nutritional qualities, HFS also possesses some phy-
siological characteristics good for human health like it stimulates the
growth of bifidobacteria in small intestine, absorbs calcium in post-
menopausal women, controls the blood sugar level, prevents colon
cancer, etc. The production of HFS started in mid-1960s with the ad-
vancements in refining and separation technologies. Later on in 1980s,
HFS completely substituted sucrose in sweet beverages like Pepsi and
Coca-Cola. The first time large-scale production of HFS was carried out
by Staley Manufacturing Company in 1987. Later on, various other
biotechnological industries have started the manufacturing of a variety
of nutritive sweeteners for processed foods. Amongst them, the demand
of HFS increased after the hindrance in the supply chain of sucrose.
Moreover, due to the unfavorable weather conditions for cultivation of
sugarcane worldwide and increase in retail price of sucrose, has opened
an opportunity to milling industries to access the new raw materials for
HFS production. Conventionally, fructose is produced by enzymatic
hydrolysis of starch. It can also be produced by acid hydrolysis, but it
has few limitations. Acid hydrolysis of starch results in the formation of
by-products like difructose anhydride which are colored compounds
and have no sweetening property. Moreover, at lower pH fructose is
easily degraded, thus making process ineffective. Enzymatic hydrolysis
of starch involves complex reactions where three enzymes (α-amylase,
amyloglucosidase and glucoisomerase) are used in the bioprocess. This
conventional approach has many disadvantages like low product yield,
requirement of multiple enzymes, labour inclusive and additional
purification and refining cost. The yield by this bioprocess is approxi-
mately 45%. Enzymatic hydrolysis of inulin and inulin-rich feedstocks
for HFS production has received considerable attention in the last few
decades (Singh et al., 2017b, 2018e). Fructose can also be produced
from inulin using inulinases. This is a single-step process which yields
R.S. Singh et al. Bioresource Technology 273 (2019) 641–653

Table 2
Inulinase production from inulin-rich plant materials.
Plant material Microorganism Type of inulinase Inulinase yield (IU/ml) Reference

Artichoke extract Pichia caribbica Exoinulinase 70 Ali et al. (2016)

Asparagus officinalis roots extract Kluyveromyces marxianus Exoinulinase 50.20 Singh and Bhermi (2008)
Aspergillus tritici Endoinulinase 25.01 Singh et al. (2016b)
Asparagus racemosus roots extract Kluyveromyces marxianus Exoinulinase 47.31 Singh et al. (2006)
Asparagus roots extract Aspergillus awamori Exoinulinase 9.58 Rawat et al. (2015a)
Aspergillus ficuum Exoinulinase 5.32 Rawat et al. (2015a)
Aspergillus niger Exoinulinase 7.52 Rawat et al. (2015a)
Penicillium citrinum Exo- and endoinulinase 1.86 Rawat et al. (2015a)
Penicillium guillermondii Exoinulinase 0.54 Rawat et al. (2015a)
Asparagus roots powder Penicillium sp. Exo- and endoinulinase 45.23a Rawat et al. (2015b)
Chicory roots extract Acinetobacter baumannii Endoinulinase 1.98 Muslim et al. (2015)
Aspergillus tamarii Exoinulinase 31.79 Saber and El-Naggar (2009)
Thielavia terrestris Exoinulinase 8.42 Fawzi (2011)
Chicory roots powder Xanthomonas sp. Endoinulinase 15 Park and Yun (2001)
Dahlia tubers extract Acinetobacter baumannii Endoinulinase 3.11 Muslim et al. (2015)
Aspergillus niger Exoinulinase 3.68 Cruz et al. (1998)
Exoinulinase 14.5 Rawat et al. (2015a)
Aspergillus tamarii Exoinulinase 36.17 Saber and El-Naggar (2009)
Cryptococcus aureus Exoinulinase 25.30a Jain et al. (2012)
Kluyveromyces marxianus Exoinulinase 55.4 Singh et al. (2007a)
Exoinulinase 140.02 Singh and Saini (2013)
Exoinulinase 1.49 Rawat et al. (2015a)
Penicillium sp. Exo- and endoinulinase 3.27 Rawat et al. (2015a)
Exo- and endoinulinase 64.54a Rawat et al. (2015b)
Dandelion tap roots extract Aspergillus niger Endoinulinase 55 Kango (2008)
Garlic extract Acinetobacter baumannii Endoinulinase 2.05 Muslim et al. (2015)
Penicillium sp. Exo- and endoinulinase 41.32a Rawat et al. (2015b)
Stenotrophomonas maltophila Exoinulinase 160 Navraj et al. (2016)
Jerusalem artichoke powder Geotrichum candidum NS 45.62 Erdal et al. (2011)
Jerusalem artichoke roots extract Aspergillius wentii Exoinulinase 3.45 Karatop and Sanal (2013)
Jerusalem artichoke tubers extract Aspergillus tamarii Exoinulinase 28.55 Saber and El-Naggar (2009)
Streptomyces sp. Endoinulinase 1.60 Laowklom et al. (2012)
Raw garlic juice Aspergillus niger Exoinulinase 5.65 Mahmoud et al. (2011)
Tithonia rotundifolia extract Arthrobacter mysorens Exoinulinase 1669.45b Kamble et al. (2018)
Yacon tubercles extract K. marxianus var. bulgaricus NS 4.10 Cazetta et al. (2005)
Yacon meal Aspergillus niger NS 4 Pivetta et al. (2016)

NS: Not specified.

a
Inulinase yield is expressed in n Kat.
b
Inulinase yield is expressed in EU/ml.

95% fructose. In this single-step enzymatic approach, either whole cells
producing inulinases or free/immobilized inulinases can be used for the
hydrolysis of inulin. Various inulin-rich feedstocks have been used for
the production of fructose using either whole cells or free/immobilized
inulinases (Table 3).
Only few reports are available on the production of HFS from inulin-
rich feedstocks using whole cells. Jerusalem artichoke tubers are potent
substrate for HFS production by Saccharomyces cerevisiae (Yu et al.,
2011) and Cladosporium cladosporioides (Ferreira et al., 1991; De
Andrade et al., 1992). The use of whole-cell technology for the pro-
duction of HFS is expensive due to the maintenance of bioprocess free
from contamination or the nutrients supplementation is required for
sustaining the microbial growth in the operating system used for HFS
production. Therefore, the use of soluble (free) inulinase can be sub-
stituted to whole cell technology for HFS production. Inulinases from a
variety of microbial strains in free form have been used for the pro-
duction of HFS. Various factors like inulinase source, enzyme con-
centration, substrate concentration, hydrolysis time, thermal stability of
biocatalyst, etc. have strong influence on the HFS production from in-
ulin-rich feedstocks. Numerous inulin-rich feedstocks like Asparagus
racemosus roots extract (Singh et al., 2007b), chicory root tubers extract
(Cruz et al., 1998; Gupta et al., 1992), agave juice (Garcia-Aguirre
et al., 2009), dahlia tubers extract (Cruz et al., 1998), Jerusalem arti-
choke tubers extract (Manzoni and Cavazzoni, 1992; Cruz et al., 1998),
Jerusalem artichoke tubers powder (Ongen-Baysal and Sukan, 1996),
Tithonia rotundifolia extract (Kamble et al., 2018), etc. have been uti-
lized for the production of HFS in batch system by inulinases from
Kluyveromyces marxianus, Aspergillus niger, Fusarium oxysporum, Ar-
throbacter mysorens, etc. Low stability, shelf-life of free enzyme and low
product yield in the bioprocess are the major drawbacks of this system.
Moreover, free enzymes cannot be recycled in the consecutive batches.
This problem can be resolved by immobilizing the inulinase on a sui-
table support/matrix which can be further used repeatedly for the hy-
drolysis of inulin in batch or continuous systems (Singh et al., 2017c).
Immobilized biocatalyst offers several advantages like easy recovery,
enhanced stability, continuous usage, reduce chances of inhibition/
catabolite repression, cost-effective, etc. Various immobilization
methods like cross-linking, gel entrapment, physical absorption and
covalent binding have been used for the immobilization of inulinases
for HFS production. A wide range of matrices/supports like duolite
A568 (Singh et al., 2007c), chitosan particles (Trivedi et al., 2015),
chitin (Kim and Rhee, 1989), DEAE-cellulose (Gupta et al., 1992), agar
gel (Bajpai and Margaritis, 1985a), sodium alginate (Parekh and
Margaritis, 1986), aminoethyl cellulose (Kim et al., 1982), etc. have
been used to develop immobilized biocatalysts using inulinases for HFS
production from chicory roots tubers, asparagus roots, Jerusalem arti-
choke tubers, etc. in a batch system. The biocatalysts which have good
stability in a batch system can be used for the hydrolysis of inulin-rich
feedstocks in continuous system for HFS production. Immobilized in-
ulinases from a wide variety of microbial strains like Aspergillus niger,
Kluyveromyces sp., Aspergillus ficuum, etc. have been used for the hy-
drolysis of inulin-rich feedstocks in continuous system for HFS pro-
duction. Continuous stirred reactor (Wenling et al., 1999) and packed-
bed reactor (Bajpai and Margaritis, 1985a,b; Singh et al., 2008; Yewale

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R.S. Singh et al. Bioresource Technology 273 (2019) 641–653

Table 3
High fructose syrup production from inulin-rich plant materials.
Plant material Inulinase source Hydrolysis system used Fructose productivity (g/l) Reference

Agave juice Kluyveromyces marxianus Batch NS Garcia-Aguirre et al. (2009)

Asparagus racemosus roots extract Kluyveromyces sp. Batch 39.2 Singh et al. (2007b)
Batch 41.3 Singh et al. (2007c)
Continuous 49.7 Singh et al. (2008)
Asparagus and chicory roots extract Aspergillus tubingensis Batch 14.25a Trivedi et al. (2015)
Chicory root tubers extract Fusarium oxysporum Batch NS Gupta et al. (1992)
Aspergillus niger Batch NS Cruz et al. (1998)
Dandelion tap roots extract Aspergillus niger Continuous NS Rawat et al. (2017)
Jerusalem artichoke tubers extract Aspergillus ficuum Continuous 61a Kim and Rhee (1989)
Batch 77.5 Kim and Rhee (1989)
Aspergillius niger Batch NS Cruz et al. (1998)
Co-culture of Aspergillus niger and Candida Batch 1.84a Prangviset et al. (2018)
guilliermondii
Fed-batch 1.03a Prangviset et al. (2018)
Cladosporium cladosporioides Batch 5 De Andrade et al. (1992)
Kluyveromyces sp. Batch 40 Bajpai and Margaritis (1985a)
Batch 42 Bajpai and Margaritis (1985b)
Batch 24 Parekh and Margaritis (1986)
Batch 34 Kim et al. (1982)
Batch NS Holyavka et al. (2018)
Continuous 199.66a Wenling et al. (1999)
Continuous 102b Kim et al. (1982)
Continuous 31a Bajpai and Margaritis (1985a)
Continuous 90a Bajpai and Margaritis (1985b)
Recombinant Saccharomyces cerevisiae Continuous 680 Kim et al. (1997)
Saccharomyces cerevisiae Continuous NS Kim et al. (1997)
Jerusalem artichoke tubers powder Co-culture of Aspergillus niger and Kluyveromyces Batch NS Ongen-Baysal and Sukan (1996)
marxianus
Kuth roots extract Aspergillus niger Batch 35 Viswanathan and Kulkarni
(1995)
a
Kuth roots powder Aspergillus niger Continuous 68 Yewale et al. (2013)
Tithonia rotundifolia extract Arthrobacter mysorens Batch 33.79c Kamble et al. (2018)

NS: Not specified.

a
Fructose productivity is expressed in g l−1 h−1.
b
Fructose productivity is expressed in mM l−1 h−1.
c
Fructose productivity is expressed in mg/ml.

et al., 2013) have been used for the preparation of HFS from asparagus
roots, dandelion tap roots, Jerusalem artichoke tubers and kuth roots in
a continuous system. Packed-bed reactor is the most investigated re-
actor for HFS production (Rawat et al., 2017). Various factors like as-
pect ratio, flow rate, residence time, hydrolytic temperature, etc. have a
strong influence on the efficiency of a continuous reactor. A continuous
system is considered successful, if it is operated for a long period
without any hindrance. During long term operation of a continuous
reactor, enzyme degeneration, enzyme leakage or blockage of the
column are the major limitations. Most of the studies on the production
of HFS from inulin-rich feedstocks are carried out at laboratory scale.
So, scale-up studies should be carried out to examine the feasibility of
the bioprocess. Fructose produced from inulin-rich feedstocks is pur-
ified using chromatographic techniques (Singh et al., 2017b).
4.3. Fructooligosaccharides production
Fructooligosaccharides are non-digestable functional food in-
gredients which are considered amongst the major group of prebiotics
with appreciable bifidogenic characteristics. Due to the prebiotic po-
tential of xylooligosaccharides (XOSs), fructooligosaccharides (FOSs)
and galactooligosaccharides (GOSs), they are considered as important
functional foods. Amongst these prebiotics, FOSs are gaining con-
siderable attention worldwide owing to their economical and functional
properties. FOSs enhances the microbiota of the colon where these are
degraded into short-chain fatty acids like acetate, propionate and bu-
tyrate. These short-chain fatty acids lower down the colon pH and si-
multaneously enhance the absorption of essential nutrients and mi-
nerals ions (Mg2+ and Ca2+) in consumer body. Moreover, anti-
tumorigenic and anti-inflammatory properties of short-chain fatty acids
have also been reported (Singh et al., 2016a). FOSs as a part of regular
diet also stimulates the host health by resistance against intestinal pa-
thogens, absorbs minerals ions in small intestine, enhance the growth of
beneficial intestinal microbiota, etc. (Singh and Singh, 2010; Singh
et al., 2016a). FOSs are also used to increase the shelf-life and taste of
various dairy and bakery products, mask bitter after taste of acesulfame
and aspartame, reduce fat content and replace sugar in ice-creams,
jellies, jams, pastries, etc. (Singh and Singh, 2010).
Naturally, FOSs are found in many inulin-rich plants like chicory,
Jerusalem artichoke, dahlia, asparagus, agave and food items like ba-
nana, onion, cereals, etc. FOSs are synthesized by three approaches: (i)
extraction from inulin-rich plants, (ii) enzymatic hydrolysis of inulin,
and (iii) enzymatic production from sucrose. Extraction of FOSs from
inulin-rich plant parts is not economical due to its high purification cost
and low product yield. Therefore, FOSs are manufactured enzymatically
either from sucrose by the hydrolytic action of fructosyltransferases
(Ganaie et al., 2014; Bali et al., 2015) or by the action of endoinulinases
on inulin (Singh and Singh, 2010; Singh et al., 2016a). Fructosyl-
transferases (E.C. 2.4.1.9) hydrolyze the β-(2 → 1) glycosidic linkages
of sucrose and transfer the fructose moiety to the receptor such as other
sucrose molecule or FOSs. The FOSs produced by this approach have α-
D-Glu(1 → 2)-[β-D-Fru(1 → 2)-]n, where ‘n’ is the repeating fructose
units which ranges between 2 and 4. Whereas, endoinulinases randomly
break the β-D-(2 → 1) glycosidic linkages of inulin to yield FOSs which
contain α-D-Glu(1 → 2)-[β-D-Fru(1 → 2)-]n, where n = 2–9 and β-D-Fru
(1 → 2)-[β-D-Fru(1 → 2)-]n, where n = 1–9. Both types of FOSs have
same physiological and functional properties. Moreover, FOSs produc-
tion from sucrose is a complex process, whereas single-step enzymatic
hydrolysis of inulin from inulin-rich feedstocks yields more than 80%
fructooligosaccharides (Chi et al., 2009). FOSs production by chemical

646
R.S. Singh et al.
glycosylation method has also been reported. But it is an expensive and
labour intensive method, where the yield of FOSs is very low.
With the advancements in industrial enzyme technology, the FOSs
production from inulin-rich feedstocks using either free endoinulinase
or immobilized endoinulinase in batch system or continuous system can
be achieved. Most of the reports on FOSs production using purified
endoinulinases are on batch system (Singh et al., 2016a). Endoinulinase
of Aspergillus ficuum yielded 80% FOSs with varied DP 2-8 from Jer-
usalem artichoke tubers (Zhengyu et al., 2005). On the other hand,
endoinulinases from Pseudomonas sp. (Park et al., 1998) and Xantho-
monas oryzae (Cho et al., 2001) produced 80% (DP 3–4) and 75.2% (DP
3–5) FOSs, respectively from chicory roots extract in a batch process.
The major advantage of batch system at laboratory scale is the utili-
zation of very small quantity of inulin-rich feedstocks for FOSs pro-
duction. Due to low stability and one-time use of biocatalyst in a batch
process, endoinulinases from different microbial strains were im-
mobilized onto a suitable matrix/support for FOSs production in batch
or continuous system. Endoinulinases from various microbial sources
has been immobilized on different matrices/supports by covalent
binding, adsorption, entrapment and encapsulation (Neeraj et al.,
2018). Endoinulinase from Pseudomonas sp. was successfully im-
mobilized on polystyrene and under optimized conditions yielded 82%
FOSs from chicory roots without any significant loss of initial enzyme
activity in a continuous system (Yun et al., 2000). In another study,
endoinulinase from Aspergillus niger immobilized in chitin was used in a
packed bed reactor for continuous production of FOSs from Jerusalem
artichoke juice. FOSs yield of 65% was obtained after the process op-
timization (Nguyen et al., 2011). Generally, the activity and thermo-
stability of endoinulinases from different microbial strains is low, which
hinders their use at industrial scale for FOSs production. To overcome
these problems, endoinulinase encoding gene from a microorganism
can be cloned and transformed into suitable host cells (lacking extra-
cellular fructosyltransferase or exoinulinase activity) which can utilize
inulin-rich feedstocks for FOSs production. Kim et al (2006) produced
the FOSs (79.8%) from Jerusalem artichoke tubers extract using re-
combinant Saccharomyces cerevisiae carrying endoinulinase encoding
Inu 1gene from Pseudomonas mucidolens. The produced FOSs consisted
of inulobiose, inulotriose, inulotetraose and inulopentaose with in-
ulotetraose as the major product. The use of genetically engineered
biocatalyst in a continuous system makes endoinulinases a potential
candidate for scale-up studies for the synthesis of FOSs from inulin-rich
feedstocks. FOSs produced by hydrolysis of inulin-rich substrates are
purified by chromatographic and membrane processes to remove glu-
cose and fructose (Singh and Singh, 2010).
4.4. Utilization of inulin-rich feedstocks for the production of other products
Inulin-rich feedstocks can also be used for the production of bio-
fuels, organic acids, single cell oils, single cell proteins, 2, 3-butanediol,
etc. (Singh et al., 2017a). These products are also produced from lig-
nocellulosic materials (bagasse, straw, etc.) and corn starch. The main
processing challenge for the production of these products from lig-
nocellulosic materials is the feedstock treatment which is complex and
very time-consuming. Saccharification of corn starch requires the use of
two enzymes i.e. α-amylase and glucoamylase which makes the process
complex and uneconomical. Comparatively, the saccharification of in-
ulin-rich feedstocks is very simple and economical.
4.4.1. Biofuels
Biofuels are products which can be processed into liquid fuels for
either heating or transport purposes. Bioethanol, acetone and butanol
are industrially important biofuels. They can be produced from a wide
variety of substrates. Their production has been reported from various
inulin-rich feedstocks (Table 4).
4.4.1.1. Bioethanol. Bioethanol is a distilled biofuel produced either
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Bioresource Technology 273 (2019) 641–653
from molasses or lignocellulosic materials. A number of substrates have
been used for the fermentative production of bioethanol. It can also be
produced from inulin-rich feedsocks. These feedstocks are ubiquitous in
both temperate and tropical countries and also have high solubility at
elevated temperature without forming a viscous solution. Various
inulin-rich feedstocks like chicory, Jerusalem artichoke, dandelion,
etc. have been used for bioethanol production. SSF of inulin-rich
feedstocks by different microorganisms have been reported for
bioethanol production. In this process, one microorganism
simultaneously saccharify and ferment inulin-rich feedstocks into
bioethanol. However, in few cases, mixed culture of two types of
microorganisms has also been used for the SSF of inulin-rich feedstocks
for bioethanol production. Various microorganisms like Kluyveromyces
marxianus (Bajpai and Margaritis, 1986; Yuan et al., 2008; Hu et al.,
2012; Gao et al., 2015), Kluyveromyces cicerisporus (Yuan et al., 2010),
Saccharomyces cerevisiae (Chang et al., 2008; Hu et al., 2012; Matias
et al., 2015), Zygosaccharomyces bailii (Paixao et al., 2018), etc. have
been used for ethanol production from Jerusalem artichoke tubers in a
batch system. The ethanol yield from Jerusalem artichoke is equivalent
to that of sugarcane and 2-time more from corn. This characteristic
makes Jerusalem an outstanding substrate for ethanol production.
Currently, it is one of the most promising crops in New Zealand,
China and Europe. Mixture of cultures of K. fragilis, S. cerevisiae and
Zymomonas mobilis have also been utilized for bioethanol production
from Jerusalem artichoke tubers (Szambelan et al., 2004). In a batch
system, the accumulation of higher concentration of ethanol activates
the feedback inhibition which causes the low product yield. To
overcome this problem, fed-batch system can be used with a low
substrate concentration and product formation can be controlled from
feedback inhibition. Bioethanol was produced from Jerusalem
artichoke tubers using a co-culture of Aspergillus niger and
Saccharomyces sp. in a fed-batch system (Ge and Zhang, 2005). Only
a few reports are available on the bioethanol production from inulin-
rich feedstocks by continuous system. Whole cells of Kluyveromyces
marxianus were used for continuous fermentation of Jerusalem
artichoke juice for ethanol production in a stirred tank reactor. Under
the optimized conditions, 7 g/l/h ethanol and 0.6 g/l/h biomass was
obtained (Margaritis and Bajpai, 1982a). In another study, alginate
entrapped whole cells of Kluyveromyces marxianus were successfully
used for continuous ethanol production from Jerusalem artichoke
tubers extract in a packed-bed bioreactor. Ethanol productivity
(104 g/l/h) under optimal conditions was 15-times higher than the
free cells of Kluyveromyces marxianus in a stirred tank reactor
(Margaritis and Bajpai 1982b). The stability of immobilized
biocatalyst hinders its prolonged use in a continuous system.
Recombinant microorganisms having higher product yield, increased
pH tolerance and thermal stability have also been used for bioethanol
production from inulin-rich feedstocks. Saccharomyces cerevisiae has
been intensively used as a genetically engineered host. Recombinant
Saccharomyces sp. carrying the inulinase gene from different microbial
strains like Aspergillus niger (Wang et al., 2016), Penicillium janthinellum
(Wang et al., 2015), Kluyveromyces marxianus (Khatun et al., 2017),
Pichia guilliermondii (Zhang et al., 2010), Arthobacter sp. (Li et al.,
2013), etc. have been used for SSF of Jerusalem artichoke tubers for
ethanol production. The primary challenge to the recombinant strategy
is heterologous expression of inulinases to permit rapid growth and
efficient conversion of biomass. The use of the inulin-rich feedstocks
has opened a new era for ethanol production. Bioethanol produced after
fermentation of inulin-rich feedstocks is separated by distillation.
4.4.1.2. Acetone and butanol. Conventionally acetone-butanol is
produced from two substrates i.e. corn and molasses. The economics
of a bioprocess is based on the cost of the substrate which accounts for
50% of its total production cost (Sarchami and Rehmann, 2014).
Therefore, it is important to identify cost-effective and easily
available raw materials for efficient acetone-butanol fermentation.
R.S. Singh et al. Bioresource Technology 273 (2019) 641–653

Table 4
Biofuels from inulin-rich plant materials.
Plant material Microorganism Hydrolysis system Product Yield (g/l) Reference
used

Chicory tubers extract Clostridium saccharobutylicum Batch ABE 12.5 Ujor et al. (2015)
Jerusalem artichoke tubers Clostridium acetobutylicum Batch Butanol 11.21 Chen et al. (2010)
extract
Batch Acetone- 23–24 Marchal et al. (1985)
butanol
Clostridium saccharobutylicum Batch ABE 0.33a Sarchami and Rehmann (2014)
Kluyveromyces marxianus Batch Ethanol 92 Bajpai and Margaritis (1986)
Batch Ethanol 1.63b Gao et al. (2015)
Continuous Ethanol 7b Margaritis and Bajpai (1982a)
Continuous Ethanol 104b Margaritis and Bajpai (1982b)
Saccharomyces cerevisiae Batch Ethanol 82 Chang et al. (2008)
Continuous Ethanol 90 Chang et al. (2008)
Batch Ethanol 1.52b Razmovski et al. (2011)
Batch Ethanol 0.458a Matias et al. (2015)
Batch Ethanol 28 Duvnjak et al. (1991)
Zymomonas mobilis Batch Ethanol 1.33b Onsoy et al. (2007)
Zygosaccharomyces bailii Batch Ethanol 3.62b Paixao et al. (2018)
Jerusalem artichoke tubers Co-culture of K. fragilis, S. cerevisiae and Batch Ethanol 74.2 Szambelan et al. (2004)
powder Zymomonas mobilis
Kluyveromyces cicerisporus Batch Ethanol 96.3 Yuan et al. (2010)
Kluyveromyces marxianus Batch Ethanol 73.6 Hu et al. (2012)
Batch Ethanol 0.467a Yuan et al. (2008)
Batch Ethanol 104.83 Charoensopharat et al. (2015)
Saccharomyces cerevisiae Batch Ethanol 13.8c Song et al. (2017)
Batch Ethanol 65.2 Hu et al. (2012)
Batch Ethanol 36.2 Lim et al. (2011)
Fed-batch Ethanol 196 Ge and Zhang (2005)
Recombinant Saccharomyces cerevisiae Batch Ethanol 3.13b Wang et al. (2016)
Fed-batch Ethanol 84.3 Wang et al. (2015)
Fed-batch Ethanol 85.2 Khatun et al. (2017)
Recombinant Saccharomyces sp. Batch Ethanol 121 Zhang et al. (2010b)
Batch Ethanol 136d Li et al. (2013)
Zymomonas mobilis Batch Ethanol 99 Szambelan and Chrapkowska
(2003)
Jerusalem artichoke stalks Saccharomyces cerevisiae Batch Ethanol 55.6 Li et al. (2016)
powder
Kazak dandelion extract Clostridium saccharobutylicum Batch ABE 8.5 Ujor et al. (2015)

ABE: Acetone butanol ethanol.

a
Yield is expressed in g/g.
b
Yield is expressed in g/l/h.
c
Yield is expressed in mg/ml.
d
Yield is expressed in ml/l.

Acetone-butanol can be produced from inulin-rich feedstocks like
Jerusalem artichoke, chicory, dandelion, etc. In a batch system under
optimized conditions, Clostridium acetobutylicum efficiently produced
23–24 g/l acetone and butanol (Marchalet al., 1985) and 11.2 g/l
butanol (Chen et al., 2010) from Jerusalem artichoke tubers. In
another consolidated bioprocess, Clostridium saccharobutylicum
produced 0.25 g/l/h acetone-butanol-ethanol from Jerusalem
artichoke tubers (Sarchami and Rehmann, 2014). Chicory and
Taraxacum kok-saghyz extract have also been reported as potent
substrates for acetone-butanol-ethanol production (Ujor et al., 2015).
Under optimized culture conditions, C. saccharobutylicum produced
8.5 g/l and 12.5 g/l of acetone-butanol-ethanol from chicory and
Taraxacum kok-saghyz extract, respectively. The recovery of acetone
and butanol has been carried out by distillation.
4.4.2. Organic acids
Organic acids are the compounds which contain one or more
functional carboxylic acid group. They have tremendous applications in
food and pharmaceutical industries. Conventionally, organic acids are
produced either by chemical synthesis or by fermentation of carbohy-
drates like glucose and sucrose. In some cases, both chemical synthesis
and fermentation are carried out sequentially for organic acid produc-
tion. Organic acids can also be produced from inulin-rich feedstocks
like chicory, Jerusalem artichoke, etc. (Table 5).
Lactic acid (2-hydroxy propionic acid or hydroxy propionic) is an
important naturally occurring organic acid. Commercially, it is pro-
duced from molasses, lignocellulosic materials, barley, whey, malt, etc.
Generally, economically cheap raw materials are preferred for a bio-
process. Therefore, inulin-rich feedstocks like chicory, Jerusalem arti-
choke, etc. has also been used for lactic acid production. In a batch
system, the production of lactic acid by SSF of chicory root tubers ex-
tract by Lactobacillus sp. (Baston and Constantin, 2012) and Jerusalem
artichoke tubers extract by Lactobacillus paracasei (Choi et al., 2012;
Petrova et al., 2015) and Lactobacillus bulgaricus (Xu et al., 2016) has
been reported. To overcome the problem of feedback inhibition in a
batch system, in a few cases fed-batch system has been preferred for
lactic acid production from Jerusalem artichoke tubers (Wang et al.,
2013a). In a fed-batch system for lactic acid production, mixed culture
of Aspergillus niger and Lactobacillus sp. has been used for SSF of Jer-
usalem artichoke tubers (Ge et al., 2009, 2010). Immobilized Lacto-
coccus lactis has also been used for the production of lactic acid from
Jerusalem artichoke tubers in a fed-batch system (Shi et al., 2012).
Now-a-days, the use of recombinant microorganisms with high product
yield is apparently increasing. Recently in a bioprocess, recombinant
Kluyveromyces marxianus has been used for lactic acid production from
Jerusalem artichoke tubers in a batch system (Bae et al., 2018).
Many reports are also available on the fermentative production of
other organic acids from inulin-rich feedstocks. In a bioprocess,

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Table 5
Organic acids from inulin-rich plant materials.
Plant material Microorganism Hydrolysis system Organic acid Yield (g/l) Reference
used

Chicory flour Lactobacillus paracasei Batch Lactic acid 123.7 Petrova et al. (2015)
Lactobacillus bulgaricus Batch Lactic acid 123.6 Xu et al. (2016)
Lactobacillus sp. Batch Lactic acid 0.11a Baston and Constantin
(2012)
Jerusalem artichoke tubers Actinobacillus succinogenes Batch Succinic acid 52.7 Gunnarsson et al. (2014)
extract
Aureobasidium pullulans Fed-batch Poly (malic acid) 117.5 Xia et al. (2017)
Bacillus amyloliquefaciens Batch Glutamic acid 39.4 Qiu et al. (2017)
Clostridium tyrobutyricum Fed-batch Butyric acid 60.4 Huang et al. (2011)
Lactobacillus paracasei Batch Lactic acid 92.5 Choi et al. (2012)
Lactobacillus sp. Batch Lactic acid 0.13a Baston and Constantin
(2012)
Lactococcus lactis Fed-batch Lactic acid 142 Shi et al. (2012)
Propionibacterium acidipropionici Fed-batch Propionic acid 26.2 Liang et al. (2012)
Yarrowia lipolytica Batch Citric acid 68.3 Wang et al. (2013b)
Co-immobilized Zymomonas mobilis and inulinase from Continuous Gluconic acid 23.4a Kim and Kim (1992)
Aspergillus niger
Jerusalem artichoke tubers Bacillus coagulans Fed-batch Lactic acid 134 Wang et al. (2013a)
powder
Co-culture of Aspergillus niger and Lactobacillus sp. Fed-batch Lactic acid 120.5 Ge et al. (2009)
Co-culture of Lactobacillus casei and Aspergillus niger Fed-batch Lactic acid 141.5 Ge et al. (2010)
Recombinant Kluyveromyces marxianus Batch L-lactic acid 130 Bae et al. (2018)
Batch D-lactic acid 122 Bae et al. (2018)

a
Yield is expressed in g/l/h.

inulinase from Aspergillus niger and whole cells of Zymomonas mobilis
were co-immobilized for continuous fermentation of Jerusalem arti-
choke tubers extract for gluconic acid production in a packed-bed re-
actor (Kim and Kim, 1992). In another bioprocess, Yarrowia lipolytica
was used for citric acid production from Jerusalem artichoke tubers
extract in a batch system (Wang et al., 2013b). Succinic acid
(Gunnarsson et al., 2014) and glutamic acid (Qiu et al., 2017) were
produced from Jerusalem artichoke tubers extract using Actinobacillus
succinogenes and Bacillus amyloliquefaciens, respectively in a batch
system. Malic acid was produced from Jerusalem artichoke tubers ex-
tract using Aureobasidium pullulans in a fed-batch system (Xia et al.,
2017). Butyric acid (Huang et al., 2011) and propionic acid (Liang
et al., 2012) has also been produced from Jerusalem artichoke tubers
extract using immobilized Clostridium tyrobutyricum and Propioni-
bacterium acidipropionici, respectively in a fed-batch system. Purification
of organic acids by chromatography with strong anionic resins is car-
ried out after the completion of fermentation process.
4.4.3. Miscellaneous applications
Apart from the production of inulinases, FOSs, HFS, biofuels and
organic acids, inulin-rich feedstocks have also been used for the pro-
duction of various other industrially important metabolites (Table 6).
Single cell oils are the lipids with a specific function and significant
properties. Traditionally, various microorganisms from yeasts, fungi,
bacteria, microalgae, etc. accumulate more than 20% lipids in their
biomass and therefore, they are considered as an oleaginous source of
single cell oils. In a bioprocess, Rhodotorula mucilaginosa was used for
single cell oil production from Jerusalem artichoke tubers extract in
batch and fed-batch systems (Zhao et al., 2010b). Co-culture of Rho-
dotorula mucilaginosa and immobilized Pichia guilliermondii has also
been used for single cell oil production from Jerusalem artichoke tubers
extract in a batch system (Zhao et al., 2011). In another batch bio-
process, recombinant cells of Yarrowia lipolytica was successfully used
for single cell oil production from Jerusalem artichoke tubers extract
(Zhao et al., 2010a).
Single cell proteins are the total proteins extracted from dried cells
of various microorganisms belonging to bacteria, filamentous fungi,
yeast, algae, etc. They are used as dietary supplements in human nu-
trition as well as in animals feed. Only a few reports are available on
single cell proteins production from inulin-rich feedstocks. Cryptococcus
aureus was efficiently grown on Jerusalem artichoke tubers extract (Gao
et al., 2007) and yacon tubercles extract (Zhao et al., 2010c) for single
cell proteins production in a batch system. Recombinant cells of Yar-
rowia lipolytica has also been used for the production of single cell
proteins from Jerusalem artichoke tubers extract in a stirred tank re-
actor (Cui et al., 2011).
2, 3-butanediol is an organic metabolite with a wide range of ap-
plications in food, cosmetics and pharmaceutical industries.
Conventionally, it is produced from molasses, whey and lignocellulosic
raw materials. Various microorganisms like Paenibacillus polymyxa (Gao
et al., 2010), Bacillus polymyxa (Fages et al., 1986), Klebsiella pneumo-
niae (Sun et al., 2009), etc. have efficiently utilized Jerusalem artichoke
tubers extract for 2, 3-butanediol production in a batch system. Both
stalks and tubers of Jerusalem artichoke have been used for 2, 3-bu-
tanediol production by Klebsiella pneumoniae in a fed-batch system. Sun
et al. (2009) successfully obtained 2, 3-butanediol from Jerusalem ar-
tichoke tubers extract using Klebsiella pneumoniae in a fed-batch system.
Sorbitol is a sugar alcohol with enormous applications in chemical
and food industry. Commercially, it is synthesized either by the cata-
lytic (nickel) hydrogenation of glucose and sucrose at high temperature
or by electrochemical reduction of glucose under alkaline conditions.
Sorbitol can also be produced from Jerusalem artichoke tubers extract.
Saccharomyces cerevisiae was successfully used for sorbitol production
from Jerusalem artichoke tubers extract in a batch system (Duvnjak
et al., 1991). In another case, inulinase from Aspergillus niger was co-
immobilized with whole cells of Zymomonas mobilis for the continuous
hydrolysis of Jerusalem artichoke tubers extract for sorbitol production
in a packed-bed reactor (Kim and Kim, 1992). The production of
mannitol (Saha, 2006) and pullulan (Xia et al., 2017) have been re-
ported from chicory root tubers extract and Jerusalem artichoke tubers
extract in batch and fed-batch systems, respectively.
5. Conclusion
Inulin-rich feedstocks are abundant and inexpensive renewable raw
material for bioprocessing industries. Only a few reports are available
on the production of HFS and FOSs from inulin-rich feedstocks at la-
boratory scale. Therefore, efforts should be made to perform scale-up

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Table 6
Miscellaneous products from inulin-rich plant materials.
Plant material Microorganism Hydrolysis system Product Yield (g/l) Reference
used

Chicory roots extract Lactobacillus intermedius Batch Mannitol 227.9 Saha (2006)
Jerusalem artichoke tuber extract Aureobasidium pullulans Fed-batch Pullulan 15.2 Xia et al. (2017)
Co-cultures of Rhodotorula mucilaginosa and Batch Single cell oil 566 Zhao et al. (2011)
immobilized Pichia guilliermondii
Cryptococcus aureus Batch Single cell proteins 5.4 Gao et al. (2007)
Paenibacillus polymyxa Batch 2,3-Butanediol 36.92 Gao et al. (2010)
Protoplast fusant of Kluyveromyces sp. and S. cerevisiae NS Sorbitol 48.7 Wei et al. (2001)
Recombinant Bacillus sp. Fed-batch 2,3-Butanediol 28.60 Park et al. (2017)
Recombinant Yarrowia lipolytica Batch Single cell oil 506 Zhao et al. (2010a)
Rhodotorula mucilaginosa Fed-batch Single cell oil 522 Zhao et al. (2010b)
Batch Single cell oil 486 Zhao et al. (2010b)
Saccharomyces cerevisiae Batch Sorbitol 46 Duvnjak et al.
(1991)
Co-immobilized Zymomonas mobilis and inulinase from Continuous Sorbitol 26a Kim and Kim (1992)
Aspergillus niger
Bacillus polymyxa Batch 2,3-Butanediol 44 Fages et al. (1986)
Klebsiella pneumoniae Batch 2,3-Butanediol 81.59 Sun et al. (2009)
Fed-batch 2,3-Butanediol 91.63 Sun et al. (2009)
Recombinant Yarrowia lipolytica Batch Single cell proteins 537 Cui et al. (2011)
Jerusalem artichoke stalks and tubers Klebsiella pneumoniae Fed-batch 2,3-Butanediol 80.5 Li et al. (2010)
powder
Yacon tubercles extract Cryptococcus aureus Batch Single cell proteins 591 Zhao et al. (2010c)

NS: Not specified.

a
Yield is expressed in g/l/h.

studies for their production using various inulin-rich feedstocks.
Saccharification of inulin-rich feedstocks is a major hindrance for their
utilization for the production of various microbial products. Scale-up
studies should be performed for SSF of inulin-rich feedstocks for the
production of various industrially important metabolites. Further, ef-
forts should also be made to develop genetically engineered microbes
for CBP of inulin-rich feedstocks.
Acknowledgement
This work was sponsored by the French Government Research
Program “Investissements d′avenir” through the IMobS3 Laboratory of
Excellence (ANR-10-LABX-16-01).
Conflict of interest
Authors have no conflict of interests.
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