CHAPTER II

LITERATURE REVIEW

INTRODUCTION

Itaconic acid is well known as a pioneer for polymer synthesis and has been
involved in industrial processes for decades but the discovery of itaconic acid has still
been a growing interest because of its potential role as a replacement for crude oil–based
products, such as acrylic acid. This chemical enables a self-polymerization to poly-itaconic
acid. Therefore, it is industrially used as a precursor for plastic polymer synthesis as well
as for resins, lattices, fibers, coatings, adhesives, thickeners and binders (Hiller et.al,
2015). In general, itaconic acid and its derivatives have wide applications in the textile,
chemical and pharmaceutical industries.
There are petroleum-based methods of itaconic acid production but the depletion
of fossil fuels and the need for sustainable development require fermentative itaconic acid
production to replace these. The increasing concern on sustainability, environmental
conservation and energy shortage drives the search for viable, renewable and
environmentally friendly alternatives to replace fossil resources as raw material in the
production of important chemicals. Plant biomass is the most feasible alternative as it is a
sustainable resource and does not liberate fossilized carbon.
Plant biomass is one of the major renewable raw material sources on earth and
the Philippines has a lot of it. It is composed of carbohydrates and aromatic polymers.
These carbohydrates (cellulose and hemicellulose) and aromatic polymers (lignin) are a
potential resource for the industrial production of value-added products and biofuel. One
under-utilized form of plant biomass is corncob.
In the Philippines, corn cobs are commonly utilized as fuel. However, even if many
people use it as fuel, there are still a lot of corn cobs and even the ash from the burnt cobs
piles up. Sadly, if not used as fuel, corn cob is disposed and destroyed by fire on the farms
to prepare for the next coming season. The dumping and burning of corn cobs on the
farms constitute gross air pollution.

13
 Therefore, there is a big reason to think of ways how to utilize corn cob. Not only
itaconic acid can be produced from corn cobs. There are other value-added chemicals like
xylitol that can be produced. The current xylitol production globally involves the
employment of high temperatures (80-140°C) and pressures (up to 50 atm) with
expensive, toxic raney nickel catalyst for the hydrogenation of xylose which is a non-
economical and non-ecofriendly process. Hence, biotechnological production of xylitol
employing lignocelluloses as substrates would be an ideal substitute for the chemical
route. As mentioned, we can conclude that it is of great significance to counterpart this
increasing concern on sustainability, environmental conservation and energy shortage by
converting corn cobs into a potential resource.
Maicon, Inc. will pursue its idea to construct and optimize the utilization of the
whole lignocellulosic biomass (corn cobs) and not only the cellulose or the
hemicellulose fraction. This idea is called “bio-refining”, which means fractionation of the
biomass, and producing value added products from each fractions. The goal is obviously
to gain the highest profit, what means that the most valuable products should be produced
with the less financial outlay.
To be successful on our objective, only the ideal strategies that will overwhelm the
concerns of the existing technologies in the current industrial production of itaconic acid
and also of xylitol will be used. This is also to satisfy the increasing demand of these
chemicals for widespread applications.

RAW MATERIALS

Corn cobs
Due to corn's chemical composition, corn residues such as corn cobs show great
potential as a renewable raw material for producing a variety of added-value chemicals,
such as lactic acid, citric acid, sugars, ethanol and itaconic acid. As a lignocellulose
biomass, corncob has an energy content of about 18.5 MJ/kg, slightly higher than that of
corn stover (17 MJ/kg) and switchgrass (18 MJ/kg). However, it has an energy density of

14
about 5,000 MJ/m3, about two times that of corn stover and switchgrass; this gives it the
significant advantage for reducing transportation cost in industrial production for value-
added chemicals.
Lignocellulosic biomasses consist of three main components: cellulose,
hemicellulose and lignin, and some minor fractions, such as proteins, lipids, and ash.
Common lignocellulosic biomasses are: wood, grass, bagasses, waste paper, and the
stalks of cereals.

Corncob is a by-product in corn (maize) production. Traditionally, it has been used

as animal beddings or returned to the field. In the Philippines, it is mainly used as fuelwood
for cooking. Four million tonnes of grain corn and 0.96 million tonnes of corn cobs are
produced yearly in the Philippines (Zafar, 2018).

Figure 2.1. Corn Cobs

Table 2.1
Chemical properties of corncob
Characteristics Percentage (wt%)

Cellulose 36.43

Hemicellulose 41.96

Lignin 9.6

Moisture 12.01

Source: Jager et. al, 2014

15
 Table 2.2
Volume of corn production by region, Philippines: 2017 (In metric ton)

Aspergillus Terreus
Aspergillus is one of the oldest named genera of fungi. By 1926, Aspergillus had
become one of the best-known and most studied mould groups. Their prevalence in the
natural environment, their ease of cultivation on laboratory media and the economic
importance of several of its species ensured that many mycologists and industrial
microbiologists were attracted to their study. Aspergilli grow abundantly as saprophytes
on decaying vegetation and have been found in large numbers in mouldy hay, organic
compost piles, leaf litter and the like. Most species are adapted for the degradation of
complex plant polymers, but they can also dine on substrates as diverse as dung, human
tissues, and antique parchment. There is even a report of an unidentified Aspergillus
species being capable of the solubilization of low rank coal (Machida, M., 2010).

16
 A. terreus is known for its pronounced sensitivity with regard to the maintenance
of sufficiently aerobic conditions (Gyamerah, 1995; Kubicek et al., 1985; Kuenz et al.,
2012; Lockwood and Reeves, 1945).

Figure 2.2. Aspergillus Terreus

Source: jp.123rf.com

Table 2.3
Comparison of different substrates
and fungal strains used for itaconic acid production
Microorganism Substrate IA titer (g L-1)
Aspergillus terreus Jatropha cake 48.7
A. terreus CECT 20365 Olive & beet waste 44
A. terreus Glucose 26.9
Glycerol 30.2
A. terreus DSM23081, NRRL Glucose 86.2
1960, NRRL 1963
A. terreus SKR10 Sago starch 48.2
A.terreus POME 5.76
A. terreus M-8 Starch hydrolysate 55
A. terreus Citric acid 1
A. terreus NRRL 1960 Various starches 18.4
A. terreus MTCC 479 Corn starch 10.3
Maize flour 6.5
Potato starch 5.8
A. terreus Sorghum bran 10
A.terreus Ast165 Corn starch 53.8
A. niger (genetically engineered) Glucose 2.5
Ustilago maydis Glucose 29
Source: Machida, 2010

Debaryomyces nepalensis
Debaryomyces nepalensis NCYC 3413, a halotolerant strain that has been
previously isolated from rotten apple, it can utilize both hexoses and pentoses; and is also

17
important in production of commercially valuable products such as arabitol, xylitol and
ethanol (Gummadi and Kumar 2006a; Kumar and Gummadi 2011; Kumdam et al. 2012).
Bioconversion of xylose to xylitol can be carried out by bacteria, fungi and yeast. Among
the reported microbial strains, Candida (Barbosa et al. 1988) and Debaryomyces (Converti
and Domı´nguez 2001; Converti et al. 2002) are the best-known yeast species for xylitol
production.

Figure 2.3. Debaryomyces nepalensis

Source: http://www.westerdijkinstitute.nl

Sulfuric Acid
Sulfuric acid (alternative spelling sulphuric acid), also known as vitriol, is a mineral
acid composed of the elements sulfur, oxygen and hydrogen, with molecular formula
H2SO4. It is a colorless, odorless, and syrupy liquid that is soluble in water, in a reaction
that is highly exothermic.

Figure 2.4. Atomic structure of sulfuric acid

Source: Quora.com

Sodium Hydroxide
Liquid sodium hydroxide is colorless and has no odor. It can react violently with
strong acids and with water. Sodium hydroxide is used in many other industrial and
manufacturing processes. It is used to manufacture rayon, spandex, explosives, epoxy
resins, paints, glass and ceramics. It is also used in the textile industry to make dyes,

18
process cotton fabric and in laundering and bleaching, as well as in metal cleaning and
processing, oxide coating, electroplating and electrolytic extracting.

Figure 2.5. Sodium Hydroxide

PRODUCTS

Itaconic Acid

Itaconic acid is also known as methylidenesuccinic acid, a naturally organic

compound which is non-toxic. It can be obtained by the distillation of citric acid but
currently produced by fermentation. From the name aconitic acid, the name itaconic acid
was derived by its anagram (Roger A. Sheldon, 2014). Using the functional carboxylic acid
groups, it can be modified or copolymerized into different polymers such as epoxy, amide,
polyesters etc. Itaconic acid also has a double bond which can undergo free radical
polymerization to produce both homo and co-polymers with other monomers (T. Werpy,
2004). It can be used as a platform chemical to produce chemicals such as itaconic
diamide, itaconic anhydride, 2-methyl-1,4-butanediol, 2-methyl-1,4-butanediamine from
which different polymers can be obtained (Geilen, F., et al., 2010).
In a recent study, IA was also found to dissolve well in several alcohols including
methanol, 2-propanol and ethanol with the solubility increasing with temperature itaconic
acid is also readily biodegradable in nature.
Itaconic acid is valuable as a monomer because of its unique chemical properties,
which derive primarily from its methylene group and its possession of two carboxylic acid
groups. Itaconic acid can take part in addition polymerization, giving polymers with many

19
 free carboxyl groups that confer advantageous properties on the polymer. It can either be
self-polymerized or can act as a co-monomer with other monomers to form
heteropolymers.
In this study we will utilize the waste corn cobs to produce a bio-based itaconic
acid which involves the fermentation of the pretreated ground corn cobs with the aid of
Aspergillus terreus as an active fungus.

Figure 2.6. Chemical Structure of Itaconic acid

Source: ChemEssen

Table 2.4
Physical and chemical properties of itaconic acid
Property Name Property Value
Molecular formula C5H6O4
Physical state and appearance Solid (white crystals)
Molecular weight 130.1g/mole
Odor Odorless
Color White crystals
Boiling point 286 C
pH (5% water) 1.5-2.0
Melting point 167-169 C
Specific Gravity 1.573 (Water =1)
IUPAC name 2-methylidenebutanedioic acid
Solubility 76.8 mg/mL at 20 °C
Source: ScienceLab.com

In the study of Okabe, M., et.al., 2009, IA has been used in a wide range of
industries. During the 1950s, IA was used in industrial adhesives. Overall, during this
period, IA was used at an industrial scale, and large amounts of it were required. The alkali
salt or sulfonated form of poly IA is used as a detergent and in shampoos. The polymerized

20
methyl, ethyl, or vinyl esters of IA are used as plastics, adhesives, elastomers, and
coatings. In the textile industry, IA was employed in nonwoven fabric binders.
Currently, the most important application of itaconic acid in terms of market size is
the production of synthetic styrene-butadiene rubber (SBR) (Weastra, 2012). SBR is, for
instance, used in coated papers, where the addition of up to 5 % itaconic acid improves
the adhesive characteristics of the rubber (Warson and Finch, 2001; Willke and Vorlop,
2001). Furthermore, itaconic acid is applied as a lubricant and dye additive, a dispersant,
as well as for the production of chemical fibers (Weastra, 2012). Aside from the already
established areas, new, high-potential applications are of considerable importance as they
could significantly influence future growth of the itaconic acid market.
Possible bulk chemicals which could be replaced by IA, are acrylic acid, acetone
cyanohydrin, maleic anhydride, and sodium tripolyphosphate. Among other investigated
processes, itaconic acid is used as a platform chemical for the production of the fuel
compound 3-MTHF. In the envisioned fuel production process, lignocellulosic biomass is
subjected to mechanical, chemical, and enzymatic pretreatment yielding glucose, pentose
sugars such as xylose, and lignin.

Xylitol

Xylitol, a pentose alcohol has been used as a sugar substitute which has
anticariogenic, microbiostatic and insulin independent metabolic nature. The xylitol
molecule consists of five carbon atoms and five hydroxyl groups (Chen et al., 2010).
A recent study on rats confirmed its antidiabetic nature. It has been approved by
the US Food and Drug Administration (FDA) as a safe food additive. The extremely low
Glycemic Index (GI) of 7 has made it a promising substituent for glucose which has GI of
100.
It has applications in sugar free gums, sugar free confectionaries, common foods
and drinks, dietetic products, oral hygiene, pharma and cosmetic products, as well as in
anti-cancer and anti-AIDS (betulin) products. As a medicine, xylitol is used to prevent
middle ear infections (otitis media) in young children, and as a sugar substitute for people

21
 with diabetes. Xylitol is added to some chewing gums and other oral care products to
prevent tooth decay and dry mouth. It is sometimes included in tube feeding formulas as
a source of energy. Xylitol is added to water for use in nasal irrigation for people with sinus
problems.
The demand-supply ratio for xylitol has been constantly rising for the past few
years. But the ecofriendly exploitation of lignocelluloses for xylitol production is still a
persistent challenge in the field of white biotechnology.

Figure 2.7. Chemical Structure of Xylitol

Source: Pubchem.com

Table 2.5
Physical and chemical properties of xylitol
Property Name Property Value
Molecular formula C5H12O5
IUPAC name (2R,3r,4S)-Pentane-1,2,3,4,5-pentol
Physical state and appearance Solid (powdered solid)
Molecular weight 152.15 g/mole
Odor Odorless
Taste Sweet
Color White
Boiling point 216 °C
Melting point 94 °C
Specific Gravity 1.52 (Water =1)
Solubility Easily soluble in cold water. Soluble in methanol
Source: Sciencelab.com

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METHODS OF MANUFACTURING

A. Pre-Treatment Processes for Lignocellulosic Biomass

The pre-treatment process is important to remove impurities from corncobs. Pre-

treatment process is mainly involved in effective separation of complex interlinked
fractions and this increase the accessibility of each individual component, thereby
becoming an essential step in a broad range of applications. It is important because it
helps in facilitating the bio-conversion processes (Laser, et al., 2009).

PHYSICAL PRE- MECHANICAL ULTRASOUND

EXTRUSION MILLING SONICATION
TREATMENT

OZONOLYSIS ACID HYDROLYSIS

CHEMICAL PRE- ALKALINE HYDROLYSIS ORGANOSOLV

TREATMENT

OXIDATIVE MICROWAVE-ASSISTED
DELIGNIFICATION PRETREATMENT
PROCESS

1. Physical pre-treatment processes for lignocellulosic biomass

1.1 Mechanical extrusion
Feedstock materials are subjected to heating process (>300 °C) under shear
mixing. This pretreatment process results mainly in production of gaseous products and
char from the pretreated lignocellulosic biomass residues. Due to the combined effects of
high temperatures that are maintained in the barrel and the shearing force generated by
the rotating screw blades, the amorphous and crystalline cellulose matrix in the biomass
residues is disrupted. However, this method requires significant amount of high energy

23
making it a cost intensive method and difficult to scale up for industrial purposes. (Zhu,
et.at., 2010).

1.2. Milling (mechanical)

It is used for reducing the crystallinity of cellulose. It mostly includes chipping,
grinding, and/or milling techniques. Chipping can reduce the biomass size to 10–30 mm
only while grinding and milling can reduce the particle size up to 0.2 mm. However, studies
found that further reduction of biomass particle below 0.4 mm has no significant effect on
rate and yield of hydrolysis (Chang et al. 1997). Chipping reduces the heat and mass
transfer limitations while grinding and milling effectively reduce the particle size and
cellulose crystallinity due to the shear forces generated during milling. The type and
duration of milling and the kind of biomass determine the increase in specific surface area,
final degree of polymerization, and the net reduction in cellulose crystallinity. Different
milling methods viz. two-roll milling, hammer milling, colloid milling, and vibratory milling
are used to improve the digestibility of the lignocellulosic materials (Taherzadeh, et.al.,
2008).

1.3. Ultrasound Sonication

It is relatively a new technique used for the pretreatment of lignocellulosic biomass.
However, studies in the laboratory have found sonication a feasible pretreatment option.
Ultrasound waves produce both physical and chemical effects which alter the morphology
of lignocellulosic biomass. Ultrasound treatment leads to formation of small cavitation
bubbles which rupture the cellulose and hemicellulose fractions thereby increasing the
accessibility to cellulose degrading enzymes for effective breakdown into simpler reducing
sugars. It is reported that the maximum cavitation was formed at 50 °C which is also the
optimum temperature for many cellulose degrading enzymes. (Yachmenev, et al., 2009).
The ultrasonic field is primarily influenced by ultrasonic frequency and duration, reactor
geometry and its type and solvent used. Furthermore, biomass characteristics, reactor
configuration, and kinetics also influence the pretreatment through sonication
(Bussemaker, et.al., 2013).

24
2. Chemical pre-treatment processes for lignocellulosic biomass
They are used to remove some of the components like lignin and hemicellulose
which form structural barrier to enzymatic accessibility of cellulose in the biomass.

2.1 Ozonolysis
Ozone treatment is one way of reducing the lignin content of lignocellulosic wastes.
This results in an increase of the in vitro digestibility of the treated material, and unlike
other chemical treatments, it does not produce toxic residues. Ozone can be used to
degrade lignin and hemicellulose in many lignocellulosic materials such as wheat straw,65
bagasse, green hay, peanut, pine,66 cotton straw,67 and poplar sawdust.68 The
degradation is mainly limited to lignin. Hemicellulose is slightly affected, but cellulose is
not.
A drawback of ozonolysis is that a large amount of ozone is required, which can
make the process expensive.

2.2. Acid Hydrolysis

Concentrated acids such as H2SO4 and HCl have also been used to treat
lignocellulosic materials. Dilute-acid hydrolysis has been successfully developed for
pretreatment of lignocellulosic materials. Sulfuric acid at concentrations usually below 4
wt %, has been of the most interest in such studies as it is inexpensive and effective.
Dilute H2SO4 is mixed with biomass to hydrolyze hemicellulose to xylose and
other sugars and then continues to break xylose down to form furfural. The dilute H2SO4
pretreatment can achieve high reaction rates and significantly improve cellulose
hydrolysis. Dilute acid effectively removes and recovers most of the hemicellulose as
dissolved sugars, and glucose yields from cellulose increase with hemicellulose removal
to almost 100% for complete hemicellulose hydrolysis. Hemicellulose is removed when
H2SO4 is added and this enhances digestibility of cellulose in the residual solids. Recently
developed dilute-acid hydrolysis processes use less severe conditions and achieve high
xylan to xylose conversion yields.

25
2.3. Alkaline Hydrolysis
Some bases can be used for the pretreatment of lignocellulosic materials, and the
effect of alkaline pretreatment depends on the lignin content of the materials. Alkali
pretreatment processes utilize lower temperatures and pressures than other pretreatment
technologies. Alkali pretreatment can be carried out at ambient conditions, but
pretreatment times are on the order of hours or days rather than minutes or seconds.
Compared with acid processes, alkaline processes cause less sugar degradation, and
many of the caustic salts can be recovered and/or regenerated. Sodium, potassium,
calcium, and ammonium hydroxides are suitable alkaline pretreatment agents. Of these
four, sodium hydroxide has been studied the most.

2.4. Oxidative Delignification

Lignin biodegradation could be catalyzed by the peroxidase enzyme with the
presence of H2O2.
Wet oxidation combined with base addition readily oxidizes lignin from wheat
straw, thus making the polysaccharides more susceptible to enzymatic hydrolysis. Furfural
and hydroxymethylfurfural, known inhibitors of microbial growth when other pretreatment
systems are applied, were not observed following the wet oxidation treatment.

2.5. Microwave-Assisted Pretreatment Process

The pretreatment results from microwave assisted process indicated that the
pretreatment and prehydrolysis of the corncob occurred in a single step due to the
combination of microwave irradiation which instantaneously promoted vibration of the
water molecules and converted the molecular motion into heat. The use of a NaOH water
solution caused an increase in the internal surface area and a decrease in the degree of
polymerization of the lignocellulose. This facilitated both the pretreatment and the
hydrolysis which involved a reaction that breaks bonds in a molecule using water. This
can be observed from the high yields of total sugars obtained from the partial hydrolysis
of the cellulose or starch or both.

26
 The total sugars are the sum of the reducing and non-reducing sugars including
several types of saccharides: monosaccharides (i.e. glucose and fructose), disaccharides
(i.e. sucrose and cellobiose), and trisaccharides (i.e. maltotriose). The pretreatment
results gave lower yields of glucose but higher yields of total sugars. The high yield of total
sugars showed that the pretreated corncob (pre-hydrolysate) was high in di- and tri- (or
bigger) saccharides which can be converted to smaller sugars or glucose by
acid/enzymatic hydrolysis. Alternatively, the amounts of glucose, total sugars, and
cellulose suggest that the pretreated corncob would be appropriate as the substrates in a
simultaneous saccharification and fermentation process for ethanol production During
pretreatment under alkaline conditions, the lignin was removed from the lignocellulose,
the crystalline structure of the cellulose was disrupted, the porosity of the lignocellulose
increased.

B. Itaconic Acid Production

The main developments in IA production (batch fermentation, free suspended
biomass) took place before about 1966. The development of new fermentation
technologies and more sophisticated bioprocess control has led to renewed interest in
improving IA production, and novel fed-batch strategies and continuous processes using
immobilised cells have now been developed and investigated.

CHEMICAL
SYNTHESIS

FUNGAL STRAINS
BIOSYNTHESIS AND INOCULUM CULTIVATION FERMENTATION
PREPARATION

1. Chemical Process
The first method of producing IA was by pyrolysing citric acid and hydrolysing the
anhydrides (Baup 1837). Another early method, discovered by Crasso, was the

27
decarboxylation of aconitic acid. Chemical synthesis is mainly performed by dry distillation
of citric acid and subsequent treatment of the anhydride with water (Blatt 1943), or using
the method of Montecatini (Italy), from propargyl chloride, carbon monoxide, nickel
carbonyl and water (Chiusoli 1962). Alternative approaches are the oxidation of mesityl
oxide and subsequent isomerisation of the formed citric acid (Berg and Hetzel 1978), or
the oxidation of isoprene (Pichler et al. 1967). But none of these processes can really
compete with fermentation by fungi and therefore none are practised commercially (Tate
1981). The main drawback of chemical synthesis is that it depends on substrates within
the same price range as IA, and a requirement for a high reaction temperature, which
makes the process more cost-intensive. Moreover, the multistep chemical synthesis of IA
has proved to be uneconomical because of high substrate cost and/or relatively low yields
and it cannot compete with fermentation process.
Itaconic acid was historically produced by various chemical methods such as
destructive distillation of citric acid, and this was the main method of producing it prior to
the 1960s; heating the calcium aconitate solution produced in the cane sugar refining
process; the patented Montecatini method involving propargyl chloride; the oxidation of
mesityl oxide and subsequent isomerization of the formed citric acid; and the oxidation of
isoprene. Maleic anhydride is also used in the production of IA, but the process has not
yet achieved industrialization. None of these processes compete favorably with the
fermentative production process, and IA is now almost entirely produced by fermentation
of sugars by Aspergillus terreus. (El-Imam AA, Du C,2014). Itaconic acid is primarily used
as a co-monomer in the production of acrylonitrile butadiene styrene and acrylate latexes
with applications in the paper and architectural coating industry.

2. Biotechnological Production
Itaconic acid biosynthesis in A. terreus is achieved via the central metabolism, i.e.
the Embden-Meyerhof-Parnas (EMP) pathway, pyruvate (de)carboxylation, and the
tricarboxylic acid (TCA) cycle. Thus, first of all glucose is converted to two pyruvate
molecules, which yield acetyl-CoA and oxaloacetate by decarboxylation and
carboxylation, respectively. Within the TCA cycle, these compounds are then transformed

28
to citrate, which is further converted to cis-aconitate, the precursor of itaconic acid. Cis-
aconitate is transported from the mitochondria to the cytosol, where it is decarboxylated
to itaconic acid via the cis-aconitate decarboxylase (CAD) (Li et al., 2011a).

Figure 2.8. Biosynthetic pathway of itaconic acid in Aspergillus terreus

Source: Huang et al., 2014

Since A. terreus is known for its pronounced sensitivity with regard to the
maintenance of sufficiently aerobic conditions, phases of insufficient oxygen supply can
thus stop product formation entirely, even if they last only several minutes. Upon
restoration of aerobic conditions, itaconic acid biosynthesis is then slowly resumed within
a time frame of several days indicating an involvement of protein synthesis. A possible
explanation for this sensitivity can be found in the export mechanism for itaconic acid. As
it requires ATP, it directly depends on oxidative phosphorylation. Hence, insufficient
oxygen supply could result in a reduced level of ATP, which hinders the export of itaconic
acid from the intracellular space (Gyamerah, 1995). In addition to oxygen supply, the pH
during the cultivation has a decisive role in itaconic acid production. Commonly, the pH
decreases from its initial value of 3.1 to approximately 2 due to the consumption of
ammonium (Hevekerl et al., 2014b; Rychtera and Wase, 1981).
Subsequently, the pH could, however, be increased to higher values without any
negative effects on itaconic acid formation. This strategy was implemented in a process
by the Pfizer company. Recently, Hevekerl et al. (2014b) furthermore combined this pH

29
shift with an optimized medium and a supply with additional glucose. This led to a process
with a productivity of 1.15 g L-1 h-1 and a final itaconic acid concentration of 129 g L-1. At
the expense of productivity, a maximum final product titer of 146 g L-1 was achieved,
which represents the current maximum in the scientific literature.

Two Types of Bioreactors

a. Stirred Tank Reactors (STR)

STR is the conventional reactor which is not suitable for filamentous
microorganisms as high shear rate is generated by the mechanical agitation that may
damage fungal cells. Hence, an optimum balance between aeration rate and agitation
speed should be maintained to avoid mechanical stress on the mycelia caused by
excessive impeller speed, or conversely, a poor oxygen transfer rate at lower speed.
Okabe et al. assessed the effect of DO concentration and agitation speed at pH 2. It was
observed that although the overall yield increased with DO, the highest yield of 0.55 g IA/
g of glucose was obtained at DO of 20% and an impeller speed of 320 rpm. Any change
in the agitation speed lowered the IA production because of either sheer stress on mycelia
or insufficient oxygen transfer rate.

b. Air Lift Reactors (ALR)

Above oxygen limitation can be overcome using ALR as it does not generate
mechanical agitation and achieves good mixing by liquid recirculation. It can provide high
oxygen transfer rates with minimal shear stress due to increased gas–liquid contact. Also,
it is easy to construct and only requires about one-third of the energy needed for an STR.
The comparison between ALR and STR for IA production showed that ALR provided
higher production rates (0.64 g L-1h-1 ) with less power input per volume (542 W m-3 )
compared to STR (0.48 g L-1h-1 at 1180 W m-3 ). In addition, the power input per volume
could be further reduced using O2- enriched air (Yusoff, 2015).

30
C. Utilization of Hemicellulose Hydrolyzates of Corn Cobs

Xylose is one of the major constitutions in the hemicellulose hydrolysate of

corncobs; hence the bioconversion of xylose to xylitol will be a useful process, which has
a wide variety of industrial application. Chemical synthesis of xylose is possible but
expensive and time consuming. The utilization of available plant biomass like corncobs
will be the potential natural resource for the production of bulk quantity of xylose with less
cost and less time. Whereas the commercial xylitol production is achieved by
aerobic(presence of oxygen) fermentation of corncob hydrolysate with yeast
(Debaryomycesnepalensis and Debaryomyceshanseni etc.,) is used as an artificial
sweetener, dental application etc. (imedpub.com).
None of the existing detoxification strategies are ideal since they neither remove
inhibitors completely nor retain fermentable sugars entirely. Hence, the use of microbes
with higher inhibitor tolerance would be economical to avoid the loss of sugars and to
exclude a costly and time consuming unit operation detoxification. Addition of external
xylose, nitrogen source, macro and micro elements to the HCH, made it diluted such that
5-HMF and furfural concentration became undetectable in the initial HCH fermentation
broth.

1. Bioconversion of Non-Detoxified Hemicellulose Hydrolysates to Xylitol

The use of a halotolerant strain D. nepalensis for the conversion of various
hemicellulose hydrolysates into value added compounds like xylitol and ethanol without
any pretreatment after hydrolysis would yield a cheaper bioprocess. In the study of
Gummadi (2014), the ability of halotolerant strain NCYC 3413 to convert non-detoxified
xylose enriched hemicellulose hydrolysates from corn cobs to xylitol was evaluated. It was
found that this strain has the capability to grow in all hemicellulose hydrolysates and
convert xylose to xylitol without detoxification of hydrolysates. The maximum xylitol
concentration of 14.6 g L-1 was obtained from corn cobs with productivities of 0.16 and
0.20 g L-1 h-1 respectively at a yield of 0.30 g g-1. Albeit, xylitol yield and productivity

31
were moderate compared to other strains (Candida strains) with non-detoxified
hydrolysates, the strain has shown high tolerance. The salt tolerance up to 2 M NaCl, 3 M
KCl and 1 M LiCl is the remarkable property of this strain, maintained by the accumulation
of glycerol, sorbitol and other polyols under hyperosmotic conditions. It could also grow in
a broad range of pH (3- 11) and temperature (8-42°C).

2. Detoxification of Corncob Hemicellulose Hydrolysate

The main objective of detoxification of corn cob hydrolysate is to minimize the
concentration of inhibitors, which can be done in many ways. Organisms which involved
in the hemicelluloses corncob hydrolysate are highly sensitive to inhibitors. Even the
presence of less concentration of inhibitors may disrupt fermentation process.
Consequently the removal is essential for complete utilization of fermentable sugars in
corncob hydrolysate. Some of the representative methods for removal of such process in
detoxification are: (1) physical methods (2) physicochemical method (3) biological method.
But the identification such a suitable method of detoxification solely depends on the source
of biomass in each method. Even though, enormous methods have been reported for
detoxification of hemicelluloses hydrolysate, still the identification of an exact method for
corncob hydrolysate detoxification in industrial level is not identified till date.

D. Xylitol Production from Xylose

1. Catalytic Hydrogenation (Chemical Process)
Several catalysts have been employed for xylose hydrogenation which
conventionally is carried out in a three phase slurry reactor using inexpensive Raney-
Nickel (Su et al., 2013). Reaction kinetics follow a semi competitive model where pentose
sugar primarily occupies the catalyst active sites with interstitial sites available for
dissociative hydrogen adsorption. Interaction between the pentose monomer and
hydrogen governs overall reaction rate as limiting step and final product yield obtained is
∼80% of the initial xylose concentration (Baudel et al., 2005). Finally, xylitol is down
streamed from reactor and crystallized (Martinez et al., 2015, Sampaioa et al., 2006).
Chemical route for xylitol production offers high yield and conversion efficiency, but suffers

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from major drawbacks in terms of requirement of specialized and expensive equipments,
extensive intermediate purification steps, product recovery, catalyst deactivation and
recycling (complicated due to homogeneity of solution) with huge energy requirements to
make the overall process very expensive.

2. Biotechnological Production
Alternative cost effective production strategies through biotechnology route have
been envisaged by the scientific fraternities for decades and attentions were given towards
cheap source of xylose and its biocatalytic conversion into xylitol under ambient
conditions. This is a potentially attractive replacement for chemical process, as it occurs
under much milder process conditions and can be based on sugar mixtures derived from
low-cost industrial and agri-waste.

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Figure 2.9. Comparison between catalytic hydrogenation and biotechnical
production for xylitol production

CHOICE OF PROCESS

Pfizer process for Itaconic Acid Production

Pfizer process is a highly competitive fermentation process of itaconic acid by
fungi. It has a grand potential for industrial production since it is economical. Several
chemical syntheses of IA have been investigated experimentally through the years and all
of them requires commercial substrate and high cost of chemicals. Moreover, chemical
synthesis of IA produces low yields as compare to biosynthesis of IA.

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Air Lift Reactors for Fermentation
Utilization of air lift reactors were expected to lower production cost. As mentioned
in the literature, ALR provided higher production rates with less power input per volume
compared to STR. This would provide a significant difference in the whole production and
an advantage over other existing reactors which uses large amount of energy.

Bioconversion of Non-Detoxified Hemicellulose Hydrolysates to Xylitol by

Halotolerant Yeast Debaryomyces nepalensis NCYC 3413
It was found that Debaryomyces nepalensis NCYC 3413 has the capability to grow
in all hemicellulose hydrolysates and convert xylose to xylitol without detoxification of
hydrolysates. The use of this novel halotolerant strain D. nepalensis for the conversion of
various hemicellulose hydrolysates into value added compounds like xylitol and ethanol
without any pretreatment after hydrolysis yields to cheaper bioprocess. D. nepalensis is a
promising strain for ecofriendly xylitol production as it exhibits broad specificity to
lignocellulose substrates, fermentation of mixed sugars and (ii) tolerance towards
lignocellulosic inhibitors making the process more economical. The maximum xylitol
concentration of 14.6 g L-1 was obtained from corn cobs. (Gummadi, 2014).

Basis on Pretreatment of Corn Cobs

The chosen process for the pretreatment of corn cobs involves alkali and
microwave assistance. The crushed corn cobs and sodium hydroxide solution will be
subjected to a microwave reactor for 20 minutes. The combination of alkali pretreatment
and acid hydrolysis has been observed with a decrease in the unwanted constituents and
increase in the sugars produced (Chongkhong et al., 2016). This alkali pretreatment step
requires only a small ratio of catalyst to the lignocellulosic material. The effect of the
microwave is also an important factor in the optimization of the process. Microwave
irradiation promotes vibration of the water molecules and converts the motion into heat
making it a good option as an optimization for the pretreatment process of the corn cobs
(Zhu et al., 2006). It has also been employed both in organic and inorganic chemical

35
reactions showing its efficiency (Kappe, 2008). Microwave heating also decrease the
consumption of chemicals and reduce the reaction time (Yoshida et al., 2010).

Basis on Cellulose Degradation to Glucose

In the formation of glucose, the process preferred is cellulose microwave assisted
acid hydrolysis. Microwave irradiation present also in acid hydrolysis optimizes the
process similar to the alkali pretreatment process. In this section, the cellulose is heated
with 1.3% w/w sulfuric acid solution with a 900W microwave power for 16 minutes. The
microwave heating is advantageous compared to conventional heating for many reasons.
The conversion of cellulose to glucose is shortened during this process and has a higher
glucose yield compared to other hydrolysis procedures. This is a preferred alternative to
both pretreatment and hydrolysis step because of the reduced cost for different catalysts
and enzymes needed, high pressure equipment and long residence time consumed in the
other options for this process (Chongkhong et al., 2016).

Basis on Hemicellulose conversion into xylose

The hemicellulosic content of the corncobs, together with the lignin, is separated
from the cellulose after the corn cob pretreatment. This hemicellulose-lignin content is
subjected to a different acid hydrolysis step consuming a different ratio of sulfuric acid to
convert the hemicellulose to different sugars. Dilute sulfuric acid hydrolysis is preferred in
this step because this significantly reduces thermal energy consumption compared with
other treatment processes. Aside from that, sulfuric acid is commercially available, easily
to purchase and is a low-cost chemical for industrial applications. This chemical also has
a low safety and environmental concerns. This process, most importantly, ensures a
hydrolysate with high xylose concentration (Wang et al., 2010).

Basis on Cultivation of Aspergillus terreus

The optimized medium composition described by Hevekerl et al. was used. It
contained 0.8 g KH2PO4, 3 g NH4NO3, 1 g MgSO4. 7H2O, 5 g CaCl2. 2 H2O, 1.67 mg
FeCl3. 6H2O, 8 mg ZnSO4.7H2O, and 15 mg CuSO4.7H2O per L. The media components

36
are prepared separately as stock solutions. The pH value is adjusted to 3.1 with 1 M
H2SO4, except the CaCl2 solution. The temperature is maintained at 33 °C with circulating
tempered water, and the broth is stirred (hanging magnetic stirrer). The initial pH value is
adjusted to 3.1 and not corrected during the cultivation.

Basis on Itaconic Acid Production

For IA production, the yield based on the amount of glucose consumed is a very
important economic consideration because a lignocellulosic material must be utilized. The
IA yield as a function of the amount of glucose consumed is at more than 0.54 (g IA/g
glucose consumed) in spite of the increase in the initial glucose concentration, which
seems to be advantageous for the commercial production of IA. The maximum yield of
glucose in the current scientific literature is .66 g IA/g glucose (Saha, 2017).
Theoretical yield (72.21%) was based on the biosynthesis of IA by A. terreus which
is via glucolysis and the tricarboxylic acid cycle. Thus, citric acid and aconitic acid are
intermediates, and IA is formed from the latter by enzymatic decarboxylation.
• C6H12O6 + O2 yields C6H8O7 + H2O + 2H+
• C6H8O7 yields C5H6O4 + CO2 + H2O

Basis on Purification of IA
The process for the industrial production of IA from the culture broth consists of
five steps. The culture broth is filtered to remove mycelia and other suspended solids. The
filtrate of the IA culture is concentrated to 98% at 30-40C and crystallized at 15°C. The
crystallized IA is washed, dried and packaged. The IA recovery yield is 95% in the filtration
process, 98% in the concentration process, and 99% in the crystallization and drying
processes. The total IA recovery yield from cultivation to final packaging is approximately
80%. IA purified from glucose had a purity of 99.0% (Okabe, et al., 2009).

Basis on Cultivation of Debaryomyces nepalensis

Semi-synthetic medium containing xylose, 100 g/l; (NH4)2SO4, 3 g/l; MgSO4, 0.1
g/l; K2HPO4, 6 g/l; Na2HPO4, 3 g/l; yeast extract, 1 g/l; CaCl2·2H2O, 147 mg/l; FeCl3, 10

37
mg/l; MnSO4·H2O, 3.4 mg/l; ZnSO4·7H2O, 8.9 mg/l; CuSO4·5H2O, 0.25 mg/l; citric acid,
6.9 mg/l, pH, 7.0 was adjusted using H3PO4 and NaOH. Among these, the parameters are
xylose, K2HPO4, ZnSO4·7H2O. The values are optimal from Kumdal et al., 2012. All the
components are autoclaved separately and mixed subsequently. Then, it is inoculated
with 2-10% (v/v) of inoculum and incubated at 33°C.

Basis on Xylitol Production

Just like IA production, the yield based on the amount of glucose consumed is a
very important economic consideration on xylitol production since a lignocellulosic material
must be utilized. On Gummadi and Pappu’s Maximum response optimization for enhanced
xylitol production by Debaryomyces nepalensis in bioreactor, the maximum actual xylitol
yield as a function of the amount of xylose consumed is at .58 g/g.
However, in this study, the actual yield used is 68.41% since fed-batch process
was also considered. According to related literature, xylitol production in batch processes
using flasks or laboratory reactors has high initial substrate concentrations at the start of
the process, but low substrate concentrations and high product concentrations and at the
end of the process. The high xylose and xylitol in the media can inhibit xylitol production
and reduce productivity (Winkelhausen et al., (1998). Continuous culture techniques have
shown better productivity and yield in many microorganisms. For continuous cultures to
achieve the high production rate a low dilution rate is essential. But the low dilution rate
makes the process impractical due to the increase in residence time. Alternatively, in a
fed-batch process (Winkelhausen et al., 1998; Horistsu et al., 1992) a constant substrate
concentration can be maintained during the fermentation. C. boidinii NRRL Y-17231
fermentations achieved 75% theoretical xylitol yield in a fed-batch process compared to
53% theoretical yield in batch process (Winkelhausen et al., 1998). The maximum
productivity (0.46 g/l.h) in the fed-batch process was twice the maximum productivity
observed in the batch process.
Theoretical yield (91.2%) was based on the biosynthesis of xylitol by Debryomyces
nepalensis.
60 xylose+ 12ADP + 12 Pi + 12 H2O + 3 O2 yields 54 xylitol + 12 ATP + 30 CO2

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Basis on Purification of Xylitol
A process of purification of Xylitol obtained by fermentation of corn hydrolysates
was adapted from Torre, et al., 2016. After biomass removal, the media were subjected
to further processing (sequential stages of adsorption, concentration, precipitation,
concentration, and crystallization) to obtain food-grade xylitol. Liquors from the adsorption
step were concentrated by vacuum evaporation at 30-40 °C to reach a xylitol concentration
of .6968 g xylitol/ g NVC. The preferred crystallization conditions corresponded to a
temperature of 15 °C. This gives .7181g xylitol crystals/gNVC. Crystallization led to
regularly shaped, well-formed, homogeneous crystals containing 98.9 wt % of xylitol,
exceeding the purity threshold required for food applications.

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