1
Introduction
The production of several useful organic components such as lactic
acid, gluconic acid, itaconic acid and several other organic acids using microorganisms has been practiced since many years as microbes are a rich source
of these components. Among them, itaconic acid is an important organic acid
which has got very good industrial applications. The itaconic acid is very
much used in the production of several suitable compounds for the
manufacture of various polymers, which was carried out in a biological way.
Itaconic acid is an unsaturated organic acid with an empirical formula
of C5H6O4.
COOH
CH2 = C – CH2 - COOH
The polymerized methyl, ethyl or vinyl esters of itaconic acid are used
as a material in manufacture of plastics, adhesives, elastomers and coatings.
Itaconic acid is also used in emulsion paints where it improves adhesion of
the polymer. Acrylic lattices supplemented with itaconic acid are used as
non-woven fabric binders.
Small amounts of itaconic acid added to
vinylidene chloride coatings lead to improved adhesion.
2
Poly acrylonitic copolymers incorporating low levels of itaconic acid
exhibit improved dye receptivity, which results in more efficient dying and
deeper shades (Tate, 1981), a process, which is used in the textile industry.
Itaconic acid may also be used as a hardening agent in organosiloxanes for
use in contact lenses (Ellis et al., 1994).
The reaction of itaconic acid with
amines results in N-substituted pyrrolidones, which can be used as thickeners
in lubricating grease (Gordon and Coupland, 1980) and in detergents,
shampoo, pharmaceuticals and herbicides.
An imidazoline derivative has
also been claimed as an active component in shampoo (Christiansen, 1980).
In detergent industry, itaconic acid compete with fumaric and malic acid
(Smith et al., 1974).
The first method of producing itaconic acid was performed by
pyrolysing citric acid and hydrolyzing the anhydrides (Luskin, 1974) and
named the product as “Itaconic acid” as an anagram of the source. Chemical
synthesis is mainly performed by dry distillation of citric acid and subsequent
treatment of the anhydride with water or nickel carbonyls (Chiusoli, 1962).
But none of these processes are proved to be efficient and can really compete
with the production of itaconic acid by fermentation process using fungi and
therefore these are not practiced commercially (Tate, 1981).
Although Aspergillus terreus is the most frequently used commercial
producer of itaconic acid, several attempts also been made to find other
3
microorganisms that are suited for efficient production of itaconic acid.
Among the filamentous fungi, some Ustilaginales species are known to
produce itaconic acid. Mutants of Candida also are reported to produce high
amounts of Itaconic acid.
An adequate oxygen supply is essential for the microbial production
of Itaconic acid because anaerobic conditions will irreversibly damage the
biomass. And itaconic acid production is strongly affected by several media
components including Fe, Mn, Mg, Cu, Zn, P and N.
Many studies have
been conducted on the influence and regulation of these substances during
the production process (Lockwood and Reeves, 1945; Batti and Schweiger,
1963; Roehr and Kubicek, 1996).
Currently, the most productive process that
has been reported is the one used by the Pfizer Company (Nubel and Ratajak,
1964) which involves a submerged fermentation process using suspended
A. terreus biomass, by inoculating the spores on pretreated molasses.
Very few investigations were made to use immobilized biomass for
improving biomass handling and itaconic acid production. Adsorption of
A. terreus was established using a porous disk reactor system (Ju and Wang,
1986), silica based material (Kautola et al., 1985) and cubes of polyurethane
foam (Kautola et al., 1991). A new and most promising immobilization
technique is by entrapment of spores or cells in polyvinyalcohol formed as
small beads (Prucssc et al., 1998) or lens shaped particles (Jekel et al., 1998).
4
The advantages of the above-mentioned materials are very good with long
term stability and ease in handling with a low price.
Itaconic acid produced by fermentation process was offered
commercially in 1955 at a price of US$ 1.3/kg.
In 1971, Pfizer supplied two
grades of itaconic acid for US $ 0.76 and US $ 0.84/kg respectively (Luskin,
1974) whereas in 1974 refined itaconic acid was sold for US$ 1.84/kg and the
industrial grade product for US$ 0.49 kg (Lockwood, 1975). In 1979, the US
price was US$ 1.92-2.04/kg (Tate, 1981). The most recent price information is
between US$ 4/kg (Cargil, 2000) and US$ 4.3/kg (Bressler and Braun, 2000).
The potential for substitution of acrylic in polymers is very high and
increasing day by day.
Therefore the market for itaconic acid is growing
rapidly. In addition, its suitable properties for use in polymer chemistry,
pharmacy and agriculture widening the applications for itaconic acid. It was
well reported that the reactions of itaconic acid and its derivatives to
polymers have not been exhausted (Rudiger, 2000).
Nonetheless it is clear
that the expansion of itaconic acid market is the only possible way for the
further reduction of its production cost.
As several attempts are made using
the low-cost substrates for production of itaconic acid with low cost, it is quite
possible that only process cost can be lowered if we can able introduce new
and novel technologies. One promising approach is by immobilization of the
biocatalyst in biocompatible polymers, which can permit sterile process
conditions and repeated use of the biocatalysts, and at the same time better
5
protection of the cells against inhibitory effects and process disturbance.
Immobilization also may ease process handing and facilitate an efficient down
stream processing. This can enormously reduce the requirement of personnel
and may definitely reduce the process and energy cost of itaconic acid
production.
Process systems for itaconic acid production
The rate of production of itaconic acid at present does not exceed
1 g l-1 h-1, accompanied by product concentration of about 80 g l-1. By
implementing various novel approaches such as screening programmes,
immobilization techniques and genetic engineering, the productivity can be
further enhanced. Also, the use of alternative substrates may reduce the cost
of its production (Willke and Vorlop, 2001).
In addition to the choice of
strain, nutritional and culture conditions, in particular the fermentation
process (Batch and Continuous) play a major role in development of
successful process for the efficient production of itaconic acid (Willke and
Vorlop, 2001).
Substrates used in itaconic acid production
Any fermentation process requires a suitable medium containing
different nutrients for efficient culture of microorganisms. The selection and
formulation of the appropriate medium containing essential components is an
important aspect in design of a fermentation process.
6
Carbon Source
A large variety of carbohydrate materials have been used for
fermentative production of itaconic acid.
The best sources of carbon are
sugars and to a lesser extent – sugar alcohols, sucrose from cane and beet
sugar, whey containing glucose, sucrose and maltose from hydrolyzed starch
are presently used in commercial production.
Refined sugars as carbon source
In general, glucose, sucrose and xylose are preferred raw materials for
itaconic acid fermentation, which are known to be utilized efficiently by most
of the Aspergillus sp.(Kautola.H 1985; Linko.Y.Y 1985 ; Yahiro et al., 1997;
Zhaoling et al., 2000).
And Kinoshita (1931) reported first time that itaconic
acid might be formed from glucose during the fermentation with Aspergillus
itaconicus.
It is reported that some molds (Candida sp.) are also reported to
utilize glucose efficiently (Tabuchi and Nakahara 1980; Hashimoto et al.,
1989).
However, the yield obtained using these species are much lower in
concentration when compared to the Aspergillus sp. Enhanced and efficient
production of itaconic acid was reported by Feng et al. (2000) by batch culture
of Aspergillus terreus in a 200-L air-lift reactor (ALR) with internal loop and a
10-L stirred tank reactor (STR). In both ALR and STR, the high itaconic acid
concentrations of
85.1 and 80.1 g/lt were obtained, and when glucose was
used, the yield of itaconic acid was 56.7% and 50% respectively.
The
fermentation kinetic models including growth of cells, consumption of
7
glucose, and formation of itaconic acid were also established. Based on the
experimental data, the model parameters were determined and the kinetic
mechanism of itaconic acid formation was analyzed. Siru (1997) studied the
production of itaconic acid by fermentation with glucose and cane sugar as
carbon source. The influence of reaction parameters (Air flow rate, nozzle
diameter, concentration of carbon source, etc.) of an air-lift bioreactor on the
yield of itaconic acid were determined, and empirical equations for oxygen
dissolution with and without sieve screen were also obtained through
regression. The results envisaged that by using air-lift bioreactor, the itaconic
acid concentration was increased by 14%, the yield by 16%, and the
productivity by 38% as compared with those by using stirred tank.
Willke and Vorlop (1998) investigated the production of itaconic acid
using various raw materials by batch and continuous fermentation with
Aspergillus terreus NRRL 1963.
The various fermentation conditions were
optimized and used sucrose as carbon source. It was observed that the cell
growth was prevented by phosphate limitation.
An yield of 60 g itaconic
acid/lt was achieved. Different species of Ustilago sp. including Ustilago
maydis, produced 53 g itaconic acid/1 within 5 days using glucose (Tabuchi
and Nakahara, 1980; Tabuchi, 1998). Since growing filamentous fungi may
cause some problems in bioreactors, yeast was also tested for itaconic acid
production.
It was reported that a Candida mutant produced up to 42 g
itaconic acid/1 after 5 days (Hashimoto et al., 1989).
Basing on these studies,
8
it was clearly evident that high production of itaconic acid can be obtained by
developing or using new species by mutagenesis process.
It was reported
that in Japan, a mutant strain of Aspergillus terreus, produced 82 g itaconic
acid/1 after 7 days of fermentation (Yahiro et al., 1997).
Complex carbohydrates as raw material
The complex carbohydrates used in fermentative production of itaconic
acid mainly include starch. In general the pretreatment of raw materials is a
requisite for all the organisms to nourish their energy, for example during the
process of utilization of starch; it is first converted into itaconic acid. In view
of this, the species possessing appreciable levels of starch hydrolytic enzymes
are preferred for commercial production of itaconic acid when starch is used
as a substrate.
The commonly used starchy materials for commercial
production of itaconic acid are corn, potato, apple, banana, sorghum.
A concentration of 60 g/lt of itaconic acid were achieved after 6 days of
fermentation at 121o C, pH 2.0 with a conversion efficiency of 56.7%. The use
of Aspergillus terreus containing high levels of amylolytic enzymes for the
direct production of itaconic acid from corn starch was reported by Yahiro et
al., (1997). Reddy and Singh (2002) investigated the metabolizing capacity of
Aspergillus terreus using apple and banana flavor and reported that it would
convert into itaconic acid and a concentration of 20 g/lt was obtained.
Petruceioli et al., (1999) reported that a number of Aspergillus sp. could
convert potato starch by treating the substrate with α-amylase and
9
glucoamylase to glucose. A concentration of 1.4 g/lt with conversion
efficiency of 36% would be achieved at the end of 6 days of fermentation
under the operating conditions of temperature 35oC and pH 3.4.
A yield of 31% and 3.3 g/lt concentration of itaconic acid was obtained
at the end of 6 days fermentation at 35oC temperature and pH 3.4 using
Aspergillus terreus (Federici et al., 1999).
Studies performed using starchy
materials such as corn starch, soft wheat flour, potato flour, cassava flour,
sorghum starch, sweet potato and industrial potato flours (Either acid or
enzymatically hydrolyzed) as substrates for itaconic acid production by
Aspergillus terreus NRRL 1960 has given encouraging results (Federici et al.,
1999).
Among all, both the production and yield were found to be highest
with corn starch (18.4 g/lt and 34.0%). In the fermentative production of
organic acids, high degree of starch saccarification is not always necessary, a
good amount of fumaric acid production and yield were reported using
starch-based materials.
Itaconic acid production using Molasses
Molasses, a by-product of sugar industry is a very convenient raw
material for itaconic acid production. Aspergillus terreus is one of the patented
microorganisms reported to utilize molasses as a good source of carbon (Kane
et al., 1945).
Many studies have been conducted on the influence and
regulation by this substance during the production process (Lockwood and
Reeves, 1945; Batti and Schweiger, 1963; Roehr and Kubicek 1996). Currently,
10
the most productive process that has been reported is the one being used by
the Pfizer Company (Nubel and Ratajak, 1964) which is involved with
submerged fermentation process using suspended A. terreus biomass
inoculated as spores on pretreated molasses.
During the initial growth
phase the original pH (5.0) was reported to drop down to less than 3.
The
second phase is characterized by phosphate-limited growth and increased
production of itaconic acid, which is substantially free from other organic
acids.
The cane molasses and cane molasses stillage are rich in aconitic and
itaconic acids.
This fermentation process, regardless of the sugar source,
produces a number of additional co-products such as carbon dioxide,
glycerin, succinic acid and their production rate can be manipulated by the
choice of the strain and operating conditions. In addition, both beet and cane
molasses are a rich source of potassium sulfate which is the main component
of molasses ash. The technology is now available for commercialization to
recover these compounds.
Itaconic acid biosynthesis
Many theories were proposed regarding the biosynthesis of itaconic
acid using fungi (Kinoshita, 1932; Eimhjellen and Larsen, 1955; Shimi et al.,
1962; Jakubowska, 1977), however according to Kinoshita (1932), the main
route of production is via glycolysis and tricarboxylic acid cycle (Bentley and
Thiessen, 1957; Winskill, 1983; Bonnarme et al., 1995). Thus citric acid and
11
aconitic acid are intermediates of this process and itaconic acid is formed from
the later by enzymatic decarboxylation (Ducrocq et al., 1995).
Itaconate biosynthesis was well studied in intact cells of high-yielding
(RC4’) and low-yielding (CM85J) species of the fungus Aspergillus terreus by
different methods that did not interfere with metabolism. Itaconate formation
using RC4’ strain requires de novo protein biosynthesis. And the Krebs cycle
intermediates were increased in both the species during the production of
itaconic acid.
The Embden-Meyerhof-Parnas pathway and the Krebs cycle
were shown to be involved in the biosynthesis using
14C-
and
13C-
labeled
substrates and nuclear magnetic resonance spectroscopy.
It is well established that glucose is broken down via the EMP pathway
during itaconate synthesis but not via the PP pathway.
This is important
since up to 16.7% of the glucose flux can be diverted to CO2 if the PP pathway
is operating alone (Bonnarme et al., 1995).
Sugars such as glucose and
sucrose are generally used in industrial production processes and several
such biosynthetic pathways leading to itaconic acid accumulation have been
postulated (Nubel et al., 1962; Cros, 1988).
12
Fig. (A). Biosynthesis of Itaconic acid
13
Fig.(B) Metabolic pathway of itaconic acid biosynthesis
14
Optimization of culture parameters
For obtaining efficient production of biomass, it is necessary to
optimize the various culture conditions and parameters during fermentation
process. In general, the optimization of fermentation media includes culture
as well as operational parameters and optimization of environmental
conditions, which can affect the growth and production of metabolites, thus it
is very important for process development.
Influence of nutrients
Microorganisms can very utilize the nitrogen either from organic or
inorganic source.
Ammonia and salts of ammonia (for eg. Ammonium
sulphate or nitrate) are common inorganic source of nitrogen salts, which
provide both the acidic and basic environments, depending upon the type of
the salt.
And amino acids, proteins or urea serve as organic sources of
nitrogen.
Microorganisms are able to grow fast in the presence of organic
nitrogen, and some of them will have an absolute need for amino acids and
usage of pure amino acids is often expensive. The influence of ammonium
phosphate and ammonium sulfate on the growth of A. terreus and production
of itaconic acid was well studied and envisaged that inorganic nitrogen
source have little influence on the growth of A. terreus (Kazutoyo et al., 1997).
Willke et al., (1998) investigated the production of itaconic acid using various
raw materials by batch and continuous fermentation using A. terreus NRRL
1960. The fermentation conditions were optimized using sucrose as the most
15
suitable C source. Cell growth was prevented by phosphate limitation and an
yield of 60 g itaconic acid/lt was achieved. Willke and Vorlop(2001) reported
that itaconic acid can easily be incorporated into polymers and may serve as a
substitute for petrochemical-based acrylic or methacrylic acid. The use of
itaconic acid has several limitations and restrictions because of its high price
(US$4/kg).
The attempts for high production and reducing the cost of
production using immobilization techniques, screening programs and
implementing genetic engineering techniques may solve this problem to a
considerable extent. The use of alternative and low cost substrates may help
in reducing the cost and provide an open market for new and increased
applications.
Kautola et al., (1991) investigated the production of itaconic
acid by immobilized A. terreus TKK 200-5-1 both using shake flask culture and
continuous column bioreactors. The influence of glucose, ammonium nitrate
and pH was studied, a high production was observed using 150 g/lt and pH
3.75 during the absence of ammonium nitrate.
Effect of pH
The pH of the medium can affect the growth and product formation by
influencing the uptake of nutrients and other physiological activities.
The
optimal pH for growth and production may vary depending on the species
and strain (Greasham, 1993).
The influence of various fermentation
conditions on the production of itaconic acid by Aspergillus terreus was
studied previously varying the different parameters (Riscaldatim et al., 2000).
16
It was reported that the production of itaconic acid using glucose based media
by A. terreus NRRL 1960 was found to be controlled by stirring rate and
phosphorous (P) level in the production medium when reduced to less than
10 mg l-1 and it was found that the fungal mycelium exhausted its primary
growth and started excreting itaconic acid and continued its secondary
growth at the expense of ammonical nitrogen (Petruceioli et al., 1999).
Further it is reported that a substantial accumulation of itaconic acid can be
obtained under acidic conditions, at a pH of below 6.0 using the cultures of
Aspergillus terreus, which is an optimum production rate (Lockwood and
Reeves, 1945; Moyer and Coghill, 1945; Nelson et al., 1946). The
fermentation pattern of A. terreus is different at pH 2.1 and 6.0.
At pH 2.1
practically all the glucose carbon fermentation accounted for as itaconic acid,
carbon dioxide and mycelium. At pH 6.0 no itaconic acid accumulated and
the glucose carbon might have been converted into the succinic and fumaric
acids, carbon dioxide and mycelium. The non-proliferating mycelia resulted
at pH 6.0 do not accumulate itaconic acid and glucose either at pH 6.0.
However mycelia grown at pH 6.0 and then transferred to a growth medium
of pH 2.1 will gradually develop stability to produce itaconic acid Eimhjellen
and Larsen 1955.
17
Effect of Temperature
Delbrueck first reported the requirement of high temperatures for
itaconic acid fermentation and temperature play a major role in itaconic acid
production (Friedkin 1945).
In general, the optimum temperature of
mesophiles is reported to be between 28-45oC and for thermopiles in the
range of 45-62oC (Kascak et al., 1986).
Tuli et al., (1985) studied the effect of
temperature on itaconic acid production using A. terreus cells and observed
no significant influence between 40-45oC for A. terreus for itaconic acid
production. Kazutoyo et al., 1997 reported an optimum temperature of 37oC
for growth and product formation by A. terreus.
Effect of inoculum size
Very little information is available on the influence of inoculum in
itaconic acid production. For efficient production of itaconic acid Hashimoto
et al., (1989) and Tabuchi (1981) used 15% inoculum of Candida sp. Kawamura
et al., (1981) observed that the increasing inoculum size (15-20% w/v) did not
increased itaconic acid production. In general, 5-10% of inoculum size has
been used in many studies for itaconic acid production.
Immobilized production of itaconic acid
As itaconic acid is an important intermediate for the manufacturing of
polyester resins or other polymeric materials as well as N-substituted
pyrrolidones (Milson and Meers, 1985), the high production of itaconic acid
18
using fungi was first attempted by Kinoshita (Kinoshita, 1931). This important
organic acid is known to be produced by many fungal species like, A. niger,
A. itaconicus, A. terreus, Ustilago zeae (Miall, 1978) and by yeasts such as
Candida sp. (Kawamura, 1981). The successful immobilized production of
itaconic acid from glucose by A. terreus immobilized on polyacrylamide or
polyurethane gels was demonstrated by Horitsu et al., (1983) and Ju.N and
Wang (1986) using porous disks as carrier for A. terreus.
The A. terreus
NRRC 1960 spores were entrapped in calcium alginate gel beads and celite R626 or in agar gel cubes and were employed both in repeated batch and
continuous column reactors to produce itaconic acid from D-xylose or Dglucose (Kawamura, 1981; Kautola et al., 1985; Welter et al., 2000; Iqbal and
Saeed, 2005).
The high itaconic acid yield was obtained in submerged culture batch
fermentation (54.5%) with a initial glucose concentration of 55 g/lt and
volumetric productivity 0.20 g/lt/h and 44.8% yield using xylose (6.7 g/lt)
with a volumetric productivity of 0.20 g/lt/h (Kautola and Linko, 1985). In
repeated batch fermentation with mycelium grown from spores immobilized
in calcium alginate gel 0.06 g/lt/h from xylose, yield was 60 g/lt. The results
further indicate that celite R-626 is a best immobilized biocatalyst system as a
carrier with volumetric productivities of 1.2 g/lt/h from glucose and 0.56
g/lt/h from xylose in continuous column operation for more than 2 weeks
(Linko and Vassilev, 1989).
19
Fermentative production of Itaconic acid
Batch processes
On commercial scale, itaconic acid is manufactured by batch
fermentation. Feng et al., (2000) demonstrated the itaconic acid production by
batch culture of Aspergillus terreus in a 200-L air-lift reactor (ALR) with
internal loop and a 10-L stirred tank reactor (STR).
In ALR and STR, the
highest concentrations were to be 85.1 and 80.1 g/lt with a yield of 56.7 and
50% respectively using glucose as substrate.
The fermentation kinetics
including growth of cells, consumption of glucose and formation of itaconic
acid were also established.
Siru.Jia (1997) established the manufacturing of
itaconic acid by fermentation with glucose and cane sugar as carbon source.
The influence of reaction parameters such as air flow rate, nozzle diameter,
concentration of carbon source, etc. using an air-lift bioreactor on the yield of
itaconic acid were also studied and empirical equations for oxygen
dissolution with and without sieve screen were calculated through regression
equation. The results envisaged that using air-lift bioreactor, the itaconic acid
concentration was increased by 14%, yield by 16%, and the productivity by
38% as compared with those obtained using stirred tank reactor.
Yahiro et al., (1997) investigated the production of itaconic acid using
2-L STR, obtained a maximum production of itaconic acid 0.48 g/h with an
20
agitation rate of 400 rpm and an aeration rate of 0.5 vv/m3. In an air-lift
reactor, the maximum production rate was 0.64 g/lt at an O2 supply rate of
0.41 L O2/lt/minute with power input per unit volume. This has yielded the
maximum production rates for STR and ALR to be 1180 and 542 w/m3
respectively.
This clearly indicates that ALR would be a suitable bioreactor
for a large scale production of itaconic acid. Siru.Jia (1997) explained the
influence of oxygen carrier in the aerobic manufacture of itaconic acid with
A. terreus. Cane molasses are rich in aconitic and itaconic acid. The alcoholic
fermentation, regardless of the sugar source, produces a number of additional
co products such as carbon dioxide, glycerin, succinic acid, etc. and their
production rate can be manipulated by judicial choice of the yeast strain and
operating conditions. In addition both beet and cane molasses are a rich
sources of potassium sulfate which is the main component of molasses.
Federici et al., (1999) stated that acid or enzymatically starchy materials such
as corn starch, soft wheat flour, potato flour, cassava flour, sorghum starch,
sweet potato and industrial potato flours used as substrate has yielded very
encouraging results for itaconic acid production by Aspergillus terreus NRRL
1960.
And Petruceioli et al., (1999) reported that when the phosphorous level
in the medium was reduced to less than 10 mg l-1, the fungal mycelium
exhausted its primary growth and started to excrete itaconic acid, while it
continued its secondary growth at the expense of ammoniacal nitrogen.
In
the batch process performed by Pfizer, after raising the pH to 3.8 with lime,
21
by using150 g of sugar, a concentration of 71 g of itaconic acid per liter within
4 days, corresponding to a productivity of 1 g/lt-hr.
Also a high product
concentration of 75, 87 and 70 g/lt under comparable conditions but only at
significantly lower productivities of 0.66, 0.66 and 0.44 g/lt-hr were reported
(Batti and Schweiger, 1963; Willke et al., 2001; Shu-Fang, 2002; Reddy and
Singh, 2002).
Continuous Processes
The present day research has been directed towards improving the
productivities by continuous process of fermentation which overcomes the
problems of end product inhibition by continuous replacement of the growth
medium. In continuous packed bed column fermentation, the substrate, feed
and aerations were introduced from the bottom of the water jacketed 130 ml
glass columns. The productivity was much higher with the celite-immobilized
mycelium than using with polyurethane foam as the carrier (Kautola, 1987).
The productivity was about 1.2 g/lt/h, almost 4 times more than that
obtained in conventional batch fermentation with free mycelium and equal to
or better than that obtained by (Horitsu et al., 1983) with polyacrylamide gel
entrapped mycelium.
Kautola (2000) have stated that a significant poor
production was obtained when both glucose and xylose when ammonium
sulphate was used as the nitrogen source.
The continuous process using
suspended biomass allow only low yields ranging between 12-16% (molar),
22
accompanied by low product concentrations of 15 and 8 g/lt and
productivities of 0.48 and 0.32 g/lt-h (Kobayashi and Nakamura, 1966).
Significance of work
Itaconic acid has got high potential for use in various industrial
applications particularly in chemical industry during the manufacture of high
molecular thermo plastics, alkyl resins, ion exchange resins and for the
production of transparent materials, high strength glass fibers and as an
intermediate for chemical synthesis of synthetic fibers. By considering the
potential importance of itaconic acid in industries, the present problem has
been designed for producing high yield of itaconic acid using various
Aspergillus species through submerged fermentation processes. And the
present investigation has been designed with the following objectives.

Selection of suitable organism as a source of itaconic acid
production.

Optimization of various physio-chemical parameters for obtaining
maximal yields of itaconic acid.

To achieve high yield of itaconic acid through solid and submerged
fermentation using selected microbes like Aspergillus sp. (free and
immobilized cells).

To design a suitable reactor for obtaining high yield of itaconic acid
for commercial application.
23
Materials and Methods
Source of microbial species
The microbial species used in this study were procured from Microbial
Type Culture Collection (MTCC), Institute of Microbial Technology
(IMTECH), Chandigarh, India.
1. Aspergillus niger
–
2. Aspergillus terreus
3. Aspergillus nidulans
–
MTCC NO. 479
–
4. Aspergillus flavus
MTCC NO.872
MTCC NO.818
–
MTCC NO.871
Culture media
Czapek Dox Medium
Czapek Dox medium was used for cultivation of all the Aspergillus
species and the medium was prepared with the following composition:
Name of the ingredient
Sucrose
Quantity (g/l)
3
Sodium nitrate
0.2
Magnesium sulphate
0.05
Potassium chloride
0.05
Ferrous sulphate
0.001
Dipotassium hydrogen
phosphate
0.1
pH
7.3
24
Production medium
Production medium was used for cultivation of Aspergillus species.
The medium was prepared as per given composition:
Name of the ingredient
Quantity
Molasses
10% (V/V)
NH4Cl
0.25% (W/V)
Mg SO4
0.095% (W/V)
KH2 PO4
0.0088% (W/V)
Cu SO4
0.0004% (W/V)
pH
5.0
Estimation of sucrose
Modified Dinitro Salicylic acid (DNS) method
Unlike other carbohydrates, sucrose is the only non-reducing common
disaccharide.
Most of the tests for sugar detection, including Benedict's
solution, Fehling's solution and DNS (3, 5-dinitrosalicylic acid) solution, result
in negative readings for sucrose.
However, these methods can still be
applied if sucrose is first hydrolyzed in an acid solution to yield glucose and
fructose. This method is a straight forward modification of the original DNS
method for glucose analysis, (Miller G.L., 1959).
25
Reagents
Dinitrosalicylic Acid Reagent Solution
- 1 ml
Dinitrosalicylic acid
- 10 g
Phenol
-2g
Sodium sulfite
- 0.5 g
Sodium hydroxide
- 10 g
Potassium sodium tartrate solution
- 40 ml
Concentrated hydrochloric acid
-11.9 N solution
Potassium hydroxide
- 5 N solution
All the reagents were dissolved in one litre of double distilled water.
Procedure
20 µl of concentrated HCl solution was added to 1 ml of the sucrose solution
and allowed for hydrolysis for 5 minutes 90ºC.
1) 0.05 ml of the 5 N KOH solutions was added to neutralize the acid,
because the DNS method must be applied in an alkaline condition to
develop the red brown color, which represents the presence of reducing
sugars.
2) DNS reagent was added and followed the DNS method henceforth.
3) The sucrose concentration was determined by measuring the absorbance
at 620 nm.
26
Determination of Growth curve
For identification of maximal production phase, the four selected
organisms were grown in their respective growth media. The itaconic acid
production rates for the four species were determined at different growth
phases as per the following procedure.
a) The cultures were inoculated in their respective growth media.
b) The Aspergillus species were propagated at room temperature.
c) The growth of the organisms was determined at regular time intervals
by measuring the absorbance at 385 nm while the medium without the
culture served as reference.
d) At the same regular intervals the rates of the itaconic acid production
for the four selected species were determined.
Itaconic acid production
The itaconic acid production was determined by rapid
spectrophotometric method (Hartford, 1962).
Itaconic acid
1.0 ml
Pyridine
1.3 ml
Acetic anhydride
5.7 ml
27
a) The grownup culture was centrifuged at 6,000 rpm for 15 minutes. 1.0
ml of the supernatant was taken in to a test tube and to this 1.3 ml of
pyridine was added.
b) To the above solution 5.7 ml of acetic anhydride was added and test
tube was placed in a water bath at 32oC.
c) The test tubes were observed for color development and this color
remains relatively constant for 15 minutes.
e) The absorbance was measured at 385 nm. The amount of itaconic acid
produced was determined using standard itaconic acid graph
Calculations
Standard Curve
▼A385nm standard = A385nm standard – A385nm Blank
A standard curve was prepared by plotting the ▼A385nm of the
standard Vs itaconic acid concentration.
Optimization of media
The different nutritional parameters that influence the itaconic acid
production were studied.
For this the crude medium was chosen and the
experiment was carried out as per the following method:
a) Different carbon sources such as molasses, potato peel, banana peel
and rice bran were prepared and supplemented to the growth medium
in the concentration of 5 g/100 ml.
28
b) All the four selected species of Aspergillus were found to grow in 3 to
10% of molasses under controlled conditions.
c) The effect of incubation time (0–144 h) on the itaconic acid production
using Aspergillus niger, Aspergillus terreus, Aspergillus nidulans and
Aspergillus flavus was determined.
d) The fermentation medium was adjusted to pH 3.0, 3.5, 4.0, 5.0, 6.0 and
7.0 to determine the level of itaconic acid production by Aspergillus
species.
e) Nitrogen sources such as urea and ammonium chloride were prepared
and supplemented at a concentration of 0.2 to 0.4% (W/V).
f) The cultures were incubated at their respective temperatures until their
maximum activity phase.
g) All the four selected species of Aspergillus were grown in the range of
150 to 250 rpm under controlled conditions.
h) All the tubes with the supplemented source were inoculated with 2%,
5% and 10% of freshly grown cultures Aspergillus niger, Aspergillus
terreus, Aspergillus nidulans and Aspergillus flavus.
i) The itaconic acid production was determined by the rapid
spectrophoto-metric method.
29
Fermentation studies
For fermentation studies, shake flask Bioreactor (100 ml) and New
Brunswick Scientific, USA Fermentor (2 L), Model BioFlo 2000 were used for
production of itaconic acid.
Immobilization studies
The influence of immobilization on itaconic acid production was also
studied.
For this the crude medium was chosen and the experiment was
carried out as per the following procedure:
a) The Polyurethane foam having an average pore size of 1.5-1.7 mm was
used for immobilization.
a) One cm cubes were autoclaved by submerging in growth medium for
sterilization.
b) Each sterilized 250 ml Erlenmeyer flask containing about 0.5 and 1.0 g
of carrier cubes was inoculated with 8-day-old Aspergillus terreus
spores grown on a Czapek-agar slant.
c) After 3 days the immobilized mycelium was washed with sterile
distilled water and transferred to the production medium.
d) The cultures were incubated at their respective temperatures until their
maximum activity phase.
e) The itaconic acid production was determined by the standard
spectrophotometric method.
30
Statistical Analysis
Statistical analysis was done for all the experimental data by means of
ANOVA. In this test, if the calculated value is less than tabulated value (FCritical value at 5% level of significance) then the assumption that all the
columns in these tables are equal, other wise accept that all the columns in
these tables are not equal. From this analysis for all the experimental data sets,
it was observed that F cal. > F crit. Hence these factors in all columns are not
same, that means all the process parameters depend on time, thus indicates
the significance of the observed results in the present study.
Regression Analysis
Using Least Square method of analysis derived the mathematical
relationship
between
itaconic
acid
concentration,
time,
substrate
concentration and biomass concentration. The second-degree polynomial was
assumed for this analysis indicating the significance of the results.
31
Aspergillus niger
Aspergillus terreus
Aspergillus nidulans
Aspergillus flavus
Fig. (C) .Photographs of Aspergillus Species