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TITLE OF THE DISSERTATION

PRODUCTION OF CITRIC ACID BY ASPERGILLUS NIGER GROWN IN PUMPKIN AS SUBSTRATES BY

SOLID- STATE FERMENTATION

Submitted in the partial fulfillment

for the award of degree in

MASTER OF SCIENCE
TO

UNIVERSITY OF SCIENCE AND TECHNOLOGY

MEGHALAYA

Submitted by

MISS NITU DEVI (

Under the guidance of

DR.

Department of Botany
University of science and technology, Megahalaya
2019-2021
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CERTIFICATE

This is to certify that this dissertation entitled “Production of Citric acid by Aspergillus niger
grown in pumpkin as substrates by solid state fermentation” submitted in partial fulfillment, for
the award of degree in Master of science degree of the University of science and technology,
Meghalaya which is the result of the bonafide. Research work carried out by Miss Nitu Devi.
I find the work complete, comprehensive and of sufficient high standard to warrant its presentation
for the examination. I further certify that the work has been carried out under my guidance and has
not been submitted earlier to any other university for the Degree or Diploma.
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DECLARATION

I Nitu Devi (Regd No: ) do hereby declare that the project report submitted to the
University of science and technology, Meghalaya in partial fulfillment for the award of degree
in Master of Science in Botany entitled “Production of Citric acid by Aspergillus niger
grown in pumpkin as substrates by solid state fermentation”, is a research work carried out
by me under the guidance and supervision of Dr.
I further declare that the information has been collected from genuine & authentic
sources and I have not submitted this project report to this or any other university for award if
diploma or degree of certificate examination.

Date: / 07 / 2021 Name of student

Nitu Devi
Place: USTM, Meghalaya
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Acknowledgement

I wish to express my heartfelt gratitude to the all the people who have played a crucial
role in the research for this project, without their active cooperation the preparation of this
project could not have been completed within the specified time limit.

I am thankful to our respected Director, Dr. , for motivating me to complete this project with
complete focus and attention.

I am also thankful to my project guide, Dr. who supported me throughout this project with
utmost cooperation and patience and for helping me in doing this project.

I also acknowledge with a deep sense of reverence, my gratitude towards my parents and
members of my family, who has always supported me morally as well as economically.
At last but not the least gratitude goes to all my friends who directly or indirectly helped me to
complete this project report. Above all my genuine gratitude and gratefulness to the Almighty
God for having bestowed me to complete this work.

CONTENTS
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SERIAL NUMBERS CONTENTS PAGE NUMBER

1. Introduction
2. Review of literature
3. Materials and methods
4. Results and discussion

5. Summary and Conclusion

6. References

LIST OF TABLES

1. Introduction

2. Review of literature
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3. Materials and method

3.1
3.2.1
3.2.2
3.3
3.3.1
3.3.2
3.4
3.4.1

4. Results and discussion

4.1 Observations
4.2.1
4.2.2
4.3.4

5. Summary and conclusion

6. References

LIST OF ABBREVIATIONS
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INTRODUCTION
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INTRODUCTION

Citric acid (2-hydroxy-propane-1, 2, 3-tricarboxylic acid) is a weak organic acid, available in two
crystalline forms: anhydrous and monohydrate with 192 and 210 g/mol molecular weight respectively. Its
traces are found in virtually all plants and animals [1]. Citric acid can be produced mechanically, chemically
and through fermentation. Fermentation technology has established its importance in processing wastes for the
production of various vital compounds e.g. enzymes, biomass, organic acids etc [2]. Food processing
and agro industrial wastes can be utilized for producing citric acid through fermentation [3].

Food industry uses about 70% of citric acid’s total production as a pH adjuster, flavor enhancer, preservative in
processed food and as an acidulant in drinks. Pharmaceutical industry consumes 12% of citric acid to
enhance flavors of medicines, as an acidulant and anti coagulator. Cosmetic industry uses citric acid
in the composition of different cosmetic products. Citric acid has also found other application such as
metal cleaning, electroplating, fabric dyeing, detergent etc.

Organic acids such as citric, acetic, tartaric, malic, lactic and gluconic acid have being utilized in
food industries for preventing food deterioration and extending shelf life of food ingredients.
Most commonly used organic acid is citric acid. Citric acid (2-hydroxy-propane-1,2,3-
tricarboxylic acid) is a weak organic acid found in citrus fruits. About 70% of total Citric acid
produced is consumed by food industry, 12% by pharmaceutical industries and rest of 18% by
other industries. It is being used in food industry to add an acidic (sour) taste to foods, soft drinks
9|Page

and other food products as jams, jellies, candies, water ice and wines. It has applications such as
acidulation, antioxidation, flavor enhancement, preservation, plasticizer and synergistic agent .

Citric acid has been produced at a commercial scale using microorganisms. Screening of microbes, selection of
suitable substrate and optimization of operating variables is crucial for citric acid production. Aspergillus niger
is highly recommended for industrial production of citric acid due to its property of citric acid
accumulation because it produces certain enzymes that help accumulate large quantities of citric acid [4]. It has
been found to produce high yields of citric acid by utilizing a variety of substrates. Previously researchers
used waste of many fruits such as pineapple waste, orange pulp, orange peel sweet lime pulp,
sweet lime peel, banana peel etc. but there is no report of using pumpkin as substrate for citric acid
production. The present study focuses on the utilization of pumpkin as substrate for the production of citric acid
and optimization of fermentation parameters.

Several microbes belonging to genia Aspergillus sp.,Bacillus sp., Penicillium sp., Candida sp.,

Arthrobacter sp., Corynebacterium sp., have been reported as efficient citric acid producers.
Fungus Aspergillus niger has received much attention recently as a potential candidate for large-
scale production of citric acid. The rapid growth rate, ability to utilize wide substrates as carbon
and nitrogen source, high yield and secretion of different metabolites enhanced the application
of Aspergillus niger in fermentation studies [7].
Aspergillus niger is most commonly used for citric acid production [3]. This is because of the
fact that this organism has capacity to utilize varieties of substrates due to its well-developed
enzymatic system [4]. A niger is normally a haploid fungus producing white septate hypha
which is profusely branched. It produces black mass of conidia, which are found in chain
arising from the secondary sterigmata. Citric acid is mainly produced by a fungus Aspergilus
niger by utilizing starchy and sugar substrates [5].

Industrial production of this chemical by fermentation using cheap raw materials will be
helpful in economic development of our country. Keeping in view the future requirements and
also the availability of cheap raw material, best to develop the process for citric acid
fermentation, based on our local discarded starchy substrate such as molasses from sugar mills.
In addition, Pumpkin and other cheap starchy raw materials can also be exploited for citric acid
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production, which will have some cost effective impact on our economy [6]. If citric acid is to
be produced in commercial level, a suitable substrate must be looked for with a view to select
the substrate, some aspect of citric acid production by microbial fermentation, using a number
of indigenous raw materials, have been carried out in present study.

Solid state fermentation (SSF) is a fermentation process which is carried out in solid matrix with
limited volume of water. Microorganisms especially fungi, can favourably grow in the solid
substrate as a carbon source and growth carrier. Solid state fermentation is economical, low
water consuming and eco-friendly. It plays an important role and has a great perspective
for bioconversion of different agro-industrial residues [7]. Solid-state fermentations refer to the
cultivation of microorganisms in a low-water-activity environment on a non-soluble material
acting as both nutrient source and physical support. The SSF is alternative to submerged
fermentation for production of value added products like antibiotics, single cell protein, poly
unsaturated fatty acids, enzymes, organic acids, etc.

The concept of using solid substrates is probably the oldest method used by man to make
microorganisms work for him. Organic wastes are comprised of materials rich in sugars,
minerals, and proteins that can be used for the cultivation of microorganisms. Organic waste is a
good cultivator to provide the appropriate conditions for the development of microorganisms as
they require carbon, nutrient, and moisture for the development [8]. In solid state fermentation,
the substrate itself acts as a carbon source and occurs in absence or near absence of free water by
employing a natural substrate or inert substrate as a solid substrate. The aim of SSF is to bring
cultivated fungi or bacteria in tight contact with the insoluble substrate and to achieve the highest
nutrient concentration from the substrate for fermentation. In pure culture SSF, individual strains
are used for substrate utilization and with mixed culture. Different microorganisms are utilized
for the bioconversion of agro-industrial residues simultaneously. Depending on the type of
organic waste, SSF can be applied with the aim of producing different valuable bio-products [9].

The main advantage of solid-state fermentation is its superior yield and the ability to utilize
inexpensive and widely available agro-industrial residues as substrates for bio-production,
making it more environmentally friendly than submerged fermentation. It requires less water and
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has lower operating costs, and does not require complex equipment. There is no need for
pretreatment as the system is less sensitive to the presence of trace elements compared to
submerged fermentation [10].

Citric acid can also be produced by purely chemical reactions. Citric acid was first synthesized
chemically in 1880 by Grimaux and Adam, using glycerol as a starting material, but this method
was not economically competitive enough compared to other production routes such as
fermentation. Therefore, microbial fermentation became the choice for commercial citric acid
fermentation as it was more successful than chemical means. Citric acid is multifunctional and
vital in many industries. Its vast and extensive range of versatile applications in various
industries since the beginning of the twentieth century has created a continuous high demand.
The worldwide production of citric acid in 2007 was 1.6 million tonnes, with an annual increase
in demand and consumption of 3.5–4.0% [11].

Consumption of citric acid is expected to multiply owing to its low prices and numerous
applications. The competitive pricing of citric acid is mainly caused by suppliers from China
being willing to sell it at the lowest price possible, and this phenomenon has led to extremely
tough competition for European suppliers [12]. ADM and Tate & Lyle have had to shut down
their citric acid plants in Ireland and the UK owing to fierce competition from China. In 2012,
China was responsible for 59% of the global production of citric acid. According to estimations,
the market value of citric acid will continue to grow and will soon exceed $2 [13].

The TCA cycle plays an important role in generating high-energy fuel(ATP) or producing
intermediates such as organic acids which can be used for various purposes in the cell through
catabolic or metabolic process. The production of high-energy fuel is depicted in which
represents the complete oxidation of glucose to carbon dioxide and water [14] .When the cell
needs high energy for cell propagation or maintenance, it initiates cellular respiration processes
including glycolysis ,the TCA cycle and electron transport/oxidative phosphorylation harnesses
all three processes for more energy generation.

Based on market trends, it is apparent that there will be a surge in the global citric acid demand.
Hence, it is crucial to optimize citric acid production by looking for alternatives that are more
economical, more environmentally friendly and have a higher production yield than current
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methods. In light of this, this review discusses the biochemistry of citric acid production, raw
material choices, selection of citric-acid producing microorganisms, production methods and
strategies, the effects of various fermentation conditions and recovery options. The aim is to
provide a thorough and comprehensive review, compared to other reviews that focus on specific
areas [15].

OBJECTIVES:

1. Isolation and selection of efficient citric acid producing Fungi.

2. Screening of the isolates for production of Citric acid.
3. Improving efficiency of Citric acid production through optimization of fermentation
conditions such as media composition, pH and incubation temperature.
4. To study the production of Citric acid through used of pumpkin by the method of solid
state fermentation.
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REVIEW OF
LITERATURE
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REVIEW OF LITERATURE

An intermediate in the tricarboxylic acid (TCA) cycle, citric acid is an importantcommercial

product with global production reaching 736,000 tons/yr. Furthermore, it is produced almost
through the submerged fermentation of the white rot fungus. Citric acid is widely used in the
food, beverage,pharmaceutical and cosmetic industries and it has other applications including
textiles, electroplating and bioremediation .Because of its numerous applications, the volume of
citric acid production by fermentation continually on the increase. The most popular white
rotfungus for large-scale production of citric acid is A. niger due to its high citric productivity at
low pH without the secretion of toxic byproducts. Besides citric acid, some strains of A.niger
also accumulate other organic acids as well such as oxalic, malic, tartaric, fumaricand pyruvic
acids under specific fermentation condition [16]. Regarding the process of citric acid
accumulation in A. niger, two main metabolic pathways have involved a major role.

Aspergillus niger is normally a haploid fungus producing white septate hypha which is profusely
branched. It produces black mass of conidia, which are found in chain arising from the secondary
sterigmata [16]. Soares et al.,2004 worked on the Aspergillus niger and found that this
microorganism was obtained from the culture collection of the Oswaldo Cruz Institute ( Rio de
Janeiro), preserved in tubes with nutrient agar and stored at 4°C. Reiss(1986) collected data on
the influence of temperature , water activity and pH on the growth of various Aspergilli. A.niger
is able to grow in the wide temperature range of 6-47°C.with a relatively high temperature
optimum at 35-37°C. The water activity limit for growth is 0.88, which is relatively high
compared with other Aspergillus species [17].
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Rippel-Baldes(1995) found that A.niger is able to grow over an extremely wide pH range : 1.4-
9.8. These abilities and the profuse production of conidiospores, which are distributed through
the air, secure the ubiquitous occurrence of the species, with a higher frequency in warm and
humid places. Raper and Fennel (1965) divided the genus Aspergillus into groups according to
the colour of the conidiospores. Aspergilli with brown to black-shaded spores constitute the
A.niger group. Although the members of this group vary considerably, only a few differ so
clearly from the majority that they can easily be classified as separate species [18]. The
apparently insignificant difference between members of the A.niger group were the decisive
reasons to classify some species, while Raper and Fennell considered them to be separate
species.

Klusters-Van-Somerenet.al.(1990,1991) introduced restriction fragment length

polymorphism(RFLP) to analyse Aspergillus taxonomy ,the ribosomal banding patterns and the
hybridization patterns of genomic digests from strains in this group. Jianlong (2000) worked on
the citric acid and found that an intermediate in the TCA cycle , citric acid is an important
commercial product with global production reaching 736,000 tons/yr. Furthermore, it is
produced almost through the submerged fermentation of the white rot fungus. The volume of
citric acid production is continually increasing day by day due to its numerous applications [18].

Sassiet et al.(1991) found that besides citric acid, some other strains of A.niger also produce
organic acids such as oxalic acid, malic, tartaric acid under specific type of fermentation but he
stated that A.nigeris the most popular white rotgungus for large scale production of citric acid
due to its high citric productivity at low pH without the secretion of toxic byproducts. According
to Wasay (1998),under optimal conditions , over 90% of glucose can be converted into citric acid
mainly, as glucose is the starting carbohydrate in glycolysis for citric acid production , it plays an
important role in citric acid production.
Majumder(2010) stated that Aspergillus niger strains biomass was increased with the increase
period of time for the production of mycelial body of the fungus and their sporulation. Without
presence of Prescott salt biomass weight was highest for fermented product of Aspergillus niger.
They observed that the production of cirtic acid was not same in all fermentation media.
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Vidya et al.(2018) worked on the citric acid production that, two isolates of A.niger AsnO and
AsnC were tested for their ability to produce citric acid from defined and crude sources.
Socol et al.(2006) demonstrated that citric acid production by fermentation has become as the
most widely used and economical process to obtain citric acid [19]. Over 90% of this organic
acid used among the globe today is obtained from fermentation. This method offers advantages
such as having simple, stable, and less complicated operations requiring less complex control
system and lower technical skill; consuming less energy;and not being affected by frequent plant
power failure.

According to Bhargav et al.(2007), most strains are unable to produce citric acid in acceptable
yields since it is a metabolite of energy metabolism. Its production rises in appreciable amounts
in drastic conditions. The main advantages of using this organism is easy to handling and its
ability to ferment a wide variety of cheap raw materials. Enhancement of production depends on
the selection of proper nutrient supplements, organism and substrates to prevent drastic changes
in pH [20].

Grewal et al.(1995) stated that the first individual process for citric acid production was the
liquid surface culture (LSC),which was introduced in 1919 by Societe des Produits Organiques
in Belgium, and in 1923 by Chas Pfizer & co. in US. After that, other methods of fermentation,
such as submerged fermentation were developed. Although this technique is more sophisticated,
surface method requied less effort in operation and installation and energy cost [21].

Rohr et al (1983)and Morgant (1988) also described that the fermentation chambers are
provided with an effective air circulation in order to control temperature and humidity.
Fermentation chambers are always in aseptic conditions, which might be conserved principally
during the first two days when spores germinate, otherwise there may be a chance of frequent
contamination by penicillia, other Aspergilli, yeast and lactic bacteria [22].

According to Kiel et al (1981), surface fermentation consists of two phases, both of which are
characterized by a rapid uptake of carbohydrates. The first phase is the development of the
fungus as mycelia on the surface of the medium and the second phase utilizes carbohydrates by
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converting them into Citric acid [23]. In the classical process of citric acid manufacture,the
culture solution is held in shallow traysan and the fungus develops as a mycelial mat on the
surface of the medium.

Pandey (1991) termed solid-strate substrate as an alternative method to produce citric acid from
agro-industrial resdues. Citric acid production by SSF was first developed in Japan and is as the
simplest method for its production. There also have been reports with yeasts. Leangon et al.
(2000) experienced on the most common process for the commercial scale production of citric
acid involves submerged fermentation using the filamentous fungus, A.niger growing on media
containing glucose or sucrose [24].

Romero-Gomez et al.(2000) studied on the use of solid substrate fermentation and observed
some potential as an alternative for the production of citric acid [25]. According to Nandakumar
et al.(1994) , the solid substrate asts as a source of carbon, nitrogen,minerals and a growth
surface which absorbs the watery necessary for microbial growth [26].

Goes(1999) experienced a microorganisms on a solid substrate that is growing under conditions

similar to their natural habitat, they may be able to produce certain enzymes, metabolites,
proteins and spores more efficiently than submerged fermentation [27].
Omoriet et al.(1994) found that solid substrate that has been widely used especially in East Asia
fortraditional food fermentations, enzyme production by the Koji process, mould-ripened cheese
and composing of agricultural residues [28]. Gutierrez et al.(1999) analyzed on the absence of a
liquid phase and low substrate humidity level allow(1) facilitated aeration through the pore
spaces between substrate particles; (2) reduction of the fermentation and the liquid effluent
volumes;(3) reduced risk of bacterial contamination because of low moisture level;(4) use of the
non-sterile solid substrate in some cases and (5) reduction in water usage and waste water
management;(6)simplified media and (7) utililization of agro-industrial sugar rich wastes or by
products [29].

According to Laboni et al.(2010) starchy substrates like pumpkin are the best for citric acid
production. Rohr et al.(1983) experienced on the submerged fermentation process that is the
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commonly employed technique for citric acid production . About 80% of world production is
obtained by SmF. Several advantages such as higher yields and productivity and lower labour
costs are the main reasons for this. Two types of fermenters, conventional stirred fermenters and
tower fermenters are employed. Fermenters are made of high-grade steel and require provision of
aeration system, which can maintain a high dissolved oxygen level. Cooling can be done by an
external water film over the entire outside wall of the fermenter. In this fermentation process,
different kinds of media are employed such as sugar and starch based media. Inoculation is
performed either by adding a suspension of spores, or of pre-cultivated mycelia. When spores
are used , a surfactant is added in order to disperse them in the medium [30].

Jianlong et al.(1998) found that citric acid production by A.niger is influenced by number of
fermentation parameters, for high production of citric acid , it is essential that the study of
influence of physical and chemical environments on citric acid production. The initial pH of the
culture medium has been reported to be important for the performance of the citric acid
fermentation [31]. Moyer (1953) got that the pH values within the range of 1.95 to 3.1 did not
have any effect on citric acid production [32]. Sanjay and Sharma (1994) reported on optimal
initial pH of 5.4 under submerged fermentation conditions. A low pH also inhibits the production
of unwanted citric acid production and this makes the recovery of citric acid from broth [33].
Gutierrez-Correa et al.,(1999) described that as Glucose does not need any future modification
for the metabolites, it is readily used by fungus. Some strains of A.niger can convert upto 90% of
the Glucose into organic acids [34].

 In view of surges in demand and growing markets, there is always a need for the discovery and
development of better production techniques and solutions to improve production yields and the
efficiency of product recovery. To support the enormous scale of production, it is necessary and
important for the production process to be environmentally friendly by utilizing readily available
and inexpensive agro-industrial waste products, while maintaining high production yields. This
article reviews the biochemistry of citric acid formation, choices of citric-acid producing
microorganisms and raw materials, fermentation strategies, the effects of various fermentation
conditions, citric acid recovery options and the numerous applications of citric acid, based on
information drawn from the literature over the past 10 years [34].
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Also, Leangon et al.,(2000) found that a substrate initiating glycolysis followed by the TCA
cycle, glucose is the crucial factor affecting acid production. An increase glucose flux through
glycolysis may be the reason forever production of citric acid in solid substrate fermentation.
According to Alvarez et al.,(2011) low glucose concentration in the solid substrate may cause
oxalic acid accumulation such as citric acid [35].

According to Goes (1999), several reports have shown the stimulatory effects of additives on
fungal citric acid accumulation and secretion. To improve citric acid production, stimulators
have been used, such as organic solvents,phytate and lipids [36]. They worked on the higher
citric acid production that can be obtained by applying stimulators, such as methanol, ethanol,
vegetable oil, oximes,etc. Jianlong and ping(1998) found that organic solvents including ethanol
and methanol stimulate the production of citric acid by increasing the permeability of the cell
membrane, decreasing cell growth or changing the activity of enzymes associated with TCA
cycle. In addition, ethanol can improve the citric acid production by converted to acetyl-CoA.
Again, they proposed that the addition of methanol increases the permeability of the cell
membrane and increases the transfer of nutrients that increases the excretion of citric acid across
the cell membrane [37].

Adham(2002) analyzed that citric acid production using A.niger is very sensitive to the
concentration of trace elements. A presence of trace elements in media can cause considerable
negative effect on citric acid production. It was propsed that phytate is also known to offer 12
replaceable protons providing the binding potential for positively charged molecules. Phytic acid
asts as a metal chealating agent , chelating free trace elements. This elements may also improve
citric acid production by using A.niger, by controlling key enzymes involved in the TCA cycle.
Adham(2002) also investigated that vegetable oils with high levels of unsaturated fatty acids,
such as olive, maize and sunflower oil, are stimulators for citric acid production, when using
both submerged and solid susbstrate fermentation by A.niger . He proposed the stimulating
mechanism of natural oil that unsaturated lipids play a role of alternative hydrogen acceptors to
oxygen [38].
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Kamini et al.,(1998) obtained that the initial moisture content influences nutrient solubility, heat
and gas transfers and substrate swelling. He found that moisture content under 60% was
determined to be found optimal for the growth of A.niger [39]. Roukas(2000) worked on the
moisrure content that moisture content between 70 and 80% increased citric acid production
[40].

MATERIALS
AND
METHODS
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MATERIALS AND METHODS

Collection and cultivation of citric acid producing fungi :

Citric acid producing fungi, A. niger, was obtained from the Botany Laboratory Department of
Life Sciences at University of science and technology, Meghalaya and then cultured on agar
media. These were incubated at 28°C for 24 hours and actively growing hyphal regions were
obtained and cultured in 100 mL of inoculum media. The inoculated inoculum media was
incubated in an incubator shaker at 120 rpm at 27 °C for 24 h.

Micr oorganism Used:

Citric acid producing strains of Aspergillus niger were used in this present study. The culture
was maintained on agar slants containing 1% malt extract, 1% yeast extract, 1.5% dextrose and
2.5% bacto agar. . By serial dilution method, these colonies were isolated from collection of soil
sample. Soil was collected from decomposing soil of university campus.

Substrates Used:
The substrate bright colored pumpkin was used in the experiments for the comparative study.
The vegetable wastes of pumpkin was used as a solid substrate for the production of citric acid
by the process of solid state fermentation. Cucurbita was purchased at street markets of Nalbari
town, Assam, India.

 Inoculum Preparation:
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The conidial inoculum was used in the present study; conidia from a 5-day-old culture were used
for inoculum preparation. The fungi were identified using lactophenol cotton blue. Inoculum
preparation was done as described by Chen- H. [15].

Preliminary Screening:

The isolate was screened qualitatively for citric acid production by plate method on agar
containing Bromocresol green as indicator 1% at pH 6. The spore suspension of the isolate was
spread on the surface of the medium plates and allowed to grow for 5 days. Yellow zones
indicated citric acid production [19].

Preparation of Pumpkin Medium:

Pumpkin was washed with tap water for several times. Their after pumpkin was sliced thinly
and dried in dryer at 50°C. The substrate was powdered by using a grinding machine. Dried
powder of pumpkin was hydrolyzed separately 300 ml solution of 0.05 N HCl and autoclaved
at 121° temperature, 15 Ibs pressure for 20 minutes. The hydrolyzed materials were
then filtered through thin cloth.

Determination of Optimum incubation time for citric acid production, cell biomass,
residual sugar and pH:

Citric Acid, cell biomass, residual sugar and pH was measured at interval of 5 days i.e. 5th, 10th
and 15th day to determine optimum incubation time based on production.

Effect of different substrates on citric acid production, cell biomass, residual sugar and
pH:

Citric Acid, cell biomass, residual sugar and pH was measured at interval of 5 days i.e. 5th, 10th
and 15th day of each substrate to determine cheap and best medium for citric acid production.

All experiments were incubated in an orbital shaker. Each assay was carried out.

Determination of Citric acid:

Determination of citric acid titrimetrically (El-Holi and Al-Delaimy, 2003) [41].

23 | P a g e

Citric acid was determined using 0.1 N NaOH and phenolphthalein indicator titrimetrically
(AOAC, 1995) [42]. % Citric acid was determined using following formula:

Normality of Citric acid = normality of NaOH × NaOH volume ÷ volume of Citric acid
Concentration of Citric acid = Citric acid normality ×equivalent ×100 ÷ volume of distillation
(Equivalent = 96, volume of distillation = 10)

Determination of citric acid by pyridine –acetic anhydride method (Marrier and Boulet ,
1958) [43]:

Filtrate was taken in clean test tubes and final volume made to 1 ml with distilled water. 8 ml
acetic anhydride was added and placed at 600 C for 10 min, which was followed by addition of 1
ml pyridine and placed at 600 C for 40 min. Color was changed to yellow and later absorbance,
was taken at 420 nm spectrophotometrically.

Determination of Residual Sugar:

Estimation of residual sugar was done by 3, 5-dinitrosalicylic acid (DNS) method (Miller 1959)
[44]. This method tests for the presence of free carbonyl group (C=O), the so-called reducing
sugars. This involves the oxidation of the aldehyde functional group present in, for example,
glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid
(DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions.

Determination of pH:

pH was measured using Anolog pH meter daily. Experiment regarding each substrate was done
in triplicates.
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RESULTS
AND
DISCUSSION
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REFERENCES
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REFERENCES

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production of bioactive substances: A comparative study,” Int. J. Sci. Nat. vol. 2, pp. 480-486,
2012.

[2]S.O. Kareem, I. Akpan, and O.O Alebiowu, “Production of citric acid by Aspergillus
niger using pineapple waste,” Malaysian J. Microbiol., vol. 6, pp. 161-165, 2010.

[3]C.R. Soccol, L.P .S. Vandenberghe, C. Rodrigues, and A. Pandey, “New

perspectives for citric acid production and application,” Food Technol. Biotechno., vol. 44,
pp. 141–149, 2006.

[4] M. Papagianni, “Advances in citric acid fermentation by Aspergillus niger: Biochemical

aspects, membrane transport and modeling,” Bio-technology Advances, vol. 25, pp. 244-263,
2007.

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