PRODUCTION& OPTIMIZATION OF CITRIC ACID BY

ASPERGILLUS NIGERUSING MAUSAMI WASTE

A DISSERTATION
Carried out at

RAPTURE BIOTECH, NOIDA

Submitted to

Department of Biotechnology
Invertis University, Bareilly, Uttar Pradesh
In partial fulfillment of the requirement for the award of the degree of

Bachelor of Technology
In
Biotechnology
Submitted by
Navjot Kaur Matharu
BT2011020

Under the guidance of

Dr. JyotiJha
Rapture Biotech, Noida

Candidates Declaration
I hereby certify that the dissertation entitled Production & Optimization

Of Citric Acid ByAspergillus Niger Using Mausami Wastewhich is being

submitted for the partial fulfillment of the requirement for the award of
degree of Bachelor of Technology in Biotechnology is a record of my
own work carried out under the Supervision of Rapture Biotech, Noida.
The matter presented in this dissertation has not been submitted
elsewhere for the award of any degree/diploma.
Date:May,2015

Navjot Kaur Matharu

Place: Bareilly

This is to certify that the above statement made by the candidate is

correct to be the best of my knowledge and belief.

Dr. Sanjeev Kumar Maurya

H.O.D
DEPARTMENT OF BIOTECHNOLOGY
Invertis Institute of Engineering and Technology (IIET)
Invertis University , NH 24 , Bareilly- 243123(UP)

Invertis
University
Department of
Biotechnology
NH-24, Lucknow-Bareilly
Highway,
Bareilly-233124, UP, India

Dr. Sanjeev Kumar

Maurya
Assistant Professor
Head of Department
Department of
Biotechnology
Email:
sanjeev.m@invertis.org
Phone: 09473909380

TO WHOM IT MAY CONCERN

It is certify that Ms Navjot Kaur Matharu D/OMr Amarjeet Singh
Matharuis a student of B.Tech Biotechnology final year from Department of
Biotechnology, Invertis University, Bareilly, UttarPradesh.
Ms Navjot Kaur Matharu has done her B.tech biotechnology final Year
dissertation work with Rapture Biotech,Noida (U.P.) entitledProduction&
Optimizationof Citric Acid By Aspergillus Niger Using Mosambie Wasteand
submitted in the Department as a part of her course curriculum.

Dr. Sanjeev Kumar Maurya

H.O.D
DEPARTMENT OF BIOTECHNOLOGY
Invertis Institute of Engineering and Technology

(IIET)
Invertis University , NH 24 , Bareilly- 243123(UP)

ABSTRACT
Citric acid is one of the very important commercial products with constant demand in the
market. Citric acid production by fungi have been reported by many workers since
1913.The present study focused on production of citric acid by Aspergillus Niger by
submerged fermentation. The major problem that is to be taken care of these days is the
management of wastes, which produce odor and soil pollution which also represents a major
threat for the food industry. Earlier submerged fermentation method was used to use these
wastes and to produce some useful products out of them. However, because of several
advantages over the submerged fermentation such as solid waste management, biomass
energy conservation, production of high value products and little risk of bacterial
contamination, the Solid State Fermentation (SSF) method have recently gained attention
using agro residues like sugarcane bagasse, coffee waste of food processing industries
including Citrus limette (mosambie) waste.
It is an integral part of Krebs cycle and therefore plays an essential role in the metabolism of
all living things. Approximately 50% of the worlds citric acid also makes it useful as a
preservative. Since, many bacteria are unable to grow in an acidic environment citric acid is
often added to jams, candies, jellies and even meat products as preservatives. These days
Citric Acid is being produced by using SSF method so that food waste can also be managed
by this and Citric Acid which is used as flavoring agent in food industries can be produced,
We have chosen a fungal strain which was isolated from soil contaminated with solid wastes
and screened for citric acid production and it was identified by Aspergillus Nigerfrom
ground soil. The solid waste substrates mosambie peel cut into pieces and 4% methanol,
70% moisture level was maintained throughout the solid state fermentation process for the
Citric Acid production from the fungal culture. And the best result of Citric acid got after 5
days of fermentation.

ACKNOWLEDGEMENTS
I would like to express my gratitude to all those who have helped me for the
completion of this project. I want to thank Mr. MayankRaj Bhardwaj (CEO Rapture
Biotech, NOIDA) for giving me permission to commence this project in the first instance
and to use departmental facilities.
I deeply indebted to my project supervisor Dr. Jyoti Jha and Project asst. Ms. Priya,
whose help stimulating suggestions and encouragement helped me in all the time of
research and writing of this thesis.
First of all,I also thank Dr. Umesh Gautam, Chancellor of Invertis University,
Bareilly for providing the facilities in the lab.
My heartful thanks are due to my Head of the Department Dr. Sanjeev Kr. Maurya
and also college teachers Dr. ShashankUpadhyay, Dr. Ravi Deval, Dr. Pankaj Tripathi,
and Er. Sachin Srivastava and Dr. Niteesh Poddhar.
I also thank my lab mate or my friends Anamika, Swadha, Neha and Vandna. All
are very helping in nature and always increased my confidence. Now there are no words in
world to explain owe of parents on children.
I pay my gratitude and reverse to my parents for their moral boosting. Without their
blessing this endeavor of my project couldnt be completed. Thanks to whole my family
Members for promoting me to do best.
I thank to God for blessing me and giving me acumen to accomplish this work. I pray to
God to bless me in future and guide me on the right path.

(NAVJOT
KAUR)

Contents

Chapters

PageNo.

1 Introduction

1-5

2 Review of Literature

6-22
6-7

2.1 History of Citric Acid

2.2 Extraction of Citric Acid from citrus fruits
2.3 Microbial production of Citric acid
2.4 Yeasts
2.5 Fungi
2.6 Growth Stages of Aspergillusniger
2.7 Biochemistry of Citric Acid Production by Aspergillusniger
2.8 Solid state fermentation
2.8.1An Overview of SSF process
2.8.2Microorganism suitable for SSF
2.8.3 Substrates used in Solid state Fermentation
2.8.4 Temperature
2.8.5Effect of pH on SSF
2.8.6 Moisture
2.8.7Application of SSF

8
8
8
9-11
11
11-12
12-21
13-14
14-15
15-16
16-17
17-18
18
18-20

2.9Citric Acid Production Process

2.9.1 Carbon Source
2.9.2 Nitrogen Source
2.9.3 Effect of temperature on the production of citric acid
2.9.4.Effect of Moisture Content

20-22
20
21
21
22

3.Objectives

23

4.Methods and Materials

4.1 Sample Collections
4.2Serial Dilutions
4.3Culture Media
4.4Isolation of Fungal Organisms
4.5 Screening for Citric Acid Production
4.6.Fermentation media for Production of Citric Acid (g/ml)
4.7.Citric Acid assay

24-31
24
24
25-26
26-27
27-28
28-30
30-31

5.Result and Discussion

32

6.Conclusion

33

7.References

34-39

LIST OF FIGURES
Fig- 1. Process of Krebs cycle.....3

Fig- 2. Serial Dilution of Soil for fungal culture....25

Fig- 3. Potato Dextrose Agar (PDA) Media....25

Fig- 4. Potato Dextrose Agar (PDA) Plates.....26

Fig- 5. Streaked Plates for Fungal Growth27

Fig- 6. Mixed culture of fungal colonies for production of citric acid....27

Fig- 7. Pure culture of fungal colony for production of citric acid28

Fig- 8. Fermentation of citric acid production using Mosambie Peels..29

Fig- 9. Fermented citric acid pH measurement.................................30

Fig-10. Pink pale color as result.....................30

List of Symbols &Abbreviations

S. No

Symbol

Meaning

Percent

Degree centigrade

Gram

mg

Microgram

mol

Molar

cm

Centimeter

CO2

Carbon di oxide

CA

Citric Acid

O2

Oxygen

10

Micrometer

11

mm

Millimeter

12

ml

Milliliter

13

Liter

14

kg

Kilogram

15

w/v

Weight per Volume

16

nm

Nanometer

17

hr

Hour