CITRIC ACID PRODUCTION BY ASPERGILLUS NIGER USING BANANA AND PLANTAIN PEELS.

ABSTRACT

In this work, the production of Citric acid by Aspergillus niger using plantain and banana peels were allowed to ferment for 7 days and the physical changes were observed.  Physical changes of banana peels showed that pH decreased from 6.5 to 2.5, while the total titrable acidity increased from 0.074% to 1.28%. The biomass increased from 64mg/L to 330mg/L. the total solid decreased from 16.86% to 9.96%. The percentage of Citric acid yielded at the end of the fermentation was increased from 0.172% to 1.304%. While the physical changes of plantain peel showed that pH decreased from 6.5 to 2.4, while the total titrable acidity increased from 0.06% to 1.24%. The biomass increased from 61mg/L to 298mg/L. The percentage of Citric acid yielded at the end of fermentation was increased from 0.156% to 1.273%. The total solid decreased from 18.32% to 93%. The properties listed above were studied during the fermentation period indicates the changes that occurred at each particular day of fermentation starting from day 1 to day 7. This work has shown that banana and plantain peel serve as a potential substrates for the production of citric acid by Aspergillus niger and its utilization will facilitate large scale production of this commercially valuable organic acid.

TABLE OF CONTENTS

Certification                                                                                                                                                                                                                                                                                        ii

Dedication                                                                                                                                                                                                                                                                                          iii

Acknowledgement                                                                                                                                                                                                                                                                              iv

Table of Content                                                                                                                                                                                                                                                                                 v

Lists of Tables                                                                                                                                                                                                                                                                                    vi

List of Figures                                                                                                                                                                                                                                                                                     vii

Abstract                                                                                                                                                                                                                                                                                              viii

CHAPTER ONE

1.0 Introduction                                                                                                                                                                                                                                                                                  1

1.1 Historical Developments                                                                                                                                                                                                                                                              2

1.2 Microorganism                                                                                                                 3               1.3 Substrates                                                                                                                                 4

1.4 Uses                                                                                                                                  5

1.5 Application of Citric Acid                                                                                                6

CHAPTER TWO     

2.0 Literature Review                                                                                                             7

2.1 Ecology                                                                                                                             8

2.2 Taxonomy                                                                                                                         9

2.3 Banana                                                                                                                              11

2.4 Plantain                                                                                                                             12

CHAPTER THREE

3.0 Materials and Method                                                                                                       18

3.1 Collection of Banana and Plantain Fruits                                                                         18

3.2 Sampling                                                                                                                           18

3.3 Sterilization                                                                                                                      18

3.4 Production of Citric Acid                                                                                                 18

3.5 Citric Acid Determination                                                                                                            19

3.6 Biomass Determination                                                                                                    19

3.7 Determination of pH                                                                                                         19

3.8 Determination of Total Solid                                                                                            20

3.9 Determination of Total Titrable Acidity                                                                          20

3.10 Determination of Sugar Content                                                                                    21

3.10.1 Lactophenol Cotton Blue Staining                                                                              22

CHAPTER FOUR

4.0 Results                                                                                                                              23

CHAPTER FIVE

5.0 Discussion and Conclusion                                                                                              29

5.1 Discussion                                                                                                                         29

5.2 Conclusion                                                                                                                        30

5.3 Recommendation                                                                                                             30

References

LIST OF TABLES

Tables      Title                                                                Page

 1  Changes in characteristics of banana peel during the fermentation process            24

 2  Changes in characteristics of banana peel during the fermentation process            25

LIST OF FIGURES

Figure          Title                                                                Page 

1      Changes in Sugar content  during the Fermentation time of Banana and

Plantain peel                                                                                                           26

2      Changes in pH level  during the Fermentation time of Banana and

Plantain peels                                                                                                          27

3      Concentration of citric acid during the fermentation of Banana and Plantain

Peels                                                                                                                  28

     CHAPTER ONE

1.0  INTRODUCTION

 Citric acid is a 6-carbon containing tricarboxylic acid which was first isolated from lemon juice. It is a natural component of many citrus fruits, and was crystallized from lemon juice by Scheele in 1784. Approximately 70% of citric acid produced is used in the food and beverage industry for various purposes, 12% in pharmaceuticals and about 18% for other industrial uses.  Commercial production of citric acid is generally by submerged fermentation of sucrose or molasses using the filamentous fungus Aspergillus niger or synthetically from acetone or glycerol (Adachi et al., 2003; Haq et al., 2004). In the recent times solid state fermentation (SSF) is considered as an alternative to submerged fermentation in the production of microbial metabolites because of higher yields, low water requirement and lower operating costs. Many microorganisms have been evaluated for the production of citric acid including bacteria, fungi and yeast. However, fungal strains of Aspergillus niger remained the organism of choice for citric acid production due to ease of handling, its ability to ferment a variety of cheap raw materials, and high yields (Schuster et al., 2002). A cost reduction in citric acid production can be achieved by using cheap agricultural wastes such as apple and grape pomace, orange peel, kiwifruit peel, cotton waste, okara soy-residue and cane molasses. Citric acid (C₆H₈O₇, 2 – hydroxy – 1,2,3 – propane tricarboxylic acid), a natural constituent and common metabolite of plants and animals, is the most versatile and widely used organic acid in the field of food (60%) and pharmaceuticals (10%). It has got several other applications in various other fields. Currently, the global production of citric acid is estimated to be around 736000 tones/year, and the entire production is carried out by fermentation. In Brazil, almost the entire demand of citric acid is met through imports. There is constant increase (3.5%-4%) each year in its consumption, showing the need of finding new alternatives for its manufacture.

1.1 HISTORICAL DEVELOPMENT

Citric acid was first isolated by Karls Scheels in 1874, in England, from the lemon juice imported from Italy. Italian manufacturers had monopoly for its production for almost 100 years, and it was sold at high cost. This led extensive attempts all over the world to find alternatives way for its production, which included chemical and microbial techniques. In 1923, Wehmer observed the presence of citric acid as a byproduct of calcium oxalate produced by a culture of Penicillium glaucum. Other investigations showed the isolation of two varieties of fungi belonging to genus Citromyces (namely Penicillium). However, industrial trials did not succeed due to contamination problems and long duration of fermentation (Rohr et al., 1983). The industrial process was first open by Currie, in 1917, who found that Aspergillus niger  had the capacity to accumulate significant amounts of citric acid in sugar based medium. He also showed that high concentrations of sugar favoured its production, which occurred under limitation of growth. In the thirties, some units were implanted in England, in Soviet Union, and in Germany for the commercial production. However, the biochemical basis was only cleared in the fifties with the discovery of the glycolytic pathway and the tricarboxylic acid cycle (TCA). Consequently, an improved process employing submerged fermentation was developed in United States (Aboud-Zeid and Ashy, 1984). Although methods were well developed to synthesis citric acid using chemical means also, better successes were achieved using microbial fermentations, and over the period of time, this technique has become the method of ultimate choice for its commercial production, mainly due to economic advantage of biological production over chemical synthesis (Mattey, 1992). Much attention has been paid on research to improve the microbial strains, and to maintain their production capacity.

1.2 CITRIC PRODUCTION

1.2.0 MICROGANISM

A large number of microorganisms including fungi and bacteria such as Arthrobacter paraffinens, Bacillus licheniformis and Corynebacterium ssp., Aspergillus niger, A.aculeatus, A. carbonarius, A. awamori, A. foetidus, A. fonsecaeus,A. phoenicis and Penicillium janthinellum; and yeasts such as Candida tropicalis, C. oleophila, C. guilliermondii, C. citroformans, and Yarrowia lipolytica have been employed for citric acid production (Grewal and kalra, 1995; Yokoya,1992). Most of them, however, are not able to produce commercially acceptable yields due to the fact that citric acid is a metabolite of energy metabolism and its accumulation rises in appreciable amounts only under conditions of drastic imbalances. Among the mentioned strains, the fungus A. niger has remained the organism of choice for commercial production because it produces more citric acid per time unit. The problem in the production of citric acid for yeasts is the simultaneous formation of isocitric acid. The main advantages of using A. niger are its ease of handling, its ability to ferment a variety of cheap raw materials, and high yields. Industrial strains which produce commercial citric acid are not freely available and only a few can be obtained from international culture collections. The improvement of citric acid producing strains has been carried out by mutagenesis and selection. The most employed technique has been by inducing mutations in parental strains using mutagens (Pandey, 2001). Mutants of Aspergillus niger are used for commercial production. Among mutagens, g-radiation, UV radiation and chemical mutagens are often used. To obtain hyper-producer strains, UV treatment can frequently be combined with some chemical mutagens. The single--spore technique and the passage method« are the principal methods of selecting strains. The first one has the disadvantage that mineral acid and organic acids (gluconic and oxalic acids) simulate the presence of citric acid (Kubicek and Röhr, 1986). Different methods of fermentation can lead to different yields of citric acid production by the same strain. Thus, a strain which produces good yields in the solid fermentation or liquid surface is not necessarily good producer in the submerged fermentation. In that way, each method and raw material of industrial interest should be tested with known producer strains (Yokoya,1992).In any technique used in citric acid production the inoculation of microorganism is done by means of spores which are added into the fermentation medium (Yokoya,1992). Spores can be inoculated either mixing them with the air, which is introduced in substrate, or in form of a spore suspension. Spores are produced in glass bottles on solid substrates at optimum temperature (Pandey et al., 2001). The type of sporulation medium and time of incubation affect spore viability and citric acid production by mycelia grown from A. niger. It was mentioned that potato dextrose agar gives high citric acid yields. Viability increases with time of incubation, but higher production of citric acid was achieved in less than 7 days of spore incubation. The capacity of germination of the spores tends to reduce with the time but in some cases, short periods of up to 7–8 days do not present significant difference in relation to spores collected after 3 days.

1.2.1 SUBSTRATES

Several raw materials such as hydrocarbons, starchy materials and molasses, have been employed as substrates for commercial submerged citric acid production (Grewal and kalra, 1995), although citric acid is mostly produced from starch or sucrose based medium using submerged fermentation. Generally, citric acid is produced by fermentation using inexpensive raw material including crude natural products, such as hydrolysate starch, sugar cane broth and by-products like sugar cane and beet molasses (Yokoya, 1992). Molasses is preferably used as the source of sugar for microbial production of citric acid due to its relatively low cost and high sugar content (40–55 %) (Grewal and Kalra, 1995). Since it is a by-product of sugar refining, the quality of molasses varies considerably, and not all types are suitable for citric acid production. The molasses composition depends on various factors like the variety of beet and cane, methods of cultivation, conditions of storage and handling (transport, temperature variations), etc. Both beet and cane molasses are suitable for citric acid production, however, beet molasses is preferred to sugarcane due to its lower content of trace metals, supplying better production yields than cane molasses, but there are considerable yield variations within each type. In the case of cane molasses, generally it contains some metals (iron, calcium, magnesium, manganese, zinc) which retard citric acid synthesis and it requires some pretreatment for the reduction of them. Palmyra jaggery, sugar syrup from the palmyra palm is a novel substrate for increasing the yield of citric acid production. The addition of phytate (an important plant constituent) at the beginning of incubation of beet molasses results in about 3-fold increase in citric acid accumulation.

1.2 USES

Citric acid is used as a food ingredient in the production of fruit products, juices, oils and fats, and for 40 many other food products where it functions as an acidulant, pH control, flavoring and sequestrant. It is also used as a dispersant in flavor or color additive products. In addition, it is used to wash processing equipment to eliminate off-flavors.

1.3 APPLICATIONS OF CITRIC ACID

Citric acid is a versatile and innocuous alimentary additive. It is accepted worldwide as GRAS (generally recognized as safe), approved by the Joint FAO/WHO Expert Committee on Food Additives (Soccol et al., 2003). The food and pharmaceutical industries utilize citric acid extensively because of its general recognition of safety, pleasant acid taste, high water solubility and chelating and buffering properties. Citric acid is used in cosmetics and toiletries as buffer, and in a wide variety of industrial applications as a buffering and chelating agent. Citric acid is also a reactive intermediate in chemical synthesis. In addition, its carboxyl and hydroxyl groups permit the formation of a variety of complex molecules and reactive products of commercial interest.

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