CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the Study

Citric acid is a weak organic acid present in all citrus fruits, with IUPAC name 2-hydroxy-

propane-1,2,3-tricarboxylic acid and molecular formula C6H8O7.H2O (Atashnoor and Anvari

et.al., 2022). The name citrus was coined from the Latin word citrus, referring to the family of

citrus fruits known as Rutaceae which includes orange, lime, lemon, citron and grapefruit (Egbe

et.al., 2022). Pure form of citric acid readily dissolves in water to give a colourless solution

(Behera et.al., 2021). It is solid at ambient temperature and melts at 153°C. The molecular

weight of citric acid is 210.14 g/mol with three distinct pKa values at pH 3.1, 4.7, and 6.4, this is

due to the presence of three carboxylic groups in the structure of citric acid (Igliński et.al., 2022).

There is continuous upsurge in demand for citric acid in various industries and it is one of the

commonly used chemical compounds (Kudu et.al., 2022). It has wide industrial applications with

great value in chemical, cosmetics, pharmaceutical, food and beverage industry as an essential

additive used as preservative, flavor, enhancer, antioxidant, acidulant, plasticizer and several

other uses combined with other chemical substances (Ben Hsouna et.al., 2023).

Citric acid is naturally obtained from citrus fruits including orange, lime, lemon amongst other, it

can also be synthesized by fermentation of various substrates or through other chemical reaction.

The extraction of citric acid from lemon juice was first engineered in the late eighteenth century

by a Swedish chemist, Karl Wilhelm Scheele in 1784 (Reena et.al., 2022). In England, the

method of extraction of citric acid began in the early nineteenth century and was commonly

employed for commercial production of citric using lemon fruits imported from Italy (Amato

et.al., 2020). The method was widely used for commercial production being the only source until

0
the late nineteenth century, when Wehmer, a German botanist in 1893 studied the production of

citric acid from fermentation of starch medium with inorganic salts Incorporated using

Penicillium glaucum as the fermenting specie (Książek et al., 2023).

In 1895, Wehmer studied the potential of various microorganisms to produce citric acid through

fermentation and successfully isolated two fungal strains which were later named Citromyces sp.

(Penicillium). Although the strains expressed unique ability, challenges were encountered in the

course of production particularly the lengthy fermentation time in concomitant with

contamination of the of the end product (Adeoye et.al., 2022). In the early twentieth century,

industrial citric acid production witnessed an enormous development particularly with the

findings of James Currie in 1916 who studied the production of citric acid using Aspergillus

niger, and observed that various strains of A. niger are capable of producing substantial amount

of citric acid (Behera et.al., 2020). Remarkable findings reported by Currie were the unique

ability of A. niger to grow in highly acidic medium (2.5-3.5), this prevented gluconic and oxalic

acid formation, and the direct proportionality of citric acid production to sugar concentration

(Igliński et.al., 2022). The study of Currie was a major breakthrough in the field and formed the

baspis for contemporary industrial production of citric acid, which was established by Pfizer

pharmaceutical company in the United States in 1923 (Börekçi et.al., 2021).

) A large number of microorganisms such as bacteria, fungi, and yeast have been harvested on a

variety of substrates for the bio-production of Citric acid. A white rot fungus A.niger is

exclusively the preferred choice of microorganism for the citric acid bio-production process.

These filamentous fungi are the most adapted and suitable microorganisms to grow on various

substrates. The fine regulation and control of glycolytic flux, secretion of citric acid from the

mitochondria and the cytosol, and the growth characteristics and adaptability of A.niger on

diverse habitats together contribute toward the massive accumulation of citric acid. The

regulation of different metabolic enzymes coupled with the effect of various positive factors on

glycolytic flux favors high CA formation and further low degradation via the citric acid cycle

1
(Finley et.al., 2023). Wehmer(1893) first observed that “Citromyces” (now Penicillium)

accumulated CA in a culture medium that contained sugars and inorganic salts. Many other

microorganisms have since been found to accumulate citric acid, including many other strains of

A.niger. Waste paper is a heterogeneous mixture of plant material, it is rich in cellulose (50-

60%), a glucose biopolymer that constitutes thousands of glucose units and hemicellulose that is

composed of two kinds of pentoses (xylose and arabinose) and three kinds of hexoses (glucose,

mannose and galactose) (jabeen et al., 2024). Lignin, which is another biopolymer in paper

materials is made up of aromatic compounds only which can’t be easily hydrolyzed as it works

as a glue cementing cellulose and hemicellulose together (Luchese et.al., 2024).

However, with advances made in biotechnology, the plant components that constitute paper

wastes (i.e. cellulose, hemicellulose, lignin) are now used as feeds stock in the production of

valuable products.The use of plant components like cellulose, hemicellulose, and lignin from

paper waste as feedstock for valuable products is a promising advancement in biotechnology.

This innovative approach repurposes waste paper into bioethanol and biochar, offering

sustainable solutions for energy and material production (Mokatse et.al., 2021). Lignocellulosic

biomass is a complex structure composed of cellulose, hemicellulose, and lignin, making it a

significant source of renewable energy and raw materials (waste Paper such as woods and

leaves) for various industries (Ojewumi et.al., 2019). Hemicellulose, a primary component of

xylose is responsible for the interlink bonds present in cellulose molecules. The structural

strength of paper and other materials is attributed to the presence of lignin, which has a highly

linked molecular structure (Pandit et al., 2024). These components of paper wastes have been

reported in the past as good sources of fermentable sugars when hydrolyzed, and can be used in

the production of value added bio-products such as bioethanol and bio-pharmaceuticals (Mokatse

et.al., 2021).

The bioconversion of organic materials by microorganisms to citric acid is however complex and

involves a series of biochemical reactions under certain conditions which must be carefully

2
controlled (Dar et.al., 2024). The fermentation process is affected by a number of factors which

include the type and concentration of carbon source, pH of the culture medium, aeration,

concentration of alcohols and trace elements, nitrogen and phosphorous source and the

morphology of the citric acid producing microorganisms (Lende et.al., 2021). The composition

of fermentation medium have defined limit, nutrients such as nitrogen, phosphorous and

manganese are added in limited quantity for excellent fermentation set up, while other nutrients

like oxygen and sugar are added in excess (Dar et al., 2024).

1.2 Statement of Research Problem

Citric acid has extensive industrial applications and is widely used in several industries like

pharmaceutical, food and beverage industry, however citric acid is imported into Nigeria from

other countries such as France, Germany, Italy, America amongst other, thereby rendering it very

expensive (Kudu et.al., 2022). Citric acid is a valuable organic acid, it's production is

predominantly reliant on the extraction process from citrus fruits, leading to supply chain

vulnerabilities and environmental concerns. In Nigeria, a country abundant in agricultural waste,

exploring alternative sustainable sources for citric acid production, such as waste paper, offers a

promising avenue for addressing both environmental and economic challenges (Ashokumar

et.al., 2022). However, the feasibility, efficiency, and scalability of citric acid production from

waste paper in Nigeria remain largely unexplored. The ingredients utilized for citric acid

production and other imported substances may be adulterated or have contain additional

additives which may not be safe for human consumption or meet the industrial standard (Jurica

et.al., 2021). Citric acid is used in all industries at one stage of processing or another to produce

valuable products (Mores et.al., 2021). The increasing demand coupled with the shortage in

supply of citric acid has led to a high cost of production using conventional production process,

which is not environmentally friendly.

Abundance of paper waste are found littering the environment across the country which are not

being efficiency utilized. These waste Paper Office paper, foolscap paper, Pick ’n Pay

3
advertising paper, Woolworths advertising paper, newspaper, brown envelope paper and filter

paper (Ndoluv et.al., 2023). These waste are gradually becoming nuisance as they contribute

greatly to environmental pollution including deterioration of land and pollution of several water

bodies. Waste Paper has negative impact on the environment, affecting the air, water and soil

(Gondal et al., 2023). The negative effects of waste paper on the environment include ecological

damage from toxins leaking into the soil, air pollution from burning waste, littering that disrupts

ecosystems, and the significant contribution to solid municipal waste (Siddiqu et.al., 2022).

Additionally, the paper industry is a major polluter, releasing toxic chemicals into the air, water,

and land, impacting freshwater, forests, and soil quality. Waste paper also leads to deforestation,

water pollution, and the generation of greenhouse gases, highlighting the urgent need for

sustainable paper production and consumption practices to mitigate these environmental impacts.

Currently, no known waste Paper management policy exist in Nigeria, Paper waste still remain

as litters or pollutants in the environment. Incineration of paper waste contributes to greenhouse

gas emissions like CO, CO2, SO, and NO, impacting the environment negatively (Yang et.al.,

2024). This method releases carbon dioxide, disturbing the atmospheric balance and contributing

to global warming. Recycling paper is a more environmentally friendly solution, saving trees,

energy, and water while reducing waste sent to landfills or incinerators ( Devi et.al., 2024).

Proper waste management, including recycling and composting, is crucial to mitigate

environmental harm caused by traditional disposal methods like incineration and landfilling

(Pathak et.al., 2024). Landfills can cause the leaching of harmful chemical substances, including

Per- and polyfluoroalkyl substances (PFAS), into underground waters, affecting life in water

bodies and ultimately affecting humans through bioaccumulation and trophic transfer. PFAS in

landfills may not degrade like other waste components and can leach from the waste over the

long-term, potentially impacting PFAS releases into the environment. Since plants are the source

of paper, efforts have been undertaken to turn the cellulolytic content of these materials into

useful products by using heat and inorganic acids like hydrochloric and sulfuric.

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1.3 Justification for the Study

Citric acid is used worldwide with countless applications due to its very low level of toxicity in

comparison to other acidulants commonly used in food and pharmaceutical industry (Massadeh

et.al., 2022). Citric acid is also utilized in detergent industry for cleaning products, in cosmetic

and toiletries amongst other (Bangare et.al., 2021). Citric acid production on a global level has

exceeded 2 million tonnes with annual growth rate of 3.5-4.0% and projected to reach 3 million

tonnes by 2024 (Kumar et.al., 2024). There is need to study the various possible ways to

potentiate the production of citric acid. The use of waste paper substrates for the production of

citric acid can be a cost-effective approach, as various inexpensive and readily available raw

materials are employed in commercial production (Dar et.al., 2024). For instance, in the study

mentioned, crude seaweed powder and 10% sucrose yielded 50 g of citric acid at a lower cost of

Rs. 35, while other media gave yields of 80 and 30 g with higher costs. Similarly, using cheaper

and less pure substrates like glycerol waste from the biodiesel industry can be preferable on a

production scale, despite lower CA ratios and mass yields. Agro- for industrial wastes like apple

pomace solid waste can also be utilized sustainable bio production of citric acid by Aspergillus

niger, leading to cost reduction.

The biotransformation of waste paper, through biological processes such as hydrolysis and

fermentation by fungal species, is indeed considered an efficient and environmentally friendly

approach to the production of high-quality citric acid (Nazir et.al., 2024). This method utilizes

inexpensive and readily available raw materials, such as paper residues from Urban Solid Waste

(USW), which can yield citric acid with high conversion efficiency. The use of other renewable

raw materials, like wood, straw, and agricultural products processing waste, can also lead to cost

reductions in citric acid production (Prawira et.al., 2024). Furthermore, the evaluation of wastes

as low-cost substrates for microbial citric acid production has been explored, demonstrating the

potential for sustainable and cost-effective bioproduction. Citric acid is a value added product

5
that can be derived from wide range of waste paper including Office paper, foolscap paper, Pick

’n Pay advertising paper, Woolworths advertising paper, newspaper, brown envelope paper and

filter paper.

Citric acid production is yet to be commercialized in Nigeria, however, vast majority of

industries including pharmaceutical, food, beverage, cosmetics, chemical, textile among other

make good use of citric acid for the manufacture of numerous products (Adeoye et.al., 2022).

The production of citric acid from waste paper through solid-state fermentation (SSF) is

considered an economical and effective approach to minimizing environmental problems (Laltha

et.al., 2022). Therefore, citric acid production from waste paper will address the problems

resulting from waste paper pollution and cost-effective approach to production of citric acid as

well as job creation.

1.4 Aim and Objectives of the Study

The study is aimed at microbial conversion of waste Paper for citric acid production.While the

objective of the study were;

i. isolate Aspergillus niger from soil samples.

ii. screen the isolates for potential to produce citric acid.

iii. identify A. niger strain with the highest potentials for citric acid production.

iv. determine the reducing sugar contents of the waste paper.

v. determine the concentration of citric acid produced.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Citric Acid

All citrus fruits, such as orange, lime, grape, lemon, and pineapple fruits, contain citric acid, a

weak organic acid (Cahardoli et.al., 2020). Additionally, it is created through the fermentation

process of different types of microorganisms, such as fungus, bacteria, or yeast, on a variety of

substrates, including cellulose, waste paper, molasses, starch, oils and fats, syrups, sucrose, and

other carbohydrates. 2-hydroxy-propane-1,2,3-tricarboxylic acid, having the chemical formula

C6H8O7.H2O, is the IUPAC designation for citric acid (Figure 2.1) (Lambros et.al., 2022). It is a

non-toxic material that helps the kidneys operate properly, keeps energy levels stable, and

detoxifies the body. Because of it sour and refreshing flavor It is used extensively in the food and

beverage sector to counteract the sweetness of juice, soft drinks, and other liquids.

Figure 2.1. Structural formula of citric acid (Bahera et.al., 2021).

2.2 Chemistry of Citric Acid

Tricarboxylic acid, as opposed to citric acid, is defined as having three molecules of carboxylic

(COOH) groups in its structure. It is a metabolite that can be found in both plants and mammals

7
and is generally present in citrus and pineapple juice (Lende et.al., 2021). Citric acid has a white

crystalline appearances and odourless in it's pure state, Citric acid comes in two different forms:

the anhydrous form, which has a molecular weight of 192.12 g/mol and the molecular formula

C6H8O7, and the monohydrate form, which has a molecular weight of 210.14 g/mol and the

molecular formula C6H8O7.H2O. While the monohydrate form is soluble in water and sparingly

soluble in ether, the anhydrous form is easily soluble in water, freely soluble in ethanol, and

sparingly soluble in ether (Behera et.al., 2021). Citric acid exists as solid at ambient temperature

and melts at 153°C with a boiling point of 310°C and gets decomposed at higher temperatures

(above 175°C) with loss of carbon dioxide (Babers et.al., 2020). It is biodegradable and

ubiquitous in nature, considered as cost-effective, eco-friendly, non-toxic and a versatile

compound used to sequester, buffer, wet, clean and for dispersion (Behera et al., 2021). It has

several applications in food and beverage sectors, confections, soft drinks, and the manufacturing

of medical citrate. It is used in dying, calico printing, engraving, and silvering (Knight et.al.,

2024).

2.3 Biochemistry of Citric Acid

The byproducts of the metabolism of carbohydrates make up the building blocks of many aerobic

and anaerobic microorganisms (Kopp et.al., 2020). According to Scanes (2022), the primary

intermediary in the metabolism of carbohydrates is citric acid. Because of a malfunction in the

tricarboxylic acid (TCA) cycle, certain fungi (like Aspergillus niger) create citric acid as an

overflow product in specific environmental conditions (Rokem et.al., 2020).

TCA cycle involves series of steps in the breaking down of complex polymers (carbohydrates,

proteins and fats) into their respective monomers (glucose, amino acids and fatty acid and

glycerol) accompanied with energy release for biochemical reactions including growth and

metabolism, luminescence and other biological processes (Tegenu 2020). Researches have

shown the essential role performed by the TCA cycle in fermentation by the action of the

enzymes present in various species of microorganisms including A. niger in accumulation of

8
citric acid (Baher et.al., 2020). Citric acid production by microorganisms is solely by the action

of microbial enzymes. The efficiency of citric acid production by microorganisms is defined by

how the enzymes that catalyze the reactions in TCA cycle under various control mechanism are

regulated, such as cofactors associated with the enzymes (Arnold et.al., 2023). Metal ions

constitute an important class of cofactors as such the activity of enzymes can be regulated by

controlling the concentration of the trace elements (Jomova et.al., 2022).

2.4 Global Citric Acid Market

As of 1989, around 0.5 million tons of citric acid and citrate salts were produced commercially

worldwide. In 2015, it reached over 2 million tonnes, and until 2024, it is expected to rise at a

rate of 3.7–4.0% year (Markit, 2015). In 2015, China emerged as the world's leading producer of

citric acid, contributing approximately 59% of the global production and 74% of the global

exports. Citric acid was widely available, and between 2004 and 2013, its manufacturing yield

nearly doubled, leading to historically low prices that reached USD 700 per tonne in 2015

(CCM, 2016). In the last few years, there is increasing demand for citric acid is various

industries, this is attributed to high demand for ready-to-drink beverages and processed foods as

a result of increasing urbanization, rise in economy, disposable incomes and busy lifestyles

(Behera et.al., 2021). Furthermore, the shift toward products of natural ingredients due to the

awareness spreading among people on the toxic effect of chemical constituents of products has

led to upsurge in citric acid production. The citric acid market worldwide is forecasted to reach

USD 3.2 billion by the year 2023 with 5.1% growth rate until 2024 (Market report world.com,

2020). Global citric acid market is classified based on the application into food and beverages,

Pharmaceutics, and others. In food and beverage industry, citric acid is extensively used for the

production of several products including juices, creams, mayonnaise, soup and other beverages.

The market is bifurcated based on the form into the anhydrous and lipid sectors. At present, large

percentage of the total share in the market is accounted for by anhydrous citric acid. Western

Europe has The largest citric acid market in the world based on region. The United States,

9
China, Middle East and Africa, Central/Eastern Europe, Brazil, and India constitute the other

major markets across the globe. Assessment of the import and export data of the market, the

United States represents the biggest importer while China represents the biggest exporter of citric

acid (Behera et.al., 2021).

In 2016, the citric acid market in the United States accounted for approximately USD 448.4

million and is projected to upsurge over the next decade. In 2014, the largest share in the market

was accounted for by the European region, this is due to high demand for citric acid from food

and beverage market of the region as functional food additive (Behera et.al., 2021). Food sector

as an application of citric acid dominates accounting for more than 75% share in terms of volume

and value. It had the most applications and is projected to rise in the coming years (Babers et.al.,

2021). The Pharmaceutics and cosmetic sector has witnessed a remarkable growth over the past

few years and forecasted to increase at a competitive growth rate in the next five years. The

introduction of various citric acid-based products aid the penetration of various sectors by

manufacturers. There are expectations for increase in supply to consumers due to the growing

demand for these products (Behera, 2020).

The focus of every industry is to increase the production capacity to suffice the growing demand

for various products. Jungbunzlauer, producer of large volume of biodegradable ingredients, in

2017, initiated the construction of new citric acid plant in Austria. WHO reported an estimate of

over 65 million cases of digestive system problem in North America. There is increase in

demand for citric acid-based food and beverages and also pharmaceutical products as a result of

the growing awareness about the non-toxic nature of citric acid. The awareness was reported by

the German Nutrition Society to have increased in Europe from 44% to 46% between 2015 and

2016 (Behera et.al., 2021). Researches have reported that the consumption of citric acid-based

products for maintenance of digestive health has significantly increased. Rapid growth in citric

acid market is recorded in the Middle East due to the increasing awareness in the region about

citric acid-based food and beverages and pharmaceutical products. Citric acid plants have been

10
constructed by various international market participants across the region in order to increase

their supply. In January 2017, a pharmaceutical manufacturing plant was constructed in Saudi

Arabia by the Pfizer pharmaceutical company. Over the coming years, it is anticipated that the

Chinese market for citric acid would grow significantly throughout Asia Pacific. Numerous

causes, including the growing population and the frequency of different health illnesses like

mental health difficulties, cardiovascular ailments, and other digestive system concerns,

contribute to the growth (Behera et.al., 2021).

2.5 Biochemical Pathway for Citric Acid Production

In the breakdown of glucose, oxidation of pyruvate occurs and the product is combined with

coenzyme A to yield Acetyl Co-A, carbondioxide and nicotinamide adenine dinucleotide +

hydrogen (NADH) (Behera et.al., 2021). Subsequently, citric acid is formed by the reaction of

Acetyl Co-A with oxaloacetate (Figure 2.2) (Behera et.al., 2021). In the biochemical pathway of

glucose breakdown, pyruvate carboxylase catalyzes the carboxylation of pyruvate to yield

oxaloacetate (Prochownik et al., 2020). Citric acid formed from the reaction of Acetyl Co-A with

oxaloacetate passes through a series of reaction which yield two molecules of carbondioxide

which in turn result in the production of four carbon oxaloacetate (Ben Hsouna et.al., 2023).

Therefore whole cycle is characterized by utilization of one molecule of acetic acid to yield two

molecules of ATP and CO2, and citric acid is produced by the reaction of Acetyl Co-A with one

molecule of oxaloacetate (Pu et.al., 2020).

11
 Figure 2.2. Biochemical pathway for citric acid production (Behera et al., 2021).

The biochemical pathway of produces citric acid is depicted in Figure 2.2 (Behera et.al., 2021).

Enzymes are essential in the production of citric acid. Numerous enzymes work together along

the glycolytic pathways to catalyze the accumulation of citric acid from the metabolization of

glucose (Chandel et.al., 2021). Börekçi et.al., (2021) reported that the reversible reaction of

Acetyl Co-A and oxaloacetate is catalyzed by the enzyme citrate synthase which favors citric

acid formation. Kim et.al., (2015) proposed that the inhibition of various enzymes including

aconitase, isocitrate dehydrogenase and succinic dehydrogenase during the TCA cycle result in

accumulation of citric acid. Contrarily, the study of Morgunov et.al., (2019) reported the

association of these enzymes in fermentation process in minute concentrations. Thus,

accumulation of citric acid is as a result of enhanced biosynthesis, rather than inhibited

degradation (Tahjib et.al., 2021).

The disaccharide sucrose is splitted into its monomers glucose and fructose by the enzyme

invertase, a membrane bound enzyme outside the cell in A. niger, the monomers are thereafter

moved inside the cell by glucose transporters (Daniele et.al., 2022). When glucose enters the

cell, the enzyme hexokinase phosphorylates it to generate glucose-6-phosphate, starting the

12
glycolytic pathway that breaks down glucose. During glycolysis by A. niger, citric acid

accumulation results from the inhibition of certain enzymes (Książek et.al., 2023). For instance,

the anabolic process of A. niger is inhibited by the insufficiency of elements such as manganese,

phosphate or nitrogen resulting in protein breakdown subsequently leading to increase in the

concentration of intracellular ammonium ion (Behera et.al., 2021).

Phosphofructokinase enzyme having magnesium as cofactor is inhibited by high concentration of

intracellular ammonium ion, This leads to in the conversion of fructose-6-phosphate to fructose-

1,6-bisphosphate during glycolysis (Figure 2) (Behera et.al., 2021). Citric acid is accumulated in

the process from the flux through glycolysis brought about by the inhibition of

phosphofructokinase. The study of Bahera et.al. (2020) showed that the presence of intracellular

ammonium pool inhibited the phosphofructokinase enzyme that result in citric acid accumulation

by A. niger. In the course of their research, they observed that phosphofructokinase will inhibited

by glucosamine which is formed by ammonium ion and glucose, then discharged into the

fermentation broth.

The presence of Mg2+ and Mn2+ activate the NADP+ dependent enzyme isocitrate

dehydrogenase located in the cytoplasm and mitochondria. Citric acid accumulation is also

triggered when α-ketoglutarate inhibits this enzyme (Lengedre et al., 2020). The synthesis of the

enzyme α-ketoglutarate dehydrogenase which is an essential enzyme involve in the regulation of

TCA cycle is repressed by high concentrations of glucose and ammonium pool as such inhibited

the citrate cycle movement which results in citric acid accumulation (Bahera et.al., 2021).

Thus, exact connectedness between citric acid accumulation, intracellular ammonium ion

concentration, glucose and the enzymes involved in TCA cycle is yet unclear and certainly needs

further research (Bahera et.al., 2020).

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2.6 Microorganisms Employed for Citric Acid Production

Citric acid is produced by several microbial species as metabolite of primary metabolism. (Odu

et.al., 2020). Vast array of microbial strains have been employed in citric acid production in the

past century, including yeast, fungal and bacterial strains as shown in Table 2.1 (Behera et.al.,

2021).

Mazinanian et.al. (2015) recounted that large amount of citric acid is produced by various

bacterial species including Arthrobacter paraffinens, Bacillus licheniformis, and

Corynebacterium sp. A number of yeast species including some species of Candida, Kloekera,

Saccharomyces, Torulopsis, Yarrowia and Zygosaccharomyces were also reported to produce

appreciable amount of citric acid (Börekçi et.al., 2021). Studies have shown the capability of

yeast species in citric acid production, however, the major setback associated with the production

of citric acid by yeasts is the production of undesired by-products such as iso-citric acid (Börekçi

et.al., 2021). In addition to yeasts and bacteria, numerous fungal species have also been studied

for their potential to produce citric acid including Aspergillus niger, A. flavus and other

Aspergillus species, species of Mucor, Penicillium citrinum and few other Penicillium species,

Talaromyces sp., Trichoderma viride and Ustulina vulgaris (Koul et.al., 2020).

Table 2.1. Citric acid producing microorganisms (Behera et.al., 2021)

____________________________________________________________________________
Microorganisms. Citric acid producing species
____________________________________________________________________________
Bacteria. Achromobacter sp., Aerobacter sp., Alkaligenes sp.,

Arthrobacter paraffinens, Bacillus licheniformis,

B. subtilis, Brevibacterium flavum, Corynebacterium sp.,

Micrococcus sp., Pseudomonas sp., Klebsiela sp.

14
Fungi. Aspergillus niger, A. flavus A. carbonarius, A. foetidus,

A. aculeatus, A. awamori, A. wentii, Mucor sp.,

Penicillium citrinum, P. janthinelum, Talaromyces sp.,

Trichoderma viride, and Ustulina vulgaris

Yeast. Candida tropicalis, C. Citroformans, C. Parapsilosis,

Kloekera sp., Saccahromicopsis lipolytica,Torulopsis sp.,

Yarrowia lipolytica and Zygosaccharomyces

_____________________________________________________________________________

A. niger, a filamentous fungus is the most commonly employed microorganism for industrial

citric acid fermentation (Bahera et.al., 2020). Researchers became interested in the accumulation

of citric acid by A. niger in the second half of the 20th century due to the tremendous

advancements in the biological sciences and the growing body of knowledge regarding

metabolism (Lende et.al., 2021). Several studies were conducted to ascertain the cause of citric

acid accumulation by A. niger and the conditions responsible for the accumulation in order to

potentiate the production through genetic modification. Various biochemical events were

reported to be associated with the overflow of citric acid, however, strains have different

potential to produce citric acid (Börekçi et.al., 2021). Researchers identified the enzymes and

metabolic process that control the build up of citric acid in the early 21st century, and this

description is now widely accepted theory (Muchowska et.al., 2020).

2.7 Strain Improvement for Citric Acid Production

The goal of strain improvement is to maximize the strain's potential to yield more of a desired

product. As demonstrated in Figure 2.3, techniques for optimizing industrially useful strains

include gene cloning, DNA recombinant technology, mutation (either by chemical or physical

15
mutagens), and protoplast fusion. (Behera et.al., 2021). The most common among these

techniques which are also considered the simpler are random mutation and protoplast fusion.

Chemical, physical and site-directed mutagenesis are the various mutagenic processes employed

in strain improvement (Behera et al., 2021). Improvement of industrially beneficial strains has

been considered in commercial production process for overproduction of industrially valued

products (Jeevanandam et.al., 2021). Modification of the metabolism of microorganisms is also a

means of improving strain, this is done by using physical or chemical mutagens to induce

mutation (Yu et.al., 2020). The most widely used chemical mutagens to cause mutation include

diethyl sulfonate, ethyl methane-sulfonate, ethidium bromide aziridine, N-methyl-N-nitroso-

guanidine, and N-nitroso-N-methyl urea. The most widely utilized physical mutagens are

ultraviolet and gamma radiation (KHURSHEED et.al., 2021).

16
 Figure 2.3 Overview of strain improvement (Behera et.al., 2021).

In 1953, Gr Pontecorvo and his research team described the parasexual cycle as a technique of

strain improvement (Behera et.al., 2021). They reported that the parent haploids yielded lower

concentration of citric acid as compared to the diploids. In 1988, Kohtaro Kirimura and his

colleagues successfully manipulated the genetic make-up of A. niger by protoplast fusion to

enhance the citric acid production (Behera et.al., 2021). Other alternative methods used for

improvement of strains are the single-spore technique and passage technique employed for

selecting improved strains (Behera et.al., 2021).

2.8 Substrates for Citric Acid Production

Substrate is an essential component in citric acid fermentation which that reduces the cost and

ensures optimal yield. According to Hesham et.al. (2020), substrate is thought to be the most

17
crucial component for productivity and fermentation yield. A. niger may ferment a wide variety

of substrates, including waste paper, molasses, starch, sucrose, syrups, hydrol, oil, and fat,

among others, to produce citric acid (Adele et.al., 2022). The purity of the substrate used

determines the yield of the finished product and the length of the fermentation process (Rawoof

et al., 2021). Pretreatment of substrate prior to fermentation for citric acid production is

immensely important to breakdown the lignin content of the paper (Laltham et.al., 2022). The

frequently used chemical for pretreatment of substrates is sodium hydroxide which effectively

breakdown the lignin content of the paper (Naicker et.al., 2022). Pretreatment can also be done

using certain enzymes. Wates paper are oftenly utilized for the production of citric acid to

minimize the cost of production (Sharma et al., 2020). Wide range of wastes can be employed in

citric acid production such as waste paper, corn cob, sugarcane bagasse, cassava bagasse, yam

peel, orange peel, pineapple peel, banana peel and among other (Dutta et al., 2019).

2.8.1 Waste Paper

Waste paper refers to paper products that are discarded and no longer needed, including items

like newspapers, magazines, office paper, cardboard, and packaging materials (Bajpai 2024).

Effective management of waste paper is essential. Using waste paper in citric acid production is

an innovative approach aimed at reducing waste and promoting sustainability. Citric acid is a

valuable organic compound widely used in food, pharmaceuticals, and cosmetics, and its

production from renewable resources like waste paper can enhance environmental sustainability

(Yadav et.al. 2022).

2.8.2 Starch

Citric acid is commonly produced from various starchy materials including wheat, corn and

tapioca (Golachowski et.al., 2020). The purity and method of hydrolysis determine the quality

and suitability of the substrates (Zhang et.al., 2023). Hydrolysis is done using Acid hydrolysis

and/or enzymatic hydrolysis (Suresh et.al., 2020). Starchy substrates are prepared on the basis of

18
their enzymatic liquefaction and saccharification. Addition of nutrients depends on the type of

starch use. The medium is acidified to pH 3-4 with hydrochloric (HCl) or sulphuric acid (H 2SO4),

and sterilized at 121°C for 30 minutes to an hour (Aboyeji et al., 2020).

2.8.6 Alkanes

Alkanes were commonly utilized for industrial fermentation of citric acid during the 1960s and

1970s owing to their cost-effectiveness and suitability for fermentation by microorganisms. A

typical example is the fermentation of alkanes for citric acid production using the yeast specie

Candida lipolytica as the producing strain (Börekçi et. al., 2021). At present, a few industrial

citric acid processes that involve the use of alkanes. These processes also result in the production

of isocitric acid that would cause problem during product recovery and also reduces citric acid

yield (Lende et.al., 2021).

2.8.7 Oils and fats

In present times, oil has been widely used as carbon source for the production of citric acid.

Citric acid yield of 145% was reported using palm oil as the carbon source and a mutant of

Candida lipolytica (Naserzadeh and Mahmoudi, 2018). Wide variety of oil exist that are often

used in minute quantities in fermentation set up and may even constitute the chief source of

carbon for A. niger fermentation (Behera et.al., 2020). Researchers have affirmed that the

production of citric acid from these substrates resulted in good yield. It has been forecasted that

alkanes previously used for citric acid fermentation may be replaced by these oils and fats,

however, there could be change in price from the current low prices (Behera et.al.., 2020).

2.9 Applications of Citric Acid

The key benefits of Citric acid's that allowed it's widely used in many different industries, are its

non-toxicity and adaptability. World Health Organization and Food and Agriculture

Organization endorsed citric acid as a beneficial food additive and globally accepted as

19
"generally recognized as safe" (GRAS). It has wide industrial applications as depicted in Table

2.2 (Börekçi et.al., 2021). Food and beverage industry accounts for over 60% of its applications,

owing to its pleasant flavor, safe and non-toxic nature coupled with its high solubility in non-

polar solvent, buffering and chelating potential (Behera et.al., 2021). Carbonated drinks are

made with the addition of citric acid to improve their flavor and taste. Citric acid is used to boost

the antimicrobial potential of certain food preservatives (Zhang et.al., 2023). It's used to enhance

flavor and modify pH in jam and jelly end products. For the best gelation, pH must be adjusted

within a specific range (Marques et.al., 2020).

A concentration of 0.5-2.0% citric acid is in confectionery industry as flowing agent (Zhang

et.al., 2021). Citric acid is employed in optimizing the shelf life of frozen food products due to

its chelating and pH adjusting properties, this is achieved by improving the antioxidant activity

and controlling the activity of enzymes. The shelf life of shellfish and frozen fish is prolonged

upon treatment with citric acid (Abd El-Fatah et.al., 2023). Deterioration and colour change of

fruits can be inhibited with citric acid (Bhat et.al., 2021). Concentration of 0.005–0.02% citric

acid is used as an antioxidant synergism in fat containing food products. It is used as a flavor

adjunct in sherbet and ice creams (Marques et.al., 2020). In Pharmaceutical industry, citric acid

is used as elixirs to mask the unpleasant taste in medicine and also as suspensions for buffering

and maintaining the stability of active ingredients and to improve the effectiveness of wide

variety of preservatives (Nangare et.al., 2021). Citric acid is employed in chemical and cosmetic

industry to adjust the pH during cosmetic formulations, and also as a metallic ion chelator in

antioxidant systems (Nangare et.al., 2021). In detergent industry, citrate is utilized as an effective

and non-toxic substituent for phosphate in non-phosphate detergent powder due to its detergent-

building properties (Behera, 2020). Citric acid-based metal cleaning solutions effectively remove

metal oxidation products from both ferrous and non-ferrous metal surfaces (Bahera et.al., 2020).

Research has demonstrated that citrates are useful for plating a variety of heavy metals, including

lead (Ali et.al., 2021), copper (Saeki et.al., 2020), nickel (Takuma et.al., 2018), and chromium

20
(Ma et.al., 2020).Many industries, including the paper and pulp, textile, and tobacco industries,

require a lot of citric acid and its salts and esters. It is utilized in photography as a part of printing

plate emulsions in different oil well treatments, cements, and bleaches, fixers, and stabilizers

(Behera,2020).

21
 Table 2.2: Applications of citric acid in various industries

___________________________________________________________________________________________________________
Food Animal feed Complement feed
___________________________________________________________________________________________________________
Confectionery Used as acidulant. It prevents sucrose from crystallizing, enables dark

coloration of sweets, sugar syrup inversion. Improves sourness.

Dairy products Used for emulsification in ice cream and processing of cheese, antioxidant and

pH stabilizer in cheese production.

Fats and oils Acts as stabilizer. Acts in synergy with other metabolites to prevent fats oxidation.

Frozen fruits Protects ascorbic acid by inactivation of trace metals. Inactivation of oxidative

enzymes by stabilizing the pH.

Jellies and jams Stabilizes pH to enable pectin act as gelling agent. Enhances flavor, tartness and

spicines. Improves the antimicrobial potential of preservatives.

Beverages Fruit and vegetable juices Acts by stabilizing pH in commercially prepared juices

22
 Soft drinks and syrups Improves tartness and flavor in fruits and carbonated and other saccharose-based

beverages.

Wines and ciders Prevents formation of brown coloration in white wines. Prevents turbidity.

Acts as antioxidant and pH stabilizer.

Pharmaceutics Pharmaceuticals Enhances active ingredients dissolution. Anticoagulant and acidifying agent

in mild astringent formulations. Acts in synergy with bicarbonate as

effervescent in tablets and powders. Solubilizes cathartics. Prevents oxidation

during vitamin preparations.

Cosmetics and toiletries Prevents oxidation and stabilizes the pH. Chelates and buffers metal ions.

Other Other Base neutralizer, sequestering of metal ions, acts as buffering agent.

Removes oxides of metal from surfaces of ferrous and non-ferrous metal.

Cleans oxides of iron and copper. Complexation of ion in ceramic making.

Coating of metals, cleaning of metal, copper plating, leather making, printing inks,

detergent formulation, cement making, fabrics, photography, treatment of waste,

23
 concrete construction, production of plaster, paper and polymers, tobacco

processing, adhesive formulation, chemical conditioner on teeth surface.

24
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Collection of Samples

3.1.1 Collection of wastes paper

Paper waste (foolscap paper l) were collected from waste bins and dumpsites in Federal

University Of Technology Minna which was used as substrate for saccharification and cellulase

enzyme production. The substrates was collected in container and taken to Microbiology

laboratory of Federal University of Technology Minna Gidan Kwano Campus.

3.1.2 Collection of soil samples

Soil samples for the substrates were collected from dumpsites of Federal University Of

Technology Minna Gidan Kwano Campus. The soil sample was aseptically collected using

spatula into sample bottles, accurately labeled and taken to Microbiology laboratory of Federal

University of Technology Minna Gidan Kwano Campus.

3.2 Preparation of media

The media minimal salt agar and potato dextrose agar was prepared for the isolation and

screening of fungal isolates with citric acid producing potential.

3.2.1 Preparation of Minimal salt agar medium

Minimal salt medium weas prepared by weighing and dispensing the require amounts of each

component (Table 3.1) in 1000 mL of conical flask and appropriately dissolved with 900 mL

distilled water.The pH of the medium was adjust around 6.0-6.5 using a suitable base (like

NaOH). Optimal pH range was maintain for the growth of microorganism capable for citric acid

25
production. The medium is sterilize by autoclave at 121°C for 15-20 minutes and was allowed to

cool to pouring temperature (45-50 °C). Then dispense into sterilize plate.

Table 3.1 Component of minimal salt medium

____________________________________________________________________________
Ingredients Composition
____________________________________________________________________________
Glucose 5g/L

Ammonium sulfate (NH₄)₂SO₄ 3g/L

Dipotassium Hydrogen phosphate (K₂HPO₄) 0.5g/L

Magnesium sulfate heptahydrate (MgSO₄·7H₂O) 0.2g/L

Calcium chloride (CaCl₂) 0.1g,/L

Ferrous sulfate heptahydrate (FeSO₄·7H₂O) 0.01g/L

pH 6.0-6.5

Agar Agar 15g

Potatoes dextrose agar was prepared by washing 200g of peeled Irish potatoes and boil them for

20 minutes in 100 ml of distilled water that has been sanitized. After the supernatant is emptied,

the volume increased to 1000 ml with sterilized distilled water in a clean 1000 cm³ Erlenmeyer

flask. Using a weighing scale, 20.0g of agar and glucose powder are added The flask's opening

was sealed with an aluminum foil-wrapped cotton wool cork, with addition of 1g of

chloramphenicol to suppress bacterial growth (Yang et.al., 2020). The prepared medium is

26
autoclave for 15 minutes at 121°C, allow to cool to pouring temperature (45–50°C), and then

aseptically pour into petri dishes.

3.3 Isolation and screening of Aspergillus niger strain

One gram of the soil sample was weigh using the weighing balance and dispensed in a clean test

tube containing 9 ml of sterilize water and thoroughly shaken. Ten fold serial dilution was

appropriately carry out using a sterile disposable syringe and using the pour plate method of

inoculation, 0.1 mL of the dilution from test tube 3, 5 and 7 with dilution factors dilution factors

103, 105 and 107, is transfer aseptically to sterile petri plates. 20 ml of molten freshly minimal salt

medium was pour over and the plate were gently swirled and allow to gel. The inoculate plates

was wrap in aluminum foil paper, inverted and place in an incubator at ambient temperature see

(25 ± 2°C) for 5 days (Chauhan et.al., 2020).

Duplicate of soil sample was incubated at 25 ± 2°C, for 5 days and examine visually (i.e colonial

morphology) as well as microscopically (i.e wet mount) daily for preliminary identification of

fungal genera. The identified genera are then sub cultured on potato dextrose agar plates for

species identification (Dutta et.al., 2019).

3.3.1 Macroscopic and microscopic identification of Aspergillus niger

The isolated Aspergillus niger strains was identify base on their cultural and morphological

features. These include colony colour, shape, margins and texture amongst other macroscopic

characteristics, as well as microscopic observation of the strains including hyphae, conidiophore

vesicle and conidia head (Šimonovičová et.al., 2021).

3.4 Pretreatment of waste paper

The pretreatment of the waste paper involves rinsed of the substrates with clean running water,

sun-drying for 5 days and milling into powder. 5 g of the powdered substrates was weigh using

the weighing balance, and soak with 250 ml of prepare NaOH solution for 10 minutes.

27
Preparation of NaOH solution was done by dissolving 20g of NaOH base in 1000 ml distilled

water and gently shaken to obtain homogeneous mixture. The substrates are thereafter

microwave for 5 minutes to delignify the substrates. Lignin has inhibitory effect on the microbial

enzyme to be use for hydrolysis. The microwave substrates is thoroughly washed using clean

water to remove the NaOH, the washing continues until neutral pH is attain, this are confirm

with the aid of pH paper. The substrates are then air-driy to have the extract which are

hydrolyzed (Ali et.al., 2016)

To pretreat waste paper for citric acid production, several steps are typically involved to ensure

the efficient breakdown of cellulose into fermentable sugars. Here are the key steps in the

pretreatment process: (i) Collection and Sorting: Collect waste paper from various sources and

sort it to remove contaminants like plastic, metal, and other non-paper materials. (ii) Shredding:

Shred the sorted waste paper into small pieces to increase the surface area for better access to

enzymes during subsequent processing. Shredding in the pretreatment of waste paper involves

cutting the paper into smaller pieces to enhance its biodegradability for processes like anaerobic

digestion. (iii) Chemical Pretreatment: Treat the shredded paper with chemicals like alkalis, or

enzymes to break down the lignin and hemicellulose components, which can inhibit enzymatic

hydrolysis of cellulose. The pretreatment involved mixing the paper samples with a 1% sodium

hydroxide (NaOH) solution, followed by sonication for 15-60 minutes. (iv)Hydrolysis: Subject

the pretreated material to hydrolysis, where enzymes break down cellulose into fermentable

sugars like glucose. Hydrolysis of pretreated waste paper involves breaking down the cellulose

and hemicellulose components of the waste paper into fermentable sugars through enzymatic

processes. The pretreatment of waste paper is crucial to make the cellulose more accessible to

enzymes during hydrolysis, enhancing the efficiency of sugar production for subsequent

processes like fermentation to citric acid. The pretreatment methods, such as chemical, physical,

or biological treatments, aim to soften the biomass, open its cellular structure, and improve the

28
enzymatic degradation of cellulose. The resulting sugars (Glucose) from the hydrolysis of

pretreated waste paper can then be used for the production of citric acid.

3.5 Cellulase enzyme production

Cellulase enzyme was produce according to the method of Deacon (1985) using Czapex dox

broth which contains several components (Table 3.2). Each of the components was measured

using weighing balance and dispensed In 1000ml conical flask, dissolved in 1000ml of distilled

water and autoclaved at 121°C for 15 minute to attain sterilization and then allowed to cool to

pouring temperature (45-50°C). 10ml of inoculant (Aspergillus niger) from potatoes dextrose

broth was inoculated into the prepared Czapex dox broth, wrapped in aluminum foil paper and

incubate for 7 days at 25°C.

Table 3.2: Components of czapek dox agar medium

____________________________________________________________________________
Ingredient Composition
____________________________________________________________________________
Sucrose 30.0 g

Sodium nitrate 2.0 g

Dipotassium phosphate 1.0 g

Magnesium sulfate heptahydrate 0.5 g

Potassium chloride 0.5 g

Iron(II) sulfate 0.1 g

Bromocresol green dye 4.0 ml

Chloramphenicol 1.0 g

29
Distilled water 1000 ml

3.6 Hydrolysis of waste paper using cellulase produced by Aspergillus nger

Waste paper was hydrolyzed accordance with Makut and Ekeleme (2018) by measuring 20g of

extract, and the result is dispense in clearly labeled 250ml conical flasks with a tight-fitting cork

filled with the substrate and 20ml of cellulase enzyme. To guarantee that the enzyme is

distributed evenly, the conical flasks was placed in a water bath at 60°C for 48 hours while being

shaken frequently. After 48 hours of incubation, the hydrolysis was stopped by increasing the

water bath's temperature to 100°C for 1 hour, which totally inhibit the activity of the enzyme.

The hydrolysate is obtained by centrifuge the culture to separate the enzyme-rich supernatant,

sieving the hydrolyzed substrates through muslin cloth and then filtering them again using

Whatsmann filter paper No 1. (Oyewale et.al., 2023).

3.7 Cellulase Activity

Cellulase activity was assayed according to method of Mandel's (1985) using Carboxymethyl-

cellulose (CMC) as substrate. The reaction mixture contained 1ml of 1.0% (w/v) CMC in 0.1M

solution of sodium acetate buffer, pH 5.0, and 0.5 ml of the cell-free culture supernatant. The

mixture will be incubated at 500°C for 30-60 minutes. The reducing sugar released by the

enzyme will be measured as glucose equivalent using dinitrosalicyclic acid reagent. A unit of

activity is defined as the amount of enzyme required to liberate 1 mol of glucose per minute

under the assay conditions.

3.8 Determination of reducing sugar concentration

After the substrate extract has fully hydrolyzed to produce hydrolysate, the reducing sugar

concentration was determine by volumetric methods using Fehling's solution, this involves

titrating the reducing sugar solution against a standard Fehling's solution copper(II) in alkaline

tartrate until the blue color disappears, indicating the endpoint.

30
3.9 Fermentation of citric acid

The fermentation of citric acid was carryout by preparing a broth solution, 9ml of sterile water in

a testube and a gram of Aspergillus niger was then inoculated vigorously shaken to obtain

homogeneous mixtures and 3.5ml of the Inoculant was dispensed into 35ml of hydrolysate,

incubated at 25°C for 72, hours.

3.10 Qualitative analysis for citric acid productions

All of the medium's components in Table 3.1 was added to create a minimal salt medium with

the exception of glucose, 20 ml of hydrolysate substrate was added in place of the glucose as the

carbon source. The medium was then dispensed in sterilize 100 ml conical flask and autoclave at

121°C for 15 minutes and allowed to cool to pouring temperature (45-50°C). After cooling, the

isolates was inoculate into the medium and incubate at ambient temperature (25 ± 2°C) for a

duration of 3 days with daily observation for growth. The isolate with the highest and most rapid

growth was subculture on a PDA slant bottle as the most efficient strain.

3.11 Determination of citric acid concentration

The citric acid concentration in a sample can be determined using quantitative analysis methods

such as titration, the sample is titrate with a standard solution of sodium hydroxide (NaOH) using

phenolphthalein as the indicator. The volume of NaOH use to reach the endpoint corresponds to

the amount of citric acid present. The amount of citric acid produce is calculate using the

formula;

C1V1 = C2V2

31
 CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 Results

4.2 Macroscopy of isolates on Czapek dox agar medium and PDA

The macroscopic examination of fungal isolates isolated from soil samples expressed differences

in the colony color, shape, borders, elevation, texture, and growth rate of the colonies (Plate).

Table 4.1 shows the morphological features of Aspergillus species isolated on minimal salt

media, which is basically Aspergillus niger. The colonies appeared initially as white colonies and

are then transformed to yellow and after a few days finally turned black and produced conidial

spore. On sub culturing on PDA medium, the isolates appeared initially white and changed to

black as the colony enlarged, irregular in shape, raised elevation and undulate margin (plate II)

shown in Table 4.2.

Plate I. Colonial morphology of isolates on minimal salt medium, A: black coloration of

32
 Aspergillus niger, B: Yellow coloration of the intermediate stage of A. niger, C: white coloration

of the initial stage of A. niger.

Plate II. Colonial morphology of isolates on PDA medium, M: PDA medium; N: Pure isolate of

A. niger appearing black.

Table 4.1: Macroscopy characteristics of isolates on minimal salt medium

___________________________________________________________________________
Characteristics Morphology
___________________________________________________________________________
Surface colour From yellow to black

Shape irregular

Texture Velvety

Growth Rapid

Elevation Raised
__________________________________________________________________________

33
Table 4.2: Macroscopy characteristics of isolates on potatoes dextrose agar medium

_________________________________________________________________________
Characteristics Morphology
__________________________________________________________________________
Surface colour Dark brown

Shape irregular

Margins Slightly undulate

Texture Velvety

Elevation Raised

Reverse side Pale yellow

Growth Rapid
__________________________________________________________________________

4.3 Microscopy of isolates

After completion of macroscopic examination, the isolated Aspergilus species were examined

using wet mount technique for their microscopic features and are shown in Plate III and Table

4.3. The hyphae were branched septate with globose conidiophore vesicle and black conidia

head.

34
M

Plate III. Microscopy of isolates. M: Hypha; N: Conidia

Table 4.3: Microscopy characteristics of isolate

___________________________________________________________________________
Characteristics
___________________________________________________________________________
Hyphae Branched septate

35
Conidiophorevesicle Globose

Conidiahead Black
__________________________________________________________________________
4.4 Enzyme activity analysis

Enzyme activity during cellulose enzyme production are depicted in Table 4.4, Table 4.4 shows

the cellulase activity and protein concentration of crude cellulase enzyme, with values expressed

as mean ± standard error of mean (SEM). The enzyme exhibited a mean cellulase activity of

11.92±0.43 U/mL, indicating its catalytic capability. Additionally, the protein concentration is

2.91±0.26 mg/mL, representing the amount of protein present.

____________________________________________________________________
Sample Activity (U/mL) Protein (mg/mL)
____________________________________________________________________
Microbe 11.85 2.96

12.71 2.43

11.21 3.34
____________________________________________________________________

4.5 Reducing sugar concentration of the substrates

TThe waste paper hydrolysate exhibited a reducing sugar concentration of approximately 20 g/L,

as quantified by the Benedict's test method, corresponding to a hydrolysis efficiency of 14.29%.

36
Table 4.5: Result on reducing sugar concentration of the substrates

______________________________________________________________________________
Substrate weight of Beneidicts test Estimated reducing hydrolysis
The paper colour change sugar(g/l) efficiency
(%)
______________________________________________________________________________
Waste paper 140 orange to red ppt 20 14.29
____________________________________________________________________________
4.6 Citric acid concentration

The absorbance reading for the substrates are given in Table 4.6. the absorbance reading was

recorded for waste paper with value of 1.2

Table 4.6 Result on spectrophotometric absorbance for citric acid concentration

____________________________________________________________________________
Substrate absorbance (420 nm)
____________________________________________________________________________
Citric acid 1.2
____________________________________________________________________________

4.7 Discussion

Aspergillus niger is considered a common natural flora found in soil and also associated with

with variety of edible products including grains, bread, vegetables, wheat bran, banana and other

fruits. Strains of A. niger form filamented hyphae (filamentous in nature) appearing like small

plants. Macroscopic observation of the isolated A. niger strains on czapek dox agar medium

37
revealed yellow zone around the colonies which appeared black producing conidial spore. This

corresponds with the result of Sawant et.al. (2018) who reported yellow coloration around the

fungal colonies on czapek dox medium containing bromocresol green indicator evincing

production of citric acid. Dutta et.al. (2019) asserted that change in pH translates into a change in

the structure of bromocresol green indicator. The production of citric acid by A. niger in the

medium results in its diffusion throughout the medium, hence reacts with the dye leading to

colour change of the dye from blue to yellow. On potatoes dextrose agar it started as white

colony and changed to black colony with irregular elevation and entire margin with pale yellow

edges, this was in line with the result obtained by Abbas et.al. (2016), pale yellow coloration was

produced around the edges of the colonies with radial fissures.

Microscopic observation of the isolated A. niger strains revealed black colored conidiophore and

conidia. Singh et.al. (2016) recounted that A. niger is a mould of the Deuteromycota class of

fungi which reproduces asexually and produces black coloured erect conidiophores having

globose columella at the tip. The conidial heads appear radial and they split into columns

(biseriate). Metulae which are sterile cells are produced by conidiophore vesicle which give

support to the phialides on the conidiophores (Alnassar et.al., 2016). The conidiophores appear

somewhat long, smooth and hyaline. Globose vesicle is formed at the tip of the conidiophore

which becomes dark. The vesicle is covered by metulae and phialides. Conidia are produced by

phialides appearing dark brown with rough texture (Singh et.al., 2016).

The waste paper showed degree reducing sugar concentration. This is attributed to the percentage

of ash content and the concentration of other nonsugar substances of the waste paper. Behera

et.al. (2021) classified raw materials commonly utilized for fermentation to produce citric acid

into raw materials having low ash content from which removal of cations could be done by

standard procedures and raw materials having considerably high content of ash and high

concentrations of other nonsugar substances.

38
The microbial growth of isolates from soil substrates showed degrees of growth. These isolate

grew more rapidly with higher absorbance. The findings were in confirmation with Khattab et.al.

(2017), who observed higher inoculum size at higher reducing sugar concentration indicating

higher amount of citric acid. In addition, maximum citric acid production at much higher

inoculum concentration was also reported by Maharani et.al. (2014).

The citric acid concentration of the substrates having higher absorbance value of translating

to g/L of citric acid. The results are in agreement with Pathania et.al. (2018) and Bhattacherjee

and Baruah (2015), who reported that the concentration of citric acid is strongly influenced by

the type and concentration of reducing sugar. A higher initial sugar concentration resulting in

increase in citric acid yield and productivity was also reported Shankar and Shivkumar (2016).

39
 CHAPTER FIVE

CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

Citric acid is produce from variety source, however the citric acid produce from waste is proven

to be a promising approach due to its cost effective, efficiency and environmental friendliness.

Aspergilus niger was isolated from soil sample, citric acid was produced from waste paper using

isolated Aspergilus niger with citric acid concentration of 1.2mg/ml.

5.2 Recommendations

From the study's conclusions led to the following recommendations being made:

Using waste paper for citric acid production offers several environmental benefits that contribute

to sustainability and waste management. This include

i. The Large scale application of waste paper as a source of reducing sugar for citric

acid production.

ii. Optimization of condition for citric acid production.

40
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 APPENDIX

Plate i: waste paper before pretreatment

Plate II: waste paper after pretreatment

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 Plate III: Benedict's test result

Plate Iv: hydrolysate for fermentation of citric acid

49