Encapsulation of Urease Inhibitor By Chitosan Biopolymer as Controlled Release

Fertiliser

by

Choo Ee Huey
18510

Extended proposal submitted in partial fulfilment of

the requirements for the
Bachelor of Engineering (Hons)
(Chemical Engineering)

SEPTEMBER 2016

Universiti Teknologi PETRONAS,

32610, Bandar Seri Iskandar,
Perak Darul Ridzuan

ABSTRACT

Controlled release fertilizer has been widely used to reduce fertilizer consumption
as well as the environmental pollutions caused by conventional fertilizers usage. The
present work deals with the synthesis of chitosan/starch polymeric film by incorporating
glutaraldehyde as the crosslinking agent. The synthesized film is subjected to Fourier
Transforms Infrared Spectroscopy (FTIR) and swelling study. Encapsulation of urease
inhibitor is done using the biopolymer film prepared. The effects imposed by the
encapsulation on the controlled urea release behavior of urea fertilizer is studied. Inhibitor
encapsulated with chitosan/starch polymeric blend is expected to exhibit higher inhibition
efficiency than garlic extracts (GE), NBPT and Allicin urease inhibitors.

TABLE OF CONTENTS
ABSTRACT .......................................................................................................................i
CHAPTER 1: INTRODUCTION ................................................................................... 1
1.1

Background ......................................................................................................... 1

1.2

Problem Statement .............................................................................................. 1

1.2.1 Nitrogen Loss through Ammonia Volatilization ....................................... 1
1.2.2 Biodegradability Issues of Polymer Coated CRF ...................................... 2

1.3

Objectives ............................................................................................................ 3

1.4

Scope of Study..................................................................................................... 3

CHAPTER 2: LITERATURE REVIEW ....................................................................... 4

2.1

Ammonia Volatilization from Urea Fertilizer ..................................................... 4

2.2

Enhanced Efficiency Nitrogen Sources ............................................................... 5

2.2.1 Urease Inhibitor .......................................................................................... 5
2.2.2 Controlled Release Fertilizer ...................................................................... 7
2.2.2.1 Overview ........................................................................................ 7
2.2.2.2 Classifications ............................................................................... 8
2.2.2.2.1 Polymer Coated Multi-Nutrient Fertilizers .................... 8
2.2.2.2.2 Coated, Nitrogen Based Fertilizers ................................ 9
2.2.2.2.3 Uncoated, Nitrogen Based Fertilizers ............................ 9

2.3

Starch/Chitosan ................................................................................................. 10

CHAPTER 3: METHODOLOGY ................................................................................ 13

3.1

Project Methodology ......................................................................................... 13

3.2

Experimental Procedures ................................................................................... 15

CONCLUSION AND RECOMMENDATION ........................................................... 20

REFERENCES ............................................................................................................... 21
APPENDICES ................................................................................................................ 25

ii

LIST OF FIGURES
Figure 2.1: Structure of Chitosan ..................................................................................... 11
Figure 2.2 Structure of Starch Consisting of Amylose and Amylopectin with Different
Linkages ........................................................................................................................... 11
Figure 3.1: Flow Chart of Project Methodology .............................................................. 13
Figure 3.2.1: Glutaraldehyde Cross-linked Reaction between Chitosan and Starch ....... 16
Figure 3.2.2: FT-IR Spectrum of Pure Chitosan, Pure Starch and Cross-linked
Chitosan/Starch Film........................................................................................................ 17

LIST OF TABLES
Table 1: Different Samples of Chitosan/starch ................................................................ 17

iii

CHAPTER 1

INTRODUCTION

1.1

Background
According to (United Nations, 2015), the worlds population is expected to reach

9.7 billion by 2050 and subsequent growth in the food production by 70% is required to
meet the surging food demand. This called for a proportional growth in the production of
fertilizers to be supplied to the crops as the main source of nutrient However, the
inefficiency of nutrient assimilation by the agriculture crops has been a deterrent in the
increased fertilizer production (Jamnongkan & Kaewpirom, 2010). The assimilation of
the most crucial nutrient in the optimization of crop yield, nitrogen is reckoned to be
between the range of 30% to 50% (Lubkowski, 2012). The application of control release
nitrogen (N) fertilizers has been adopted in order to reduce nitrate losses from the plants
and subsequently curtailing the environmental pollution resulting from the usage of
nitrogen fertilizers. The fertilizers enable gradual release of nitrogen into the soil in
accordance to the crop nutrient needs (Lubkowski, 2012).

Controlled released nitrogen (N) fertilizers production technologies have been

firmly developed since the 20th century. Control release nitrogen (N) fertilizers can be
categorized into uncoated nitrogen based fertilizers, coated nitrogen based fertilizers and
polymer coated fertilizers. Polymer coated fertilizers advance over other fertilizers in
terms of the regulation of coated product longevity and efficiency of nutrient transfer for
plant absorption (Jacobs, 2005). These fertilizers deliver a more uniform and steady

nutrient release pattern over an extended period. There are various polymer coated
fertilizers in the market at present including Osmocote, Multicote, Plantacote and Meister.
(Lubkowski, 2012). The polymer coating materials used to produce these fertilizers
include alkyd resins, polyurethane and polyolefin (Trenkel, 1997).

Coating material is the main factor affecting the production of polymer coated
fertilizers. Chitosan (CS) is a natural polysaccharide of interest in this scope as it possesses
high biocompatibility and good film forming capability (Maitra & Singh, 2014). The
physical and chemical attributes of chitosan can be enhanced through blending with other
polymers including starch followed by the incorporation of cross-linking agents to form
strong polymeric blend (Okolo, Akakuru, Osuoji, & Jideonwo, 2014; N. Singh & Maitra,
2015). In this work, starch/chitosan polymeric film through crosslinking with
glutaraldehyde is prepared. The wide application of glutaraldehyde as the cross-linking
agent is due to its low cost and high availability. Besides, it possesses high solubility in
aqueous solutions (N. Singh & Maitra, 2015). The verification of film structure is
evaluated with Fourier Transforms Infrared Spectroscopy (FTIR) and swelling behavior
of the film is studied. Finally, the effect of controlled release of urea on soil will also be
conducted.

1.2

Problem Statement

1.2.1

Nitrogen Loss through Ammonia Volatilization

Nitrogen fertilizer sources are susceptible to ammonia volatilization when surfaceapplied to the soil. Gradual and sustained supply of nutrients to the crops for a prolonged
period can be ensured through the application of controlled release fertilizers (CRF). At
present, the most widely used soil urease inhibitor, Agrotain is able to impede ammonia
volatilization from two weeks up to ten weeks and reduces the ammonia volatilization to
approximately 30% of the loss from conventional type of urea. On the other hand, the
most technically developed CRF type, polymer coated fertilizer (PCF) is able to

progressively provide nutrient to the crops at release rates ranging from 3 months up to 18
months long. The efficiency of nitrogen assimilation by the crops has yet to be maximized
by the present enhanced efficiency nitrogen sources. will be stepped up through the
synthesis of chitosan/starch biopolymer. Modifications to the polymer is required to step
up the ability to hinder instantaneous urea escape from the surface of coating.

1.2.2

Biodegradability Issues of Polymer Coated CRF

The current technology of controlled release fertilizers particularly polymer coated

CRF has potential adverse effects on the field conditions. The futile residues of synthetic
polymer remain in the soil following the application.

The disintegration and

decomposition of polymer coating the fertilizer occur at slow pace in the field. This may
result in the buildup of an approximate of 50 kg/ha per year of harmful plastic residues in
the soil (Lubkowski, 2012). Subsequently, the soil composition may undergo undesirable
changes in cases where the decay of polymer shell fragmented particles does not take
place. These polymer scraps may be of smaller size compared to the soil particles and
they would integrate into the soil and become the constituents of the soil. Thus, the
production of controlled release fertilizers using biodegradable and natural polymer in
which its degradation in soil would not result in the generation of substances that are toxic
to the crops are of paramount importance. At present, manufacturing of biopolymer based
controlled release fertilizers has not been done on a technological scale. There have been
comprehensive studies conducted to access the potential of chitosan usage in
agrochemical industry owing to its high biodegradability property but there are limited
literatures done regarding the interest of using chitosan as CRF so far.

1.3

Objectives

The general intentions of this project are manifested through the objectives established
as following:
To synthesize and characterize starch/chitosan based biopolymer using glutaraldehyde
as cross-linking agent.
To examine and analyze the water adsorption through the swelling behavior of the
synthesized starch/chitosan polymer.
To encapsulate urease inhibitor in the synthesized starch/chitosan biopolymer in order
to further enhance the present controlled release property of urea fertilizer.
To make a comparison of the effect of as-synthesized starch/chitosan biopolymer and
chitosan encapsulated urease inhibitor on the controlled release behavior of urea
fertilizer in soil.

1.4

Scope of Study

In this study, a starch/chitosan polymeric film which manifests biodegradability

and microbial activity will be synthesized with glutaraldehyde as the crosslinking agent.
Several potential biopolymers to blend with chitosan will be shortlisted and accessed in
terms of their compatibility with chitosan prior to the synthesizing process.
Characterization of the covalent bonding in the film will be performed through Fourier
Transforms Infrared Spectroscopy (FTIR) in order to validate the molecular structure of
crosslinked film. Besides, characterization of the film will be performed by studying and
analyzing its swelling behavior in distilled water. Encapsulation of urease inhibitor using
the biopolymer film prepared will be carried out to further enhance the efficiency of
fertilizer use. In addition, the effect on controlled release manner of urea fertilizer brought
about by the coating of biopolymer film will be investigated by accessing the amount of
urea remaining in soil after 30 days

CHAPTER 2

LITERATURE REVIEW

2.1

Ammonia Volatilization from Urea Fertilizer

Ammonia volatilization is described as the loss of nitrogen in the form of ammonia

gas into the atmosphere (Jones, Brown, Engel, Horneck, & Olson-Rutz, 2013). NH3
volatilization is most likely to occur when urea fertilizers are applied onto the soil surfaces
without any incorporations. Surface-applied urea fertilizers are subjected to immediate
granular dissolution due to the soil setting (Rawluk, Grant, & Racz, 2001). However, the
movement of urea fertilizer into the soil is confined. The possibility of the risk of ammonia
volatilization falls in between of 0% to 65% of the applied nitrogen (Bishop & Manning,
2008). It is also stated that 0 to 50% of the applied nitrogen is released from the surfaceapplied fertilizers ("Nitrogen Notes,"). The liberation of nitrogen usually happens during
a two to three weeks period after the fertilizer applications (Jones et al., 2013).

Ammonia volatilization results from the hydrolysis of urea once it is dissolved in

water. Hydrolysis process involves the rapid reaction between surface-applied urea
fertilizers with water under the presence of urease enzymes, creating ammonium
carbonate, (4 )2 3 , an alkaline product (Mikkelsen, 2009). Urease enzyme is
produced by microorganisms found abundantly in soil. Since ammonium carbonate is an
unstable compound, it undergoes rapid decomposition to produce 3 along with carbon
dioxide and water. Urea fertilizer will be transformed into ammonium carbonate through
the following reactions shown in Equation 1, 2 and 3 regardless of the method of

application to the soil either in dry granules (solid) or fluid (liquid) form ("Nitrogen
Notes,").

The significant factors which affects the occurrence of ammonia volatilization

includes the placement of urea fertilizers, soil pH and properties, moisture and temperature
regime and nitrogen source (Rawluk et al., 2001). The volatilization process can be
alleviated by adopting a few mitigation approaches which include the placement of urea
fertilizer below the surface of the soil, addition of urease inhibitors in order to decelerate
the hydrolysis process and application of urea fertilizer before the rainfall (Bishop &
Manning, 2008).

2.2

Enhanced Efficiency Nitrogen Sources

2.2.1

Urease Inhibitor

A few products are established to slow down the transformation process of

nitrogen in urea fertilizers including nitrification inhibitor and urease inhibitor. The
presence of these inhibitors shorten the time difference between the time in which nitrogen
is available for plant uptake and the time the crops actually requires the nitrogen, hence
enhancing the efficiency of urea usage (Franzen, 2011). They extend the period of time
available for rainfall adequate enough to shift the surface applied urea fertilizers into the
soil (Watson, Stevens, & Laughlin, 1990). In addition, they permit urea diffusion far off

from the location of application advocating urea uptake by crops in a whole and intact
form (Zanin, Tomasi, Zamboni, Varanini, & Pinton, 2015).

Urease inhibitors hampers the rate at which the enzymatic hydrolysis of urea take
place by decreasing the activity of urease enzyme ("Nitrogen Notes,"). There are a number
of scientific researches and laboratory experiments conducted to look into the inhibiting
properties of several compounds including N-(n-butyl) thiophosphoric triamide (NBPT),
thiophosphoryl triamide (TPT), phenyl phosphorodiamidate (PPD/PPDA) and cyclohexyl
thiophosphoric triamide (CHTPT) (Trenkel, 1997). NBPT is found to be the most effective
soil urease inhibitor in which it is able to exhibit consistent ability to decrease the
occurrence of ammonia volatilization from urea (Watson et al., 1990). Two distinct studies
conducted shown that the amount of ammonia liberated from urea at Brandon, Canada
following the urea application reduces significantly from 40 mg to 2 mg and 88 mg to 12
mg over a seven days period (Franzen, 2011). Urea treated with NBPT is able to achieve
50% to 90% reduction in ammonia loss ("Nitrogen Notes,").

NBPT is also known commercially as Agrotain. It functions through a lock and

key mechanism whereby the inhibitor molecule hooks onto the binding site of urease
catalytic enzyme (Franzen, 2013). With that, the urease enzyme is inactivated and this
leads to the inhibition of ammonia volatilization for up to two weeks. NBPT should be
applied onto the soil when rainfall is not likely to occur at any moment from the time of
application since rainfall surpassing 0.75 inches would obstruct the performance of the
inhibitor (Jones et al., 2013). N-(n-butyl) thiophosphoric triamide constituted 25% of
NBPT as its active ingredient and is listed as a TSCA (Toxic Substances Control Act)
substance (Trenkel, 1997). The inhibitor is found to affect the transport mechanism and
urea assimilation in plants which are grown with urea despite of its general role of
inhibiting urea hydrolysis. The presence of NBPT limits the induction of genes
necessitated for the assimilation of urea (Zanin et al., 2015).

Since there are uprising concerns relating to the physiological effects brought by
conventional chemical based inhibitors on the plants following their applications, bio
6

inhibitors have been garnering attentions and are becoming more sought after due to their
higher potential in terms of environmental sustainability (Mansor, Herng, Samsudin,
Sufian, & Uemura, 2016). A few studies have been conducted to investigate and explore
the potential and suitability of plant derived natural products to be pursued as
urease inhibitors in fulfilling the agriculture interest. These products include extracts
carrying polyphenolics retrieved from the Acacia decurrenss bark, the waste of C.
sinensis, a black tea. Terminalia chebulas seed coat and Allicin have shown as potential
urease-inhibiting property (Modolo, de Souza, Horta, Araujo, & de Ftima, 2015).

In this study, Allicin is opted as the urease inhibitor. Allicin is found in the garlic
cloves that is freshly crushed (Marchese et al., 2016). Enzymatic reaction occurs following
the tissue damage in which alliinase enzyme converts alliin into allicin and produces
thiosulfinates compounds (Borlinghaus, Albrecht, Gruhlke, Nwachukwu, & Slusarenko,
2014). Allicin possesses great capacity of inhibiting the hydrolysis process driven by
urease enzyme in which the activity of enzyme undergoes complete depletion when it is
incubated with garlic extract just for a period of 18 minutes (Modolo et al., 2015). The
inhibition outcome exhibited by thiosulfinates is attributed to the -SH group located at the
active site of urease enzyme to form covalent disulphur compound. Besides, it exhibits
great extent of antibacterial activity and is capable of inhibiting the growth of Gramnegative and Gram-positive bacteria (Juszkiewicz, Zaborska, apta, & Olech, 2004).

2.2.2
2.2.2.1

Controlled Release Fertilizer

Overview

Fertilizer which gradually releases nutrients required for crops uptake in order to
lengthen its period of availability for plants absorption compared to the conventional
nutrient fertilizer including urea and ammonium phosphate in order to reduce the loss of
nutrients between the time of application and absorption is termed as controlled release
fertilizer (CRF) (Liu et al., 2014). CRF eliminated the inconvenience to apply the
fertilizers more than once throughout the entire vegetation period attributed to its slow
7

releasing pattern. Conventional fertilizing method involving urea requires twice or thrice
of fertilizer application for plants uptake to sustain throughout the crops growth cycle
(NAVIA et al., 2014). The nutrient release from CRF is regulated by two crucial factors
which are the coating thickness/weight and temperature. The shelf life of CRF increases
with coating but decreases with temperature (Fan, 2009). Urea is a popular choice for the
applications of CRF since it is cheap, abundant and contains high nitrogen content (Trinh,
Shaari, Basit, & Azeem, 2014).

The nutrients release mechanism of CRF is initiated with the encapsulation of

fertilizer granule with an inert layer. Water particles make way into the inner part of the
granule through penetration across the hydrophobic membrane following the application
of fertilizers. Fertilizer nutrients then disintegrated and dissolved. The increasing osmotic
pressure developed in the granule results in the expansion of membrane, leading to the
incomplete shred of the membrane. Movement of ions across the membrane is mediated
to ions transport into the soil. Coating diffusion coefficient manages the rate at which the
nutrients release take place (Lubkowski & Grzmil, 2007).

2.2.2.2

Classifications

Generally, there are three types of CRF which are polymer coated CRF, coated
and uncoated nitrogen based fertilizers. They differ in terms of the nature of coating and
their respective nutrients composition (Landis & Dumroese, 2009).

2.2.2.2.1

Polymer Coated Multi-Nutrient Fertilizers

The manufacture of fertilizers encapsulated with polymeric coating material

reflects the state of art in the agricultural technology. A core detaining the nutrients for
plant uptake in soluble form is enclosed within the polymeric (Trenkel, 1997). Polymeric
coating particularly its thickness, temperature and moisture permeability meticulously
8

regulates the mechanism of nutrient release from the fertilizer. PCRF is capable of
imparting all the fundamental nutrients of soil fertilizer namely NPK comprising of
nitrogen, phosphorus and potassium along with micronutrients unlike the singular
nitrogen contribution from other groups of CRF. The coating constructed with the latest
technological advancement distinguishes PCRF from others since nutrients are steadily
fed to the plants over a prolonged period ranges from 3 months to 18 months (Landis &
Dumroese, 2009). Examples of PRCF products marketed include Osmocote, Agriform,
Multicote and Nutricote (Lubkowski, 2012).

2.2.2.2.2

Coated, Nitrogen Based Fertilizers

Sulfur coated urea (SCU) is one of the significant examples of coated, nitrogen
based fertilizer (Landis & Dumroese, 2009). The coating is described as an impermeable
membrane whereby it will undergo degradation once subjected to any microbial, chemical
and physical activities (M.E.Trenkel, 2010). The thickness of sulfur coating has an impact
on the nutrient release of SCU (Kang & Banga, 2013). This can be observed through the
occurrence of urea dissolution from the fertilizer into soil when the coating degenerates
or due to presence of micropores and cracks on the fertilizer (Trenkel, 1997). Besides,
temperature factor plays a dominant role in the nutrient release since microbial activities
particularly the microbial attack of sealant surrounding the coating is reliant on
temperature (Trenkel, 1997). The disadvantages of using SCY fertilizers include the
damages caused to the crop when the initial release of nutrients is too quick. Besides,
excessive thickness of the sulfur coating the SCU granules might impeded the nutrients
release to the crops during crops demand period.

2.2.2.2.3

Uncoated, Nitrogen Based Fertilizers

This group of fertilizers had advanced in years compared to the other two types of
fertilizers (Landis & Dumroese, 2009). They are made up of chemically-bound urea and
9

are compounds which possess low solubility. These fertilizers are condensation products
of urea and aldehydes (Azeem, KuShaari, Man, Basit, & Thanh, 2014). There are three
types of products under this category of high market importance include ureaformaldehyde (UF) and urea-isobutyraldehyde (IBDU) and urea-crotonaldehyde. Ureaformaldehyde fertilizers contain 38% of nitrogen and they exhibit notable effect on the
gradual release of nitrogen liberation into the atmosphere. Besides, there is great
compatibility between the fertilizers and the plants since urea-formaldehyde fertilizers
low solubility nature prevents the vegetation from being burned (Trenkel, 1997). The
disadvantage of using urea-formaldehyde fertilizers is that a fraction of the nitrogen
nutrients might be released very slowly or not at all into the soil.

2.3

Starch/Chitosan

Chitosan is a polysaccharide derived from the partial deacetylation of chitin which

is a polymer that is insoluble in water (Phuong, Quyen, Thi, & Khoa, 2014). It is
comprised of -(1-4)-linked Dglucosamine (deacetylated unit) and N-acetylDglucosamine (acetylated unit) (Okolo et al., 2014). It possesses high biodegradability,
biocompatibility, hydrophilicity as well as bio adhesiveness (Okolo et al., 2014).
However, the rate at which the polymer chains relax is low resulting in weak water
absorbency. Blending between chitosan and hydrophilic polymers would enhance its
capacity to swell (Phuong et al., 2014). The existence of amine side group in chitosan
enables it to exhibit polymeric cationic character and to form intermolecular complexes
with carboxylic acid and poly carboxylic acid (V. Singh & Kumari, 2014). Besides,
favorable interaction with biomolecules through binding take place due to the presence of
primary amino groups (Okolo et al., 2014).

10

Figure 2.1: Structure of Chitosan

Starch comprises of two repeating homopolymers of 1,4--D-glucopyranosyl.

They are amylose of (1, 4)-linked glucose and branched amylopectin (1, 6) glycosidic
linkages of (V. Singh & Kumari, 2014). It is a polymer in semi-crystalline state made up
of varying percentage of amylopectin and amylose depending on the source of starch.
Starch is generally made up of 10-20 % of amylose and 80-90 % of amylopectin (N. Singh
& Maitra, 2015). Amylose contributes to the film-forming ability of the starch. The
advantages associated with the application of starch films in producing biodegradable
materials include its high abundance, low production cost, biodegradability and
renewability (Phuong et al., 2014). Despite that, biodegradable products based on starch
are prone to many drawbacks which are attributed to its water solubility and fragile
characteristic (Mathew, Brahmakumar, & Abraham, 2006).

Figure 2.2 Structure of Starch Consisting of Amylose and Amylopectin with Different
Linkages
11

Glycosidic linkage is the principal differentiation between chitosan and starch in

which it is (1,4) glycosidic linkage for starch and (1,4) glycosidic linkage for chitosan
(Fahanwi, 2014). The detrimental properties owned by chitosan and starch respectively
are solved through the utilization of polymer blending technique. Polymer blending
enables the material to be manufactured and tailored in accordance with the requirements
compared to the limited freedom a single element polymeric material is confined to
(Okolo et al., 2014). With that, the mechanical properties of chitosan/starch polymeric
blend particularly its strength can be further enhanced due to chitosans more dominant
hydrophobic characteristic than starchs (V. Singh & Kumari, 2014). Desired
chitosan/starch coating which manifests robust film forming property and mechanical
stability is then formed (Okolo et al., 2014). Chitosan/starch blend possesses excellent
film forming property due to the presence of hydrogen bonding between amino groups
and hydroxyl groups of chitosan and starch respectively (Lu, 2009). Chitosan has
antimicrobial nature and it has been widely applied as an antimicrobial agent. The
common method is to apply chitosan alone or mix it with other natural biodegradable
polymers. Chitosan/starch polymeric blend has been proven to be effective for its control
activity against Gram-negative pathogenic and non pathogenic bacteria which are
Salmonella enterica and Escherichia coli respectively (N. Singh & Maitra, 2015). The
blend also exhibits high microbial activity against E. coli, Aspergillus niger and F.
oxysporum (Phuong et al., 2014).

However, it is highly advocated to incorporate other polysaccharides or cross

linking agents during the manufacture of starch/chitosan polymeric blend instead of direct
usage since native starch and chitosan has poor dimensional stability and
physicomechanical properties respectively (Maitra & Singh, 2014).

12

CHAPTER 3

METHODOLOGY

3.1

Project Methodology

The following figure shows the flow chart of the project particularly the
sequencing of research activities conducted in accordance with the objectives of project.

Synthesizing starch/chitosan crosslinked

biopolymer by using starch solution and chitosan
solution
Characterization of starch/chitosan crosslinked
biopolymer by applying the Fourier Transform
Infrared Spectroscopy analytical technique and
conducting swelling study
Encapsulation of Allicin using the starch/chitosan
crosslinked polymer synthesized

Study on N nutrient release from urea through

UV-Visible Spectroscopy

Data analysis and documentation of report

Figure 3.1: Flow Chart of Project Methodology

13

3.2

Experimental Procedure

3.2.1

Synthesis of Starch/Chitosan Biopolymer

3.2.1.1 Preparation of Starch Solution

1. 2gm of soluble starch was weighed using weigh balance.

2. Weighed starch was transferred into a 250mL beaker. 100mL of distilled water was
added to the beaker to produce 2% w/v starch solution.
3. The starch solution was heated at temperature ranging between 353K to 363K using
magnetic stirrer for a period of 30mins.
4. After 30 minutes, a homogenous and decolorized solution was obtained.

3.2.1.2 Preparation of Chitosan Solution

1. Chitosan was weighed at a precision of 2g using weigh balance.

2. Weighed chitosan was transferred into a 250mL beaker. 100mL of 2% v/v acetic acid
was added to the beaker to create 2% w/v chitosan solution.
3. The solution was stirred continuously utilizing the magnetic stirrer for a period of 3
hours at room temperature (25)
4. After 3 hours, a homogenous solution was obtained.

3.2.1.3 Starch/Chitosan Hydrogel Membrane Synthesis (Crosslinking)

1. A total amount of 10mL 2% w/v chitosan solution and 10mL 2% w/v starch solution
was gathered respectively.
2. Both solutions are combined together in a 50mL beaker.
3. The combined solution was mixed by utilizing the magnetic stirrer set at 450 rpm
throughout a 15 minutes period.
14

4. A homogenous solution was obtained after 15 minutes.

5. 0.4mL of 25%wt glutaraldehyde was pipetted to the homogenous solution.
6. The resultant solution was left to undergo thorough magnetic stirring to allow the
occurrence of crosslinking.
7. Observations were done at different time intervals to look out for the changes in the
viscosity of the gel.
8. Gels viscosity intensified within 30 minutes and a solid gel was formed after 30
minutes.
9. The solid gel was poured onto a leveled and non-stick flat tray/ Petri plates with
parafilms to settle.
10. The crosslinked hydrogel was dried at 60 in a vacuum oven overnight/ at room
temperature for 72 hours.
11. The hydrogel was peeled off from the tray/parafilm after drying finished and are
stored in polybegs.
12. The hydrogel was subsequently kept in dessicator at 60% relative humidity to enable
future usage.

Figure 3.2.1: Glutaraldehyde Cross-linked Reaction between Chitosan and Starch

15

3.2.2

Characterization of Starch/Chitosan Biopolymer

3.2.2.1 FT-IR Spectroscopy

1. 1.8 inch of the solid sample of starch/chitosan crosslinked hydrogel and 0.2 0.5 inch
of KBr was taken with a microspatula.
2. The sample was pulverized in a mortar and mixed thoroughly with KBr to reduce the
particle size to fewer than 5mm diameter.
3. The mixture just sufficient to cover the pellet die was placed in a hydraulic press and
pressed to cast thin pellets.
4. Pressed sample was removed from pellet die.
5. Pressed sample was placed in the FT-IR sample holder for FT-IR analysis and study.

Figure 3.2.2: FT-IR Spectrum of

Pure Chitosan, Pure Starch and Cross-linked

Chitosan/Starch Film
3.2.2.2 Swelling Study

1. Dry crosslinked film was weighed using weight balance.

2. Dry crosslinked film was placed in distilled water at room temperature for 60
minutes.
16

3. Wet film was taken out from the water and surface water was removed using
blotting paper at 10 mins intervals. The film was weighted and the final weight
was recorded.
4. Swelling ratio was calculated based on the formula:

% =

100

Table 1: Different Samples of Chitosan/starch

Sample Name

Volume of

Volume of

Chitosan

Starch (ml) Crosslinking

(ml)

Volume of

Agent

chitosan/starch 5

2% (v/v)

solution

Crosslinked

Uncrosslinked

4 ml of

chitosan/starch

3.2.3

Encapsulation of Allicin

1. Allimax Pro capsules were crushed to obtain Allicin.

2. Allicin was dissolved in distilled water at room temperature to obtain Allicin
solution concentration.
3.

Hydrogel was immersed in Allicin solution in a beaker.

17

3.2.4

Urea Release Study

3.2.4.1 Preparation of Soil Samples

1. 720g of soil was air-dried to reduce the moisture content to 15%.

2. Soil was sieved through a 2mm screen.

3.2.4.2 Preparation of Assay

3.2.4.2.1 Application of Urea to Soil Samples

1. Urea was added at a quantity of 1000 g urea-N per g of soil (equals to 2164.5 g urea
per g soil).
2. 1.56 g urea was added to the assay containing 720 g of soil.
3. Urea was applied onto the soil by using the mechanical shaker throughout the 1-hour
period.
4. The mixture was stored in the refrigerator at approximately 4C if immediate usage is
not required.

3.2.4.2.2 Preparation of KCl-PMA Solution

1. 50 mg of PMA was dissolved in 1 liter of water to produce phenylmercuric acetate
(PMA) solution.
2. 1500 g of KCl was combined together with 9 liters of water.
3. 1 liter of PMA solution prepared earlier was added to the KCl solution obtain a 2M
KCl-PMA solution.
4. The mixture was stored in the refrigerator at approximately 4C if immediate usage is
not required.

18

3.2.4.2.3 Preparation of Color Reagent

1. The acid reagent was prepared by adding 300 ml phosphoric acid to 10 ml sulfuric
acid.
2. The mixture was diluted using distilled water to make up to 500 ml.
3. 25 ml of 0.2473 M diacetylmonoxime (DAM) solution and 10 ml of 0.02743 M
thiosemicarbazide (TSC) solution were added to 500 ml of acid reagent to formulate
the color reagent.

3.2.4.2.4 Colorimetric Method

1. 10 g of the urea treated soil was placed in a proper glassware and 100 ml of 2M KClPMA solution was added to the soil.
2. The mixture was shaken on a mechanical shaker for one hour.
3. The suspension was then filtered using Whatman no. 42 filter paper.
4. The extract was stored in the refrigerator at approximately 4C if immediate analysis
is not needed.
5. 10 ml of aliquots was pipetted from the extract into a 50 ml volumetric flask.
6. The volume of aliquots was made up to 10 ml through the addition of 2M KCl-PMA
solution.
7. 30 ml of color reagent was added to the mixture.
8. The mixture was briefly swirled and was placed in oven 120C for 30 minutes.
9. The mixture was taken out after 30 minutes, cooled under running water for 15
minutes and added with water to dilute the contents to 50 ml.
10. A 10 ml sample was taken from the resulting solution to be analyzed by utilizing a
UV-Vis spectrophotometer.

19

CHAPTER 4

CONCLUSION AND RECOMMENDATION

The focus of this project is to synthesize chitosan based biopolymer by

crosslinking chitosan and starch with glutaraldehyde as the cross linker. Characterization
of chitosan/starch biopolymer is studied by applying the FT-IR analytical technique.
Swelling study is conducted to determine the swelling ratio of chitosan/starch
biopolymer with varying amounts of crosslinking agents and chitosan/starch ratio.
Allicin urease inhibitor is then encapsulated using the chitosan/starch biopolymer.
Colorimetric estimation of urea in soil is performed to access the efficiency of the
encapsulated inhibitor through quantitative measure of urea left in soil after 30 days.

The following results should be obtained by the end of this project:

FTIR spectrum exhibits the presence of functional groups in chitosan/starch
polymeric blend.
Swelling ratio of crosslinked chitosan/starch biopolymer is higher than that of
uncrosslinked chitosan/starch biopolymer.
Swelling ratio of chitosan/starch blend is the highest at the ratio 5:5.
Inhibitor encapsulated with chitosan/starch polymeric blend exhibits higher
efficiency than garlic extracts (GE), NBPT and Allicin urease inhibitors. The
amount of urea content released over 30 days in soil is less when encapsulated
inhibitor is applied compared to the other three inhibitors.
This research project is feasible within the timeframe given according to the
Gantt Chart which is set out to ensure systematic completion of the project.
20

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24

APPENDICES
APPENDIX 1-1: Gantt Chart of FYP I

2016
September
Tasks

October

November

December

Status
I

II

III

IV

II

III IV

II

III IV

10 11 12 13 14

Selection and Confirmation of Project Completed

Topic
Preliminary Background Research

Completed

Literature Review

Completed

Selection of Biopolymer

Completed

Development and Review of Methodology Ongoing

Submission of Extended Proposal Draft

Ongoing

Submission of Extended Proposal

Pending

Synthesis of Starch/Chitosan Biopolymer

Pending

25

II

III IV
-

Pending

Proposal Defense Presentation

Characterization

of

Starch/Chitosan Pending

Biopolymer
Encapsulation of Allicin Inhibitor

Pending

Swelling Study

Pending

Submission of Interim Report Draft

Pending

Submission of Interim Report

Pending

APPENDIX 1-2: Gantt Chart of FYP 2

2016
January
Tasks

February

March

April

Status

Test on Soil

Data Analysis and Interpretation

Progress Report Write-up

II III IV

II

III IV

II III IV

26

10 11

II

III

IV

12

13

14

Submission of Progress Report Draft

Submission of Progress Report

Preparation of Pre-SEDEX
Pre-SEDEX

Final Report Write-up

Submission of Final Report Draft

Submission of Project Dissertation in Soft Bound Copy

Technical Paper Write-up and Viva
Submission of Technical Paper Draft
Submission of Technical Paper

Viva Presentation

Submission of Project Dissertation in Hard Bound Copy

27

APPENDIX 2: Key Milestone

FYP

Milestone

Task Allocated

Part

Date of
Completion

Selection and Confirmation of Topic

12/09/16

Literature Review for Data Gathering

05/10/16

Research on Project Background

Selection of Biopolymer

Development of Methodology

Review and Finalization of Methodology

Extended Proposal Submission

Synthesis of Starch/Chitosan Biopolymer

07/11/16

Proposal Defense Presentation

11/11/16

Characterization of Starch/Chitosan

14/11/16

3
FYP I

12/10/16

28/10/16

Biopolymer

FYP

Encapsulation of Allicin Inhibitor

21/11/16

Swelling Study

28/11/16

10

Interim Report Submission

06/12/16

11

Test on Soil

Week 3

12

Data Analysis and Interpretation

Week 6

13

Progress Report Submission

Week 7

14

Pre-Sedex

Week 10

15

Final Report Draft Submission

Week 11

16

Submission of Project Dissertation in Soft

Week 12

II

Bound Copy
17

Technical Paper Submission

Week 12

18

Viva Presentation

Week 13

16

Submission of Project Dissertation in Hard

Week 14

Bound Copy

28