Polysaccharides 05 00004

Article
Exploring the Potential of 3D-Printable Agar–Urea Hydrogels

as an Efficient Method of Delivering Nitrogen in
Agricultural Applications
Wathsala Dissanayake 1 , Hossein Najaf Zadeh 1,2 , Ali Reza Nazmi 1,2 , Campbell Stevens 1 , Tim Huber 3
and Pramuditha L. Abhayawardhana 1,2, *
1 School of Product Design, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand;
[email protected] (W.D.);
[email protected] (H.N.Z.); [email protected] (A.R.N.);
[email protected] (C.S.)
2 Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand
3 Luxembourg Institute of Science and Technology, 5 Av. des Hauts-Fourneaux,
4362 Luxembourg, Luxembourg; [email protected]
* Correspondence: [email protected]; Tel.: +64-21467442
Abstract: Amidst population growth and challenges with existing fertilizers, the development of
smart and environmentally friendly agrochemicals is imperative. While 3D printing is widespread, its
potential in slow-release agrochemicals remains unexplored. This proof-of-concept study employed
solvent casting and 3D printing to develop agar–urea structures. These structures, comprising 2.5%
(w/w) agar, incorporated either 7% (w/w) or 13% (w/w) urea as nitrogen nutrients. Rheological,
mechanical, and morphological properties and sorption capabilities were explored. Rheological
analysis revealed a substantial impact of urea, enhancing material resistance to deformation. In
mechanical tests, inclusion of urea showed no significant impact on compressive strength. SEM
analysis confirmed the successful entrapment of urea within the agar matrix. The inclusion of urea
resulted in a diminished water sorption capacity, attributed to the urea–water interactions disrupting
Citation: Dissanayake, W.; Najaf
Zadeh, H.; Nazmi, A.R.; Stevens, C.; the hydrogen bonding ability of agar. Agar–urea inks were employed in 3D printing utilizing the
Huber, T.; Abhayawardhana, P.L. direct-ink writing technique, and the nitrogen release behavior was investigated. Results revealed
Exploring the Potential of nearly complete urea release in the positive control within 48 h. In contrast, agar–urea formulations
3D-Printable Agar–Urea Hydrogels as with 7% (w/w) and 13% (w/w) achieved nitrogen release rates of 88.8% and 94.4%, respectively,
an Efficient Method of Delivering suggesting potential for 3D-printed agar formulations to modify the immediate release behavior seen
Nitrogen in Agricultural Applications. in conventional urea fertilizers.
Polysaccharides 2024, 5, 49–66.
https://doi.org/10.3390/ Keywords: hydrogel; agar; urea; additive manufacturing; 3D printing; slow-release
polysaccharides5010004
Academic Editor: Luminita Marin
Received: 30 January 2024

1. Introduction
Revised: 23 February 2024
Accepted: 1 March 2024
According to estimations, the population of our planet will approach 8.6 billion by
Published: 7 March 2024 2030, 9.8 billion by 2050, and 11.2 billion by 2100, which forecasts a noticeable increase
from the current global population of 8.1 billion people [1]. Synchronically, there will be a
pressing need for a 70% expansion in worldwide food production by the year 2050, which
will draw attention to the significant demands on sources including land, water, labor,
Copyright: © 2024 by the authors. utilities, fuel, and nutrient fertilizers [2]. Nitrogenous fertilizers are chemical fertilizers
Licensee MDPI, Basel, Switzerland. widely employed to enhance agricultural productivity [3] and urea [CO(NH2 )2 ] stands out
This article is an open access article as the foremost nitrogenous fertilizer applied worldwide on account of its rich nitrogen
distributed under the terms and
content of 46%. Projections indicate that by 2050, the annual application of urea in agricul-
conditions of the Creative Commons
ture is expected to increase by approximately 130–150 million tons to meet the prevailing
Attribution (CC BY) license (https://
agricultural demand [4]. This surge can result in a considerable economic burden, given
creativecommons.org/licenses/by/
4.0/).
Polysaccharides 2024, 5, 49–66. https://doi.org/10.3390/polysaccharides5010004 https://www.mdpi.com/journal/polysaccharides

Polysaccharides 2024, 5 50
the substantial cost of fertilizer, particularly impacting countries primarily reliant on agri-
culture [5]. Furthermore, it will have serious detrimental effects on the natural ecosystem
and the environment [6].
The inefficient nutrient utilization of nitrogenous fertilizers often emerges from con-
ventional fertilizer release rates exceeding the rate at which nutrients are absorbed by plants,
or the conversion of nutrients/fertilizers into forms that are not readily usable by crops [7].
Hence, the Nutrient Utilization Efficiency (NUE) of urea is constrained to a range of 30–35%,
with a substantial portion of nitrogen escaping from the fertilizer through processes such as
volatilization and leaching into the soil and surrounding environment. The latter pathway
substantially contributes to eutrophication in aquatic systems, which is specifically an
obstacle to the pursuit of reasonable agricultural sustainability [8]. Noticeably, chemical
fertilizers identified as a major source of rising N2 O emissions [9] contribute significantly to
stratospheric ozone depletion and pose a dual challenge to global environmental issues [10].
Despite advancements in slow-release fertilizer mechanisms within the 4R nutrient manage-
ment concept, challenges persist in optimizing nutrient release through these formulations.
Elevated costs and detrimental environmental impacts are particularly associated with
non-degradable organic/inorganic polymer coating materials [11] and may lead to lasting
soil pollution spanning decades or centuries [12]. In anticipation of increased demand
for slow-release fertilizers, the European Union (EU) implemented mid-2021 restrictions,
primarily addressing the use of microplastics as urea coating materials. From 2026 onwards,
only biodegradable materials will be approved for urea slow-release coatings [13].
Within the realm of biodegradable materials, hydrogels stand out as highly promis-
ing nutrient carrier substances for transporting fertilizer nutrients, which can be released
in a slow manner [14]. Hydrogels are crosslinked three-dimensional network architec-
tures made from an assembly of artificial and/or natural polymers. They are renowned
for their remarkable ability to absorb and retain water to a substantial degree [15]. The
water-holding capability of a hydrogel is primarily attributed to the prevalence of certain
hydrophilic groups in its polymer network particularly carboxyl, hydroxyl, amide, and
others [16]. Owing to their well-established biocompatibility, facile synthesis, and broad
spectrum of applications, hydrogels have secured a greater degree of attention in recent
decades [17]. Specifically, they have demonstrated extensive applications in regenerative
medicine [18], in drug delivery [19], in controlled-release devices in agricultural applica-
tions [20], as a bio-adsorbent [21], in wound healing [22], in food packaging [23], and in
separation systems [24]. In slow-release fertilizer applications, hydrogels demonstrate the
ability to release nitrogen in a controlled manner through various mechanisms including
diffusion, swelling, and degradation [16]. Their proven properties make them promising
candidates for combining with nitrogenous fertilizers, thereby reducing irrigation needs,
adverse environmental effects, and fertilizer loss while facilitating the gradual release
of nitrogen [25,26]. Recently, super-absorbent hydrogels and synthetic hydrogels have
shown significant promise in the market, whereas natural hydrogels have drawn more
attention because of their availability, environmental safety, and cost-effectiveness [27,28].
Among these natural hydrogels, chitosan- [29], starch- [3], cellulose- [30], gelatin- [31], and
algae-based hydrogels including alginate [32], carrageenan [33], agar [34] and agarose [35]
have attracted great research interest towards slow-release fertilizer applications.
In the pool of naturally available hydrogels, agar is a natural polysaccharide-based hy-
drocolloid seaweed with strong gelling properties [36]. Agar has numerous applications in
food [37], drugs [38], beauty products [39], healthcare [40] and biotech industries [41]. Due
to its high demand, global agar production increased from 6800 tons in 2002 to 9600 tons
in 2009 [42]. A global growth rate of 4.6% is forecast for the next five years [43]. Agar
comprises two polysaccharides, namely, the gelling component agarose and the sulfated
nongelling component agaropectin. At around 85 ◦ C, agar liquefies in both water and
organic solvents and forms a gel upon cooling [44]. It encompasses galacto-pyranose
dimers that alternate between galactose and 3,6-anhydro-α-galactopyranose units and are
linked by alternating α-1,3 and β-1,4-linkages [45]. It has the capability to form semi-rigid,
thermo-reversible gels that are hydrophilic by dispersing the powder agar in water, then
heating and finally cooling. The final stage involves arranging agar chains into an orga-
nized structure, with aggregates of co-axial helices forming the connections of the 3D gel
network [46]. Furthermore, agar contains numerous hydroxyl groups that can potentially
create intermolecular or intramolecular hydrogen bonds with urea molecules [47]. Nu-
merous studies have discovered that agar can be employed as a matrix for the prolonged
release of pharmaceuticals [48–50]. The study conducted by Sampson et al. [34] explored
the use of agar as a coating material for soluble NPK fertilizer in both capsular and granu-
lar slow-release formulations. In contrast, agar hydrogel is utilized in a double network
hydrogel system with starch for herbicide control release, and it has been shown that agar
helps promote soil health [51].
3D printing, also known as Additive Manufacturing (AM), is a technique for fabricat-
ing engineered structures guided by a 3D model. It involves layer-by-layer assembly of
materials using a 3D printer, in contrast to conventional fabrication techniques focused on
material removal and shaping [52]. AM generates a complex structured product with excel-
lent accuracy and significantly reduced material waste and contrasts with the limitations
of conventional machining, forging, and casting methods [53]. Due to their exceptional
properties, hydrogels hold great promise for diverse 3D printing applications [54], includ-
ing biosensors [55], electronics [56], biomedicine [57], and food packaging [58], and have
prominently advanced tissue engineering applications [59]. Notably, hydrogels based on
chitosan/alginate have been specifically designed for use as active wound dressings [60].
Furthermore, in response to the increasing demand for personalized food products in
recent decades, agar-based hydrogel substances also have garnered popularity in 3D food
printing [61]. For instance, agar is combined with gelatin hydrogel in a 3D jet printer to
formulate tailored chewable soft meals [62]. Konjac glucomannan is utilized in extrusion-
based 3D food printing with agar to upgrade rheological characteristics and smooth the
extrusion [63]. Within an agricultural framework, 3D printing has been used to manufac-
ture equipment for irrigation and urban farming practices in recent decades [64]. However,
to the best of our knowledge, the application of 3D printing for fabricating customized
agricultural formulations with a specific focus on slow release has not yet been comprehen-
sively explored. This study aims to evaluate the potential of agar–urea hydrogel structures
for delivering nitrogen nutrients. Through solvent casting and extrusion-based 3D printing,
agar–urea structures were developed, and their rheological, mechanical, morphological,
sorption capabilities and nitrogen release behavior were assessed.
2. Materials and Methods

2.1. Materials
Urea (ACS reagent 99.0–100.5%) and agar (high gel strength, plant cell culture tested)
powder were purchased from Sigma-Aldrich, St. Louis, MO, USA. Carrageenan powder
was purchased from Go Native New Zealand Ltd. (Auckland, New Zealand). Clear and
unflavored gelatine powder was purchased from a local supermarket. Agarose powder was
purchased from Purolite New Zealand Ltd., Auckland, New Zealand Washed Sea sand was
purchased from a local garden store. Deionized (DI) water was used for all the experiments.
2.2. Preparation and Evaluation of Natural Hydrogels for Initial Screening

A preliminary screening study was conducted to identify the most suitable natural
hydrogel material for exploring the formulation of 3D-printed slow-release fertilizers.
Four naturally occurring hydrogels, namely, carrageenan, gelatin, agarose, and agar, were
selected for this purpose. Approximately 50 g of each hydrogel formulation was prepared
by adding 1.25 g of hydrogel material into 48.75 g of DI water, resulting in 2.5% (w/w)
hydrogel formulations. The formulations were subjected to mechanical stirring at 150 rpm
using a stirrer at room temperature (RT). The temperature was gradually increased to
95 ◦ C using a hot plate while continuously stirring at 150 rpm to ensure proper dissolution
of hydrogel in water. Once a homogeneous hydrogel formulation was achieved, it was
removed from the hot plate and cooled down to 60 ◦ C. Then, 3.50 g and 6.50 g of urea
were added into the 2.5% (w/w) hydrogel formulation to prepare 2.5% (w/w) hydrogel
combined with 7% (w/w) and 13% (w/w) urea, respectively. Temperature was maintained
at 60 ◦ C under continuing constant stirring at 200 rpm to ensure thorough dissolution. The
prepared formulations consisting of hydrogel, urea, and DI water were cooled down to
40 ◦ C and the solvent cast into pre-prepared cube-shaped molds and allowed to be set for
20 min. Subsequently, the molded formulations with dimensions of ~10 × 10 × 10 mm
were transferred to a refrigerator at 4 ◦ C for 10 min. These formulations were then weighed
and stored in labeled air-tight containers until initial screening tests were conducted. These
tests, which were aimed at selecting the most suitable natural hydrogel for the intended
function, included visual observation for microbial contamination at RT with varying
humidity, tolerability tests under various temperatures and humidity, extrudability using
five different needle sizes: 22G ¼′′ , 22G ½′′ , 25G ¼′′ , 25G ½′′ , 27G ¼′′ , 27G ½′′ , and water
sorption. The water sorption capacity was measured by drying the hydrogel samples at
room temperature for 7 days and then placing the dried samples in 200 mL of DI water.
After 6 h, they were retrieved from the DI water, and their weights were measured after
careful wiping to remove any excess surface water. The water sorption capacity was
calculated using the following formula [29]:
(Ws − Wd)
Water Sorption Capacity = (1)
Wd
where Wd and Ws represent the weight of the initial dried sample and swollen sample,
respectively.
2.3. Solvent Casting of the Agar-Based Hydrogel Formulations

Results of the preliminary screening suggested the suitability of agar for the design of
the next experiments. A control formulation of 2.5% w/w agar (A2.5 ) and two test formula-
tions: 2.5% w/w agar–7% w/w urea (AU7 ) and 2.5% w/w agar–13% w/w urea (AU13 ) were
prepared and solvent cast following the same methodology described above (Section 2.2).
2.4. Characterizations of Solvent Cast Agar-Based Hydrogel Formulations

2.4.1. Differential Scanning Calorimetry
A DSC8500 (PerkinElmer, Waltham, MA, USA) Differential Scanning Calorimeter
(DSC) was employed to explore the thermal behavior and phase shifts of A2.5 gel samples
in order to obtain their gelation and melting temperatures. Approximately 7 mg of agar
gel samples was utilized. Tests were conducted across a temperature range spanning
20–105 ◦ C, employing a heating/cooling rate of 20 ◦ C/min. A continuous nitrogen stream
flowing at a volume rate of 20 mL/min was supplied during the DSC analysis. To avoid
thermal memory effects, the specimens underwent a preliminary thermal cycling procedure
involving both heating and cooling phases. Hermetic pans were utilized throughout the
study and the pans were sealed after loading with the sample prior to testing.
2.4.2. Rheological Analysis

Rheological measurements were executed directly on the liquid formulations (Section 2.2),
using an Anton Paar Modular Compact Rheometer MCR 302 (Anton Paar, Graz, Austria).
The employed measurement system consisted of a cone plate (CP50-1), with a Peltier plate
serving as the cell. Prior to loading the samples onto the lower plate, the system was heated
to 60 ◦ C to avoid solidification of the formulation onto the plate. For each test, a freshly
prepared mixture was heated to 60 ◦ C and carefully deposited onto the lower plate. The
upper element was then lowered to achieve a gap distance of 0.102 mm, and any excess
material was removed. The system temperature was then adjusted to the measurement
temperature of 30 ◦ C. The tests were conducted at a low temperature of 30 ◦ C using freshly
prepared samples for each test to avoid sample dehydration. To understand the viscoelastic
properties of the formulations, amplitude sweeps were conducted on each sample, em-
ploying a fixed frequency of 1 Hz and a strain ranging from 0.0001 to 1%. This allowed for
an understanding of the complex moduli and overall flow properties of the formulation,
including the storage modulus (G′ ) and loss modulus (G′′ ), and the loss factor (tanδ) in
response to applied shear strain.
2.4.3. Mechanical Properties

Compression tests were carried out using an MTS Criterion Model 43 Universal
Testing machine (MTS in Eden Prairie, MN, USA) to explore the mechanical properties of
the hydrogel samples. All tests were conducted at room temperature using solvent-cast
samples with dimensions of ~10 × 10 × 10 mm. Cylindrical clamps with a diameter of
40 mm were employed. Force application was accomplished with a 0.5 kN load cell, and a
consistent crosshead speed of 1 mm/min was maintained. Prior to the testing, excess water
on the samples was effectively removed using microfiber wipe paper (Kimberly-Clark,
Irving, TX, USA). To prevent dehydration during the compression tests and eliminate
the possibility of shear stress between the machine clamp and the part, a thin layer of
low-viscosity silicon oil (Silk One drop 3054 silicone lubricant oil, Auckland, New Zealand)
was applied. Additionally, the tests and replicates were conducted at room temperature
with swift execution to minimize the duration between tests, ensuring minimal exposure of
the hydrogel samples to air. A preload of 0.1 N was introduced to the samples positioned
between the compression clamps and yield tests were then conducted. The test was
conducted in triplicate for each formulation, and the results are reported as the mean value
with the standard deviation.
2.4.4. Scanning Electron Microscopy (SEM)

SEM was conducted to explore the morphological characteristics of the A2.5 , AU7 ,
and AU13 formulations. Hydrogel samples were subjected to freeze-drying for SEM
analysis. Rapid freezing was achieved by immersing the hydrogel samples in liquid
nitrogen. Subsequently, freeze-drying was conducted at −50 ◦ C for 36 h, utilizing a
Labconco freeze dryer (Labconco Corporation, Kansas City, MO, USA). Following the
freeze-drying process, hydrogel samples underwent manual fracturing to prepare fracture
surfaces suitable for SEM analysis. A carbon coating process was applied in two cycles for
90 s at 25 mA using an Emitech K975X coater (Quorum Technologies Ltd., East Grinstead,
UK). Field Emission Scanning Electron Microscopy (FE-SEM) JEOL 7000F FE-SEM (JEOL
Ltd., Tokyo, Japan) was employed with a probe current of 10 mA and under an acceleration
voltage of 5 kV. Additionally, the pore diameters of all samples were calculated using
ImageJ software (Version 1.54, 2023, Wayne Rasband, NIH, Madison, WI, USA).
2.4.5. Water Sorption Capacity

The water sorption capacity of hydrogel samples was assessed using a standard weigh-
ing method. The samples were subjected to freeze-drying by immersing in liquid nitrogen
followed by freeze-drying at −50 ◦ C for 36 h using a Labconco freeze drier (Labconco
Corporation, Kansas City, MO, USA). The freeze-dried and pre-weighed hydrogel samples
were then immersed in DI water. At certain time intervals (30, 60, 90, and 120 min), the
hydrogel samples were retrieved from DI water and their weights were measured after
careful wiping to remove any excess surface water. The experiment was conducted in
triplicate for each formulation and the results are reported as the mean value accompanied
by the standard deviation. The water sorption capacity was calculated using Equation (1)
(Section 2.2).
2.5. Preparation of Agar-Based Hydrogel Formulations for 3D Printing

Approximately 100 g samples of the control A2.5 and two test formulations; AU7 and
AU13 were prepared for 3D printing using the same method of formulation just prior to
molding as described above (Section 2.2).
2.6. Determination of Ideal 3D Printing Parameters

To achieve optimal 3D printing, it was imperative to ascertain the optimal temperature
range. Printing at room temperature was deemed ineffective, as the solution would rapidly
gelate, leading to the obstruction of the printer nozzle. To overcome this challenge, a
temperature-controlled print head was employed, and the gelation temperature of the
various formulations was determined. This involved preparing the formulation mixtures
as outlined in Section 2.5 and subjecting them to different temperatures, starting at 50 ◦ C
and systematically decreasing the temperature. Subsequently, the formulation was hand-
extruded onto a small plate using a small syringe to observe the extrusion behavior. The
plate was maintained at room temperature throughout the experiment. The optimal
temperature range for printing was identified to be between 37 ◦ C and 44 ◦ C. Additionally, it
was determined that the formulation mixtures needed approximately 1 h at 37 ◦ C to achieve
readiness prior to initiating the 3D printing process. Ambient conditions played a crucial
role in influencing the viscosity of the formulation mixture, necessitating temperature
adjustments on hotter and cooler days to achieve optimal viscosity.
Furthermore, continuous adjustments to the printing pressure were required during
the printing process due to inconsistent temperatures throughout the barrel (printing
reservoir). This adjustment was facilitated using the pressure regulator of the Cellink
Inkredible + 3D Bioprinter to maintain a constant extrusion rate. Maintaining control over
printing parameters and temperature within the specified ideal ranges resulted in more
consistent printing behavior, characterized by uniform shape and precision. Consequently,
the ideal 3D printing parameters were identified as a temperature range of 37 ◦ C to 44 ◦ C,
pressure ~3 kPa, printing head speed of 10 mm/s, and line height of 1 mm.
2.7. 3D Printing
After identifying the ideal 3D printing parameters as described in the above Section 2.6,
the prepared formulations (Section 2.5) were poured directly into two Cellink barrels
(BICO, Gothenburg, Sweden). These barrels were immediately placed in the temperature-
controlled holders of the 3D bioprinter and kept for approximately 60 min to equilibrate
to the desired temperature. Then, 3D printing was performed using the 18 gauge32 mm
nozzle. During printing, the printing pressure was continuously adjusted to ensure a
consistent extrusion rate. After successfully 3D printing cylindrical-shaped A2.5 , AU7 , and
AU13 samples with dimensions of ~5 mm radius (r) and ~10 mm height (h), the samples
were immediately transferred to individual airtight containers and stored in a refrigerator
at 4 ◦ C until characterization.
2.8. Nitrogen Release Studies of 3D Printed Agar-Based Hydrogel Formulations

The method for urea slow-release study described by Gungula et al. [65] was adapted
and modified for this study. A 250 mL cylindrical, graduated glass separatory funnel was
used to test the nitrogen release behavior of the following; 3D printed AU7 , 3D printed
AU13 , 3D printed A2.5 (negative control), and conventional urea powder (positive control).
Triplicates of each of the above samples were tested. Each separatory funnel was filled
with 150 g of washed and autoclaved sea sand. 1 L of DI water was added to the column
to wash the sand until clear water was collected. The weighed test or control samples
were added to the surface of the sand column covering a central circular area of about
one-inch diameter. Approximately 50 g of separately washed sea sand was added to the
top of each set-up covering the test/control samples. The weight of the conventional urea
powder was calculated based on the amounts of urea estimated in the AU13 formulation.
Approximately 50 mL of DI water was gently introduced to the top surface of the sand
column, facilitating the collection of leachates from the soil into 15 mL centrifuge tubes.
The same draining procedure was repeated for each sand column with 50 mL of DI water
each time at the following time intervals: 12, 24, 48, 72, 96, 120, 144, 168, 264, and 312 h. All
collections of leachates were stored in a freezer until the total nitrogen content was analyzed
using an Elementar Vario TOC cube (Elementar Analyse Systeme GmbH, Langenselbold,
Germany). Finally, the cumulative nitrogen release % of the AU7 and AU13 formulations
was calculated and analyzed compared to negative and positive control samples. The
cumulative release of nitrogen was calculated as follows [66]:
Ms
Qn (%) = × 100% (2)
Mt
where Qn represents the cumulative nitrogen %, Ms denotes the accumulated mass of
total nitrogen in leachate, and Mt indicates the mass of total nitrogen content of the
formulation, respectively.
3. Results and Discussion

3.1. Initial Screening for the Selection of Most Suitable Natural Hydrogel
The visual appearance of hydrogel samples was assessed at room temperature and
varying humidity conditions over a 10-day period to identify any physical changes and po-
tential microbial contamination. The observations suggested that carrageenan and agarose
hydrogel samples showed a significantly shriveled, dried, and reduced size compared to
agar and gelatin hydrogel samples. It was evident that all urea-containing hydrogel sam-
ples showed no visible microbial growth compared to the respective non-urea-containing
samples. The antimicrobial study conducted by Arafa et al. [67] demonstrated the inhibitory
effects of urea-loaded hydrogel formulations on microbial growth. Tolerability tests were
conducted at 25 ◦ C with 60% humidity and at 40 ◦ C with 75% humidity over a 10-day pe-
riod. These tests also confirmed that carrageenan and agarose hydrogel samples displayed
a shriveled, dried appearance and were reduced in size compared to agar hydrogel samples
when temperature and humidity conditions were increased.
To assess the 3D printability of the hydrogels, extrudability tests were carried out
and the results confirmed that all urea-containing samples could easily extrude through
the 22G ¼′′ and the 22G ½′′ . Carrageenan, gelatin, and agar hydrogel samples were
able to be extruded through the 22G ¼′′ needle, but not the agarose hydrogel sample. It
was concluded that mainly urea-containing agar and carrageenan hydrogel samples were
compatible with varying needle sizes and demonstrated a favorable extrudability capacity,
thus confirming their suitability for 3D printing even without any heating.
During water sorption tests, it was noted that agarose and agar hydrogel samples
without urea exhibited notably higher sorption capacities compared to their respective
counterparts containing urea. This observation suggests that urea molecules may in-
terfere with the hydrogen bonding ability of hydrogels, emphasizing their potential for
slow-release applications in agricultural formulations. While agarose and agar samples
containing urea demonstrated favorable water sorption behaviors, carrageenan and gelatin
samples containing urea exhibited unexpectedly high levels of swelling, making them
unsuitable candidates for the intended purpose. Gelatin, a soluble protein compound
obtained through partial collagen hydrolysis, can undergo denaturation under certain
conditions [68]. Given its frequent use as an additive in protein denaturation, urea is pre-
sumed to contribute to gelatin denaturation by interfering with its secondary and tertiary
structures. The behavior of carrageenan with urea in water compared to carrageenan alone
can be influenced by various factors, including the concentrations of carrageenan and
urea, the type of carrageenan, and the specific conditions of the water sorption experiment.
Urea could have disrupted the hydrogen bonding network within the carrageenan gel
under our experimental conditions. It is important to note that the specific interactions
between gelatin or carrageenan and urea can be complex and may vary based on the type of
hydrogel (e.g., kappa, iota, lambda carrageenan) and the specific experimental conditions.
Experimental studies, such as water sorption tests conducted under controlled conditions,
would be needed to provide detailed insights into the unusually high swelling behavior of
the two gels with urea.
After assessing the initial results related to microbial contamination tolerance, adapt-
ability to temperature and humidity variations, extrudability capacity, and water sorption
After assessing the initial results related to microbial contamination toler-
ance, adaptability to temperature and humidity variations, extrudability capacity,
and water sorption potential, over a tested period, it was concluded that among
the tested hydrogels, agar emerged as the most suitable hydrogel material for the
subsequent experimental phase. This phase would delve into investigating its sol-
vent casting and 3D printability attributes, physical, rheological, mechanical, and
morphological
potential, over aproperties, andit nitrogen
tested period, release
was concluded potential
that as atested
among the matrix for slow-re-
hydrogels, agar
lease fertilizer formulation.
emerged as the most suitable hydrogel material for the subsequent experimental phase.
This phase would delve into investigating its solvent casting and 3D printability attributes,
3.2. Characterizations
physical, rheological, of Solvent Cast
mechanical, andAgar-Based Hydrogel
morphological Formulations
properties, and nitrogen release
potential as a matrix for slow-release
3.2.1. Differential Scanning Calorimetryfertilizer formulation.
DSC was performed
3.2. Characterizations to determine
of Solvent the Hydrogel
Cast Agar-Based (Tm) and gelation (Tg) temper-
melting Formulations
atures of the agarScanning
3.2.1. Differential hydrogel (A2.5) as illustrated in Figure 1 These values are crucial
Calorimetry
for identifying the idealtotemperature
DSC was performed determine therange for(Tmixing
melting agar with urea during 3D
m ) and gelation (Tg ) temperatures of
printing while shielding
the agar hydrogel against inurea
(A2.5 ) as illustrated decomposition.
Figure 1 These values are The A2.5 for
crucial formulation
identifying
showed
the ideal a pronounced
temperature rangeendothermic peak
for mixing agar at urea
with 83.8 during
°C with 3Daprinting
meltingwhile
enthalpy of
shielding
against
0.44 J/g urea
which decomposition.
represents the Themelting
A2.5 formulation
process showed a pronounced
of the agar hydrogel.endothermic peak
The Tm of pure
at 83.8 ◦ C with a melting enthalpy of 0.44 J/g which represents the melting process of the
agar was reported to be 83.9 °C [69]. The slight variation in Tm values of pure agar
agar hydrogel. Theattributed
Tm of puretoagar ◦ C [69]. The slight variation in
samples is likely the was reported
various to be 83.9
seaweed sources/origins and processing
Tm values of pure agar samples is likely attributed to the various seaweed sources/origins
conditions of natural agar [70]. This discrepancy has been observed to result in
and processing conditions of natural agar [70]. This discrepancy has been observed to
the identification of distinct endothermic peaks in agar gels from diverse sources,
result in the identification of distinct endothermic peaks in agar gels from diverse sources,
occurring
occurring at attemperature
temperature intervals
intervals ranging
ranging fromfrom
75 ◦ C75to°C
90 to
◦ C90 °C [71].
[71].
Figure
Figure1.
1.DSC
DSCthermographs
thermographsof
ofAA2.5 formulation during heating (blue) and cooling (red) trace.
2.5 formulation during heating (blue) and cooling (red) trace.
In order
In order totodissolve
dissolve thethe
agarose fraction
agarose in water,
fraction heatingheating
in water, it to temperatures exceeding
it to temperatures
80–90 ◦ C is required to break down the robust interactions among the agarose chains.
exceeding 80–90 °C is required to break down the robust interactions among the
Following
agarose dissolution,
chains. it adopts
Following a uniform solution
dissolution, it adoptsstate capable of
a uniform transitioning
solution state into a gel
capable
upon cooling below its Upper Critical Solution Temperature (UCST) [72]. This singular
of transitioning into a gel upon cooling below its Upper Critical Solution Temper-
exothermic peak observed in the cooling curve is associated with the sol–gel transition
ature (UCST) [72]. This singular exothermic peak observed in the cooling curve is
of pure agar [73]. When considering the exothermic peak associated with A2.5 , which
associated with the profile
exhibits a narrower sol–gelcompared
transitiontoofthe
pure agar [73].peak,
endothermic When it considering the ex-
becomes apparent at
othermic peak associated with A , which exhibits a narrower profile
34.7 C with an enthalpy change of 2.21 J/g. Pino-Ramos et al. [74] identified Tg at 33 ◦ C,
◦ 2.5 compared
attributable to the presence of agarose and agaropectin within the agar hydrogel. This
observed peak may be ascribed to the gelation process in agarose solutions, which arises
from the accumulation of the double helices of agarose, aided by intermolecular bonding of
hydrogen atoms and the generation of microcrystalline junction regions [75]. In summary,
it is evident that the observed endothermic and exothermic peaks, occurring at 83.8 ◦ C and
34.7 ◦ C, are respectively the establishment and disruption of hydrogen bonds among agar
polymer strands, leading to the formation of physical gels and, primarily, the aggregation
mary, it is evident that the observed endothermic and exothermic peaks, occur-
ring at 83.8 °C and 34.7 °C, are respectively the establishment and disruption of
hydrogen bonds among agar polymer strands, leading to the formation of physi-
cal gels and, primarily, the aggregation or disintegration of the agar double he-
Polysaccharides 2024, 5 lixes. By analyzing the obtained results, it was determined that 60 °C, representing57
within the range between the obtained Tm and Tg of A2.5, is the ideal temperature
for the 3D printing experiments.
or disintegration of the agar double helixes. By analyzing the obtained results, it was
determined that 60 ◦Analysis
3.2.2. Rheological C, representing within the range between the obtained Tm and Tg of
A2.5 , isAmplitude
the ideal temperature
sweeps werefor the 3D printing
employed experiments.
to elucidate the viscoelastic behavior of
hydrogel formulations, with the dependence of viscoelastic moduli (G′ and G″)
3.2.2. Rheological Analysis
and loss factor (tanδ) vs. applied shear strain depicted in Figure 2. These strain
Amplitude sweeps were employed to elucidate the viscoelastic behavior of hydrogel
sweep tests were used to determine the linear viscoelastic (LVE) region and to
formulations, with the dependence of viscoelastic moduli (G′ and G′′ ) and loss factor
comprehend how viscoelastic properties are influenced by the incorporation of
(tanδ) vs. applied shear strain depicted in Figure 2. These strain sweep tests were used
tourea into thethe
determine agar hydrogel
linear network.
viscoelastic (LVE)The region
region andoftoconstant
comprehendG′, G″how andviscoelastic
tanδ de-
fines the LVE range [76]. This region signifies the range in which
properties are influenced by the incorporation of urea into the agar hydrogel network. tests can be con- The
ductedof without
region constant compromising
G , G and tanδthe
′ ′′ sample
defines structure.
the LVE Beyond
range [76]. Thisthe critical
region strain
signifies the
threshold,
range in which thetests
gel can
structure breaks without
be conducted down with G′ decreasing
compromising at the end
the sample of theBeyond
structure. LVE
region,
the indicating
critical dependence
strain threshold, the gelon the applied
structure breaksstrain
down[77].
with A G′ longer LVEatregion
decreasing the end
ofimplies
the LVE region,stability
greater indicating dependence on
to deformation. Allthe applied strain
hydrogel [77]. A longer
formulations, LVE region
including A2.5,
implies
AU7, and greater
AU13stability
, exhibittoadeformation.
pronouncedAll hydrogel formulations,
dominance of G′ over G″, including
confirming A2.5 , the
AU7,
and AU , exhibit a pronounced dominance of G ′ over G′′ , confirming the prevalence of
prevalence of elastic characteristics over viscous properties. This observation un-
13
elastic characteristics
derscores a robust gel over viscous across
behavior properties. This observation underscores a robust gel
all formulations.
behavior across all formulations.
′ ) and loss modulus (G′′ ) graphs and (b) tanδ graphs as a function of
Figure2.2.(a)
Figure (a)Storage
Storagemodulus
modulus(G (G′) and loss modulus (G″) graphs and (b) tanδ graphs as a function
shear strain for for
A2.5A,2.5AU ◦ C. G′ , G′′ , and tanδ are denoted by filled,
of shear strain 7 and
, AU 7 andAUAU 1313formulations
formulations at 30 °C. G′, G″, and tanδ are denoted by filled,
unfilled,and
unfilled, anddiamond
diamond shapes,shapes, respectively.
respectively.
The A2.5 formulation displays a higher G′ value at nearly 40 kPa and is closely aligned
with the findings of the study by Norziah et al. [78] on 1.5% (w/w) agar at 30 ◦ C, and
demonstrates a more rigid and denser gel structure of agar hydrogel. This occurrence is
likely a result of enhanced molecular aggregation of agar polymer chains, contributing to
the formation of a rigid structure. In the case of other samples where agar is mixed with
urea, the behavior is slightly different. When compared to the A2.5 , the AU13 formulations
exhibit a lower G′ due to the presence of urea, resulting in a more flexible gel structure. The
network becomes less rigid with the presence of urea molecules in the agar matrix. The
diminished gel strength (G′ and G′′ ) in hydrogel formulations with urea (AU7 and AU13 )
can be attributed to both reduced crosslinking density in their networks and an increased
plasticization effect of urea on the network chains. A similar observation was noted in a
study by Wei et al. [79] during the synthesis of urea-embedded grafted starch hydrogels.
Nevertheless, the G′ value of the AU7 formulation closely approximates that of the A2.5 .
As depicted in Figure 2, the presence of urea has a notable impact on the extension
of the LVE region in AU7 and AU13 formulations, resulting in a material more resistant
to deformation with increasing urea concentration. In the case of the A2.5 , the G′ value
begins to decrease at 0.01% strain, signifying the LVE region between 0.0001% and 0.01%.
In contrast, AU7 and AU13 samples exhibit longer LVE regions of 0.0001–0.03% and 0.0001–
0.14%, respectively, compared to the A2.5 sample. This phenomenon is likely due to the
entrapment of urea, which enhances the gel network’s elasticity through the physical
entanglement of urea molecules in the agar hydrogel matrix. At strain values of 0.04%,
0.6%, and 0.3%, G′′ crosses G′ for the A2.5 , AU7 , and AU13 samples, respectively. This
outcome suggests a loss of network strength with increasing strain. Within the two agar–
urea formulations, AU7 manifests a pronounced elastic characteristic in contrast to AU13 ,
as indicated by the absence of crossover until reaching a 0.6% strain value. Outside the LVE
region, the material’s viscous properties become dominant, and it begins to behave like
a fluid.
Furthermore, upon examining tanδ (G′′ /G′ ) values within the LVE range, the sub-
stantial difference in tanδ between agar–urea (AU7 and AU13 ) formulations and the A2.5
underscores the profound influence of urea on the interactions among agar chains. Typ-
ically, the loss factor tanδ serves as an indicator of the dynamic viscoelastic behavior of
samples. When tanδ > 1, G′′ dominates, indicating mainly fluid properties, whereas tanδ < 1
signifies samples with predominantly elastic properties [63]. Across all our formulations,
tanδ remains below 1, indicating their predominantly elastic nature and gel behavior.
After analyzing tanδ values within the LVE range, it becomes evident that the inclusion
of urea in agar hydrogel formulations leads to a reduction in the loss factor, signifying
an enhancement in the strength of the hydrogel network. In the case of AU7 and AU13 ,
tanδ increases with higher urea concentration, indicating a progressively less developed
network of hydrogels. A parallel observation was noted in a study on the synthesis of
urea-embedded grafted starch hydrogels conducted by Wei et al. [79].
3.2.3. Mechanical Properties

The mechanical characteristics of A2.5 , AU7 , and AU13 formulations were assessed
through uniaxial compression tests. The samples underwent compression loading until
the propagation of cracks resulted in structural failure, allowing for the calculation of
compressive strength and Young’s modulus. Young’s modulus is calculated within the
strain range of 0.4% to 13% and compressive strength between 1 kPa and 33 kPa. The
formulation exhibits clear rupture damage when the tension approaches the maximum
bearing stress, after which the stress swiftly decreases (Figure 3). The maximum compres-
sive strength of the A2.5 formulation was 92 kPa. Considering that 97.5% (w/w) of the
formulation is water, the plasticizing effect of water molecules in the agar hydrogel [80] and
the physical entanglements of 2.5% (w/w) agar polymer chains and intermolecular forces
of the polymers contribute to the compressive strength of the 2.5% (w/w) agar hydrogel.
The inclusion of 7% (w/w) urea does not remarkably impact the compressive strength of
the agar–urea hydrogel structures. Moreover, an increase in urea content from 7% (w/w) to
13% (w/w) leads to a slight reduction in the strength of the formulation (refer to Table 1).
The higher amount of urea molecule content potentially causes slight denaturation in the
agar biopolymer, which could account for the slight compressive strength reduction [81].
Furthermore, at higher urea loading levels, the lack of consistent urea dispersion caused
an unequal distribution of stress throughout the agar matrix, hence the reduction in com-
pressive strength. This reduction in compressive strength could also be influenced by the
existence of pores, particularly at higher urea loading levels.
the slight compressive strength reduction [81]. Furthermore, at higher urea load-
ing levels, the lack of consistent urea dispersion caused an unequal distribution
of stress throughout the agar matrix, hence the reduction in compressive strength.
Polysaccharides 2024, 5
This reduction in compressive strength could also be influenced by the existence
59
of pores, particularly at higher urea loading levels.
Stress–straincurve
Figure3.3.Stress–strain
Figure curveof
ofAA2.52.5 , AU
, AU , andAU
7, 7and AU formulations.
formulations.
13 13
Table 1. Compressive strength and Young’s modulus of A2.5 , AU7 , and AU13 formulations.
The Young’s modulus for A2.5 was 238 kPa, which is a value similar to the
Young’s moduli of 2% (w/v) pureAverage
agar atCompressive
RT reported by Thompson
Averageet al. [73] and
Young’s
Formulation
Strength (kPa) Modulus (kPa)
2% (w/w) agar reported by Nayar et al. [82]. As Table 1 illustrates, the introduction
of urea intoAthe
2.5 agar matrix shows92.0 slightly
± 6.2 higher Young’s 238
modulus
± 10.1 values,
though statistically not significant, imparting gradually enhanced ±stiffness
AU 7 92.2 ± 1.5 256 8.4 and
AU13 86.4 ± 1.3 260 ± 2.5
making the agar formulation slightly stiffer.
Table The Young’s modulus

1. Compressive for A
strength and 2.5 wasmodulus
Young’s 238 kPa,ofwhich is7,aand
A2.5, AU value
AU13similar to the Young’s
formulations.
moduli of 2% (w/v) pure agar at RT reported by Thompson et al. [73] and 2% (w/w) agar
by Nayar et al. [82]. AsAverage
reportedFormulation Compressive
Table 1 illustrates, Average
the introduction Young’s
of urea into $$$
the agar
matrix shows slightly higher Young’sStrength
modulus(kPa) Modulusnot
values, though statistically (kPa)
significant,
imparting gradually enhanced stiffness and making the agar formulation slightly stiffer.
3.2.4. SEM Morphology

SEM was performed to study the morphological surface characterization of A2.5 , AU7 ,
and AU13 urea formulations (Figure 4) using freeze-dried samples. According to the SEM
image of A2.5 (Figure 4a), it exhibited a uniform, regular porous structure and a slightly
smooth surface when compared to agar–urea surface morphology behavior. Interestingly,
it represents a well-known porous, three-dimensional structure that encompasses the
assembly of agar spherical networks [83]. However, the addition of urea molecules steadily
enhanced the surface roughness of the A2.5 formulation. When analyzing the average pore
size, initially A2.5 showed pores with dimensions of 148 ± 26 nm, followed by AU7 and
AU13 exhibiting average diameter values of 90 ± 19 nm and 153 ± 32 nm, respectively.
This pore size distribution confirmed that the porosity of AU7 was moderately reduced and
consisted of urea crystals that guaranteed urea entrapment, as shown in Figure 4b, which
would then have an impact on the porous structure of the network as well as nutrient
release behavior [67]. Conversely, after incorporating urea into the hydrogel matrix, a
porous structure can still be observed in the hydrogel (as shown in Figure 4b,c). This
suggests that urea molecules are not interacting or bonding with the hydrogel via any
covalent/hydrogen bonds, as the urea entrapment occurred within the porous matrix of
the hydrogel.
shown in Figure 4b, which would then have an impact on the porous structure of
the network as well as nutrient release behavior [67]. Conversely, after incorpo-
rating urea into the hydrogel matrix, a porous structure can still be observed in
the hydrogel (as shown in Figure 4b,c). This suggests that urea molecules are not
Polysaccharides 2024, 5
interacting or bonding with the hydrogel via any covalent/hydrogen bonds, as the60
urea entrapment occurred within the porous matrix of the hydrogel.
Figure4.4.SEM
SEMimages
imagesof of(a)
(a)A
A2.5 (b) AU7 and (c) AU13 formulations (scale bar: 1 μm, magnification:
Figure 2.5 (b) AU7 and (c) AU13 formulations (scale bar: 1 µm, magnification:
×10,000, voltage: 15.0 kV).
×10,000, voltage: 15.0 kV).
Comparatively,
Comparatively, thethe
AUAU 7 formulation showed a slightly rough nature and po-
7 formulation showed a slightly rough nature and porous sur-
rous
face surface
(Figure 4b)(Figure
compared4b)to
compared to the AU13 formulation
the AU13 formulation (Figure 4c),
(Figure 4c), indicating indicating
the homogenous
the homogenous
trapping trapping
of urea inside of ureanetwork.
the hydrogel inside the hydrogel
However, fewnetwork.
lumps andHowever, few
a less homoge-
neous
lumpsnature
and awere
less seen on its rough
homogeneous surface
nature when
were theon
seen urea
itsconcentration
rough surfacewas raised
when theto
13%.
urea When the urea concentration
concentration was raised towas 13%.raised
When from
the7% to concentration
urea 13%, some crystallized
was raisedurea
particles were recognized on the irregular porous structure of agar with
from 7% to 13%, some crystallized urea particles were recognized on the irregular no uniformities;
this phenomenon
porous structuremayof be
agarduewith
to thenoprecipitation
uniformities;of saturated urea. Similar
this phenomenon maymorphological
be due to
observations due to the precipitation of saturated urea were reported by Wei et al. [79]
the precipitation of saturated urea. Similar morphological observations due to the
when the urea loading rate increased from 40% to 60% within a starch hydrogel matrix.
precipitation of saturated urea were reported by Wei et al. [79] when the urea
Conclusively, the urea concentration in hydrogel may affect how the urea is dispersed
loading rate increased from 40% to 60% within a starch hydrogel matrix. Conclu-
within the agar hydrogel matrix, which may have a significant effect on the slow-release
sively,and
ability thewater
ureapermeability
concentration in hydrogel
properties of themay affect how
synthesized the ureaformulations.
agricultural is dispersed
within the agar hydrogel matrix, which may have a significant effect on the slow-
3.2.5. Water Sorption Capacity
The water sorption capacity was investigated using freeze-dried agar-based hydrogel
compositions (Table 2), initially focusing on A2.5 and then also exploring the influence
of urea addition with AU7 and AU13 formulations. The results, as illustrated in Table 2,
indicate the water sorption behavior of the respective compositions over different time
intervals. It is observed that the water sorption capacity increased significantly after
120 min compared to 30 min. This finding is consistent with the well-established property
of hydrogels, which possess a high capacity to absorb water owing to the abundance of
hydrophilic groups within their structure. This phenomenon underscores the potential of
hydrogels for slow-release fertilizer applications, where controlled water absorption and
retention over time are essential for providing gradual nutrient release to plants.
Table 2. Water sorption capacities of A2.5 , AU7 , and AU13 formulations at 30, 60, 90, and 120 min
time intervals.
Average Capacity (∆g/ginitial )

Formulation
30 min 60 min 90 min 120 min
A2.5 17.82 ± 2.10 18.30 ± 1.68 18.28 ± 1.55 18.77 ± 1.62
AU7 6.52 ± 0.04 6.61 ± 0.10 6.58 ± 0.33 6.71 ± 0.13
AU13 3.61 ± 0.05 3.58 ± 0.08 3.77 ± 0.02 3.76 ± 0.11
A2.5 exhibited notably higher water sorption capacity across all time intervals (30,
60, 90, and 120 min) compared to AU7 and AU13 . The higher water sorption capacity
of the agar formulation (A2.5 ) aligns with the well-documented hydrophilic nature of
agar, stemming from its abundant hydroxyl groups capable of forming hydrogen bonds
with water molecules [84]. Agar hydrogel shows more hydrophilicity as the agaropectin
present in agar hydrogel comprises a heterogeneous collection of smaller branching and

sulfated molecules [85]. Moreover, agar is a sulfated polysaccharide, and unlike other
polysaccharides, its charged group allows its chains to be more elongated and boosted in
water-attracting capabilities [86]. Consequently, A2.5 demonstrated a superior capacity for
water absorption and retention, underscoring the pivotal role of agar in determining water
sorption capabilities.
For further analysis of the A2.5 , AU7 , and AU13 formulations, the average water
sorption capacity after 120 min was explored. The influence of urea addition on agar
hydrogel was another critical aspect of this study. The introduction of urea into the agar
matrix resulted in a significant reduction in water sorption capacity. This decline became
more pronounced as urea concentrations increased from 0% (A2.5 ) to 7% (AU7 ) and 13%
(AU13 ). Specifically, AU7 exhibited a diminished water sorption capacity of approximately
6.71 ∆g/ginitial compared to the agar-only counterpart (A2.5 ). Similar trends were observed
in the third formulation of AU13 . AU13 demonstrated the lowest water sorption capacity
among the tested samples, measuring only about 3.76 ∆g/ginitial .
The sorption capacity of AU7 and AU13 steadily reduced, feasibly due to the disso-
lution of non-crosslinked urea and polymer molecules. Additionally, this decline could
be attributed to the hydrolysis of the hydrogel structure [29]. Furthermore, this reduction
could be attributed to the presence of urea while interacting with water molecules, which
lack the extensive hydrogen bonding capabilities of agar. Consequently, urea disrupts the
hydrogen bonding network within the agar matrix, leading to a diminished capacity to
retain water. Analyzing the data over varying time intervals revealed minor fluctuations
in water sorption capacity. While these variations were not substantial, they indicated a
slight time-dependent aspect to water sorption in agar-based hydrogels. Water sorption
capacity tended to marginally increase with longer exposure times, although the overall
trends associated with agar concentration and urea addition remained consistent. These
findings have significant implications for potential applications of agar-based hydrogels,
particularly in scenarios where controlled water sorption is crucial, such as in smart and
slow-release applications in agriculture. The capability to customize agar–urea formula-
tions facilitates the attainment of distinct water sorption characteristics, aligning with the
needs of various industries.
3.3. Nitrogen Release Studies of 3D Printed Agar-Based Hydrogel Formulations in Soil Medium
Figure 5 illustrates the cumulative release rates of the positive control (conventional
urea powder), AU7 , and AU13 in soil medium. The results demonstrated that conventional
urea powder released its nitrogen at a quicker pace than other formulations due to its high
solubility. Within 72 h, 99% of the urea of the positive control had been released into the
soil, which is consistent with previously reported urea release characterizations in a soil
medium by Gungula et al. [65]. Typically, within the soil environment, a minor fraction
of urea undergoes hydrolysis; the majority, decomposed by microorganisms, transforms
into ammonia gas, resulting in urea loss within a few days [87]. In the context of agar–urea
hydrogel formulations, both AU7 and AU13 formulations demonstrated a gradual and
cumulative release of nitrogen nutrients when compared to the positive control, mainly
from 24–48 h to around 72 h (Figure 5). This observation affirms the viability of our
agar–urea formulations. Evidently, the capability of absorbing a higher amount of water
within the hydrogel matrix might be substantially accountable for the improvement in the
gradual-release feature of the prepared agar–urea formulations [88]. The cumulative release
rates of the formulations after 12 h were 13.6%, 85.4%, 39.0%, and 45.3% for the negative
control (A2.5 ), positive control, AU7 , and AU13 respectively. In the experimental setup, the
soil itself was subjected to autoclaving. Nevertheless, throughout the study, the presence of
any residual microbes in the soil could have led to the generation of nitrogen-containing
compounds. The interactions or contaminations of nitrogen-containing compounds in the
soil medium might be responsible for the slight amounts of nitrogen released from the
negative control. In the agar–urea formulations, the weakly entrapped/bonded urea on
Polysaccharides 2024, 5, FOR PEER REVIEW 19

the exterior surface of the agar hydrogel caused them to swiftly diffuse or dissolve into
the solution, which may have affected the rapid release within the first 12 h. Then, agar–
urea formulations exhibited slow nitrogen release behavior from 24 h to around 48–72 h.
state. Despite
According to thethat, AU13 exhibited
hydrogel–urea study aconducted
speedy release
by Song pattern
et al. [87],when comparedwere
urea molecules to
AU 7. The cumulative
embedded nitrogen
as small molecules release
in the percentage
swelled of AUthe
hydrogel when 7 was
soil 39%, 70%, 88.8%,
and water-retentive
hydrogel
96.3%, and were blended.
99.1% after Due to the
12, 24, 48, concentration
72, and 96 h,gradient between
respectively, the water-absorbing
which indicates that
material
in AU7, the andhydrogel
the surrounding soil, nitrogen
had certain release
slow-release achieves a consistent
characteristics, making state. Despite
it suitable
that, AU 13 exhibited a speedy release pattern when compared to
to be considered for a potential nutrient carrier in agricultural formulations. In AU 7 . The cumulative
nitrogen release percentage of AU7 was 39%, 70%, 88.8%, 96.3%, and 99.1% after 12, 24,
contrast, AU13 emerged with quicker cumulative nitrogen release rates of 45.3%,
48, 72, and 96 h, respectively, which indicates that in AU7 , the hydrogel had certain slow-
82.3%,
release 94.4%, 98.5%, making
characteristics, and 99.6% at theto
it suitable same time intervals
be considered indicated
for a potential above.
nutrient Inter-in
carrier
estingly, our morphological analysis shows a consistent relationship
agricultural formulations. In contrast, AU13 emerged with quicker cumulative nitrogen with release
data,
releaseand in of
rates accordance with
45.3%, 82.3%, the SEM
94.4%, 98.5%,images,
and 99.6%theatAU formulation
the13same encountered
time intervals indicated
aabove.
crystalInterestingly,
structure of oururea,morphological
revealing theanalysis
precipitation
shows of saturated relationship
a consistent urea crystalswithin
release
the data, and
hydrogel in accordance
substrate. with the
Therefore, outSEM images,
of the two the
testAU 13 formulation
formulations, encountered
the AU7 for-
a crystal structure
mulation of urea, revealing
can be considered the precipitation
the optimum formulationof saturated urea crystals
for slow-release in the
fertilizer
hydrogel substrate. Therefore, out of the two test formulations,
applications. Optimization of these formulations through architecture and 3D the AU 7 formulation can be
considered the optimum formulation for slow-release fertilizer applications. Optimization
printing parameters should be aimed at achieving a reliable prolonged release
of these formulations through architecture and 3D printing parameters should be aimed at
pattern
achieving over an extended
a reliable period
prolonged of pattern
release time. over an extended period of time.
Cumulativenitrogen
Figure5.5.Cumulative
Figure nitrogenrelease
release%
%of
ofurea
ureapowder
powder (positive
(positive control),
control), AU
AU7,7,and
andAU
AU1313formula-
formula-
tionsininsoil
tions soilmedium.
medium.
4. Conclusions
4. Conclusions
Agar hydrogel was blended with urea in two different weight ratios to formulate
Agar hydrogel
a slow-release was blended
agricultural with The
formulation. ureaagar–urea
in two different weightsolvent
inks underwent ratios to for-
casting,
mulate
and theira rheological,
slow-releasemechanical,
agricultural formulation.and
morphological, The agar–urea
water sorptioninks underwent
capabilities were
solvent casting,analyzed.
systematically and theirRheological
rheological,results
mechanical,
unveiledmorphological, and on
the impact of urea water sorp-
extending
the LVE
tion region. Incorporating
capabilities urea did not
were systematically yield a significant
analyzed. Rheological impact on the
results mechanical
unveiled the
properties.
impact SEMon
of urea characterization
extending theconfirmed the successful
LVE region. entrapment
Incorporating urea of urea
did notmolecules
yield a
in the porous
significant agar on
impact hydrogel matrix. The
the mechanical AU7 and AU
properties. SEM13 formulations exhibited
characterization dimin-
confirmed
ished water sorption capacities, compared to A2.5 . Simultaneously, an extrusion-based
the successful entrapment of urea molecules in the porous agar hydrogel matrix.
3D printing technique was employed to fabricate agar–urea hydrogel formulations, and
The AU7 and AU13 formulations exhibited diminished water sorption capacities,
their nitrogen release behavior was assessed. After approximately 72 h, the cumulative
compared
nitrogen release . Simultaneously,
to A2.5rates an extrusion-based
were 99.9%, 96.3%, and 98.5% for urea3D powder
printing(positive
technique was
control),
employed
AU7 , and AUto 13fabricate agar–urea
, respectively. hydrogel
The findings formulations,
of this andstudy
proof-of-concept theirunderscore
nitrogen re-the
lease behavior was assessed. After approximately 72 h, the cumulative nitrogen
promise of 3D-printable agar–urea hydrogel structures, necessitating further exploration
release rates were 99.9%, 96.3%, and 98.5% for urea powder (positive control),
AU7, and AU13, respectively. The findings of this proof-of-concept study under-
score the promise of 3D-printable agar–urea hydrogel structures, necessitating
as slow-release agricultural formulations. Moving forward, the next phase of the project
will focus on optimizing 3D printing parameters and refining the architectural design of
structures to enhance formulation efficiency, cost-effectiveness, and scalability, all of which
are critical factors for the practical implications of the outcomes.
Author Contributions: Methodology/formal analysis/investigation, W.D., P.L.A., A.R.N., H.N.Z.

and C.S.; writing—original draft preparation, W.D., P.L.A., A.R.N. and H.N.Z.; writing—review,
editing, and supervision, P.L.A., A.R.N., H.N.Z. and T.H.; funding acquisition, P.L.A. All authors
have read and agreed to the published version of the manuscript.
Funding: Funding was provided to P. Abhayawardhana by BIC Seed Funding (reference code:
41700:5009:417SEED:0:1) from Biomolecular Interaction Centre, University of Canterbury and by
the UC Early Career Research Accelerator Fund 2022 (grant code: 23-09161) from University of
Canterbury. W. Dissanayake is a recipient of a PhD Scholarship funded by the School of Product
Design, University of Canterbury, Christchurch, New Zealand (reference code: ECRAF2309161).
Institutional Review Board Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article.
Acknowledgments: The authors would like to express their gratitude to the funding sources men-
tioned above as well as Gemma Thompson and Min Min for their contributions associated with the
preliminary stages of the study. Additionally, the authors acknowledge D. Holland for sharing his
expertise to help advance the project.
Conflicts of Interest: The authors declare no conflict of interest.
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Article

Exploring the Potential of 3D-Printable Agar–Urea Hydrogels

Academic Editor: Luminita Marin

Received: 30 January 2024

Polysaccharides 2024, 5, 49–66. https://doi.org/10.3390/polysaccharides5010004 https://www.mdpi.com/journal/polysaccharides

2. Materials and Methods

2.2. Preparation and Evaluation of Natural Hydrogels for Initial Screening

2.3. Solvent Casting of the Agar-Based Hydrogel Formulations

2.4. Characterizations of Solvent Cast Agar-Based Hydrogel Formulations

2.4.2. Rheological Analysis

2.4.3. Mechanical Properties

2.4.4. Scanning Electron Microscopy (SEM)

2.4.5. Water Sorption Capacity

2.5. Preparation of Agar-Based Hydrogel Formulations for 3D Printing

2.6. Determination of Ideal 3D Printing Parameters

2.8. Nitrogen Release Studies of 3D Printed Agar-Based Hydrogel Formulations

3. Results and Discussion

3.2.3. Mechanical Properties

Table The Young’s modulus

3.2.4. SEM Morphology

Average Capacity (∆g/ginitial )

present in agar hydrogel comprises a heterogeneous collection of smaller branching and

Polysaccharides 2024, 5, FOR PEER REVIEW 19

Author Contributions: Methodology/formal analysis/investigation, W.D., P.L.A., A.R.N., H.N.Z.

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