Industrial Crops & Products 144 (2020) 112062

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Industrial Crops & Products

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Cassava-based biocomposites as fertilizer controlled-release systems for T

plant growth improvement
Florencia Versinoa,b,*, Marina Urrizaa, María A. Garcíaa,c,*
a
Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), UNLP-CONICET-CICPBA, 47 y 116, La Plata, Buenos Aires, 1900, Argentina
b
Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad Nacional de La Plata (UNLP), 47 y 115, La Plata, Buenos Aires, 1900, Argentina
c
Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), 47 y 115, La Plata, Buenos Aires, 1900, Argentina

A R T I C LE I N FO A B S T R A C T

Keywords: One of the main problems caused by intensive agriculture, is the consequent water pollution resulting from
Starch fertilizer leaching, especially as a result of fertilizers overuse. Therefore, in the present study an evaluation of the
Renewable materials efficiency of different urea controlled-release systems, based on cassava starch and bagasse, over tomato plants
Biodegradable growth was carried out. All fertilized systems showed a tendency towards a greater general development,
Fibrous fillers
photosynthetic pigments content and nitrogen status of the plant. However, significant differences were ob-
Urea
served regarding macronutrient dosage, being unsupported biocomposite films with 25 wt.% urea content the
systems with a better performance. Thus, an ecofriendly, efficient and easy to implement urea dosage alternative
system for greenhouse seedling has been developed.

1. Introduction this regard, it is necessary to develop new methods to reduce the

Research and development related to the functionalization of bio-
degradable materials through the addition of active compounds is a
widespread approach in the pharmaceutical and biomedical field, with
few agronomic applications: the use of biodegradable composites as
pots (Horinouchi et al., 2008) and mulching has been reported (Bilck
et al., 2010; Moreno and Moreno, 2008). The design of functionalized
materials allows the controlled-release of the active component in-
cluded in the formulation and consequently its dosage. However, ap-
plications in the agronomic area that include fertilizers have mostly
focused in urea encapsulation (Chen et al., 2008; Rychter et al., 2016)
or its inclusion in mixed matrices (Riyajan et al., 2012).
The use of fertilizers controlled-release systems arises as an alter-
native for a rational and adequate use of nutrients, which increase the
efficiency in the consumption of each nutritive element and, in turn, the
productivity with lower cost and environmental impact. This alter-
native clearly constitutes a resource towards a cleaner and more sus-
tainable agriculture, since it intends to avoid the excessive use of fer-
tilizers in crops mitigating the environmental issues the latter causes,
especially nitrate water pollution (Baccaro et al., 2006). Therefore, new
agricultural production systems with less use of agrochemicals need
innovative technologies to ensure the yields and quality of agricultural
products, while minimizing the negative effects on the environment. In
amounts of agrochemicals that enter the environment, helping to
maintain biological activity at a desirable level. Accordingly, the in-
clusion of urea in the formulation of biodegradable films would allow
the gradual release of fertilizer, minimizing the contamination of soil
and water associated with overdose as opposed to the traditional way of
application(Rychter et al., 2016).
The present work is focused on the effect on plant growth of dif-
ferent forms of applications of cassava starch and bagasse biocomposite
films plasticized with urea, which have been previously developed and
studied (Versino and García, 2019, 2018, 2014).
2. Materials and experimental methods
2.1. Materials and films preparation
Cassava (Manihot esculenta) starch from Cooperativa de Productores
de Jardín América Ltda. (Misiones, Argentina) and bagasse derived
from starch extraction of cassava roots cultivated by the INTA
Montecarlo Experimental Station farm (Misiones, Argentina), were used
as described in a previous work (López et al., 2010; Versino et al.,
2015). The fibrous residue was dried, crushed and sieved (through a
500 μm mesh sieve). Distilled water and urea (CAS# 57-13-6, Biopack,
Argentina) were used as additives, while kraft paper and raft wood

⁎
Corresponding authors at: Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), UNLP-CONICET-CICPBA, 47 y 116, La Plata, Buenos
Aires, 1900, Argentina.
E-mail addresses: florencia.versino@ing.unlp.edu.ar, florenciaversino@gmail.com (F. Versino), magarcia@quimica.unlp.edu.ar (M.A. García).

https://doi.org/10.1016/j.indcrop.2019.112062
Received 9 May 2019; Accepted 16 December 2019
0926-6690/ © 2019 Published by Elsevier B.V.
F. Versino, et al.
Fig. 1. Diagram of treatments preparation of controlled urea release with cas-
sava starch and bagasse biocomposite functionalized with urea: A) control; B)
discs of 5 cm in diameter of film 25U; C) 4.7 cm diameter film disc 37.5U; D) 5
cm diameter kraft paper disc coated with the film-containing 25U suspension;
and E) 4 sticks with 25U.
sticks where used as support material.
Aqueous suspensions of 3 wt.% starch with 1.5 wt.% of the fibrous
filler were gelatinized (90 °C for 20 min) and cast on acrylic molds or on
top of kraft paper and later dried in a forced convection oven (50 °C for
4 h). Urea was added to the gelatinized suspensions as plasticizer and
active compound in the films in two concentrations: 25 and 37.5 g/100
g of starch (named 25U and 37.5U, respectively). The urea content was
selected considering the mechanical properties, as well as biodegrada-
tion and urea release kinetics in soil determined in previous studies
(Versino et al., 2019; Versino and García, 2019, 2018).
2.2. Plants growth evaluation
2.2.1. Seedlings and treatments preparation
Since urea contributes approximately with 46% of nitrogen, a re-
quirement of 50 mg N was estimated considering this requirement and a
complete release: A) control; B) discs of 5 cm in diameter of film 25U;
C) 4.7 cm diameter film disc 37.5U; D) 5 cm diameter kraft paper disc
coated with the film-containing 25U suspension; and E) 4 raft wood
sticks with 25U (Fig. 1).
As shown in the diagram (Fig. 1) the discs of films used in treat-
ments B, C and D were placed at 3.5 cm from the base of the pot to
ensure that the radicle was not in direct contact with the material. For
treatment E, 5 cm × 1 cm pieces of 25U film were fixed with a com-
mercial adhesive on the wood sticks, which were placed in a way that
the active films were at 3.5 cm from the base pot (same height as for the
discs of treatments B, C and D). A completely randomized experimental
design was used with 8 replicates per treatment. The aim was to analyze
the effect of the biocomposites biodegradation rate and urea dosage (B
and C) and the directions of diffusion of the fertilizer in the medium
using different support systems (B, D and E), in comparison with a
control with no urea addition (A).
2
Industrial Crops & Products 144 (2020) 112062
2.2.2. Morphological parameters of plant growth
After 28 days the plants were uprooted with extreme care to avoid
root rupture or any other damage. The roots were washed with tap
water to remove substrate remains. Then they were placed on a white
panel provided with a millimeter scale and a circular red pattern of
known dimensions and photographed. With the provided scale stem
height (SH, cm) and root length (RL, cm) were determined and the stem
diameter was measured (SD, mm) using a digital millimeter gauge
(Fowler ProMax, USA). The leaves were separated one by one, placed
on the white panel and photographed to determine the total leaf area
(AF, cm2) and quantify the number of sheets (NH, #) by image pro-
cessing with the Free ImageJ Software (Image Processing and Analyzes
in Java, National Institutes of Health, USA). Leaves, stems and washed
roots were dried with absorbent paper and the fresh weights were re-
corded (FLW, FSW, FRW, respectively). Once dried in an oven at 65 °C
under vacuum to constant weight, the dry matter of the fractions:
leaves, stems and roots (DLW, DSW, DRW, respectively) was also de-
termined with a Mettler Toledo AB204- analytical balance (USA) with
an accuracy of ± 0.1 mg (Pérez et al., 2016).
Finally, the shoot (which consists of two types of organs: stem and
leaves) to root ratio (Sh/R) and the leaves to stem ratio (L/S) both on
dry basis and the total dry matter content expressed as percentage
(%TDM) were determined as growth parameters (De Grazia et al., 2011;
Vagnoni et al., 2014).
2.2.3. Chlorophylls and total carotenoids content
Photosynthetic pigments are vital substances for plants. In leaves of
higher plants the total pigments content comprises chlorophyll-a,
chlorophyll-b and carotenoids(Sumanta et al., 2014). Accordingly,
chlorophyll-a, chlorophyll-b, and total carotenoids were determined by
extraction with 5 mL of DMF (CAS# 68-12- 2, Anedra, Argentina) for 24
h at 4 °C, from 9 discs of 3 mm diameter from different leaves of a
sample plant (equivalent to approximately 250 mg of sample). Samples
were later centrifuged for 10 min at 4500 rpm (Rolco CM 2036, Ar-
gentina) and the colored extract absorbance was thereof measured at
663.8, 646.8 and 480 nm in a spectrophotometer U-1900 HITACHI
(Japan). The total content of each pigment was determined as was
specified by Porra (2002):
Chla = 12 * A663,8 + 3,11 * A646,8 (1)
Chlb = 20,78 * A646,8 − 4,88 * A663,8 (2)
Cx + c = (1000 * A 480 − 1,12 * Chla − 53,78 * Chlb)/245 (3)
Where Chla, Cb and Cx+c represent the concentrations of chlorophyll a
and b, and total carotenoids expressed in g/L and A663.8, A646.8 and A480
represent the absorbance of the sample at 663.8, 646.8 and 480 nm,
respectively. The tests were carried out at least in quadruplicate and the
results were expressed as μg of pigment per leaf mass (g) and leaf area
(cm2).
2.2.4. Nitrogen content
The total nitrogen content of the plant is related to the protein
synthesis by the plant as well as the capacity of this macronutrient
metabolization (Taiz and Zeiger, 2002) and it is a relatively good in-
dicator of N status in plants (Ferreira et al., 2015). The nitrogen content
of both the leaves and the roots of the tomato plants treated was as-
sessed by the micro-Kjeldahl method (Allen, 1931). Firstly, a digestion
of the sample with 5 mL of concentrated sulfuric acid (H2SO4 98%,
CAS# 7664-93-9, Anedra, Argentina) and 1 g of catalyst (10 g of
Na2SO4+1 g of CuSO4, CAS# 7757-82-6 and 7758-98-7 respectively,
both products Anedra, Argentina). The nitrogen content of the sample is
later determined by colorimetry, since the ammonium ions obtained in
the digestion, react with sodium salicylate (C7H5NaO3, CAS# 54-21-7,
Riedel-de Haën, Germany) and sodium hypochlorite (NaClO with 6-
14% active chloride, CAS# 7681-52-9, Merck, Germany), in the
F. Versino, et al.
Table 1
Characteristics and properties of cassava starch and bagasse biocomposites
plasticized with different urea concentrations used for tomato plant growth.
Biocomposite 25U 37.5U
Thickness (μm) 164 ± 12 110 ± 17
Moisture content (%) 12.10 ± 0.74 16.87 ± 1.74
Water uptake (water g/g DB*) 0.4092 ± 0.008 0.5266 ± 0.006
Tensile strength
Stress at break (MPa) 2.9 ± 0.1 1.1 ± 0.1
Strain at break (%) 10.8 ± 1.0 25.8 ± 2.6
Biodegradation rate
t50 (days) 24.64 ± 0.6 27.33 ± 1.4
dt (days) 4.07 ± 0.4 6.68 ± 1.3
Urea release kinetics
k 0.029 ± 0.009 0.045 ± 0.017
n 0.884 ± 0.098 0.793 ± 0.107
*DB = dry basis. Reported values correspond to the mean ± standard devia-
tion.
presence of sodium nitroprusside catalyst (Na2[Fe(CN)5NO], CAS#
13755-38-9, Anedra, Argentina) to form indophenol (C12H9NO2). The
intensity of the green color compound formed, absorbance read at 660
nm, is proportional to the concentration of nitrogen in the sample
(Scheiner, 1976).
2.3. Statistical analysis
In the analyses of variance (ANOVA) of the data, Fisher's minimum
square difference (LSD) test was used for means comparison, using a
significance level of α = 0.05 and the Statistical Software. InfoStat (Di
Rienzo et al., 2011). In addition, in order to analyze the inter-
dependence and variability of the results obtained, a Cluster Analysis
using Euclidean-distance measure method and Principal Components
Analysis (PCA) was also carried out.
3. Results and discussion
In Table 1 are summarized the main characteristics and properties
evaluated of cassava starch and bagasse biocomposites films plasticized
with 25 and 37.5 wt.% of urea with respect to starch content.
The selection was based on a previous study were urea content was
optimized, urea content over 37.5 % showed signs of urea surface mi-
gration, and urea contest below 25 % resulted in higher amounts of the
biocomposite material needed for a certain N dosage. As is shown in
Table 1, films with higher urea content were more flexible and pre-
sented higher biodegradation and urea release rate.
The urea release data were fitted to a semiempirical model were k is
a proportional constant related to the active compound release rate (the
higher this parameter the faster is urea released) and n is a parameter
indicative of the imperative transference mechanisms of the active
agent, in this case displaying an anomalous transport mechanism cor-
responding to the superposition of both diffusion and relaxation/ero-
sion mechanism, as described by Rivero et al. (2013).
As explained before, different strategies for the use of functionalized
films as fertilizer controlled-release systems were proposed and as-
sessed after 28 days of treatment, studying the tomato plant growth
morphological parameters (Fig. 2).
All plants presented a good global growth, though significant dif-
ferences (p < 0.05) in the plant size, total weight, leaves number and
development, and in the relative growth of roots, stems and leaves were
found, as summarized in Tables 2 and 3.
In general, except for treatment with films discs supported over kraft
paper (D), plant subjected to fertilization with biocomposites presented
-as expected- a more vigorous growth than the control plants (A). Not
only did D treatment present similar (p > 0.05) total fresh leaves, stems
and roots weight than A, but also the lowest total dry matter content
(Table 2) indicating that this treatment derived in higher water
3
Industrial Crops & Products 144 (2020) 112062
retention and the poorer urea dosage. On the contrary, treatment with
25U unsupported biocomposite films (B) exhibited the best growth
overall results, while treatments with 37.5U films (C) and 25U sup-
ported on raft wood (E) presented similar results though the latter with
better radical development as shown as well in Table 3 in root length
(RL) and stem and leaves to root ratio (Sh/R) parameters.
Correspondingly, treatment B presented the greatest leaves devel-
opment, accounted for the maximum leaf number (LN) and total leaf
area (LA), as well as the maximum leaf to stem weight ratio (L/S), while
this ratio was significantly lower for treatment C (p < 0.05) reflecting a
greater stem development (Table 3).
As it is widely known, photosynthetic pigments -which are con-
tained in chloroplasts within mesophyll tissue cells- are specialized in
light-energy absorbing used for carbon fixation (Sujatha, 2015). Their
content and composition have a direct impact on the plants photo-
synthesis capacity and therefore on its nutrients level and growth
(Jianfeng et al., 2015). Hence, both the content and proportion of
chlorophyll-a and chlorophyll-b affect the selective absorption of light
and its use by the plant.
In all cases a high chlorophyll-a to chlorophyll-b ratio is observed,
which indicates that the plants were well illuminated (Sumanta et al.,
2014). From Fig. 3 it can be observed that this ratio was not affected by
the treatment. However, a significant increase (p < 0.05) in total
chlorophylls and carotenoids content per leaf cm2 for treatments B, D
and E was observed, being this increment greater for treatment B
(Fig. 3). This parameter, on the contrary, resulted significantly lower
(p < 0.05) for treatment C. This behavior was attributed to the fact that
the 25U discs (B) leaded to a more controlled dosage of the fertilizer
over time, leading to a greater and more balanced growth of the tomato
seedlings, while the 37.5U film discs (C) promoted an uneven growth of
the plant, favoring the development of the stem with respect to leaf
development (see Table 3) and therefore exhibiting lower photo-
synthetic pigments content.
On the other hand, Fig. 3 shows that the contents of photosynthetic
pigments vary if evaluated in respect to leaf area or weight (fresh). Such
variances are significant for samples C, D and E, which are related to
completely different factors. In comparison, plants treated with C pre-
sented thinner and more fragile leaves that correlated with a higher leaf
development in number (NL) and area (LA) -shown in Table 3- but not
in weight (FLW and DLW) – shown in Table 2. Likewise, plants treated
with D did not exhibit a great leaf development, but leaves were
spongier that matches with higher moisture content, which was re-
flected in the lower total dry matter content (Table 2). Finally, and
contrary to C samples, E samples showed thicker and more dense leaves
which were attributed to similar leaves weight (FLW and DLW, in
Table 2) for a lesser leaf development (lower NL and LA, in Table 3).
Considering that N contents in the plant could be indicative of the
plants absorption of this macronutrient, beings its concentration higher
in leaves and roots than in stems, total N content of the tomato plants
leaves and roots was evaluated to compare the effect of the treatment
on the N status in the plant. Results expressed as total N content in mg
are shown in Fig. 4, indicating a significant increase (p < 0.05) for all
treatments with respect to the control (A). Leaves N content presented
the following tendency: B > C > D > E > A, while N content in roots
resulted significantly lower (p < 0.05) for D treatment, which could be
attributed to the poorer root development in these plants.
Nonetheless, it should be remarked that if the percentage of N ab-
sorption is compared, no significant differences (p > 0.05) were ob-
served between E samples and A with approximately 3.06 ± 0.2 % and
1.95 ± 0.2 % of N in leaves and roots for both samples, indicating a
poorer distribution of N for E systems. Besides, even though samples
treated with B, C and D showed greater N absorption the tendency was
inverted being roots N content similar for the three treatments (circa
2.45 ± 0.1%) and leaves N content higher for D (4.90 ± 0.2%) than C
(3.83 ± 0.2%) and B (3.41 ± 0.2%), in that order. Since the Kjeldahl
test was carried out on dry sample, these results are explained by both
F. Versino, et al. Industrial Crops & Products 144 (2020) 112062

Fig. 2. Plant growth morphological

parameters determination for control
tomato plants and plants subjected to
different urea controlled-release sys-
tems after 28 days of treatment: A)
control; B) discs of 5 cm in diameter of
film 25U; C) 4.7 cm diameter film disc
37.5U; D) 5 cm diameter kraft paper
disc coated with the film-containing
25U suspension; and E) 4 sticks with
25U.

the inferior leave development and higher moisture content for D plants
than C and B. The greater N content in leaves for C however is attrib-
uted to a high urea availability in a shorter time term, provided by 37.5
U higher biodegradation and urea release kinetics (Table 1).
Finally, the clustering and PCA of the tomato seedlings growth
parameters after 28 days of the transplant with different fertilizing
treatments showed the impact of the material used for the urea con-
trolled-release systems on the general plant growth that is intimately
related to the systems fertilizer efficiency.
On the one hand, hierarchical clustering dendrogram shown in
Fig. 5.a shows that there was no significant effect of E treatment when
compared to the control A, though all the other treatments presented
significant differences.
On the other hand, PCA presented in Fig. 5.b describes to main
components: CP1 that explains 43.5 % of the total variance and CP2
that explains 33.5 %, with a cophenetic correlation parameter of 0.932
indicating that the data clustering adequately describes the experi-
mental variables under study.
The first principal component (CP1) was attributed to the urea-re-
lease rate from the material, with treatments B and E having a positive
impact, while C, A and D on the contrary, having little or -for the latter-
a rather drastically negative effect (Fig. 5.b). Comparing the effect of
films B and C: a higher biodegradation rate and urea release kinetics of
the 37.5U materials than in the 25U films resulting in a more rapid
release of the urea to the soil favoring its wash-off in time. In addition,
the high availability of urea in the first growth-period promoted a
disproportionated growth of the stem in detriment of root development
(high Sh/R and low L/S and DRW parameters).
A different effect was observed between B and D samples: although
both materials were obtained from the same formulation (25U), the
swelling of the paper with soil-moisture could have delayed the urea
release to the soil due to the diffusion of this component to the support
matrix. A similar effect could be observed in E systems, though raft
wood sticks swelling is expected to be lesser than kraft papers, thus
having still a positive effect on plant growth.
The second main component (CP2) is associated with the type of
support material (with or without support) and therefore urea dis-
tribution. In this case, the effect was positive for treatments B and C that
only had the functionalized film based on cassava starch and bagasse,
due to the diffusion of the fertilizer to the medium from both sides of
the film (B and C), whereas when a support was used (D and E) the
diffusion is unidirectional. This effect was more noticeable in total N
content and the growth parameters of the shoot.
Even though the use of raft wood sticks supported films (E) was

Table 2
Fresh and dry weight of tomato plant fractions for control and different urea controlled-release systems.
SAMPLES FRESH PLANT WEIGTH (g) DRY PLANT WEIGHT (g) TOTAL DRY MATTER (%)

LEAVES STEM ROOTS LEAVES STEM ROOTS

a a ab a b
A 4.34 ± 0.61 7.20 ± 1.01 2.65 ± 0.79 0.48 ± 0.10 0.52 ± 0.13 0.28 ± 0.09bc 9.25 ± 0.87b
B 6.16 ± 0.85b 9.50 ± 0.78b 3.24 ± 0.68ab 0.85 ± 0.17c 0.64 ± 0.09b 0.34 ± 0.11bc 9.68 ± 1.09b
C 5.50 ± 1.30b 8.33 ± 0.92a 2.34 ± 0.77a 0.70 ± 0.14b 0.60 ± 0.10b 0.25 ± 0.10ab 9.64 ± 0.87b
D 4.38 ± 0.30a 7.58 ± 0.55a 2.23 ± 0.91a 0.47 ± 0.05a 0.40 ± 0.06a 0.13 ± 0.04a 7.71 ± 0.72a
E 5.48 ± 0.50b 7.98 ± 0.94a 3.92 ± 1.20b 0.64 ± 0.09b 0.52 ± 0.08b 0.37 ± 0.10c 9.72 ± 0.68b

Different letters in the same column indicate significant differences (p < 0.05) among samples.

4
F. Versino, et al. Industrial Crops & Products 144 (2020) 112062

Table 3
Plant growth morphological parameters for control and different urea controlled-release systems.
SAMPLES SH (cm) SD (mm) RL (cm) Sh/R (DB) L/S (DB) LA (cm2) NL (#)

a a ab a b a
A 43.11 ± 5.4 4.34 ± 0.2 12.69 ± 0.6 3.56 ± 1.3 1.15 ± 0.1 414.23 ± 45.0 46.71 ± 5.4a
B 46.65 ± 3.6b 4.75 ± 0.3b 16.36 ± 1.1c 5.76 ± 1.8bc 1.27 ± 0.1c 522.88 ± 50.5b 57.75 ± 4.1c
C 50.18 ± 1.5c 4.18 ± 0.4a 12.17 ± 1.9a 6.24 ± 1.9c 1.05 ± 0.1a 496.50 ± 99.1b 59.71 ± 4.7c
D 44.50 ± 2.9ab 4.00 ± 0.2a 14.20 ± 1.1b 5.63 ± 1.9bc 1.13 ± 0.1ab 420.99 ± 51.1a 51.50 ± 6.4ab
E 44.62 ± 2.0ab 4.73 ± 0.3b 16.13 ± 1.7c 4.15 ± 0.5ab 1.20 ± 0.1b 430.53 ± 35.1a 53.75 ± 7.4bc

Different letters in the same column indicate significant differences (p < 0.05) among samples.
Nomenclature: SH = stem height; SD = stem diameter; RL = roots length; Sh/R = shoot to root ratio on dry basis (DB); L/S = leaves to stem ratio on dry basis (DB);
LA = leaf area; NL = number of leaves.

Fig. 3. Leaves chlorophylls (a and b) and total carotenoids content expressed
per cm2 of leaf area or g of leaf on dry basis, for control tomato plants and
plants subjected to different urea controlled-release systems after 28 days of
treatment: A) control; B) discs of 5 cm in diameter of film 25U; C) 4.7 cm
diameter film disc 37.5U; D) 5 cm diameter kraft paper disc coated with the
film-containing 25U suspension; and E) 4 sticks with 25U.
expected to lead to a more uniform availability of fertilizer towards the
root and gradual urea dosage, the treatment did not have a significant
impact (p > 0.05) on the vegetative development of the seedlings,
probably due to the quality of the substrate used. Finally, it could be
observed that treatment B had the greatest positive impact on most of
the plant growth parameters, resulting this the most promising system
for urea controlled-release in soil.
In general, the differences between the samples that included the
films with fertilizer and the control differ according to the dosage
system used and in some cases (for example, D and E) are not very
marked, since the nutrients load of substrate used was good and pos-
sibly sufficient for a the initial development of the plant. However, a
tendency towards a greater leaf area, size and weight of the plant is
observed in fertilized systems. Undoubtably, the best results were ob-
tained for unsupported biocomposite films, being 25U films (B) the
most efficient urea dosage systems.
4. Conclusions
Since urea content affects the mechanical behavior and biodegrad-
able kinetics of the material, for this work cassava starch and bagasse
biocomposite films with 25 and 37.5% urea were selected. Different
strategies for the use of the developed materials as controlled fertilizer
Fig. 4. Total Nitrogen content in leaves and roots of tomato plants subjected to
different urea controlled-release systems after 28 days of treatment: A) control;
B) discs of 5 cm in diameter of film 25U; C) 4.7 cm diameter film disc 37.5U; D)
5 cm diameter kraft paper disc coated with the film-containing 25U suspension;
and E) 4 sticks with 25U.
release systems were proposed.
In general, the differences between the samples that included the
films with fertilizer and the control differ according to the dosage
system used and in some cases (for example, D and E) are not very
marked, since the nutrients load of substrate used was good and pos-
sibly sufficient for a the initial development of the plant. However, a
tendency towards a greater leaf area, size and weight of the plant is
observed in fertilized systems.
Undoubtably, the best results were obtained for unsupported bio-
composite films, being 25U films (B) the most efficient urea dosage
systems.
In summary, an ecofriendly, efficient and simple urea dosage al-
ternative system for greenhouse seedling with relatively short growth
times has been developed. Yet further investigations are needed to
develop longer lasting release systems.
Acknowledgements
This work was supported by the Agencia Nacional de Promoción
Científica y Tecnológica (ANPCyT, Project PICT 2011–1213 and 2015-
0921) and the Consejo Nacional de Investigaciones Científicas y
Técnicas (CONICET). Florencia Versino wishes to thank CONICET as
well for a Doctoral and Postdoctoral Fellowship.

5
F. Versino, et al.
Fig. 5. a) Hierarchical clustering dendrogram using Euclidean distances and b)
Biplot graph resulting from the Principal Components Analysis (PCA) of the
tomato seedlings growth parameters after 28 days of the transplant with dif-
ferent fertilizing treatments: A) control; B) discs of 5 cm in diameter of film
25U; C) 4.7 cm diameter film disc 37.5U; D) 5 cm diameter kraft paper disc
coated with the film-containing 25U suspension; and E) 4 sticks with 25U.
References
Allen, W.F., 1931. A micro-Kjeldahl method for nitrogen determination. Oil Fat Ind. 8,
391–397. https://doi.org/10.1007/BF02640022.
Baccaro, K., Degorgue, M., Lucca, M., Picone, L., Zamuner, E., Andreoli, Y., 2006. Calidad
de agua para consumo humano y riego en muestras del cintrurón hortícola de Mar del
Plata. RIA. Rev. Investig. Agropecu. Inst. Nac. Tecnol. Agropecu. Argentina 35,
95–110.
Bilck, A.P., Grossmann, M.V.E., Yamashita, F., 2010. Biodegradable mulch films for
strawberry production. Polym. Test. 29, 471–476. https://doi.org/10.1016/j.
polymertesting.2010.02.007.
Chen, L., Xie, Z., Zhuang, X., Chen, X., Jing, X., 2008. Controlled release of urea en-
capsulated by starch-g-poly(l-lactide). Carbohydr. Polym. 72, 342–348. https://doi.
Industrial Crops & Products 144 (2020) 112062
org/10.1016/j.carbpol.2007.09.003.
De Grazia, J., Tittonell, P.A., Chiesa, A., 2011. Fertilización nitrogenada en plantines de
pimiento (Capsicum annuum L.) cultivados en sustratos con diferentes proporciones
de materiales compostados : efecto sobre los parámetros de calidad del plantín. Rev.
FCA UNCUYO Tomo 43, 175–186.
Di Rienzo, J.A., Casanoves, F., Balzarini, M.G., Gonzalez, L., Tablada, M., Robledo, C.W.,
2011. Infostat Software.
Ferreira, E.V., de, O., Novais, R.F., Médice, B.M., Barros, N.F., Silva, I.R., 2015. Leaf total
nitrogen concentration as an indicator of nitrogen status for plantlets and young
plants of eucalyptus clones. Rev. Bras. Ciência do Solo 39, 1127–1140. https://doi.
org/10.1590/01000683rbcs20140689.
Horinouchi, H., Katsuyama, N., Taguchi, Y., Hyakumachi, M., 2008. Control of Fusarium
crown and root rot of tomato in a soil system by combination of a plant growth-
promoting fungus, Fusarium equiseti and biodegradable pots. Crop Prot. 27,
859–864. https://doi.org/10.1016/j.cropro.2007.08.009.
Jianfeng, W., Dongxian, H., Jinxiu, S., Haijie, D., Weifen, D., 2015. Non-destructive
measurement of chlorophyll in tomato leaves using spectral transmittance. Int. J.
Agric. Biol. Eng. 8, 73–78. https://doi.org/10.3965/j.ijabe.20150805.1931.
López, O.V., Viña, S.Z., Pachas, A.N.A., Sisterna, M.N., Rohatsch, P.H., Mugridge, A., Lo,
O.V., Fassola, H.E., García, M.A., 2010. Composition and food properties of
Pachyrhizus ahipa roots and starch. Int. J. Food Sci. Technol. 45, 223–233. https://
doi.org/10.1111/j.1365-2621.2009.02125.x.
Moreno, M.M., Moreno, A., 2008. Effect of different biodegradable and polyethylene
mulches on soil properties and production in a tomato crop. Sci. Hortic.
(Amsterdam). 116, 256–263. https://doi.org/10.1016/j.scienta.2008.01.007.
Pérez, D., Lovisolo, M., Chiesa, A., 2016. Estudio morfoanatómico de plantines de Tomate
(Solanum lycopersicum L.) con agua de riego salinas. Rev. Divulg. Técnica Agropecu.
Agroindustrial y Ambient. Fac. Ciencias Agrar. Univ. Nac. Lomas Zamora. 3, 3–8.
Porra, R.J., 2002. The chequered history of the development and use of simultaneous
equations for the accurate determination of chlorophylls a and b. Photosynth. Res.
73, 149–156. https://doi.org/10.1023/A:1020470224740.
Rivero, S., Giannuzzi, L., García, M.A., Pinotti, A., 2013. Controlled delivery of propionic
acid from chitosan films for pastry dough conservation. J. Food Eng. 116, 524–531.
https://doi.org/10.1016/j.jfoodeng.2012.12.025.
Riyajan, S.A., Sasithornsonti, Y., Phinyocheep, P., 2012. Green natural rubber-g-modified
starch for controlling urea release. Carbohydr. Polym. 89, 251–258. https://doi.org/
10.1016/j.carbpol.2012.03.004.
Rychter, P., Kot, M., Bajer, K., Rogacz, D., Siskova, A., Kapusniak, J., 2016. Utilization of
starch films plasticized with urea as fertilizer for improvement of plant growth.
Carbohydr. Polym. 137, 127–138. https://doi.org/10.1016/j.carbpol.2015.10.051.
Scheiner, D., 1976. Determination of ammonia and Kjeldahl nitrogen by indophenol
method. Water Res. 10, 31–36. https://doi.org/10.1016/0043-1354(76)90154-8.
Sujatha, B., 2015. Photosynthesis. In: Bahadur, B., Venkat Rajam, M., Sahijram, L.,
Krishnamurthy, K.V. (Eds.), Plant Biology and Biotechnology. Volume I: Plant
Diversity, Organization, Function and Improvement. Springe, New Delhi, pp.
569–591. https://doi.org/10.1007/978-81-322-2286-6_22.
Sumanta, N., Imranul Haque, C., Nishika, J., Suprakash, R., 2014. Spectrophotometric
analysis of chlorophylls and carotenoids from commonly grown fern species by using
various extracting solvents. Res. J. Chem. Sci. 4, 63–69. https://doi.org/10.1055/s-
0033-1340072.
Taiz, L., Zeiger, E., 2002. Mineral nutrition. Plant Physiology. Oxford University Press Inc,
Oxford, UK, pp. 67–86.
Vagnoni, R., Buyatti, M., Favaro, J.C., 2014. Efecto del tamaño de celda de bandejas de
siembra sobre la morfología y fisiología de plantines de tomate (Lycopersicon escu-
lentum Mill.). Hortic. Argentina 30, 15–19.
Versino, F., García, M.A., 2019. Particle size distribution effect on cassava starch and
cassava bagasse biocomposites. ACS Sustain. Chem. Eng. 7, 1052–1060. https://doi.
org/10.1021/acssuschemeng.8b04700.
Versino, F., García, M.A., 2018. Starch films for agronomic applications : comparative
study of urea and glycerol as plasticizers. Int. J. Environ. Agric. Biotechnol. 3,
1854–1864. https://doi.org/10.22161/ijeab/3.5.38.
Versino, F., García, M.A., 2014. Cassava (Manihot esculenta) starch films reinforced with
natural fibrous filler. Ind. Crops Prod. 58, 305–314. https://doi.org/10.1016/j.
indcrop.2014.04.040.
Versino, F., López, O.V., García, M.A., 2015. Sustainable use of cassava (Manihot escu-
lenta) roots as raw material for biocomposites development. Ind. Crops Prod. 65,
79–89. https://doi.org/10.1016/j.indcrop.2014.11.054.
Versino, F., Urriza, M., García, M.A., 2019. Eco-compatible cassava starch films for fer-
tilizer controlled-release. Int. J. Biol. Macromol. https://doi.org/10.1016/J.
IJBIOMAC.2019.05.037.