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Cassava-based biocomposites as fertilizer controlled-release systems for plant growth improvement
Industrial Crops & Products 144 (2020) 112062
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Cassava-based biocomposites as fertilizer controlled-release systems for plant growth improvement
T
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 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 b
A R T I C LE I N FO
A B S T R A C T
Keywords: Starch Renewable materials Biodegradable Fibrous fillers Urea
One of the main problems caused by intensive agriculture, is the consequent water pollution resulting from fertilizer leaching, especially as a result of fertilizers overuse. Therefore, in the present study an evaluation of the efficiency of different urea controlled-release systems, based on cassava starch and bagasse, over tomato plants growth was carried out. All fertilized systems showed a tendency towards a greater general development, photosynthetic pigments content and nitrogen status of the plant. However, significant differences were observed 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 amounts of agrochemicals that enter the environment, helping to maintain biological activity at a desirable level. Accordingly, the inclusion 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 different 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).
Research and development related to the functionalization of biodegradable 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 included in the formulation and consequently its dosage. However, applications 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 alternative 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 alternative clearly constitutes a resource towards a cleaner and more sustainable agriculture, since it intends to avoid the excessive use of fertilizers 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
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: fl[email protected], fl[email protected] (F. Versino), [email protected] (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.
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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 processing 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 recorded (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 determined 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, Argentina) 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):
Fig. 1. Diagram of treatments preparation of controlled urea release with cassava 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 biodegradation and urea release kinetics in soil determined in previous studies (Versino et al., 2019; Versino and García, 2019, 2018).
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. Plants growth evaluation 2.2.1. Seedlings and treatments preparation Since urea contributes approximately with 46% of nitrogen, a requirement 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 treatments 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 commercial 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).
2.2.4. 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 indicator 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 assessed 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 614% active chloride, CAS# 7681-52-9, Merck, Germany), in the 2
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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 supported 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 development, 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 contained 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 photosynthesis 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 photosynthetic 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 presented 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 reflected 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 absorption is compared, no significant differences (p > 0.05) were observed 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
Table 1 Characteristics and properties of cassava starch and bagasse biocomposites plasticized with different urea concentrations used for tomato plant growth. Biocomposite Thickness (μm) Moisture content (%) Water uptake (water g/g Tensile strength Stress at break (MPa) Strain at break (%) Biodegradation rate t50 (days) dt (days) Urea release kinetics k n
DB*)
25U 164 ± 12 12.10 ± 0.74 0.4092 ± 0.008
37.5U 110 ± 17 16.87 ± 1.74 0.5266 ± 0.006
2.9 ± 0.1 10.8 ± 1.0
1.1 ± 0.1 25.8 ± 2.6
24.64 ± 0.6 4.07 ± 0.4
27.33 ± 1.4 6.68 ± 1.3
0.029 ± 0.009 0.884 ± 0.098
0.045 ± 0.017 0.793 ± 0.107
*DB = dry basis. Reported values correspond to the mean ± standard deviation.
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 interdependence 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 migration, 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 presented 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 corresponding to the superposition of both diffusion and relaxation/erosion 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 assessed after 28 days of treatment, studying the tomato plant growth morphological parameters (Fig. 2). All plants presented a good global growth, though significant differences (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
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Fig. 2. Plant growth morphological parameters determination 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.
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 distribution. 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
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 attributed 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 controlled-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 experimental variables under study. The first principal component (CP1) was attributed to the urea-release 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 lattera rather drastically negative effect (Fig. 5.b). Comparing the effect of
Table 2 Fresh and dry weight of tomato plant fractions for control and different urea controlled-release systems. SAMPLES
FRESH PLANT WEIGTH LEAVES
A B C D E
(g)
STEM a
4.34 ± 0.61 6.16 ± 0.85b 5.50 ± 1.30b 4.38 ± 0.30a 5.48 ± 0.50b
ROOTS a
7.20 ± 1.01 9.50 ± 0.78b 8.33 ± 0.92a 7.58 ± 0.55a 7.98 ± 0.94a
ab
2.65 ± 0.79 3.24 ± 0.68ab 2.34 ± 0.77a 2.23 ± 0.91a 3.92 ± 1.20b
DRY PLANT WEIGHT
(g)
LEAVES
STEM a
0.48 ± 0.10 0.85 ± 0.17c 0.70 ± 0.14b 0.47 ± 0.05a 0.64 ± 0.09b
Different letters in the same column indicate significant differences (p < 0.05) among samples. 4
TOTAL DRY MATTER ROOTS b
0.52 ± 0.13 0.64 ± 0.09b 0.60 ± 0.10b 0.40 ± 0.06a 0.52 ± 0.08b
0.28 ± 0.09bc 0.34 ± 0.11bc 0.25 ± 0.10ab 0.13 ± 0.04a 0.37 ± 0.10c
9.25 ± 0.87b 9.68 ± 1.09b 9.64 ± 0.87b 7.71 ± 0.72a 9.72 ± 0.68b
(%)
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Table 3 Plant growth morphological parameters for control and different urea controlled-release systems. SAMPLES A B C D E
SH
SD
(cm) a
43.11 ± 5.4 46.65 ± 3.6b 50.18 ± 1.5c 44.50 ± 2.9ab 44.62 ± 2.0ab
RL
(mm) a
4.34 ± 0.2 4.75 ± 0.3b 4.18 ± 0.4a 4.00 ± 0.2a 4.73 ± 0.3b
Sh/R
(cm) ab
L/S
(DB) a
12.69 ± 0.6 16.36 ± 1.1c 12.17 ± 1.9a 14.20 ± 1.1b 16.13 ± 1.7c
3.56 ± 1.3 5.76 ± 1.8bc 6.24 ± 1.9c 5.63 ± 1.9bc 4.15 ± 0.5ab
LA
(DB) b
1.15 ± 0.1 1.27 ± 0.1c 1.05 ± 0.1a 1.13 ± 0.1ab 1.20 ± 0.1b
(cm2)
NL a
414.23 ± 45.0 522.88 ± 50.5b 496.50 ± 99.1b 420.99 ± 51.1a 430.53 ± 35.1a
(#)
46.71 ± 5.4a 57.75 ± 4.1c 59.71 ± 4.7c 51.50 ± 6.4ab 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. 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.
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.
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 possibly 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 biocomposite films, being 25U films (B) the most efficient urea dosage systems. In summary, an ecofriendly, efficient and simple urea dosage alternative system for greenhouse seedling with relatively short growth times has been developed. Yet further investigations are needed to develop longer lasting release systems.
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 possibly 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 biocomposite films, being 25U films (B) the most efficient urea dosage systems.
Acknowledgements This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Project PICT 2011–1213 and 20150921) and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Florencia Versino wishes to thank CONICET as well for a Doctoral and Postdoctoral Fellowship.
4. Conclusions Since urea content affects the mechanical behavior and biodegradable 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 5
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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/s0033-1340072. 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 esculentum 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 esculenta) 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 fertilizer controlled-release. Int. J. Biol. Macromol. https://doi.org/10.1016/J. IJBIOMAC.2019.05.037.
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 different 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 encapsulated by starch-g-poly(l-lactide). Carbohydr. Polym. 72, 342–348. https://doi.
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Cassava-based biocomposites as fertilizer controlled-release systems for plant growth improvement
T
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 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 b
A R T I C LE I N FO
A B S T R A C T
Keywords: Starch Renewable materials Biodegradable Fibrous fillers Urea
One of the main problems caused by intensive agriculture, is the consequent water pollution resulting from fertilizer leaching, especially as a result of fertilizers overuse. Therefore, in the present study an evaluation of the efficiency of different urea controlled-release systems, based on cassava starch and bagasse, over tomato plants growth was carried out. All fertilized systems showed a tendency towards a greater general development, photosynthetic pigments content and nitrogen status of the plant. However, significant differences were observed 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 amounts of agrochemicals that enter the environment, helping to maintain biological activity at a desirable level. Accordingly, the inclusion 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 different 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).
Research and development related to the functionalization of biodegradable 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 included in the formulation and consequently its dosage. However, applications 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 alternative 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 alternative clearly constitutes a resource towards a cleaner and more sustainable agriculture, since it intends to avoid the excessive use of fertilizers 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
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: fl[email protected], fl[email protected] (F. Versino), [email protected] (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.
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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 processing 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 recorded (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 determined 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, Argentina) 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):
Fig. 1. Diagram of treatments preparation of controlled urea release with cassava 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 biodegradation and urea release kinetics in soil determined in previous studies (Versino et al., 2019; Versino and García, 2019, 2018).
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. Plants growth evaluation 2.2.1. Seedlings and treatments preparation Since urea contributes approximately with 46% of nitrogen, a requirement 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 treatments 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 commercial 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).
2.2.4. 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 indicator 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 assessed 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 614% active chloride, CAS# 7681-52-9, Merck, Germany), in the 2
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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 supported 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 development, 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 contained 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 photosynthesis 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 photosynthetic 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 presented 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 reflected 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 absorption is compared, no significant differences (p > 0.05) were observed 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
Table 1 Characteristics and properties of cassava starch and bagasse biocomposites plasticized with different urea concentrations used for tomato plant growth. Biocomposite Thickness (μm) Moisture content (%) Water uptake (water g/g Tensile strength Stress at break (MPa) Strain at break (%) Biodegradation rate t50 (days) dt (days) Urea release kinetics k n
DB*)
25U 164 ± 12 12.10 ± 0.74 0.4092 ± 0.008
37.5U 110 ± 17 16.87 ± 1.74 0.5266 ± 0.006
2.9 ± 0.1 10.8 ± 1.0
1.1 ± 0.1 25.8 ± 2.6
24.64 ± 0.6 4.07 ± 0.4
27.33 ± 1.4 6.68 ± 1.3
0.029 ± 0.009 0.884 ± 0.098
0.045 ± 0.017 0.793 ± 0.107
*DB = dry basis. Reported values correspond to the mean ± standard deviation.
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 interdependence 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 migration, 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 presented 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 corresponding to the superposition of both diffusion and relaxation/erosion 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 assessed after 28 days of treatment, studying the tomato plant growth morphological parameters (Fig. 2). All plants presented a good global growth, though significant differences (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
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Fig. 2. Plant growth morphological parameters determination 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.
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 distribution. 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
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 attributed 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 controlled-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 experimental variables under study. The first principal component (CP1) was attributed to the urea-release 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 lattera rather drastically negative effect (Fig. 5.b). Comparing the effect of
Table 2 Fresh and dry weight of tomato plant fractions for control and different urea controlled-release systems. SAMPLES
FRESH PLANT WEIGTH LEAVES
A B C D E
(g)
STEM a
4.34 ± 0.61 6.16 ± 0.85b 5.50 ± 1.30b 4.38 ± 0.30a 5.48 ± 0.50b
ROOTS a
7.20 ± 1.01 9.50 ± 0.78b 8.33 ± 0.92a 7.58 ± 0.55a 7.98 ± 0.94a
ab
2.65 ± 0.79 3.24 ± 0.68ab 2.34 ± 0.77a 2.23 ± 0.91a 3.92 ± 1.20b
DRY PLANT WEIGHT
(g)
LEAVES
STEM a
0.48 ± 0.10 0.85 ± 0.17c 0.70 ± 0.14b 0.47 ± 0.05a 0.64 ± 0.09b
Different letters in the same column indicate significant differences (p < 0.05) among samples. 4
TOTAL DRY MATTER ROOTS b
0.52 ± 0.13 0.64 ± 0.09b 0.60 ± 0.10b 0.40 ± 0.06a 0.52 ± 0.08b
0.28 ± 0.09bc 0.34 ± 0.11bc 0.25 ± 0.10ab 0.13 ± 0.04a 0.37 ± 0.10c
9.25 ± 0.87b 9.68 ± 1.09b 9.64 ± 0.87b 7.71 ± 0.72a 9.72 ± 0.68b
(%)
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Table 3 Plant growth morphological parameters for control and different urea controlled-release systems. SAMPLES A B C D E
SH
SD
(cm) a
43.11 ± 5.4 46.65 ± 3.6b 50.18 ± 1.5c 44.50 ± 2.9ab 44.62 ± 2.0ab
RL
(mm) a
4.34 ± 0.2 4.75 ± 0.3b 4.18 ± 0.4a 4.00 ± 0.2a 4.73 ± 0.3b
Sh/R
(cm) ab
L/S
(DB) a
12.69 ± 0.6 16.36 ± 1.1c 12.17 ± 1.9a 14.20 ± 1.1b 16.13 ± 1.7c
3.56 ± 1.3 5.76 ± 1.8bc 6.24 ± 1.9c 5.63 ± 1.9bc 4.15 ± 0.5ab
LA
(DB) b
1.15 ± 0.1 1.27 ± 0.1c 1.05 ± 0.1a 1.13 ± 0.1ab 1.20 ± 0.1b
(cm2)
NL a
414.23 ± 45.0 522.88 ± 50.5b 496.50 ± 99.1b 420.99 ± 51.1a 430.53 ± 35.1a
(#)
46.71 ± 5.4a 57.75 ± 4.1c 59.71 ± 4.7c 51.50 ± 6.4ab 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. 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.
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.
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 possibly 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 biocomposite films, being 25U films (B) the most efficient urea dosage systems. In summary, an ecofriendly, efficient and simple urea dosage alternative system for greenhouse seedling with relatively short growth times has been developed. Yet further investigations are needed to develop longer lasting release systems.
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 possibly 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 biocomposite films, being 25U films (B) the most efficient urea dosage systems.
Acknowledgements This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Project PICT 2011–1213 and 20150921) and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Florencia Versino wishes to thank CONICET as well for a Doctoral and Postdoctoral Fellowship.
4. Conclusions Since urea content affects the mechanical behavior and biodegradable 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 5
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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 different 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.
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