Encapsulation of Biological and Chemical Agents for Plant Nutrition

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Encapsulation of Biological and Chemical Agents for Plant Nutrition and Protection: Chitosan/Alginate Microcapsules Loaded with Copper Cations and Trichoderma viride Marko Vincekovic, Nenad Jalšenjak, Snježana TopolovecPintari#, Edyta #ermi#, Marija Bujan, and Slaven Juri# J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02879 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 8, 2016

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Journal of Agricultural and Food Chemistry

Encapsulation of Biological and Chemical Agents for Plant Nutrition and Protection: Chitosan/Alginate Microcapsules Loaded with Copper Cations and Trichoderma viride Marko Vinceković1,*, Nenad Jalšenjak,1 Snježana Topolovec-Pintarić,2 Edyta Đermić,2 Marija Bujan,1 Slaven Jurić1 Department of Chemistry1, University of Zagreb Faculty of Agriculture, 10000 Zagreb Croatia Department of Plant Pathology2, University of Zagreb Faculty of Agriculture, 10000 Zagreb Croatia

ACS Paragon Plus Environment


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ABSTRACT: Novel chitosan/alginate microcapsules simultaneously loaded with copper

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cations and Trichoderma viride have been prepared and characterized. Information about

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intermolecular interactions between biopolymers and bioactive agents were obtained by

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Fourier transform infrared spectroscopy. Encapsulation of Trichoderma viride spores and the

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presence of copper cations in the same compartment do not inhibit their activity. Dependence

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of microcapsule loading capacity and efficiency, swelling behavior and releasing depend on

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both, the size of the microcapsule and bioactive agents. The in vitro copper cations release

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profile was fitted to Korsmeyer–Peppas empirical model. Fickian diffusion was found to be a

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rate-controlling mechanism of release from smaller microcapsules, whereas anomalous

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transport kinetics controlled release from larger microcapsules. Trichoderma viride spores

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releasing profile exhibited exponential release over the initial lag time. Results obtained

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opened up perspectives for the future use of chitosan/alginate microcapsules simultaneously

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loaded with biological and chemical agents in the plant nutrition and protection.

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KEYWORDS: chitosan/alginate microcapsules, encapsulation, copper cations, Trichoderma

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viride spores, sustainability


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1. INTRODUCTION

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The encapsulation of bioactive agents has been developed in recent years as a new potential

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tool for ecological and sustainable plant production. Encapsulation in biopolymer matrices

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has been recognized as an effective method for controlled release of a bioactive agent used for

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plant protection.1 Biopolymer based microcapsules with a single bioactive agent have

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extensive applications in agriculture and became one of the standard capsule formulations.2 and

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references therein

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chemical agents, there are no data in the literature about simultaneous encapsulation and

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delivery of biological and chemical agents.

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Polysaccharides such as chitosan (abbreviation, CS) and alginate (abbreviation, ALG) are

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biopolymers that easily create capsules in which an active ingredient can be incorporated

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using an aqueous system at ambient temperature. Alginate is an anionic polysaccharide

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composed of two repeating carboxylated monosaccharide units (manuronic and guluronic

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acids) the ratio of which influences the properties of the biopolymer. Chitosan is a partially

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deacetylated polymer of N-acetylglucosamine obtained after alkaline deacetylation of chitin.

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The N-deacetylation is almost never complete and the fraction of N-acetylglucosamine

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determines the degree of acetylation which serves as a base to classify the biopolymer as

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chitin or chitosan. When the degree of N-acetylation (defined as the average number of N-

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acetylglucosamine units per 100 monomers expressed as a percentage) is less than 50%, chitin

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becomes soluble in aqueous acidic solutions (pH < 6.0) and is called chitosan. The

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electrostatic attraction between the cationic amino groups of chitosan and the carboxylic

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groups of the alginate lead to the formation of the polyelectrolyte complexes of various

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structures. The structure and physicochemical properties of these complexes may be tailored

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by controlling the degree of association among the functional groups.3 There are many

Despite the array of methodologies for simultaneous encapsulation of two



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possible applications for CS and ALG complexes in the microcapsule form such as for

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immobilization and the controlled release of various chemical or biologically active agents.4

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It is well known that chemical elements (primary, secondary and micronutrients) are

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important for plant growth and survival. Copper cations are an essential micronutrient for

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plants and it is used for the management of a wide range of fungal and bacterial diseases in

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various crops. Plants require copper cations for normal growth and development, and when it

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is not available, plants develop specific deficiency symptoms, most of which affect young

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leaves and reproductive organs.5 Thus, a deficiency in the copper cations supply can alter the

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essential functions in plant metabolism. Copper cations have traditionally been used in

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agriculture as plant pathogens control, and it is also extensively released into the environment

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by other human activities that often cause environmental pollution. Therefore, it is important

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to control the level of copper cations added to the plant. It is worth to notice that positive

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charge of chitosan microcapsules loaded with copper cations facilitate better bioadhesion on

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leaves that enables prolonged presence of copper cations on the leaf surface.6,7 The prolonged

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release of copper cations from microcapsules and its prolonged presence on the leaves may

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lead to reduced levels of copper cations needed for sufficient crop protection showing

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microencapsulation is a better alternative to traditional applying of copper cations formulation

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in plant protection.

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Plants at all stages of their development, as well harvested products in storage, are susceptible

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to attack by many pathogens that cause severe damage to plants. Biological agents

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(biopesticides) may represent a new approach to plant protection.8,9 Biopesticides are based

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on microorganisms such as bacteria, viruses, fungi, nematodes or natural substances,

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including plant extracts and semi chemicals (e.g. insect pheromones). However, applications

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of biopesticides are still limited to only a few percent of all active agents used for plant

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protection.


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Filamentous fungi Trichoderma viride (abbrevation, TV) is an opportunistic avirulent plant

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symbiont as well as mycoparasite of plant pathogenic fungi. Its agricultural importance is

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good antagonistic abilities against soil born plant pathogenic fungi thanks to different

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mechanisms of antagonism, the production of antifungal metabolites (antibiosis), competition

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for space and nutrients, induction of defense responses in plant and mycoparasitism.

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Investigations revealed also the ability of T. viride to promote plant growth that was first

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treated as a side effect of suppression of plant pathogenic fungi.10 Today is considered that the

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direct effects of these fungi on plant growth and development are crucially important for

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agricultural uses and for understanding the roles of T. viride in natural and managed

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ecosystems.10,11 The dual roles of antagonistic activity against plant pathogens and promotion

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of soil fertility make Trichoderma species a promising alternative to standard plant protection

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and nutrition technologies.

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The benefits of biopolymer microcapsules simultaneously loaded with copper cations and T.

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viride are delayed and controlled release of bioactive agents, as well as prolonged fertilization

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effect in order to supply plant with the protecting/nutrient agents during the whole vegetation.

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Finally, only one application would be enough to cover plant active agent needs in the

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vegetation period. There is not important economic aspect only, but also agroecological

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because a reduced number of mechanization passes will result with lower soil compaction,

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and thus the root would have much better conditions for growth and development.

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In the light of the importance of T. viride as a biocontrol agent, showing the high ability to

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accumulate and compatibility with macro- and micronutrients,12,13 simultaneous encapsulation

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of copper cations and T. viride could give wider opportunities in both, the plant nutrition and

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protection. The aim of this work was to prepare chitosan/alginate microcapsules

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simultaneously loaded with copper cations and T. viride (abbreviation, CS/(ALG/(Cu+TV))



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for plant protection and nutrition. Copper cation concentrations and microcapsule size were

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considered as variables with possible influence on the essential microcapsule parameters.

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2. MATERIALS AND METHODS

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2.1 Materials. Low viscosity sodium alginate (CAS Number: 9005-38-3; Brookfield viscosity

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4 - 12 cps (1% in H2O at 25oC) was purchased from Sigma Aldrich (USA). Low molecular

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weight chitosan (CAS RN: 9012-76-4, molecular weight: 100 000–300 000) was obtained

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from Acros Organic (USA). A commercially available product copper sulphate pentahydrate,

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CuSO4 · 5H2O was used as a copper donating substance (Kemika Croatia). All other

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chemicals were of analytical grade and used as received without further purification.

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Indigenous isolate of T. viride (abbreviation, STP) originated from parasited sclerotia of

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Athelia rolfsii was used in all experiments.

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2.2. Preparation of Trichoderma viride isolates and spore suspension. The culture of the

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isolate was grown in Petri dishes of 10 cm in diameter containing 20 ml of potato dextrose

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agar (PDA, Biolife, Italy) plates and incubated in an incubator at 25 °C for 7 days until

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conidiation occurred. To obtain spore suspensions, the STP was grown in Erlenmeyer flasks

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containing potato dextrose broth (PDB). Flasks were inoculated with 5 PDA mycelial plugs.

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Incubation took place at 22 ºC under the constant aeration for 10 days under illumination.

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After this incubation period the liquid cultures with fungal biomass, consisting of hyphal

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segments, chlamydiospores and conidia were filtrated by suction through the filter paper (595

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Schleicher & Schuell; Whatman International, Ltd., Kent, England) so that the major parts of

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mycelium was removed. Nebulisation of microencapsulated spores was checked in vitro using

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an ultrasonic nebuliser (Omron Healthcare Europe, Netherlands).

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2.3. Methods of microencapsulation. 2.3.1. Preparation of microcapsules viscous

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dispersion. Copper cations loaded microcapsules (abbrevation, CS/(ALG/Cu)) viscous


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dispersions (abbreviation, CCVD) were prepared by modifying known procedure7 as

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described in Supporting Information (S2.3.1.).

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2.3.2. Preparation of CS/(ALG/Cu) and CS/(ALG/(Cu+TV) microcapsules. Microcapsules

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were prepared by the ionic gelation technique at ambient temperature. Preparation is rapid and

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reliable, and microcapsules were obtained spontaneously under very mild conditions in two

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stages. The first stage comprises the formation of core microcapsules loaded with copper

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cations (abbrevation, ALG/Cu) or loaded with T. viride (abbrevation, ALG/(Cu+TV)), and the

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second stage includes the coating of core microcapsules by chitosan. Details of preparation

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are presented in Supporting Information (S2.3.2.1. and S2.3.2.2.).

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2.4. Methods. 2.4.1. Influence of copper cations on Trichoderma viride growth and survival.

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The cultures of fungus T. viride were maintained on potato dextrose agar (PDA, Biolife, Italy)

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plates at 25 °C. Influence of copper cations concentration was examined by spraying CCVD

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loaded with copper cations. Mycelial growth was observed visually.

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The viability of spores was monitored according to ability of germination that is by measuring

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changes in the concentration of T. viride expressed as the number of spores (NS) per 1 g of

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dry microcapsule. Samples for measurements were prepared by dissolving 4 g of

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microcapsules in 100 ml of a mixture (0.2 mol dm-3 NaHCO3 and 0.06 mol dm-3 Na2C6H5O7 x

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2H2O). Dissolved microcapsules were mixed for 30 minutes with a magnetic stirrer at room

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temperature. Five milliliters of a sample were filtered through the sterilized muslin cloth.14

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Aseptic conditions were provided throughout the assay. The number of spores was

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determined spectrophotometrically by a method of Waghunde et al.15 Stock solution of T.

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viride spores (adjusted to 1.4 x 106 of spores/ml) used for calibration curve was diluted with

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sterilized water. The number of spores in stock solution was determined with a

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hemocytometer, using Neubauer counting chambers (Hirschmann EM Techcolor, Germany).



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Absorbance was measured at 550 nm using UV-VIS spectrophotometer (UV-1700, Shimadzu,

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Japan).

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2.4.2. Determination of Trichoderma viride spores charge. The zeta potential (ζ/mV) of T.

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viride spore was measured by Zetasizer Nano ZS (Malvern, UK). The zeta potential was

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estimated from electrophoretic measurements using Henry equation: Ue = 2ε ζ (fκa/3η)

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(1),

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where ζ is the zeta potential, ε is the dielectric constant, Ue is the electrophoretic mobility and

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η is the viscosity. fκa is in this case 1.5 and is referred to as the Smoluchowski

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approximation.16 Deviations ranged within ±1 mV.

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2.5. Characterization of delivery systems. 2.5.1. Microscopic observations.The size of

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prepared microcapsules was measured using a light binocular. Diameters of about 100

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microcapsules were measured. Microphotographs were taken by a Leica DFC295 digital

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camera on a trinocular mount of a Leica MZ16a stereo-microscope (Leica Microsystems Ltd.,

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Switzerland). Microcapsules were also observed by confocal laser scanning microscope

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(CLSM, TCP SP2, Leica Lasertechnik, Germany). Samples for CLSM were stained with

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eosin (0.01 % (w/v)). The microscope was operated in fluorescence and transmitted mode at

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an acceleration voltage of 80 kV. All sample preparations for microscopic observation were

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performed at room temperature.

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2.5.2. Fourier Transform Infrared Spectroscopy. The Fourier transform infrared spectroscopy

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(FTIR) spectra of the samples were recorded with the FTIR Instrument - Cary 660 FTIR

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(MIR system) spectrometer (Agilent Technologies, USA). Dry microcapsules were crushed

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with potassium bromide to get pellets. Spectral scanning was done in the range of 400-4000

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cm-1.


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2.5.3. Encapsulation efficiency, loading capacity, swelling degree and in vitro active agent

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release. Encapsulation efficiency (EE) and bioactive agent loading capacity (LC)

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determination were performed to obtain information on the yield and amount of copper

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cations and T. viride spores encapsulated in microcapsules. The concentration of copper

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cations was determined at λ = 795 nm and concentration of T. viride at 550 nm by a

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spectrophotometer (Shimadzu, UV-1700).

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The swelling degree (Sw) was determined in both CS/(ALG/Cu) and CS/(ALG/(Cu+TV))

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microcapsules loaded with various amounts of copper cations to better understand

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mechanisms of bioactive agents releasing from microcapsules. All measurements were made

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in triplicate.

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Detailed procedures of sample preparation for measurements and calculations are presented in

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Supporting Information (from S2.5.3.1. to S2.5.3.4.).

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3. RESULTS AND DISCUSSION

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3.1. Copper cations effect on the growth and viability of Trichoderma viride spores. One

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of the problems that should be solved in the simultaneous encapsulation of biological and

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chemical agents is that the presence of active agents in the same compartment should not

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diminish their activities. It is well known that T. viride belong to the group of

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microorganisms, which can survive in high concentrations of different metals and have the

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potential to bind them.12,13 However, this has been well explored only in the light of the

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possible application of fungi in metal removal from solid and liquid substrates and industrial

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wastes.

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Visual observation after 10 days of CCVD application revealed high resistance of T. viride

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spores to applicated copper cations concentrations. The presence of copper cations did not

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inhibit the growth of fungi, i.e. none of the applied concentrations caused inhibition of

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mycelial growth. On the contrary, the presence of copper cations promotes growth, e.g. at the



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highest copper cation concentration the growth of T. viride was the most homogeneous (Fig.

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1). It is in accordance with investigations showing that the copper cations concentration at

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0.75 mmol dm-3 promotes the growth of Trichoderma species.13

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Experiments performed on T. viride spores survival in CS/ALG/(Cu+TV) revealed that during

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storage at the room temperature the number of spores was almost constant in the smaller

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microcapsule, whereas in larger microcapsules somewhat increased (Table 1). It seems that

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smaller microcapsules were fully loaded with T. viride spores and there was no space for their

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germination. Smaller loading capacity (see later) and available internal volume in larger

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microcapsules allowed spores to germinate indicating that copper does not inhibit their

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activity.

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Chemical analyses of the fungal cell walls have revealed a very complex chemical

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composition that may be different in the several taxonomic groups.17 Cell walls have been

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shown to be composed of polysaccharides (mainly glucan and chitin, up to 90%) and

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glycoproteins, as well as of lipids and other minor components (pigments and inorganic salts).

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Melanin, which in other fungi is associated with chitin, was shown to replace this polymer in

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the spore wall of Trichoderma species and chitin was found only in mycelial cell walls. FTIR

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spectrum of T. viride is presented in Fig. 2. Characteristics of T. viride spectrum are strong

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and a broad peak at 3321 cm-1 assignable to amino (-NH) group superimposed on the side

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hydroxyl (-OH) groups, frequencies at 2921 and 2854 cm-1 indicative of alkyl (>CH2) and

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hydroxyl (-OH) groups, small peak at 2367 cm-1 assigned to the asymmetric stretching of the

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isocyanate group (-N=C=O),18 the strong peak at 1625 cm-1 as well as several small peaks

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between 1452 and 1200 cm-1 caused by stretching mode of carbonyl group (C=O) conjugated

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to -NH deformation mode (indicative of amide bond formation).19 The small peak at 1545 cm-

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1

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deformation mode,20 and a strong peak at 1030 cm-1 represents C-F and C-Br vibrations. The

represents amine group stretching vibration resulting from -NH deformation mode to C=N


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functional groups detected in the FTIR spectrum relate well with the the chemical structure of

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the cell wall.

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The characteristic peaks of copper sulfate pentahydrate at 3200, 1667, 1067 and 860 cm-1 are

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presented in Fig. 2. The peaks over 3000 cm-1 represent the crystal water in the structure,

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whereas peaks at lower band values correspond to the vibrations between O and nonmetal

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atoms. A broad stretching frequency at 3200 cm-1 and a band of medium intensity at 1667 cm-

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1

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characteristic bands of inorganic sulfates ((SO4)2- stretching region).21

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The spectrum of T. viride with bind copper cations shows much more intense and broad -OH

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and -NH stretching vibrations band, the disappearance of T. viride bands at 2921, 2854 and

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1545 cm-1, and the absence of small peaks between 1452 and 1200 cm-1. The disappearance of

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bands and shifting of peaks towards the lower frequency (from 3321 to 3274 cm-1) or towards

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higher frequency (1625 to 1635 cm-1, 1072 to 1087cm-1, and 887 to 981 cm-1) have suggested

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that at least amine, hydroxyl, carbonyl and amide bonds are the major sites for binding of

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copper cations.

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Observation conducted by electron microscopy and cell fractionation studies revealed copper

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cations location on the cell wall of T. viride spores indicating this is the place where the

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interaction between T. viride and copper cations occurred.22 Our preliminary zeta potential

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measurements performed on T. viride spores dispersed in water showed the broad charge

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distribution (including negative and positive) with the average zeta potential of -35.1 mV. It

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seems that the interaction between copper cations and T. viride spores primarily occurred due

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to the electrostatic attractions.

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3.2. Guidelines for the preparation of microcapsules simultaneously loaded with

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biological and chemical agents. When a solution of sodium alginate comes into contact with

represent the bending modes of the hydroxyl group. Frequencies at 1067 and 860 cm-1 are



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divalent or trivalent cations, a rapid, strong and irreversible formation of a gel takes place, e.g.

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cations cooperatively interact with alginate blocks of guluronic acid residues forming a gel

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network. Due to very rapid and irreversible binding reaction of gelling cations to alginate

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chains, they should be mixed under controlled conditions.23,24 There are wide variations in the

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procedure of alginate microcapsules preparation. The essential properties of all microcapsules

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include high loading capacity, high mechanical and chemical stability, controllable releasing

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and swelling properties, low toxicity, etc. The ideal microcapsules which meet all the

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requirements do not exist, but in accordance with intended use, procedures of preparation

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must be adapted. To obtain the well-designed microcapsules efficient for copper cations and

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T. viride encapsulation, and prolonged release, it is important to choose an alginate with high

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content of guluronic acid residues (a high guluronic acid content develop stiffer, more porous

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gels, which maintain their integrity for longer periods of time) as well as the proper

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concentration of alginate and gelling cation.23-25 The ratio between gelling cations and

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alginate quantities in the gelation system determines the kinetics of gelation and the

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characteristics of the gel formed.25 It is shown that the decrease of alginate concentration from

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18 mmol dm-3 to ~ 7 mmol dm-3 increase the mechanical stability of alginate microcapsule,

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but when the alginate concentration was further lowered to 4.5 mmol dm-3 mechanical

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stability of capsules decreased.24,26 Another important parameter is the viscosity of alginate

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solution. Goosen et al.27 showed that the minimum viscosity of alginate solution in order to

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form a spherical microcapsule is 30 cPs. Spherical microcapsules are obtained over a wide

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range of viscosity of the sodium alginate solution. Usually, the viscosity of the aqueous

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sodium alginate solution does not exceed about 1000 cps. Having in mind that both amounts

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of alginate and gelling cations concentration in solution affect markedly the cation binding

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and kinetics of gel formation,26 we have investigated the impact of copper cations


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concentration on essential microcapsule properties at an adjusted constant alginate

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concentration convenient for uniform spherical microcapsules formation.

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Chosen procedure took place in two stages by method, relying on ionic gelation and

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polyelectrolyte complexation.28 The first stage involves the preparation of alginate core

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microcapsules loaded with copper cations or simultaneously loaded with copper cations and

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T. viride, and the second stage includes a coating of alginate core microcapsules by chitosan

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(Scheme 1).

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The aim of the second step was to reduce the porosity, improve stability and encapsulation

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efficiency, and delay the release behavior.28,29 By dispersing core microcapsules in chitosan

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acidic solution the chitosan rapidly bind onto their surface by electrostatic interactions

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between protonated amino groups on chitosan and ionized carboxylic acid groups on

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alginate.30 The electrostatic interaction between chitosan and alginate tightens and stabilizes

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the surface of the microcapsules.31 Once the chitosan bind to the core microcapsules

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competing ions (H+, Na+) have minor influence on the stability of the polyelectrolyte

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complex,32 and the chitosan diffusion into the inner core is limited.33

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3.3. Characterization of microcapsules loaded with bioactive agents. 3.3.1. Microscopic

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observation. Examination of prepared microcapsules under a light binocular revealed

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formation of spherical microcapsules which size was determined by the diameter of the funnel

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or needle, respectively (Figs. 3a,b,c). They were colored due to the copper cations presence.

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Microphotographs of two closely spaced spherical microcapsules taken under CLSM in

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fluorescence mode and transmitted light clearly showed the existence of the chitosan layer on

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the surface of microcapsules (Figs. 4a,b). Chitosan layer thickness became visible by staining

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with eosin, which binds to the amino groups of chitosan. The thickness of coating layer is ~ 7

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µm on microcapsules loaded only with copper cations (Fig. 4c) and ~ 11 µm loaded with



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copper cations and T. viride (Fig. 4d), respectively. Obviously, the different core content

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affected the thickness of coating layer, e.g. microcapsules loaded only with copper cations

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exhibited somewhat thinner coating layer indicating the lower extent of reaction between

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chitosan and sodium alginate molecules. It seems that electrostatic interaction between

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protonated amino groups on chitosan and ionized carboxylic acid groups on alginate is

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diminished due to the binding of T. viride to sodium alginate (see later in the section 3.3.2.).

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Microphotographs of the core matrix in transmitted mode revealed almost homogenous

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matrix texture of CS/(ALG/Cu) (Fig. 4e) and distributed T. viride spores throughout the core

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matrix in CS/(ALG/(Cu+TV)) microcapsule (Figs. 4f,g). On air drying, the sphericity of

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microcapsule was lost and its size somewhat decreased (Fig. 4h). The surface of the

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microcapsules becomes rough containing many wrinkles. The occurrence of wrinkles on the

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surface of the microcapsules can be explained by the syneresis.

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3.3.2. Fourier transform infrared spectroscopy (FTIR). Information on intermolecular

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interactions between alginate and active agents as well as in microcapsules coated with

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chitosan was obtained using FTIR. FTIR spectra of sodium alginate, the mixture of sodium

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alginate and T. spores, and core microcapsules ALG/Cu and ALG/(Cu + TV) are presented in

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Fig. 5a, whereas spectra of chitosan and microcapsules coated with chitosan, CS/ALG/Cu and

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CS/(ALG/(Cu TV), are presented in Fig. 5b.

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Characteristic FTIR bands of sodium alginate with assignements listed in Table 2 are in

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accordance with literature data.34 The spectrum of T. viride and alginate shows most intense

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change of broad band from 3700 to 3000 as well as shifting of the other characteristic bands

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of sodium alginate at 1405, 1295, 1125, 1081 and 1025 cm-1 to 1415, 1146, 1094 and 1038

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cm-1. The disappearance of some bands (at 3198, 2925, 1595 cm-1 as well as bands attributed

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to the ALG saccharide structure) is an indication of at least interactions with amine,

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carboxylate and CO groups. Broader and somewhat shifted to a lower wave number as well


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more intense (almost five times higher intensity) the -OH stretching vibrations band (around

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3400 cm-1) suggested enhanced intermolecular hydrogen bonds.

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The spectrum of ALG/Cu showed the most significant changes in the alginate functional

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groups region: carboxylate (COO-), ether (COC) and hydroxyl (OH). It can be seen from the

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Fig. 5a that the intensities of main alginate peaks become reduced and shifted to lower or

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somewhat higher wavenumbers. Shifting of alginate broad band around 3400 cm-1 to lower

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frequencies could be ascribed to copper cations interaction with hydroxyl groups. The

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characteristic peak for asymmetric COO- stretching vibrations shifted from 1595 to 1590 cm-1

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and the peak for symmetric COO- stretching vibrations shifted to higher wavenumber from

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1405 to 1411 cm-1. Additional observed shifts are at 2925, 1081, 950 and 813 cm-1 to 2926,

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1068, 944 and 811 cm-1, respectively. In agreement with FTIR data, X-ray diffraction

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evidence and the egg-box model, Papageorgiou et al.35 proposed in the metal-alginate

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complexes a "pseudo-bridged" unidentate coordination with intermolecular hydrogen bond in

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polyguluronic regions and the bidentate bridging coordination the polymannuronic region.

322

In comparison with ALG/Cu spectrum, all peaks in ALG/(Cu+TV) spectrum are somewhat

323

more intense. Small shifts of alginate peaks (from 3400, 2925, 1405, 1295, 1125, 1025, 950

324

and 606 cm-1 to 3328, 2927, 1411, 1301, and 1026, 947 and 599 cm-1, respectively) and

325

disappearance of alginate peaks at 1125 and 1081 cm-1 indicated complex interactions

326

between all components in the microcapsule.

327

Characteristic FTIR bands of chitosan with assignements listed in Table 3 are in accordance

328

with literature data.36,37 Spectra of microcapsules with added chitosan (Fig. 5b) show some

329

peaks disappeared or become weaker due to the interaction between or superposition of the

330

groups of chitosan and alginate (at 2875, 1648, 1582, 1373 and 1150 cm-1).36 In comparison

331

with ALG/Cu spectrum (Fig. 5a), both spectra, CS/(ALG/Cu) and CS/(ALG/Cu+TV)) show a

332

little broader band around 3265 cm-1 due to enhanced hydrogen bonding. In comparison with



333

peaks in ALG/Cu spectrum all peaks in CS/(ALG/Cu) spectrum are less intense, but more

334

intense in CS/(ALG/Cu+TV)) spectrum. The absence of chitosan band at 1582 cm-1 (-NH

335

bending vibration) and shifting of alginate carboxylate region bands, from 1595 to 1585 cm-1

336

for CS/(ALG/Cu) and to 1590 cm-1 for CS/(ALG/Cu+TV), and from 1405 to 1409 cm-1 for

337

CS/(ALG/Cu) and to 1411 cm-1 for CS/(ALG/Cu+TV), respectively) indicated electrostatic

338

interactions between two oppositely charged polyelectrolytes.

339

3.3.3. Encapsulation efficiency and loading capacity. Results on copper cations encapsulation

340

efficiency and loading capacity are presented in Figs. 6a and 6b, respectively. Apparently, the

341

increase of copper cations concentration decreases encapsulation efficiency for CS/(ALG/Cu)

342

microcapsules, whereas microcapsules loaded with both active agents exhibits the increase in

343

encapsulation efficiency (Fig. 6a). Results are in accordance with the values of microcapsules

344

loading capacity (Fig. 6b). The decrease in encapsulation efficiency of microcapsules loaded

345

with copper cations is a consequence of loading capacity constancy, that is, microcapsules

346

reached maximum loading capacity even at the lowest copper cations concentration. On the

347

contrary, the loading capacity and consequently encapsulation efficiency by simultaneously

348

loaded microcapsules increased with copper cations concentration reaching values of

349

microcapsules without TV only at the highest copper cations concentration. The smaller

350

loading capacity of CS/(ALG/(Cu + TV)) in comparison with CS/(ALG/(Cu) microcapsules

351

can be explained by copper cations binding to T. viride, that is, the decrease in active copper

352

cations available due to binding to T. viride in solution. It is in accordance with results of

353

Bespalova et al.38 who have found that T. viride addition leads to a decrease in the amount of

354

copper cations bound to organic matter (by about 1.1-1.2-fold). The size of microcapsules

355

does not significantly affect the mode of changes in EE and LC with copper cations

356

concentration.


Page 16 of 39

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357

Spores of T. viride were not detected in filtrate after core microcapsule separation showing

358

the encapsulation efficiency was almost 100%. Data on the loading capacity expressed as the

359

number of spores per 1 g of dry microcapsule are listed in Table 1.

360

3.3.4. Swelling degree. On in vivo applying, alginate microcapsules coated with chitosan

361

usually swell thus influencing the release of active agents from them. In comparison with

362

uncoated alginate microcapsules, those coated with chitosan exhibit higher swelling degree

363

due to highly hydrophilic nature of polyelectrolyte complex formed.31,39-42 The swelling

364

degree of alginate-chitosan complex depends on the extent of electrostatic interaction between

365

the alginate carboxylate groups and protonated amine groups of chitosan. Both, sodium

366

alginate and chitosan are weak polyelectrolytes and the degree of dissociation of their

367

functional groups strongly depends on the pH of the solution. Sodium alginate chains with

368

pKa values of 3.38 and 3.65 for mannuronic and guluronic acids, respectively, are negatively

369

charged across a wide range of pH values, whereas chitosan is positively charged (protonated)

370

below its pKa of 6.5.43 Swelling degree of coating layer is low in acidic medium due to the

371

dense polyelectrolyte complex structure as a result of strong interactions between protonated

372

amino groups of chitosan and carboxylate anions of alginate. With pH increasing the

373

carboxylic acid groups become more ionized. Close to pH ~ 6.5 chitosan become

374

deprotonated and consequently the extent of complexation is reduced forming a less dense

375

structure of coating layer which alows higher swelling.43

376

When chitosan/alginate microcapsules loaded with copper, and with copper and T. viride were

377

dispersed in deionized water (pH ~ 6) they started to swell like a microcapsule consisting of a

378

chitosan wall and Ca-alginate core.40,43 During three hours of exposure in deionized water the

379

pH of solutions decreased from pH 6 to ~ 5. The decrease of pH may be attributed to the

380

small quantity of H+ ions released due to ionization of carboxylic groups.31



381

The effect of increasing copper cations concentration on swelling degree is presented in Fig.

382

7. It can be clearly seen that higher copper cations concentration promoted swelling, but there

383

existed obvious differences regarding loaded agents and microcapsule size. The higher

384

swelling extent of microcapsules loaded with copper cations than those simultaneously loaded

385

with copper cations and T. viride may be attributed to the difference in chitosan layer

386

thickness (Figs. 4c,d), e.g. the increase of coating layer thickness resulted in a decrease of

387

swelling extent. Bartkowiak et al.29 have shown that the chitosan coating layer thins with an

388

increase in microcapsule size prepared under the same conditions. Accordingly, the

389

differences in swelling behavior between differently sized microcapsules can be also

390

explained by differences in chitosan layer thickness.

391

3.3.5. In vitro release of active agents. The possible use of biopolymer microcapsules in the

392

plant imposes research regarding their release capacity in certain physicochemical conditions.

393

In this direction, the kinetics of the copper cations ion release was studied on microcapsules

394

prepared at the highest copper cations concentration. This concentration was used because of

395

its positive affect on the growth and behavior of T. viride spores. The release profiles of

396

copper cations from different types of sodium alginate/chitosan microcapsules are presented

397

in Fig. 8. A set of curves for copper cations release exhibit rapid initial release followed by

398

slower release obeying power law equation.

399

It can be clearly seen that the amount of copper cations released depends on microcapsule size

400

and loaded active agents. The microcapsules of different sizes prepared under the same

401

conditions differ in surface layer thickness and it should be possible to correlate most

402

microcapsule properties by taking into the account the changes in microcapsule

403

surface/volume ratio.29 It was observed that chitosan layer thins with the increase of

404

microcapsule diameter causing some changes in mechanical and permeability properties.

405

Faster releasing from the large microcapsules may be attributed to the thinner surface layer


Page 18 of 39

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406

allowing easier penetration of both the dissolution medium (swelling) and the copper through

407

the layer. Large microcapsules released a larger amount of copper cations, because there were

408

more copper cations loaded per microcapsule than in smaller microcapsules (Fig. 6b). Despite

409

to the somewhat thinner surface layer (Fig. 4e), both small and large microcapsules loaded

410

with copper and T. viride released smaller amount of copper. This may be attributed to the

411

binding of copper cations to T. viride.

412

Various mechanisms such as desorption from the surface, diffusion through the pore of the

413

core and wall, microcapsule disintegration, dissolution or erosion of the structure, or on their

414

combination, may be included in the release of active agents from microcapsules. Mechanism

415

of active agents releasing primarily depends on the characteristics of core material and active

416

agents as well as on the microcapsule size.44

417

To identify the type of mechanism involved in copper cations releasing a semi-empirical

418

Korsmeyer–Peppas model was applied.45,46 According to Korsmeyer–Peppas, the release

419

exponent n can be characterized by three different mechanisms (Fickian diffusion, (n)

420

anomalous (non Fickian diffusion), or Type II transport). Values of n < 0.43 indicates the

421

release is controlled by classical Fickian diffusion, n > 0.85 is controlled by Type II transport,

422

involving polymer swelling and relaxation of the polymeric matrix, whereas values of n

423

between 0.43 and 0.85 shows the anomalous transport kinetics determined by a combination

424

of the two diffusion mechanisms and Type II transport.

425

All curves presented in Fig. 8 can be described by the equation:

426

ோ೟

݂ ሺ‫ݑܥ‬ሻ = ோ

= ݇‫ ݐ‬௡

(2),

೟೚೟

427

where f(Cu) represents the fraction of released copper cations, Rt is the amount of copper

428

cations released at time t, Rtot is the total amount of Cu loaded in capsules, k is a constant

429

characteristic of the active agents/polymer system that considers structural and geometrical

430

aspects of the system, and the value of the exponent (n) is an exponent which characterize the



431

transport mechanism of active agents through microcapsule. The values of the release

432

constants and exponents are listed in Table 4.

433

Lower n values than 0.43 for larger microcapsules indicated that the release mechanism

434

involved is controlled by a classical Fickian diffusion, whereas higher n values for smaller

435

microcapsules indicated copper cations release followed non-Fickian kinetics, due to probable

436

rapid swelling and partial dissolution of microcapsules.

437

Change of released T. viride amount (expressed as spore number, NS/g) over time presented

438

in Fig. 9. shows that after initial lag time the amount of spores in the medium increases

439

exponentially. The processes involved during the lag phase are penetration of water and

440

filling of the microcapsule surface pore with water, as well as transport of T. viride through

441

the alginate matrix and the water-filled chitosan pores, and finally diffusion in the

442

surrounding media. Slower T. viride diffusion through alginate matrix and water filled pores

443

can be ascribed to higher size of T. viride spores in comparison with the size of copper cations

444

as well to their intermolecular interactions with alginate and copper cations. As the swelling

445

process progressed, transport of T. viride toward water phase occurred through the swollen

446

layer. The exponential increase in the amount of T. viride and much higher concentration

447

detected in water than concentration loaded in microcapsule indicated that spores germinated

448

and germ tube biomass was formed in the surrounding medium. Actually, copper cations

449

released from microcapsule promote germination in water. The increasing amount of T. viride

450

in the dispersing medium is closely related with two processes, one is the release of T. viride

451

from microcapsules and the other is germination. It can be seen that smaller microcapsules

452

deliver larger amount of T. viride to the surrounding medium and consequently exhibited

453

greater yield of biomass.

454

3.3.6. Concluding remarks


Page 20 of 39

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455

We reported, for the first time, the possibility to encapsulate both, biological and chemical

456

bioactive agents in a chitosan/alginate microcapsule. Encapsulation of T. viride spores and the

457

presence of copper cations in the same compartment do not inhibit their activity and even it

458

promoted T. viride germination. Investigation of intermolecular interactions between

459

oppositely charged biopolymers and bioactive agents using FTIR spectroscopy revealed an

460

interaction between copper cations and T. viride spores functional groups as well between

461

alginate and bioactive agents.

462

Loading capacity and efficiency, swelling behavior and releasing of active agent depend on

463

both, the kind of encapsulated agents and microcapsule size. The increase in copper cations

464

concentration promoted swelling, but there exist obvious differences regarding to the loaded

465

agents and microcapsule size. Higher swelling extent and greater amount of released copper

466

cations from microcapsules loaded only with copper cations than those simultaneously loaded

467

with copper cations and T. viride spores can be attributed to the differences in chitosan layer

468

thickness.

469

The in vitro copper cations release profile was fitted to Korsmeyer–Peppas empirical model.

470

Fickian diffusion was found to be the rate-controlling mechanism at smaller microcapsules,

471

whereas anomalous transport kinetics (combination of the diffusion mechanisms and Type II

472

transport) controlled release from larger microcapsules. The copper cations release exhibited

473

initial burst followed by a slower release, but Trichoderma viride spores releasing profile

474

showed exponential increasing over initial lag time. Much slower release of T. viride spores at

475

the early stage may be ascribed to their higher size in comparison with copper cations, and

476

intermolecular interactions with alginate and copper cations.

477

Results showed chitosan/alginate microcapsules can simultaneously incorporate T.viride

478

spores and chemical bioactive agent without inhibiting their activities. With all these results it

479

seems that copper cations and T. viride spores loaded in chitosan/alginate microcapsules can



480

be suitable for plant nutrition and protection and that is worth to preform further research. Our

481

future investigations are directed to test CS/ALG microcapsules simultaneously loaded with

482

chemical and biological agents on plants under greenhouse conditions as well as in an open

483

field.

484 485 486 487 488 489 490

ASSOCIATED CONTENT Supporting Information. Additional information on microcapsule preparation and methods of characterization as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION * Corresponding author

491 492 493 494

Marko Vinceković Department of Chemistry, University of Zagreb Faculty of Agriculture Svetošimunska cesta 25, 10000 Zagreb, Croatia E-mail: [email protected] ; Tel: + 385 1 239 3953

495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512

Authors’ contributions The corresponding author designed the research work and wrote the manuscript. All the authors carried out this research work under the guidance of the corresponding author. The final manuscript was read, corrected, improved and approved by all coauthors. Funding We acknowledge the financial support of Croatian Science Foundation (Project: UIP-201409-6462). Notes The authors declare no competing financial interest. ABBREVIATIONS USED ALG, alginate; CS, chitosan; TV, Trichoderma viride (T. viride); ALG/Cu, alginate core microcapsule loaded with copper cations; CS/(ALG/Cu), chitosan/alginate microcapsule loaded with copper cations, CS/(ALG/(Cu+TV)), chitosan/alginate microcapsule loaded with copper cations and T. viride; PDA, potato dextrose agar; PDB, potato dextrose broth; STP, indigenous isolate of T. viride; EE, encapsulation efficiency, LC, loading capacity, Sw, swelling degree; Rt, the amount of Cu released at time t; Rtot, the total amount of Cu loaded in capsules.


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Figure Caption Scheme 1. Schematic diagram of the experimental setup corresponding to ionic gelation (the first stage - I) and polyelectrolyte complexation (the second stage - II). Figure 1. Microphotographs of the mycelial growth of Trichoderma viride spores sprayed with CCVD prepared at increasing initial copper cations concentration (ci) = 4.5, 9 and 18 mmol dm-3 (from left to right side) taken after spraying. Figure 2. FTIR spectra of Trichoderma viride spores (black line), copper sulfate pentahydrate (cyan line) and their mixture (red line). Figure 3. Microcapsules prepared with (a) needle (0.45 mm) and (b) funnel (2 mm), (c) enlarged picture of a microcapsule prepared with funnel (black bar in the background = 0.5 mm). Figure 4. CLSM microphotographs: parts of two closely spaced microcapsules in fluorescence (a) and transmitted mode (b); part of the CS/(ALG/Cu) microcapsule in fluorescence (c) and transmitted mode (d); part of the CS/(ALG/(Cu + TV)) microcapsule in fluorescence (e) and transmitted mode (f); cross-section of CS/(ALG/(Cu + TV) microcapsule matrix obtained by optical microscopy (g); image of dried CS/(ALG/(Cu + TV)) microcapsule obtained by optical microscopy (h). Microcapsules are prepared at the initial copper cations concentration, ci = 18 mmol dm-3. Bars are indicated. Figure 5. FTIR spectra of (a) sodium alginate (ALG - black line), alginate and Trichoderma viride (ALG/TV - red line), core microcapsule (ALG/Cu - green line), core microcapsule with copper cations and Trichoderma viride (ALG/(Cu+TV) - blue line); (b) chitosan (CS - black line), core microcapsule with copper cations ((CS/ALG/Cu) - blue line), and core microcapsule with copper cations and Trichoderma viride coated with chitosan (CS/ALG/(Cu+TV) - red line).


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Figure 6. Variation of copper cations (a) encapsulation efficiency (EE) and (b) loading capacity (LC) of CS/(ALG/Cu) (open signs) and CS/(ALG/(Cu+TV)) (full signs) microcapsules with initial copper cations concentration (ci). Microcapsule diameters are denoted in brackets. The error bars indicate the standard deviation of the means. Figure 7. Variation of the swelling degree (Sw) of CS/(ALG/Cu) (open signs) and CS/(ALG/(Cu + TV)) (full signs) microcapsules in deionized water with initial copper cations concentration (ci). Microcapsule diameters are denoted in brackets. The error bars indicate the standard deviation of the means. Figure 8. Fraction of released copper cations, f(Cu), from CS/(ALG/Cu) (open signs) and CS/((ALG/(Cu+TV)) (full signs) microcapsules at initial copper cations concentration c(Cu)i = 18 mmol dm-3 with time (t). Microcapsule diameters are denoted in brackets. The error bars indicate the standard deviation of the means. Figure 9. Variation of a number of Trichoderma viride spores (NS g-1) with time (t). Microcapsule diameters are denoted in brackets. The error bars indicate the standard deviation of the means.



Table 1. Number of Trichoderma Viride Spores (NS) per 1 g of CS/(ALG/(Cu+TV)) after 1 and 10 Days of Storage at Room Temperature. size / mm

NS/g (1 day)

NS/g (10 days)

0.45

14444

14188

2

6500

7750

Table 2. FTIR Bands of Sodium Alginate with Assignments. sodium alginate vibration (cm-1)

assignment

3700 - 3000

O-H stretching

3198 2925

O-H stretching (intermolecular hydrogen bond) C-H stretching

1595

COO- stretching (asymmetric)

1405

COO- stretching (symmetric)

1295

C-O stretching

1125

C-C stretching

1081- 1027

assymmetric C-O-C stretching

1033

C-O stretching

950 cm-1

C-O stretching of uronic acid


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Table 3. FTIR Bands of Chitosan with Assignments. Reversed chitosan-alginate chitosan vibration (cm-1)

assignment

3750-3000

O-H and N-H stretching

2925

C-H in CH2 stretching

2875

C-H in CH3 stretching

1648 1582

C=O stretching of the secondary amide (amide band I) N-H bending of amine and amide II

1425

CH2 bending

1373

CH3 smmetrical deformation

1150

assymmetric C-O-C and C-N stretching

1026

skeletal vibration of C-O stretching

Table. 4. The Values of the Release Constant (k) and Exponent (n) of Copper Cations Encapsulated in CS/(ALG/Cu) and CS/(ALG/(Cu+TV)) microcapsules. microcapsule

size (mm)

k (day-1)

n

CS/(ALG/Cu)

0.45

0.167

0.45

CS/(ALG/Cu)

2.0

0.551

0.23

CS/(ALG/(Cu + TV))

0.45

0.081

0.68

CS/(ALG/(Cu + TV))

2.0

0.436

0.27



Scheme 1.

Figure. 1.


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1,1 1,0

Transmittance

0,9 0,8 0,7 0,6 0,5 0,4

Trichoderma viride CuSO4 x 5H2O

0,3

Trichoderma viride + CuSO4 x 5H2O

0,2 3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

Figure 2.

(a)

(b) Figure 3.


(c)


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Figure 4.


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1,1

(a)

1,0

Transmittance

0,9 0,8 0,7 0,6 ALG ALG/TV ALG/Cu ALG/(Cu+TV)

0,5 0,4 0,3 0,2 3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

Figure 5a.

1,00

(b)

0,95

Transmittance

0,90 0,85 0,80 0,75

CS/(ALG/Cu) CS/(ALG/(Cu+TV)) CS

0,70 0,65 0,60 3500

3000

2500

2000

1500

1000

-1

Wavenumber (cm )

Figure 5b.


500


CS/(ALG/(Cu) (0.45 mm)

(a)

CS/(ALG/Cu ) (2 mm)

80

CS/(ALG/(Cu + TV)) (0.45 mm)

EE / %

CS/(ALG/(Cu + TV)) (2 mm)

60

40

8

10

12

14

16

ci / mmol dm

18

-3

Figure 6a.

3,0

(b)

CS/(ALG/Cu) (0.45 mm)

2,5

CS/(ALG/Cu) (2 mm)

LC / mmol g-1

CS/(ALG/(Cu + TV)) (0.45 mm) CS/(ALG/(Cu + TV)) (2 mm)

2,0

1,5

1,0

0,5

8

10

12

14

ci / mmol dm

16

-3

Figure 6b.


18

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CS/(ALG/(Cu) (0.45 mm)

90

CS/(ALG/Cu ) (2 mm) CS/(ALG/(Cu + TV)) (0.45 mm)

80


Sw / %

70 60 50 40 30 8

10

12

14

16

18

-3

ci / mmol dm

Figure 7.

1,0

0,8

f(Cu)

0,6

0,4 CS/(ALG/(Cu) (0.45 mm)

0,2

CS/(ALG/Cu ) (2 mm) CS/(ALG/(Cu + TV)) (0.45 mm)

0,0


0

2

4

6

8

10

12

t / days

Figure 8.


14


2x105 CTS/(ALG/(Cu + TV)) (2.0 mm)

NS g-1

CTS/(ALG/(Cu + TV)) (0.45 mm)

1x105

0 0,0

0,5

1,0

1,5

2,0

2,5

t / week

Figure 9.


3,0

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For Table of Contents Only

Figure caption


Encapsulation of Biological and Chemical Agents for Plant Nutrition

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