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Constructing slow-release formulations of ammonium nitrate fertilizer based on degradable poly(3-hydroxybutyrate) Anatoly Nikolayevich Boyandin, Eugenia Andreevna Kazantseva, Daria Eugenievna Varygina, and Tatiana Volova J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01217 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 20, 2017

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Journal of Agricultural and Food Chemistry

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Title: CONSTRUCTING SLOW-RELEASE FORMULATIONS OF AMMONIUM

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NITRATE FERTILIZER BASED ON DEGRADABLE POLY(3-

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HYDROXYBUTYRATE)

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Authorship: Anatoly Nikolayevich Boyandin*1,2, Eugenia Andreevna Kazantseva2, Daria

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Eugenievna Varygina2, Tatiana Grigorievna Volova1,2

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1

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Akademgorodok, Krasnoyarsk 660036, Russia

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2

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*Corresponding author. E-mail: [email protected]

Institute of Biophysics of Siberian Branch of Russian Academy of Sciences, 50/50

Siberian Federal University, 79 Svobodny pr., Krasnoyarsk 660041, Russia

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Keywords: ammonium nitrate, nitrogen fertilizers, embedding, degradable poly-3-

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hydroxybutyrate, fillers, tablets

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ACS Paragon Plus Environment


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ABSTRACT

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The present study describes construction and investigation of experimental

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formulations of ammonium nitrate embedded in a matrix of degradable natural polymer

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poly-3-hydroxybutyrate [P(3HB)] and P(3HB) blended with wood flour shaped as tablets,

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some of them coated with P(3HB). Kinetics of ammonium release into soil as dependent on

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the composition of the polymer matrix was investigated in laboratory experiments. The

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rates of fertilizer release from formulations coated with a biopolymer layer were

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considerably (two months or longer) slower than the rates of fertilizer release from

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uncoated formulations, while release from polymer and composite (polymer/wood flour)

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formulations occurred with comparable rates. The use of the experimental formulations in

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laboratory ecosystems with wheat (Triticum aestivum L.) was more effective than

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application of free ammonium nitrate. The advantage of the slow-release fertilizer

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formulations is that they are buried in soil together with the seeds, and the fertilizer remains

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effective over the first three months of plant growth. The use of such slow-release

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formulations will reduce the amounts of chemicals released into the environment, curbing

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their accumulation in food chains of ecosystems and mitigating their adverse effects on the

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biosphere.

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Introduction

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The use of mineral and organic fertilizers is a necessary part of modern intensive

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farming practices. Nitrogen is one of the major elements supplied by mineral fertilizers. Its

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compounds have high mobility in soil, which causes nitrogen losses through runoff.

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Moreover, nitrogen available for plant use is present in soil in very small amounts, and this

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limits crop development. Therefore, application of nitrogen fertilizers favorably affects

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crops. However, because of the high mobility of mineral nitrogen, application of nitrogen

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fertilizers by traditional methods has some disadvantages: high (sometimes too high)

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concentration of the fertilizer in soil immediately upon application, which declines over a

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certain time.

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A possible solution may be to construct slow-release formulations for agriculture

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and develop ways to deliver them.1 Binding nitrogen fertilizers with substances that slow

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down nitrogen release to soil will enable maintaining relatively stable concentrations of the

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fertilizer for a required time period. The fertilizers can be bound with various biodegradable

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materials and composites based on them, which are slowly degraded by soil microflora.

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Different technologies of preparing such formulations have been developed by now.

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The simplest way is to encapsulate them in a polymeric (e.g., graft-copolymerized starch,

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poly(L-lactide),2 polyvinyl alcohol (PVA), chitosan3) or mineral (montmorillonite,4,5

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bentonite,6 etc.) matrix. Another approach to preparing slow-release formulations is to

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cover fertilizers with coatings of degradable polymers: plasticized polyurethanes, plant-

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derived materials (lignin, cellulose, starch),7 sulfur, wax, aldehyde condensates, and resins,8

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polysulfone, polyacrylonitrile, and cellulose acetate,9 polyolefins,10 polyacrylic acid latex,11

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phosphogypsum mixed with neem oil and alkyl benzene,12 and modified polyethylene

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terephthalate.13 Some of coated fertilizers are available on the market.

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The higher efficacy of such formulations compared to ordinary fertilizers has been

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demonstrated in very many experiments. For instance, the use of ESN (Agrium Inc.,

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Canada) – polymer-coated urea – increased yields of corn,14,15 wheat, canola, and barley16

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compared with the yields achieved by applying fertilizers by traditional methods. The study

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by Medina et al.17 showed the effectiveness of using CitriBlen (Everris, ICL Specialty

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Fertilizers, the Netherlands) to fertilize citrus trees. The effectiveness of the commercial

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nitrogen formulation Meister programmed release N fertilizer T15 Meister (Chisso

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Corporation, Japan) for cotton production was shown in field experiments in Arkansas and

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Tennessee.18 Polyolefin-coated urea, Meister 70, maintained higher nitrogen concentration

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in soil than the uncoated urea and, in some of the experiments, enabled production of higher

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yields.8

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In some instances, however, the non-optimal rate of release of the fertilizer canceled

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out its beneficial effects. In field experiments with the polyolefin-coated urea formulation

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Meister 270 used as a fertilizer for cotton, nitrogen release was too slow.8 In another study,

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the effectiveness of the fertilizer Osmocote was low because of the too quick release of

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active ingredients.19 The use of Meister 7, urea with dicyandiamide, and polyolefin-coated

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urea to fertilize barley, potatoes, and corn did not show any positive effect of urea

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encapsulation compared with the use of ordinary urea.20 Thus, scientific research aimed at

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increasing the efficacy of fertilizers embedded in degradable materials should involve

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development of new processes for producing fertilizer formulations intended for different

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environmental conditions and meeting requirements of various crops.

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Polyhydroxyalkanoates, which are biodegradable in natural media and have

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physicochemical properties making them suitable for processing by various techniques, are

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promising materials for constructing carriers for slow-release fertilizer systems. However,

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very few studies have investigated the possibility of using them as a matrix or coating for

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nitrogen fertilizers. In the study by Costa et al.,22 a solution of poly-3-hydroxybutyrate in

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chloroform was used to coat urea. Urea granules were either immersed into the solution and

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then dried on the glass surface or sprayed with the solution and dried. However, urea was

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released to distilled water in about five minutes, suggesting low effectiveness of such

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coatings. The authors of the present study previously showed effectiveness of compressed

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pellets of P(3HB) and urea for fertilizing fast-growing plants (lettuce and a model crop –

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creeping bentgrass) for 22-28 days.23 Obviously, more complex approaches should be

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developed for effective use of P(3HB) as a carrier of nitrogen fertilizers, which will enable

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slower release of nitrogen. Slower fertilizer release can be achieved by both using a more

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complex basis (P(3HB) composites with other degradable materials) and covering the

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fertilizer with additional biopolymer coatings, which will enable delayed release of active

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ingredients.

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The purpose of this study was to develop slow-release formulations of ammonium

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nitrate based on poly-3-hydroxybutyrate and its composite with wood flour with different

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rates of release of the active ingredient, with and without an additional coating, and to

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investigate the effectiveness of these formulations in laboratory experiments with wheat

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plants.

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Materials and Methods

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Materials. Poly(3-hydroxybutyrate) [P(3HB)] (the weight average molecular

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weight Mw 920 kDa; polydispersity 2.52) was synthesized according to previously

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described technology.24 Poly-ε-caprolactone (PCL) with the number average molecular

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weight Mn 80 kDa in the form of granules was produced by Aldrich (U.S.). Wood flour was

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produced by pulverizing birch wood with an MD 250-85 wood-carving workbench

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(Stanko-Premier, Russia). Then it was dried at 60°C for 120 h until it reached constant

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weight, and 0.5 mm mesh was used to separate the particle size fraction.

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Ammonium nitrate, NH4NO3, manufactured by Chudovoagrokhimservis (Russia)

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was used as the fertilizer. Chemically pure chloroform manufactured by Ekos-1 (Russia)

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was used as a solvent to prepare polymer coatings. Nitrogen measurement (by the

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ammonium ion) was conducted using Nessler’s reagent (K2HgI4×NaOH) manufactured by

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Reakhim (Russia), chemically pure potassium chloride (KCl) manufactured by Reakhim

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(Russia), and chemically pure Rochelle salt (potassium sodium tartrate tetrahydrate,

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C4H4KNaO6×4H2O) manufactured by Kupavnareaktiv.

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Preparation of P(3HB)/filling material mixtures. P(3HB)/filling material

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mixtures were prepared as follows: polymers (P(3HB) and PCL) were ground using a ZM

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200 mill (Retsch, Germany) and particle fractions under size of 1 mm were selected.

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Particle size distribution of the powder thus prepared was determined with an AS 200

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analytical sieving machine (Retsch, Germany): particle fraction of size under 0.50 mm

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constituted 60% and that of size between 0.50 and 1.00 mm 40%.

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Pure P(3HB) powder and its mixtures with birch flour and PCL powder at a source

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ratio of 1:1 (i.e. 50% of P(3HB) and 50% of the other component) were used for

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construction of tablets loaded with 25% ammonium nitrate. Specimens prepared by mixing

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90 mg filler powders (90 mg of pure P(3HB) or 45 mg of P(3HB) and 45 mg of the second

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component) with 30 mg of NH4NO3 were cold-pressed using a Carver Auto Pellet 3887

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press (Carver, U.S.) to tablets, 10 mm in diameter and 1.5 mm thick; pressing force was

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2000 lbf (about 8.90 kN).

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To further decrease the fertilizer release rate, some of the specimens were coated

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with a layer of P(3HB) by dipping them in a 5% solution of the polymer in chloroform and

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drying for 30 min in the open air at a temperature of 25°C and humidity no more than 10%.

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To investigate coating formation, this procedure was repeated from one to six times. To

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further investigate nitrogen release kinetics in water and soil, we used specimens that had

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been dipped in the polymer solution six times.

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Studying release kinetics of ammonium nitrate from slow-release formulations in water.

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To estimate stability of formulations and their ability to release the fertilizer in

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abiotic conditions, a seven day water dissolution experiment was carried on. The specimens

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were sterilized using the Sterrad NX system (Johnson & Johnson, U.S.) and were put into

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100-ml sterile conical flasks filled with 25 ml of sterile distilled water. The experiment was

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conducted at 30°C. After specified intervals (one hour, one, three and seven days), water

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samples were analyzed for ammonium concentration by the colorimetric method, using

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Nessler’s reagent; the calibration curve was plotted for the ammonium ion as the reference

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standard (Russian Federal Standard 7259-96).

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Biodegradation of composite materials based on P(3HB) and release kinetics of ammonium nitrate in soil.

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Field soil (collected at the village of Minino, the Krasnoyarsk Territory) was

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cryogenic-mycelial agricultural black soil characterized by high humus level, weakly

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alkaline reaction of media (рН 7.1-7.8), and organic carbon 5.1%. Nitrate nitrogen (NO3–)

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concentration was 6 mg/kg, Р2О5 – 60 mg/kg, K2О – 220 mg/kg soil. Soil density had

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normal and mellow topsoil configuration (0.85-1.11g/cm³). The total counts of

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organotrophic bacteria were (16.3±5.1)×106 Colony Forming Units per ml.

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To measure the weight loss of the experimental formulations and kinetics of

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nitrogen release from the polymer matrix into soil, the specimens were placed into

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containers filled with 100 g soil per container. The containers with the soil and specimens

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were kept in a temperature-controlled cabinet at a constant temperature of 20±0.1°С and

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soil moisture content of 50%. Tablets were incubated in soil for 35 days at a temperature of

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20ºС. Several tablets were extracted from the soil every 7 days. To measure weight loss,

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they were rinsed to remove soil, kept in a thermostat at 40ºС to dry for 24 h until constant

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weight was obtained, and weighed on analytical scales (Ohaus Discovery, Switzerland).

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Nitrogen concentration in the soil was measured by the colorimetric method, using

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Nessler’s reagent; the calibration curve was plotted for the ammonium ion as the reference

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standard (Russian Federal Standard 7259-96). A 2% KCl solution (100 ml) was added to 10

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g of soil. The flask was shaken for one hour, and, then, the mixture was filtered through

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White Ribbon filter paper. Then, a 5 ml aliquot of the extract was diluted with distilled

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water (35 ml). After that, we added 2 ml of a 50% solution of Rochelle salt (to eliminate the

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adverse effects of the ions Ca2+ and Mg2+), 2 ml of Nessler’s reagent, and 6 ml of distilled

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water, the volume of the final solution reaching 50 ml. After 2-3 min, the solution was

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analyzed using a photocolorimeter (wavelength 425 nm) in 3-cm-long cuvettes relative to

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distilled water. Nitrogen concentration (mg/ml) in the solution was determined using a

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calibration curve.

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Laboratory test-systems with higher plants. Soft spring wheat Triticum aestivum

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cv. Altaiskaya 70 produced by double individual selection from the hybrid Altaiskaya 98

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and Altaiskaya 32525 was used to test the fertilizer formulations. The cultivar has been

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tested for frost tolerance in the harsh environments of Siberia and Russian Far East. The

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crop yield of this cultivar reaches 3.59-4.51 t/ha (the highest being 5.63 t/ha). This is a

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medium-maturing cultivar, with 74-79 days from emergence to waxy maturity. Good

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tillering capacity and resistance to the frit fly of this wheat variety result in the formation of

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the dense crop. This cultivar is resistant to wheat smut, lodging, and grain shedding. It is

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rarely damaged by powdery mildew. Triticum aestivum cv. Altaiskaya 70 responds to

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intensive management using mineral fertilizers. The cultivar has been included in the State

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Register of Breeding Achievements Approved for Use in 2009.

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Experimental formulations of the nitrogen fertilizer based on P(3HB) and a

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P(3HB)/wood flour blend were placed in bags of fine-meshed synthetic cloth and then

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buried in field soil (300 g on dry weight basis per container) in 350-ml containers. In each

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container, there were two specimens of the same type, which contained a total of 60 mg

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ammonium nitrate, which corresponded to 13.5 mg pure ammonium (Fig. 1a). The free

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form of the fertilizer was simultaneously buried in other containers (positive control). Soil

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without any fertilizer was the negative control. Twelve seeds of the soft wheat (Triticum

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aestivum L. cv. Altaiskaya 70) were buried in the soil in each container (Fig. 1b). To collect

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irrigation water, each container was placed into a larger 500 ml-container.

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The experimental formulations were buried in the field soil before sowing wheat seeds.

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Plants were grown in a Conviron A1000-AR environmental chamber, under

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conditions that simulated diurnal variations in temperature and lighting (Table 1). Plants

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were watered when the soil was dry, usually twice a week, with 50 ml of tap water. Every 7

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days, we used 150 ml of water to irrigate the plants and collected the irrigation solution.

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Kinetics of ammonium nitrogen release to soil was determined based on its concentration in

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the irrigation solution, and the total ammonium released to soil was calculated for the total

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volume of the solution. Every 7-14 days, we removed plants and (if present) tablet

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formulations from some of the containers of each group (positive control, negative control,

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coated and uncoated fertilizer formulations based on P(3HB) and P(3HB)/wood flour).

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Plants were dried at 105°C and tablet formulations at 40°C for 24 h, until they reached

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constant weight. The aboveground and belowground parts of the plants and the remaining

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formulations were weighed. Soil was analyzed for dissolved ammonium nitrogen. Polymer

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biodegradation was evaluated from the weight loss of the specimens.

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Statistical analysis. Statistical analysis of results was performed by conventional

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methods, using the standard software package of Microsoft Excel 2003. Results were

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presented as arithmetic means with standard deviations. The significance of results was

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determined using Student’s t test (significance level р≤0.05).

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Results

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In the preliminary stage, we developed formulations of different compositions

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(using P(3HB) or P(3HB)/wood mixture as polymeric fillers) and structures (uncoated and

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coated ones). To slow down fertilizer release from the experimental formulations, we

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coated them with a layer of P(3HB) by dipping them in a polymer solution in chloroform.

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Previously, we estimated the effect of the repeated immersion – drying treatment of

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samples on their weight increase (Table 2) and calculated the thickness of the coating after

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each immersion using the formula for the surface area of a cylinder S = 2 π r (h + r), the

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weight added after immersion, diameter (1 cm) and thickness of the tablet, and poly-3-

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hydroxybutyrate density – 1.25 g/cm3. On average, every immersion increased the mass of

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the tablets by 10-13 mg (about 10% of the mass of the initial sample) and the thickness of

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the coating by 4-5 µm, which shows sustainability of the dipping process. In the

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comparative study, we used tablets that had been dipped in the polymer solution six times,

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with the mass of the polymer constituting at least 50% of the initial mass of the tablet and

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the calculated thickness of the coating of at least 25 µm.

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In the first phase of the study, we estimated the interaction between nitrogen and the

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polymer matrix by dissolving the fertilizer in distilled water for one hour, one, three and

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seven days (Table 3). Under these conditions, the uncoated tablets lost about 40% of

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ammonium nitrate after 24 h and almost all of it after seven days. By contrast, no more than

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25% of the initially embedded fertilizer was released from the tablets with polymer coating

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even after seven days of incubation in water, suggesting the effectiveness of using the

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polymer coating.

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In the second stage, we investigated degradation behavior of experimental nitrogen

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fertilizer formulations incubated in soil and ammonium release kinetics. We used

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ammonium nitrate rather than urea because it contains both ammonium and nitrate nitrogen

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forms. We tested not only pure P(3HB) but also P(3HB) composites with wood (birch)

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flour or polycaprolactone (Fig. 2).

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Dynamics of changes in the weight of formulations and nitrogen concentration in

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soil are shown in Figure 3. Investigation of degradation dynamics revealed (Fig. 3a)

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comparable degradation rates of different formulations (reaching 70.3 – 74% of the initial

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mass over 7 days). Measurements of ammonium nitrogen concentrations in soil indicated

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high release rates even in the first seven days (Fig. 3b). Throughout the experiment,

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ammonium nitrogen concentration in soil increased gradually, with no dramatic changes

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and with relatively similar patterns observed for formulations with different compositions.

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In the treatment with uncoated specimens, 30-35% of the embedded nitrogen was

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released to soil in two weeks (Fig. 3b), while the amount of the fertilizer released from the

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coated formulations was no greater than 4.4-5.8 mg (15-20%) after six weeks of incubation

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in soil (Fig. 4). No significant differences were noted in the kinetics of degradation of

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formulations and ammonium nitrate release between treatments with matrices of different

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compositions. Later, however, the rate of ammonium release to soil increased rapidly, and

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the largest amounts of the fertilizer were released to soil over three weeks after Week 6 of

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the experiment.

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In the second phase of the study, we estimated the effectiveness of the experimental formulations in the laboratory experiment, in an environmental chamber with wheat plants.

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Formulations based on pure P(3HB) and P(3HB)/wood flour composites showed

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comparable biodegradation rates (Fig. 5a), degradation rates of uncoated specimens being

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considerably faster than those of coated tablets. This difference was first observed in Week

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2 of the experiment, by which time uncoated tablets had lost 11-12% and coated ones 4-6%

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of their weight. By Week 12, the weight loss had reached 68-79% and 52-57%,

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respectively. Coated tablets with a P(3HB)/wood powder core demonstrated the slowest

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degradation.

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As plants were growing and regularly watered, as described in the Methods section,

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at the beginning of Week 2 of the experiment, the amounts of ammonium in soil began to

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decline, but the patterns of decline were different (Fig. 5b). In the negative control, the total

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amount of ammonium was originally below 0.5 mg, decreasing gradually over the course of

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the experiment and dramatically dropping (to 0.01 mg or lower) after Week 8. In the

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positive control, ammonium decreased rather slowly, from 7.3 mg after two weeks of the

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experiment to 0.02 mg by Week 12. In the treatments with uncoated formulations that had

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different compositions of the cores, about 5 mg of ammonium was contained in the soil

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after two weeks. Later, the ammonium level decreased but at a slower rate, and by the end

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of Week 5, it was higher than the ammonium level in the positive control (1.08-1.12 mg

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and 0.86 mg, respectively). This difference was retained throughout the rest of the

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experiment, reaching its maximum at the end of Week 8 (0.42-0.45 mg and 0.14 mg,

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respectively).

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In the treatment with coated formulations, ammonium was released at a

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considerably slower rate. After two weeks of the experiment, ammonium content in the soil

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(0.44-0.50 mg) was comparable to that in the negative control. Then, the amount of

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ammonium in soil increased gradually, reaching its maximum (1.2 – 1.3 mg) by the end of

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Week 4. After that, it decreased slowly, but remained higher than the levels obtained in the

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treatments with uncoated formulations.

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The amount of ammonium in the irrigation solution (Fig. 5c) generally correlated

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with its amount in soil. The lowest, gradually decreasing, values were obtained in the

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negative control. Similar gradual changes were observed in the positive control, although

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the initial values were two orders of magnitude higher and the decrease rate was faster. In

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the treatments with uncoated formulations, dynamics was more intricate, with the initial

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concentration peak at 0.04-0.07 mg, a decrease by a factor of ten (to 0.004-0.006 mg), an

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increase to 0.01-0.02 mg (over 3-7 weeks), and a gradual decline throughout the rest of the

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experiment. In the treatments with coated formulations, a similar development was

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observed, but the second concentration peak occurred later (after 6-10 weeks).

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Evaluation of the increase in the biomass of spring wheat in the 12-week experiment

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showed that the use of the experimental fertilizer formulations was more effective than

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application of the fertilizer by the traditional method (Table 4). In the treatments with

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uncoated formulations, the biomass increase was 12-20% higher than in the positive control

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at Week 4, and that was the greatest difference observed; by the end of the experiment, it

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had decreased to 8%. The effect of the coated formulations became evident later, by Week

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8, but it was more pronounced, and in these treatments, at the end of the experiment, the

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biomass increase was more than 25% higher than in the positive control. Thus, the use of

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slow-release formulations alleviated nitrogen deficiency in wheat plants in the second half

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of the experiment, when the soil with the free fertilizer applied to it was actually nitrogen

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depleted.

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Discussion

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In a previous study, we evaluated soil degradation of P(3HB) composites with wood

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flour, PCL, or polyethylene glycol (PEG) that did not contain a fertilizer, based on the

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weight loss of the specimens.26 Compressed specimens were placed in containers with soil

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with constant (50%) moisture content. Analysis of degradation behavior of four types of the

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specimens (pure polymer and composites) showed that it depended on their composition.

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P(3HB)/PEG specimens had the highest degradation rate, and their residual weight at Day

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35 of the experiment constituted 37.7% of their initial weight. Some of the weight loss

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could be caused by PEG being dissolved in water and washed away. At the end of the

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experiment, the weight of P(3HB)/PCL specimens constituted 50.6% of their initial weight,

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and the weight of P(3HB) and P(3HB)/wood flour specimens decreased to 69.7% and

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72.9% of their initial weight, respectively. Thus, blending of P(3HB) with fillers affected

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degradation rates of the specimens.

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Degradation of polymer/fertilizer formulations (Fig. 3) investigated in this study

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occurred in soil with a much higher rate than degradation of pure polymer and composite

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specimens. After seven days of incubation in soil, the weight loss of fertilizer formulations

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reached 32-35 mg (26-30%), depending on the composition of the polymer matrix, and 8.7-

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9.6 mg (29-32%) of fertilizer per tablet was released. Nevertheless, this study proved that

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both pure poly-3-hydroxybutyrate and blends thereof could be used to prepare slow-release

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fertilizer formulations. Although P(3HB) and composite matrices were comparable in their

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degradation and fertilizer release rates, the use of less expensive fillers (such as wood flour)

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can reduce the cost of slow-release fertilizer formulations.

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As in the first experiment, compressed fertilizer/polymer formulations were

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degraded at a high rate and ammonium nitrate had been almost completely released by the

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end of the experiment (Day 35), the formulations were coated with a layer of P(3HB) to

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slow down fertilizer release. Application of the coating considerably decreased the rates of

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degradation of the tablets with all types of the core and release of ammonium nitrogen from

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them. Thus, this study showed the possibility of constructing slow-release nitrogen

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formulations with degradable poly-3-hydroxybutyrate used as a matrix and proved that the

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rate of nitrogen release to soil could be regulated by changing the type of formulation and

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production technique.

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As it was shown in the water experiments, formulations based both on pure P(3HB)

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and P(3HB)/wood composite as fillers demonstrated similar release kinetics of the

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fertilizer. In the soil experiments, the results were also similar in the experiments with and

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without plants, but the release rates were slower. The dense soil structure might have

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prevented self-destruction of samples and made the release slower even in the case of

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uncoated tablets. Coating had much higher importance in all experiments.

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In the experiment with plants, biodegradation rates of formulations based on pure

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P(3HB) and P(3HB)/wood flour were similar to each other (Fig. 5a) while the rates of

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degradation of coated and uncoated formulations differed considerably. After the first two

340

weeks, 14.3-15.5 mg (12.0-12.9%) of the uncoated and 9.0-11.2 mg (4.3-5.4%, taking into

341

account the mass of the polymer coating) of the coated tablets were degraded. After six

342

weeks, the weight loss of uncoated tablets reached 30.2-31.3% and the weight loss of the

343

coated ones – 15.9-17.8%, although the absolute values of the loss, taking into account the

344

mass of the coating, were comparable (36.3-37.5 mg and 33.2-37.0 mg, respectively).

345

These differences were caused by both the effect of the additional mass of the coating and

346

slower degradation of the core protected by the coating. In this experiment, degradation

347

rates of uncoated formulations were substantially slower than in the previous experiment,

348

without plants (Fig. 3). A possible reason for that may different temperature conditions of

349

the experiments: in the first experiment, specimens were incubated at a constant

350

temperature of 20°C while in the second experiment, the temperature was 2-10°C lower

351

than that for the greater part of the 24-h period, which created less favorable conditions for

352

the development of biodegrading microorganisms.

353

Measurements of soil ammonium nitrogen, performed after Week 1, showed that it

354

gradually decreased in both the controls and soil with the uncoated experimental

355

formulations (Fig. 5b). In the treatments with coated formulations, ammonium release was

356

noticeably delayed. After two weeks, measurements showed release of 75-78% (5.1-5.3 mg

357

ammonium which is equivalent to 22-24 mg NH4NO3) and 6.6-7.3% (0.44-0.50 mg

358

ammonium) of the initially embedded fertilizer, respectively. The ammonium concentration

359

peak was observed at Weeks 4-6 of the experiment. However, ammonium concentrations

15



360

(1.2-1.3 mg per container at the most) never reached the maxima obtained in the positive

361

control (7.3 mg) and in the treatments with uncoated formulations. This could be caused by

362

the delayed release and the higher loss due to ammonium accumulation in plant biomass.

363

Although ammonium concentration in soil gradually decreased in all laboratory

364

ecosystems, as the fertilizer was washed out and consumed by plants, the level of

365

ammonium nitrogen remained an order of magnitude higher in the treatments than in the

366

positive control throughout the rest of the experiment.

367

Nitrogen concentration in soil correlated with the amount of nitrogen released to the

368

irrigation solution (Fig. 5c). During Week 1 of the experiment, the highest amount of

369

ammonium nitrogen in water was observed in the positive control (0.36 mg) and much

370

lower content (0.037-0.065 mg) – in the treatment with uncoated formulations. In the

371

treatment with coated formulations, initial amount of ammonium in the irrigation solution

372

(0.004-0.006 mg) was comparable with that in the negative control (0.002 mg). In the

373

treatment with uncoated formulations, ammonium noticeably dropped at Weeks 3-4 of the

374

experiment (to 0.003-0.006 mg) but temporarily rose later (up to 0.018 mg). The first peak

375

evidently resulted from the passive washing of the fertilizer off the tablet surface while the

376

second indicated the beginning of biological degradation of the specimens and release of

377

the fertilizer from deeper layers of the formulation. Another proof of this is that the first

378

peak of nitrogen concentration in water and soil was observed after two weeks of

379

incubation of experimental formulations, when their weight loss was relatively small (about

380

10%) (Fig. 5). By the end of the experiment, nitrogen concentration in the irrigation water

381

in the positive control had dropped by more than three orders of magnitude (to 9×10–5 mg),

382

the obvious reason being that almost all of it was washed out of the soil. In the treatments

383

with the experimental formulations, initial nitrogen concentration in the irrigation water

384

was considerably lower, but at Week 7, it began to increase, reaching levels about one order

16


Page 16 of 38

Page 17 of 38


385

of magnitude higher than in the positive control, which suggested that the fertilizer was still

386

being released to soil.

387

These differences correlated with the increase in the biomass of experimental plants

388

(Table 4). Even after the first three weeks, plant biomass increase in the treatment with

389

uncoated tablets was higher than in the positive control. By the end of the experiment,

390

however, that difference had been considerably reduced, probably because of complete

391

release of nitrogen from the uncoated tablets followed by slower growth of the plants in that

392

treatment. In the treatment with the coated fertilizer tablets, a considerable plant biomass

393

increase was first observed later, at Week 8, but that treatment showed the most striking

394

differences by the end of the experiment.

395

The experimental formulations prepared and tested in this study had significantly

396

greater longevity than those previously described by other researchers. The time necessary

397

for complete release of the fertilizer varied from 24 h2 to 10 days.5 Only one study reported

398

an about 15% weight loss over 20 days for the urea/chitosan/bentonite system.6 A possible

399

reason for the short lifetime of the formulations could be the use of relatively hydrophilic

400

fillers, which were quickly saturated with water, leading to more rapid dissolution and

401

release of the fertilizers.

402

Neata et al.27 reported the use of systems containing starch (10%), wood flour

403

(40%), and an NPK fertilizer (50%) to increase the growth of petunia plants by about 10%

404

over 28 days of the experiment; the systems containing other proportions of the components

405

were effective compared to control in shorter experiments (lasting two or three weeks). In

406

the previous study authors also showed effectiveness of compressed pellets of pure P(3HB)

407

with urea for comparable period, 22-28 days.23

408

The fertilizer can be retained in the granule by applying non-biodegradable coatings,

409

but this may result in accumulation of polymer waste, which will persist in the soil for

17



410

years. In a study by Yang et al.,7 the authors coated urea granules or compressed tablets

411

with foam from non-biodegradable polystyrene: the fertilizer was sprayed with the ethyl

412

acetate solution of polystyrene or polystyrene foam and additionally coated with wax or

413

castor oil with diphenylmethane diisocyanate. The authors showed that such coatings could

414

be used to prepare slow-release fertilizers, with nitrogen release time reaching 100 days.

415

Liang and Liu28 reported preparation of a double-coated fertilizer. Polystyrene was used to

416

make the inner coating, and urea with cross-linked polyacrylamide created the outer

417

coating. Over 30 days, about 60% nitrogen was released from the double-coated granules.

418

A few studies described preparation of fertilizer formulations that combined

419

embedding of the fertilizer into the matrix of natural and biodegradable materials and

420

coating of the formulation, but no sufficiently slow release was achieved, which may be

421

also caused by increased water permeability of materials used. Ni et al.29 developed three

422

types of nitrogen fertilizer formulations, which were based on urea, ammonium sulfate, and

423

ammonium chloride. The fertilizer was mixed with clay, and that mixture was then blended

424

with urea granules and some water. The next step was to spray a mixture of ethyl cellulose

425

and stearic acid (5:2) in ethanol on polymer granules; the formulation was additionally

426

coated with carboxymethylcellulose/hydroxyethylcellulose hydrogel. The peak of nitrogen

427

release to soil was observed for five days, followed by slow release over 20 days.

428

In the present study, we used the method of cold pressing of nitrogen fertilizer with

429

the polymer and composite components alone and together with application of the

430

additional polymer coating to prepare fertilizer formulations that differed considerably in

431

the kinetics of release of the active ingredient to soil. These methods are not technologically

432

difficult and can be scaled up for commercial manufacture. In the future, simultaneous

433

application of various types of formulations could enable maintaining stable nitrogen levels

434

in soil throughout the growing season of crops. The use of a hydrophobic and relatively

18


Page 18 of 38

Page 19 of 38


435

slowly biodegraded coating will considerably slow down fertilizer release, without

436

contaminating soil with polymer waste.

437

Development of new processes and production optimization have decreased the

438

minimal cost of P(3HB) from US$ 10-12/kg in 2006 to €1.5/kg in 2010,30 making the cost

439

of the polymer comparable to the cost of plastics derived from oil products.31 Also, to

440

decrease the cost of the final fertilizer, it is necessary to collect and reuse the evaporated

441

chloroform, which can be achieved in the case of scaling up the process. Because of its

442

toxicity, this is also important in order to maximize eco-friendliness of the technology.

443

Thus, controlled slow-release fertilizer formulations can be prepared by embedding

444

nitrogen fertilizers into matrices of poly-3-hydroxybutyrate and composites based on it.

445

These may be uncoated compressed tablets or tablets additionally coated with a layer of

446

polymer based on the core/shell principle, with the core also stabilized by using the

447

polymer or composite matrix. These formulations are capable of maintaining higher

448

concentrations of nitrogen in soil compared to fertilizers applied by usual methods over

449

long time periods (up to several months). Laboratory experiments showed that these

450

formulations had a more favorable effect on plant growth than the free fertilizer and did not

451

inhibit soil microflora. These experimental formulations can be used as prototypes for

452

developing controlled-release agricultural formulations.

453 454

The study was supported by the State budget allocated to the fundamental research at the Russian Academy of Sciences (project No AAAA-A17-117013050028-8).

455 456

References

457

1. Sopeña, F.; Cabrera, A.; Maqueda, C.; Morillo, E. Controlled release of the

458

herbicide norflurazon into water from ethylcellulose formulations. J. Agr. Food Chem.

459

2005, 53, 3540–3547.

19



460 461

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2. Chen, L.; Xie, Z.; Zhuang, X.; Chen, X.; Jing, X. Controlled release of urea encapsulated by starch-g-poly (L-lactide). Carbohydrate polymers. 2008a, 72, 342–348.

462

3. Jamnongkan, T.; Kaewpirom, S. Potassium release kinetics and water retention of

463

controlled-release fertilizers based on chitosan hydrogels. J. Polym. Environ. 2010, 18,

464

413–421.

465

4. Pereira, E.I.; Minussi, F.B.; da Cruz, C.C.; Bernardi, A.C.C.; Ribeiro, C. Urea–

466

montmorillonite-extruded nanocomposites: a novel slow-release material. J. Agr. Food

467

Chem. 2012, 60, 5267–5272.

468 469

5.

Wanyika,

H.

Controlled

release

of

agrochemicals

intercalated

into

montmorillonite interlayer space. Scientific World J. 2014, 2014, Article ID 656287 (9 p.).

470

6. Hamid, N.N.A.; Mohamad, N.; Hing, L.Y.; Dimin, M.F.; Azam, M.A.; Hassan,

471

M.H.C.; Ahmad, M.K.S.M.; Shaaban, A. The effect of chitosan content to physical and

472

degradation properties of biodegradable urea fertilizer. J. of Sci. Inno. Res. 2013, 2, 893–

473

902.

474

7. Yang, Y.C.; Zhang, M.; Li, Y.; Fan, X.; Geng, Y. Improving the quality of

475

polymer-coated urea with recycled plastic, proper additives, and large tablets. J. Agr. Food

476

Chem. 2012, 60, 11229–11237.

477

8. Chen, D.; Freney, J.R.; Rochester, I.; Constable, G.A.; Mosier, A.R.; Chalk, P.M.

478

Evaluation of a polyolefin coated urea (Meister) as a fertilizer for irrigated cotton. Nutr.

479

Cycl. Agroecosyst. 2008b, 81, 245–254.

480 481

9.

Jarosiewicz,

A.;

Tomaszewska,

M.

Controlled-release

NPK

fertilizer

encapsulated by polymeric membranes. J. Agr. Food Chem. 2003, 51, 413–417.

482

10. Zvomuya, F.; Rosen, C.J.; Russelle, M.P.; Gupta, S.C. Nitrate leaching and

483

nitrogen recovery following application of polyolefin-coated urea to potato. J. Environ.

484

Qual. 2003, 32, 480–489.

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11. Lan, R.; Liu, Y.; Wang, G.; Kan, C.; Jin, Y. Experimental modeling of polymer latex spray coating for producing controlled-release urea. Particuology. 2011, 9, 510–516. 12. Vashishtha, M.; Dongara, P.; Singh, D. Improvement in properties of urea by phosphogypsum coating. Int. J. ChemTech Res. 2010, 2, 36–44.

489

13. Anghel, A.; Lăcătuşu, A.-R.; Lăcătuşu, R.; Iancu, S.; Lungu, M.; Lazăr, R.;

490

Vrînceanu, A.; Bălăceanu, C. Testing kinetic of nutrients release from complex mineral

491

fertilizers coated with co-polyester films from pet waste recycling and effect on soil

492

chemical properties. Scientific Papers. Series A. Agronomy. 2012, 55, 13–18.

493 494 495 496

14. Nash, P.R.; Nelson, K.A.; Motavalli, P.P. Corn yield response to polymer and non-coated urea placement and timings. Int. J. Plant Prod. 2013, 7, 373–392. 15. Nelson, K.A.; Nash, P.R.; Dudenhoeffer, C.J. Effect of nitrogen source and weed management systems on no-till corn yields. J. Agr. Sci. 2013, 5, 87–96.

497

16. Grant, C.A.; Wu, R.; Selles, F.; Harker, K.N.; Clayton, G.W.; Bittman, S.;

498

Zebarth, B.J.; Lupwayi, N.Z. Crop yield and nitrogen concentration with controlled release

499

urea and split applications of nitrogen as compared to non-coated urea applied at seeding.

500

Field Crop. Res. 2012, 127, 170–180.

501

17. Medina, L.C.; Obreza, T.A.; Sartain, J.B.; Rouse, R.E. Nitrogen release patterns

502

of a mixed controlled-release fertilizer and its components. HortTechnology. 2008, 18,

503

475–480.

504 505

18. Oosterhuis, D.M.; Howard, D.D. Evaluation of slow-release nitrogen and potassium fertilizers for cotton production. Afr. J. Agric. Res. 2008, 3, 68–73.

506

19. Adams, C.; Frantz, J.; Bugbee, B. Macro- and micronutrient-release

507

characteristics of three polymer-coated fertilizers: Theory and measurements. J. Plant Nutr.

508

Soil Sci. 2013, 176, 76–88.

21



509

20. Shoji, S.; Delgado, J.; Mosier, A.; Miura, Y. Use of controlled release fertilizers

510

and nitrification inhibitors to increase nitrogen use efficiency and to conserve air and water

511

quality. Commun. Soil Sci. Plant Anal. 2001, 32, 1051–1070.

512

21. Huett, D.O.; Gogel, B.J. Longevities and nitrogen, phosphorus, and potassium

513

release patterns of polymer-coated controlled-release fertilizers at 30°C and 40°C.

514

Commun. Soil Sci. Plant Anal. 2000, 31, 959–973.

515

22. Costa, M.M.; Cabral-Albuquerque, E.C.; Alves, T.L.; Pinto, J.C.; Fialho, R.L.

516

Use of polyhydroxybutyrate and ethyl cellulose for coating of urea granules. J. Agr. Food

517

Chem. 2013, 61, 9984–9991.

518 519

23. Volova, T.G.; Prudnikova, S.V.; Boyandin, A.N. Biodegradable poly-3hydroxybutyrate as a fertilizer carrier. J. Sci. Food Agr. 2016, 96, 4183–4193.

520

24. Volova, T.G.; Kiselev, E.G.; Vinogradova, O.N.; Nikolaeva, E.D.; Chistyakov,

521

A.A.; Sukovatyi, A.G.; Shishatskaya, E.I. A glucose-utilizing strain, Сupriavidus eutrophus

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В-10646: growth kinetics, characterization and synthesis of multicomponent PHAs. PLOS

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ONE. 2014, 9, e87551.

524

25. Korobeinikov, N.I.; Peshkova, N.V.; Boradulina, V.A.; Musalitin, G.M.;

525

Valekzhanin, V.S.; Kvasnik, E.V. Pshenitsa myagkaya yarovaya (Triticum aestivum L.),

526

sort ALTAISKAYA 70 (Soft spring wheat (Triticum aestivum L.), cultivar ALTAISKAYA

527

70). Patent of the Russian Federation for selection invention No. 4758 of 25.05.2009

528

(http://reestr.gossort.com/reg/cultivar/10443) (in Russian)

529

26. Boyandin, A.N.; Zhila, N.O.; Kiselev, E.G.; Volova, T.G. Constructing slow-

530

release formulations of metribuzin based on degradable poly(3-hydroxybutyrate). J. Agr.

531

Food Chem. 2016, 64, 5625–5632.

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532

27. Neata, G.; Popa, M.; Mitelut, A.; Guidea, S.; Pukanski, B. Bio-based composite

533

use in fertilization of petunia and carnation culture. Scientific Bulletin. Series F.

534

Biotechnologies. 2012, 16, 30–35.

535 536 537 538 539 540

28. Liang, R.; Liu, M. Preparation and properties of a double-coated slow-release and water-retention urea fertilizer. J. Agr. Food Chem. 2006. 54, 1392–1398. 29. Ni, B.; Liu, M.; Lü, S.; Xie, L.; Wang, Y. Environmentally friendly slow-release nitrogen fertilizer. J. Agr. Food Chem. 2011, 59, 10169–10175. 30. Chanprateep S. Current trends in biodegradable polyhydroxyalkanoates. J. Biosci. Bioeng. 2010, 110, 621–632.

541

31. Lea-Smith, D.J.; Howe, C.J. The Use of Cyanobacteria for Biofuel Production.

542

In Biofuels and Bioenergy; Love, J.; Bryant, J.A., Eds.; Wiley-Blackwell: Chichester, UK;

543

2017; p 147.

544

23



545

Page 24 of 38

Table 1. Conditions in the Conviron environmental chamber Time

t, °C, Weeks 1- t, °C, Weeks 8- Minimal

Irradiance,

7

µmol/m2/s

12

humidity, %

1

0:00

10°C

14°C

50

0

2

6:00

12°C

16°C

50

100

3

9:00

14°C

18°C

40

200

4

12:00

18°C

22°C

20

300

5

16:00

16°C

20°C

30

200

6

20:00

13°C

17°C

30

100

7

23:00

10°C

14°C

40

0

546 547

24


Page 25 of 38


548

Table 2. Mass and thickness of P(3HB) coatings depending on the number of immersions

549

of tablets into the P(3HB) solution Number of Coating mass, mg

Coating weight, % of Coating thickness, µm

immersions

the initial weight

(calculated)

1

13.2±2.2

11.0±1.8

5.2±0.9

2

23.3±4.1

19.4±3.4

9.1±1.6

3

34.6±4.0

28.8±3.3

13.6±1.6

4

47.0±5.3

39.2±4.4

18.4±2.1

5

59.2±5.8

49.3±4.8

23.2±2.3

6

71.1±6.7

59.3±5.6

27.9±2.6

550

25



Page 26 of 38

551

Table 3. Fertilizer release from formulations of different types into sterile water during the

552

7-day experiment, % of the initial ammonium nitrate (30 mg per tablet) Filler, coating

1 hour

1 day

3 days

7 days

P(3HB), uncoated

23.3±7.4

37.0±3.9

44.0±3.9

97.9±2.3

P(3HB), coated

1.1±0.1

5.8±3.1

7.4±5.3

18.3±7.9

P(3HB)/wood, uncoated

36±2

43±2

45.5±1.0

98.9±1.1

P(3HB)/wood, coated

1.0±0.0

2.2±0.9

2.4±1.0

13.4±2.8

553 554

26


Page 27 of 38


555

Table 4. Increase in the total dry biomass of soft wheat (Triticum aestivum L.) in the

556

experiment with different formulations of ammonium nitrate (mg per plant) Week

Negative

Positive

P(3HB),

P(3HB),

P(3HB)/wood

P(3HB)/wood

control

control

uncoated

coated

flour, uncoated

flour, coated

2

27.4 ± 0.3

34.8 ± 0.5

32.3 ± 1.6

36.8 ± 0.6

30.8 ± 1.3

33.1 ± 0.9

3

42.3 ± 2.9

45.0 ± 2.3

52.0 ± 2.2

47.3 ± 3.3

55.8 ± 1.9

44.9 ± 2.6

4

54.7 ± 3.1

69.3 ± 4.3

77.9 ± 4.1

51.7 ± 3.9

81.8 ± 3.8

61.0 ± 2.1

5

54.8 ± 3.7

127.1 ± 6.9

142.1 ± 5.3

101.4 ± 8.1

145.2 ± 10.6

110.0 ± 6.4

6

86.0 ± 6.2

138.0 ± 8.7

169.8 ± 6.2

123.4 ± 10.0

179.6 ± 12.1

150.4 ± 8.8

8

106.0 ± 9.8

195.0 ± 11.1

239.0 ± 10.9

257.2 ± 16.9

193.2 ± 10.9

275.0 ± 13.9

10

179.2 ± 11.2

295.8 ± 14.2

356.1 ± 10.7

454.4 ± 26.8

333.1 ± 12.3

412.7 ± 19.5

12

180.8 ± 16.7

362.6 ± 12.6

393.3 ± 15.8

463.8 ± 23.3

392.8 ± 16.3

460.0 ± 29.0

557

27



558 559

Figure 1. Constructing an experimental container: a) – placement of a fertilizer-containing bag; b) –

560

sowing of wheat seeds

561

28


Page 28 of 38

Page 29 of 38


1

2

3

4

562

7 days

14 days

21 days

28 days

35 days

563

Figure 2. Ammonium nitrate embedded in the polymer matrices of different compositions during

564

degradation: 1 – P(3HB); 2 – P(3HB)/wood flour; 3 – P(3HB)/PCL; 4 – P(3HB)/PEG

29


100 90 80 70 60 50 40 30 20 10 0

П(3ГБ)/ПКЛ NH 4NO3/P(3HB)/PCL NH П(3ГБ)/опилки 4NO3/P(3HB)/sawdust

566

b

70 60 50 40

NH П3ГБ 4NO3/P(3HB)

30

NH П3ГБ/ПКЛ 4NO3/P(3HB)/PCL

20

NH П3ГБ/опилки 4NO3/P(3HB)/sawdust

10 0

0

565

80

NH NH4NO3/П(3ГБ) 4NO3/P(3HB)

a

Ammonium output, % initial

Residual polymer mass, %


7

14

21

28

35 Time, days

0

7

14

21

28

35 Time, days

Figure 3. Changes in the weight of formulations (a) and ammonium nitrogen release to soil (b)

567


Page 30 of 38


a

Residual polymer mass, %

100 90

П3ГБ NH4NO3/P(3HB) П3ГБ:ПКЛ 1:1 NH4NO3/P(3HB)/PCL

80

П3ГБ:опилки 1:1 NH4NO3/P(3HB)/sawdust

70 60 50 40 30 20 10 0

90

П3ГБ NH 4NO3/P(3HB)

80

NH П3ГБ:ПКЛ 1:1 4NO3/P(3HB)/PCL

70

NH П3ГБ:опилки 1:1 4NO3/P(3HB)/sawdust

60 50 40 30 20 10 0

0

568

b

100 Ammonium output, % initial.

Page 31 of 38

20

40

60

80 Time, days

0

20

40

60

80 Time, days

569

Fig. 4. Kinetics of degradation of polymer-coated fertilizer formulations (a) and ammonium release to soil (b) as dependent on the composition of the

570

polymer matrix

571

31 ACS Paragon Plus Environment


Page 32 of 38

572

Total ammonium in soil, mg.

Sample mass, % initial.

80

60

40 П3ГБ без покрытия NH 4NO 3/P(3HB) П3ГБ с 3покрытием NH /P(3HB) with coating 4NO П3ГБ/опилки без покрытия NH 4NO3/P(3HB)/sawdust П3ГБ/опилки с покрытием NH with coating 4NO3/P(3HB)/sawdust

20

1.00

0.10

Отриц. контроль Negative control Полож. контроль Positive control П3ГБ без покрытия NH 4NO 3/P(3HB) П3ГБ с покрытием NH 4NO3/P(3HB) with coating П3ГБ/опилки без покрытия NH 4NO3/P(3HB)/sawdust П3ГБ/опилки с покрытием NH 4NO3/P(3HB)/sawdust with coating

0.01

0

2

4

6

8

10 12 Time, weeks

c

1.00000

0.00

0

573

b

10.00

Total ammonium in water, mg.

a

100

0.10000

0.01000

0.00100

0.00010

0.00001 0

2

4

6

8

10 12 Time, weeks

0

2

4

6

8

574

Fig. 5. Changes in the weight of fertilizer tablets during the experiment with soft wheat (a), and kinetics of ammonium release to soil (b) and to the

575

irrigation solution (c)

32 ACS Paragon Plus Environment

10 12 Time, weeks

Page 33 of 38


576

TOC Graphic

577 No fertilizer

No fertilizer

Free fertilizer Fertilizer/P(3HB)

Free fertilizer

Coated fertilizer/P(3HB)

NH4NO3

Biomass

Free fertilizer

Poly-3-hydroxybutyrate [P(3HB)] tablets with NH4NO3

P(3HB)/ fertilizer H3 C

H

O

...

O

...

n Poly-3-hydroxybutyrate

(+ sawdust)

P(3HB) coated P(3HB) tablets with NH4NO3 0

2

4

6

8

10

12

Time, weeks


P(3HB)coated P(3HB)/ fertilizer system


Figure 1. Constructing an experimental container: a) – placement of a fertilizer-containing bag; b) – sowing of wheat seeds 1083x479mm (72 x 72 DPI)


Page 34 of 38

Page 35 of 38


Figure 2. Ammonium nitrate embedded in the polymer matrices of different compositions during degradation: 1 – P(3HB); 2 – P(3HB)/wood flour; 3 – P(3HB)/PCL; 4 – P(3HB)/PEG 1083x815mm (72 x 72 DPI)



Figure 3. Changes in the weight of formulations (a) and ammonium nitrogen release to soil (b) 1083x343mm (72 x 72 DPI)


Page 36 of 38

Page 37 of 38


Fig. 4. Kinetics of degradation of polymer-coated fertilizer formulations (a) and ammonium release to soil (b) as dependent on the composition of the polymer matrix 1083x358mm (72 x 72 DPI)



Fig. 5. Changes in the weight of fertilizer tablets during the experiment (a), and kinetics of ammonium release to soil (b) and to the irrigation solution (c) 1504x330mm (32 x 32 DPI)


Page 38 of 38

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