DIVISION OF FERTILIZERS | Annual Report 1996

We aim to develop integrated technology for sustainable agriculture by maximizing fertilizer efficiency and promoting material recycling in agro-ecosystems. In FY 1996, we developed a new analytical system for macronutrients using hollow fibers. We also improved the recovery of macronutrients from livestock urine for the use of fertilizer. As to micronutrients, in particular, the research activities stress boron. The isolation and characterization of boron complexes in plants were carried out by using high performance liquid chromatography (HPLC), inductively coupled plasma - mass spectrometry (ICP-MS), and nuclear magnetic resonance (NMR) techniques. In the cooperative work, boron distribution in Arabitopsis was investigated by using ¹¹B tracer/ICP-MS technique. Concerning the utilization of organic matter, we studied energy use of animal wastes,control of rare metals from organic wastes, and microbiological effects of organic matter application.Rare metals whose concentrations increase in soil by the application of sewage sludge to soil were identified.Bacteria carrying nitrogen-fixing genes were found to proliferate in soil by the addition of straw.

To obtain comprehensive information on the concentration of nutrients, such as NO₃^-, Cl^-, K⁺, Ca²⁺ and Mg²⁺, in heterogeneous soil solutions in soil layers,a new method, using a hollow fiber, was developed. The fiber is made of an Ultra filtration membrane which can pass molecules such as water, inorganic ions and organic molecules with low weight. In this method, the hollow fiber was laid as scroll state in soil (Andosol). Deionized water flowed into the fiber successively through a peristaltic pump at a constant flow rate (Fig.1 a). Solutes in the external solution easily entered the inner water by the diffusion force resulting from the differences in concentrations between inner and external solutions (Fig.1 b). The outlet solution which passed through the fiber was fractionated and inorganic ions in the solution were analyzed.

When the soil moisture was higher than ca. 30% on the volume basis,a linear correlation was found between nutrient contents (x) in the soil solution which was obtained by centrifugation and those (y) in the initial portion of outlet solution from the hollow fiber. It was also shown that the coefficient (a: y = ax) depended on a flow rate of deionized water passing through the fiber, length of the fiber, ion species and soil moisture. The coefficients of NO₃^-, Cl^-, K⁺, Ca²⁺ and Mg²⁺were shown respectively in Table 1, when the water content by volume was 37% and 29%,the length of a fiber was 110cm and flow rate was 0.03 ml/r n.

The proposed method using a hollow fiber is suitable for line or plane sampling, because of their thin and long shape. Another merit is that since the sampling of nutrients in the soil solution is based on diffusion, the sampling can be performed without suction which very likely brings about the clogging of the sampling device, such as a ceramic filter. This merit would be more pronounced in clayey soil which is too sticky to obtain the soil solution by the suction method.

Boron (B) has been recognized as an essential microelement for higher plants. However, its physiological and biochemical roles in plants are not yet well understood.The principal reason for this uncertainty of the boron function is that the chemical forms of boron in plants are not well known.We have revealed the boron chemical forms in plants using modern analytical techniques such as NMR and ICP-MS.

In vivo ¹¹B NMR spectra of plant tissue, namely, radish roots, apple fruit, cabbage leaves and komatsuna plant leaves and roots, were successfully obtained. In the spectra, the signals of borate monoester and diester, together with boric acid,were observed, which shows that the In vivo ¹¹B NMR technique provides direct evidence that the water-soluble borate esters exist in plant tissue.

It has been demonstrated that part of boron in plants is located in cell walls as a water-insoluble form. We have isolated a borate-rhamnogalacturonan-II complex (B-RG-II) from Driselase digests of sugar beet (dicot) and bamboo (monocot) cell walls. RG-II is a component of pectin, one of the cell-wall matrix polysaccharides. Instrumental analysis showed one molecule of boric acid cross-links two RG-II molecules through borate-diol ester bonding. We further demonstrated that Sr²⁺, Ba²⁺ and Pb²⁺ are specifically bound to B-RG-II using HPLC/ICP-MS. The cation binding capacity of the complex may be a mechanism allowing plants to tolerate these toxic metals. In plants,absorbed boric acid binds with cis-diol compounds to make borate esters with a negative charge, which may be concerned with mobility or storage of boron. Then the water-soluble boron would be incorporated in the B-RG-II to cross-link pectin molecules and hence stabilize cell wall structure. (Fig.2)

Animal wastes have increased extremely as the livestock industry developed. They pose a serious treatment problem. It is urgent to develop new usages of the wastes in addition to promoting their use as fertilizer. We tried to convert livestock excrement to oil, which is one of the most convenient forms of fuel.

A simple technique was developed for converting bovine excrement to oil. The technique included only a process of the material under high temperature and high pressure. No drying of excrement or no addition of other materials was necessary. Twenty minute treatment at 300°C and 10 MPa was the optimum condition for bovine raw excrement. The produced oil was extracted with dichloromethane.

The oil yield was 27% on the organic matter basis or 42% on the carbon basis. The product was a brownish black oil with a high viscosity at room temperature. The chemical structure of the product was determined to be mainly paraffin hydrocarbon by ¹³C NMR. The elemental composition of the oil was 74% carbon, 5% hydrogen, 4% nitrogen and 17% oxygen. The calorific value of the oil was estimated to be 30 MJ kg^-1 from the elemental composition and was equivalent to 70% of that of the marine fuel oil from petroleum.

Though the developed technique is simple, the cost for producing oil is still high. The combustion energy in the oil was about the same magnitude as the electric energy used for heating the material during processing. Improvement of oil yields and processing equipment is required before practical applications of the technique. This technique would be applicable to excrement of different animals only by determining the optimum conditions. Fig.3)

Fig.1 Schematic diagram of the extraction of inoraric ions from soil sohrtion using a hollow fiber.
A bollow fiber: made of potyacrylocitrile, internal diamerer. 0.8 mm, extemal diameter: 1.4 mm, moleculer weight cutoffs of ca13,000.

Table 1. The coefficients (a: y = ax) between nutrient contents (x) in the soil solution obtained by centrifugation and those (y) in the initial portion of outlet solution from the hollow fiber.

Fig.2 Chemical forms of boron in plants

Fig.3 Oil produced from bovine excrement