Ken-ichiro Suehara

Application of image analysis with neural network for plant somatic embryo culture

Abstract A method of classifying celery embryos and nonembryos using image analysis with a neural network to decide the time for transfer to the next culture stage in plant somatic embryo culture is presented. Since the image database of celery cells is vast, four key input parameters (area, ratio of length to width, circularity and distance dispersion) were selected. Among these four parameters, use of the first three was found to be satisfactory for classification between embryos and nonembryos. Using the three parameters, the trained neural network was also able to classify globular, heart- and torpedo-shaped embryos at a level comparable with estimation by a human expert. By using the trained neural network, the number of plantlets that would be formed after the second regeneration culture of 14 d could be successfully predicted from number of heart- and torpedo-shaped embryos at the end of the first regeneration stage.

Effects of C N ratio and pH of raw materials on oil degradation efficiency in a compost fermentation process.

Waste oil treatment was attempted using a compost fermentation process. To develop a simple method for waste oil treatment, cheap and simple materials were used as compost materials. The fermentation experiment was performed using a domestic composter to determine the optimum conditions of the fermentation. Adjustment of the pH value during the compost fermentation was also important for progression of the oil degradation. When the pH value was not controlled, the pH value decreased quickly and reached about 2 and the oil degradation was stopped. Adding caustic lime to the raw materials caused the pH value of the compost to stabilize at approximately 7. The addition of a nitrogen source had a large effect on oil degradation during the compost fermentation. The optimum value of the C N ratio of the raw materials with pH control was between 10 and 40. When the C N ratio of the materials was adjusted to 10, 20, and 40, the rate constants for oil degradation were very similar. The rate constants for NH4+ consumption were also similar. Oil degradation efficiency reached 83.5% relative to the initial oil content in the compost materials. Repeated batch operation of the compost fermentation was carried out and the compost system could maintain good efficiency for oil degradation over several repeated batch operations. Finally, the compost system was applied to the treatment of recalled mayonnaise, with favorable results being obtained.

Simultaneous measurement of carbon and nitrogen content of compost using near infrared spectroscopy

Near infrared (NIR) spectroscopy was used to determine the carbon and nitrogen content of compost during compost fermentation of tofu (soybean-curd) refuse. Reflectance measurements in the wavelength range between 400 and 2500 nm were made at 2 nm intervals. The NIR absorption of the carbon components was observed at two wavelengths, 1584 and 1024 nm in the second derivative spectrum. The NIR absorption of nitrogen components was observed at two wavelengths, 2174 and 900 nm in the second derivative spectrum. To formulate a calibration equation, a multiple linear regression analysis was carried out between the NIR spectral data and the carbon and nitrogen content in the calibration sample set (sample number, n = 60) obtained using a Pregie–Dumas combustion method. The values of the multiple correlation coefficient were 0.988 and 0.984, respectively. To validate the calibration equations obtained, the carbon and nitrogen content in the prediction sample set (n = 48), not used for formulating the calibration equation, were calculated using the calibration equations and compared with the values obtained using the Pregie–Dumas combustion method. Good agreement was observed between the results of the Pregie–Dumas combustion method and the NIR method. The values of the simple correlation coefficient were 0.986 and 0.972, respectively. The study indicates that NIR is a useful method for process management of the compost fermentation of tofu refuse.

Optimal expression of GUS gene from methyl jasmonate-inducible promoter in high density culture of transformed tobacco cell line BY-2

The optimal expression of the β-glucuronidase (GUS) gene was studied in a tobacco cell line, BY-2, that was transformed with the GUS gene under the control of the cathepsin D inhibitor (CDI) promoter. In batch culture, the optimal induction time was in the late growth phase, and the optimal concentration of methyl jasmonate (MJ) was 0.7 mM. In fed-batch culture, the GUS specific activity when MJ was added several times was about 1.7-fold that when MJ was added only once. However, a significant decrease in the growth rate was observed after MJ addition. During the fed-batch culture, no medium components were depleted, and the presence of inhibitory metabolite(s) was observed. To remove inhibitory metabolite(s) from the medium, filtration culture was carried out, which gave a cell growth rate faster than that of the fed-batch culture. The final cell concentration and total GUS activity reached 480 g-fresh weight/l and 5.2 μkat/l, which were 1.3-fold and 1.5-fold the amounts obtained in fed-batch culture. The GUS specific activity using the CDI promoter was about 17-fold that obtained in a rbcS-promoter system studied previously. Efficient foreign gene production from transformed BY-2 cells was thus performed on a fermentor scale.

Rapid measurement and control of the moisture content of compost using near-infrared spectroscopy

In a compost fermentation of soybean-curd (tofu) refuse, the effects of the moisture content of the compost on the compost reaction were studied. The moisture content of the compost was a very important factor for good fermentation. Near-infrared spectroscopy (NIRS) was applied to the determination of the moisture content of the compost. The reflected rays in the wavelength range between 400 and 2500 nm were measured at 2 nm intervals. The absorption of water was observed at three wavelengths, 960, 1406 and 1888 nm. To formulate a calibration equation, a multiple linear regression analysis was carried out between the near-infrared spectral data at 960 nm (sample number, n = 50) and on the moisture content obtained using a drying method. The values of the simple correlation coefficient and the standard error of calibration were 0.987 and 1.33%, respectively. To validate the calibration equation obtained, the moisture content in the prediction sample set (n = 35) not used for formulating the calibration equation was calculated using the calibration equation, and compared with the values obtained using the drying method. Good agreement was observed between the results of the drying method and those of the NIRS method. The simple correlation coefficient and standard error of prediction were 0.979 and 1.85%, respectively. Then, the NIRS method was applied to a practical situation in which the moisture content was measured and controlled during the compost fermentation, and good results were obtained. The study indicates that NIRS is a useful method for measurement and control of the moisture content in the compost of soybean-curd refuse.

Determination of the Content of Water and Rice Bran in Solid Media Used for Mushroom Cultivation Using Near-Infrared Spectroscopy

Abstract Near-infrared spectroscopy (NIR) was employed for the simultaneous determination of the water and rice bran content in solid media used for mushroom cultivation. The solid media were prepared by addition of water to a mixture of sawdust with rice bran and wheat bran. The medium packed in a polyethylene bag was placed in a near-infrared spectrophotometer. The reflected rays, in the wavelength range between 400 and 2500 nm, were measured at 2 nm intervals. To obtain a calibration equation for water content, a simple linear regression was carried out on the near-infrared spectral data at 1450 nm and on the water content of a calibration sample set (sample number, n=113) obtained using dry-weight method. The values of the simple correlation coefficient and the standard error of calibration were 0.995 and 1.33%, respectively. On the basis of the result of a multiple linear regression on the content of rice bran in the solid media, a calibration equation using the second-derivative reflectance data at the wavelengths of 672 and 2100 nm was obtained, with the values of the multiple correlation coefficient and standard error of 0.978 and 1.73%, respectively. To validate the calibration equations obtained, water and rice bran content in the prediction sample set (n=56) not used for formulating the calibration equations were calculated using the calibration equations, and compared with the values obtained using the dry-weight method based on the mixing ratio. For both the water and rice bran content, excellent agreement was observed between the results of the conventional method and those of NIR method. The simple correlation coefficient and standard error of prediction were 0.995 and 1.33% for water content and 0.975 and 1.84% for rice bran content. The content of water and rice bran in the solid media could be analyzed simultaneously by NIR. The NIR procedure was simple, and the operation time required to determine the content was only 5 min. These results indicate that NIR may be a useful method for monitoring the content of water and rice bran in solid media used for mushroom cultivation.

Efficient Production of Celery Embryos and Plantlets Released in Culture of Immobilized Gel Beads

Abstract Celery embryos and plantlets were found to be selectively released in a culture of immobilized Ca-alginate gel beads in which celery callus was entrapped under regeneration conditions. We studied the feasibility of use of this process for celery embryogenesis in an artificial seed system. The cells released from the gel beads were larger than those obtained in suspension culture. The optimal concentration of alginate gel for embryo and plantlet production was 2% for the immobilized cell culture. Considering the maintenance of the gel bead structure and detrimental effect of CaCl2 on plantlet development, 5 mM CaCl2 supplementation gave the best result in terms of the number of heart and torpedo embryos and plantlets. The ratio of the number of heart embryos, torpedo embryos and plantlets to total number of cells in the immobilized cell culture was higher than that in the suspension culture. Repeated batch culture with 5 mM CaCl2 provided long-term (more than 154 d) embryo and plantlet production without gel beads disruption. Productivity of plantlets in the immobilized cell culture with 5 mM CaCl2 was 2.2-fold as high as that in the suspension culture.

X-Ray Fluorescent and Mid-Infrared Spectroscopic Analysis of Tomato Leaves

This study aims to develop a simple, rapid, simultaneous and quantitative method for evaluating nutrimental elements of the plant vigor using X-ray fluorescent (XRF) and mid-infrared (MIR) spectroscopy. Tomato, which is one of the typical agricultural products cultivated by hydroponics, was used as the sample, and the leaves would be an index of the nutrimental conditions. We developed the quantitative methods to analyze the XRF and MIR spectroscopic information, and studied the effects of the cultivation conditions on the nutrient information between branches of a trunk. The experimental results presented a great potentiality to obtain the nutrient information on the chemical elements such as Ca and the organic components such as the nitrogen in the plural modes using the both spectroscopic methods. This study presents an important step in the development of XRF and MIR spectroscopic analysis of the plant vigor in the agricultural fields

Measurement of the concentrations of glucose and citric acid in the aqueous solution of a blood anticoagulant using near infrared spectroscopy

Near infrared (NIR) spectroscopy was employed for the simultaneous prediction of the concentrations of glucose and citric acid in the aqueous solution of a blood anticoagulant solution. The solution was placed in an NIR spectrophotometer and the absorbance at wavelengths between 400 and 2500 nm was measured at 2 nm intervals. Multiple linear regression (MLR) was used to obtain calibration equations relating the NIR spectral data and the glucose and citric acid concentrations of a calibration sample set obtained by enzymatic methods. The value of the multiple correlation coefficient (R) was 0.993 when using the wavelengths of 2274 and 1674 nm for glucose. The value of the simple correlation coefficient (r) for citric acid was 0.993 when using the wavelength 1690 nm. To validate the calibration equations obtained, glucose and citric acid concentrations in a prediction sample set were compared with the concentrations measured by the enzymatic methods. Excellent agreement between the results of the enzymatic method and those of NIR spectroscopy was observed for both constituents. The procedure of NIR spectroscopy was simple and the operation time required to predict the concentrations was a few minutes. These results indicate that NIR spectroscopy may be a useful method for monitoring the production process of the blood anticoagulant.

Rapid and simple determination of oil and urea concentrations and solids content to monitor biodegradation conditions of wastewater discharged from a biodiesel fuel production plant

To prepare and maintain the optimal biodegradation conditions of wastewater discharged from a biodiesel fuel (BDF) production process with alkali–catalysis transesterification, rapid and simple methods were investigated to measure oil (carbon source of microorganisms) and urea (nitrogen source) concentrations and solids content (indicator of growth inhibition of microorganism) of the wastewater. Two non-destructive methods were investigated. First, physical properties of the BDF wastewater such as electric conductivity (EC), specific gravity (Sp.Gr.) and Brix values were measured and the values were analysed with multiple liner regression (MLR). Only solids content could be predicted by measured physical properties. However, adjustment of the C/N ratio of the BDF wastewater was also necessary to prepare the optimal condition for the microorganism growth in the wastewater treatment process. Therefore, near infrared (NIR) spectroscopy was applied to simultaneous, non-destructive and rapid measurement of the constituents of the wastewater. The dominant absorptions caused by oil, urea and solids material in the wastewater were observed at 1718, 2154 and 2286 nm, respectively. These were used as the first wavelength to formulate calibration equations, a MLR analysis was carried out between the NIR spectral data and the values of conventional analyses, such as hexane extraction, enzymatic and oven-drying methods, in the calibration sample set (sample number, n = 50). To validate the calibration equations obtained, the predicted values of the oil, urea and solids in the validation sample set (n = 40), which was not used for formulating the calibration equations, were calculated using the calibration equations. Good agreement was observed between the values of the conventional analyses and the values predicted using NIR; the multiple correlation coefficients of determination (r2) for the validation equations for oil, solids and urea were 0.993, 0.877 and 0.960, respectively. Prediction of the solids content of the BDF wastewater using EC and Brix values is also possible with the handy EC and Brix meters which are inexpensive and easy to handle. However, these do not allow total management of the BDF production and BDF wastewater treatment processes. On the other hand, the NIR method is potentially suitable for automated process management of a BDF wastewater treatment system.