László Kocsányi

Optimizing Phytoremediation of Heavy Metal-Contaminated Soil by Exploiting Plants' Stress Adaptation

Soil phytoextraction is based on the ability of plants to extract contaminants from the soil. For less bioavailable metals, such as Pb, a chelator is added to the soil to mobilize the metal. The effect can be significant and in certain species, heavy metal accumulation can rapidly increase 10-fold. Accumulation of high levels of toxic metals may result in irreversible damage to the plant. Monitoring and controlling the phytotoxicity caused by EDTA-induced metal accumulation is crucial to optimize the remedial process, i.e. to achieve maximum uptake. We describe an EDTA-application procedure that minimizes phytotoxicity by increasing plant tolerance and allows phytoextraction of elevated levels of Pb and Cd. Brassica juncea is tested in soil with typical Pb and Cd concentrations of 500 mg kg−1 and 15 mg kg−1, respectively. Instead of a single dose treatment, the chelator is applied in multiple doses, that is, in several small increments, thus providing time for plants to initiate their adaptation mechanisms and raise their damage threshold. In situ monitoring of plant stress conditions by chlorophyll fluorescence recording allows for the identification of the saturating heavy metal accumulation process and of simultaneous plant deterioration.

Spectrometer for fast measurements of in vivo reflectance, absorptance, and fluorescence in the visible and near-infrared

A new type of VIRAF spectrometer (Visible Infrared Reflectance Absorptance Fluorescence), the VIRAF II, is presented. The VIRAF spectrometer has been developed and constructed for ground truthing of remote sensing of terrestrial vegetation and for other types of ecophysiological measurements. It is a multipurpose instrument with the capability to measure reflectance, absorptance, and fluorescence in the visible and near-infrared (400–910 nm). The position of the sample is unchanged throughout the measurements, which guarantees a good comparison between the different types of spectra. Since leaf samples can be left intact and attached to the plant, real in vivo measurements as well as long-term and repetitive studies using different additional techniques can be carried out. In contrast to conventional spectrometers, the sample is illuminated with strong white (or blue) light and only a narrow angle of the reflected, transmitted, or emitted light is detected. This mimics the outdoor situation in remote sensing. Furthermore, the strong light maintains the physiological activity of the leaf sample. The new VIRAF II described here has a diode array detector and a shutter which enables to measure within 20 ms. This opens the possibility for studies of kinetic changes in the range of seconds or minutes. Results are presented from a comparison of light green leaves of an Aurea mutant and dark green leaves of a wildtype tobacco plant (Nicotiana tabacum L.). The data are interpreted in terms of difference in chlorophyll concentration, leaf tissue structure, epidermis structure, and photosynthetic activity. Slight differences between the 20-ms reflectance spectra of a dark-adapted leaf taken directly after onset of illumination and after 60 s or 180 s of continuous illumination (decrease between 650 nm and 800 nm and increase between 400 nm and 600 nm) may be attributed to changes of the chlorophyll fluorescence and to chloroplast movements, respectively, reflecting physiological activity of the sample.

Two-wavelength, multipurpose, truly portable chlorophyll fluorometer and its application in field monitoring of phytoremediation

In this paper a compact, portable instrument is presented for the measurement of full chlorophyll fluorescence kinetics of plants at two different wavelengths. The instrument uses a 635 nm laser diode as a light source with variable gain driving that allows excitations at selectable actinic levels. The plant fluorescence is detected, at 690 nm and 735 nm, through a specially mixed three-branch optical fibre bundle. Large scale field monitoring of vegetation is made possible by the utilization of PC/104-form embedded electronics including a low power, IBM PC/386-compatible single board computer (SBC) with non-volatile flash memory. Application of a general purpose SBC and task oriented programming offers in situ data evaluation making process control possible. The capabilities of the instrument were demonstrated in monitoring soil phytoremediation processes.

Excitation kinetics during induction of chlorophyll a fluorescence

We present a chlorophyll fluorometer module system which adapts the intensity to the individual leaf sample by adjusting the quantum flux density of the excitation light so that the fluorescence signal is kept constant. This is achieved by means of a feedback power adjustment of the fluorescence exciting laser diode. Thus, the intensity of the excitation light is adapted to the actual need of a particular sample for quantum conversion without applying exaggeratedly high quantum flux density. We demonstrate the influence of the initial laser power chosen at the onset of irradiation and kept constant during fluorescence rise transient within the first second. Examples are shown for measuring upper and lower leaf sides, a single leaf with different pre-darkening periods, as well as yellow, light green and dark green leaves. The novel excitation kinetics during the induction of chlorophyll fluorescence can be used to study the yield and regulation of photosynthesis and its related non-photochemical processes for an individual leaf. It allows not only to sense the present state of pre-darkening or pre-irradiation but also the light environment the leaf has experienced during its growth and development. Thus, the individual physiological capacity and plasticity of each leaf sample can be sensed being of high importance for basic and applied ecophysiological research which makes this new methodology both innovative and informative.

Excitation kinetics of chlorophyll fluorescence during light-induced greening and establishment of photosynthetic activity of barley seedlings

Excitation kinetics based on feedback regulation of chlorophyll (Chl) fluorescence of leaves measured with the chlorophyll fluorometer, FluoroMeter Modul (FMM), are presented. These kinetics showed the variation of excitation light (laser power, LP) regulated by the feedback mechanism of the FMM, an intelligent Chl fluorometer with embedded computer, which maintains the fluorescence response constant during the 300-s transient between the dark- and lightadapted state of photosynthesis. The excitation kinetics exhibited a rise of LP with different time constants and fluctuations leading to a type of steady state. The variation of excitation kinetics were demonstrated using the example of primary leaves of etiolated barley seedlings (Hordeum vulgare L. cv. Barke) during 48 h of greening in the light with gradual accumulation of Chl and development of photosynthetic activity. The excitation kinetics showed a fast rise followed by a short plateau at ca. 30 s and finally a slow constant increase up to 300 s. Only in the case of 2 h of greening in the light, the curve reached a stable steady state after 75 s followed by a slight decline. The final LP value (at 300 s of illumination) increased up to 12 h of greening and decreased with longer greening times. The active feedback mechanism of the FMM adjusted the excitation light during the measurement to the actual photosynthetic capacity of the individual leaf sample. In this way, the illumination with excessive light was avoided. The novel excitation kinetics can be used to characterize health, stress, disease, and/or product quality of plant material.

Eliminating the Interference Pattern in Near-Infrared Spectra Used for Identification of Thin Plastic Foils

A Fourier type filtering method is proposed for the pretreatment of near-infrared (NIR) spectra of thin (<100 μm) transparent plastic foils before their identification by means of multivariate calibration methods. The interference of multiply reflected beams from the boundary surfaces of the foil causes a disturbing signal component in the spectrum and the identification becomes impossible. The purpose of the filtering is to eliminate the interference pattern from the spectrum. In the Fourier transformed NIR spectrum against the wavenumber there appears a discrete spectral component caused by the interference. This component can be recognized and cut off. After inverse Fourier transformation of such pretreated spectra, absorption peaks are free from interference modulation, so application of multivariate calibration methods is much more effective. With principal component analysis (PCA) on cluster plots, visual distinction between different plastics becomes possible. Correct class membership is provided by use of the Mahalanobis distance.

SNR analysis of the stripe illuminator based on linear emitter and cylindrical lens against solar irradiance

Achieving good signal-to-noise ratio (SNR) against solar illumination is a basic requirement for the light source of active vision sensors in outdoor applications. The article examines the high power stripe-lighting setup, constructed with a cylindrical lens and a rectangular emitting source placed at the focal line of the lens. This source type is capable of illuminating a very high total power because of the large surface size of the emitter; thus it is advantageous for outdoor applications. A detailed radiometric analysis of this setup is presented resulting in an integral for the calculation of the spatial irradiance distribution within the stripe. The achievable SNRs against solar illumination are calculated by applying the radiometric integral for different rectangular emitters built of LED arrays, laser diode arrays and incandescent sources. The article proposes stripe illuminators applying all the analyzed emitter types that are suitable for operation in direct sunshine.

Demonstration of plant fluorescence by imaging technique and Intelligent FluoroSensor

Photosynthesis is a process that converts carbon-dioxide into organic compounds, especially into sugars, using the energy of sunlight. The absorbed light energy is used mainly for photosynthesis initiated at the reaction centers of chlorophyll-protein complexes, but part of it is lost as heat and chlorophyll fluorescence. Therefore, the measurement of the latter can be used to estimate the photosynthetic activity. The basic method, when illuminating intact leaves with strong light after a dark adaptation of at least 20 minutes resulting in a transient change of fluorescence emission of the fluorophore chlorophyll-a called ‘Kautsky effect’, is demonstrated by an imaging setup. The experimental kit includes a high radiant blue LED and a CCD camera (or a human eye) equipped with a red transmittance filter to detect the changing fluorescence radiation. However, for the measurement of several fluorescence parameters, describing the plant physiological processes in detail, the variation of several excitation light sources and an adequate detection method are needed. Several fluorescence induction protocols (e.g. traditional Kautsky, pulse amplitude modulated and excitation kinetic), are realized in the Intelligent FluoroSensor instrument. Using it, students are able to measure different plant fluorescence induction curves, quantitatively determine characteristic parameters and qualitatively interpret the measured signals.

Novel, fourier filtering method that reuses interference-patterned spectra to extend the calibration set for thickness determination

Determining the thickness of plastic sheets on the basis of near-infrared spectra by building a multivariate calibration model requires a relatively large sample set. In the thickness region, where just a few noninterference-patterned samples are available, it is a waste of information if interference-patterned spectra are excluded. After eliminating the interference pattern from the spectra (filtering), the calibration set can be extended with these filtered spectra. Fourier transformation of an interference-patterned spectrum versus wavenumber leads to a Fourier spectrum as a function of the optical path length containing an easily recognizable interference peak. Unfortunately, this peak coincides with components of the spectral information of absorbance, on which multivariate calibration is based. Hence, replacing the interference peak is a cardinal step of the filtering process. Since the Fourier spectrum versus optical path length function is not known, it has been shown that interpolated data over the remaining Fourier components can be substituted for the missing part of the spectrum. In this paper, a novel method is proposed that uses a linear approximation between the Fourier spectra and the thickness values so that the regression coefficients are calculated on components of all but the interference-patterned Fourier spectra and the corresponding thicknesses, and then the deleted components in the filtered spectrum are replaced. This latter method yields more detailed Fourier spectra. Reducing the disturbing effect of scattering is also discussed. The effectiveness of the filtering was tested on low-density polyethylene sheets. The performance of different calibration models with or without filtering was compared by significance tests on standard error of prediction values. Application of the new Fourier type filtering technique led to significant improvements in the calibration performance.