Nitrogen detection in the vegetation of prototype constructed wetlands using chlorophyll fluorescence

Edison Roseroa, Lucero Plazasa, Efraín Solartea, Adrián Fernándezb, Enrique Peñab, Miguel Peñac

aGrupo de Óptica Cuántica, Universidad del Valle, A.A. 25360, Cali, Colombia; bGrupo de Fisiologia Vegetal, Departamento de Biología, Universidad del Valle, Cali, Colombia;

cFundación CINARA, Facultad de Ingeniería, Universidad del Valle, Cali, Colombia.

ABSTRACT

Constructed wetlands are a very efficient, clean and economical way to remove organic contaminants from waste water. In the whole water cleaning process, some other complex processes, such as physical sedimentation, filtration, chemical precipitation, and material absorption by vegetation, are involved. The Nitrogen absorption efficiency by heliconnia psitacorumm, was studied at laboratory scale in a small reactor simulating a subsurface flow constructed wetland. Chlorophyll increasing was measured by fluorescence, using blue LED, 460 [nm] as excitation light source. Besides, spectral differences were observed in the spectral signal and in its derivative, indicating changes in the plant physiological response.

Keywords: Fluorescence, Chlorophyll, Nitrogen, Plant Physiology.

1. INTRODUCTION The constructed wetlands are areas formed by a porous subsurface (usually gravel) that let wastewater flow through this substrate. Usually there are natural plants on the surface of wetlands. These systems have been designed to improve treatment of wastewater and take advantage of natural methods to cleanse water. According to the flow pattern, constructed wetlands are divided into two types: the surface flow (SF) and subsurface flow (SSF) systems. The characteristic of SSF wetlands is that water level remains below the substrate. A prototype constructed wetland consists of a properly designed pot that contains wastewater in motion, substrate, and plants [1]. Wetlands are an economical way to treat water contaminated by organic compounds, which are removed from wastewater using a variety of complex physicochemical (eg, mineralization, denitrification, plant uptake, settling particulate matter suspension and filtration) and biological processes. Therefore, to enhance removal of contaminants, it should require that each active process in the system is analyzed, taking into account, and the results are integrated into a complete model.

Nitrogenous compounds (ammonia, nitrite, and nitrate) are known to be the most contaminant substances in wastewater; for that reason, one of the main goals of wastewater treatment is nitrogen elimination. The principal mechanisms for nitrogen removal are both heterotrophic and autotrophic microbes [2]. Regarding to the function of plants in the wetland, it is quite necessary to obtain performance data about the contribution of plants in the overall nitrogen removal process, and evaluate the potential for nutrients uptake and storage by plants. The main goal this research work was to develop an optical method to determine nitrogen absorption by plants in a wetland, with the aim to follow the changes in nitrogen concentration accumulated by live plants without destroying the plant leaves. The efficacy of the technique was evaluated through its application at lab-scale on prototype constructed wetlands, planted with helicconia psitacorum.

2. THEORETICAL CONSIDERATIONS 2.1. Chlorophyll fluorescence The total of nitrogen absorbed by plants is used for protein, enzymes and chlorophyll synthesis. It makes possible to quantify the number of structures inside leaves that take nitrogen to its processes. As spectral absorption characteristics and chlorophyll emission lead to an accurate quantification of this molecule through spectroscopic techniques, it is feasible to establish in an indirect way, the content of nitrogen in leaves by observing the fluorescence of chlorophyll molecule. The photosynthetic system in a green leaf consists of chlorophyll molecules, which at normal environmental

Ultraviolet and Visible Ground- and Space-based Measurements, Trace Gases, Aerosols and Effects VI, edited by Jay R. Herman, Wei Gao, Proc. of SPIE Vol. 7462, 746208 · © 2009 SPIE

CCC code: 0277-786X/09/$18 · doi: 10.1117/12.826714

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temperatures reveal a spectrum emission. This emission is characterized by a narrow maximum in the red region, between 685-690 nm, and a wide maximum in the far-red region, about 730-740 nm approximately [3]. In addition, there are differences regarding spectrum intensity between the emission measured at the upper side and the measured at the lower side of the leaf. Leaves have a higher chlorophyll density on palisade parenchyma cells, mostly located in the upper side. Hence, fluorescence spectrum obtained from adaxial (upper epidermis) should have a greater intensity than abaxial (lower epidermis). Nevertheless, what is observed is that the intensity measured on abaxial side is bigger than on adaxial. That is because of the overlapping of the absorption and the emission spectra in the red region. So that the light emitted by a molecule is re-absorbed by other molecules surrounding it. The process depends on chlorophyll content differences. Through the time, the plants grow by absorbing nitrogen compounds present in wastewater. These plants reach better performance; they exhibit a great ability to increase its biomass and its number of chlorophyll molecules. It has been observed that, on advanced stages of development, the measured fluorescence intensity is not proportional to the number of molecules, since a high density of chlorophyll leads to increasing of re-absorption of radiation.

2.1. Determination of the effective fluorescence spectrum The effective fluorescence spectrum that one leaf emits, when it is excited by a spectral energy density of radiation, can be determined taking into account the transmission and the reflection properties of vegetal tissue. Reflectance and transmittance spectra of a leaf are mainly determined by superficial properties and its inner structures. The percentage of reflected light R is calculated experimentally as:

100)()()()(

)(0

×−−

=λλλλ

λDRDS

R R (1)

Where SR (λ) is the measured intensity of the reflected light by leaf, R0 (λ) is reference intensity taken with a diffuse reflectance material (99%), D (λ) is the background radiation or “dark current”. A similar rule applies to transmittance determination.

Diverse methods to calculate the effective fluorescence spectrum have been developed and evaluated, concluding that the best accurate model to correct the emitted fluorescence spectrum of a unique green leaf is the one proposed by G. Agati [4]. This model states that spectral energy density above surface of a leaf is reduced exponentially by the interaction with infinitesimal layers of vegetal tissue. Likewise, it establishes an isotropic distribution of fluorescence emission inside the leaf, which is exponentially diminished by re-absorption process in the tissue. The scattering of radiation is considered by assuming a given effective length for the optical path into the tissue. The properties of reflection and transmission are included to establish the fraction of radiation that has not been absorbed by leaf. This fraction has to be the same as the sum of both quantities R (λ) and T (λ).

According to this model, the effective fluorescence spectrum IFe is obtained from the measured fluorescence spectrum

IFm, by considering the experimentally obtained reflection and transmission, through the following equation:

( )( )⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢

⎣

⎡

++−−−

⎟⎟⎠

⎞⎜⎜⎝

⎛+

⎟⎠⎞

⎜⎝⎛

++⎟⎟

⎠

⎞⎜⎜⎝

⎛+

=TRTR

TR

TRLn

TRLn

TRLn

II mF

eF

00

00

00

00

11

1

11

(2)

Where R0 and T0 represent the amount of radiation reflected and transmitted by the leaf in the spectral region corresponding to excitation. R and T respectively regards with reflection and transmission of light into the spectral region in which the process of fluorescence emission is observed. It is possible to get the effective fluorescence spectrum by using the equation (2) that includes the measured fluorescence spectrum and the correction factor containing experimental reflectance and transmission. This calculated value allows the estimation of fluorescent chlorophyll molecular concentration inside the leaf. As absorbed nitrogen is used in the synthesis of chlorophylls, an increase of this kind of molecules evidences nitrogen absorption by the plant; so, as the effective fluorescence spectrum makes possible

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to assess a quantity proportional to the amount of molecules present on the leaf, it makes possible to determine nitrogen absorption by the plant, that means nitrogen removed from wastewater.

2.2. Fraction of ligth absorbed The absorption coefficient α(λ), corresponding to the fraction of light absorbed by vegetal tissue, can be determined using the measured reflectance and transmittance values. The method was developed by Allen and Richardson [5] given the following equations:

α λ log b (3)

a 1 R λ T λ ∆2R λ b 1 R λ T λ ∆2T λ

∆ 1 R λ T λ 1 R λ T λ 1 R λ T λ 1 R λ T λ The absorption coefficient depends on the effective absorption cross section per unit volume of the tissue. The effective absorption cross section can vary when the concentration of absorbing molecules change. Proteins and chlorophylls are the primary nitrogenous compound in leaves; therefore the variation of absorption coefficient shows changes in the concentrations of these compounds and in turn, changes in the nitrogen content.

3. EXPERIMENTAL METHODS 3.1. Prototype constructed wetland

Fig 1 Schematic representation of the prototype SSF constructed wetland is shown on left. On right shows a picture of the real treatment system. Tank feeding and six constructed wetland are shown in this picture.

Every wetland has an area of 491 cm2 and it is 40 cm high. The wastewater was fed continuously at a rate of 14.5 ml/minute, which corresponds to a hydraulic retention time of 1 day. In three of these wetlands, labeled as 2, 4, and 6, five plants with similar characteristics to each other were placed; on average there were 3 leaves per plant. Spectroscopic measures were performed on each leaf at all the plants in the wetland, during four months (March-June). Due to the large amount of results only the most important ones, concerning to the validation of the nitrogen detection method, will be shown.

3.1. Fluorescence spectroscopy technique Fluorescence emission spectra were taken in vivo, on leaves of Heliconia psíttacorum, to establish variations on the effective fluorescence spectrum of the leaves’ chlorophylls. As various non-radiative processes lead to decay from the excited state, reducing the fluorescence emission, some care must be taken to minimize them. To diminish the effects on

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the photosynthetic process and those due to energy dissipation as heat, leaves were adapted to darkness for one hour, before the corresponding measurements. So, the measurements were taken at night. For each leaf, six spectra from different zones of the leaf were acquired. The molecular excitation was generated by irradiate the leaf with a high luminescence diode (LED), emitting in the blue region with a maximum peak on 470 [nm] and a half width of 30 [nm]. The diode operation voltage (2.90 [V]) was controlled to keep it constant during the measurement period. The average LED emission power was 0.01 [mW]. The experimental set up, used to measure fluorescence, is shown on Figure 2.

2a)

2b)

Fig 2. Schematic representation of the experimental setup is shown on Figure 2a. Equipment used for obtaining fluorescence spectra and details of in situ measurement, are presented on Figure 2b. The SSF wetlands prototypes can be identified in the picture on the right side.

To collect both the transmitted excitation light and the emitted fluorescence light from the leaves, an optical system was designed and built. This system consists of a coupler for optical fibers, made of aluminum, that permits to influence on leaves with an excitation light (LED light) in a direction 45° from the leaf normal, and grasp the fluorescence emission in a 90 º angle in regard to direction of incidence. Besides, to collect the fluorescence emission, it is necessary a compound system consisting of a small focal convergent lens and color filter, to reduce blue radiation intensity spread in the direction of the collector lent. This lens is coupled to a 200 [μm] optical fiber, whose function is to send the fluorescence signals to a spectrophotometer system (USB4000® controlled by the Spectra Suite® software from Ocean Optics). In addition, a simple electronic circuit to switch the LED was built. This allows synchronizing the LED switching with the spectrometer working in the rapid acquisition mode, so that all of the spectra were taken during a time of 50 [ms]. The measurements were performed at the Greenhouse facility from Biology Department, Universidad del Valle.

3.2. Spectroscopy of Reflection and Transmission The reflection and transmission spectral responses were taken after the fluorescence measurements, allowing a one hour rest interval between the two measurements. During this time, the plants were kept in darkness. The light reflection measurements were done using a 200 [μm] diameter, reflectance/backscattering fiber optics probe, consisting of six illumination fibers around one read fiber bundle, with an additional fiber to monitor the illumination source and provided with a stainless steel ferrule probe. The light transmitted through the leaf was collected by means of an optical fiber of 200 [μm] diameter. As illumination source a halogen tungsten lamp was used, with a spectral range from 350 [nm] to 1000 [nm]. To hold the reflectance probe and the collecting fiber, together with the leaf, a mechanical system was designed and built. It consists of an aluminum cube block, with exact collinear holding holes for the probe and the fiber, and a movable plate to press the leaf in place. This system allows attaching the reflectance probe and the detector fiber directly on the leaf. Details of this setup are shown on Figure 3.

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Figure 3 The photograph shows the setup used for Reflectance and Transmittance spectra acquisition. On the bottom side, the detail presents the adapter designed to attach optical fibers on the leaf.

4. RESULTS AND DISSCUSION In Figure 4 typical reflection spectra from a leaf, collecting during one month, are presented. These data corresponds to the leaf number 6 of the plant 28. As expected the reflection acquired in the blue region is near to cero, due to the strong absorption of chlorophyll a and b molecules, whereas there is a reflectance maximum in the green region, latter changes as the tissue chlorophyll content increases, but in the long term it declines as the plant increases its density, as it is evidenced by the dark green color of mature leaves.

Fig 4. Reflection spectra obtained from the leaf number 6 of plant 28. The data were collected during a month. In the upper square the first derivative in the range between 680 and 750 nm is presented. The maxima of this derivative correspond to a given chlorophyll content.

In the red edge (680-750 nm), an absorption zone can be identified, which is generated by the chlorophyll content. In the nearest infrared, most of the incident light is scattered and reflected by the spongy layer, which holds lower chlorophyll content. As the absorption region of chlorophyll, is wider for leaves with more chlorophyll content, the first derivative of reflection spectrum can be used to make evident these changes. Due to the form of the reflectance spectrum a maximum is expected, that corresponds to a given biological situation in the leaf. Changes in the chlorophyll content are

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(%)

Wavelength [nm]

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Plant 28, Leaf 6

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observed as broadening and shifts of these maxima, this makes possible to determine the increasing or diminishing of chlorophyll content, and therefore to identify an increase or a decrease in the amount of absorbed nitrogen.

Figure 5 presents a set of typical leaf absorbance spectra. The measurements were carried out in different days. In Figure 6, the corresponding absorption coefficients are displayed. The data show that in June the highest percentage absorption was achieved, indicating that, in that month, the plant increased the concentration of absorbing pigments, and therefore its rate of nitrogen absorption.

Fig 5. Absorption spectra set obtained from the leaf number 1 of plant 31. In this figure the changes in the percentage of absorbed light during the experimental period from April to June can be observed.

Fig 6. Absorption coefficients calculated in the chlorophyll emission spectral region. Data corresponding to leaf number 1 of plant 31.

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Plant 31, Leaf 1

640 660 680 700 720 740 760 780 800 8200.0

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α (λ

)

Wavelength [nm]

α (λ) 13 April α (λ) 27 April α (λ) 12 May α (λ) 24 May α (λ) 31 May α (λ) 7 June

Plant 31, Leaf 1

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Figure 7 illustrate typical results of measured fluorescence spectra, and shows the corresponding effective spectra. Both types of spectra are related by equation (2). The area of the effective fluorescence spectrum, acquired from each measured one, is proportional to the number of chlorophyll a and b molecules. Therefore, it gives the nitrogen concentration at the measurement time. Hence, percentage variations of this quantity can be interpreted as variations of concentration nitrogen. The calculated effective spectral areas from data in Figure 7, give the following results: 410821[nm.s-1] ± 6.5%, in April 9 and 507364 [nm.s-1] ± 4.2%, in April 13. These areas correspond to nitrogen concentration in the live leaves, and it is possible to establish a percent variation of the content of nitrogen, in this case of (23 ± 2) %, in April 9 to 13, for plant number 6 on its leaf numbered 1.

Fig 7. Typical fluorescence results from wetland plants chlorophyll. IF

m is the measured fluorescence spectrum, and IF

e the calculated effective fluorescence.

Fig 8. Effective fluorescence spectra obtained from the leaf number 1 of plant 31. It shows a similar variation to the absorbance (fig 5) and fraction of light absorbed (fig 6). In this case, the area under the curve is proportional to the chlorophyll concentration.

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640 660 680 700 720 740 760 7800

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nsity

[

s-1]

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IeF 13 April

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Plant 31, Leaf 1

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Proc. of SPIE Vol. 7462 746208-8

[3] Buschmann C, "Variability and application of the chlorophyll fluorescence emitted ratio red/far-red in leaves" Photosynth Res. 92, 261-271, (2007).

[4] Agati G., Fusi, F., Mazzinghi P., "A simple Approach to the Evaluation of the Reabsorption of Chlorophyll Fluorescence Spectra in Intact Leaves" Photochemical and Photobiological Sciences B. 17,163-171, (1993).

[5] Allen, A., Richardson, A., "Interaction of Light With a Plant Canopy" Journal of the Optical society of America. 58, 8, (1968).

[6] Fernández A., "Evaluación de la Tasa de Asimilación y Remoción de Nitrógeno por heliconnia psitacorumm en Humedales Artificiales Subsuperficiales Para el Tratamiento de Agua Residual" Undergraduate thesis, Universidad del Valle, (2008).Davis , L [A Handbook of Constructed Wetlands], EPA publications, vol 1. http://www.epa.gov/OWOW/wetlands/pdf/hand.pdf

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