Claudia Hernandez-Aguilar

Arturo Dominguez-Pacheco

Mariana Palma Tenango

Carmen Valderrama-Bravo

Marcos Soto Hernández

Alfredo Cruz-Orea

Jose Ordonez-Miranda

Characterization of Bean Seeds, Germination, and Phenolic Compounds of Seedlings by UV-C Radiation

Keywords: 00+52+55+91086594 bean seeds, secondary metabolites, UV-C radiation, photoacoustic spectroscopy

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

One of the challenges of the humanity is to improve food production in quantity and quality for a current population over 7 billion and projected for 2050 of 9 billion (FAO 2009a;2009b;[START_REF] Baulcombe | Reaping the benefits: science and the sustainable intensification of global agriculture[END_REF][START_REF] Godfray | Food security: the challenge of feeding 9 billion people[END_REF]Araujo et al. 2016), reducing the environmental impact [START_REF] Edmondson | Urban cultivation in allotments maintains soil qualities adversely affected by conventional agriculture[END_REF][START_REF] Hernández-Aguilar | Bioestimulación láser en semillas y plantas[END_REF]. Therefore, it is necessary to reduce the use of chemical products in the production process of food (Altieri 2014). For this purpose, in this century has been intensified research into alternative physical methods that are called "emerging technologies" in response to the needs of the modern world [START_REF] Rifna | Emerging technology applications for improving seed germination[END_REF]. Which could help stop the destructive processes of the soil, the environment and protect the production of food and consequently the health and quality of life of the population [START_REF] Vasilevski | Perspectives of the application of biophysical methods in sustainable agriculture[END_REF][START_REF] Hernández-Aguilar | Bioestimulación láser en semillas y plantas[END_REF].

In this way, there are various physical methods, used in different phenological stages, by them could interest the pre-sowing stage [START_REF] Hernández-Aguilar | Laser irradiation effects on maize seed field performance[END_REF]2007;[START_REF] Fao | High Level Expert Forum -How to Feed the World in 2050, Global agriculture towards[END_REF][START_REF] Aladjadjiyan | Physical methods in agro-food chain[END_REF]. The pre-sowing methods allow a conditioning of the seed; producing beneficial effects in early stages of growth (Thomas and Pothur 2016;

Aladjadjiyan 2012), as in germination, which conditions the vigor of the plants and could be the basis for intensification of crop production [START_REF] Mariz-Ponte | Moderate UV-A supplementation benefits tomato seed and seedling invigoration: a contribution to the use of UV in seed technology[END_REF]. Physical methods for seed invigoration have advantages over conventional treatments based on chemical substances, e.g. reduce the use of fertilizers, fungicides and pesticides, etc., thus could decreasing pollution [START_REF] Rifna | Emerging technology applications for improving seed germination[END_REF][START_REF] Aladjadjiyan | Physical factors for plant growth stimulation improve food quality. Food productionapproaches, challenges and tasks[END_REF]. Within these emerging physical methods, are those based on technology laser, led, magnetic and electric fields, nonthermal plasma, ultrasound, ionizing radiation (gamma rays and x-rays), microwave, ozone, magnetically treated water, electrolyzed oxidizing water, and Ultraviolet (UV) radiation, among others (Vasilevsky 2003;[START_REF] Marinković | Use of biophysical methods to improve yields and quality of agricultural products[END_REF]Hernandez et al. 2010;2016;[START_REF] Aladjadjiyan | Physical factors for plant growth stimulation improve food quality. Food productionapproaches, challenges and tasks[END_REF][START_REF] Occhipinti | Magnetoreception: an unavoidable step for plant evolution?[END_REF][START_REF] Maffei | Magnetic field effects on plant growth, development, and evolution[END_REF]Araujo et al. 2016;Thomas and Pothur 2016;[START_REF] Rifna | Emerging technology applications for improving seed germination[END_REF].

Ultraviolet radiation to low levels for treatment of seeds is an ecologically safe method, then, an environmentally-friendly alternative to improve plant productivity, yield quality and as well as to induce tolerance of plants to various biotic and abiotic stresses [START_REF] Forges | Impact of UV-C radiation on the sensitivity of three strawberry plant cultivars (Fragaria x ananassa) against Botrytis cinerea[END_REF]Thomas and Puthur, 2016;[START_REF] Ehsanpour | Effect of UV-C radiation on drought tolerance of alfalfa (Medicago sativa) callus[END_REF]. UV light is an electromagnetic radiation of wavelengths shorter than the visible region, but longer than X-rays [START_REF] Shetta | Impact of ultraviolet-C radiation on seed germination and chlorophyll concentration of some woody trees grown in Saudi Arabia[END_REF]. UV radiation has been classified as: UV-A (320-400 nm), called black light, UV-B (280-320 nm), called medium wave and UV-C (200-280 nm), called short wave or germicidal [START_REF] Katerova | Endogenous polyamines lessen membrane damages in pea plants provoked by enhanced ultraviolet-C radiation[END_REF][START_REF] Shetta | Impact of ultraviolet-C radiation on seed germination and chlorophyll concentration of some woody trees grown in Saudi Arabia[END_REF]. The damaging effect of UV radiation increases towards shorter wavelengths: UV-A is less effective than UV-B, while UV-C photons are highly energetic, and quickly create high levels of damage [START_REF] Katerova | Endogenous polyamines lessen membrane damages in pea plants provoked by enhanced ultraviolet-C radiation[END_REF][START_REF] Barta | Differences in the ROS-generating efficacy of various ultraviolet wavelengths in detached spinach leaves[END_REF][START_REF] Stapleton | Ultraviolet radiation and plants: burning questions[END_REF]).

The UV light continuous or pulsed has been studied to have a broad application in the food and agriculture industry [START_REF] Elmnasser | Pulsed-light system as a novel food decontamination technology: a review[END_REF]Gómez-López et al. 2007;[START_REF] Alexieva | The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat[END_REF][START_REF] Mukhopadhyay | Application of emerging technologies to control Salmonella in foods: A review[END_REF]. In particular, in the agriculture, the UV-C radiation non-ionizing continuous have shown to stimulate positive impact on seed health, germination (G), and seedling vigor (SV), also has been applied in different fruit, vegetable and legume crops, among others [START_REF] Sukthavornthum | UV-C irradiation induced alterations in shoot proliferation and in vitro flowering in plantlets developed from encapsulated and non-encapsulated microshoots of Persian violet[END_REF][START_REF] Dawar | Fungicidal effect of ultraviolet-C radiations in prevention of mycoflora of castor bean seeds[END_REF]. In legumes, most applications have focused on the health-related effects of seed, seedlings, seedling roots and / or crops (fungal control and sterilizing effects) [START_REF] Dawar | Fungicidal effect of ultraviolet-C radiations in prevention of mycoflora of castor bean seeds[END_REF][START_REF] Siddiqui | Role of ultra violet (UV-C) radiation in the control of root infecting fungi on groundnut and mung bean[END_REF], as well as the effects at the level of seedlings and plants radiated with UV-C (Da Silva 2019; Kara 2013) and to a lesser degree, physiological effects (G and SV) and / or of secondary metabolites caused by UV-C light when is used as presowing treatment to the seed and as well the possible degradation produced in it. Among the legumes, most consumed in various countries of the world, e.g. Africa, India, Latin America and Mexico is the bean (Chávez-Mendoza and Sánchez 2017).

In this way, the present investigation experimental aims are: a) to investigate the degradation of bean seed treated with UV-C light using Photoacoustic spectroscopy (PAS), as well as to evaluate their morphological changes by means of scanning electron microscopy, b) evaluate the physiological quality of the seed (through the germination, seedling length and dry weight) and the phenolic compounds (phenolic acids and flavonoids) of the seedlings grown from seed treated with UV-C radiation prior to sowing.

Globally 800 million people are malnourished [START_REF] Broughton | Beans (Phaseolus spp.)model food legumes[END_REF], being one problems in Mexico and other countries in the world. Beans are considered important because they are a source of protein, fiber, iron, complex carbohydrates, minerals and vitamins [START_REF] Grela | Chemical composition of leguminous seeds: part I-content of basic nutrients, amino acids, phytochemical compounds, and antioxidant activity[END_REF]). In addition, characteristics of Nutraceuticals compounds have been identified as polyphenols, flavonoids and anthocyanins [START_REF] Lin | The polyphenolic profiles of common bean (Phaseolus vulgaris L.)[END_REF]). In such a way that it is a healthy option for the consumption of the human population. For this reason, it is one of the crops that requires improving its production.

MATERIALS AND METHODS

Biological material

In the present research project, bean seeds "Oti" (Phaseolus vulgaris L.) of the cycle productive vegetation period 2017 were used. These have been provided by the Mexican Genetic Quality control assurance institute-Seed Program of the "Instituto de Recursos Genéticos y Productividad del Colegio de Postgraduados"-(IREGEP). The seed lot was standardized and 100-seed weight averages were 31.84 g and length, width and thickness (ls), were about 11.508 ± 0.56, 7.6 ± 0.39 and 5.24 ± 0.5 mm, respectively. The measurements were realized by digital micrometer (Mitutoyo).

.

Treatment of UV-C radiation

After homogenizing the bean seed, it was treated pre-sowing, with UV-C radiator system (UV-C/RS-Homemade). The system consisted of an arrangement of 4 lamps (UV-C, 254 nm) accommodated on the top and bottom of a cylindrical stainless steel base (Figure 1). The lamps when turned on emits light towards a rotating wire-mesh container. Bean seeds were positioned in this container. Several radiation times were applied to the seeds: 0, 2, 5, 10 and 15 min, programmed through a timer. The light intensity measured by the UV-C/254 measuring equipment was of 700 w/cm 2 .

Figure 1 UV-C radiator system

PAS EXPERIMENTAL SETUP

The experimental setup of PAS consists of a Xenon lamp used as the light excitation source, a monochromator applied to obtain a monochromatic light beam, and a mechanical chopper employed to modulate the light beam at the fixed frequency f = 17 Hz, as shown in Fig. 2. The modulated beam was focused onto an optical fiber to guide it to the Photoacoustic (PA) cell, whose signal was then detected by an electret microphone (Photoacoustic sensor) connected to the cell through a fine channel (1 mm diameter). The PA signal was amplified by means of a Lock-in amplifier (EG&G, Mod. 5210) before being recorded on a personal computer, as a function of beam wavelength. Experiments were performed for wavelengths between 275 to 700 nm and by placing the samples into the PA cell with a diameter of 6 mm. The samples of bean seeds of each treatment UV-C were prepared on a circular form and diameter of 5 mm, such that they do not obstruct the microphone sensor and can be measurements when located inside the Photoacoustic cell.

Calculation of absorption coefficient and optical penetration length.

The determination of the absorption coefficient () and optical penetration length (l) of the bean seed were determined from the equation of Poulet et al. (1980), as has been indicated by Hernández et al. (2011a;2011b) for the characterization of agricultural plant seed. It is possible to observe in equation (1),  is a function of the PA signal of amplitude (q) obtained by the PAS

2 2 1 2 q q q q s - - + =   , (1)
Which it was used after verification of the condition thermal thickness of the seed: ls/µs >> 1, where s = f   is the thermal diffusion length for the frequency of 17 Hz; finally, α and ls the sample thermal diffusivity and thickness, respectively.

The q = PA signal of normalized amplitude,  = thermal diffusivity. In this case, the value corresponding to simple starch was used = 4.44x10 -3 cm 2 /s (Sanchez et al. 2014). Finally, the optical penetration length l was calculated from the inverse of , being l = 1/.

Scanning electron microscopy

The morphological changes of the beans seeds, control and treated with the exposition times of UV-C radiation (2, 5, 10 and 15 min) were analyzed with a scanning electron microscope (JEOL JSM-6010LA, Tokyo, Japan) under high vacuum. The whole bean seeds were cut in half, placed in an aluminum sample holder, and fixing them with a carbon tape. The samples were covered with gold nanoparticles. The micrographs were obtained from the surface of the whole bean seeds at 5000 x. In the bean cut in half, the test and starches were analyzed at 1000x. The conditions of analysis in the equipment were 15 kV of voltage of acceleration of electrons.

Germination test

Germination test was established under a randomized complete block design, with five repetitions and 20 seeds as an experimental unit. Seeds after irradiation were seeded on absorbent paper (double 24x23.2cm) moistened; with 40 ml of distilled water.

The germination stage was considered finished 12 days after the experiment was established. The temperature and humidity conditions corresponded to an average temperature of 24.9 °C and 26% humidity. At the end of the germination, fresh weight of seedlings was obtained, and subsequently their dry weight. Roots and foliage were separated to obtain biomass dry weight after drying in an oven (Felisa® Model 293) at 45 °C for 48 h.

After drying the bean seedling organs were milled and sieved (U.S.A. Standard testing sieve NO°50 300 micrometer 0.117 inches). Then were perform the analysis by HPLC.

Analysis by HPLC

Extracts were obtained with 50 mg of dry and pulverized material in 1 mL of methanol of HPLC grade (Sigma-Aldrich number 36860) at 80%, incubated for 20 min in an ultrasound bath (BRANSON at Smithkline company 50/60 Hz, model B-220, USA) [START_REF] Meneses-Reyes | Optimización del proceso de extracción de flavonoides de flor de manzanilla (Matricaria recutita L.)[END_REF]. The crude extracts were centrifuged at 731 g (Centrifuge 5804 eppendorf, model 5804) for 10 min [START_REF] Irakli | Optimization of ultrasound-assisted extraction of phenolic compounds: Oleuropein, phenolic acids, phenolic alcohols and flavonoids from olive leaves and evaluation of its antioxidant activities[END_REF]. Supernatants were filtered with 25 mm diameter acrodiscs with nylon membrane and 0.45 mm pore size (Titan). These extracts were injected immediately for the analysis by HPLC of phenolic acids and flavonoids. For the analysis of free flavonoids additionally a hydrolysis was made exposing the extracts at 70 °C for one hour. Each extract was realized in duplicate.

Phenolic acids

The samples were analyzed in a Hewlett Packard® chromatograph mod. 1100 provided with diode array detector and an Agilent Technologies automatic injector model 1200. The column was a Nautilus 125 x 4.0 mm d. i., 5 μm. The mobile phase was distilled water adjusted to pH 2.5 with trifuoracetic acid and acetonitrile. The analysis was by gradient: T1 0.10 min (85% A) (15% B); T2 20 min (65% A) (35% B) and T3 23 min (65% A), (35% B), λ = 280 nm, column temperature 30 ° C and flow of 1 mL min -1 (Bilia et al. 2001). Calibration curves were performed for the galic acid, chlorogenic acid, syringic acid, vanillic acid, p-hydroxybenzoic acid, caffeic acid, ferulic acid and p-coumaric acid standards (Sigma-Aldrich).

Flavonoids

Flavonoid glycosides

A Nucleosil 100-5 C-18 Nautilus 125 x 4.0 mm d. i., 5 µm column was used. The mobile phase was distilled water adjusted to pH 3 with H3PO4 (A) and acetonitrile (B). The analysis was by gradient: T1 0.10 min (88% A) (12% B); T2 10 min (82% A) (18% B), T3 15 min (82% A), (18% B), T4 30 min (55% A), (45% B) and T5 32 min (0% A), (10% B), λ= 254, 316, 365 nm, column temperature 30 °C and flow of 1.3 mL min -1 ; λ= 350 nm and the column temperature was 30 °C (Bilia et al. 2001). Calibration curve was performed for apigenin-7-0-glucoside, phlorizin, rutin and naringin (Sigma-Aldrich).

Free flavonoids

A Hypersil ODS HP column of 125 mm length and 4 mm internal diameter, 5 μm particle size was used. The mobile phase was distilled water adjusted to pH 2.5 with trifluoroacetic acid (A) and acetonitrile (B). The analysis was by gradient: T1 0.10 min (85% A) (15% B); T2 20 min (65% A) (35% B) and T3 25 min (65% A), (35% B), λ= 254, 316 and 365 nm, Column temperature 30 ° C and flow of 1 mL min -1 . Calibration curves were performed for standards: rutin, phorizine, quercetin, naringin, naringenin, floretin, galangin (Sigma-Aldrich).

The interpolations of all the extracts were calculated with ChemStation software © Agilent Technologies, Inc.

2004.

RESULTS AND DISCUSSION

By the application of photoacoustic spectroscopy and scanning electron microscopy for evaluate the effects of UV-C radiation on bean seeds was found than there were differences between the control and treated samples.

According to PAS, there is a degradation of the seed due to the radiation, there were differences between the photoacoustic spectra obtained for the control seeds (T0= 0 min) and for each one of the radiation treatments applied (T1 = 2, T2 = 5, T3 = 10 and T4 = 15 min) prior to sowing.

Figure 3a shows the optical absorption spectra where it is noted that the level of the photoacoustic signal decreases with the increase of the radiation time of the bean seed (Oti). Note that the control seeds presented the highest photoacoustic signal intensity level when compared with the spectra obtained from the seeds treated with UV-C. the ultraviolet region (280-400 nm); note that from 445, there were no significant statistical differences.

It is observed that the absorption region of 280-430 nm, significant statistical differences were found in the wavelengths of 280, 295, 310, 325, 340, 355, 370, 385, 400, 415 and 430 nm. Being the maximum decrease in the level of photoacoustic signal at the wavelengths of 295 and 340 nm, there being a percentage of decrease of 58.33 %.

In the range of 280 -430, the percentage of decrease in the value of maximum intensity of photoacoustic signal (control seeds) and the lowest value obtained in seeds T4 (15 min) varied between 52 and 58%. The higher decrease in the photoacoustic signal was found in the UV region range. For wavelength values above 430 nm, a variation between the maximum and minimum photoacoustic signal value between 40 and 50% was obtained.

In this way, significant changes in the photoacoustic absorption spectra are related, due to the changes in the bean seed produced by the different radiation times applied in the bean seed.

Other authors evaluating milk powder by photoacoustic spectroscopy found changes in optical absorption spectra due to different times of UV-C radiation. They related the optical absorption spectra with changes in their oxidation levels [START_REF] Dóka | Assessing the extent of degradation in the UV radiation and heat-catalyzed oxidized whole milk powder: The UV photoacoustic and diffuse reflectance spectroscopies versus the peroxide value[END_REF]. In this research, is demonstrated that the content of secondary metabolites such as phenolic acids and flavonoids are modified in seedlings from seed radiated by UV-C light.

Then, there is a decrease in the level of intensity of the photoacoustic signal. Since there is a relationship between the level of the photoacoustic signal and the presence of secondary metabolites in the bean seeds, which in bean seeds has been reported by other authors [START_REF] Camacho | El consumo de frijol común (Phaseolus vulgaris L.) y su efecto sobre el cáncer de colon en ratas Sprague-Dawley[END_REF]. If the photoacoustic signal level is increased, it is because there are more chromophores associated at those wavelengths. The chromophores would be the secondary metabolites present in the bean seed, which are affected by UV-C radiation.

Figure 3b represents the spectra of the optical absorption coefficient and the optical penetration length. It is observed that the highest values of  correspond to the spectra with the highest level of PA signal; it was the spectrum corresponding to the control seeds (without radiation). The bean seeds were thermally thick, which means that ls/µs = ls /(α/f) ½ = 577 >> 1. By using the eq. 1, were calculated  and l . Figure 3b shows the optical absorption coefficient () and optical penetration length (l ), as a function of the wavelength. The sample average thickness ls = 5.24 mm ± 0.5, according to the measurements of 100 seeds made by digital micrometer.

It is possible to observe that bean seeds are optically opaque in the 270 to 750 nm range, which means that l < ls for all wavelengths and treatments applied to the seeds in the different radiation times UV-C (0, 2, 5,10, and 15 min). Fulfilling this condition since the values of the optical penetration length were lower than 1.2 mm. Also, it is observed that the optical penetration length increases as the radiation time of the seed increases for the entire range of wavelength evaluated (270 -750 nm).

The l values decrease towards the ultraviolet region; the control seeds had the lowest optical penetration values.

It was found that to the higher degradation of the bean seed, the penetration length increases in the UV-A region (320-400 nm) and with the tendency to decrease toward the UV-B (280-320 nm) and UV-C (200-280) nm. The portion of the UV-C spectrum that is observed in Figure 3b, is the corresponding to 270-280 nm.

The value of the optical penetration length of the control seeds and those radiated at the different times (T1, T2, T3 and T4) varied from 0.22-0.65 mm, at wavelength 270 nm, corresponding to the UV-C range. According to the trend of the curve, these values tend to decrease towards the wavelength value of 254 nm (maximum peak of UV-C lamps). The value in the control seeds is lower and the higher penetration value corresponds to the case of the seeds treated with UV-C for 15 min, which is the maximum time of exposure of the seed to radiation in this study; also, showed the higher degradation to this time of radiation (T4). It should be mentioned that the thermal diffusion length (s = 0.009 mm) is smaller than the optical penetration length.

In this investigation it was found that the degradation of the bean seed did not cause a decrease in the physiological quality of the seed; expressed through germination, length and dry weight of seedlings. Variables which, when compared with control, did not find significant statistical differences (

05 . 0  p ) (Table 1-Section b).
Although there is a tendency to increase and then decrease in the germination of seeds irradiated by 10 and 15 min, respectively. The results show that PAS is sensitive to detect changes in bean seed, observed as a color degradation in the seed itself. These changes were verified in this investigation, through the scanning electron microscopy.

Analysis of micrographs

Figure 4 shows micrographs of control bean seed (T0 = 0 min) and exposed with UV-C light at different radiation times (T1 = 2, T2 = 5, T3 = 10 and T4 = 15 min), taken at 5000x from the surface of beans (first row) and 1000x in half of the bean seed (second and third row). In the image of the surface of the bean seed (first row) it is observed that the treatment with UV-C at different times affected significantly, because there is evidence of surface rupture notoriously observed in T3 = 10 and T4 = 15 min. In the second row it is observed that for the control sample (T0), the cell wall (sclereides), mainly cellulose, hemicellulose and lignin are found Torres (1985) mentions that the cell wall of the cotyledon is mainly composed of cellulose, hemicellulose and pectic substances, which bind to most cells and provide the physical resistance of the tissue. Figure 4 could assume that the network that holds the starches radiated at the highest dose contains mostly pectic substances because it shows a gel texture, more flexible than the cell wall of the "testa". [START_REF] Singh | Food Microstructure and starch digestión[END_REF] found that the stability of cell walls components and the rearrangement of starch granules in cells can affect the free access of amylolitic enzymes during digestion (when beans are used for consumption), since the starch granules enclosed within the intact cells are compacted. [START_REF] Hussain | Gamma irradiation induced modification of bean polysaccharides: Impact on physicochemical, morphological and antioxidant properties[END_REF], reported that by applying gamma radiation to bean starch the proportions of rapidly digestible starch were increased.

Likewise, Hyun-Jung et al. ( 2014) and [START_REF] Gani | Modification of bean starch by -irradiation: Effect on functional and morphological properties[END_REF] showed that by applying gamma radiation the degradation of amylose and amylopectin is induced, which alters the morphological, crystalline and physicochemical properties of starch. Starches in their native form are sometimes not functional for some processes. However, when applying UV-C radiation, the bean starches and the cell wall of the cotyledon undergo changes, as was demonstrated in the present investigation. These changes, as mentioned by other authors, could modify their functional properties, which would benefit the food industry as an additive. The advantage of applying UV-C could be in which does not induce a significant increase in temperature, minimum preparation of the sample, without dependence on some type of catalysis [START_REF] Farkas | Irradiation as method for decontaminating food: a review[END_REF], as well as a low cost.

For the condition of bean as a seed and not as grain of consumption, UV-C radiation has been reported with stimulating effects on germination, both stimulant and inhibitory effects [START_REF] Urban | Understanding the physiological effects of UV-C light and exploiting its agronomic potential before and after harvest[END_REF]. As the case described by [START_REF] Lazim | The effect of magnetic field and ultraviolet-C radiation on germination and growth seedling of sorghum (Sorghum bicolor L. Moench)[END_REF] who treated sorghum seed (Sorghum bicolor L.) with UV-C for different times (0, 30 and 60 min) did not find significant effects on germination percentage and the length of the seedling roots. While showed a significantly negative impact on speed germination, seedling length and the number of leaves. Similar results in the length of the seedlings germinated were found in black beans by [START_REF] Guajardo-Flores | Effect of Germination and UV-C Radiation on the Accumulation of Flavonoids and Saponins in Black Bean Seed Coats[END_REF], who did not find significant statistical differences in the length of the seedlings; however, changes were found in the speed of germination.

In the present investigation, the length of the seedlings varied in a range of 9.48 to11.06 cm after 11 days of germination; under the conditions in which the experiment was elaborated and it did not have statistical differences. In this way the physiological quality is not modified, within the radiation time interval used in this investigation. It is possible to note (Table 1b) that the smallest and the maximum length of the seedlings were from control seed and radiated for 15 min.

The emergent physical methods have been reported as pre-sowing treatments harmless for the environment; but these treatments must be applied with suitable parameters in order to improve the seed vigour and ensure better plant development at later stages [START_REF] Hernández-Aguilar | Alternating magnetic field irradiation effects on three genotype maize seed field performance[END_REF]. In this way is necessary to explore the optimal radiation parameters: exposure time, the radiation intensity, the power of the light source, the radiation regimes, etc. Likewise, the conditions and location of the seeds: the separation distance of them respect to the light, the placement (on moving or fixed), the container on which it is placed, etc. It is important use the appropriate treatment and conditions to produce the expected beneficial result.

In the present investigation, there were no significant differences between the treatments applied, but there was a tendency to reduce the germination at 15 min. Other authors with a longer exposure time (up to 180 min) found a stimulating effect on wheat seeds, improving germination [START_REF] Rupiasih | Effect of UV-C radiation and hypergravity on germination, growth and content of chlorophyll of wheat seedlings[END_REF]. Other studies also indicated a tendency to decrease and increase the percentage of germination, according to the times of exposure to UV-C radiation. [START_REF] Neelamegam | UV-C irradiation effect on seed germination, seedling growth and productivity of groundnut (Arachis hypogaea L.)[END_REF], found a decrease in germination and then an increase at 5 and 60 min of exposure in groundnut seed (Arachis hypogaea L.) by UV-C, respectively at 10 days of germination.

Depending on the genotype and characteristics of the seed, the effect will be produced. In the present investigation, it is observed that germination tends to decrease (T2) and then to present a slight increase (T3)

and then decrease again (T4). In experiments carried out with sunflower seeds by [START_REF] Torres | Sunflower seed deterioration from exposure to UV-C radiation[END_REF] it was reported that the UV-C radiation between the time of 5-60 min reduced the percentage of normal seedlings.

Other authors have found that depending on the genotype of red bean seeds are the effects obtained on germination [START_REF] Peykarestan | UV irradiation effects on seed germination and growth, protein content, peroxidase and protease activity in redbean[END_REF].

According to [START_REF] Solecka | Role of phenylpropanoid compounds in plant responses to different stress factors[END_REF], exposure of plant tissues to UVC increases the concentration of reactive oxygen species (ROS), which induces seed germination [START_REF] Guajardo-Flores | Effect of Germination and UV-C Radiation on the Accumulation of Flavonoids and Saponins in Black Bean Seed Coats[END_REF]. UV-C irradiation treatment on agricultural seeds has generally been reported promoted the seed germination. In experiments carried out on Mung bean (Vigna radiata) seeds, they reported an increase in germination at times of exposure to UV-C radiation of 2, 4 and 6 h [START_REF] Hamid | Influence of seed pre-treatment by UV-A and UV-C radiation on germination and growth of Mung beans[END_REF]. Although there were no significant differences in germination in this investigation, a tendency to be modified by UV-C was found depending on the time of exposure to radiation to this type of bean seed used.

In the case of the dry weight of the seedlings from the radiated seed and control (without radiation) (Table 1b), it was found that the highest weight was obtained in the control seedlings and those radiated at 2 min. While the lowest weight was for seedlings from seed radiated for 5 min (T2).

In a separate way the weight of root and foliage; it was found that at two minutes the foliage weight tends to increase, with a tendency to decrease at 5 min and then increase again at 10 min. In the case of the weight of the roots, a slight tendency to decrease was obtained as the pre-sowing UV-C radiation time increases. Also, other authors have indicated a modification of the weight of the components of seedlings (root and foliage). [START_REF] Siddiqui | Role of ultra violet (UV-C) radiation in the control of root infecting fungi on groundnut and mung bean[END_REF] observed increment in shoot weight when seeds of groundnut (Arachis hypogaea L.) and mung bean (Vigna radiata L.) were treated with UV-C for 10, 15, 30 and 60 min. Biomass as a measure of growth makes it possible to know the distribution of dry matter among the various organs, as well as to estimate the magnitude of the demand for assimilates (Hunt 1990). In the different times exposed to UV-C radiation there were significant differences with respect to the dry weight accumulated in roots and foliage (Figure 5). Other applications of UV-C radiation on agricultural seeds before sowing, is the improvement of sanitary quality, due to its sterilizing and antifungal effects [START_REF] Dawar | Fungicidal effect of ultraviolet-C radiations in prevention of mycoflora of castor bean seeds[END_REF]Ferreira 2011;[START_REF] Santos | Wavelength dependence of biological damage induced by UV radiation on bacteria[END_REF][START_REF] Siddiqui | Role of ultra violet (UV-C) radiation in the control of root infecting fungi on groundnut and mung bean[END_REF]Rodriguez et al. 2012). UV-C radiation has been used in the control of fungal growth of castor bean seeds [START_REF] Dawar | Fungicidal effect of ultraviolet-C radiations in prevention of mycoflora of castor bean seeds[END_REF]. In this way it is important to define the appropriate radiation times because UV-C radiation degrades the seed and could modify (positive, null or negative) their physiological quality variables.

Phenolic compounds

The phenolic acids identified in the seedling from bean seeds treated pre-sowing by UVC and control (without radiation) were similar between root and foliage: gallic, chlorogenic, syringic, vanillinic, p-hydroxybenzoic, caffeic, ferulic , p-cumaric ; to exception of the ferulic acid which was not identified in foliage.

The phenolic acid most abundant in seedling from seed control (without radiation) was chlorogenic (18.7 µg/mL), followed by p-cumaric (2.8554 µg/mL) and caffeic (2.72 µg/mL). In the case of foliage, the most abundant were caffeic acid (78.27 µg/mL), followed by chlorogenic (18.71 µg/mL).

The phenolic acids of the bean (Oti) seedlings from of seed treated by UV-C for different exposure times (0, 2, 5, 10 and 15 min) showed significant statistical differences (P ≤ 0.05) in both roots and foliage. There were significant differences according to the treatment in p-hydroxybenzoic, syringic, caffeic and ferulic of the roots (Table 2). The highest concentrations of p-hydroxybenzoic acid (2.26 µg/mL), syringic acid (1.61 µg/mL) were obtained at 10 min of radiation exposure of the seed prior to sowing. While at 15 minutes of exposure to UV-C radiation was found the highest concentration of phenolic acids: caffeic acid (10.6 µg/mL) and ferulic acid (2.6 µg/mL). For phenolic acids of foliage there were significant differences in gallic acid, chlorogenic, syringic acid, vanillinic, p-hydroxybenzoic, caffeic acid and p-cumaric. Being the highest concentration at the time of 10 min for: gallic acid (6.65 µg/mL), vanillinic (0.83 µg/mL) and p-hydroxybenzoic acid (2.23 µg/mL). While seeds radiated for 15 min produced seedlings with higher concentration levels of: chlorogenic acid (23.74 µg/mL), p-cumaric (7.8 µg/mL) and caffeic (6.75 µg/mL). Studies carried out by other authors have coincided that the application of UV radiation can induce the accumulation of phenolic compounds in legume seedlings. [START_REF] Shetty | Effects of UV treatment on the proline-linked pentose phosphate pathway for phenolics and L-DOPA synthesis in dark germinated Vicia faba[END_REF] exposing seeds of faba beans (Vicia faba) to UV-C light for 2, 5 and 10 h and germinated for 8 days in the dark indicated an increase in nutraceutically-relevant phenolic metabolites with enhanced antioxidant activity in response to UV stress.

In our results there was modification of phenolic acids, which in general, increased their concentration with the increase of exposure times to UV-C radiation; without stress condition. Aboul et al. (2014), evidenced that the Green bean seed treatment with UV-C improved plant growth under stressful and non-stressful conditions.

Growth variables were modified through exposure times of 30 and 60 minutes; e.g. the authors found significantly enhanced shoot fresh weight of grown plants by 33.9 and 82.5%, respectively. In other studies, applying UV-C in fruit epidermal tissue has been shown that UV light induces the accumulation of UV absorbing flavonoids and other phenolics compounds [START_REF] Strack | Phenolic Metabolism[END_REF].

Flavonoids

Regarding to phenolic compounds of the flavonoid type, it can be observed (Table 3), that the flavonoids identified in roots of control and radiated seedlings were: rutin, phorizine, quercetin, naringenin, floretin, galangin, naringin, hespiridin, apigenin-7-0-glucoside. In foliage there were similars flavonoids identified in the roots, except the phloretin that was not identified. The flavonoid most abundant in this type of bean seed (Oti) was Phlorizin (460 µg/mL).

Flavonoids of the seedlings from of seed treated by UV-C for different exposure times (0, 2, 5, 10 and 15 min) showed significant statistical differences (P ≤ 0.05) in both root and foliage. It is possible observe that there were significant statistical differences in the case of the flavonoids of the seedling roots for rutin, phorizine, quercetin, galangin, and apigenin-7-0-glucoside. The highest concentrations of flavonoids: Phlorizin (737.1 µg/mL), quercetin (39.7 µg/mL), galangin (25.4 µg/mL), and apigenin-7-0-glucoside (9.95 µg/mL) were found in the seedling roots whose seed was treated during 15 min. In the case of flavonoids rutin (3.4 µg/mL) was found the maximum concentration in the radiation time of the seed for 5 min (Table 3-section a).

In relation to flavonoids of foliage it is possible observe that there were significant differences in phorizine, quercetin, naringenin, galangin, hespiridin and apigenin-7-0-glucoside. Being, the highest concentration obtained in the foliage of the seedlings of the control seeds (without radiation pre-sowing). For all cases, UV-C radiation caused a decrease of this type of flavonoids in the foliage of the seedlings (Table 3-section b) In this way, this research demonstrates that by means of UV-C radiation, applied pre-sowing to the bean seed (Oti), it is possible to modify phenolic compounds (phenolic acids and flavonoids) of the seedlings from the radiated seeds. Similar results have been reported by [START_REF] Guajardo-Flores | Effect of Germination and UV-C Radiation on the Accumulation of Flavonoids and Saponins in Black Bean Seed Coats[END_REF], who radiated the black bean seed during 20 h in germination period, reporting higher accumulation of flavonoids during germination in radiated seed. In this investigation, some of the flavonoids of the roots of the seedlings from radiated seed were increased to 5 and 15 min of UV-C exposure.

As is known, secondary metabolites have a relevant role in the physiological and plant development processes, included to stress responses and to the protection against pathogens (Katerova et al. 2013).

The results coincide with other authors who have detected the presence of flavonols, flavones and anthocyanins in bean plants [START_REF] Deckmyn | The importance of the ratio UV-B/photosynthetic active radiation (PAR) during leaf development as determining factor of plant sensitivity to increased UV-B irradiance: Effects on growth, gas exchange and pigmentation of bean plants (Phaseolus vulgaris cv. Label)[END_REF]. Especially the flavonoids are linked with regulatory mechanisms with symbiosis of bacteria (M. [START_REF] Hungria | Effects of flavonoids released naturally from bean (Phaseolus vulgaris) on nodD-regulated gene transcription in Rhizobium leguminosarum bv Phaseoli[END_REF]. Coinciding the presence of naringin and naringenin in the exudates of bean roots. the UV-C radiation time increases from 2 to 15 min, the morphological changes at the surface of the bean seeds exhibits micro-holes and detachment of the cotyledon sclereids. The phenolic acids and flavonoids of the bean seedlings of seeds treated by UV-C for different exposure times (0, 2, 5, 10 and 15 min) exhibit significant statistical differences (P ≤ 0.05) in both roots and foliage. For both the roots and foliage, the highest concentration of phenolic acids has been found at 10 and 15 minutes, while for root flavonoids, it appears at 5 and 15 minutes.

Figure 2

 2 Figure 2 Scheme of the PAS experimental setup used in our experiments.

Figure 3

 3 Figure 3 Spectra of optical absorption obtained by PAS in control seeds (without UV-C radiation) and seeds treated with UV-C at 2, 5, 10 and 15 min: a) Photoacoustic signal b) Optical absorption coefficient () and optical penetration length (l ) as a function of wavelength for seed samples.

  attached to the testa and body of the cotyledon. As the dose of UV-C increases, the presence of micro-holes in the testa, and detachment of the sclereids of the cotyledon is evident. In the T4 treatment, changes of structural conformation are clearly shown. Regarding the change of the starches (third row), when applying higher radiation treatments to the seed (T3 and T4), the starch granules are compacted, such that the cell wall of the cotyledon swells.

Figure 4

 4 Figure 4 Micrographs of control bean seed (T0 = 0 min) and exposed with UVC light at different radiation times (T1 = 2, T2 = 5, T3 = 10 and T4 = 15 min),

Figure 5 .

 5 Figure 5. Cumulative dry weight of seed bean seedlings exposed to different times of shortwave ultraviolet radiation. Different letters mean that there are significant differences according to the average comparisons (Tukey α= 0.05). Time refers to different minutes of UV-C irradiation (T0, T1, T2, T3 and T4).

  (α = 0.05). T0, T1, T2, T3 and T4 equals to radiation exposure time of the pre-sowing seed : 0, 2, 5, 10 and 15 min of radiation UV-C.

  T3 and T4 equals to 2, 5, 10 and 15 min of radiation UV-C.

Table 1

 1 Averages of parameters obtained of seeds and seedlings of bean a) level of photoacoustic signal obtained by PAS at different wavelengths b)

	germination variables of seedlings from treated seed.
	a. Photoacoustic signal at different wavelengths

Table 2 .

 2 Concentration of phenolic acids in seedlings of bean seeds exposed at different times of shortwave

	ultraviolet radiation.
		a) Roots
	Phenolic acids	T0
	(µg/mL)	(Control)	T1	T2	T3	T4	DMS	R 2	Mean
	Gallic acid
		0.89a	0.68a	38.62a	9.05a	0a	43.144	0.79030	9.848445
	Chlorogenic acid
		18.7a	10.054a 10.086a 5.241a	8.886a	17.487	0.673174 10.59106
	Syringic acid
		0b	0b	0b	1.61a	0b	1.0947	0.91773	0.322246
	Vanillinic acid
		0a	0a	1.4177a 0a	0a	3.5968	0.44444	0.283534
	p-hydroxybenzoic
	acid	0c	0c	2.1b	2.26a	0c	0.1058	0.9999	0.871215
	Caffeic acid	2.72a	3.1a	0b	0b	10.684a 9.8393	0.83602	3.300694
	Ferulic acid	0c	0c	0c	1.60b	2.6826a 0.4097	0.995743 0.857896
	p-Coumaric acid	2.8554a	1.5117a 0.6051a 0.0763a 0a	3.9911	0.69776	1.009725
		b) Foliage
	Gallic acid	0a	0a	0b	6.65a	0b	0.2243	0.999780 1.331945
	Chlorogenic acid	18.71a	0c	6.037c	9.682b	23.74a	12.261 0.94020	11.63444
	Syringic acid	0b	3.5091a 0b	0.813b	0b	1.0323	0.97825	0.792902
	Vanillinic acid	0 b	0 b	0 b	0.838 a 0 b
							0.0196	0.9999	0.167695
	p-hydroxybenzoic acid	0b	0b	0 b	2.237 a 0 b	0.0149	0.9999	0.447427
	Caffeic acid	78.27b	0.317b	5.01a	4.77a	6.755a	2.0191	0.98061	3.530364
	Ferulic acid	0b	0b	7.559a	6.813a	7.893a	1.5868	0.99417	0.004453
	DMS is significant minimum difference. The different letters indicate significant differences between the
	Tukey means test

Table 3

 3 Concentration of flavonoids in bean seedlings from seeds radiated by UV-C: a) Roots, b) Foliage. least significant difference and the same letter are statistically equal (Tukey, P ≤ 0.05). T0, T1, T2,

			a) Root
	Flavonoids	T0							Media
	(µg/mL)	(Control) T1	T2	T3	T4	DMS	R 2
	Rutin	1.9 a	0 b	3.4 a	0 b		1.9 a 1.5	0.958	1.45
	Phlorizin	460 c	398 c	553.75 b	269.6 d	737.1 a 77.24	0.992	483.7400
	quercetin	0 b	0 b	0 b	0 b		39.7 a 4.3294	0.997	7.936310
	Naringenin	26.3 a	0 a	4.74 a	0 a		10.9 a 52.792	0.526	8.386
	Phloretin	1.2594 a	0 a	0 a 1.6884 a		0 a 3.9597	0.525	0.589561
	Galangin	17.32 b	7.52 d	15.85 b	11.5 c	25.48 a 4.608	0.982	15.53901
	Naringin	185.8 a	48.2 a	41.57 a	39.82 a	26.64 a 190.1	0.756	68.41133
	Hespiridin	198.7 a 185.69 a	139.24 a 117.15 a 195.21 a 214.8	0.430	167.2037
	Apigenin-7-0-
	glucoside	0 c 5.4151 b	4.9966 b	3.57 b		9.95 a 0.0022	0.9857	4.788967
			b) Foliage
	Rutin	2.37 a 0.9368 a	0.1305 a	0 a 0.4258 a 2.7431	0.760633	0.773029
	Phlorizin	532.9 a	515.1 a	247.2 c	411.2 b	268.4 c 62	0.991702	394.9824
	Quercetin	162 a	54.46 b	5.36 b	0 b		0 b 101.6	0.93561	44.39249
	Naringenin	3.53 a	1.02 b	1.89 b	13.0 a		0 b 10.506	0.865709	3.890745
	Galangin	18.41 a	12.1 b	3.8 c	13.48 a	10.18 b 6.0233	0.952677	11.58393
	Naringin	0 a	66.85 a	28.14 a	32.90 a		0.76 a 79.045	0.757594	25.73127
	Hespiridin	249.32 a	82.75 b	0 b	1.67 b	11.27 b 163.66	0.915828	69.00097
	Apigenin-7-0-
	glucoside	11.646 a	2.162 b	2.79 b	1.189 b	1.454 b 5.187	0.948951	3.859616
	DMS is the

Acknowledgements

I thank Dr. Estrada for providing the bean seed and Ing. Esther Ayala for the technical assistance at the Cinvestav Photothermal Techniques Laboratories. Claudia Hernandez thanks the support for SIP research projects.

Other authors have agreed on the effects of UV-C radiation, but in another type of biological sample. As in the case of callus culture; e.g. in callus culture (Vitis vinifera L.) where a higher accumulation secondary metabolites was reported [START_REF] Cetin | Induction of secondary metabolite production by UV-C radiation in Vitis vinifera L. Öküzgözü callus cultures[END_REF]. Other authors applying the UV-C in callus culture of Bacopa monnier have suggested that UV-C radiation is a useful technique which can be used to enhance the accumulation of phytochemicals [START_REF] Meenashree | Estimation of Metabolites and Antioxidant Activity in UV-C Treated Callus Cultures of Bacopa monnieri (Linn.) Pennell[END_REF].

Likewise, the effects due to UV-C on secondary metabolites in epidermal tissues of fruit have been evaluated.

In tomatoes irradiated by UV-C, significantly increased total phenolic content was described when were compared treated and untreated samples [START_REF] Bravo | The influence of post-harvest UV-C hormesis on lycopene, β-carotene, and phenolic content and antioxidant activity of breaker tomatoes[END_REF]).

According to our findings it has been demonstrated that the seed treated with UV-C light is degraded as a function of the time of exposure to UV-C light, demonstrated through photoacoustic spectroscopy and scanning electron microscopy. But, the seeds radiated could produce seedlings with higher concentration of some secondary metabolites, as the flavonoid and phenolic acid type. These results could impact on different applications, in the seed: to activate defense mechanisms and improve germination. Other future applications could concentrate on the products consumed by the population, since they could modify the nutritional qualities of the products consumed and have better nutritional quality of them.

For this reason, several recent studies have examined the importance of UV-C radiation with regard to different quality parameters of treated plant foods, evaluating possible effects on their functional properties. A recognized advantage of applying UV-C is the capability to improve the nutraceutical properties of plant foods, and some studies have reported the possibility of increasing the antioxidant content through UV-C irradiation as a postharvest treatment. In this research, secondary metabolites were modified both in seed and in the seedling from radiated seed. Then, in the case of agricultural seeds, UV-C radiation could serve not only to improve physiological quality, but also physiological and nutritional, the latter being beneficial when the plant is consumed in the form of sprouts.

In this way, UV-C radiation on seeds produce their degradation as a function of the radiation time. It is possible that the UV-C light interacts with different structural levels of the seed and in this way to photostimulate chromophores that intervene in metabolic processes of the seed. Future applications should be optimized towards the most convenient method; in relation to the conditions of the seed, the characteristics of the seed itself as well as the expected effect produced by UV-C light.

CONCLUSIONS

Variations of the photoacoustic signal produced by bean seeds exposed to UV-C radiation during 2, 5, 10 and 15 minutes, have been measured within the spectral band (270 -400) nm. Based on scanning electron microscopy, it has been shown that the decrease of this signal is generated by the seeds' degradation, such that its maximum reduction from 0.24 to 0.10 mV shows up at 295 and 340 nm, after 15 minutes of exposure. As

Conflict of Interest

There is no conflict of interest in relation to the article presented. Taking into consideration that the author and the coauthors of the article mention that an original article is presented, not published and does not infringe any legal copyright, or any property right or any other right whatsoever.


Jamal Chenouf

Mourad Boutahir
email: mourad.boutahir@fis.puc.cl

B Fakrach

Abdelali Rahmani

H Chadli

Patrick Hermet

Jose Mejía-López

| Fakrach

| Rahmani

| Chadli

| Mejía-López

Encapsulation effect of π -conjugated

Keywords: Thiophene, CNTs, Raman spectroscopy, Charge transfer

come

| INTRODUCTION

Organic solar cells have received a lot of attention in recent years because they make it possible to diversify renewable energy sources thanks to their simple preparation, lightweight, low technological cost and large-area flexible fabrication 1;2;3;4;5 . However, their energy efficiency and durability need to be improved for the development of sustainable technology. In these devices, photovoltaic conversion is based on the separation of photogenerated charges at an interface between electron donor and acceptor materials, which imposes some constraints on the photoactive layer of the cells.

Fullerene derivatives are among the most popular materials used in organic or related photovoltaic devices 6;7;8;9;10;11 .

In particular, organic photovoltaic (OPV) solar cells using a bulk-heterojunction (BHJ) of conjugated polymer donors and fullerene derivative acceptors have shown the best performance with a solar power conversion efficiency (PCE) of 11.5% 12;13 . Single-walled carbon nanotubes (SWCNT) are also interesting potential candidates to replace the electronaccepting and transport fullerene phase in BHJ OPV systems 14;15 . Indeed, carbon nanotubes (CNTs) offer distinctive properties that are not found in fullerenes, such as: (i) the very broad spectral coverage with band-gap electronic transitions well into the near-IR spectral region to match the solar spectrum [START_REF] Kataura | [END_REF] , (ii) a charge mobility (about 105 cm 2 /V.s) 17 that may facilitate charge carrier transfer and/or dissociation and transport over an extended distance because of their large surface area (above 400 2 /g) and (iii) electron-accepting properties 18;19 .

Despite the fact that graphene has emerged to become the center of attention in the field of carbon nanomaterials in recent years 20 , there are still distinct advantages for the use of CNTs in energy conversion applications. Several studies on blends of conjugated polymers and SWCNT suggest that efficient exciton quenching occurs at these interfaces and many studies have shown direct evidence for the generation of mobile carriers in polymer-SWCNT composites 21;22;23;24 .

After exciton dissociation, ballistic carrier transport in single tubes and extremely high conductivity in networks suggest that SWCNT may yield higher mobilities and form more conductive percolation networks than fullerenes. OPV cells based on CNTs as acceptor on the concept of BHJ is one of the most promising. Nevertheless, the efficiency of this kind of device is limited because of the incomplete exciton dissociation 25;26 .

In addition, the electronic diversity of nanotubes produced which are typically a mixture of metallic and semiconducting CNTs 27;28 may affect the device efficiency due to the presence of the amount of metallic CNTs. As metallic SWCNTs do not have a true bandgap, one would naturally suspect these species to act as charge carrier or exciton recombination centers, lowering the efficiency of charge separation, and, by extension, the efficiency of devices. Thus, only semiconducting SWCNT are interesting for OPV as different authors report three-times long-lived charge carriers for the photocurrent generation by eliminating metallic CNTs 29;30 . Exciton separation across this interface could be possible, but it has been revealed that energy transfer rates limiting the ability of the Donor/Acceptor interface to separate excitons in free charge carriers, decrease with the diameter of the tubes 31 . A stable structure of the hybrid system with an optimal diameter to decrease energy transfer and allows the exciton separation at the interface is principal.

The inferior OPV device stability still remains a great challenge. Many factors limit the stability of organic solar cells such as: metastable morphology and heating 32 , oxygen and water 33 , and irradiation 34;35 . Indeed, the degradation of -conjugated molecules is a bottleneck impeding their application in OPV devices 36 . The use of these molecules as device materials in OPV technologies therefore requires the development of an innovative technique that can overcome this degradation problem. An efficient technique consists in encapsulating -conjugated molecules inside the hollow core of nanotubes 37 . This strategy is among the best methods to combine the physical properties of both systems without any mechanical or chemical damaging 38;39;40 and offers opportunities in achieving high efficiency and high stability of organic solar cells towards future industrial manufacture. With this perspective, the encapsulation of CNTs with photoactive molecules would have a major impact on OPV technology particularly thanks to the scaling-up of a variety of deposition and patterning methods available today allowing encapsulate the conjugated polymers inside CNTs 41;42 In this regard for important feedback on the PCE optimization, we report a reliable computational method to correlate chirality and diameter of SWCNT with characteristics of a photoactive nanocomposite. In this context, a non-covalent functionalization is employed and we choose the 4T@SWCNT as model system where the 4T is encapsulated into a SWCNT (Figure 1). We developed an hybrid approach, combining the density functional theory and molecular mechanics, to build the dynamical model of 4T@SWCNT. For this hybrid system, we performed total energy minimization to derive a stable structure with an optimal tube diameter. The knowledge of this structure allows to estimate the exciton separation. We also calculated the Raman spectra, using the bond polarizability model combined with the spectral moments method, of the free 4T molecule, empty SWCNTs with different diameters and chiralities, and 4T@SWCNT to analyze the influence of the encapsulation on the Raman fingerprints. From these Raman calculations, we discuss on an eventual charge transfer between the 4T oligomer and the nanotube.

| MODEL, METHOD AND AP-PROACH

Calculation of Raman spectra requires the knowledge of the polarizability tensor (mainly for the intensity of Raman lines) and the dynamical matrix (mainly for the frequency position of Raman lines) of the system.

The dynamical matrix of a system has a 3Nx3N dimension, where N is the number of atoms in the primitive unit cell. Taking the symmetry of the system into account significantly decrease the number of matrix elements to be calculated.

However, in the case of the 4T@SWCNT hybrid system, we have no symmetry elements, except the trivial identity operation, and all the elements of the dynamical matrix have to be calclated. Fortunately, we can work with a lower or upper triangular matrix because the dynamical matrix is hermitian by definition.

First principles based methods are very accurate to compute the dynamical matrix (frozen phonon or density functional perturbation theory) but their computational cost is very high for large system (like 4T@SWCNT) and especially without any symmetry. Molecular mechanics could be an excellent alternative to build the 4T@SWCNT dynamical matrix but such a force field still remains unknown. Thus, we adopted three-step methodology taking advantage of the densiy functional theory and the molecular mechanics. The idea consists to decompose the 4T@SWCNT hybrid system into two independent subsystems (SWCNT and 4T), which will be coupled by an adequate empirical intermolecular potential.

The dynamical matrix of the oligomers molecules will then be calculated using DFT (first step) and the dynamical matrix of SWCNT will be computed from the force constant model introducted by Saito et al [START_REF] Dresselhaus | Physical properties of carbon nanotubes[END_REF] (second step). The interactions between 4T-SWCNT will be described by a van der Waals potential (third step). We choose a Lennard Jones type:

( ) = 4 12 - 6 (1)
with parameters: = 2.964 meV and = 0.3407 nm for carbon atoms. = 10.7 meV and = 0.3560 nm for sulfur atoms. = 0.67 meV and = 0.1069 nm for hydrogen atoms.

The Carbon-Sulfur and Hydrogen-Carbon parameters were calculated using Lorentz -Berthelot rule:

= √ et = ( + )∕2.
The DFT calculations on 4T molecule have been performed with the SIESTA package, which use pseudo-potentials and localized atomic basis [START_REF] José | [END_REF] . We used the local density approximation (LDA). The valence electrons were described by a double-singly polarized basis set. The localization of the basis is controlled by an energy shift of 50 meV and the real space integration was performed on a regular grid corresponding to a plane wave cutoff around 360 Ry. We considered a vaccum size of 12Å to avoid interactions between adjacent systems.

The Raman susceptibilty of phonon modes is calculated according to the bond polarizability model of SWCNTs 45 combined with the one of free 4T molecules 46 . This approach has been successfully used to predict the nonresonant Raman spectra of SWCNTs and double-walled carbon nanotubes (DWCNTs) as a function of their lenghts 47;48;49;50 .

Calculations of Raman spectra of our studied systems are based on the spectral moment's method. For more details on this method, see Ref. 51 .

| RESULTS

| Stability of 4T@SWCNT

We determined the range of tube diameters in which the 4T@SWCNT hybrid system is stable. Such a study can guide experimentalists as they provide reliable estimates about reasonable nanotube sizes and the steric hindrance of the 4T oligomer inside the tube. We assume in our calculations that the 4T size is strongly reduced under nanoconfinement by aligning all the thiophene rings to form a planar structure.

The optimized configuration corresponds to an isolated chain inserted into the carbon nanotube and centred along the nanotube main axis. In practice, the structure relaxation was achieved by minimizing the distances between atoms associated to Lennard-Jones potential. Figure 2 shows the Lennard Jones potential for 4T@SWCNT as a function of the tube diameter ranging from 0.8 to 2.5 nm. We observe a minimum at 0.94 nm corresponding to the equilibrium distance between the SWCNT and 4T molecule. Thus, the insertion of a single 4T oligomer, aligned along the axis of the carbon nanotube and located at its center, is favorable for SWCNTs whose diameter is close to 0.94 nm. This diameter is in agreement with the experimental observations of Alvarez et al. 52 for a single 4T molecule. Note that a distribution of SWCNTs ranging from 0.9 to 1.0 nm in diameter with corresponding S1 optical bandgaps ranging from 1.0 to 1.2 eV 16;53;54 , could be employed to capture photons of energy h > 1 eV, which is almost the same spectral range captured by a silicon solar cell. Indeed, on the concept of BHJ , following photon absorption of either 4T or SWCNT, the excitation of 4T followed by electron transfer, or the excitation of SWCNT followed by hole transfer lead to the same charge separation state, and therefore, the spectral coverage realized by a particular mixture of SWCNTs with diameter around 0.94nm can be exploited to optimize lightharvesting and, by extension, the power conversion efficiency of organic solar cells (OSCs).

| Raman spectra of isolated 4T

The calculated Raman spectrum of 4T oligomer is reported in

| Raman spectra of 4T@SWCNT

Raman lines of pristine (empty) carbon nanotubes can be divided into two ranges of frequencies: below 400 cm -1 where are located the radial breathing modes (RBM) and above 1500 cm -1 where are located the tangential modes (G-band). These modes are especially relevant to probe an eventual charge transfer between 4T and SWCNT. This possibility (or not) of a charge transfer is of major interest in the nanotube community workers and constitutes the main question of this work.

To answer to this question, we considered six 4T@SWCNT with different tube chirality ( , ) and whose the diameters are around 0.94 nm (the optimal value found in section 3.1 to encapsulate only one 4T). Among them, three are metallic [(12,0), (11,2) and (7,7)] and three are semiconducting [ (8,6), (9,5) and (10,3)]. The ZZ-polarized Raman spectra of empty SWCNTs and hybrid systems after minimizations are displayed in Figures 4 (metallic tubes) and 5 (semiconducting tubes). For each SWCNT and 4T@SWCNT, the intensities of the different spectra are normalized and can be therefore compared. At first glance and whatever the nature (metallic or semiconducting) of the nanotube, Raman spectra of the 4T@SWCNT hybrid systems seem to be to the superposition of the 4T and SWCNT spectra. However, a more careful analysis shows several differences which are characteristic of the interactions between the 4T and SWCNT in hybrid systems. Indeed, the libration modes (8 and 20 -1 ) of 4T oligomer and evidenced in Figure 3 disappear in Figures 4 and5 after encapsulation. This type of reciprocating motion, in which the free 4T repeatedly rotates slightly back and forth, is deactivated after encapsulation. This absence of libration can explains a certain stability of 4T inside the SWCNT cavity.

In addition, an upshift of all Raman lines between 1 and 9 cm -1 is observed in the 4T@SWCNT spectra with respect to corresponding ones in the free 4T oligomer. This upshift associated to van der Waals interaction is another hint of a significant interaction between 4T and the nanotubes. The radial breathing modes (RBM) clearly exhibit a frequency upshift after encapsulation by about 1 cm -1 for 4T@(7,7) nanotube (Figure 6, bottom). Let us recall that, charge transfer (positive or negative) onto individual metallic nanotubes has been shown to induce a small RBM up-shift of about 2 cm -1 58 . The shift of a few to a higher frequency indicates that the diameter of the nanotube is energetically favorable for the encapsulated molecules as predicted by Okada and co-workers for nanotubes encapsulating fullerenes 59 . For 4T@(11,2) and 4T@(12,0) hybrid systems, no shift of RBM position is observed in Figure 6. Such a behavior is observed when rubidium atoms (electron donor molecule) are encapsulated into bundles of CNT 60 . Conversely, in the case of semiconducting nanotubes, the RBM observed in empty tubes is downshifted by about 2 cm -1 for both 4T@(10,3) and 4T@(9,5) and by about 1 cm -1 for 4T@(8,6) as shown in figure 6. In order to discuss the origin of this mode softening, it is worth mentioning that the dispersive interactions (vdW) lead to a small downshift (2 or 3 cm -1 ) for fullerene molecules 61 . Generally, the RBM shifts are at most -3 -1 for dispersive interactions 59;61;62 and 2 -1 for charge transfer 58;63 .

In addition, the lower shift of the RBM frequencies originates from modulation of the charge distribution of -electrons in the nanotubes induced by the encapsulation 59 . By contrast, insertion of lithium (electron donor molecule) gives rise to a small down-shift 64 . However, the physical interactions of thiophene molecules with the nanotubes are probably dispersive and combined to a slight charge transfer as reported by Alvarez et al 52 . using Raman and infrared spectroscopies.

F I G U R E 7

The G-band Raman spectra of empty SWCNT (black curve) and 4T@SWCNT (red curve). (left) Metallic SWCNT. (right) Semiconducting SWCNT. peak assigned to A 1 (ZZ) tangential modes. This peak is centered at 1582 cm -1 in the (7,7) SWCNT and at 1591 cm -1 in the (12,0) SWCNT. In the following, we use the terms G - and G + when referring to the lower-frequency peak and the higher-frequency peak, respectively. When increasing from 0 to 30 • , we observe the appearance of two peaks (G -and G + ) at frequencies close to the frequency of the zigzag and armchair tangential modes (see for instance the (11,2) whose the chiral angle is 8 • ). Consequently, the G + (resp.G -) peak is dominant in the zigzag (resp. armchair) tube, while both the G + and G -peaks appear in chiral tubes (0

• < < 30 • ).
The intensity of these peaks (G -and G + ) shifts from the one located around the frequency of the zigzag to that located around the frequency of the armchair when increasing . In all our calculations, the G -and G + features were assigned to the transverse optical (TO) and the longitudinal optical (LO) phonon modes, respectively, independent of whether the nanotube is metallic or semiconducting. With the 4T encapsulation, the 4T@SWCNT spectra (red curves) exhibit not only the G-bands of the nanotubes (G -and G + ) between 1560-1620 cm -1 ) but also the C-C stretching vibrations of the confined 4T molecule located around 1575 cm -1 . For metallic SWCNT (left hand part of the figure), the influence of the confinement shows that the G -peak (TO mode) position shows a downshift by 1 cm -1 in the 4T@(7,7), the G + peak (LO mode) shows a significant upshift by 4 cm -1 in the 4T@(12,0), and the 4T@(11,2) spectrum clearly exhibits the same shift of these two G-bands. For semiconducting SWCNT (right hand part of Figure 7), the three considered tubes, (10,3), (9,5) and (8,6), are chiral with ∼ 13, 20 and 25 • , respectively. Both the G + and G -peaks appear in the Raman spectra of empty tubes. After encapsulation, all spectra exhibit a G -peaks (TO modes) downshifted by 1 cm -1 . At the opposite, the G + peaks (LO modes) shows a significant upshift by 3 cm -1 for both the 4T@(10,3) and 4T@ (8,6), and by 2 cm -1 for the 4T@(9,5). Based on these observations, The G-band behavior for both metallic and semiconducting SWCNT show similar trends under the confinement effect. The G -(TO-modes) of SWCNT show a slight downshift of about 1 -1 , whereas the G + (LO-modes) demonstrate a larger upshift between 2 and 4 cm -1 . These shifts to higher frequencies of the G + peaks, which corresppond to axial displacement of the atoms (see Indeed, the literature reports that p-doping always leads to the contraction of bonds and phonon stiffening 65 . In the other hand, for the slight downshift of the G -peaks, which refers to a circumferential displacement of the atoms (see Figure 8(top)), there is probably a competition between dispersive interactions of 4T molecules with the nanotubes which cause phonon softening and the downshift of the G -peaks, and the effect of slight charge transfer which results in the upshift of the G -peaks. Both effects may compensate each other, and the shift of the G-band is expected to be smaller or even to have the opposite sign than for p-doping.

| 4. DISCUSSION

To make a comparison with the experimental data as realistic as possible, we discuss in this section the result of our calculations on the G + and G -peaks to those obtained over the years by various theoretical and experimental works.

One method to distinguish between metallic and semi-F I G U R E 8 Calculated eigen displacement vectors for the tangential modes in (11,2) tube (left) and 4T@(11,2) (right).

conducting SWCNT is based on the distinct differences in the Raman line shape of the tangential G-band. The Gband of SWCNT consists of two main peaks due to the curvature-induced inequality of the two bond-displacement directions: a lower-frequency peak (G -) in the 1550-1570 cm -1 range and a higher-frequency peak (G + ) near 1580-1590 cm -1 . In semiconducting tubes both peaks are sharp (narrow Lorentzian peaks), while in metallic tubes the G -is extremely broad 66;67;68 . However, optical phonon modes in nanotubes were studied theoretically by various methods, such as zone-folding schemes 69 , force-constant models 70;71 and first-principles based methods 72;73;74;75;76;77;78;79 , and different models have been developed for the description of the peculiar line shape of the Raman G-band in metallic carbon nanotubes. In refs. 80 for example, Kempa proposed phononplasmon interactions as the origin of the metallic line shape where the G -and G + features were respectively assigned to the TO and LO phonon as in the case of semiconducting tubes. In contrast, other models treated this broad G -peak through a Breit-Wigner-Fano (BWF) line shape ascribed to the strong coupling between the LO phonon and electronic excitations 68;72;81;82 . In these models, the LO frequency is below the TO frequency due to the strong electron-phonon coupling, and consequently, the G -peak is assigned to the LO phonon. As a result, the frequency of the LO phonon drops below the frequency of the TO phonon in metallic nanotubes. However, the assignment of the G -and G + Raman peaks to the TO and LO phonons for metallic nanotubes is still under debate with contradicting conclusions appearing in the literature, even if there is accumulating consensus that the broad G -peak is a softened and broadened LO phonon feature arising from Kohn anomalies.

In view of these results from the literature, the splitting of the G -and G + peaks (two narrow Lorentzian peaks) in our calculations, and their assignment to the TO and LO phonons respectively, seems to be in good agreement with the experimental data for semiconducting nanotubes. In the case of metallic nanotube, our results are more problematic as the asymmetric G-band line-shape in Raman spectra is related to Fano resonance (BWF) lines. However we highlight that our calculations does not consider the eletron-phonon coupling responsible for the BWF line shape in metallic SWCNT.

This will be studied in a future works towards improving the detailed comparison of the theoretical and experimental frequency shifts in hybrid systems with metallic SWCNT.

Nevertheless, even with the present limitations, our predictions concerning the number and frequency of Raman lines obtained in the framework of a nonresonant Raman theory do not depend on the resonant process of Raman scattering and can be helpful to interpret the frequencies shifts in experimental data of hybrid systems.

| 5. CONCLUSIONS

In this study, we use an orignal approach to study the encapsulation of a 4T molecule inside SWCNT. The dynamical model of the 4T and the nanotube have been respectively described by DFT and molecular mechanics whereas the coupling between the 4T molecule and the nanotube have been described by a Lennard-Jones potential. First, we found that the configurations energetically favorable for the insertion of a single molecule aligned along the nanotube main axis and located at its center, are those with a tube diameter around 0.94 nm.

Then, for the obtained configurations of 4T@SWCNT hybrid systems with metallic and semiconducting nanotubes, the nonresonant Raman spectra have been calculated using the bond-polarizability model combined with the spectral moments method. The charge transfer in the 4T@SWCNT has been investigated by analysing the Raman-active modes of the 4T molecule, as the RBM and G-band modes of SWCNT, before and after encapsulation. Finally, we conclude that a possible charge transfer between the 4T and the SWCNTs independently of their metallic or semiconducting character.

In this context, the encapsulation of organic molecules inside carbon nanotube, thus creating a new hybrid system, appear to be of great interest for solar cell applications.

F I G U R E 1

 1 Schematic representation of the 4T encapsulation into a SWCNT. The yellow color indicates the Sulfur, grey the Carbon, and white the Hydrogen atoms.

2

 2 Minimization energy calculations of 4T@SWCNT with different diameters.

Figure 3 3

 33 Figure 3 up to 1600 cm -1 and shows several distinct lines at 8, 20, 97,160, 315, 369, 463, 633, 697, 844, 877, 1055,1201, 1266, 1283, 1344, 1412, 1503, 1521 and 1575 cm -1 . We assigned these lines according to the various experimental and theoretical Raman studies on polythiophene reported in literature 55;56;57 . The couple of lines at 8 and 20 cm -1 are libration modes. The lines at 97 and 315 cm -1 are assigned to in-plane vibrations characterized by the C-S-C bending modes (the ring deformation C-S-C). The in-plane vibrations characterized by torsional modes S-C are evidenced at 160 cm -1 . The line at 369 cm -1 is assigned to in-plane vibrations characterized by torsional modes C-C and out-of-plane vibrations characterized by torsional modes S-C. The line at 463

F

  I G U R E 4 Calculated Raman spectra of a 4T encapsulated into a metallic SWCNT.

F I G U R E 5

 5 Calculated Raman spectra of a 4T encapsulated into a semiconducting SWCNT.

6

 6 Calculated Raman spectra of empty SWCNT (black curve) and 4T@SWCNT (red curve) in the RBM range. (left) Metallic SWCNT. (right) Semiconducting SWCNT.

Figure 7

 7 Figure 7 displays the calculated ZZ-polarized Raman spectra within the G-band range (1560-1620 cm -1 ) of the empty SWCNT (black curves) with different chirality. For the (12,0) zigzag tube (chiral angle = 0 • ) and the (7,7) armchair (chiral angle = 30 • ) tubes, the spectra are featured by a single

Figure 8 (

 8 Figure 8(bottom)), indicate a possible charge transfer from the SWCNT to the 4T molecule.

Funding information

CONICYT Posdoctodado 2020, Grant/Award Number: 3200046; Financiamiento basal para centros científicos y tecnológicos de excelencia, Grant/Award Number: AFB180001 approach, combining the density functional

AC K N OW L E D G M E N T

The work was supported by Moulay Ismail University Research Support (13-16) and CONICYT Posdoctodado 2020 Nº 3200046. JML acknowledges support from Financiamiento basal para centros científicos y tecnológicos de excelencia AFB180001.

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