**Effects of pH and Phosphorus Concentrations on the Chlorophyll Responses of** *Salvia chamelaeagnea* **(Lamiaceae) Grown in Hydroponics**

Kerwin Lefever, Charles P. Laubscher, Patrick A. Ndakidemi and Felix Nchu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67610

#### **Abstract**

*Salvia chamelaeagnea* (Lamiaceae) is a slow growing water‐wise evergreen shrub originat‐ ing from the western province of South Africa. It is an attractive landscape, and *S. chamelaeagnea* is a medicinal plant. It is important to develop enhanced cultivation protocols that could result in high yield and high‐quality medicinal materials. Chlorophyll is a fundamental part of the light‐dependent reactions of the photosynthesis process. This chapter investigates the effects of four phosphorus concentrations and three pH levels of supplied irrigated water on the production of chlorophyll A, chlorophyll B, total chloro‐ phyll, leaf colour and the nutrient uptake of *S. chamelaeagnea* grown in hydroponics over an 8‐week period at the Cape Peninsula University of Technology. The treatments of pH 4, pH 6 and pH 8 at 31, 90, 150 and 210 ppm of phosphorus were received by 12 groups of plants and were replicated 10 times. The results indicated that at pH 4, P fertilization sig‐ nificantly (*P* < 0.05) induced a higher chlorophyll production of *S. chamelaeagnea* grown in hydroponics compared to other pH treatments (pH 8 and pH 6).

**Keywords:** hydroponics, pH, chlorophyll production, medicinal plants, *Salvia chamelaeagnea*

### **1. Introduction**

*Salvia chamelaeagnea* P.J. is a member of the Lamiaceae family. Plant species in this fam‐ ily include many culinary and medicinal herbs like *Salvia officinalis, Salvia verbenacea* and *Salvia libanotica,* which have been used for many years against diarrhoea, indigestion, colic, abdominal trouble, influenza, bacterial infections, tuberculosis, cough, cold and many other

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ailments [1–3]. Some of these uses date back to medieval times [4]. Many of the Lamiaceae secondary metabolites are of commercial interest to the food industry as sources of natural preservatives, flavourants and antioxidants [2, 5], as well as to the pharmaceutical industry as sources of antioxidants, anti‐inflammatories [6], antibacterials and anti‐mycobacterials [7].

Salvias are renowned for their variety and their many uses around the home and garden; they have beautiful flowers and attract birds [8]. In its natural habitat, *S. chamelaeagnea* will develop into attractive foliage and flowering landscape plants, with small mid‐green egg‐shaped leaves and masses of bright blue or white flowers borne at the tops of each stem, which are suitable for the cut flower trade [8–10]. *S. chamelaeagnea* also has value in the medicinal plant trade as it contains the phenolic compounds carnosol, rosmarinic acid and caffeic acid, which exhibits antioxidant and anti‐bacterial activities [2, 6, 11].

Unfortunately, very little information has been documented on the cultivation of this species. Cultivation of medicinal plants is gaining traction worldwide; it is seen as a tool for biodiver‐ sity conservation, poverty alleviation and cultural preservation [12]. However, good knowl‐ edge of plant physiology must be attained in order to develop enhanced cultivation protocols that could result in high yield and high‐quality medicinal materials. Effects of nutrients and nutrient ratios on many food and medicinal crop plants, such as soya bean, thyme, wheat cul‐ tivars, barley, spinach and pelargoniums, have been studied. In most cases, a positive result in growth is noticed with the addition of some macro‐nutrients such as N, P, K, Mg or Ca [13–21]. It is therefore crucial that adequate plant nutrition and soil pH levels are met for any given plant so that the cell's functioning is not impeded. Chlorophyll is a fundamental part of the light‐dependent reactions of the photosynthesis process, capturing light rays from the sun and producing energy‐storing ATP molecules that are essential for the functioning of a healthy plant [22, 23]. The effects of poor nutrition, be it through infertile soils or incorrect soil pH level, directly affect the production of chlorophyll molecules resulting in chlorosis of leaves and a reduced photosynthetic rate, thus inhibiting some biological processes and decreasing the general health of the plants [23–25]. There are plausible mechanisms through which the production of chlorophyll could be affected, for example, the pH level of a growing medium affects the uptake of P [26] and the P level influences the nutrient uptake by plants [27]. The relationship between the nutrient P and chlorophyll is not fully understood. According to Nicholls and Dillon [28], there are substantial variations of the published phosphorus‐chlo‐ rophyll relationship, which they ascribed to variations in sampling and analytical techniques.

This chapter aims to investigate the effects of P and pH on the chlorophyll production, leaf colour and the nutrient uptake of medicinal *S. chamelaeagnea* in hydroponics, in order to determine a fertilizer regime that will promote the development of *S. chamelaeagnea* without degrading soils and leaching nutrients into the water table.

#### **2. Materials and methods**

#### **2.1. Experimental process**

The experiment took place in the research glasshouse at the Cape Peninsula University of Technology (CPUT), Cape Town campus, South Africa, latitude and longitude S33°55′ 58 E18°25′ 57, from June 2012 to August 2012. Inside the glasshouse was a 40%‐Aluminet shade cloth, raised 2 m above the floor, resulting in light intensities ranging from 10 to 13 Klx, determined by using a Toptronic T630 light meter. The climate was controlled between 16 and 28°C during the day while 10–20°C during the night, with an average relative humidity of 42%.

ailments [1–3]. Some of these uses date back to medieval times [4]. Many of the Lamiaceae secondary metabolites are of commercial interest to the food industry as sources of natural preservatives, flavourants and antioxidants [2, 5], as well as to the pharmaceutical industry as sources of antioxidants, anti‐inflammatories [6], antibacterials and anti‐mycobacterials [7]. Salvias are renowned for their variety and their many uses around the home and garden; they have beautiful flowers and attract birds [8]. In its natural habitat, *S. chamelaeagnea* will develop into attractive foliage and flowering landscape plants, with small mid‐green egg‐shaped leaves and masses of bright blue or white flowers borne at the tops of each stem, which are suitable for the cut flower trade [8–10]. *S. chamelaeagnea* also has value in the medicinal plant trade as it contains the phenolic compounds carnosol, rosmarinic acid and caffeic acid, which

Unfortunately, very little information has been documented on the cultivation of this species. Cultivation of medicinal plants is gaining traction worldwide; it is seen as a tool for biodiver‐ sity conservation, poverty alleviation and cultural preservation [12]. However, good knowl‐ edge of plant physiology must be attained in order to develop enhanced cultivation protocols that could result in high yield and high‐quality medicinal materials. Effects of nutrients and nutrient ratios on many food and medicinal crop plants, such as soya bean, thyme, wheat cul‐ tivars, barley, spinach and pelargoniums, have been studied. In most cases, a positive result in growth is noticed with the addition of some macro‐nutrients such as N, P, K, Mg or Ca [13–21]. It is therefore crucial that adequate plant nutrition and soil pH levels are met for any given plant so that the cell's functioning is not impeded. Chlorophyll is a fundamental part of the light‐dependent reactions of the photosynthesis process, capturing light rays from the sun and producing energy‐storing ATP molecules that are essential for the functioning of a healthy plant [22, 23]. The effects of poor nutrition, be it through infertile soils or incorrect soil pH level, directly affect the production of chlorophyll molecules resulting in chlorosis of leaves and a reduced photosynthetic rate, thus inhibiting some biological processes and decreasing the general health of the plants [23–25]. There are plausible mechanisms through which the production of chlorophyll could be affected, for example, the pH level of a growing medium affects the uptake of P [26] and the P level influences the nutrient uptake by plants [27]. The relationship between the nutrient P and chlorophyll is not fully understood. According to Nicholls and Dillon [28], there are substantial variations of the published phosphorus‐chlo‐ rophyll relationship, which they ascribed to variations in sampling and analytical techniques. This chapter aims to investigate the effects of P and pH on the chlorophyll production, leaf colour and the nutrient uptake of medicinal *S. chamelaeagnea* in hydroponics, in order to determine a fertilizer regime that will promote the development of *S. chamelaeagnea* without

The experiment took place in the research glasshouse at the Cape Peninsula University of Technology (CPUT), Cape Town campus, South Africa, latitude and longitude S33°55′ 58

exhibits antioxidant and anti‐bacterial activities [2, 6, 11].

80 Chlorophyll

degrading soils and leaching nutrients into the water table.

**2. Materials and methods**

**2.1. Experimental process**

The experiment was laid out in a randomized block design with plants being spaced 30 cm apart and consisted of 12 treatments of four differing nutrient solutions offering a low con‐ centration of P, a balanced concentration of supplementary P, a moderate concentration of supplementary P and a high concentration of supplementary P at three differing pH levels. The control treatment of 31 ppm was chosen due to the nature of fynbos soils being low in available P [29–31].

Hoagland solution, a well‐known hydroponic nutrient solution modified by Hershey [32, 33], offering all the necessary macro‐ and micro‐nutrients for healthy plant growth, was used as a base nutrient and supplemented with P.

The plants for the experiment were rooted tip cuttings sourced from healthy mother stock plants at the CPUT Glass House Nursery. The rooted cuttings were gently rinsed in deion‐ ized water to remove any rooting media from the root's zone. They were then weighed and planted into 25‐cm plastic pots filled with leca clay and placed into a recirculating closed hydroponics system at a spacing of 30 cm, where their heights were recorded (**Figure 1**).

**Figure 1.** *S. chamelaeagnea* rooted cuttings exposed to varied combinations of pH and P treatments in hydroponics under greenhouse conditions (Picture: K. Lefever).

The plants were irrigated with the treatments 15 times per day at equal timed intervals for the duration of the experiment. For each treatment, there were 10 plants. The treatments were as follows:

	- Hoagland hydroponic nutrient solution with 31 ppm of P at a pH of 6.
	- Hoagland hydroponic nutrient solution with 31 ppm of P at a pH of 8.
	- Hoagland hydroponic nutrient solution supplemented with 90 ppm of P at a pH of 6.
	- Hoagland hydroponic nutrient solution supplemented with 90 ppm of P at a pH of 8.
	- Hoagland hydroponic nutrient solution supplemented with 150 ppm of P at a pH of 6.
	- Hoagland hydroponic nutrient solution supplemented with 150 ppm of P at a pH of 8.
	- Hoagland hydroponic nutrient solution supplemented with 210 ppm of P at a pH of 6.
	- Hoagland hydroponic nutrient solution supplemented with 210 ppm of P at a pH of 8.

#### **2.2. pH level**

The pH levels of the nutrient solutions were monitored using a Martini Instrument PH55 pH probe and were adjusted accordingly using either hydrochloric acid (HCl) to lower the pH or sodium hydroxide (NaOH) to raise the pH.

#### **2.3. Irrigation**

The treatments were set to irrigate 15 times daily for a duration of 15 min using a 1350 L/h Boyu submersible pump and a Tedelex analogue timer to regulate irrigation frequencies.

#### **2.4. Data collection**

#### *2.4.1. Measurement of leaf colour*

Green leaf colour intensity was measured using a hand‐held, dual‐wavelength SPAD meter (SPAD 502, chlorophyll meter, Minolta Camera Co., Ltd., Japan). Readings were taken from the top three fully developed leaves of each plant. For each treatment, 30 fully developed leaves were used weekly. The SPAD meter stored and automatically averaged the recordings to generate one reading per plant.

#### *2.4.2. Measurement of chlorophyll content in leaves*

The plants were irrigated with the treatments 15 times per day at equal timed intervals for the duration of the experiment. For each treatment, there were 10 plants. The treatments were as

**1.** Hoagland hydroponic nutrient solution with 31 ppm of P at a pH of 4.

– Hoagland hydroponic nutrient solution with 31 ppm of P at a pH of 6. – Hoagland hydroponic nutrient solution with 31 ppm of P at a pH of 8.

**2.** Hoagland hydroponic nutrient solution supplemented with 90 ppm of P at a pH of 4.

**3.** Hoagland hydroponic nutrient solution supplemented with 150 ppm of P at a pH of 4.

**4.** Hoagland hydroponic nutrient solution supplemented with 210 ppm of P at a pH of 4.

– Hoagland hydroponic nutrient solution supplemented with 150 ppm of P at a pH of 6. – Hoagland hydroponic nutrient solution supplemented with 150 ppm of P at a pH of 8.

– Hoagland hydroponic nutrient solution supplemented with 210 ppm of P at a pH of 6. – Hoagland hydroponic nutrient solution supplemented with 210 ppm of P at a pH of 8.

The pH levels of the nutrient solutions were monitored using a Martini Instrument PH55 pH probe and were adjusted accordingly using either hydrochloric acid (HCl) to lower the pH or

The treatments were set to irrigate 15 times daily for a duration of 15 min using a 1350 L/h Boyu submersible pump and a Tedelex analogue timer to regulate irrigation frequencies.

Green leaf colour intensity was measured using a hand‐held, dual‐wavelength SPAD meter (SPAD 502, chlorophyll meter, Minolta Camera Co., Ltd., Japan). Readings were taken from the top three fully developed leaves of each plant. For each treatment, 30 fully developed leaves were used weekly. The SPAD meter stored and automatically averaged the recordings

– Hoagland hydroponic nutrient solution supplemented with 90 ppm of P at a pH of 6. – Hoagland hydroponic nutrient solution supplemented with 90 ppm of P at a pH of 8.

follows:

82 Chlorophyll

**2.2. pH level**

**2.3. Irrigation**

**2.4. Data collection**

*2.4.1. Measurement of leaf colour*

to generate one reading per plant.

sodium hydroxide (NaOH) to raise the pH.

The extraction of leaf chlorophyll using dimethylsulphoxide (DMSO) was carried out as described in Hiscox and Israelsta [34]. A third of plant leaves from the tip were collected from each plant. About 100 mg of the middle portion of the fresh leaf slices was placed in a 15‐mL vial containing 7 mL DMSO and incubated at 4°C for 72 h. After the incuba‐ tion, the extract was diluted to 10 mL with DMSO. A 3‐mL sample of chlorophyll extract was then transferred into curvets for absorbance determination. A spectrophotometer (UV/Visible Spectrophotometer, Pharmacia LKB. Ultrospec II E) was used to determine absorbance values at 645 and 663 nm, which were then used in the equation proposed by Arnon [35] to determine the total leaf chlorophyll content against DMSO blank, expressed as mg L‐1 as follows: Chl *a* = 12.7D663 ‐ 2.69D645, Chl *b* = 22.9D645 ‐ 4.68D663 and Total Chl = 20.2D645 + 8.02D663.

#### *2.4.3. Measurement of the levels of macro‐ and micro‐nutrients in dry plant material*

The measurements of macronutrients (N, P, K, Ca and Mg) and micronutrients (Cu, Zn, Mn, Fe and B) were determined by ashing a 1 g ground sample in a porcelain crucible at 500°C overnight. This was followed by dissolving the ash in 5 mL of 6 M HCl and putting it in an oven at 50°C for 30 min; 35 mL of deionized water was added, and the extract was filtered through Whatman no. 1 filter paper. Nutrient concentrations in plant extracts were deter‐ mined using an inductively coupled plasma (ICP) emission spectrophotometer (IRIS/AP HR DUO Thermo Electron Corporation, Franklin, Massachusetts, USA) [36].

#### **2.5. Statistical analysis**

Data collected was analysed for statistical significance using the two‐way analysis of vari‐ ance (ANOVA), with the computations being done using the software program STATISTICA. Fisher's least significance difference (LSD) was used to compare treatment means at *P* ≤ 0.05 level of significance [37].

#### **3. Results**

#### **3.1. Effects of pH and phosphorus concentrations on the chlorophyll content of**  *S. chamelaeagnea* **grown in hydroponics**

Treatment significantly (*P* ≤ 0.001) affected the chlorophyll A, chlorophyll B and total chloro‐ phyll contents of *S. chamelaeagnea* grown hydroponically (**Table 1**). The chlorophyll A (10.9–12.2), chlorophyll B (3–3.4) and total chlorophyll (13.9–14.7) values of the plants exposed to phospho‐ rus at pH 4 treatments were significantly (*P* ≤ 0.001) higher compared to the corresponding values at pH 6 (chlorophyll A [8.3–10.3], chlorophyll B [2.2–2.8] and total chlorophyll [10.7–13.4] and at pH 8—chlorophyll A [3.5–10.17], chlorophyll B [0.91–2.7] and total chlorophyll [4.4–12.9]) treatments (**Table 1**). Leaf chlorosis of plants grown at pH 8 was observed.


a‐j Means followed by same lowercase letters in the same column are not significantly different (*P* > 0.05) following comparison using Tukey test.

\*\*\*represents a statistical significance of (*P* ≤ 0.001) according to Fisher's least significant difference.

**Table 1.** The effects of pH and Phosphorus concentrations on the chlorophyll content of *S. chamelaeagnea* grown in hydroponics.

#### **3.2. Effects of pH and phosphorus concentrations on the leaf colour of** *S. chamelaeagnea* **grown in hydroponics**

Effects of various P treatments at differed pH levels induced varied colour intensities, rang‐ ing from 16 to 31.7 from week 1 to week 8 on the leaf colour of *S. chamelaeagnea* (*P* ≤ 0.001) (**Table 2**, **Figure 2**). While treatment 1 offering a pH level of 4 at 31 ppm P generally yielded the highest leaf colour values over the 8‐week growth period, these values did not differ significantly from that of the other pH 4 treatments receiving supplementary P. Of these treatments receiving supplementary P, the highest results were recorded at pH 4 receiv‐ ing 210 ppm P closely followed by pH 4 at 90 ppm P and pH 4 at 150 ppm P treatments, respectively.

#### **3.3. Effects of pH and phosphorus concentrations on the uptake of macro‐nutrients in**  *S. chamelaeagnea* **grown in hydroponics**

Macro‐nutrient uptake of P, K and Mg was significantly (*P* ≤ 0.001) affected by the treatment (**Table 3**). There was a noticeable higher tissue P content (1.07 ± 0.08%) at pH 8, 150 ppm of P (**Table 3**). Tissue nitrogen content (4.41 ± 0.20%) was significantly higher in plants in treat‐ ment (90 ppm of P) at pH 6. Highest uptake of Ca was recorded at a pH of 8 at 90 ppm of P.

Effects of pH and Phosphorus Concentrations on the Chlorophyll Responses of *Salvia chamelaeagnea*... http://dx.doi.org/10.5772/67610 85


NS represents no statistical significance,

**3.2. Effects of pH and phosphorus concentrations on the leaf colour of** *S. chamelaeagnea*

\*\*\*represents a statistical significance of (*P* ≤ 0.001) according to Fisher's least significant difference.

46.757\*\*\* 43.425\*\*\* 46.388\*\*\*

Means followed by same lowercase letters in the same column are not significantly different (*P* > 0.05) following

**Table 1.** The effects of pH and Phosphorus concentrations on the chlorophyll content of *S. chamelaeagnea* grown in

**Treatments Chlorophyll A Chlorophyll B Total chlorophyll** pH 4, P 31 ppm 12.242 ± 1.7a 3.446 ± 0.5a 15.684 ± 2.2a

pH 8, P 31 ppm 10.173 ± 1.1cde 2.784 ± 0.3ef 12.954 ± 1.5cde pH 4, P 90 ppm 11.419 ± 0.5ab 3.233 ± 0.2ab 14.649 ± 0.6ab pH 6, P 90 ppm 8.348 ± 1.1g 2.227 ± 0.3hi 10.574 ± 1.4g pH 8, P 90 ppm 9.327 ± 1.3ef 2.600 ± 0.4g 11.924 ± 1.7ef pH 4, P 150 ppm 10.929 ± 0.7bc 3.014 ± 0.3bcd 13.941 ± 0.9bc pH 6, P 150 ppm 8.463 ± 1.4fg 2.282 ± 0.4h 10.744 ± 1.8fg pH 8, P 150 ppm 7.063 ± 0.6h 1.988 ± 0.2i 9.049 ± 0.7h pH 4, P 210 ppm 10.900 ± 0.7bc 3.108 ± 0.3bc 14.005 ± 0.9bc pH 6, P 210 ppm 9.817 ± 1.0de 2.650 ± 0.3g 12.465 ± 1.3de pH 8, P 210 ppm 3.547 ± 0.5i 0.910 ± 0.2j 4.456 ± 0.7i

10.384 ± 1.0cd 2.848 ± 0.3cde 13.229 ± 1.3cd

Effects of various P treatments at differed pH levels induced varied colour intensities, rang‐ ing from 16 to 31.7 from week 1 to week 8 on the leaf colour of *S. chamelaeagnea* (*P* ≤ 0.001) (**Table 2**, **Figure 2**). While treatment 1 offering a pH level of 4 at 31 ppm P generally yielded the highest leaf colour values over the 8‐week growth period, these values did not differ significantly from that of the other pH 4 treatments receiving supplementary P. Of these treatments receiving supplementary P, the highest results were recorded at pH 4 receiv‐ ing 210 ppm P closely followed by pH 4 at 90 ppm P and pH 4 at 150 ppm P treatments,

**3.3. Effects of pH and phosphorus concentrations on the uptake of macro‐nutrients in** 

Macro‐nutrient uptake of P, K and Mg was significantly (*P* ≤ 0.001) affected by the treatment (**Table 3**). There was a noticeable higher tissue P content (1.07 ± 0.08%) at pH 8, 150 ppm of P (**Table 3**). Tissue nitrogen content (4.41 ± 0.20%) was significantly higher in plants in treat‐ ment (90 ppm of P) at pH 6. Highest uptake of Ca was recorded at a pH of 8 at 90 ppm of P.

**grown in hydroponics**

comparison using Tukey test.

pH 6, P 31 ppm (Control)

84 Chlorophyll

One‐way ANOVA (*F*‐statistic)

hydroponics.

a‐j

respectively.

*S. chamelaeagnea* **grown in hydroponics**

\*represents a statistical significance of (*P* ≤ 0.05),

\*\*represents a statistical significance of (*P* ≤ 0.01) and

\*\*\*represents a statistical significance of (*P* ≤ 0.001) according to Fisher's least significant difference.

**Table 2.** The effects of pH and phosphorus concentrations on the leaf colour of *S. chamelaeagnea* grown in hydroponics.

#### **3.4. Effects of pH and phosphorus concentrations on the uptake of micro‐nutrients in**  *S. chamelaeagnea* **grown in hydroponics**

The micro‐nutrient uptake of Na, Mn, Fe, Cu, Zn and B was significantly (*P* ≤ 0.001) affected by the treatments (**Table 4**). The highest nutrient uptake values of Na (867.67 ± 131.72%) and Zn (46.78 ± 7.31%) were recorded at pH 8, 210 ppm of P treatment. The Fe uptake value (175.00 ± 14.42%) in the treatment at pH 4 of 210 ppm was the highest value. Highest recorded uptake values of Cu were obtained in plants receiving a pH of 4 at 31 ppm of P closely followed by the plants receiving a pH of 4 at 210 ppm.

**Figure 2.** Observable variations in the leaf's green colour among plants (*S. chamelaeagnea*) following exposure to varied combinations of pH and P treatments in hydroponics under greenhouse conditions (Picture: K. Lefever).


NS represents no statistical significance,

\*represents a statistical significance of (*P* ≤ 0.05),

\*\*represents a statistical significance of (*P* ≤ 0.01) and

\*\*\*represents a statistical significance of (*P* ≤ 0.001) according to Fisher's least significant difference.

**Table 3.** The effects of pH and Phosphorus concentrations on the uptake of macro‐nutrients in *S. chamelaeagnea* grown in hydroponics.


NS represents no statistical significance,

\*represents a statistical significance of (*P* ≤ 0.05),

\*\*represents a statistical significance of (*P* ≤ 0.01) and

\*\*\*represents a statistical significance of (*P* ≤ 0.001) according to Fisher's least significant difference.

**Table 4.** The effects of pH and phosphorus concentrations on the uptake of micro‐nutrients in *S. chamelaeagnea* grown in hydroponics.

#### **4. Discussions**

**Figure 2.** Observable variations in the leaf's green colour among plants (*S. chamelaeagnea*) following exposure to varied

4.35\*\*\* 21.34\*\*\* 31.67\*\*\* 68.64\*\*\* 89.74\*\*\*

**Table 3.** The effects of pH and Phosphorus concentrations on the uptake of macro‐nutrients in *S. chamelaeagnea* grown

\*\*\*represents a statistical significance of (*P* ≤ 0.001) according to Fisher's least significant difference.

combinations of pH and P treatments in hydroponics under greenhouse conditions (Picture: K. Lefever).

**Treatments N (%) P (%) K (%) Ca (%) Mg (%)** pH 4, P 31 ppm 4.18 ± 0.29bcd 0.64 ± 0.06g 4.23 ± 0.29g 1.13 ± 0.07a 0.28 ± 0.01h pH 6, P 31 ppm 4.24 ± 0.55abc 0.73 ± 0.06f 4.41 ± 0.23fg 1.12 ± 0.10a 0.36 ± 0.03e pH 8, P 31 ppm 4.19 ± 0.18abcd 0.62 ± 0.08g 4.47 ± 0.26efg 1.10 ± 0.11ab 0.43 ± 0.04c pH 4, P 90 ppm 4.27 ± 0.26abc 0.77 ± 0.08ef 4.64 ± 0.36cdef 1.10 ± 0.10ab 0.31 ± 0.02g pH 6, P 90 ppm 4.41 ± 0.20a 0.82 ± 0.08cde 4.53 ± 0.13defg 1.08 ± 0.07ab 0.38 ± 0.03d pH 8, P 90 ppm 4.20 ± 0.12abcd 0.80 ± 0.07def 4.45 ± 0.24efg 1.14 ± 0.05a 0.48 ± 0.03b pH 4, P 150 ppm 4.37 ± 0.19ab 0.82 ± 0.04cde 4.79 ± 0.58cd 1.07 ± 0.06abc 0.32 ± 0.03fg pH 6, P 150 ppm 4.09 ± 0.22cd 0.88 ± 0.07bc 4.87 ± 0.19c 1.01 ± 0.07cd 0.36 ± 0.02de pH 8, P 150 ppm 4.00 ± 0.08de 1.07 ± 0.08a 6.29 ± 0.39a 0.77 ± 0.03e 0.55 ± 0.02a pH 4, P 210 ppm 4.13 ± 0.18cd 0.84 ± 0.06bcd 4.73 ± 0.32cde 1.05 ± 0.06bc 0.31 ± 0.02g pH 6, P 210 ppm 4.05 ± 0.18cd 0.87 ± 0.08bcd 4.23 ± 0.35g 0.97 ± 0.06d 0.35 ± 0.02ef pH 8, P 210 ppm 3.77 ± 0.16e 0.91 ± 0.13b 5.71 ± 0.38b 0.46 ± 0.03f 0.48 ± 0.03b

One‐way ANOVA (*F*‐statistic)

86 Chlorophyll

in hydroponics.

NS represents no statistical significance, \*represents a statistical significance of (*P* ≤ 0.05), \*\*represents a statistical significance of (*P* ≤ 0.01) and In this chapter, the significantly (*P* ≤ 0.001) higher chlorophyll values recorded in the treat‐ ments at a pH of 4 with supplementary P show that phosphorous fertilization under an acidic condition of chlorophyll production by *S. chamelaeagnea* will largely increase in hydroponic production. Also, high leaf colour intensity values were recorded in treatments with a pH of 4 compared to that of treatments with a higher pH of 6 or 8. On the other hand, it seems that a higher P concentration had a minimal effect on leaf colour intensity. It is worth noting that studies have shown high correlations between chlorophyll meter readings, that is, the leaf's green colour intensity and extractable leaf chlorophyll [38]. The effect of P on chlorophyll could be indirect and complex. P fertilization may indirectly influence or hinder the uptake of other nutrients [39], which in turn affects chlorophyll production in plants. Indigenous plants, especially those occurring in the fynbos biome, are expected to be adapted to nutrient‐poor and low‐pH soils and tend to have low critical levels for most of the nutrients. Therefore, exposing these species to high P concentration may have a minimal effect on plant physiology and can even have detrimental effects on plant growth.

Despite the relatively high nutrient uptake values in plants receiving a nutrient solution with a pH 8, chlorosis of their leaves was apparent during the growth period. This suggests that the uptake of some essential nutrients responsible for chlorophyll development was affected at this pH level, namely the mineral nutrients Cu, B, N and Fe which are directly involved in photosynthesis, respiration, cell division and protein formation [23, 40]. In soil‐less media, the affinity of soluble nutrients to negatively charged surfaces and the interactions between charged cations can have a profound effect on nutrient availability and subsequently, the uptake of nutrients by plants. For example, fertilization with phosphorous increases the soil's nitrogen absorption in young plants of *Eucalyptus grandis* [39]. Silber [41] argued that a con‐ tinuous decline of soluble P concentration during fertilization can be explained through two mechanisms, a rapid electrostatic reaction and adsorption of the onto substrate and a slow formation of solid metal‐P compounds with Al and Fe under acidic conditions and Ca and Mg under basic‐to‐neutral conditions. Therefore, the substrate used in hydroponic setups could affect the availability of micro‐ and macro‐nutrients. Shen et al. [42] suggested that the avail‐ ability of soil P is extremely complex and needs to be systemically evaluated. Previously, Wu et al. [43] showed that under phosphorus stress, no significant changes in chlorophyll A and B, total chlorophyll and carotenoid contents were found, and phosphorus stress generally had no effect on photosynthesis. The highest nutrient uptake values were recorded in nine of the 12 treatments receiving supplementary P, with only Cu and Mn yielding the highest values in treatments receiving no supplementary P. Thus, it is evident that phosphorus treatments had a significant effect on nutrient uptake in *S. chamelaeagnea* grown hydroponically [44].

#### **5. Conclusions**

In conclusion, this chapter gives insight into the unknown cultivation requirements of the leaf's chlorophyll development of *S. chamelaeagnea* and shows that the use of a hydroponic nutrient system offering little to no supplementary phosphorus at a pH level of 4 significantly correlated with the chlorophyll development of *S. chamelaeagnea* grown in hydroponics. Based on the results obtained in the chapter, it is plausible to assume that P has an indirect effect on chlorophyll production in *S. chamelaeagnea*.

#### **Acknowledgements**

This study was funded by Cape Peninsula University of Technology through CPUT Bursary and University Research Funds.

#### **Author details**

studies have shown high correlations between chlorophyll meter readings, that is, the leaf's green colour intensity and extractable leaf chlorophyll [38]. The effect of P on chlorophyll could be indirect and complex. P fertilization may indirectly influence or hinder the uptake of other nutrients [39], which in turn affects chlorophyll production in plants. Indigenous plants, especially those occurring in the fynbos biome, are expected to be adapted to nutrient‐poor and low‐pH soils and tend to have low critical levels for most of the nutrients. Therefore, exposing these species to high P concentration may have a minimal effect on plant physiology

Despite the relatively high nutrient uptake values in plants receiving a nutrient solution with a pH 8, chlorosis of their leaves was apparent during the growth period. This suggests that the uptake of some essential nutrients responsible for chlorophyll development was affected at this pH level, namely the mineral nutrients Cu, B, N and Fe which are directly involved in photosynthesis, respiration, cell division and protein formation [23, 40]. In soil‐less media, the affinity of soluble nutrients to negatively charged surfaces and the interactions between charged cations can have a profound effect on nutrient availability and subsequently, the uptake of nutrients by plants. For example, fertilization with phosphorous increases the soil's nitrogen absorption in young plants of *Eucalyptus grandis* [39]. Silber [41] argued that a con‐ tinuous decline of soluble P concentration during fertilization can be explained through two mechanisms, a rapid electrostatic reaction and adsorption of the onto substrate and a slow formation of solid metal‐P compounds with Al and Fe under acidic conditions and Ca and Mg under basic‐to‐neutral conditions. Therefore, the substrate used in hydroponic setups could affect the availability of micro‐ and macro‐nutrients. Shen et al. [42] suggested that the avail‐ ability of soil P is extremely complex and needs to be systemically evaluated. Previously, Wu et al. [43] showed that under phosphorus stress, no significant changes in chlorophyll A and B, total chlorophyll and carotenoid contents were found, and phosphorus stress generally had no effect on photosynthesis. The highest nutrient uptake values were recorded in nine of the 12 treatments receiving supplementary P, with only Cu and Mn yielding the highest values in treatments receiving no supplementary P. Thus, it is evident that phosphorus treatments had

a significant effect on nutrient uptake in *S. chamelaeagnea* grown hydroponically [44].

In conclusion, this chapter gives insight into the unknown cultivation requirements of the leaf's chlorophyll development of *S. chamelaeagnea* and shows that the use of a hydroponic nutrient system offering little to no supplementary phosphorus at a pH level of 4 significantly correlated with the chlorophyll development of *S. chamelaeagnea* grown in hydroponics. Based on the results obtained in the chapter, it is plausible to assume that P has an indirect effect on

This study was funded by Cape Peninsula University of Technology through CPUT Bursary

and can even have detrimental effects on plant growth.

**5. Conclusions**

88 Chlorophyll

**Acknowledgements**

and University Research Funds.

chlorophyll production in *S. chamelaeagnea*.

Kerwin Lefever<sup>1</sup> , Charles P. Laubscher1 \*, Patrick A. Ndakidemi2 and Felix Nchu1

\*Address all correspondence to: laubscherc@cput.ac.za

1 Faculty of Applied Sciences, Cape Peninsula University of Technology, Bellville, South Africa

2 School of Life Sciences and Bio‐engineering, Nelson Mandela Institute of Science and Technology, Arusha, Tanzania

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[15] Ichir, L.L., Ismaili, M. & van Cleemput, O. 2003. Effect of organic and mineral fertilizers on N‐use by wheat under different irrigation frequencies. *Competes Rendus Biologies*.

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**Provisional chapter**

### **Light‐Emitting Diodes: Progress in Plant Micropropagation Micropropagation**

**Light**‐**Emitting Diodes: Progress in Plant** 

Jericó J. Bello‐Bello, Juan A. Pérez‐Sato, Carlos A. Cruz‐Cruz and Eduardo Martínez‐Estrada Carlos A. Cruz‐Cruz and Eduardo Martínez‐Estrada

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Jericó J. Bello‐Bello, Juan A. Pérez‐Sato,

http://dx.doi.org/10.5772/67913

#### **Abstract**

[42] Shen, J., Yuan, L., Zhang, L., Li, H., Bai, Z., Chen, X., Zhang, W. & Zhang, F. 2011.

[43] Wu C., Fan Z. & Wang, Z. 2004. Effect of phosphorus stress on chlorophyll biosynthesis, photosynthesis and biomass partitioning pattern of *Fraxinus mandchurica* seedlings]. *The* 

[44] Naeem, M., Khan, M.M.A., Idrees, M. & Aftab, T. 2010. Phosphorus ameliorates crop productivity, photosynthetic efficiency, nitrogen‐fixation, activities of the enzymes and content of nutraceuticals of *Lablab purpureus* L. *Scientia Horticulturae*. 126:205–214.

Phosphorus dynamics: From soil to plant. *Plant Physiology*. 156:997–1005.

*Journal of Applied Ecology*. 15(6):935–940.

92 Chlorophyll

In commercial micropropagation laboratories, the light source is one of the most important factors controlling plant morphogenesis and metabolism of plant cells and tissue and organ cultures. Lamp manufacturers have begun to rate lamps specifically for plant needs. The traditional light source used for in vitro propagation is fluorescent lamps (FLs). However, power consumption in FL use is expensive and produces a wide range of wavelengths (350–750 nm) unnecessary for plant development. Light‐emitting diodes (LEDs) have recently emerged as an alternative for commercial micropropagation. The flexibility of matching LED wavelengths to plant photoreceptors may provide more optimal production, influencing plant morphology and chlorophyll content. Although previous reports have confirmed physiological effects of LED light quality on morphogenesis and growth of several plantlets in vitro, these study results showed that LED light is more suitable for plant morphogenesis and growth than FLs. However, the responses vary according to plant species. This chapter describes the applications and benefits of LED lamps on chlorophyll in plant micropropagation. Two study cases are exposed, Anthurium (*Anthurium andreanum*) and moth orchids (*Phalaenopsisis* sp.), both species with economic importance as ornamental plants, where LEDs have a positive effect on in vitro development and chlorophyll content.

**Keywords:** in vitro cloning, light quality, tissue culture, chlorophyll

#### **1. Introduction**

Micropropagation or in vitro plant cloning is being widely used for large‐scale plant multi‐ plication. This method enables the identical reproduction of the selected parents, following

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the maintenance of genetic fidelity. In commercial micropropagation laboratories, the light source is one of the most important factors controlling plant morphogenesis and growth cells, tissue and organ cultures. Lamp manufacturers have begun to rate lamps specifically for plant needs. The traditional light source used for in vitro propagation is fluorescent lamps (FLs). Nevertheless, the power consumption in FLs is expensive and produces a wide range of wavelengths (350–750 nm) unnecessary for plant development. Light‐emitting diodes (LEDs) have recently emerged as an alternative for commercial micropropagation. LEDs possess advantages such as less heat radiation, a monochromatic spectrum, greater durability, and low power consumption. The LED illumination system for in vitro culture provides light in the spectral region that is involved in photosynthesis and in the photomorphogenic responses in plants.

LED colors or combinations commonly used for in vitro culture are white, red, blue, and mixture rates of blue and red. It has been reported that red light is important for shoot and stem elongation, phytochrome responses and changes in plant anatomy [1]. In contrast, blue light is important in chlorophyll biosynthesis, stomatal opening, chloroplast maturation, and photosynthesis [2]. Blue and red combination LEDs have been used for studies in many areas of photobiological research such as photosynthesis [3] and chlorophyll synthesis [4].

In addition, several studies have shown positive effects of LED lamps on plant development during in vitro culture of different species such as *Fragaria* × *ananassa* [5, 6], *Musa* spp. [7], *Solanum tuberosum* [8], *Chrysanthemum* [9, 10], *Vitis riparia* × *V. vinifera* [11], *Brassica napus* [12], *Populus euroamericana* [13], and *Saccharum* spp. [14], among others. However, the response in LED systems depends on the wavelength to which the plants are exposed and varies according to the species [15].

This chapter describes the applications and benefits of LED lamps on chlorophyll in plant micropropagation. Two study cases are exposed, Anthurium (*Anthurium andreanum* Lind.) and moth orchids (*Phalaenopsisis* sp.), both species with economic importance as ornamental plants, where LEDs have had a positive effect on in vitro development and chlorophyll content.

#### **2. Plant micropropagation**

Micropropagation is the asexual propagation of plants using the techniques of plant tis‐ sue culture (PTC). Plant tissue culture refers to growing and differentiation of cells, tis‐ sues, and organs isolated from the mother plant, on artificial solid or liquid media under aseptic and controlled conditions. The small organs or pieces of tissue plants used in PTC are called explants. Plant tissue culture medium provides inorganic nutrients and usually a carbohydrate to replace the carbon which the plant normally fixes from the atmosphere by photosynthesis. When carbon is supplied with sucrose and kept in low light conditions, micropropagated plantlets are not fully dependent on their own photosynthesis.

The PTC techniques provide a new approach to plant propagation, being the best way to produce uniform plant germplasm and the regeneration of pathogen‐free plants. To date, commercial plant micropropagation has shown great productive potential; it is being used in hundreds of commercial laboratories for the propagation of species of agricultural and for‐ estry importance. Commercial micropropagation of different species of economic importance is shown in **Figure 1**.

The commercial micropropagation process is carried out in the following stages:

the maintenance of genetic fidelity. In commercial micropropagation laboratories, the light source is one of the most important factors controlling plant morphogenesis and growth cells, tissue and organ cultures. Lamp manufacturers have begun to rate lamps specifically for plant needs. The traditional light source used for in vitro propagation is fluorescent lamps (FLs). Nevertheless, the power consumption in FLs is expensive and produces a wide range of wavelengths (350–750 nm) unnecessary for plant development. Light‐emitting diodes (LEDs) have recently emerged as an alternative for commercial micropropagation. LEDs possess advantages such as less heat radiation, a monochromatic spectrum, greater durability, and low power consumption. The LED illumination system for in vitro culture provides light in the spectral region that is involved in photosynthesis and in the photomorphogenic responses

LED colors or combinations commonly used for in vitro culture are white, red, blue, and mixture rates of blue and red. It has been reported that red light is important for shoot and stem elongation, phytochrome responses and changes in plant anatomy [1]. In contrast, blue light is important in chlorophyll biosynthesis, stomatal opening, chloroplast maturation, and photosynthesis [2]. Blue and red combination LEDs have been used for studies in many areas

In addition, several studies have shown positive effects of LED lamps on plant development during in vitro culture of different species such as *Fragaria* × *ananassa* [5, 6], *Musa* spp. [7], *Solanum tuberosum* [8], *Chrysanthemum* [9, 10], *Vitis riparia* × *V. vinifera* [11], *Brassica napus* [12], *Populus euroamericana* [13], and *Saccharum* spp. [14], among others. However, the response in LED systems depends on the wavelength to which the plants are exposed and varies

This chapter describes the applications and benefits of LED lamps on chlorophyll in plant micropropagation. Two study cases are exposed, Anthurium (*Anthurium andreanum* Lind.) and moth orchids (*Phalaenopsisis* sp.), both species with economic importance as ornamental plants, where LEDs have had a positive effect on in vitro development and chlorophyll

Micropropagation is the asexual propagation of plants using the techniques of plant tis‐ sue culture (PTC). Plant tissue culture refers to growing and differentiation of cells, tis‐ sues, and organs isolated from the mother plant, on artificial solid or liquid media under aseptic and controlled conditions. The small organs or pieces of tissue plants used in PTC are called explants. Plant tissue culture medium provides inorganic nutrients and usually a carbohydrate to replace the carbon which the plant normally fixes from the atmosphere by photosynthesis. When carbon is supplied with sucrose and kept in low light conditions,

The PTC techniques provide a new approach to plant propagation, being the best way to produce uniform plant germplasm and the regeneration of pathogen‐free plants. To date,

micropropagated plantlets are not fully dependent on their own photosynthesis.

of photobiological research such as photosynthesis [3] and chlorophyll synthesis [4].

in plants.

94 Chlorophyll

content.

according to the species [15].

**2. Plant micropropagation**

**Stage 0: Mother plant selection.** Donor plants are selected and conditioned to be used to initi‐ ate in vitro cultures.

**Stage I: In vitro establishing.** The choice of the explant and its disinfection is carried out to initiate an aseptic culture.

**Stage II: Multiplication.** It is at this stage that mass propagation is performed, obtaining a large number of new individuals from minimal amounts of tissue.

**Stage III: Elongation and rooting.** The shoots must form their root system and at the same time increase their size to facilitate their manipulation and adaptation to the acclimatization conditions.

**Stage IV: Acclimatization.** It consists of a slow reduction of the relative humidity and gradual increases in the luminous intensity for a better adaptation to the external environment.

**Figure 1.** Commercial micropropagation of different species. (a) *Stevia rebaudiana*, (b) *Ananas comosus*, (c) *Vanilla planifolia* and (d) *Anthurium andreanum*.

Requirements for the completion of each stage of micropropagation vary according to the method being utilized; it is not always necessary to follow each of the prescribed steps.

However, there are factors that affect the micropropagation process, including:

**Factors that depend on the explant:** Size, physiological age of the tissue, and explant position.

**Factors that depend on the culture medium:** Growth regulators, macro‐ and micronutrients, organic nitrogen, and carbon source.

**Factors related to the incubation environment:** Photoperiod, temperature, humidity, and light source.

Factors related to the incubation environment refer to incubators or growth rooms where temperature, humidity, and light can be controlled. In commercial micropropagation laboratories, the light source is one of the most important factors controlling plant development. Light quality (spectral quality), quantity, (photon flux) and photoperiod have a profound influence on the morphogenesis, growth and chlorophyll contents of a plant cell, and tissue and organ cultures.

The illumination systems allow wavelengths to be matched to plant photoreceptors to provide more optimal production and to influence plant morphology and metabolic composition [16]. Plants use energy between 400 and 700 nm and light in this region is called photosynthetically active radiation (PAR).

The growth and development of plants is dependent on light for:

**Photosynthesis:** The process whereby light energy is converted to chemical energy in the biosynthesis of chemicals from carbon dioxide and water.

**Photomorphogenesis:** The light‐induced development of structure or form.

**Phototropism:** The growth response of plants which is induced by unilateral light.

In recent years, LEDs have emerged as an alternative for commercial micropropagation. LEDs possess various advantages such as less heat radiation, small mass, a monochromatic spectrum, greater durability, low power consumption, and specific wavelength. The flexibility of matching LED wavelengths to plant photoreceptors may provide more optimal production, influencing plant morphology and metabolism.

#### **3. Spectral quality of LEDs**

The traditional light source used for in vitro propagation is fluorescent lamps (FLs). However, power consumption in FL use is expensive and produces a wide range of wavelengths (350–750 nm) unnecessary for plant development, whereas monochromatic light‐emitting diodes (LEDs) emit light at specific wavelengths. In this sense, LEDs can be fine‐tuned to only produce the spectrums that plants need for morphogenic responses [17]. The response to LED light in micropropagation systems depends on light irradiance, photoperiod, and wavelength. The wavelength to which in vitro plants are exposed varies according to the species. Recent studies compare the effect of FLs (545–610 nm) vs white LEDs (460 and 560 nm), red LEDs (660 nm), blue LEDs (460 nm), and the combination of blue and red LED (460 and 660 nm) treatments. LEDs affect in vitro rooting, number and length of new shoots, chlorophyll and carotenoid pigments, and other characteristics in plants. The spectral irradiance of LEDs is shown in **Figure 2**.

**Figure 2.** Spectral curves distribution in relative response of the LEDs and fluorescent lamps.

#### **4. LEDs affect chlorophyll content**

Requirements for the completion of each stage of micropropagation vary according to the method being utilized; it is not always necessary to follow each of the prescribed steps.

**Factors that depend on the explant:** Size, physiological age of the tissue, and explant position. **Factors that depend on the culture medium:** Growth regulators, macro‐ and micronutrients,

**Factors related to the incubation environment:** Photoperiod, temperature, humidity, and

Factors related to the incubation environment refer to incubators or growth rooms where temperature, humidity, and light can be controlled. In commercial micropropagation laboratories, the light source is one of the most important factors controlling plant development. Light quality (spectral quality), quantity, (photon flux) and photoperiod have a profound influence on the morphogenesis, growth and chlorophyll contents of a plant cell,

The illumination systems allow wavelengths to be matched to plant photoreceptors to provide more optimal production and to influence plant morphology and metabolic composition [16]. Plants use energy between 400 and 700 nm and light in this region is called photosynthetically

**Photosynthesis:** The process whereby light energy is converted to chemical energy in the

In recent years, LEDs have emerged as an alternative for commercial micropropagation. LEDs possess various advantages such as less heat radiation, small mass, a monochromatic spectrum, greater durability, low power consumption, and specific wavelength. The flexibility of matching LED wavelengths to plant photoreceptors may provide more optimal production,

The traditional light source used for in vitro propagation is fluorescent lamps (FLs). However, power consumption in FL use is expensive and produces a wide range of wavelengths (350–750 nm) unnecessary for plant development, whereas monochromatic light‐emitting diodes (LEDs) emit light at specific wavelengths. In this sense, LEDs can be fine‐tuned to only produce the spectrums that plants need for morphogenic responses [17]. The response to LED light in micropropagation systems depends on light irradiance, photoperiod, and wavelength. The wavelength to which in vitro plants are exposed varies according to the species. Recent

The growth and development of plants is dependent on light for:

**Photomorphogenesis:** The light‐induced development of structure or form.

**Phototropism:** The growth response of plants which is induced by unilateral light.

biosynthesis of chemicals from carbon dioxide and water.

influencing plant morphology and metabolism.

**3. Spectral quality of LEDs**

However, there are factors that affect the micropropagation process, including:

organic nitrogen, and carbon source.

and tissue and organ cultures.

active radiation (PAR).

light source.

96 Chlorophyll

Several studies have shown important effects of LEDs on photosynthetic pigments during micropropagation of different species. Studies show that blue LEDs are a good light source for chlorophyll induction and that red LEDs decrease chlorophyll content. Dewir et al. [15] found that blue LEDs showed greater growth, vigor, and chlorophyll content in *Euphorbia milli*. Jao et al. [18] reported that blue LEDs promote growth and increase chlorophyll content in *Zantedeschia jucunda*. The same effect was observed by Li et al. [19, 20] during in vitro culture of *Gossypium hirsutum* and *Brassica campestris*, respectively. Kim et al. [9] and Moon et al. [21] emphasized the role of blue light on chlorophyll formation and chloroplast development in their work with *Chrysanthemum* and *Tripterospermum japonicum*, respectively. Monochromatic red LEDs with narrow peak emissions may cause an imbalance in the distribution of light energy between photosystems I and II, and thus be responsible for a reduction in net photosynthesis [3]. According to Li et al. [19], it has been observed that plantlets with lower chlorophyll content utilize the chlorophyll more efficiently than plantlets with higher chloro‐ phyll content under red LEDs.

According to Soebo et al. [22], the possibility exists that red light may inhibit the translocation of photosynthetic products thereby increasing the accumulation of starch. Goins et al. [23] observed higher photosynthetic rates and an increase in stomatal conductance in wheat leaves under mixed red and blue LEDs. Plant growth and development by increasing net photosynthetic rate was also observed in *Chrysanthemum* under mixed red/blue LED treatments and has been attributed to the similarities of the spectral energy distribution of red/blue to chlorophyll absorption [9].

The importance of blue light in stomatal opening has already been studied. It has been proposed that blue light received by phototropins activates a signaling cascade, resulting in fast stomata opening under a red light background [19]. The effect of light quality on stomatal characteristics has not yet been clearly determined, and differential stomatal behavior could be related to photosynthetic activity and plant growth.

According to Topchiy et al. [24], light quality also plays an important role in photosynthesis, influencing the way in which light is absorbed by chlorophyll. According to George [25], the level of chlorophyll so far obtained in tissue cultures is well below that found in mesophyll cells of whole plants of the same species, and the rate of chlorophyll formation on exposure of cultured cells to the light is extremely slow compared to the response of etiolated organized tissues. The greening of cultures also tends to be unpredictable, and even within individual cells, a range in the degree of chloroplast development is often found. In the carbon dioxide concentrations found in culture vessels, green callus tissue is normally photomixotrophic and growth is still partly dependent on the incorporation of sucrose into the medium [25]. However, green photoautotrophic callus cultures have been obtained from several different kinds of plants.

#### **5. Study cases**

Anthurium (*A. andreanum* Lind.) and moth orchids (*Phalaenopsis*is sp.) are tropical species with worldwide economic importance as ornamental plants and cut flowers. These species are commonly propagated by suckers; however, this propagation method is relatively slow and can cause disease transmission. Micropropagation has emerged as an alternative for fast mass production of *A. andreanum* and *Phalaenopsisis* plants of high phytosanitary quality.

For *A. andreanum*, nodal segments were excised from in vitro‐derived adventitious shoots and were used as explants. For in vitro culture of *Phalaenopsis*is, protocorms were used as explants. The explants were placed in a 500 ml jar containing 40 ml of MS [26] medium with‐ out growth regulators. The pH of the culture medium was adjusted to 5.8 with 0.1 N sodium hydroxide, 0.25% (w/v) Phytagel was added as a gelling agent and then it was autoclaved for 15 min at 120°C and 117.7 kPa. The nodal segments were exposed to white LEDs (460 and 560 nm), red LEDs (660 nm), blue LEDs (460 nm), the combination of blue and red LEDs (460 and 660 nm, respectively), and FLs (545–610 nm) as a control. The LED system (model: 5050–1M‐RGB, 3M, MN, USA) consisted of strips remotely controlled with a 12 V DC power adapter (model: SDK‐0605, 3M, MN, USA). The explants were incubated at 24 ± 2°C and for 16 h light photoperiod. In all treatments, the photosynthetic photon flux density (PPFD) was maintained to 25 μmol m−2 s−1. PPFD was measured using a FieldScout Quantum Light Meter®.

After 60 days of in vitro culture, shoot length (cm), number of leaves, rooted shoots, and chlorophyll *a*, chlorophyll *b*, and total chlorophyll contents were evaluated. Chlorophyll content was determined according to the method of Harborne [27]. For experimental design and data analysis, a completely randomized experimental design was used for all experiments. For each treatment, ten culture vessels, containing three explants each, were used. An analysis of variance (ANOVA) and Tukey's comparison of means test (*p* ≤ 0.05) were performed for each species using SPSS statistical software (version 22 for Windows).

observed higher photosynthetic rates and an increase in stomatal conductance in wheat leaves under mixed red and blue LEDs. Plant growth and development by increasing net photosynthetic rate was also observed in *Chrysanthemum* under mixed red/blue LED treatments and has been attributed to the similarities of the spectral energy distribution of

The importance of blue light in stomatal opening has already been studied. It has been proposed that blue light received by phototropins activates a signaling cascade, resulting in fast stomata opening under a red light background [19]. The effect of light quality on stomatal characteristics has not yet been clearly determined, and differential stomatal behavior could

According to Topchiy et al. [24], light quality also plays an important role in photosynthesis, influencing the way in which light is absorbed by chlorophyll. According to George [25], the level of chlorophyll so far obtained in tissue cultures is well below that found in mesophyll cells of whole plants of the same species, and the rate of chlorophyll formation on exposure of cultured cells to the light is extremely slow compared to the response of etiolated organized tissues. The greening of cultures also tends to be unpredictable, and even within individual cells, a range in the degree of chloroplast development is often found. In the carbon dioxide concentrations found in culture vessels, green callus tissue is normally photomixotrophic and growth is still partly dependent on the incorporation of sucrose into the medium [25]. However, green photoautotrophic callus cultures have been obtained from several different

Anthurium (*A. andreanum* Lind.) and moth orchids (*Phalaenopsis*is sp.) are tropical species with worldwide economic importance as ornamental plants and cut flowers. These species are commonly propagated by suckers; however, this propagation method is relatively slow and can cause disease transmission. Micropropagation has emerged as an alternative for fast mass production of *A. andreanum* and *Phalaenopsisis* plants of high phytosanitary quality.

For *A. andreanum*, nodal segments were excised from in vitro‐derived adventitious shoots and were used as explants. For in vitro culture of *Phalaenopsis*is, protocorms were used as explants. The explants were placed in a 500 ml jar containing 40 ml of MS [26] medium with‐ out growth regulators. The pH of the culture medium was adjusted to 5.8 with 0.1 N sodium hydroxide, 0.25% (w/v) Phytagel was added as a gelling agent and then it was autoclaved for 15 min at 120°C and 117.7 kPa. The nodal segments were exposed to white LEDs (460 and 560 nm), red LEDs (660 nm), blue LEDs (460 nm), the combination of blue and red LEDs (460 and 660 nm, respectively), and FLs (545–610 nm) as a control. The LED system (model: 5050–1M‐RGB, 3M, MN, USA) consisted of strips remotely controlled with a 12 V DC power adapter (model: SDK‐0605, 3M, MN, USA). The explants were incubated at 24 ± 2°C and for 16 h light photoperiod. In all treatments, the photosynthetic photon flux density (PPFD) was maintained to 25 μmol m−2 s−1. PPFD was measured using a FieldScout Quantum Light

red/blue to chlorophyll absorption [9].

kinds of plants.

98 Chlorophyll

**5. Study cases**

Meter®.

be related to photosynthetic activity and plant growth.

For *A. andreanum,* treatments with white LEDs, blue LEDs, and the combination of blue and red LEDs showed the greatest plantlet length and number of leaves. The FL and red LED treatments showed similar responses in promoting the formation of plantlets and their leaves. All shoots were rooted and the highest root number was induced in cultures incubated in FLs and blue LEDs with 6.6 and 6.0 roots, respectively. The lowest root number (1.5) was recorded in cultures incubated in red LEDs (**Table 1**). Chlorophyll a, b, and total chlorophyll content was significantly higher in the blue LED treatment (0.692 mg g−1 fresh weight), while the lowest total chlorophyll content was found in the red LED and FL treatments with 0.327 and 0.375 mg g−1 fresh weight, respectively (**Figure 3a**).

In *Phalaenopsisis,* treatments with FLs, white LED and the combination of blue and red LEDs showed the greatest plantlet length and number of leaves (**Table 1**) The white, red and blue LEDs showed similar responses in promoting the formation of plantlets and their leaves. All protocorms were rooted and had the same root number. Chlorophyll a content was significantly higher in the blue LED treatment (0.2813 mg g−1 fresh weight), while chlorophyll b content was higher in blue and the combination of blue and red LED treatments, with 0.1368 and 0.1468 mg g−1 fresh weight, respectively. Total chlorophyll (0.421875 mg g−1 fresh weight)


**Table 1.** Effect of LEDs on in vitro growth and rooting of *Anthurium andreanum* cv. Rosa and *Phalaenopsis* sp after 60 days of culture.

was higher in blue LED. The lowest total chlorophyll content was found in FL treatments and white LEDs with 0.1810 and 0.2500 mg g−1 fresh weight, respectively (**Figure 3b**).

Our results indicate that FLs can be replaced by LEDs. The same effect was observed by Kurilčik et al. [10] and Lin et al. [28] during in vitro development of *Chrysanthemum* plantlets and *Dendrobium officinale* protocorms, respectively. In *Phalaenopsis*, LEDs had no effect on the number of roots, while in *A. andreanum* the highest number of roots was obtained in FLs and

**Figure 3.** Effect of light quality on chlorophyll content in *Anthurium andreanum* (a) and *Phalaenopsis*is sp. (b) after 60 days of culture in vitro. Different letters denote statistically significant differences according to Tukey's multiple range test at *p* ≤ 0.05. Bars represent mean ± SE.

blue LEDs. Similar results were reported by Cybularz‐Urban et al. [29] and Waman et al. [7] in *Cattleya* and *Musa* spp., respectively.

According to Topchiy et al. [24], light quality also plays an important role in photosynthesis, influencing the way in which light is absorbed by chlorophyll. The present results demon‐ strated that the chlorophyll a, chlorophyll b, and total chlorophyll content appeared greater in plantlets growing under treatments containing blue light. Similar results were reported by Dewir et al. [15] where blue LEDs showed greater growth, vigor, and chlorophyll content in *E. milli*. Jao et al. [18] reported that blue LEDs promote growth and increase chlorophyll content in *Zantedeschia jucunda*. Our results are consistent with these studies in that the blue LEDs have an important role in the synthesis of photosynthetic pigments. This suggests that LEDs can also be used for improving the quality of *ex vitro* plantlets of *A. andreanum* and *Phalaenopsisis* sp.

In conclusion, the use of light‐emitting diodes (LEDs) as a radiation source for plants has attracted considerable interest for commercial micropropagation. The flexibility of match‐ ing LED wavelengths to plant photoreceptors may provide more optimal production, influencing plant morphology, and chlorophyll content. Although previous reports have confirmed physiological and morphological effects of LED light quality on metabolism and development of several plantlets in vitro, in our experience, LED light is more suitable for plant morphogenesis and growth than FLs. However, the responses vary according to plant species.

#### **Author details**

was higher in blue LED. The lowest total chlorophyll content was found in FL treatments and

Our results indicate that FLs can be replaced by LEDs. The same effect was observed by Kurilčik et al. [10] and Lin et al. [28] during in vitro development of *Chrysanthemum* plantlets and *Dendrobium officinale* protocorms, respectively. In *Phalaenopsis*, LEDs had no effect on the number of roots, while in *A. andreanum* the highest number of roots was obtained in FLs and

**Figure 3.** Effect of light quality on chlorophyll content in *Anthurium andreanum* (a) and *Phalaenopsis*is sp. (b) after 60 days of culture in vitro. Different letters denote statistically significant differences according to Tukey's multiple range test at

*p* ≤ 0.05. Bars represent mean ± SE.

100 Chlorophyll

white LEDs with 0.1810 and 0.2500 mg g−1 fresh weight, respectively (**Figure 3b**).

Jericó J. Bello‐Bello<sup>1</sup> \*, Juan A. Pérez‐Sato<sup>1</sup> , Carlos A. Cruz‐Cruz<sup>2</sup> and Eduardo Martínez‐Estrada<sup>1</sup>


#### **References**


[18] Jao R. C., Lai C. C., Fang W., Chang S. F. Effects of red light on the growth of Zantedeschia plantlets in vitro and tuber formation using light‐emitting diodes. HortScience. 2005;**40**(2):436–438.

[4] Tripathy B. C., Brown C. S. Root‐shoot interaction in the greening of wheat seedlings

[5] Nhut D. T., Takamura T., Watanabe H., Okamoto K., Tanaka, M. Responses of strawberry plantlets cultured in vitro under superbright red and blue light‐emitting diodes (LEDs).

[6] Hung C. D., Hong C. H., Jung H. B., Kim S. K., Van Ket N., Nam M. W., et al. Growth and morphogenesis of encapsulated strawberry shoot tips under mixed LEDs. Scientia

[7] Waman A. A., Bohra P., Sathyanarayana B. N., Umesha K., Gowda B., Ashok T. H. In vitro shoot multiplication and root induction in Silk banana variety Nanjanagud Rasabale as influenced by monochromatic light spectra. Proceedings of the National Academy of

[8] Jao R. C., Fang W. Effects of frequency and duty ratio on the growth of potato plantlets

[9] Kim S. J., Hahn E. J., Heo J. W., Paek K. Y. Effects of LEDs on net photosynthetic rate, growth and leaf stomata of chrysanthemum plantlets in vitro. Scientia Horticulturae.

[10] Kurilčik A., Miklušyte‐Čanova R., Dapkūniene S., Žilinskaite S., Kurilčik G., Tamulaitis G., et al. In vitro culture of chrysanthemum plantlets using light‐emitting diodes. Central

[11] Poudel P. R., Kataoka I., Mochioka R. Effect of red‐and blue‐light‐emitting diodes on growth and morphogenesis of grapes. Plant Cell, Tissue and Organ Culture.

[12] Li H., Tang C., Xu Z. The effects of different light qualities on rapeseed (*Brassica napus* L.) plantlet growth and morphogenesis in vitro. Scientia Horticulturae. 2003;**150**:117–124.

[13] Kwon A. R., Cui H. Y., Lee H., Shin H., Kang K. S., Park S. Y. Light quality affects shoot regeneration, cell division, and wood formation in elite clones of *Populus euramericana*.

[14] Ferreira L. T., de Araújo Silva M. M., Ulisses C., Camara T. R., Willadino L. Using LED lighting in somatic embryogenesis and micropropagation of an elite sugarcane variety and its effect on redox metabolism during acclimatization. Plant Cell, Tissue and Organ

[15] Dewir Y. H., Chakrabarty D., Kim S. J., Hahn E. J., Paek K. Y. Effect of light‐emitting diode on growth and shoot proliferation of *Euphorbia millii* and *Spathiphyllum cannifo-*

[17] Gupta S. D., Jatothu B. Fundamentals and applications of light‐emitting diodes (LEDs) in in vitro plant growth and morphogenesis. Plant Biotechnology Reports. 2013;**7**(3):211–220.

*lium*. Horticulture, Environment, and Biotechnology. 2005;**46**(6):375–379. [16] Morrow R. C. LED lighting in horticulture. HortScience. 2008;**43**(7):1947–1950.

grown under red light. Plant Physiology. 1995;**107**(2):407–411.

Sciences, India Section B: Biological Sciences. 2015;**86**(3):577–584.

European Journal of Biology. 2008;**32**(2):161–167.

Acta Physiologiae Plantarum. 2015;**37**(3):1–9.

Culture. 2017;**128**(1):211–221.

in vitro using light‐emitting diodes. HortScience. 2004;**39**(2):375–379.

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102 Chlorophyll

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2008;**92**(2):147–153.


**Provisional chapter**

#### **Chlorophyll as Photosensitizer in Dye-Sensitized Solar Cells Cells**

**Chlorophyll as Photosensitizer in Dye-Sensitized Solar** 

Abdul Kariem Arof and Teo Li Ping

Abdul Kariem Arof and Teo Li Ping Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67955

#### **Abstract**

Chlorophyll, being the most abundant pigment that commonly found in plants, bacteria, bryophytes and algae, plays a vital role in photosynthesis. Chlorophylls are natural pigments and therefore safe, environmental friendly, easily available and cheap. Chlorophyll has been experimented to function as a photosensitizer in dye-sensitized solar cells (DSSCs) as DSSCs mimic the photosynthesis process in green plants. DSSC was first developed by Gratzel in 1991 and since then has gained tremendous attention as its fabrication is cheap and easy. A DSSC basically comprises a semiconductor that has been soaked in sensitizing dye (chlorophyll), a counter electrode, and an electrolyte containing a redox mediator. The dye absorbs light, which is transformed into electricity. Chlorophyll can be extracted from the leaves of pomegranate, bougainvillea, papaya, *Pandanus amaryllifolius*, spinach, green grasses, seaweeds, algae and bryophytes. Chlorophyll from these sources has been studied as possible photosensitizers for DSSCs. Most researches done in chlorophyll DSSC use the extracted natural pigments. The type of solvent and pH of the dye solution will also affect the stability of chlorophyll and subsequently the performance of the DSSCs. This chapter will present an inexhaustive overview on DSSCs using chlorophyll as dye.

**Keywords:** chlorophyll, photosensitizer, light adsorption, dye-sensitized solar cells, efficiency

#### **1. Introduction**

Over billions of years, Mother Nature has been converting light from the sun into energy via photosynthesis. Sunlight is the most abundant and sustainable energy source that is free.

The Earth receives energy from the sun at the rate of ~12 × 1017 J s−1 [1]. This has exceeded the yearly worldwide energy consumption rate of ~1.5 × 1013 J s−1 [1]. Therefore, it is a challenge to devise an approach for the effective capture and storage of solar energy for our consumption since fossil fuels such as oil and gas will be depleted in the years to come. In order to imitate the photosynthesis process, Gratzel and coworkers have developed dye-sensitized solar cells (DSSCs) based on the similar working mechanism [2]. Nevertheless, one main difference between photosynthesis of plants and DSSCs is that the energy can be stored in plants for later use but DSSC is unable to store energy. Ever since the birth of DSSCs, they have become the spotlight of attention among scientists and researchers around the world as they are much cheaper, easier to fabricate, and more environmental friendly when compared with conventional silicon solar cells [3, 4]. A DSSC is an electrochemical device that comprises a transparent-conducting oxide (TCO) glass over which is deposited a semiconductor. The semiconductor will be soaked in a dye solution. An electrolyte with reduction-oxidation (redox) mediator and cathode are the other remaining components. The fluorine-doped tin oxide (FTO)/semiconductor/dye assembly is referred to as photoanode. Indium-doped tin oxide (ITO) and FTO are two TCOs used commonly in DSSCs. Titanium dioxide (TiO<sup>2</sup> ) is one of the popular semiconductors used for DSSC since it is cheap, non-toxic, and possesses a large bandgap [5]. TiO<sup>2</sup> is deposited on the TCO substrate in the form of TiO<sup>2</sup> nanoporous particle network to increase the coverage area for the sensitizing dye. The cathode is made up of another TCO on top of which platinum is deposited. Carbon and conducting polymers can also be employed as counter electrode. If a gel polymer electrolyte is used, it is sandwiched between the photoanode and cathode. The dye, on the other hand, can be categorized into two groups: synthetic and natural. The most frequently used synthetic dye is the ruthenium (Ru)-based dyes but they are not environmental friendly since Ru is a heavy metal [6]. Such dyes are also very expensive due to the scarcity of Ru. By contrast, natural dyes are readily available and thus cheap besides being non-toxic, environmental friendly, biodegradable, easily extracted as well as can be used without any purification [6]. Since DSSC mimics the photosynthesis of green plants, therefore chlorophyll can also function as photosensitizer for DSSC. In fact, report on chlorophyll as photosensitizer on zinc oxide (ZnO) semiconductor was first published by Tributsch in 1972 [7].

#### **2. Basic working principle of chlorophyll-sensitized DSSC**

In this chapter, discussion is based on the TiO<sup>2</sup> semiconductor photoanode. However, occasionally we refer to zinc oxide (ZnO) and tin dioxide (SnO<sup>2</sup> ). The dye is chlorophyll extracted from various sources including leaves, grasses, flowers, seaweeds, and algae. The electrolyte is generally in the form of liquid and quasi-solid state. The commonly used mediator is the *I*− /*I*3 − redox couple and the counter electrode chosen in the preceding discussion is platinum (Pt) or carbon (C). Upon shining light on the cell, the molecules of the chlorophyll dye (*D*) will be excited (*D*\* ) after absorbing photons (*hν*) and inject electrons into the semiconductor conduction band as described in the equation below:

$$hv + D \to D^\* \tag{1}$$

The excited chlorophyll molecules (*D*\* ) will inject electrons into the TiO<sup>2</sup> conduction band and the excited dye will then be oxidized or ionized (*D*<sup>+</sup> ). The reaction process involved is as follows:

The Earth receives energy from the sun at the rate of ~12 × 1017 J s−1 [1]. This has exceeded the yearly worldwide energy consumption rate of ~1.5 × 1013 J s−1 [1]. Therefore, it is a challenge to devise an approach for the effective capture and storage of solar energy for our consumption since fossil fuels such as oil and gas will be depleted in the years to come. In order to imitate the photosynthesis process, Gratzel and coworkers have developed dye-sensitized solar cells (DSSCs) based on the similar working mechanism [2]. Nevertheless, one main difference between photosynthesis of plants and DSSCs is that the energy can be stored in plants for later use but DSSC is unable to store energy. Ever since the birth of DSSCs, they have become the spotlight of attention among scientists and researchers around the world as they are much cheaper, easier to fabricate, and more environmental friendly when compared with conventional silicon solar cells [3, 4]. A DSSC is an electrochemical device that comprises a transparent-conducting oxide (TCO) glass over which is deposited a semiconductor. The semiconductor will be soaked in a dye solution. An electrolyte with reduction-oxidation (redox) mediator and cathode are the other remaining components. The fluorine-doped tin oxide (FTO)/semiconductor/dye assembly is referred to as photoanode. Indium-doped tin oxide (ITO) and FTO are two TCOs used commonly in DSSCs. Titanium dioxide (TiO<sup>2</sup>

one of the popular semiconductors used for DSSC since it is cheap, non-toxic, and possesses

particle network to increase the coverage area for the sensitizing dye. The cathode is made up of another TCO on top of which platinum is deposited. Carbon and conducting polymers can also be employed as counter electrode. If a gel polymer electrolyte is used, it is sandwiched between the photoanode and cathode. The dye, on the other hand, can be categorized into two groups: synthetic and natural. The most frequently used synthetic dye is the ruthenium (Ru)-based dyes but they are not environmental friendly since Ru is a heavy metal [6]. Such dyes are also very expensive due to the scarcity of Ru. By contrast, natural dyes are readily available and thus cheap besides being non-toxic, environmental friendly, biodegradable, easily extracted as well as can be used without any purification [6]. Since DSSC mimics the photosynthesis of green plants, therefore chlorophyll can also function as photosensitizer for DSSC. In fact, report on chlorophyll as photosensitizer on zinc oxide (ZnO) semiconductor

from various sources including leaves, grasses, flowers, seaweeds, and algae. The electrolyte is generally in the form of liquid and quasi-solid state. The commonly used mediator is the

 redox couple and the counter electrode chosen in the preceding discussion is platinum (Pt) or carbon (C). Upon shining light on the cell, the molecules of the chlorophyll dye (*D*)

) after absorbing photons (*hν*) and inject electrons into the semiconductor

is deposited on the TCO substrate in the form of TiO<sup>2</sup>

a large bandgap [5]. TiO<sup>2</sup>

106 Chlorophyll

*I*− /*I*3 −

will be excited (*D*\*

was first published by Tributsch in 1972 [7].

In this chapter, discussion is based on the TiO<sup>2</sup>

sionally we refer to zinc oxide (ZnO) and tin dioxide (SnO<sup>2</sup>

conduction band as described in the equation below:

**2. Basic working principle of chlorophyll-sensitized DSSC**

) is

nanoporous

semiconductor photoanode. However, occa-

). The dye is chlorophyll extracted

$$D^\* + \text{TiO}\_2 \rightarrow D^\* + e^-\_{\phi} \text{(TiO}\_2\text{)}\tag{2}$$

The oxidized chlorophyll dye molecules (*D*<sup>+</sup> ) will accept electrons from an iodide ion (*I*<sup>−</sup> ) in the electrolyte when the *I*<sup>−</sup> ions were released to the oxidized molecules and in turn oxidized to triiodide ions (I<sup>3</sup> − ) according to the equation below:

$$2D^{+} + 3\,\mathrm{I^{-}} \rightarrow \mathrm{I\_{3}^{-}} + 2D \tag{3}$$

The electron in the TiO<sup>2</sup> conduction band flows out of the device through the load to reach the counterelectrode and reduced the triiodide ion as follows:

$$I\_3^- + 2 \, e^- \to \, 3 \, I^- \tag{4}$$

The iodide ion is now restored, the electron circuit is completed, and the whole system is back to its original state to start a new cycle. These processes will continue as long as there is light and current is produced in the external circuit continuously. Under illumination, the voltage generated is given by the energy difference between the photoanode's Fermi level and the electrolyte's redox potential. **Figure 1** illustrates the schematic diagram of the chlorophyllsensitized DSSC and its operating principle.

The light to electricity conversion efficiency (*η*) of the chlorophyll DSSC can be calculated from the equation below:

$$\eta = \frac{J\_{\text{sc}} \times V\_{\text{oc}} \times FF}{P\_{\text{in}}} \times 100\% \tag{5}$$

Here *J*sc is the short circuit current density (unit: mA cm−2), which is obtained without any external applied voltage or potential, *V*oc is the open circuit voltage (unit: mV) obtained under the condition of open circuit when there is no current, *P*in is the input power (total incident light power density), and *FF* is the fill factor which can be expressed as

$$FF = \frac{J\_{\text{max}} \times V\_{\text{max}}}{J\_{\text{sc}} \times V\_{\text{oc}}} \tag{6}$$

**Figure 1.** Operating principle of a chlorophyll-sensitized DSSC.

Here *J*max and *V*max are the photocurrent density and voltage in the *J-V* curve at the maximum power output. Each single component in a DSSC is important to ensure good performance. The main focus in this chapter is the chlorophyll dye. The dye is to absorb light, injects electrons into the semiconductor, and receives electrons from the redox mediator in the electrolyte. The cycle continues. An efficient dye sensitizer should display unique characteristics as listed below [8, 9]:


#### **3. Performance of chlorophyll-sensitized DSSCs**

**Table 1** summarizes the performance of some DSSCs employing chlorophyll as photosensitizer reported by researchers worldwide. Herein, the illumination of the chlorophyllsensitized DSSCs was carried out under intensity of 100 mW cm−2 unless stated otherwise.

It is evident that the condition of leaves whether fresh or dried affects the adsorption of chlorophyll onto the photoanode surface and consequently the performance. Taya et al. [10] observed that DSSCs having chlorophyll extracted from fresh leaves of *Anethum graveolens* (Indian traditional medicinal herb and spice) and arugula (arugula salad leaves) exhibited better performance than DSSCs with *A. graveolens* and arugula leaves that have been dried for 1 week. On the other hand, higher efficiencies were detected in DSSCs with dried parsley, spinach, and green algae as compared to fresh ones. However, the authors did not discuss the reason behind this. Among parsley, arugula, *A. graveolens*, *Spinach oleracea*, and green algae, chlorophyll extracted from spinach produced the best efficiency of 0.290 % [10]. The efficiency of the DSSCs depends on the soaking temperature and time. The 0.290% efficiency was obtained when the TiO<sup>2</sup> photoanode was soaked in the spinach extract solution at 60°C for 12 h [10]. Decreasing the temperature yielded low efficiency with *η* = ~0.0380% at 30°C. Beyond the optimum temperature, the efficiency decreased to ~0.175% (70°C) and ~0.0190% (80°C), respectively. Reducing the TiO2 soaking time in the chlorophyll spinach solution to 2 h gave poor efficiency of ~0.021% [10]. Beyond 12 h of soaking, no obvious change in efficiency was observed from the spinach chlorophyll-sensitized DSSC [10]. Therefore, it can be inferred that the freshness of chlorophyll leaves, the soaking temperature of TiO<sup>2</sup> in chlorophyll solution, and its duration influenced the DSSC performance. From **Table 1**, comparison has been


Here *J*max and *V*max are the photocurrent density and voltage in the *J-V* curve at the maximum power output. Each single component in a DSSC is important to ensure good performance. The main focus in this chapter is the chlorophyll dye. The dye is to absorb light, injects electrons into the semiconductor, and receives electrons from the redox mediator in the electrolyte. The cycle continues. An efficient dye sensitizer should display unique characteristics as

• Good attachment at the surface of photoelectrode to ensure fast electron transfer.

• Good interfacial properties and high stability to enable good absorption to TiO2

**Table 1** summarizes the performance of some DSSCs employing chlorophyll as photosensitizer reported by researchers worldwide. Herein, the illumination of the chlorophyllsensitized DSSCs was carried out under intensity of 100 mW cm−2 unless stated otherwise.

It is evident that the condition of leaves whether fresh or dried affects the adsorption of chlorophyll onto the photoanode surface and consequently the performance. Taya et al. [10] observed that DSSCs having chlorophyll extracted from fresh leaves of *Anethum graveolens* (Indian traditional medicinal herb and spice) and arugula (arugula salad leaves) exhibited better performance than DSSCs with *A. graveolens* and arugula leaves that have been dried for 1 week. On the other hand, higher efficiencies were detected in DSSCs with dried parsley, spinach, and green algae as compared to fresh ones. However, the authors did not discuss the reason behind this. Among parsley, arugula, *A. graveolens*, *Spinach oleracea*, and green algae, chlorophyll extracted from spinach produced the best efficiency of 0.290 % [10]. The efficiency of the DSSCs depends on the soaking temperature and time. The 0.290% efficiency

for 12 h [10]. Decreasing the temperature yielded low efficiency with *η* = ~0.0380% at 30°C. Beyond the optimum temperature, the efficiency decreased to ~0.175% (70°C) and ~0.0190%

gave poor efficiency of ~0.021% [10]. Beyond 12 h of soaking, no obvious change in efficiency was observed from the spinach chlorophyll-sensitized DSSC [10]. Therefore, it can be inferred

tion, and its duration influenced the DSSC performance. From **Table 1**, comparison has been

that the freshness of chlorophyll leaves, the soaking temperature of TiO<sup>2</sup>

photoanode was soaked in the spinach extract solution at 60°C

soaking time in the chlorophyll spinach solution to 2 h

in chlorophyll solu-

• Easily accepting replacement electron from electrolyte.

• Excited state of dye must be slightly above the TiO<sup>2</sup>

level below the redox potential of the electrolyte.

• Lifetime of the dye must be consistent with device life.

**3. Performance of chlorophyll-sensitized DSSCs**

• Stable enough to sustain about 20 years exposure to natural light.

.

conduction band and its ground-state

listed below [8, 9]:

108 Chlorophyll

• Absorb light in the visible region.

was obtained when the TiO<sup>2</sup>

(80°C), respectively. Reducing the TiO2



**Dye** Papaya leaves Jatropha leaves

Ipomoea leaves extract

**Photoanode**

TiO2/FTO TiO2/ITO TiO2/ITO TiO2/ITO TiO2/ITO TiO2/ITO TiO2/ITO TiO2/ITO TiO2/ITO

*Azadirachta indica* (Neem) leaves

*Ziziphus jujuba* leaves (dried)

Basil leaves (dried)

Basil flower Mint flower Mint leaves (dried)

Lemon leavesa

Morula leavesa

Fig leaves (dried)

Berry leaves (dried)

*Pandanus amaryllifolius* leaves

TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/ITO TiO2/FTO TiO2/FTO

GPE

Pt/FTO

GPE

Pt/FTO

GPE

Pt/ITO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

C/FTO

I−/I3

− LE

C/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

C/FTO

I−/I3

− LE

Pt/ITO

I−/I3

− LE

Pt/ITO

I−/I3

− LE

Pt/ITO

I−/I3

− LE

Pt/ITO

I−/I3

− LE

Pt/ITO

I−/I3

− LE

Pt/ITO

0.850 0.914 0.825 1.120 0.982 0.915 0.430 0.230 3.180 1.398 1.120 0.450 0.980 1.080 0.059 2.091 3.573 1.610 1.190 1.910

0.480

0.560

0.510

[31]

0.490

0.630

0.390

[30]

0.360

0.410

0.240

[29]

0.595

0.441

0.939

[26]

0.596

0.515

0.642

[26]

0.472

0.050

0.001

[28]

0.592

0.100

0.036

[28]

0.579

0.400

0.227

[26]

0.560

0.380

0.090

[27]

0.600

0.400

0.270

[27]

0.581

0.499

0.409

[26]

0.652

0.519

1.077

[26]

0.467

0.392

0.050

[25]

0.404

0.401

0.720

[24]

0.510

0.552

0.253

[19]

0.543

0.564

0.292

[19]

0.565

0.592

0.318

[19]

0.533

0.548

0.259

[19]

0.540

0.563

0.278

[19]

0.495

0.536

0.233

[19]

I−/I3

− LE

C/FTO

I−/I3

− LE

C/FTO

I−/I3

− LE

Pt/FTO

**Electrolyte**

**Counter electrode**

*J***sc (mA cm−2)**

0.360 0.060 mA 0.042 mA

0.350

0.250


[23]

0.394

0.250


[23]

0.325

0.560

0.070

[22]

*V***oc (V)**

*FF*

*η* **(%)**

**Ref.**

110 Chlorophyll


**Table 1.** The photovoltaic performance of some chlorophyll-sensitized DSSCs. made between TiO<sup>2</sup> and ZnO photoanodes for chlorophyll spinach DSSCs where the former gave better performance than the latter [10].

In the case of chlorophyll extract from ipomoea leaves (leaves of morning glory flower), 50°C is the optimum temperature for TiO<sup>2</sup> immersion with efficiency of 0.278% [19]. Lower efficiencies of 0.233 and 0.259% were obtained when the TiO2 -soaking temperature in ipomoea leaves extract solution were at 30 and 80°C, respectively for 24 h [19]. Other than temperature, pH of the dye solution is another factor influencing the efficiency. Maintaining the soaking temperature at 50°C, the pH of ipomoea dye solutions was adjusted to pH 1, 2, and 3 [19]. However, there was no mention on the type of acid used. Thus, it is not known whether the anion of acid had any influence on the DSSC performance. Nevertheless, improvement in efficiency can be seen when the acidity of the dye solution was adjusted to pH 1 and 2 with efficiencies of 0.318 and 0.292%, respectively. However, further increasing the pH to 3 decreased the efficiency (*η* = 0.253%) [19].

The type of solvent used for pigments extraction can also give different results in the absorption spectrum [45–47]. From the work of Al-Alwani et al. [45], it has been reported that the UV-vis absorption spectra of chlorophyll extracted from *Pandanus amaryllifolius* (screwpine leaves) and *Cordyline fruticosa* (commonly known as Ti plant or cabbage palm) in ethanol and methanol solution displayed highest intensity absorption peaks among other solvents such as n-butyl alcohol, ethyl-acetate, n-hexane, chloroform, acetonitrile, ethyl-ether, and petroleum ether. Then, 1 g TiO2 powder was added in chlorophyll extracted from *P. amaryllifolius* and *C. fruticosa* in respective solvents (ethanol for Pandanus leaves and methanol for *Cordyline* leaves) and water at different ratios. The addition of an appropriate quantity of water into the respective alcohol solvent increased the polarity of solution for better dye adsorption on the TiO<sup>2</sup> surface [45]. For solution containing TiO<sup>2</sup> and *P. amaryllifolius* chlorophyll, the best absorption spectrum was obtained at 2:1 of ethanol to water ratio, whereas the optimum ratio for TiO<sup>2</sup> -*C. fruticosa* solution with mixture solvents of methanol and water was 3:1. Better dye absorption onto TiO<sup>2</sup> surface is said to improve the performance of DSSCs but there is no DSSC results in Ref. [45]. Nonetheless, in recent publication [12], the same group of authors have turned to response surface methodology (RSM) approach to investigate the various parameters involved in the chlorophyll extraction process from *C. fruticosa* simultaneously and then predict their response in order to obtain the optimized condition for its extraction. After taking three factors into consideration, that is, boiling temperature for organic solvents (acetonitrile, ethanol, and methanol), different pH ranging from 4 to 8 and temperature for chlorophyll extraction from 50 to 90°C, it was found that chlorophyll can be best extracted from the cordyline leaves under the condition where the solvent was ethanol (boiling point 78°C), pH of 7.99, and at temperature of 78.33°C [12]. As a result, the efficiency of 0.500% was obtained for DSSC with *C. fruticosa* chlorophyll as listed in **Table 1**.

It should be noted that both betalain and chlorophyll pigments can be extracted from the flowers of *Amaranthus caudatus* (common name: love-lies-bleeding, velvet flower) and *Bougainvillea spectabilis* using different solvents. Chlorophyll pigments have been obtained when 0.1 mol L−1 hydrochloric acid (HCl) was used to dissolve the amaranthus and bougainvillea flowers, whereas ethanol as solvent will yield betalain pigments from the same flowers. Surprisingly,

**Dye** Kelp (brown algae)

*Undaria pinnatifida* (brown seaweed)

TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO TiO2/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

*Cladophora* sp. (green algae)

Green algae (fresh)

Green algae (dried)

*Chlorella vulgaris* (microalgae)

aIntensity 80 mW cm−2.

bIntensity 45 mW cm−2.

**Table 1.**

LE: liquid electrolyte; GPE: gel polymer electrolyte; PEDOT: poly(3,4-ethylenedioxythiophene).

The photovoltaic performance of some chlorophyll-sensitized DSSCs.

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

I−/I3

− LE

Pt/FTO

**Photoanode**

TiO2/TCO

I−/I3

− LE

Pt/TCO

**Electrolyte**

**Counter electrode**

*J***sc (mA cm−2)**

0.433 0.800 10.700 13.800

8.600 9.000 0.145 0.134 0.397 2.530

0.551

0.650

0.900

[44]

0.559

0.440

0.100

[10]

0.416

0.210

0.010

[10]

0.585

0.590

0.055

[43]

0.470

0.610

2.600

[42]

0.470

0.600

2.500

[42]

0.570

0.580

4.600

[42]

0.530

0.600

3.400

[42]

0.360

0.690

0.178

[41]

0.441

0.620


[40]

*V***oc (V)**

*FF*

*η* **(%)**

**Ref.**

112 Chlorophyll

DSSCs having chlorophyll from amaranthus and bougainvillea flowers in **Table 1** demonstrated better performance as compared to DSSCs with betalain from the same flowers. The *J* sc, *V*oc, *FF*, and *η* values of betalain DSSC from *B. spectabilis* were 0.081 mA cm−2, 0.450 V, 0.483, and 0.018%, respectively [11]. DSSCs using betalain extracted from *A. caudatus* flower gave the *J* sc of 0.102 mA cm−2, *V*oc of 0.530 V, *FF* of 0.610, and *η* of 0.033% [11]. The efficiencies exhibited by chlorophyll-sensitized DSSCs from amaranthus (*η* = 0.610 %) and bougainvillea (*η* = 0. 325 %) flowers were surprisingly high considering that gel polymer electrolyte and PEDOT counterelectrode were used instead of conventional liquid electrolyte and Pt electrode [11].

Khan and coworkers [20] have examined the effect of acid treatment on TiO2 nanoparticles in the making of TiO<sup>2</sup> paste to be coated on ITO glass substrate via the doctor blade method. It is found that chlorophyll from red spinach leaves-sensitized DSSC without any acid treatment on TiO<sup>2</sup> photoanode exhibited the efficiency of 0.296% which is lower than the TiO2 acidtreated DSSC with chlorophyll extracted from the same source under intensity of 50 mW cm−2 [20]. The presence of acid can prevent agglomeration of TiO<sup>2</sup> nanoparticles and results in better TiO2 dispersion and thereby offer more adsorption sites for the dye molecules [20]. Khan et al. [20] used citric acid (organic acid) and nitric acid (inorganic acid) to prevent TiO<sup>2</sup> agglomeration in the DSSC fabricated with chlorophyll from red spinach leaves, and TiO<sup>2</sup> treated with citric acid gave higher efficiency of 0.583% compared to that using nitric acid treatment on TiO<sup>2</sup> electrode (*η* = 0.357%). The lower efficiency yielded by nitric acid treatment could be due to nitric acid being a strong oxidizing acid and its corrosive nature may ruin the TiO<sup>2</sup> surface [48]. From **Table 1**, the *J*sc and *FF* of the DSSC with chlorophyll extracted from red spinach leaves can also be seen to increase with efficiency following the order *η* (TiO<sup>2</sup> ) < *η* (nitric acid treated TiO<sup>2</sup> ) < *η* (citric acid treated TiO<sup>2</sup> ), whereas the *V*oc values decreased in the same order.

It can be noted from **Table 1** that SnO<sup>2</sup> was employed as photoanode instead of TiO<sup>2</sup> in the cell having chlorophyll extracted from *Coccinia indica* (ivy gourd) leaves [32]. Comparison with TiO<sup>2</sup> revealed that SnO<sup>2</sup> is chemically stable and has larger bandgap of 3.6 eV and higher electron mobility [49, 50]. Due to its wide bandgap, it is less sensitive to UV degradation and thus possesses better stability as compared to TiO2 [51]. Nonetheless, its large bandgap will also cause SnO<sup>2</sup> to have lower open circuit voltage. SnO<sup>2</sup> having high electron mobility can yield fast electron transport and therefore electron recombination can be decreased. From the table, the cells with chlorophyll from *C. indica* leaves exhibited the efficiencies of 0.260, 0.290, and 0.310% using three photoanodes, that is, SnO2 , La-doped SnO2, and La-Cu-doped SnO<sup>2</sup> , respectively [32]. Doping elements into DSSC photoanode improved the performance since more dye molecules can be adsorbed in the working electrode due to larger surface area owing to increased roughness and pores after doping [52].

Chang et al. [17] have investigated the plasmonic effect of gold (Au) nanoparticles with an average size of 27 nm in TiO2 DSSC using chlorophyll from bougainvillea leaves. An efficiency of 0.618% was obtained. The Au nanoparticles showed localized surface plasmon resonance behavior when the frequency of the incident light came close to the surface plasmon frequency of Au and consequently improved light absorption leading to a considerably high efficiency of 0.618% as listed in **Table 1**. Also, the interface between Au and TiO<sup>2</sup> formed a Schottky barrier where electrons will be blocked from re-entering the dye or electrolyte, which decreased electron recombination and improved the DSSC performance. Earlier report on TiO<sup>2</sup> loaded with Au nanoparticles prior to chlorophyll sensitization was published by Lai and coworkers [33]. Instead of using *I*<sup>−</sup> /*I*3 − LE, the authors employed water-based electrolyte at ethanol:water ratio of 7:13 with Ce4+/ 3+ as redox couple since water-based DSSC can be totally free from toxic and is biologically friendly. The chlorophyll was extracted from herbal plant *Rhoeo spathacea* (Sw.) Stearn. Unexpectedly, an efficiency of more than 1% was produced by the chlorophyll *R. spathacea* water-based DSSC which is higher than that of a similar cell but with one of the earliest synthetic dyes, that is, crystal violet (*η* = 0.010%) [33]. The authors attributed the aggregation of crystal violet dyes as the culprit behind this based on its photocurrent density value (*J* sc = 2.040 mA cm−2), which is lower than that of *R. spathacea* cell (*J* sc = 10.900 mA cm−2) [33]. In fact, the other two water-based DSSCs using chlorophyll extracted from *Ficus retusa* Linn. (common name: bonsai plant) and *Garcinia subelliptica* (common name: happiness tree) have also demonstrated higher efficiency than that of crystal violet-sensitized DSSC with efficiencies of 1.180 and 0.691%, respectively [33].

DSSCs having chlorophyll from amaranthus and bougainvillea flowers in **Table 1** demonstrated better performance as compared to DSSCs with betalain from the same flowers. The *J*

*V*oc, *FF*, and *η* values of betalain DSSC from *B. spectabilis* were 0.081 mA cm−2, 0.450 V, 0.483, and 0.018%, respectively [11]. DSSCs using betalain extracted from *A. caudatus* flower gave the *J*

of 0.102 mA cm−2, *V*oc of 0.530 V, *FF* of 0.610, and *η* of 0.033% [11]. The efficiencies exhibited by chlorophyll-sensitized DSSCs from amaranthus (*η* = 0.610 %) and bougainvillea (*η* = 0. 325 %) flowers were surprisingly high considering that gel polymer electrolyte and PEDOT counter-

found that chlorophyll from red spinach leaves-sensitized DSSC without any acid treatment

treated DSSC with chlorophyll extracted from the same source under intensity of 50 mW cm−2

Khan et al. [20] used citric acid (organic acid) and nitric acid (inorganic acid) to prevent TiO<sup>2</sup> agglomeration in the DSSC fabricated with chlorophyll from red spinach leaves, and TiO<sup>2</sup> treated with citric acid gave higher efficiency of 0.583% compared to that using nitric acid

could be due to nitric acid being a strong oxidizing acid and its corrosive nature may ruin the

cell having chlorophyll extracted from *Coccinia indica* (ivy gourd) leaves [32]. Comparison

electron mobility [49, 50]. Due to its wide bandgap, it is less sensitive to UV degradation and

yield fast electron transport and therefore electron recombination can be decreased. From the table, the cells with chlorophyll from *C. indica* leaves exhibited the efficiencies of 0.260,

Chang et al. [17] have investigated the plasmonic effect of gold (Au) nanoparticles with an aver-

of 0.618% was obtained. The Au nanoparticles showed localized surface plasmon resonance behavior when the frequency of the incident light came close to the surface plasmon frequency of Au and consequently improved light absorption leading to a considerably high efficiency of

, respectively [32]. Doping elements into DSSC photoanode improved the performance since more dye molecules can be adsorbed in the working electrode due to larger surface area

red spinach leaves can also be seen to increase with efficiency following the order *η* (TiO<sup>2</sup>

) < *η* (citric acid treated TiO<sup>2</sup>

to have lower open circuit voltage. SnO<sup>2</sup>

surface [48]. From **Table 1**, the *J*sc and *FF* of the DSSC with chlorophyll extracted from

photoanode exhibited the efficiency of 0.296% which is lower than the TiO2

dispersion and thereby offer more adsorption sites for the dye molecules [20].

electrode (*η* = 0.357%). The lower efficiency yielded by nitric acid treatment

paste to be coated on ITO glass substrate via the doctor blade method. It is

electrode were used instead of conventional liquid electrolyte and Pt electrode [11].

Khan and coworkers [20] have examined the effect of acid treatment on TiO2

[20]. The presence of acid can prevent agglomeration of TiO<sup>2</sup>

the making of TiO<sup>2</sup>

on TiO<sup>2</sup>

114 Chlorophyll

better TiO2

TiO<sup>2</sup>

same order.

with TiO<sup>2</sup>

SnO<sup>2</sup>

also cause SnO<sup>2</sup>

age size of 27 nm in TiO2

treatment on TiO<sup>2</sup>

(nitric acid treated TiO<sup>2</sup>

It can be noted from **Table 1** that SnO<sup>2</sup>

revealed that SnO<sup>2</sup>

thus possesses better stability as compared to TiO2

0.290, and 0.310% using three photoanodes, that is, SnO2

owing to increased roughness and pores after doping [52].

0.618% as listed in **Table 1**. Also, the interface between Au and TiO<sup>2</sup>

sc,

sc

nanoparticles in

nanoparticles and results in

), whereas the *V*oc values decreased in the

[51]. Nonetheless, its large bandgap will

having high electron mobility can

, La-doped SnO2, and La-Cu-doped

formed a Schottky barrier

was employed as photoanode instead of TiO<sup>2</sup>

is chemically stable and has larger bandgap of 3.6 eV and higher

DSSC using chlorophyll from bougainvillea leaves. An efficiency

acid-

) < *η*

in the

From **Table 1**, it can be observed that most of the DSSCs employ liquid electrolytes based on *I*<sup>−</sup> /*I*3 − redox mediator. The maximum power conversion efficiencies of liquid electrolytebased DSSCs using synthetic dyes have reached around 14% [53]. Liquid-based electrolytes are desired since they can infiltrate into the TiO2 nanopores network to make contact with the dye molecules for dye regeneration. Still, liquid electrolyte-based cells have limited durability due to the possibility of leakage and volatility of solvents. Some of the solvents are flammable as well. In an attempt to avoid these complications, researchers worldwide are focusing on developing polymer-based electrolytes for DSSCs. Solid polymer electrolytes can exhibit reasonable ionic conductivities but have poor interfacial contact with electrodes. Hence, gel-type polymer electrolytes (GPEs) are being developed. GPEs, which are basically liquid electrolyte trapped in the polymer matrix, have good flexibility and conductivities comparable to those of liquid electrolytes. It can be clearly seen from **Table 1** that the DSSC using chlorophyll extracted from moss bryophyte and gel polymer electrolyte exhibited exceptionally high efficiencies of ~2–2.620% under different conditions. The bryophyte cell with *η* = 1.970% was obtained with GPE having polyacrylonitrile (PAN) as polymer host, tetrapropylammonium iodide (TPAI) salt, iodine, ethylene carbonate (EC), and propylene carbonate (PC) as solvent and plasticizer [37]. Efficiencies of 1.770 and 2.000% were attained for bryophyte cells using GPE based on poly(vinyl alcohol) (PVA) with single [potassium iodide (KI)] and double salts (KI and TPAI), respectively [38]. Higher efficiency and *J* sc values observed in the cell having binary salts GPE could be most probably due to the higher number of iodide ions contained in the GPE. As for the second best performing chlorophyll bryophyte DSSC (*η* = 2.170 %), it is acquired using GPE based on poly(vinyl alcohol) (PVA) and double salts of potassium iodide (KI) and TPAI with the addition of 0.7 M tert-butylpyridine (TBP) [38]. TBP can be used either by incorporating it in electrolyte or photoanode in order to improve the *V*oc and subsequently *η*. However, in this case where TBP was added into GPE, the TBP effect is insignificant as compared to the bryophyte cell when the working electrode was immersed in TBP for 1 h (*η* = 1.690%) [38]. Nonetheless, the efficiency of the latter is lower than the former owing to its lower *J* sc value which might be due to lesser photon harvesting as a result of reflection and light scattering by TBP [38]. The most efficient chlorophyll bryophyte DSSC having *η* of 2.620% was attained when a co-adsorbent, that is, chenodeoxycholic acid (CDCA), was added in the moss bryophyte [38]. The GPE used was PVA-based double salt without TBP. CDCA served as spacer to prevent the self-aggregation of chlorophyll molecules, diminish electron recombination, and stabilize the chlorophyll, thereby improving the efficiency.

It is worth mentioning from **Table 1** that the cell having the efficiency of 0.590% with chlorophyll extracted from shiso leaves used copper iodide (CuI) as hole transport material (HTM) instead of conventional liquid electrolyte [16]. Therefore, the DSSC has the configuration of FTO/TiO<sup>2</sup> /chlorophyll dye/CuI. CuI, a p-type semiconductor, has bandgap of 3.1 eV and good optical transparency [54, 55]. The p-CuI was coated onto the chlorophyll/TiO<sup>2</sup> /FTO using dipand spray-coating technique as this method involves low calcination temperature and thus the degradation of dye will not occur [16, 54]. The p-CuI solid-state DSSC has similar working principle with conventional DSSC except that after photon absorption, the dye molecules will be excited and then inject electrons and holes into TiO<sup>2</sup> and p-CuI, respectively. This indicates that the dye at ground state must be positioned below CuI valence band and the dye-excited state should be above the TiO<sup>2</sup> conduction band in order to ensure proper functioning of chlorophyll CuI DSSC. With the usage of HTM, there will be no issue on pigment deterioration since natural pigment is unstable against the oxidized species in electrolyte with iodine as redox mediator [16].

Most of the reports on chlorophyll-sensitized DSSCs summarized in **Table 1** do not contain information on the type of chlorophyll used. Among the six chlorophylls, chlorophyll *a*, which plays vital role in photosynthesis process, shows poor adsorption and sensitization on TiO<sup>2</sup> due to its structure that contains phytyl and alkyl groups causing steric hindrance that obstruct the chlorophyll molecules to bind efficiently with TiO2 molecules [40, 56, 57]. The structure of chlorophyll *b* only differs from chlorophyll *a* by the aldehyde group (–CHO) rather than methyl group (–CH<sup>3</sup> ). On the other hand, chlorophyll *c*, which consists of chlorophyll *c*<sup>1</sup> and chlorophyll *c*2, has carboxyl group (–COOH) that can effectively attach to TiO2 as reported in Ref. [41]. Chlorophyll *c*, which is the main pigment in *Undaria pinnatifida* (brown seaweed) yielded the efficiency of 0.178% when applied in DSSC as listed in **Table 1**. Wang et al. [42] have purified the pigments in *U. pinnatifida* to obtain chlorophyll *c* and remove chlorophyll *a* and carotenoids. Then, the purified chlorophyll *c* was subjected to polyethylene column chromatography to isolate chlorophyll *c*<sup>1</sup> and chlorophyll *c*<sup>2</sup> . Using the same method, chlorophyll *c*1 and chlorophyll *c*<sup>2</sup> in oxidized form can also be obtained. As a result, efficiencies of 3.400 and 4.600% have been obtained from the chlorophyll *c*1- and chlorophyll *c*<sup>2</sup> -sensitized DSSCs with liquid electrolyte [42]. Decrement in efficiency can be seen in DSSCs employing oxidized chlorophyll *c*<sup>1</sup> (designated as chlorophyll *c'*<sup>1</sup> ) and oxidized chlorophyll *c*<sup>2</sup> (chlorophyll *c'*<sup>2</sup> ) with the values of 2.500 and 2.600%, respectively [42]. Nonetheless, to the best of our knowledge, the chlorophyll-sensitized DSSC utilizing chlorophyll *c*<sup>2</sup> extracted from *U. pinnatifida* exhibited the highest efficiency among other chlorophyll-sensitized DSSCs till date. However, there remains the stability issue encountered by chlorophyll-sensitized DSSCs. Therefore, further work must be done to enhance the stability as well as improving the chlorophyll-sensitized DSSCs performance before they can be put into practical usage.

#### **4. Summary**

and light scattering by TBP [38]. The most efficient chlorophyll bryophyte DSSC having *η* of 2.620% was attained when a co-adsorbent, that is, chenodeoxycholic acid (CDCA), was added in the moss bryophyte [38]. The GPE used was PVA-based double salt without TBP. CDCA served as spacer to prevent the self-aggregation of chlorophyll molecules, diminish electron

It is worth mentioning from **Table 1** that the cell having the efficiency of 0.590% with chlorophyll extracted from shiso leaves used copper iodide (CuI) as hole transport material (HTM) instead of conventional liquid electrolyte [16]. Therefore, the DSSC has the configuration of

and spray-coating technique as this method involves low calcination temperature and thus the degradation of dye will not occur [16, 54]. The p-CuI solid-state DSSC has similar working principle with conventional DSSC except that after photon absorption, the dye molecules will

that the dye at ground state must be positioned below CuI valence band and the dye-excited

chlorophyll CuI DSSC. With the usage of HTM, there will be no issue on pigment deterioration since natural pigment is unstable against the oxidized species in electrolyte with iodine

Most of the reports on chlorophyll-sensitized DSSCs summarized in **Table 1** do not contain information on the type of chlorophyll used. Among the six chlorophylls, chlorophyll *a*, which plays vital role in photosynthesis process, shows poor adsorption and sensitization on TiO<sup>2</sup> due to its structure that contains phytyl and alkyl groups causing steric hindrance that obstruct

of chlorophyll *b* only differs from chlorophyll *a* by the aldehyde group (–CHO) rather than

Ref. [41]. Chlorophyll *c*, which is the main pigment in *Undaria pinnatifida* (brown seaweed) yielded the efficiency of 0.178% when applied in DSSC as listed in **Table 1**. Wang et al. [42] have purified the pigments in *U. pinnatifida* to obtain chlorophyll *c* and remove chlorophyll *a* and carotenoids. Then, the purified chlorophyll *c* was subjected to polyethylene column chro-

and chlorophyll *c*<sup>2</sup>

with liquid electrolyte [42]. Decrement in efficiency can be seen in DSSCs employing oxidized

the values of 2.500 and 2.600%, respectively [42]. Nonetheless, to the best of our knowledge,

ited the highest efficiency among other chlorophyll-sensitized DSSCs till date. However, there remains the stability issue encountered by chlorophyll-sensitized DSSCs. Therefore, further work must be done to enhance the stability as well as improving the chlorophyll-sensitized

chlorophyll *c*2, has carboxyl group (–COOH) that can effectively attach to TiO2

and 4.600% have been obtained from the chlorophyll *c*1- and chlorophyll *c*<sup>2</sup>

). On the other hand, chlorophyll *c*, which consists of chlorophyll *c*<sup>1</sup>

in oxidized form can also be obtained. As a result, efficiencies of 3.400

) and oxidized chlorophyll *c*<sup>2</sup>

/chlorophyll dye/CuI. CuI, a p-type semiconductor, has bandgap of 3.1 eV and good

/FTO using dip-

and

) with

as reported in


(chlorophyll *c'*<sup>2</sup>

and p-CuI, respectively. This indicates

molecules [40, 56, 57]. The structure

. Using the same method, chlorophyll

extracted from *U. pinnatifida* exhib-

conduction band in order to ensure proper functioning of

recombination, and stabilize the chlorophyll, thereby improving the efficiency.

optical transparency [54, 55]. The p-CuI was coated onto the chlorophyll/TiO<sup>2</sup>

be excited and then inject electrons and holes into TiO<sup>2</sup>

the chlorophyll molecules to bind efficiently with TiO2

(designated as chlorophyll *c'*<sup>1</sup>

the chlorophyll-sensitized DSSC utilizing chlorophyll *c*<sup>2</sup>

DSSCs performance before they can be put into practical usage.

state should be above the TiO<sup>2</sup>

as redox mediator [16].

methyl group (–CH<sup>3</sup>

and chlorophyll *c*<sup>2</sup>

chlorophyll *c*<sup>1</sup>

*c*1

matography to isolate chlorophyll *c*<sup>1</sup>

FTO/TiO<sup>2</sup>

116 Chlorophyll

It has been shown that chlorophyll has good potential to serve as photosensitizer in dyesensitized solar cells. Moreover, they are cheap, non-toxic, biodegradable, easily found, and easy to use as sensitizer. Although the efficiency is still considerably low with highest efficiency to date being only 4.600% from DSSC with *U. pinnatifida* chlorophyll *c*<sup>2</sup> , there remains the possibility and room for improvement to further enhance the performance and improve stability of chlorophyll-sensitized DSSCs for practical applications.

#### **Acknowledgements**

The authors thank the University of Malaya and Malaysian Ministry of Higher Education (MOHE) for the UMRG grant no. RP024C-14AFR and PRGS grant no. PR001-2014A.

#### **Author details**

Abdul Kariem Arof\* and Teo Li Ping

\*Address all correspondence to: akarof@um.edu.my

Centre for Ionics University of Malaya, Physics Department, Faculty of Science, Kuala Lumpur, Malaysia

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### *Edited by Eduardo Jacob-Lopes, Leila Queiroz Zepka and Maria Isabel Queiroz*

Chlorophyll presents an authoritative and comprehensive overview of the biology, biochemistry and chemistry of chlorophylls in photosynthetic organisms. Divided into seven discreet parts, the book covers topics on basic science and applied technology of chlorophyll molecules. Chlorophyll provides an insight into future developments in each field and extensive bibliography. It will be an essential resource for researchers and academic and industry professionals in the natural pigment field.

Chlorophyll

Chlorophyll

*Edited by Eduardo Jacob-Lopes,* 

*Leila Queiroz Zepka and Maria Isabel Queiroz*

Photo by Anna Babich / iStock