The effect of palm oil (Elaeis guineensis Jacq) solid waste on the stomatal density and chlorophyll content of the pepper (Piper nigrum L.)

This study investigates the impact of palm oil (Elaeis guineensis Jacq) solid waste on stomatal density and chlorophyll content in pepper plants (Piper nigrum L.). Employing a Randomized Block Design (RBD) comprising four treatments and five replications, the research used the mixed of the waste and soil with the ratios: P0 (1 kg soil, control), P1 (333 g palm oil solid waste and 666 g soil, 1:2 ratio), P2 (500 g soil and 500 g palm oil solid waste, 1:1 ratio), and P3 (666 g palm oil solid waste and 333 g soil, 2:1 ratio). Our findings indicate that the stomatal density in the adaxial part of pepper’s leaves was highest in treatment P2, whereas stomatal density in the abaxial part was highest in treatment P3. Additionally, the highest chlorophyll a content was observed in treatment P2, while chlorophyll b and total chlorophyll content were highest in treatment P3. These results underscore the complex interplay between waste composition and plant physiological responses.


Introduction
Pepper (Piper nigrum L.) is well-known as the 'King of Spices', widely used in the world, and contributes a lot to the country's foreign exchange [1,4].It is originating from South Asia and also was the first product offered between West and East area in the Middle Ages [5].This herb is a commercial spice with high economic value due to its pungency and flavor.Three main types of peppers that are valuable in the market are; black, white, and green.The black pepper is the most widely used in the world, commonly for healthy food preparation.Blackening is the most important step for coloring and flavoring in black pepper processing [3].
Oil palm now plays a significant role in the world, particularly Indonesia and Malaysia, from which significant amounts of its products are exported in the form of oil, meal, cooking spices, cosmetics, medicine, and other derivatives [6,7,8,11,12].Oil palm is now more frequently grown in plantations throughout the humid tropics of Asia, Africa, and the Americas, from which its goods are sold to international markets.The majority of oil palm is still grown in the two adjacent countries in Southeast Asia, Indonesia and Malaysia, which produce almost 85% of the world's total production [8,9,10].This spice belongs to the Piperaceae family, a woody climber that reaches up to 10 m in height, each spike consists of 40-50 blossoms, rounded leaves, oblong and elongated, compound fruit, and single seed.It recorded about 4226 plant species, used as spices, medicinal plants, and ornamental plants [2].
Indonesia is a tropical country that grows a lot of beneficial plants, it is also rich in flora diversity.One of those is the pepper plant (Piper nigrum L.) [5].The rapid growth of the global human population and the increasing demand for food resources have driven the expansion of agricultural and plantation areas [13,14,16].One prominent agricultural commodity that garnered significant attention is palm oil (Elaeis guineensis Jacq), which has become a primary contributor to the global vegetable oil supply [15].However, the growth of the palm oil industry has also led to a surge in solid waste generation, encompassing fibres and other organic materials [16].An eco-sustainable approach to addressing this waste issue involves repurposing it in agriculture as organic fertilizer [17].In this context, the present study aims to investigate the function of palm oil solid waste as fertilizer on stomatal density and chlorophyll content in pepper (Piper nigrum L.).
The primary issue in cultivating pepper plants in Aceh is soil fertility and insufficient soil nutrient content for this crop, applying organic fertilizers can help solve this problem [42].Utilizing palm oil solid waste as a potential fertilizer presents an opportunity to enhance soil fertility and nutrient accessibility, potentially resulting in improved growth and yield of pepper plants [18].Therefore, understanding the effects of applying palm oil solid waste on stomatal density and chlorophyll content in pepper plants holds significance.
Stomata and chlorophyll are the main important parts of plants, especially for photosynthetic reactions.Stomata, which are tiny holes dispersed throughout the leaf epidermis, act as dynamic gatekeepers for the exchange of gases essential for regulating transpiration and photosynthesis.As the key component of photosynthetic efficiency, chlorophyll, the green pigment found in chloroplasts, uses solar radiation to start the energy conversion process in the creation of glucose and other organic compounds.Stomata and chlorophyll work together, highlighting their crucial roles in coordinating the essentials for plant growth [19,20,21] 2. Materials and methods

Plant materials
The study was conducted from August to December 2021 in the Biological Garden, Biology Department, Science and Technology Faculty, Ar-raniry State Islamic University, Banda Aceh, Indonesia.The plant material used was stem cutting of Pipper (Piper nigrum L.) collected from the nursery garden in Banda Aceh.

Palm oil solid waste preparation
Fresh fruit bunches (FFB) waste is collected from oil palm plantations in Nagan Raya District, Aceh, Indonesia.Next, the waste undergoes a grinding process to reduce its size and consistency, making it easier to handle and use later.easier.The finely ground waste then undergoes controlled fermentation lasting for 20 days to break down the organic matter present in the waste and lower the temperature, thereby improving the nutrient content and transforming it into a form more suitable for use for a mixture of plant growth medium [22].

Pepper cultivation
Pepper plant cuttings, measuring 10 cm in length, were transplanted into polybags with a growing medium composed of a mixture of soil and palm oil solid waste in the following treatments: P0 (1:0) = 1000 g of soil + 0 g of palm oil solid waste, P1 (1:1) = 500 g of soil + 500 g of palm oil solid waste, P2 (1:2) = 333 g of soil + 666 g of palm oil solid waste, and P4 (2:1) = 666 g of soil + 333 g of palm oil solid waste.The seedlings were watered twice a day.After four months of growth, pepper leaves were collected to determine stomatal density and chlorophyll content

Stomatal density calculation
Leaf number three from the top was used to calculate the stomatal density.The replica method was used to prepare the slide to observe the stomatal type, stomatal number, stomatal density, and epidermal cell under the microscope.The steps are as follows: (1) Cleaned the upper and lower surfaces of the leaves, (2) Applied the nail polish and left it for 10 minutes to be dried (3) Dried spreads were attached with transparent tape and flattened, (4) the transparent tape was peeled and removed slowly from the leaves surfaces, then attached to the object-glass, (5) flattened and labelled with a description of the plant type, and (6) Calculated the stomatal density by using a light microscope with same magnification (40x) [23] The stomatal density calculation was conducted with three repetitions for each species with the following formulas: Stomatal density = Number of stomata Field of view under the microscope The number of stomata was calculated under the microscope with 10x magnifications, while the field of view was calculated with the same formula with circle area formulas.

Chlorophyll content measurement
The chlorophyll content measurement was done by preparing the leaf extract of pepper, and the results were examined using Perkin Elmer Spectrophotometer Lambda 365 UV/Vis (Fig. 1) with wavelengths of 663 nm and 645 nm, and chlorophyll content was calculated with the following formulas:

Experimental design and data analysis
The experiments were laid out in a randomized block design with 5 replications.Each experimental treatment combination consisted of soil and palm oil solid waste.The parameters recorded Stomatal density and chlorophyll content.The data were subjected to analysis of variance (ANOVA) and significant differences among the treatments were tested using two-way ANOVA and means were separated by Duncan multiple range test (DMRT) at P ≤ 0.05.

Results and discussion
Many researchers have applied palm oil waste to improve plant growth and yield.Decanter cake derived from waste produced by palm oil mills was employed as an organic material source to enhance soybean yield [31].Palm oil solid waste also influenced the plant height, flowering speed, and fruit fresh weight of green eggplant (Solanum melongena L.) [30,32].The utilization of palm oil solid waste fertilizer had an impact on flowering age, flower diameter, flower weight, and cauliflower (Brassica oleracea var.botrytis L.) yield per hectare [33].The use of palm oil empty bunch (POEB) waste also affected the vegetative and generative growth of three pineapple accessions cultivated on post-tin mining land, palm oil empty bunches also resulted in the highest chlorophyll content, which was significantly distinct from both the control and palm oil empty ash treatments [34].In this study, FFB waste of palm oil significantly affected the stomatal density (Table 1) and chlorophyll content (Table 2) of the pepper plant.

Stomatal density
The data obtained from the study showed a significant variation in stomatal density on the abaxial and adaxial sides of plant leaves across different treatments (Table 1).In the P0 treatment (1:0), where only pure soil was used, the stomatal density on the adaxial side was recorded at 184.40 mm -2 while on the abaxial side, it was 982.63 mm -2 .In the P1 treatment (1:1), with the application of 500 grams of palm oil waste, the stomatal distribution on the adaxial side increased to 220.89 mm -2 .On the abaxial side, it substantially increased to 1.444.03mm -2 .Furthermore, the P2 treatment (1:2), with 666 grams of palm oil waste, resulted in a stomatal density of 208.30 mm -2 on the adaxial side and 1.177.96mm -2 on the abaxial side.Lastly, the P3 treatment (2:1), involving 333 grams of palm oil waste, exhibited a stomatal density of 223.13 mm -2 on the adaxial side and 783.83 mm -2 on the abaxial side.The highest stomatal density in the abaxial part of the leaves showed in the combination of P3 treatment which is 223.13 mm -2 while in the abaxial part in the combination of P1 treatment which is 1.444.03mm -2.Stomata are essential organs in the process of photosynthesis as well as transpiration in plants.Stomata serve as sites for the exchange of CO2 in leaves for the process of photosynthesis and as locations for water evaporation in the transpiration process [35].The results obtained from this study showed that the additional palm oil solid waste was increasing the stomatal density which is higher than the control.The highest number of stomatal densities was on the abaxial part of the leaves, in the P2 treatment where the amount of the soil and palm oil waste were in a balanced composition.Stomata have a crucial function in controlling gas and water exchange in plants by aiding various physiological processes like transpiration, stomatal conductance, and photosynthesis [38].
An increase in stomatal density within plant leaves can lead to several conditions.Higher stomatal density can enhance the plant's photosynthetic capacity by facilitating increased carbon dioxide (CO2) uptake, ultimately resulting in improved photosynthesis rates and potentially higher biomass production.However, this may also lead to elevated transpiration rates, increasing water loss through the stomata.While this can aid in cooling the plant, excessive transpiration without sufficient water uptake may risk desiccation and water stress.Moreover, an elevated stomatal density can affect leaf temperature regulation; if transpiration rates become too high, leaf temperature might decrease, potentially leading to cold stress in certain conditions.Therefore, the impact of increased stomatal density is contextdependent, influenced by factors such as plant species, environmental conditions, and water availability, and necessitates a balance between efficient photosynthesis and water management [36,37,39] IOP Publishing doi:10.1088/1755-1315/1297/1/0120135 The study obtained those different treatments involving palm oil waste significantly influenced chlorophyll (Chl) content in plants.The application of palm oil waste led to substantial increases in chlorophyll a (Chl a), chlorophyll b (Chl b), and the total chlorophyll (Chl) content compared to the control (P0), indicating a potential stimulatory effect on chlorophyll synthesis.The observed variations in chlorophyll content among treatments (P1, P2, and P3) suggest a dose-dependent response.

Chlorophyll content
The highest chlorophyll a content was recorded in the P1 treatment (1.5457 mg/L), where the amount of the soil and palm oil waste were in a balanced composition, this condition was also applied for the stomatal density on the abaxial part (Table 1).The highest chlorophyll b and the total chlorophyll content were recorded in the P3 treatment which is 2.09267 mg/L and 2.26633 mg/L respectively.While the use of decanter solid waste of palm oil showed no significant effect on the chlorophyll content of palm oil seedlings itself [41].
These findings underscore the importance of considering the environmental impact of palm oil waste on plant physiology and highlight the potential for using such waste as a nutrient source for plant growth, while also emphasizing the need for further research to elucidate the underlying mechanisms driving these changes in chlorophyll content.The increasing number in chlorophyll content in this research is attributed to the impact of potassium (K) nutrients.Palm oil waste, with each ton containing 1.5% nitrogen (N), 0.5% phosphorus (P), 7.3% potassium (K), and 0.9% magnesium (Mg), can serve as a viable alternative fertilizer for plants.[40,34].
FFB is an organic waste consisting of 1.5% N, 0.5% P, 7.3% K, and 0.9% Mg.It has sufficient potency large enough to be used as a substitute fertilizer by applying the waste above the land around the plantations [27].Nitrogen functions as a component found in amino acids and proteins, which are essential elements in the composition of chlorophyll, a crucial component in the process of photosynthesis [34].Another research mentioned that EFB consists of 52.93% cellulose, 15.27% hemicellulose, and 17.43% lignin [28].Organic matter can be utilized to enhance soil fertility, including soil physical attributes such as soil structure and porosity.Essentially, any solid organic material can undergo composting, including household organic waste, livestock manure, agricultural residues, agroindustrial byproducts, paper mill leftovers, sugar factory residues, palm oil mill residues, and various others [29].The term "solid" refers to waste generated during the processing of fresh fruit bunches (FFB) at palm oil mills to produce crude palm oil (CPO).This raw solid waste has a consistency similar to tofu dregs, possesses a brownish hue, emits a sour odor, and still retains approximately 1.5% CPO oil content [29,30].

Conclusions
The combination of soil and palm oil solid waste as a growth medium significantly affected the stomatal density of the pepper (Piper nigrum L.) plant especially on the adaxial part in the P3 treatment and on the abaxial part in the P1 treatment.The same condition applied for the chlorophyll content, whereas the highest Chl a obtained in the P1 treatment, Chl b and total Chl obtained in the treatment P3.In terms of stomatal density, these treatments induced diverse effects on the density of stomata on both the upper and lower leaf surfaces, reflecting the plant's ability to adapt to the presence of the waste material.Furthermore, the examination of chlorophyll content underscored the potential of palm oil waste as a source of vital nutrients, notably potassium (K), contributing to heightened chlorophyll levels within plants.These findings underscore the intricate relationship between palm oil waste treatments, the distribution of stomata, and chlorophyll production, emphasizing the necessity for further investigation to elucidate the underlying mechanisms and their implications for plant physiology and sustainable environmental management.

Acknowledgement
The authors would like to thank the Biology Department of Muhammadiyah Aceh University for providing laboratory services in stomatal distribution analysis and the financial support of publication, and the Biology Department of Ar-raniry State Islamic University for laboratory assistance in chlorophyll content analysis.

Table 1 .
The stomatal density of the pepper plants Means followed by the same letter in each column do not differ by Duncan's multiple range test at P < .05

Table 2 .
Chlorophyll content of the pepper plantsMeans followed by the same letter in each column do not differ by Duncan's multiple range test at P < .05