Growth evaluation of several types of energy crops from tropical shrubs species

Background: Few species of tropical shrubs potentially produce biomass to replace fossil fuels for heat production and electricity. The aims of this study were to determine the growth and nutrient status of leaves of several types of energy crops from tropical shrub species with NPK fertilizer application. Methods: Randomized block design was used with ten replications of four levels of fertilizer treatment: T0 = 40 g, T1 = 80 g, T2 = 120 g and T4 = 160 g per plant. Results: The results indicated that fertilization increased plant growth and the quantity of nutrients in leaves. The plants accumulated a lot of potassium, followed by nitrogen and phosphorus. The species of tropical shrubs with the best growth were Vernonia amygdalina, Calliandra calothyrsus and Gliricidia sepium, which are all potentially cultivated as sustainable energy crops. Conclusions: Serious attention must be paid to the availability of soil nutrients in order to sustain the cultivation of these plants.


Introduction
The Indonesian government has issued a national energy policy to encourage the development of alternative energy. This policy targets the replacement of diesel and gasoline with biodiesel and bio-ethanol by 5% in Indonesia by 2025. The Government commissioned the Ministry of Forestry to play an active role in the development of biomass production. The use of raw material biomass to replace fossil fuels for heat and electricity production is prioritized, including permits for the utilization of plantations in the form of unproductive areas and permits for natural forest utilization 1,2 .
Different types of forest plants and lignocellulose weeds have the potential to be developed as biomass feedstock for electricity production. Woody shrub species, such as Vernonia amygdalina Delile, Piper aduncum L., Gliricidia sepium (Jacq.) Kunth ex Walp., Calliandra calothyrsus Meissner., Bridelia tomentosa Blume, Vitex pinnata L., Vernonia arborea Buch.-Ham. and Bauhinia purpurea var. corneri de Wit., are suitable for use as sustainable raw materials for electrical energy 3 . This type of bush plant is a pioneer species that can easily be grown in secondary forests and open lands that have formerly been cultivated and logged. Energy crops are defined as cultivated plants that are developed and grown specifically for fuel and are rapidly growing, resistant to pests and droughts and can quickly be harvested, so they could have competitive prices if used as fuel 4 .
Wood is a renewable resource as it is sustainable and its supply will always be available. Meanwhile, cellulotic bio-fuel is produced from non-food (feed) stocks that play a critical role in reducing dependence on oil imports 5 . Woody biomass is produced, among others, by establishing plantations of energy crops. Coppice is a tree process regenerated with new shoots from the stumps that have been harvested and the Short Rotation Coppice (SRC) from hardwoods is more promising in generating biomass for bio-energy because it consists of fast-growing tree species and high-yield planted varieties (5000-15000 stems per ha), harvested in a two to six year rotation cycle.
This study focused on the growth and nutrient status of leaves of several types of energy crops from tropical shrub species with NPK fertilizer application, as a first step in the preparation of ready-to-plant if they are cultivated in the future as raw materials for biomass for renewable electricity.

Site and time
This study was conducted from January to September 2018 in a secondary forest located at Suka Damai Village, Muara Badak Sub-district, Kutai Kartanegara District, East Kalimantan province, Indonesia (00°17' to 18°2" S latitude and 117°14' to 14°39.5" E longitude). The wet season varies from 9 to 12 months and the dry season varies from 0 to 3 months. The average monthly temperature is 27.5°C and average air humidity is 82% 6 .

Plant materials
Seedling plants (Vernonia amygdalina, Piper aduncum, Gliricidia sepium, Symplocos fasciculata, Vitex pinnata, Bauhinia purpurea, Melastoma malabathricum, and Calliandra calothyrsus) were taken from the Mulawarman University Botanical Garden. Seedlings are included in polyethylene bags and maintained for 3 months. Those with uniform height are selected before being planted in the research plot.

Experimental design
Complete randomized block design (CRBD) was used as the experimental design. There were 8 plant species and their growth responses were evaluated based on 5 treatments using NPK fertilizer (commercial by YARA International ASA, Oslo, Norway; 16% N, 16% P 2 O 5 , 16% K 2 O 5 , 1.5% MgO, and 5% CaO). Five groups in five plots (each plant species): T0: with no fertilizer as control group (0 g/plant); T1: supplemented with NPK 40 g/plant; T2: supplemented with NPK 80 g/plant; T3: supplemented with NPK 120 g/plant; T4: supplemented with NPK 160 g/plant. The fertilizer treatments were conducted 2 weeks after sowing. There were 10 sample plants (as replication) for each treatment, a total of 50 plants for each species (divided into 5 experimental plots). Therefore, in total, there were 40 experimental plots, each plant was separated 1 m in length within each plot while it was separated 3 m in length between each plot ( Figure 1).

Measured parameters
All data were recorded after 5 months of planting. Stem height was measured by retractable tape measure (Shiro, Japan), basal stem diameter was measured by Vernier digital caliper (Mitutoyo, Japan), and the number of leaves and branches of each plant were counted.
All 10 replications of each treatment were pooled into one composited plant sample. Plant materials were analyzed at Forest Soil Science Laboratory, Faculty of Forestry, Mulawarman University to determine the total nitrogen, phosphorus, potassium, calcium and magnesium concentration. Total nitrogen was estimated using the Kjeldahl method 7 . Briefly, leaves were extracted using the wet destruction method using concentrated H 2 SO 4 (MERCK, Germany). The extract was distilled and added to 20 ml of 0.05 N NaOH (MERCK, Germany).
To measure the elements of P, K, Ca and Mg, the plant materials were extracted using high pressure digestion method at a temperature of 180 0 C for 10 hours with HNO 3 65% (MERCK Milipore, Germany) as a reductant. The calorimetric technique used nitric acid-molybdate-vanadate (MERCK, Germany) as a coloring agent and it was determined by spectrophotometer (GENESYS™20 Visible Spectrophotometer, Thermo Scientific™, Thermo Electron North America LLC, 14-385-445) at a wavelength 470 nm. Meanwhile potassium, calcium and magnesium concentration were measured by Atomic Absorption Spectrophotometer (Trace 1800, Aurora Biomed, Canada) at wavelengths of 766.5, 489.5 and 245.2 nm, respectively 5 .

Data analysis
The plant growth data were expressed as mean ± standard error. The data were subjected to ANOVA, followed by Duncan's Multiple Range Test (DMRT) to evaluated significant differences among the groups of treatment. All analysis was done using SPSS 22 (SPSS Inc. USA) and all significant tests were set at p≤0.05. The data of leaves' nutrient concentration were analyzed descriptively.

Results
The first part was the result of planting several types of tropical shrubs, including height growth, stem diameter, number of branches and leaves, while second part included the results of the analysis of nutrient content that was accumulated on the leaves of several types of tropical shrubs, namely nitrogen, phosphorus, potassium, calcium and magnesium. The results show that fertilization treatment affects the growth of superior energy tropical shrub plants. In plants V. amygdalina, P. aduncum, V. pinnata, C. callothyrsus, S. fasciculata and G. sepium, the best growth was found in T4 treatment, whereas in M. malabathricum and B. purpurea, the best growth was obtained by T1 treatment. This indicates that the response of plants varies according to the fertilization treatment. Stem height and stem diameter of the various plants after 5 months of planting are presented in Figure 2.
According to Figure 2, the highest stem height was observed in V. amygdalina while the lowest was in V. pinnata, S. fasciculata and B. purpurea. Moreover, the large stem diameter was measured in P. aduncum, while the lowest was in V. pinnata and B. purpurea.
The accumulation of nutrients in the leaves of the tropical shrub plants varied widely. The highest nitrogenous nutrients accumulated in the leaves of M. malabatricum, C. calothyrsus and V. pinnata, while the highest phosphorus and potassium nutrients accumulated in the leaves of V. amygdalina, P. aduncum and G. sepium. Calcium nutrients accumulated the most in the leaves of P. aduncum, G. sepium and S. fasciculata. On the other hand, the highest accumulation of magnesium nutrients occurred in the leaves of B. purpurea, V. amiqdalina and G. sepium (Figure 3). Full data of the effect of fertilizer applications on tropical shrubs growth (height, diameter, and Table 1. Fertilization effect on the average number of plant height, stem diameter, and leaf and branch number. T0 = 0 g (control), T1 = 40 g, T2 = 80 g, T3 = 120 g and T4 = 160 g of NPK. Number followed by the same letter in the same column show no significant difference in the DMRT test (p=0.05) after analysis by ANOVA.   leaf and branch number) and leaves' nutrient concentration (N, P, K, Ca and Mg) are available 8 .

Discussion
In this study, the growth and accumulation of nutrients in the leaves of tropical shrub plants varied greatly. The tropical shrubs that grew the best were V. amygdalina, G. sepium and C. calothyrsus, which accumulated mostly phosphorus and potassium nutrients in the leaves, while nitrogen, calcium and magnesium were least accumulated. These plants responded to NPK fertilizer up to 160 g per plant, resulting in best growth and high production of biomass, showing their suitable as raw material for biomass to create electricity. A previous study has shown that V. amygdalina provides 2.25 MWh, G. sepium 2.08 MWh and C. calothyrsus 2.01 MWh per ton of dry biomass 3 . As reported by Susanto and Amirta, fast-growing pioneer species such as M. gigantea absorbs the most potassium reaching 35% in every ton of plant biomass 6,9 . For V. amygdalina, growth parameters are positively correlated to rainfall, relative humidity and cloud cover 10 . On the other hand, G. sepium can be harvested at a residual height of 70 cm, with better agronomic characteristics and chemical composition occurring in the fall 11 . C. calothyrsus also has good growth in planting plots in previous research 12 , and it was previously reported that mycorrhizae such as Glomus sp. and Acaulospora sp. have significant influence on its height. In Colombia, planted fallows using C. calothyrsus have an additional benefit of producing large quantities of wood for household use 13 . Based on the growth and nutrient analysis in the present study, we believe that these tree plants species can be developed widely to support a sustainable supply of biomass feedstock for the green electricity program in Indonesia.
The growth of five plants species, namely M. malabatricum, P. aduncum, V. pinnata, B. purpurea, S. fasciculata was lower (less than half) than the three plants species discussed earlier (Table 1). In the present study, several plant species that grew slower actually accumulated more nutrients of nitrogen and phosphorus in the leaves, such as S. fasciculata, P. aduncum, V. pinnata, B. purpurea and M. malabatricum, P. aduncum and S. fasciculata also accumulated a large amount of potassium and calcium nutrients in their leaves. Moreover B. purpurea accumulated magnesium mostly in its leaves ( Figure 3). For M. malabathricum, the availability of phosphorus and aluminum in the rhizosphere increases its growth, it can also adapt to low soil pH 14 and absorb heavy metals in contaminated soils 15 . On the other hand, the mean foliar aluminum concentration in wild plants of M. malabathricum had positive correlation with foliar calcium, total nitrogen, calcium and magnesium concentrations within this species 16 while Symplocos sp. mean foliar aluminum concentrations were detected at 4107 (±1474 mg kg -1 ) and 4290 (±4025 mg kg -1 ) for seedlings and saplings, respectively 17 . P. aduncum can be propagated with seeds and shoot cuttings 18 and can accumulate large amounts of potassium, as previously reported 19 ; at 23 months, it had accumulated 222 kg N, 50 kg P, 686 kg K, 255 kg Ca, 75 kg Mg, and 24 kg S ha − 1 . More than half of the P, K, Ca and Mg nutrients were found in the stem (wood). Its leaf litter is significant and becomes an easily decomposable source of potassium, but G. sepium's leaf litter contains much nitrogen 18 while B. purpurea, which is a light-demanding tree, only grew 25% in a shady house with full sunlight 20 . Therefore, each tropical shrubs species in this study varies in accumulating plant nutrients N, P, K, Ca and Mg in their leaves.
Based on this study, it is necessary to pay attention to the fast developing plant type, especially for tropical shrubs that have the potential as raw materials for biomass energy. Many beneficial nutrients also accumulated mostly in the leaves. This accumulation reflects the nutrient requirements of those plants, which will be cultivated as energy raw materials; therefore at the initial stage, the demand for plant fertilizer can be predicted. The carrying capacity of soil nutrients requires serious attention for the sustainment of plant cultivation. These wood shrubs species (V. amygdalina, G. sepium and C. calothyrsus) were also able to re-grow naturally by generation more than single shoots on their coppice trees. The scheme of Short Rotation Coppices (SRC) was an effort to achieve forest energy plantation using fast growing trees and wood shrubs species for aiming for a sustainable cycle 3 .

Conclusion
Of the eight types of tropical shrubs in this study, three species, namely V. amygdalina, G. sepium and C. calothyrsus, had the best growth and could potentially be developed as Energy Crops. The most accumulated nutrients in the leaves of these three species of plants are phosphorus and potassium.

Data availability
Open and roots? It is recommended to write a few lines. Also write about the biomass of each plant part.
The entire discussion section is lacking an in-depth look at what the driving factors for the results were. Most of this section appears to be a literature review of what others have done and does not draw strong arguments to the data presented. This is in direct contradiction with several statements. This section needs a complete rewrite. 6.