Effects of nitrogen addition on Eucalyptus globulus growth and carbon sequestration potential under various CO₂ climatic conditions

ABSTRACT

The catastrophic and enormous increase in atmospheric greenhouse gas levels leads to global climate change, and an immediate need to investigate the effects of increased CO₂ levels on natural vegetation is complex. The goal of this study was to see how nitrogen addition affected Eucalyptus globulus Labill. species development in both ambient (408 ppm) and different elevated (450 ppm, 500 ppm, 550 ppm, and 600 ppm) carbon dioxide environments with OSC S-1000 L plant growth chamber. In this investigation, nitrogen was added in the form of potassium nitrate in various amounts (0, 25, 50, 100, and 200 mg/kg of soil). The plant’s growth responses (leaf length, leaf width, and shoot length) were investigated. To calculate the carbon content, a multiple regression equation was employed to fit the model. The maximum carbon content value 52.37 ± 0.03% was recorded at 200 mg/kg nitrogen addition level for an elevated CO₂ condition of 600 ppm. The results of this study conclude that the nitrogen addition had a positive impact on the plant growth and carbon sequestration potential of Eucalyptus globulus under ambient as well as elevated CO₂ conditions.

KEYWORDS:

• Agroforestry
• climate change
• plant growth
• model fit equation
• carbon content

Introduction

Global climate change is associated with increased atmospheric carbon dioxide (CO₂) concentrations generated by fossil fuel burning and increased nitrogen (N) deposition induced by human activities (Martinez et al., 2017). Specific plant communities and their associations are likely to be affected by these factors (Churkina, 2016). Most vegetation has the well-established effects of increasing the level of CO₂ on photosynthesis, biomass production, and plant water-use efficiency, and these effects vary depending on vegetation and nutrient levels (Bhargava & Mitra, 2021). The increasing N restriction produced by higher CO₂ growth stimulation is thought to limit the sustainability of ecological responses to CO₂ (Z. Xu et al., 2016). As a result, scientists from all around the world have been studying the relationship between CO₂ and soil N availability, with varying results reported in several studies (Farhate et al., 2018).

N emissions and deposition rates are expected to quadruple by 2050 due to anthropogenic activities dominating global N cycles, resulting in additional regions receiving potentially dangerous levels of N inputs. The quantity of N deposited across the planet varies dramatically between locations (Verma & Sagar, 2020). Enhancement of biologically reactive N by human activity alters the nutrient cycles in soils and vegetation structure and limits biological diversity (Kenkel et al., 2016). In many terrestrial habitats, N is the most important nutrient for plant development. As a result, N fertilizer is frequently employed in terrestrial N-limited habitats to boost plant growth and production by increasing soil N availability (Ganesh & Pragasan, 2019). The pace and duration of N deposition can affect cellular and soil N status, and deposition rates can surpass plant N absorption capability (Carter et al., 2017). N deposition has been shown to have varied impacts on many plant species (Boutin et al., 2017), depending on whether N saturation occurs at a given deposition rate (Lu et al., 2016). However, the physiological adaptations of the species present influence the responses of forest trees and shrubs to N deposition (Penuelas et al., 2020).

Nature has its own “reservoirs” or “sinks” for sequestering and storing carbon (C). Over 50% of the CO₂ discharge produced by humans has been stored in natural terrestrial and ocean ecosystems. Forests plays a critical role in the C cycle and C storage, and they store more than half of all terrestrial C and have a high potential to absorb atmospheric CO₂ (Abeysekara et al., 2018). Trees are an important component of forest ecosystems because they function as a significant CO₂ sink, collecting CO₂ from the atmosphere and eliminating it through photosynthesis in fixed woody biomass (Martinez et al., 2017).

Agroforestry practices have a long history in India. C capture occurs both belowground and aboveground in many forest ecosystems, as in forms of soil C and root growth augmentation, as well as C deposited in standing biomass. In India, the forestry system consists of farm trees, social forestry, and a variety of indigenous forest conservation and ethno forestry approaches. The practice of planting trees on farms dates back centuries and has mostly stayed constant. These trees are multifunctional, serving as shade, fodder, fuelwood, fruit, vegetables, and medicinal purposes (Basu, 2014).

Agroforestry has been estimated to store 9, 21, 50, and 63 Mg C per hectare (ha) of C in semiarid, sub-humid, humid, and temperate areas, respectively. Early research of effective sequestering carbon in forest ecosystems and other land-use systems in India indicated a capacity of 68 to 228 Mg C per hectare (Murthy et al., 2013), equivalent to 25 tonnes C per ha over 96 million ha of land (Rao et al., 2016). However, depending on biomass production, this number varies by area (Sun & Liu, 2020). Eucalyptus provides a variety of medical benefits, including antibacterial characteristics, effectiveness as an insect repellent, acting as a painkiller, triggering an immunological response, treating skin wounds and fungi, and acting as an expectorant to release mucus and alleviate congestion (Khan et al., 2020).

With the primary objective of reducing atmospheric C concentration by trapping it in plant biomass to mitigate greenhouse effect, which causes global warming and climate change. Although one cannot accurately foresee the climate of the future, it is anticipated that the amount of CO₂ would rise; thus, how would the current species store C under this circumstance? or, to put it another way, how would the species lower the level of C in the atmosphere? Whether they would sequester more, less, or not at all in proportion to rising levels of atmospheric C? An extensive literature search found that only a few research studies on the impact of N on C storage have been conducted across the world. Hence, to fill the knowledge gap, the current work was conducted to determine the effects of N addition (N_add) on the C storage capacity of Eucalyptus globulus under ambient and different elevated CO₂ environment conditions.

Materials and methods

Study species

The current study was focused on the tree species Eucalyptus globulus (Myrtaceae). A pot experiment was conducted at ambient (408 ppm) and different elevated CO₂ (450 ppm, 500 ppm, 550 ppm, and 600 ppm) conditions using a plant growth chamber (OSC S-1000 L) at Environmental Ecology Laboratory, Environmental Sciences Department, Bharathiar University.

Experimental design and treatments

This work was conducted in an OSC S-1000 L plant growth chamber with various levels of CO₂ conditions. The plants were kept alive by a 16-/8-hour light/dark cycle. The chambers were kept at a temperature of 25 ± 3.1°C, which was close to room temperature. The seeds of the Eucalyptus globulus species were procured from Tamil Nadu Agricultural University (TNAU) in Coimbatore and were germinated on seedling trays. After 3 weeks of germination, 25 productive plants were selected and each of them was transplanted separately in a pot with 1 kg of soil under ambient CO₂ (408 ppm) and different elevated CO₂ (450 ppm, 500 ppm, 550 ppm and 600 ppm) environment conditions for 98 days. The characteristics of soil were analysed before and after the experiment. For each experimental condition, N was supplied in the form of potassium nitrate at rates of 0, 25, 50, 100, and 200 mg/kg soil, with five replicates for each experimental setup (Pragasan & Ganesh, 2021). The pots were sprinkled with water at regular intervals to keep the soil moisture at 30–35%.

Plant growth analysis

Growth parameters such as shoot length (SL), leaf length (LL), and leaf width (LW) were recorded once in two weeks. The plants were then collected and utilized for further investigation. SL, LL, and LW were measured using a measuring scale, and C content was calculated using the loss on ignition method.

Model fit equation

A model fit equation was developed using linear regression analysis to predict the C content of the Eucalyptus globulus species under various N_add levels and different levels of CO₂ environmental conditions. For the model fit, a multiple regression equation was employed (Abdullah et al., 2012),

(1) $\mathrm{Y}={\beta }_0+{\beta }_1{\mathrm{X}}_1+{\beta }_2{\mathrm{X}}_2,$(1)

where Y is the dependent variable C content, X₁ is the independent variable as N_add levels, X₂ is the independent variable as different levels of CO₂ conditions, and β₀, β₁, and β₂ are the coefficients.

Statistical analysis

Analysis of Variance (ANOVA, F-test) was made to check statistically significant changes between treatments by using SPSS (ver. 21). The F-test was used to reveal significant differences in the C content of the Eucalyptus globulus species (p < 0.05) among the various N_add levels and CO₂ conditions. In addition, an equation for determining the C content was developed by using linear regression analysis.

Results

Plant growth analysis

Figures 1–5 depict the growth of Eucalyptus globulus in ambient (408 ppm) and elevated (450, 500, 550, and 600 ppm) CO₂ environments with various levels of N_add. N promotes the growth of SL, LL, and LW of the transplanting seedlings for all the environmental setups throughout the study. The SL, LL, and LW were recorded maximum for N_add at 200 mg/kg for the transplanted Eucalyptus globulus seedlings to the pots. At 408 ppm, the values of SL, LL, and LW were 24.74 ± 0.50 cm, 4.42 ± 0.42 cm, and 2.46 ± 0.11 cm, respectively (Figure 1). Likewise, for elevated CO₂ conditions 450 ppm, 500 ppm, 550 ppm, and 600 ppm, the values were 23.92 ± 0.61 cm, 3.68 ± 0.22 cm, and 2.36 ± 0.31 cm (Figure 2); 22.94 ± 0.64 cm, 4.26 ± 0.34 cm, and 3.54 ± 0.15 cm (Figure 3),;24.3 ± 1.22 cm, 4.32 ± 0.28 cm, and 3.02 ± 0.31 cm (Figure 4), and 24.42 ± 1.18 cm, 4.28 ± 0.27 cm, and 2.7 ± 0.27 cm (Figure 5).

Figure 1. Effect of various levels of N_add on the growth of Eucalyptus globulus Labill under ambient CO₂ conditions (408 ppm).

Figure 2. Effect of various levels of N_add on the growth of Eucalyptus globulus Labill under 450 ppm eCO₂ conditions.

Figure 3. Effect of various levels of N_add on the growth of Eucalyptus globulus Labill under 500 ppm eCO₂ conditions.

Figure 4. Effect of various levels of N_add on the growth of Eucalyptus globulus Labill under 550 ppm eCO₂ conditions.

Figure 5. Effect of various levels of N_add on the growth of Eucalyptus globulus Labill under 600 ppm eCO₂ conditions.

Carbon content

Figure 6 depicts the C content of Eucalyptus globulus under various degrees of N_add and CO₂ conditions. Table 1 shows the results of the F-test ANOVA values for C content. The F-test findings show that varying amounts of N_add, CO₂ environmental conditions and the combination of N_add and CO₂ conditions all has a significant impact on the C content of Eucalyptus globulus. We reject the null hypothesis because the alpha value (0.05) is larger than the p-value (0.000; Table 1). Likewise, a significant difference exists across the impacts of different CO₂ environmental setups on C content, as revealed by the ANOVA value. Furthermore, the C content differs significantly between N_add and various CO₂ situations.

Figure 6. Effect of various levels of nitrogen addition on the carbon content of Eucalyptus globulus Labill at different CO₂ environmental conditions.

Table 1. F-test ANOVA values for the carbon content of Eucalyptus globulus under various levels of N_add and different CO₂ environmental conditions.

Source	Type III Sum of Squares	Df	Mean Square	F	P Value
N	6.548	4	1.637	464.353	0.000
CO2	77.353	4	19.338	5485.526	0.000
N * CO2	8.101	16	0.506	143.624	0.000
Error	0.176	50	0.004
Total	92.179	74

Model fit equation

To assess the C content of the Eucalyptus globulus species, a multiple regression equation was applied for model fit. Linear regression analysis was used to determine the coefficient values (Table 2) for the model fit equation and model summary (Table 3) to estimate the C content with various levels of N_add and CO₂ environmental setups. Multiple regression R² values for Eucalyptus globulus are shown in the scatterplot diagram (Figure 7).

Figure 7. Multiple regression scatterplots for Eucalyptus globulus.

Table 2. Values of model coefficients for the dependent variable (carbon content).

Model	Unstandardized Coefficients		Standardized Coefficients	T	P Value
	β	Std. Error	Beta
Constant Nitrogen addition Carbon dioxide level	47.547	0.142		333.747	0.000
	0.187	0.032	0.238	5.864	0.000
	0.712	0.032	0.908	22.342	0.000

Table 3. Model summary for the dependent variable (carbon content).

Model	R	R Square	Adjusted R Square	Std. Error of the Estimate
1	0.939	0.881	0.878	0.39015

The coefficient values of β0 (47.547), β1 (0.187), and β2 (0.712) were implemented to the model fit to find out C content in the Eucalyptus globulus species by using (1),

(2) $\mathrm{Y}=47.547+0.187{\mathrm{X}}_1+0.712{\mathrm{X}}_2,$(2)

where Y is the C content, X₁ is the N_add level, and X₂ is the CO₂ level.

Discussion

Climate change has emerged as the most significant environmental issue in recent years, owing to growing CO₂ levels in the atmosphere. These problems are linked to several issues, which comprise a variation in primary production and natural ecosystem carbon dynamics. The current work is focused on studying the impacts of N_add on the growth and C sequestration potential of Eucalyptus globulus under ambient and elevated CO₂ environmental conditions. The outcome of this experimental work reveals that the impact of N_add had a positive impact on the growth and CO₂ potential of Eucalyptus globulus species at ambient and elevated CO₂ environment conditions. Under ambient CO₂ conditions, the C content has recorded a maximum of 49.6 ± 0.07 at the 50 mg/kg N_add level. For the elevated CO₂ condition 450 ppm, the maximum C content 50.02 ± 0.05% was recorded at the 50 mg/kg N_add level. While, for the elevated CO₂ condition at 500 ppm, the maximum carbon content 50.07 ± 0.05% was recorded at 0 mg/kg N_add level, for the elevated CO₂ conditions at 550 ppm and 600 ppm, the maximum C content values (51.87 ± 0.03% and 52.37 ± 0.03%, respectively) were recorded at the 200 mg/kg N_add level. Similarly, plant growth parameters such as SL, LL, and LW showed positive growth along with an increase in N_add and CO₂ concentrations.

Also, our earlier studies on Azadirachta indica (Ganesh & Pragasan, 2019) and Syzygium cumini (Pragasan & Ganesh, 2021) have revealed that the C sequestration potential of plants species had positive effects with N_add and increasing CO₂ environment conditions. Similar results were seen on enhanced photosynthetic capacity, and biomass production included how plants grown in elevated CO₂ environments would react to CO₂ enrichment (Oliveira & Marenco, 2019). Plant growth and terrestrial ecosystem development can have positive effects from increased N availability (W. Li et al., 2015; H. Zhang et al., 2018). The plant’s response to CO₂ is influenced by several factors that include air temperature, N, phosphorous (P), various nutrients, growing period, symbiosis with mycorrhiza and availability of water (Baslam et al., 2012; Bicharanloo et al., 2021). CO₂ impacts on growth are only seen in completely watered plants; those that are just moderately watered have no CO₂ effects (Irigoyen et al., 2014). Plants grow rapidly when subjected to a result of high CO₂ concentrations and high-temperature circumstances (Barickman et al., 2021).

In this study, Eucalyptus globulus species recorded a maximum C content of 52.37 ± 0.03% at the 200 mg/kg N_add level for the elevated CO₂ condition 600 ppm. Similar results were reported for Azadirachta indica (54.82 ± 0.53%, Ganesh & Pragasan, 2019) and for Syzygium cumini (53.3 ± 0.09%, Pragasan & Ganesh, 2021). The application of N has complex impacts on seedling growth and survival (Razaq et al., 2017). N enhancement has a significant impact on the shoot morphology and nutritional status of nursery seedlings in prior research (F. Xu et al., 2020). Moreover, N supplementation increases the seedling’s height as well as the diameter of the root collar (Hu et al., 2019). Similarly, the results of this study found that supplementing with N enhances Eucalyptus globulus growth (SL, LL, and LW). This demonstrates that N fertilizers are required for maximum plant development. Brienen et al. (2020) have revealed that the responses of plants to increased CO₂ levels in the atmosphere have enhanced net photosynthesis, development, and biomass production of plants, and increased CO₂ stimulates faster growth rates in young seedlings than in older trees, while Eastman et al. (2021) stated that the addition of N encourages the storage of additional C from the environment in respective plant species. Furthermore, the most prevalent inorganic cation is potassium (K), which is crucial for promoting healthy plant development. It contributes significantly to the development of yield and quality enhancement. In addition to being crucial for plant development and function, K is also crucial for cell proliferation (F. Xu et al., 2020).

Multiple regression analysis was used in this study to develop a model fit equation to assess the C content. The R² value is 0.881 (Table 3 and Figure 7), and this indicates that the equation has an accuracy of 88.1% in predicting the C content of Eucalyptus globulus species under various N addition levels (0–200 mg/kg) and different CO₂ environmental conditions (408–600 ppm). This equation is suitable for predicting the C content of Eucalyptus globulus species within the prescribed ranges of N_add and CO₂ levels.

The ability of elevated CO₂ to enhance plant development is highly dependent on the growth potential of the plant’s species (Silva et al., 2020). Every species has responded to elevated CO₂ environments in a distinctive manner (Gagne et al., 2020), including changes in aboveground growth (Gupta et al., 2020). Table 4 represents how N addition and CO₂ levels affect the growth of plant species reported earlier. A positive relationship between increased CO₂ and N implies that soil N limitations may gradually reduce beneficial photosynthetic carbon uptake and biomass reactions with elevated CO₂ conditions (Wang, 2007). Species that can enhance their nutritional supply in the presence of an increased CO₂ environment either via root absorption and mycorrhizal symbiosis or through increased N fixation will react very strongly to rising environmental conditions (Diagne et al., 2020).

Table 4. Growth response of plant species to N_add/CO₂ levels reported so far.

Species name	Place	Impact	Reference
Azadirachta indica	India	Positive	Ganesh & Pragasan, 2019
Brassica rapa	Australia	Positive	Thomson et al., 2013
Calluna vulgaris	Norway	Positive	Sabo et al., 2001
Caragana microphylla	China	Positive	Zhang et al., 2011
Cucumis sativus	China	Positive	X. Li et al., 2020
Fraxinus mandshurica	China	Positive	M. Wang et al., 2012
Fraxinus rhynchophylla	South Korea	Negative	Cha et al., 2017
Impatiens hawkeri	China	Positive	F. F. Zhang et al., 2012
Kobresia pygmaea	Tibet	Negative	Wang et al., 2015
Leymus chinensis	China	Positive	Bai et al., 2020
Leymus chinensis	China	Positive	Zhang et al., 2010
Ligularia virgaurea	Tibet	Negative	Wang et al., 2016
Lindera glauca	China	Positive	Hu & Wan, 2019
Paliurus ramosissimus	South Korea	Positive	Jeong et al., 2018
Quercus acutissima	South Korea	Negative	Cha et al., 2017
Quercus gilva	South Korea	Neutral	Jeong et al., 2018
Stipa purpurea	Tibet	Positive	Wang et al., 2015
Symplocos chinensis	China	Positive	Hu & Wan, 2019
Syzygium cumini	India	Positive	Pragasan & Ganesh, 2021
Taraxacum mongolicum	Tibet	Negative	Wang et al., 2016
Vitex negundo	China	Positive	Hu & Wan, 2019

Generally, plants acclimatize and adjust to their surroundings to maximize their survival and growth. Some plant species that grow well in high CO₂ environments when cultivated alone have been found to show less growth in different plant communities (Velasquez et al., 2018). This impact is most probably because the direct benefits of increased CO₂ are offset by the negative consequences of promoting competition growth. As CO₂ levels rise in the atmosphere, plant communities may alter, as certain species benefit more than others from the increasing atmospheric CO₂ concentrations. When the CO₂ level in the atmosphere rises, the first effect is on photosynthetic enzymes. This main impact is the source of all subsequent modifications. Plants adapt their physiological functions as well as their biochemical processes in response to environmental factors. Variations in biochemical composition, carbonic anhydrase, and nitrate reductase activity have been reported in CO₂ enriched studies, and the responses are species-specific (Aswathi et al., 2018).

Conclusion

We conclude from this experimental study that the effect of N_add had a positive impact on the plant growth (SL, LL, and LW), and C sequestration potential of Eucalyptus globulus species under ambient and different elevated CO₂ environmental conditions. The current study was performed under constrained environments, in terms of N_add and different CO₂ setups. Furthermore, a model fit equation was developed to assess the C content of Eucalyptus globulus species grown at various N_add levels (0–200 mg/kg soil) and CO₂ concentrations (408–600 ppm). Thus, the current study provides valuable information on the growth and C sequestration potential of Eucalyptus globulus under N_add and different CO₂ conditions. We recommend plantation of Eucalyptus globulus, not only for its medicinal purposes but also for its C sequestration potential in mitigation of global warming and climate change.

Author contributions

Dr L. Arul Pragasan, Assistant Professor, the corresponding author contributed by conceptualization, funding acquisition, supervision, and final approval of the paper. Mr K.P. Ganesh is a research scholar who contributed by carrying out the work, data collection, laboratory analysis, and drafted the paper.

Acknowledgments

The authors sincerely thank DST-SERB, Government of India for the financial support for this research work.

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Funding

This work was supported by the Science & Engineering Research Board (SERB), Department of Science & Technology, Government of India (EMR/2016/003254).