Development and evaluation of biochar formulated fertilizers for climate-smart sustainable crop production

Abstract

Climate-smart agriculture represents a sustainable approach that promotes food production and soil fertility while reducing greenhouse gas (GHG) emissions. Within this framework, organic agriculture serves as a vital strategy as it increases soil carbon, lowers GHG emissions, and supports long-term sustainability. Biochar is a key innovation in this context, enhancing soil carbon sequestration and contributing to GHG mitigation. However, despite its role in soil conditioning, biochar was found to have some limitations related to improving soil nitrogen. Hence, this study was initiated with the aim of developing biochar-formulated fertilizer to complement what biochar lacks and evaluating it on model winter and summer crops. A Randomized Complete Block Design (RCBD) experimental setup was employed, with 17 treatments (including four organic amendments, twelve formulated fertilizers, and a control), each replicated three times. The research followed a two-phase experimental design: an initial screening during the winter season of 2024 using mustard greens as a winter model crop, followed by a summer 2025 trial with sorghum as a summer model crop. Measurements included soil parameters, plant growth performance, and soil GHG emissions (nitrous oxide, N₂O), methane (CH₄), and carbon dioxide (CO₂), analyzed using state-of-the-art methodologies. Among the 17 treatments, five (T6, T7, T8, T15, and T17) demonstrated notable improvements in soil nitrogen content, plant growth, and emission reduction. These five amendments were subsequently compared to standard NPK fertilizer during the summer trial. The findings demonstrated that treatments T7 (40% biochar, 20% chitosan, 40% chicken manure) and T17 (75% biochar, 25% cow manure) markedly improved sorghum growth, yielding results comparable to those observed with the positive control group that received NPK fertilizer. It is evident that these two formulations are substantially mitigating GHG emissions from the soil and improving crop performance. This study suggests that these two biochar-based formulations should be tested on larger farms and with both winter and summer crops to determine their effectiveness and potential for broader use.

1 Introduction

Sustainable soil management is essential for maintaining agricultural productivity and mitigating climate change. However, soil degradation, declining organic matter, and nutrient depletion continue to threaten crop production worldwide (1, 2). Organic soil amendments have gained increasing attention as strategies to improve soil fertility, enhance nutrient retention, and reduce reliance on synthetic fertilizers while supporting environmental sustainability (3–5).

Among these amendments, biochar has emerged as a promising material because of its porous structure, high surface area, and stable carbon composition. Numerous studies have demonstrated that biochar improves soil physical and chemical properties, enhances water and nutrient retention, promotes beneficial microbial activity, and contributes to long-term carbon sequestration (6–8). These attributes have led to increased interest in biochar as a climate-smart agricultural amendment capable of improving soil health while mitigating greenhouse gas (GHG) emissions (9–11).

Despite these advantages, biochar has an important limitation as a direct nutrient source. Most biochars, particularly those produced from woody feedstocks, contain relatively low concentrations of nitrogen (N) and may temporarily immobilize soil N because of their high carbon-to-nitrogen ratio (12, 13). Consequently, biochar alone often cannot supply sufficient nitrogen to meet crop requirements, limiting its effectiveness as a stand-alone fertility amendment (14).

One approach to overcome this limitation is to combine biochar with nitrogen-rich organic materials. Organic amendments such as animal manures provide readily available nutrients, while biochar can enhance nutrient retention and reduce nutrient losses through adsorption and improved soil structure (3, 15, 16). Similarly, chitosan has received attention as a multifunctional organic amendment as it supplies nitrogen, stimulates beneficial microbial activity, and may enhance nutrient-use efficiency and plant growth (17). Integrating biochar with manure and chitosan therefore has the potential to create synergistic effects that improve both soil fertility and crop productivity.

In addition to agronomic benefits, biochar-based formulations may influence soil greenhouse gas dynamics. Previous studies have shown that biochar and organic amendments can alter emissions of carbon dioxide (CO₂), methane (CH₄), and nitrous oxide (N₂O) by modifying soil physical conditions, nutrient availability, and microbial processes (18, 19). However, improvements in crop productivity do not always correspond with reductions in GHG emissions, making it important to evaluate both agronomic performance and environmental trade-offs simultaneously.

Although biochar-compost systems have been extensively investigated, fewer studies have examined optimized biochar formulations incorporating manure and chitosan under field conditions (20, 21). Existing studies in Texas have evaluated broad combinations of manure and biochar application rates (22), yet information remains limited regarding optimized biochar-based formulations tailored to Texas soils and cropping systems. Furthermore, many previous studies have focused primarily on crop productivity without simultaneously assessing soil carbon, nitrogen dynamics, and greenhouse gas emissions.

Therefore, following nitrogen recommendations from Texas A&M AgriLife Extension (23), this study developed and evaluated a series of biochar-based formulations containing manure and chitosan for winter and summer cropping systems. The objective was to identify optimized biochar-formulated fertilizers by assessing their effects on soil carbon and nitrogen dynamics, crop performance, and greenhouse gas emissions under Texas field conditions. We hypothesized that integrating biochar with nitrogen-rich organic amendments would improve soil fertility and crop productivity while reducing greenhouse gas emissions compared with individual amendment applications.

2 Materials and methods

2.1 Methodology

The research utilized two-phase, sequential experimental approaches to assess biochar formulations. During the first phase (fall-winter season, 2024), a screening trial was conducted with 17 different biochar formulations, employing a randomized complete block design (RCBD) with three replications and mustard greens (Brassica juncea (L.) Czern.) serving as the model winter crop. In the second phase (summer season, 2025), the top-performing blends identified from the mustard green trial were further evaluated against a standard NPK fertilizer, using sorghum as the model summer crop. This adaptive approach enabled systematic refining of treatments followed by rigorous validation, ensuring robust identification of the best formulations compared to conventional NPK fertilizer.

2.2 Study domain

The study was conducted at the Prairie View A&M University (PVAMU) CAFNR Research Bill and Vara Daniels Ranch and Farm, located approximately 45 miles northwest of Houston, Texas. According to the data from the parameter-elevation regressions on the independent slopes model (PRISM), the site receives an average of 1,118 mm of annual rainfall, mainly from June to October. Summers are hot, with July averaging 35 °C, while winters are cooler with January averaging 3 °C (24). The soil is classified as a Wockley fine sandy loam, with a surface of fine sandy loam and a base of sandy clay loam, that allows for effective water infiltration and retention, making it suitable for agriculture (25).

2.3 Mustard green project

This was the first phase, conducted in winter 2024, aimed to evaluate treatments for their impact on soil nitrogen and carbon, mustard green growth, and greenhouse gas emissions.

2.3.1 Experimental field and plot design

The experimental trial for the mustard green project was performed in a 35 m by 5 m plot. This was divided into three blocks (RA, RB, RC), each containing 17 smaller plots of 1m by 1m to see the effects of the treatments on crop growth, soil nutrient content, and their effect on GHG emission. Beds were raised 30 cm, with 1 m gaps between plots and 50 cm between replications to prevent cross-contamination from water or wind. Each replication was equipped with irrigation lines and a reliable water supply throughout the study period. The organic amendments and biochar formulations were manually applied to the soil. For GHG emissions, soil collars of 5 cm diameter were installed in the center of each experimental bed after the treatments were thoroughly mixed with the soil (Supplementary Figure 1).

2.3.2 Formulation development and treatment application

Treatment formulations were systematically developed by blending biochar with various manures and chitosan, leveraging these components’ value as reliable sources of nitrogen. To maintain consistency, facilitate handling, and comply with regulatory requirements, commercially processed and composted manures were utilized in place of raw manures, as recommended (26). Commercially available manures deliver consistent nutrient profiles, minimize the risk of pathogen contamination, and are easily accessible and transportable, typically meeting established safety and quality standards. The biochar used in this study was previously characterized in detail by Shembo et al. (27), including feedstock source, pyrolysis conditions, elemental composition, and physicochemical properties.

Biochar was applied at a rate of 14 t/ha following our previous study on optimal biochar application rate (27). This rate is also aligned with the application recommendation set by the International Biochar Initiative (28). All other organic amendments were measured and applied to each experimental plot on a dry weight basis, considering the specific nitrogen content of each amendment and the recommended soil nitrogen application rate of 56 kg N/ha for mustard crops, as specified by Texas A&M AgriLife Extension guidelines. The nitrogen concentrations employed for chicken manure, cow manure, and chitosan were 3%, 0.5%, and 1.5%, respectively (Table 1). Based on these values, 12 distinct biochar-based organic fertilizers (also referred to as biochar formulation) were created by blending varying proportions of biochar with the organic amendments (Table 1). The selected formulation ranges (10–20% chitosan and 20–40% manure) were chosen to balance nitrogen supplementation, product scalability, and resource availability under Texas conditions, while maintaining biochar as the dominant component of the formulation.

2.3.3 Soil parameters

After plot preparation, soil samples were collected before treatment and at three subsequent intervals: during seed germination, and at one month and two months after germination (Supplementary Figure 2). Plots were routinely hand-weeded to minimize weed interference with soil physico-chemical properties (Supplementary Figure 2). For each treatment, soil was sampled at a 15 cm depth from each plot using a Mascaro soil profile sampler (Model 3KHF3, Turf-Tec International, Florida, USA), air-dried, sieved to 2 mm, packed, and clearly labeled.

Total soil carbon (C) and nitrogen (N) were determined using an elemental analyzer (CHNS, Elementar Americas Inc., New York). Anionic nutrients such as chloride (Cl^-), nitrate (NO₃^-), sulfate (SO₄²^-), and phosphate (PO₄³^-), were quantified by ion chromatography (Metrohm 930 Compact IC, Metrohm, United States). Total organic carbon (TOC) was measured using a TOC analyzer, while macro- and micronutrient concentrations were quantified using inductively coupled plasma–optical emission spectroscopy (ICP-OES; Agilent 5100, Agilent Technologies). Soil pH and EC were assessed using a portable pH meter and a PCTSTestr™ 50 Waterproof Pocket Tester (Oakton Instruments, Vernon Hills, IL, United States) to evaluate ionic strength and nutrient availability, respectively.

2.3.4 Soil flux measurements

Soil collars were installed one week before the first GHG measurement to allow soil stabilization. Soil gas flux was determined by monitoring diffusion gradients between atmospheric and soil air (29). Soil greenhouse gas fluxes (CO₂, CH₄, and N₂O) were quantified using a portable smart chamber system. Specifically, the LI-COR 8100A survey chamber was placed over each polyvinyl chloride soil collar and connected to the corresponding gas analyzers: LI-7810 for simultaneous CO₂ and CH₄ detection, and the LI-7820 for N₂O measurement. This configuration enabled precise and repeatable in situ assessment of soil emissions across all experimental plots. Measurements were conducted weekly between 9:00 a.m. and 11:30 a.m. over a three-month period (Supplementary Figure 3). During each measurement session, a two-minute accumulation phase was employed to allow for the establishment of a stable diffusion gradient (30).

2.3.5 Plant parameters

We assessed the effect of the 17 treatments on nutrient content, growth, and physiological parameters of mustard greens. The contents of anionic and cationic nutrients (macro and micronutrients) of mustard plants were thoroughly analyzed. For this purpose, plant tissue samples were oven-dried at 60 °C for 48 h, ground to a fine powder, and digested with concentrated HNO₃ and H₂O₂ using a microwave digestion system (ETHOS Microwave Digestion System, Milestone, Italy) with digestion tubes, ensuring complete recovery of all elements. The digests were diluted to volume with ultrapure water and filtered through a 0.25 µm PTFE syringe filter (Millex-LG, Merck Millipore, Burlington, MA, USA) before analysis. Elemental concentrations of macro- and micronutrients were determined by ICP-OES using multi-element calibration standards and quality control with certified reference materials.

The chlorophyll content was determined using a SPAD-502 Plus chlorophyll meter (Konica Minolta, Inc., Japan), with measurements taken from the midpoint of fully expanded leaves, avoiding major veins. For each leaf, three readings were recorded and averaged to yield a representative SPAD value. Leaf area (LA) was measured by recording leaf length and width with a ruler and calculating area in square centimeters, with all measurements taken consistently in the morning (9:00 a.m.-11:30 a.m.) when light intensity is stable enough to account for its influence on chlorophyll dynamics (17). All measurements were conducted on healthy, mature leaves at a consistent growth stage to ensure reliability and comparability.

2.4 Sorghum research

To further evaluate the most promising BC-based formulations for enhancing soil nitrogen while also leveraging BC as a climate mitigation amendment, we conducted additional research using sorghum as a summer model crop. In this second phase field experiment, selected BC-formulated fertilizers among the 17 treatments of mustard green research were validated for their effectiveness.

2.4.1 Plot preparation and management for sorghum research

For this validation study, the experimental plots were meticulously prepared. First, the previous mustard green plots were tilled, and the topsoil was removed to eliminate any residual effects from earlier treatments. The plots were re-tilled and prepared for the sorghum experiment, which employed a randomized complete block design (RCBD) in which all treatments were randomly arranged within each block and replicated three times to ensure reliable results.

To maintain consistency with the mustard research, each sorghum experimental unit measured 1 m × 1 m and was situated on a 30 cm raised bed, with soil collars installed at the center of each plot. Irrigation lines were set up for every replication to provide a uniform water supply throughout the study. In the sorghum experiment, NPK synthetic fertilizer was applied as a positive control to enable direct comparison with the recommended standard rate. An untreated plot was included as a negative control.

Before applying for treatments, soil samples were collected from each bed. The formulated fertilizers were incorporated into the top 15 cm of soil, according to established protocols. Planting and management procedures were based on guidelines from the Texas AgriLife Extension (31). Sorghum plots were seeded in June 2025, with an estimated seeding depth of approximately 1.5 inches as recommended by Trostle et al. (31).

2.4.2 Data from sorghum experimental plot

Sorghum growth and physiological responses were evaluated to determine the impact of the top-performing formulated fertilizers on crop development (Supplementary Figure 4). Growth parameters included plant height, leaf area, and biomass accumulation. Plant height was measured at physiological maturity from the soil surface to the tip of the panicle using a measuring tape (cm) on five randomly selected plants per plot, and the average value was calculated.

Leaf area of sorghum was determined by measuring the length of fully expanded leaf lamina from the base to the tip and the maximum leaf width using a transparent ruler, following the method of Stickler et al. (32) (Equation 1).

For biomass assessment, plants were harvested, oven-dried at 60 °C until a constant weight was achieved and then weighed to determine dry matter yield.

Physiological performance was assessed by measuring photosynthetic rate (A), transpiration rate (E), Stomatal conductance (g_sw), and relative water content (RWC). A, E, and g_sw were measured weekly between 9:00 a.m. and 11:30 a.m. Measurements were taken on fully expanded leaves at the mid-reproductive stage, ranging from the flag leaf to early grain fill. All measurements were conducted under appropriate environmental conditions, including light levels of approximately 1500 μmol m^-² s^-¹, CO₂ concentration near 400 ppm, and leaf temperature between 25–30 °C. The LI-COR 6800 Portable Photosynthesis System (LI-COR Inc., Lincoln, NE, USA) was used for these measurements, following the manufacturer’s protocols (33).

Instantaneous water use efficiency (WUE) was calculated as the ratio of A to E, reflecting the balance between carbon gain and water loss (34). RWC was determined by sampling leaf discs from fully expanded leaves at the mid-reproductive stage (35). Fresh weight (FW) was recorded, discs were saturated in distilled water to obtain turgid weight (TW), and then oven-dried at 60 °C to determine dry weight (DW). RWC was calculated using the formula (Equation 2):

2.5 Data analysis

Soil GHG emissions (CO₂, CH₄, and N₂O) were processed using SoilFluxPro™ software. Both linear and non-linear models were evaluated, and the model with the lowest coefficient of variation (CV) and highest coefficient of determination (R²) was selected. Only flux estimates with R² ≥ 0.85 were retained for analysis, while poorly fitted measurements were excluded as part of quality control. Treatment effects on GHG emissions were assessed by ANOVA and post-hoc tests, with changes in gas concentrations calculated accordingly. Further data analysis and visualization were carried out in base R and ggplot.

The relative differences in soil emissions of CO₂, CH₄, and N₂O between the applied treatments and the control treatment were calculated following Kalu et al. (36) (Equation 3).

Where F_T is the flux due to the treatments added, and F_C is the flux from the control. The positive values indicate the treatment’s emissions effect, whereas the negative values indicate the treatment’s sequestration effect. The changes in CO₂, CH₄, and N₂O were analyzed and presented on a single map to clearly illustrate which treatments are making more emissions across all the studied fluxes. Besides, treatment impacts on soil nitrogen and carbon were determined following Hedges et al. (37) (Equation 4):

Where percentage improvement refers to the improvement due to treatment application, T_n is the measured value due to treatment effect, and T_o is the control value or baseline.

Pearson correlation analyses were used to assess which soil fluxes were correlated with one another to infer potential underlying mechanisms. Correlation analyses were also conducted among total nitrogen (TN), leaf SPAD values, and leaf area (LA) of sorghum to identify potential relationships underlying plant physiological responses and growth.

Cluster analysis was performed using K-means clustering to classify treatments based on soil CO₂ flux responses, with the optimal number of clusters (K = 4) determined using the Simple Structure Index (SSI). Hierarchical clustering with Ward’s linkage method was used to visualize and confirm the resulting cluster structure. The analysis was performed using soil CO₂ fluxes since it was the dominant and most consistently detected greenhouse gas across treatments and provided sufficient variation for treatment discrimination. Similar approach have been used in previous studies (38, 39). The impacts of biochar formulated fertilizers on both soil and plant nutrient status, as well as the collected sorghum growth and physiological data, were statistically assessed using ANOVA, and mean separations were done using Tukey’s Honest Significant Difference (HSD) test, to determine the most effective fertilizer formulations.

2.6 Results and discussions

2.6.1 Treatment effects on soil flux of mustard greens

2.6.1.1 Effect of formulated fertilizer on CO₂ flux

Our results showed that both organic amendments (T1–T4) and formulated fertilizers (T5–T17) significantly increased soil CO₂ flux compared to the control (T11) (Figure 1). This rise in CO₂ emissions is due to the addition of labile carbon, which enhances mineralization and microbial activity in the soil (40). These findings are consistent with previous research showing that organic amendments accelerate carbon cycling and decomposition compared to untreated soils (41).

Figure 1

Chicken manure (T2) produced the highest CO₂ emissions (Figure 1), reflecting its high content of readily mineralizable carbon that rapidly stimulates microbial respiration, consistent with evidence that amendments rich in labile substrates generate stronger respiratory responses than more recalcitrant organic inputs (42). Treatments formulated with chicken manure consistently outperformed their cow-manure counterparts (e.g., T5 vs. T6; T7 vs. T8; T12 vs. T15), demonstrating their stronger carbon-priming effect and enhanced microbial activity (43). Recent studies have also shown that organic amendments with relatively higher labile fractions significantly increase soil CO₂ flux relative to controls, confirming the role of substrate quality in driving carbon mineralization dynamics (44).

Furthermore, as illustrated in Figure 1, the control treatment (T11) demonstrated the lowest CO₂ flux, serving as an indicator of baseline soil respiration in the absence of amendments. In contrast, treatments including T2 (100% chicken manure), T4 (100% chitosan), T10 (70% biochar, 10% chitosan, 20% cow manure), T12 (25% biochar, 75% chicken manure), T13 (50% biochar, 50% chicken manure), and T16 (75% biochar, 25% chicken manure) resulted in markedly elevated CO₂ emissions. These results highlight the pronounced effect of both organic and formulated amendments containing labile carbon substrates on stimulating microbial activity and accelerating carbon mineralization, thereby increasing soil CO₂ flux relative to the control.

The lower CO₂ flux in the control (T11) treatment might reveal the baseline microbial activity in the absence of additional carbon inputs. Whereas treatments such as T2 (100% chicken manure) and biochar formulates like T12, T13, and T16 showed substantially elevated emissions, indicating that they contain more labile organic substrates that stimulate microbial respiration for rapid mineralization of readily decomposable carbon fractions from these treatments (45). This aligns with evidence that high-quality substrates rich in labile carbon, such as manure-derived compounds, accelerate microbial enzyme activity and enhance soil organic matter decomposition, thereby increasing CO₂ release (46).

Conversely, formulations with higher proportions of biochar and chitosan (Figure 1) tended to produce lower CO₂ fluxes, reflecting the more recalcitrant nature of these blends. This phenomenon may be explained by biochar’s recalcitrant aromatic carbon structure and strong nutrient sorption capacity, which together can limit microbial access to readily degradable substrates (47). Moreover, the polymeric network in chitosan restricts diffusion and substrate availability, resulting in slower microbial decomposition in soil (48). These mechanisms are well recognized, as recalcitrant substrates typically slow microbial activity and can, in some cases, result in weaker or even negative priming effects on native soil organic carbon compared to more readily mineralizable organic inputs (49).

The study also indicates that chitosan (T4), when applied at 100%, substantially increased CO₂ emissions (Supplementary Figure 7). However, formulations incorporating chitosan (T5-T9) generally reduced CO₂ flux compared to those without (T12–T16). In most instances, treatments with higher chitosan proportions (20%; T7, T8) showed lower emissions than those with 10% (T9, T10) (Supplementary Figure 7). This reduction may result from chitosan’s amino groups interacting with the acidic biochar surfaces, enhancing adsorption and immobilization of labile carbon compounds (50, 51). Overall, the observed treatment differences underscore the central role of substrate variation, particularly carbon lability and chemical structure, in governing microbial activity and driving carbon mineralization dynamics in amended soils.

2.6.1.2 Treatments’ effect on CH₄ flux

The treatments had relatively small effects on CH₄ flux. Among them, cow manure (T3) tended to exhibit higher CH₄ fluxes than biochar (T1), chicken manure (T2), and chitosan (T4), although these differences were primarily descriptive (Figure 2). Biochar formulations containing cow manure (T6, T14, T15, T17) comparatively produced lower CH₄ flux than when the cow manure is used alone for soil amendment. This clearly indicates the role of formulating cow manure into biochar to lower cow manure’s role in methanogenesis (45). Most treatments exhibited net CH₄ uptake like the control (T11), except for a few formulations (e.g., T10, T12, T13) that showed slight emissions () (Figure 2) suggesting that our formulation does not largely contribute to CH₄ emissions.

Figure 2

2.6.1.3 Treatment effect on nitrous oxide emission

Our results also demonstrated that unamended soil (T11) acts as a stronger sink for N₂O flux (Figure 3; Supplementary Figure 5). This is primarily because unamended soils typically have limited nitrogen sources and reduced microbial activity (52). Without organic amendments, these soils contain less nitrate and ammonium, which are the main substrates for nitrification and denitrification pathways responsible for N₂O production (53). Furthermore, the innate mineral nitrogen present in the unamended soil is insufficient to significantly stimulate microbial activity, especially because the soil is less aerated in the absence of pore-forming materials like biochar (54). As a result, the limited availability of nitrogen, lower microbial stimulation, and potentially enhanced microbial reduction of N₂O to N₂ may have contributed to the reduced N₂O flux observed in unamended soils (T11). This negative flux means that the soil tends to absorb N₂O rather than release it. However, because N₂O fluxes were relatively small, the observed sink strength should be interpreted with caution.

Figure 3

The higher N₂O emission by T1 (100% BC amendment) might be due to its porosity in improving soil structure and aeration that favors nitrifiers to ultimately drive nitrification and partial denitrification, thus releasing more N₂O (55). The higher N₂O emission by T2 (chicken manure) could be because chicken manure has more nitrogen sources that can be mineralized by soil microbes and release higher N₂O (45). Moreover, the higher N₂O emission from T8 should be further investigated, but it is likely due to additional sources of nitrogen. However, treatments like T8, and other treatments that contain cow manure were revealed to have higher N₂O emission than their chicken manure counterpart respectively (e.g. T6 Vs T5, T8 vs T7, T14 vs T13, T17 vs T16) (Figure 3). The average N₂O release from the cow manure component (0.03 ± 0.05 nmol m^-² s^-¹) showed comparatively higher emission level compared to its chicken manure counterpart (-0.01 ± 0.01 nmol m^-² s^-¹). This suggests cow manures when it is formulated with other organic amendments create more favorable environments such as nitrification than chicken manure blends under similar conditions (56–58).

The results further show that adding chitosan to cow manure blends generally increased N₂O fluxes compared to blends without chitosan (Figure 3). Specifically, treatments containing biochar (BC), cow manure, and chitosan exhibited a higher average N₂O flux (T6, T8, T10; 0.048 ± 0.061 nmol m^-² s^-¹) than cow manure blends without chitosan (T15 and T17; 0.015 ± 0.023 nmol m^-² s^-¹). Studies also showed that amendments with higher structural carbon (e.g., cow manure) when combined with nitrogen-rich additives (e.g., chitosan) can increase N₂O emissions due to enhanced microbial turnover (59, 60).

Most formulates notably reduced N₂O emissions relative to biochar application alone (T1), cow manure alone (T3), chitosan alone (T4), and chicken manure alone (T2), despite these formulations inducing greater soil nitrogen content (Figure 3; Supplementary Table 1). This finding indicates that N₂O mitigation can be achieved not only by reducing soil nitrogen content but also through the biogeochemical effects of biochar- and biochar–chitosan–based formulations. This might be due to the synergistic effects of biochar and other amendments (61–63). These effects may arise from the adsorption of N₂O onto the porous biochar surface and from restricted N₂O diffusion due to electrostatic interactions between chitosan amine groups and oxygen-containing functional groups on biochar (64–66). Other researchers have also studied blends of biochar and chitosan for their potential to stabilize N₂O released from manure mineralization (67, 68).

2.6.2 Treatment effects on the relative change of soil emission

The control (T11) acted as a net sink for both CH₄ and N₂O but still emitted CO₂ due to root and microbial respiration. Our results on the effects of treatment on soil GHG fluxes reveal highly variable greenhouse gas responses. All treatments except the near-zero control T11 showed increases (above 0%) in soil CO₂, CH₄, and N₂O fluxes, though the magnitude and balance of these increases differed markedly (Figure 4). The chicken manure treatment (T2) caused the most pronounced overall stimulation of gas emissions, with the largest relative increases in CO₂, whereas some formulations, such as T12 and T13, caused higher CH₄ emissions, and T8 caused the highest N₂O among all treatments (Figure 4).

Figure 4

The higher yield of soil CO₂ and CH₄ flux by T12 (25%BC and 75%ChM) and T13 (50%BC and 50%ChM) might indicate stimulated carbon mineralization and methanogenesis while only moderately raising N₂O, whereas T1 and T8 triggered N₂O-dominant responses. N₂O flux rose to 185% (T8) and 143% (T1) of the control compared to the corresponding CO₂ or CH₄ increases, which suggesting a shift toward nitrogen-centric processes (compare to control soil) likely due to intensified nitrification or incomplete denitrification under more aerobic soil microsites. In contrast, T4 and T13 were notable for primarily elevating CO₂, suggesting enhanced soil respiration or organic matter decomposition. Moreover, T10 strongly boosted CH₄, pointing to conditions favoring methanogenic activity. T9, stood out as a high multi-gas responder, exceeding the median increase for all three gases (CO₂ +92%, CH₄ +88%, N₂O +115%), thus consistently stimulating carbon cycling and nitrogen transformations simultaneously. Table 2 summarizes the top responders for each GHG flux, highlighting treatments with broad multi-gas stimulation versus those with single-gas dominant effects.

Moreover, the correlation analysis of interrelationships among gas emissions revealed that amendments driving carbon release often also promote methane emissions (Figure 5). There was a significant positive correlation between the treatment-induced changes in CO₂ and CH₄ fluxes (r = 0.65, p< 0.01), indicating that treatments that stimulates soil CO₂ efflux (microbial respiration) tended to simultaneously enhance CH₄ production. This positive association is consistent with the possibility that a greater supply of labile (readily degradable) carbon, together with favorable redox conditions, may have concurrently stimulated aerobic decomposition processes leading to CO₂ production and anaerobic microbial pathways responsible for CH₄ generation (69). Co-elevations of CO₂ and CH₄ have also been observed in other studies under experimental warming and high labile-C availability (70).

Figure 5

In contrast, the effects of treatments on N₂O emissions showed no significant correlation with changes in CO₂ (r = 0.26, p = 0.31) or CH₄ (r = 0.35, p = 0.17), highlighting a partial decoupling of N₂O from CO₂ and CH₄ across treatments (Figure 5). This suggests that N₂O emissions may be influenced by factors different from those governing CO₂ and CH₄ fluxes and are relatively independent from the processes that drive CO₂ and CH₄ production (71, 72). This suggests that N₂O emissions may be influenced by factors different from those governing CO₂ and CH₄ fluxes and are relatively independent from the processes that drive CO₂ and CH₄ production (71, 72). Although the present study did not conduct microbial analysis, findings from other studies support the fact that nitrification and denitrification are basic microbial processes related to N₂O emission, which do not always occur alongside carbon mineralization (73–75).

For instance, studies on fertilized upland soils indicate that most N₂O is produced through nitrification (about 60–68%), while denitrification was the dominant source in wetter environments (76). This demonstrates that N₂O emissions are controlled by nitrogen availability and the types of microbes present, rather than by the overall rates of soil CO₂ and/or CH₄ release. Other studies showed that N₂O emissions from soil increased with the abundance of genes related to ammonia-oxidizing microbes (nitrifiers), but decreased with higher levels of denitrifying genes, which convert N₂O to harmless nitrogen gas (N₂) (77, 78). Several studies showed that incomplete denitrification is common in soils with high nitrate availability or suboptimal conditions since denitrifying genes such as nosZ are often scarce or inactive in aerated topsoil, leading to N₂O emission (79).

These distinct microbial processes imply that N₂O levels can occur even when CO₂ and CH₄ levels do not rise. Specifically, treatments that add nitrogen or improve soil aeration, such as biochar or formulated fertilizers, create favorable conditions for nitrifiers, resulting in elevated N₂O emissions that are independent of carbon breakdown rates (80). This pattern aligns with other studies showing that N-focused amendments can boost N₂O production without affecting CO₂ or CH₄ emissions (80–82).

2.6.3 Treatment performance and implications for climate mitigation

Cluster analysis provides a robust framework for identifying management strategies based on treatment emission profiles. Accordingly, the performances of the seventeen treatments were classified into four clusters based on weekly CO₂ emissions, as indicated by the Simple Structure Index (k = 4; SSI = 0.27), which reflects high within-cluster similarity (Supplementary Figure 6). This clustering facilitates interpretation by grouping treatments with similar emission dynamics (83).

Agglomerative hierarchical clustering showed that the control treatment (T11) formed a distinct group (Cluster 3). Treatments with very low emissions were grouped into Cluster 1, characterized by emissions closely comparable to the control. Cluster 2 comprises low-emission treatments with intermediate emissions between the very low- and high-emission groups and is therefore considered suitable for mitigation. In contrast, high-emission treatments formed Cluster 4, exhibiting significantly greater emissions than the control and thus warranting caution and further investigation (Figure 6; Supplementary Figure 7). The results indicate that low-emission clusters, particularly those involving biochar blends, represent promising options for climate mitigation, whereas high-emission clusters highlight practices requiring further optimization. These insights support targeted soil amendment strategies to reduce greenhouse gas emissions and improve sustainability in agricultural systems (4, 15).

Figure 6

Applying organic amendments (T1, T2, T3, T4) generally revealed higher CO₂ emissions performance compared to the control (T11). Of these amendments, ANOVA indicated that biochar (T1) and cow manure (T3) produce significantly lower CO₂ emissions (p< 0.05) compared to the chicken manure (T2) and chitosan (T4) (Figure 7).

Figure 7

The biochar lowers the soil’s CO₂ emissions primarily because of its aromatic, highly recalcitrant carbon structure which can resist microbial decomposition, thereby slowing overall carbon mineralization (27). Cow manure also emits less CO₂ since its lignin-rich, fibrous organic matter decomposes slowly, limiting carbon mineralization. In addition, its relatively low nitrogen content constrains microbial activity, further suppressing CO₂ release (13, 84). Conversely, chicken manure (T2) and chitosan (T4) significantly elevated CO₂ emissions (p< 0.05, Figure 7), possibly because they might enhance the soil labile carbon that mineralizes to emit CO₂ (85, 86). Such observation is supported by our results, which show a significant positive correlation (r = 0.60, p< 0.05) between soil TOC and soil CO₂ flux (Supplementary Figure 8). However, blending these amendments with biochar reduces emissions (Figure 7). This may be attributed to the occupation of biochar’s porous structure and high surface area by nutrients from the formulated manure, which limits microbial access and activity, thereby reducing organic matter decomposition and CO₂ release (87).

2.6.4 Treatment effect on soil carbon and nitrogen improvement

Organic amendments and formulated fertilizers have enhanced soil total organic carbon (TOC) and total nitrogen (TN) levels (Figure 8). Treatments such as T2, T12, T13, T7, T6, T10, T15, T8, T6, T17 enhanced TOC in the soil (Figure 8A). However, T2, T12, T13, and T10 exhibited significantly elevated CO₂ emissions, which raises concerns about their suitability for sustainable agriculture (Figure 7).

Figure 8

Treatments were screened using a multi-criteria evaluation framework. Treatments were considered promising when they (i) did not significantly increase soil CO₂ emissions relative to the control (Figure 7), (ii) significantly enhanced soil total nitrogen (Figure 8B), and (iii) maintained moderate improvements in soil total organic carbon (Figure 8A). Using these criteria and analyzing the combined results in Figures 7, 8A identifies the five most promising BC formulated fertilizers (T6, T7, T8, T15, T17) that improved soil carbon content without significantly increasing GHG emissions. Blends of biochar and manure have been reported in other studies for the same effect (88, 89).

The findings of this study revealed contrasting effects of biochar and its co-formulations on soil nitrogen dynamics and greenhouse gas emissions. Treatments with biochar alone (T1) did not significantly enhance soil nitrogen content, aligning with previous reports that biochar’s nutrient contribution to a soil is often limited due to its recalcitrant nature and low inherent nitrogen content (27, 90). Moreover, other treatments such as cow manure (T3) and certain blends such as T9 showed insignificant improvement in soil nitrogen, probably due to their poor mineralization effect and/or negative priming effect (91–93). In contrast, most of the biochar formulations improved nitrogen availability by 5-65%, suggesting their roles to enhance biochar’s nitrogen limitations through mineralization and microbial activity (93). However, some formulations, such as T12, were noted to significantly contribute to CO₂ emission, although it significantly enhances soil nitrogen content, suggesting that they are not a smart formulated fertilizer (Figure 8).

Hence, from a climate-smart agriculture perspective, treatments such as T6, T7, T8, T15, and T17 emerged as promising candidates. These formulations provided moderate-to-high nitrogen enhancement while maintaining relatively low CO₂ emissions, aligning with principles of sustainable soil management. However, the results also revealed important agronomic–environmental trade-offs. For example, treatments such as T12 substantially improved soil carbon and nitrogen content but were associated with elevated CO₂ emissions, while T8 improved soil fertility indicators yet exhibited relatively higher N₂O emissions than several other formulations. These findings suggest that maximizing soil carbon and nitrogen enhancement does not necessarily coincide with minimizing greenhouse gas emissions. Therefore, treatment selection should consider both agronomic benefits and environmental impacts rather than relying on a single performance indicator (94, 95). Other studies also revealed that integrating biochar with manure not only improves soil fertility but can also contribute to lower carbon emissions under appropriate conditions (20, 90).

2.6.5 Effects of formulated fertilizers on nutrient enhancement in mustard greens

Our findings reveal that plants grown under the tested treatments exhibited higher nitrogen and other nutrient (Supplementary Table 3) content than those grown in unamended soil (T11), suggesting that they were also grown on improved soil (Supplementary Table 2). However, plants cultivated solely with biochar treatment (T1) contained less nitrogen (21.39 ± 5.15) than those in the control treatment (28.57 ± 2.43) (Table 2), suggesting that biochar alone may act primarily as a soil conditioner rather than a direct nutrient source. This result is consistent with previous studies showing that unamended biochar reduces plant-available nitrogen through microbial immobilization and adsorption (96, 97). Among the organic amendments, cow manure (T3) contributed the least to plant nitrogen uptake relative to chicken manure (T2) and chitosan (T4). Such a difference is consistent with the inherent nitrogen content of these materials, which varies widely, with cow manure containing approximately 0.5% nitrogen, compared to ~3% in chicken manure and ~1.5% in chitosan (98, 99). These results emphasize the importance of selecting amendments with higher nutrient density to optimize crop performance.

Moreover, the formulated fertilizers (T6, T7, T8, T15, and T17) demonstrated a dual benefit: enhancing primary macronutrients (P and K) (Table 3) and secondary macronutrients (Ca, Mg, and S) (Supplementary Table 3), while also improving micronutrient (Supplementary Table 4) and anionic nutrient (Supplementary Table 5) profiles without significantly increasing carbon emissions. This suggests that biochar formulations can serve as a climate smart fertilizer by improving nutrient availability and reducing environmental impacts.

2.6.6 Mustard green chlorophyll content and leaf area

The chlorophyll content of mustard greens, expressed as SPAD values, ranged from 24.23 ± 1.47 to 27.02 ± 1.75 across treatments (Figure 9A). Plants grown exclusively with biochar amendments exhibited significantly lower SPAD readings (24.23 ± 1.47) compared to all other amendments and formulated fertilizers, including the control (24.76 ± 2.06). This observation is consistent with the reduced total soil nitrogen (TN) (Supplementary Table 1) and plant nitrate (NO₃^-) concentrations (Supplementary Table 5) in plant tissues under biochar-only treatments, which were lower than those in the control and other treatments. In fact, SPAD meter readings are widely used as nondestructive proxies for leaf chlorophyll concentration and plant nitrogen status across crop species (100, 101). Treatments T2, T5, T6, T7, T8, T12, T13, T16, and T17 recorded higher SPAD values (Supplementary Table 6), indicating improved chlorophyll content. Among these, T6, T7, T8, T15, and T17 showed modest but significant improvements in chlorophyll content while maintaining a relatively low carbon footprint (Figures 7, 9A).

Figure 9

Leaf area (LA) analysis revealed that all organic treatments contributed significantly to an increase in leaf size compared to those grown on untreated soils (Figure 9B). Importantly, those five treatments (T6, T7, T8, T15, T17) that improved chlorophyll content also significantly promoted larger LA, highlighting their dual benefit for crop productivity and environmental sustainability (102). While treatment effects on SPAD values were relatively small, the substantial increases in leaf area suggest that the formulated fertilizers exerted a stronger influence on plant growth and canopy development than on chlorophyll concentration alone.

The variation in chlorophyll content among treatments reflects their differences in nutrient availability (103). The significantly lower SPAD values observed in mustard green grown with biochar alone suggest that unfortified biochar has a limited contribution to nitrogen uptake as reported in other studies (104, 105). The lower SPAD values also align with the reduced TN and NO₃^- concentrations in plant tissues under biochar-only treatments, indicating insufficient nitrogen supply for optimal chlorophyll synthesis. SPAD readings were strongly correlated with leaf total nitrogen and leaf size (Figure 10), consistent with previous studies demonstrating close associations between SPAD values, leaf nitrogen status, and growth-related traits (101, 106). Conversely, treatments incorporating nitrogen-rich formulates (T6, T7, T8, T15, T17) demonstrate enhanced chlorophyll content and leaf area while maintaining a low carbon footprint. These underscore the potential of biochar formulations to simultaneously improve photosynthetic capacity and plant growth without exacerbating greenhouse gas emissions (107, 108).

Figure 10

The relationships observed among TN, SPAD chlorophyll readings, and LA highlight the central role of nitrogen nutrition in regulating leaf development and photosynthetic capacity in mustard greens. Analysis of the data showed that TN exhibited a moderate positive correlation with LA (r = 0.59, p< 0.05), suggesting that increased nitrogen availability promotes leaf expansion by stimulating cellular growth and metabolic processes associated with leaf development (109, 110). These results align with other studies, which show that improved nitrogen availability promotes greater leaf surface development across nutrient-amended treatments (103, 111). SPAD was also found to be significantly correlated with leaf nitrogen content in mustard green plants (r = 0.68, p< 0.05). Moreover, the study indicated a strong and significant positive correlation between SPAD values and leaf area (r = 0.89, p<0.05). These combined results clearly suggest the physiological linkage between nitrogen content, chlorophyll, and leaf expansion. This is because higher chlorophyll content boosts the plant’s photosynthetic capacity, allowing it to capture more light energy and fix more carbon, which directly fuels cell expansion (112, 113).

Based on the multi-criteria screening framework developed from the mustard green study that considered (i) non-significant increases in CO₂ emissions relative to the control, (ii) significant improvements in soil total nitrogen, and (iii) moderate improvements in soil total organic carbon, treatments T6, T7, T8, T15, and T17 were identified as the most promising biochar-based formulations, demonstrating favorable improvements in soil nitrogen and organic carbon while maintaining relatively low greenhouse gas emissions.

2.7 Results from sorghum project

Following the findings from the mustard green project, the study further extended to evaluate the five more refined formulations (T6, T7, T8, T15, T17) using sorghum (Sorghum bicolor (L.). The detailed treatment tests and their formulation are shown in the supporting table (Supplementary Table 7). Both physiology and growth-related data were generated.

2.7.1 Effects of refined treatments on sorghum physiology

2.7.1.1 Relative water content and chlorophyll content

Application of biochar formulated fertilizers generally improved sorghum physiological traits compared with the untreated control (Figure 11). Among the tested formulations, treatments T7 and T17 exhibited the most pronounced effects. Based on representative data, the mean relative water content (RWC) values were T7: 84.75 ± 3.49, T17: 83.06 ± 4.47, and T18 (synthetic NPK): 78.76 ± 3.41. Chlorophyll concentration, assessed through SPAD readings, showed T7: 42.60 ± 3.82, T17: 43.97 ± 2.07, and T18: 40.13 ± 3.19 units. A one-way ANOVA confirmed significant differences among treatments for both RWC (p<0.05) and SPAD (p<0.05). Tukey’s HSD post-hoc test showed that T17 exhibited the highest mean RWC and was significantly higher than the untreated control (T0) and several formulated treatments. However, T17 did not differ significantly from the NPK treatment (T18). Likewise, the remaining formulated treatments (T6, T7, T8, and T15) did not differ significantly from one another in terms of RWC (Figure 11A).

Figure 11

Results on chlorophyll content of sorghum crops reveal that T7, T17, and T18 recorded significantly higher SPAD values than the other treatment (Figure 11B). This indicates T7 and T17 improved the chlorophyll contents that were statistically similar to those observed under the NPK treatment (T18).

2.7.1.2 Stomatal conductance and assimilation rate

Stomatal conductance to water vapor (g_sw) is a key physiological trait that regulates transpiration and CO₂ assimilation, directly influencing plant water-use efficiency and photosynthetic capacity. Treatments T7 and T17 exhibited among the highest stomatal conductance and assimilation rates, with mean g_sw values of 0.34 ± 0.07 and 0.33 ± 0.05 mol H₂O m^-² s^-¹, respectively, and assimilation rates of 47.95 ± 4.08 and 47.67 ± 4.35 µmol CO₂ m^-² s^-¹ (Figure 12; Supplementary Table 10).

Figure 12

2.7.1.3 Transpiration and water use efficiency

Our findings show that applying soil treatments led to a significant reduction in water loss through crop transpiration (Figure 13A). Notably, T7 and T17 were especially effective at lowering evapotranspiration compared to the other groups. In addition, all formulated fertilizers and positive control enhanced the water use efficiency (WUE) of sorghum. Among the tested treatments, T7 and T17 produced the highest WUE values, with responses that were statistically similar to or greater than those observed under the synthetic fertilizer treatment (Figure 13B).

Figure 13

Overall, the sorghum physiological results (Figures 11-13; Supplementary Table 10) showed that the two biochar-formulated fertilizers, T7 and T17, consistently ranked among the best-performing treatments across the measured physiological parameters, including relative water content (RWC), chlorophyll content (SPAD), stomatal conductance (gsw), photosynthetic rate (A), transpiration rate (E), and water-use efficiency (WUE). The superior RWC and relative chlorophyll level performance of plants treated with T7 and T17 can be attributed to their enhanced soil nutrient status (Supplementary Table 8), which exceeded that of the other treatments, except the synthetic NPK fertilizer. Elevated nutrient availability is known to strengthen crops’ RWC and SPAD values by improving nutrient uptake and photosynthetic function (106, 114). Recent studies confirm that biochar formulates substantially improve soil fertility by increasing nutrient retention and microbial activity, thereby enhancing overall nutrient-use efficiency, and subsequently improve RWC and SPAD values (115, 116). The improvement of such physiological performance of T17 could be consistent with its higher biochar concentration (75%), which likely contributed to greater porosity and surface area. Biochar’s intrinsic physicochemical traits, including high microporosity and functional group abundance, have been shown to reduce nutrient leaching and improve the soil’s physicochemical environment (9, 117). This results in better nutrient retention and more favorable hydraulic properties that ultimately improve crops’ RWC and chlorophyll contents (118).

Similarly, the richer and more diverse organic matter composition in T7, as reflected by higher TOC (Supplementary Table 9), likely improved soil structure and water-holding capacity. Organic amendments, particularly those rich in stabilized carbon, are well documented to enhance soil aggregation, water retention, and plant-available moisture (118). These improvements in soil moisture status help maintain higher RWC in plant tissues, a trend consistent with studies showing that biochar and other organic amendments significantly increase RWC in crops (27, 119).

The elevated nitrogen content measured in both formulations might have contributed to the higher SPAD chlorophyll readings observed in plants grown in T7 and T17. Improvements in chlorophyll concentration following organic and biochar formulations have been widely reported and are associated with improved nitrogen availability and enhanced photosynthetic capacity (119).

Higher RWC in these treatments could also have supported increased stomatal conductance (gsw) and consequently enhanced assimilation rate (A). Studies indicate that optimized biochar formulations improve leaf water status, thereby promoting stomatal opening, increasing CO₂ diffusion, and enhancing photosynthesis (120).

The study shows that the combined effects of improved nutrient availability, greater water retention, and enhanced physiological activity resulted in increased WUE in plants using T7 and T17. Elevated assimilation coupled with moderated evapotranspiration aligns with existing evidence that appropriate formulation in organic fertilizers improves WUE by optimizing gas exchange dynamics and maintaining favorable plant water relations (121).

2.7.2 Treatment effects on sorghum growth

Sorghum plant height did not differ significantly among treatments compared to the control (T0 and T18), with values ranging from 1.21 ± 0.12 m to 1.52 ± 0.22 m (Supplementary Figure 9). The lack of significant differences in plant height across treatments suggests that the tested amendments under the given conditions did not strongly influence sorghum growth. This stability is consistent with the inherent height range of stay green sorghum genotypes commonly grown in Texas environments (31). In contrast, LA and biomass exhibited notable variation among treatments, particularly compared to the negative control (T0) (Figure 14). Specifically, treatments T7 and T17 produced significantly higher LA and biomass compared to the control and other treatments (Figure 14). However, no significant difference was observed between these treatments and the positive control, implying the comparable performance of T7 and T17 with T18 in promoting leaf expansion and biomass accumulation. This could be since larger LA is often associated with greater photosynthesis. This response aligns with other studies showing that moderate biochar or organic amendment formulates improved sorghum leaf morphology, nutrient uptake, and chlorophyll content by enhancing soil structure and nutrient retention (115, 116).

Figure 14

Biomass accumulation, which is an integrative indicator of photosynthetic capacity and resource use efficiency, was found significantly higher in sorghum grown under T7 and T17. This enhancement is consistent with the improved soil physicochemical conditions associated with these formulates, including increased nutrient availability and higher cation exchange capacity (9, 118). Recent studies confirm that biochar formulates applied at optimal rates can substantially increase sorghum biomass by improving LA, chlorophyll concentration, and shoot growth (98). Similarly, biochar combined with organic amendments or balanced mineral fertilizers has been shown to increase biomass by up to 50% due to enhanced soil structure, microbial activity, and nutrient cycling (122).

Interestingly, the significant increases in leaf area and biomass were not accompanied by corresponding increases in plant height. This suggests that the positive effects of T7 and T17 were expressed primarily through enhanced canopy development and biomass accumulation rather than stem elongation. Larger leaf areas can increase light interception and photosynthetic surface areas, thereby contributing to greater dry matter production even when plant height remains unchanged. These findings indicate that biomass response in sorghum was more closely associated with leaf development and resource-use efficiency than with vertical growth.

Overall, these findings highlight the potential of T7 and T17 to promote sorghum biomass production and canopy development at levels statistically similar to those observed under conventional fertilization.

3 Conclusion and recommendations

This study demonstrates that thoughtfully formulated blends of biochar, organic manures, and chitosan can improve soil fertility and crop performance while influencing greenhouse gas emissions. Treatments T6, T7, T8, T15, and T17 consistently enhanced soil carbon and nitrogen, improved nutrient uptake, and supported robust growth in mustard greens and sorghum. Among these, T7 and T17 showed physiological and growth responses comparable to those achieved with conventional NPK fertilization. However, the results also revealed important agronomic–environmental trade-offs, as some formulations improved soil fertility while increasing certain greenhouse gas emissions. Given the relatively short duration of this study, the observed greenhouse gas responses should be interpreted as short-term effects rather than definitive evidence of long-term climate-mitigation performance. Future research should evaluate these formulations across multiple seasons, soil types, and cropping systems to assess their long-term impacts on soil health, crop productivity, greenhouse gas dynamics, and overall climate-smart agricultural potential. Moreover, cumulative emissions or CO₂-equivalent metrics should be studied to provide a more comprehensive assessment of climate impacts and should be considered in future studies.

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