Nitrogen and phosphorus uptake efficiency in Indian mustard cultivated during three growth cycles in a copper contaminated soil treated with biochar

Biochar has been used worldwide as an efficient soil amendment due to its beneficial interaction with soil particles and nutrients; however, studies on the effect of biochar on the availability of nutrients such as N and P in tropical soils are still missing. The objective of the study was to evaluate the effect of different types and doses of biochars on the concentration and uptake of N and P in Indian mustard plants (Brassica juncea L.) grown in a Cu contaminated soil during three successive growth cycles. The greenhouse experiment was set up as randomized block design in a 3x3 factorial scheme, with 3 types of biochars (coconut shell, orange bagasse and sewage sludge) and three rates of application (0, 30 and 60t ha-1), and 4 replicates. Biochar increased plant growth by approximately 30 to 224%; however, the orange bagasse biochar was the most effective. Biochar reduced plant N concentration in approximately 15-43%, regardless of the rate of application, indicating the need to carefully adjust N fertilization. In the last growth cycle, biochar from coconut shell and orange bagasse improved the N uptake efficiency suggesting a better amelioration effect with ageing in soil. Biochar did not affect P nutrition in Indian mustard to a great extent; however, it significantly decreased the N:P ratio in the plant.


INTRODUCTION
Use of biochar as soil amendment has been considered as an environmentally friendly strategy to improve soil fertility while reducing negative impacts caused by the inappropriate disposal of organic wastes.Effect of biochar on soil fertility is believed to be related to its high porosity, surface area and CEC, which increase the soil retention capacity for water and nutrients.However, many other factors such as the biochar liming effect and the presence of a variety of oxygenated functional groups make it difficult to predict all the benefits of biochar to plant growth and productivity (NGUYEN et al., 2017), specially the 1 Departamento de Engenharia Agronômica, Universidade Federal de Sergipe (UFS), 49100-000, São Cristóvão, SE, Brasil.E-mail: mariaisisilva@gmail.com.* Corresponding author. 2 Pós-graduação em Recursos Hídricos, Universidade Federal de Sergipe (UFS), São Cristóvão, SE, Brasil.
ABSTRACT: Biochar has been used worldwide as an efficient soil amendment due to its beneficial interaction with soil particles and nutrients; however, studies on the effect of biochar on the availability of nutrients such as N and P in tropical soils are still missing.The objective of the study was to evaluate the effect of different types and doses of biochars on the concentration and uptake of N and P in Indian mustard plants (Brassica juncea L.) grown in a Cu contaminated soil during three successive growth cycles.The greenhouse experiment was set up as randomized block design in a 3x3 factorial scheme, with 3 types of biochars (coconut shell, orange bagasse and sewage sludge) and three rates of application (0, 30 and 60t ha -1 ), and 4 replicates.Biochar increased plant growth by approximately 30 to 224%; however, the orange bagasse biochar was the most effective.Biochar reduced plant N concentration in approximately 15-43%, regardless of the rate of application, indicating the need to carefully adjust N fertilization.In the last growth cycle, biochar from coconut shell and orange bagasse improved the N uptake efficiency suggesting a better amelioration effect with ageing in soil.Biochar did not affect P nutrition in Indian mustard to a great extent; however, it significantly decreased the N:P ratio in the plant.Key words: biochar, pyrogenic carbon, copper contamination, plant nutrition, Brassica juncea.

SOIL SCIENCE
Ciência Rural, v.49, n.1, 2019.Gonzaga et al. effect of biochar on the behavior of N and P in the soil and in the plant (KAMMANN et al., 2015;DEENIK & COONEY, 2016).
Nitrogen and P are very important plant macronutrients and their availability in soil as well as their interaction limits plant growth (GROOT et al., 2003).Studies about the effect of biochar on the availability of N in soil have generated many contradictory results varying from positive, negative and even no effect (CLOUGH et al., 2013;RAHMAN et al., 2013;NGUYEN et al., 2017).The same different outcomes are observed during the evaluation of the effects of biochar on plant N absorption (SAARNIO et al., 2013).Additionally, biochar has also been related to changes in the P cycle in highly weathered acidic soils (ABDELHAFEZ et al., 2017), where the availability of P is mostly controlled by the presence of reactive Al and Fe surfaces that present high affinity for P at low soil pH, causing P to be chemically adsorbed and leading to low agronomic P use efficiency (ANTONIADIS et al., 2015).In that case, the liming effect of biochar to certain extent can increase P availability by reducing P adsorption (VANEK & LEHMANN, 2015).
Plant growth in metal contaminated soils can be seriously impaired not only by high concentrations of heavy metals but also by availability of nutrients and nutritional status, including heavy metal tolerant ones such as Indian mustard (MOURATO et al., 2015) that are often used as food crop as well as to remediate contaminated soils (RODRÍGUEZ-VILA et al., 2015).Therefore, the objective of this study was to evaluate the concentration of N and P in plant tissue as well as N and P use efficiency in Indian mustard plants cultivated in a copper contaminated soil and treated with different types and doses of biochar, during three successive growth cycles.
The biochars were produced from coconut shell, orange bagasse and sewage sludge in a slow pyrolysis reactor, at 500 o C. Biochar was ground, sieved to 2mm particle size and evaluated through proximate analysis (Ash, volatile matter and fixed C), and ultimate analysis (C, N, H and O) according to the American Society for Testing and Materials (ASTM) D1752-84.Elemental composition of C, H, and N was determined using an elemental analyzer.Cation exchange capacity was determined by ammonium acetate method (THOMAS, 1982).Biochar pH and electrical conductivity were also determined.Biochar characteristics are presented in table 1.

Experimental set up
The experiments were carried out in a greenhouse environment at a temperature of 28 o C and as a completely randomized design in a 3x3 factorial scheme, with 4 replicates.Treatments were three types of biochar (coconut shell-CSB; orange bagasse -OBB; and sewage sludge -SSB) and three rates of application (0, 30 and 60t ha -1 ).Experimental units were comprised of 5L plastic pots.Soil was spiked with copper sulfate three months prior the experiment.Biochar (37,5g or 75g) was mixed with the contaminated soil (3.5kg), transferred to pots, watered to field capacity and incubated during one week before planting in order to equilibrate.One healthy Indian Mustard seedling with four leaves was transferred to each pot and allowed to grow for 60 days.Plants were watered throughout the study to keep the soil at approximately 70% of its field capacity.Indian mustard plants were cultivated for three successive growth cycles (April to June; June to August; August to October, 2016).At the beginning of each growth  cycle, 1g of mineral fertilizer (osmocote:14-14-14) was applied to each pot.Before harvesting, chlorophyll content of the mid-section, fully expanded leaves was measured, using a non-modulated fluorimeter OS-30P model (ADC BioScientific Ltd., UK).After each harvesting, plants were washed with tap water, rinsed with DI water and dried in a 60 o C oven for approximately 48 hours, weighed for the determination of the dry biomass and ground to pass through a 2mm screen for N and P analysis.Samples were digested in a hot block digestion system, according to the TKN protocol for N determination (BREMNER, 1996).Total P concentration in plant samples was determined in the TKN extracts using the molybdenum blue method (MURPHY & RILEY 1962).Soil samples were collected at the beginning of each growth cycle from each experimental pot, dried, sieved to 2mm screen and analyzed for pH, EC, organic C and concentration of extractable Cu, N, P, K and Na.Soil pH was determined in a 1:5 (w/w) biochar:water ratio after 1.5h shaking in a reciprocating shaker and 1h equilibration.Concentrations of extractable Cu, P, K and Na were determined following extraction with 1% citric acid (WU et al., 2004).Soil plant available N was extracted with 2M KCl.The uptake efficiency of N or P was calculated by dividing N or P uptake by the root biomass dry weight (GOOD et al., 2004).

Statistical analysis
Statistical analysis was performed separately on each crop cycle because the experiments were not run concurrently.Results are reported as the means of four replicates.Data obtained were subjected to two-way Analysis of Variance (ANOVA) for assessing the significance of quantitative changes in the variables as a result of biochar treatments using SISVAR software package (FERREIRA, 2011).When a significant difference was observed between treatments, multiple comparisons were made by the Tukey-test at P<0.05.

Effect of biochar in N nutrition
The increase in plant biomass caused by the presence of biochar was not followed by an increase in the concentration of N in the plant aboveground part, which varied from 15.1 -46.9g kg -1 .Considering that the concentration of N commonly ranges from 23.9 -55.1g kg -1 for plants of the Brassica genus, the presence of biochar, especially in the GC2, caused a reduction in N concentration below the lower limit in most plants.In fact, all biochars reduced plant N (Table 2) in approximately 15-43%, regardless of the rate of application, which could be observed by the chlorotic appearance of leaves.Considering that all the plants were equally fertilized with N, the yellow discoloring of the leaves observed in the treatments with biochar is supposed to be related to the effect of biochar on the fate of N and not to the effect of the high Cu concentration in the contaminated soil.Therefore, N fertilization in soils treated with biochar should be a concern.Reduction in plant N concentration due to biochar was also confirmed by the reduction in the chlorophyll content (Table 3) and could have been a result of the improved growth and consequently increased demand for this essential macronutrient.ARGENTA et al. (2004) also reported good correlations between plant N and Chlorophyll content in maize plants.
Additionally, it was reported that during the ageing of biochar in the soil, NO 3 -N chemisorption through H-bonding may be enhanced because biochar becomes more hydrophilic (HAMMES & SCHMIDT, 2009); however, in the present study, the evaluation of N availability through KCl extraction showed little influence of the biochars (Table 4), except in the OBB treatment which significantly decreased extractable N.This effect could also be related to the increase in pH when the OBB was applied to soil (Table 4), leading to N loss through volatilization (NGUYEN et al., 2017).Therefore, different biochars may likely influence different mechanisms of N interaction in soil for which more investigation is needed.

Effect of biochar in P nutrition
The concentration of P in the shoot varied from 3.40 to 5.93g kg -1 (Table 2).Biochar did not affect P nutrition in Indian mustard, except for the CSB and OBB applied at a rate of 30t ha - 1 , in the first GC, which increased P concentration in the aboveground biomass by approximately 72% and 61%, respectively.These results are not likely related to the concentration of citric acid extractable P in the soil since application of CSB had no effect on P availability, regardless of the increase in soil pH caused by this biochar.In fact, application of OBB and SSB even increased the concentration of extractable P (Table 4), which could be related not only to the increase in soil pH but also to the concentration of P in the biochar (Table 1).A reasonable explanation for the lack of plant response related to P nutrition is the lower P requirement by Indian mustard plants which have higher demand for N.

Effect of biochar on the N:P ratio in the plant
Biochar addition to soil significantly (P<0.05)reduced the N:P ratio in the plants, regardless of the type and rate of application (Table 2).In the control treatment, N:P ratio varied from 7.23 to 9.04 across the three growth cycles whereas in the biochar treatments this variation was from 3.83 to 8.38, with mean values of 5.85, 5.00 and, 6.24 for CSB, OBB and SSB, respectively, with lowest values mostly associated with the OBB.According to GARRISH et al. (2010), the N:P ratio is an effective tool to evaluate the availability of these nutrients to plants; values < 14 indicates N limitation whereas values > 16 are a sign of P restriction to plant productivity.Therefore, our values confirmed N limitation in plants treated with biochar.

CONCLUSION
All biochars increased plant growth and reduced plant N concentration; however, the most effective was the orange bagasse biochar, indicating the need for a more cautious management of the N fertilization.Biochar increased plant N uptake efficiency which could explain the improved plant growth.
Considering that the plants had poor growth in the first cultivation cycle and; therefore, should not be taken into account, the effect of biochar on the plant N and P use efficiency seemed to improve as biochar aged in the soil from the second to the third cycle.

ACKNOWLEDGMENTS
We thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for supporting our research.

Figure 1 -
Figure 1 -N (a) and P (b) uptake efficiency of Indian mustard plants after growing in the copper contaminated soil treated with 30 and 60t ha -1 of biochar from coconut shell (CSB), orange bagasse (OBB) and sewage sludge (SSB), during three cycles (1, 2 and 3).

Table 4 -
Extractable concentrations of P, N, K and Na, pH and EC in the copper contaminated soil treated with biochar from coconut shell (CSB), orange bagasse (OBB) and sewage sludge (SSB).Uppercase letters compare means among different biochars at the same dose.Lowercase letters compare means among different doses at the same biochar type ((P < 0.05) (n = 4 per treatment).
Gonzaga et al.