Combined Use of Biochar with 15 Nitrogen Labelled Urea Increases Rice Yield, N Use Efﬁciency and Fertilizer N Recovery under Water-Saving Irrigation

: Biochar is a potential carbon-rich soil amendment that improves the physicochemical properties of soil, besides acting as a controlled release fertilizer. An experiment was conducted to investigate the effect of biochars on rice yield, fertilizer use efﬁciency and recovery under water-saving irrigation by 15 N isotopic tracer study. Two types of irrigation as alternate wetting and drying (AWD) and continuous ﬂooding (CF), and four types of biochar treatments such as rice husk biochar (RHB) with 15 N urea, oil palm empty fruit bunch biochar (EFBB) with 15 N urea, 15 N urea alone and control, were applied to assess their impact on rice. About 4% reduced grain yield with 18% improved water productivity was achieved by the AWD regime over the CF, whereas RHB and EFBB signiﬁcantly increased rice yield compared to unamended soil. RHB and EFBB enhanced the water productivity up to 25.3%. The fertilizer N uptake and recovery were boosted by RHB and EFBB up to 18.8% and 24.5%, respectively. RHB and EFBB accelerated the agronomic use efﬁciency and partial factor productivity of N (up to 21% and 8%, respectively). RHB and EFBB profoundly enhanced the pH, the total C and N and the available N (NH 4+ and NO 3 − ) of the post-harvest soil. This study suggests that adding RHB and EFBB with urea improves fertilizer N utilization and soil N retention, and their combination with AWD could enhance rice yield with better water productivity due to their porous structure and controlled N release capacity.


Introduction
Rice is considered one of the vital food crops of the world after corn and wheat. Notably in Asia, rice is consumed as the staple food [1] and the consumption of rice is expected to increase until 2025 due to the expanding population [2]. Continuously flooded lowland irrigation systems account for approximately 75% of overall rice productivity [3], utilizing around 24% to 30% of the world's established freshwater resources [4]. As a remedy, extensive international programs led by the IRRI (International Rice Research Institute) and other organizations are pushing alternate wetting and drying (AWD) as a way to reduce water demands [5]. If AWD water-saving measures are to be extensively adopted, one of the most significant considerations will be their impact on grain production. It is stated that AWD irrigation can sustain or even improve grain output compared to constantly flooded condition [6][7][8][9] and that there have been occasional declines in yield [10,11]. Furthermore, Belder et al.'s [12] and Bouman and Tuong's [3] findings also highlighted the fact that the yield reduction of rice is frequently noticed with AWD irrigation compared to continuously flooded irrigation practice; however, AWD usually enhanced water productivity with total irrigation input while the yield reduced due to the amount of water saved [6]. The regenerating agriculture concept incorporates agricultural systems that are more sensitive to biological and ecological interactions rather than just yields and production [13]; in this regard, AWD irrigation is classified as a type of sustainable agricultural system. AWD irrigation causes a significant alteration in the water regime in the soil, which transforms the soil from an anaerobic to an aerobic condition, which regulates water and nutrient availability as well as crop growth [14]. Although AWD irrigation can significantly reduce water input at the field level, there is a concern about unexpected excess water inputs in heavy clayey soils since percolation rates may rise with soil drying or due to the formation of fractures that permit for quick bypass flow [3,15]. AWD promotes more oxidizing conditions in the soil compared to flooded irrigation, which promotes the decomposition of plant residue and organic materials in soil [16]. Moreover, during the dry period of AWD, heterotrophic respiration occurs in the soil, resulting in the increased mineralization of soil organic carbon (SOC) [17]. This soil transition affects N availability and leaching by regulating microbial activities such as mineralization, nitrification and denitrification [18]. The paddy soil that comprises greater clay content during drying cycles of AWD irrigation soils may consequently shrink and create cracks termed macrospores in a well-puddled paddy field. Solutes may be carried quickly to the subsurface and groundwater by preferential flow via these fractures, which is responsible for N leaching loss [19,20]. Previous research found that nitrogen loss from paddy soil was accelerated by denitrification during AWD irrigation [21,22]; as a result, the crop obtains inadequate N. Moreover, AWD irrigation increases the release of N 2 O (nitrous oxide) from rice fields by the denitrification process [23]. This emission of N 2 O must be minimized since it is a greenhouse that contributes to global warming [23].
Biochar is a carbon-rich substance formed from the incomplete combustion (300-800 • C) of diverse organic materials in an oxygen-limited condition; it is currently gaining popularity worldwide for its role in supporting agricultural and environmental sustainability [24]. Biochar has the potential to raise soil carbon stocks, retain plant nutrients, improve soil fertility and enhance crop yield [25][26][27][28]. Incorporating biochar into soil effectively maintains inorganic N content, regulating N mineralization and, ultimately, plant growth [29]. Denitrifiers accelerate the depletion of the available soil nitrates by transforming them into gases such as N 2 O (nitrous oxide), NO 2 (nitrogen oxide) and N 2 (nitrogen gas) [30]. At the same time, biochar incorporation increases soil porosity and water retention capacity, resulting in reduced denitrifier activity and increased nitrate content in the soil [31]. Biochar has been shown to have the ability to decrease soil nitrate leaching [32,33], consecutively retain ammonium ions [30,31]. Biochar inclusion was also found to improve rice N absorption in several experiments [24,34,35] and exhibited soil ameliorating properties.
Increased rice yield is also an indicator of better nitrogen uptake and indicates greater N availability and reduced loss from soil. Khan et al. [36] reported that the addition of sewage sludge biochar at the rate 50 and 100 (g kg −1 ) significantly enhanced above ground biomass around 71% and 92%, respectively, compared to the unamended treatment. The addition of rice straw biochar at the 10.5 t ha −1 in a cold waterlogged paddy increased the grain yield up to 10.12% over control also reported by Liu et al. [27].
In enhancing rice production generally, N deficiency is considered the major yieldconstraining nutrient [37]. For achieving improved rice yields, farmers frequently used various types of simple and fast-acting chemical fertilizers, particularly N fertilizer [38]. Therefore, the application rate of N fertilizer in the rice field has surpassed 300 kg nitrogen per hectare, far beyond the adequate level [39]. Previous studies especially concentrated on the N transformation, losses and rice yield under AWD irrigation due to its periodic Sustainability 2022, 14, 7622 3 of 21 drying and wetting, but few studies focus on the biochar incorporation in this water-saving irrigation and its effect on the rice growth performance, water use efficiency, fertilizer N use efficiency (NUE) and recovery. Rice processing sectors and oil palm extraction factories in Malaysia lead to a lot of rice husk and empty fruit bunch, therefore, converting these byproducts into biochar has a lot of potential, and the addition of these biochars enriches the nutrients and moisture content of soil [40].
Seeing the nutrient retention properties and productivity characteristics of the biochar in this study, we applied rice husk and empty fruit bunch biochar with 15 N labelled urea assuming it will boost N uptake, efficiency and recovery which improves rice yield. Considering the foregoing, the current research was carried out to assess the impact of biochar on rice production, water use efficiency, NUE and fertilizer nitrogen recovery using a 15 N tracer with AWD irrigation regimes.

Collection and Analysis of Soil and Biochar Samples
The soil samples for this study were obtained from an abandoned wetland paddy field in TanjungKarang (UTM Easting 742873.39 and UTM Northing 379289.29, UTM Zone 47N). The field belongs to the TanjungKarang Rice Irrigation Scheme region, located in the Kuala Selangor district of Selangor state, Malaysia. Collected soil samples were air-dried, crushed and sieved through a 2 mm screen to analyze their initial properties.
The soil belongs to the clay textural class (sand 6.60%, silt 29.62% and clay 63.79%) according to USDA taxonomy [40]. The pH of the soil was measured by a glass electrode digital pH meter from the 1:2.5 ratio soil and water solution [41]. A CNS analyzer (LECO, Corporation, Saint Joseph, USA was used for the dry combustion method to measure total carbon, nitrogen and sulphur content in the soil. The amount of available N (ammonium and nitrate N) was measured from fresh soil by 1:4 ratio of soil and 2 M KClphenylmercuric acetate (KCl-PMA) mixture and titrating it against 0.01 N HCl [42]. Available phosphorus was extracted by the Bray and Kurtz II method [43] using 0.03 N NH 4 F and 0.1 N HCl as the extracting solution and P concentration was determined by inductively coupled plasma spectrometry (ICP-OES). Determination of exchangeable forms of various basic cations such as potassium (K + ), calcium (Ca 2+ ) and magnesium (Mg 2+ ) was extracted by leaching method using ammonium acetate Schollenberger [44]. Atomic absorption spectrophotometer (AAS, PerkinElmer Analyst 400) was used to determine exchangeable K, Ca and Mg in the collected leachate. The soil properties presented in Appendix A (Table A1).
There were two types of commercially produced biochar used in this study: rice husk biochar (produced from rice husk pyrolyzed at 300 • C) and oil palm empty fruit bunch biochar (produced from oil palm bunch pyrolyzed at 450 • C). A CNS analyzer (LECO, Corporation, Saint Joseph, MO, USA) was used to analyze the total carbon, nitrogen and sulphur content in the biochar. Total phosphorus, potassium and calcium in the biochar was analyzed by dry ashing [45], followed by using atomic absorption spectrophotometer (AAS, PerkinElmer Analyst 400). The pH of the biochar was measured from the 1:10 (w/w) ratio solution of biochar and water using a glass electrode digital pH meter [46]. The properties of biochars are presented in Appendix A (Table A2).

Scanning Electron Microscopy (SEM) of Biochar
The biochars were dried followed by metalizing through-TECB sputter coater system (SCD 005, BALZERS) to get the ideal conductive surface. Next, the metalized biochar samples were analyzed and amplified to 1000X by an SEM (LEO 1455VP, Oxford instrument and INCA software, London, UK) at 15 kilovolts.  (Table A3). The experiment was laid out in a factorial randomized complete block design (RCBD) with four replications, where two irrigation treatments namely AWD and CF were assigned as the first factor and four biochar and 15 N labelled urea combinations were assigned as the second factor such as (i) Rice husk biochar + 15 N urea, (ii) Oil palm empty fruit bunch biochar + 15 N urea and (iii) 15 N urea (iv) Control (no biochar and 15 N urea). The rice variety used in this study was MR297 developed in the year 2017 by the Malaysian Agricultural Research and Development Institute (MARDI).

Experimental Design and Pot Set-Up
A total of 32 experimental pots (45 cm × 52 cm) were filled with 50 kg of air-dried soil; furthermore, for AWD irrigation regimes (16 pots), a 4 cm diameter and 30 cm long perforated PVC pipe was inserted in soil up to 20 cm by keeping 10 cm above the surface and keeping others pot normal. After the pot preparation, RHB and EFBB were applied at the rate of 4% of the soil (wt/wt) in the respective treatments. The biochars were mixed thoroughly with soil 10 days before the transplanting and irrigated for proper seedling establishment. In this study, we used 10% atom excess 15 N labelled urea. Nitrogen (3.1 g pot −1 ), phosphorus (0.7 g pot −1 ) and potassium (2.5 g pot −1 ) were applied from urea, triple superphosphate and muriate of potash, respectively, as recommended by MARDI.
Four seedlings with 15 cm plant spacing were transplanted in a single pot, and further 3-5 cm ponding water depth was retained for the seedlings' establishment for 15 days. Moreover, in the individual AWD treated pots, a perforated PVC pipe was inserted up to 20 cm below the soil surface to monitor the water levels in the soil. In the AWD pots, the soil is allowed to dry until the water levels reach 15 cm from the surface, and after that, the pots were flooded to 5 cm above the soil surface ( Figure 1). This periodical drying cycle continued to the whole growth period, except in the flowering stage. In the continuously flooded irrigation pots, water levels were kept above 3-5 cm from the soil surface during the whole growth period of the rice. Other intercultural operations such as weeding and spraying pesticides were performed when necessary. At maturity, rice was harvested after 98 days of transplanting.
(UPM), Serdang, Selangor, Malaysia from April 2020 to July 2020. The meteorologica for the months of the research are referred to in Appendix A (Table A3). The exper was laid out in a factorial randomized complete block design (RCBD) with four re tions, where two irrigation treatments namely AWD and CF were assigned as th factor and four biochar and 15 N labelled urea combinations were assigned as the s factor such as i. Rice husk biochar + 15 N urea, ii. Oil palm empty fruit bunch biocha urea and iii. 15 N urea iv. Control (no biochar and 15 N urea). The rice variety used study was MR297 developed in the year 2017 by the Malaysian Agricultural Researc Development Institute (MARDI).
A total of 32 experimental pots (45 cm × 52 cm) were filled with 50 kg of air soil; furthermore, for AWD irrigation regimes (16 pots), a 4 cm diameter and 30 cm perforated PVC pipe was inserted in soil up to 20 cm by keeping 10 cm above the s and keeping others pot normal. After the pot preparation, RHB and EFBB were app the rate of 4% of the soil (wt/wt) in the respective treatments. The biochars were thoroughly with soil 10 days before the transplanting and irrigated for proper se establishment. In this study, we used 10% atom excess 15 N labelled urea. Nitrogen pot −1 ), phosphorus (0.7 g pot −1 ) and potassium (2.5 g pot −1 ) were applied from urea, superphosphate and muriate of potash, respectively, as recommended by MARDI.
Four seedlings with 15 cm plant spacing were transplanted in a single pot, an ther 3-5 cm ponding water depth was retained for the seedlings' establishment days. Moreover, in the individual AWD treated pots, a perforated PVC pipe was in up to 20 cm below the soil surface to monitor the water levels in the soil. In the AWD the soil is allowed to dry until the water levels reach 15 cm from the surface, and that, the pots were flooded to 5 cm above the soil surface ( Figure 1). This periodical d cycle continued to the whole growth period, except in the flowering stage. In the c uously flooded irrigation pots, water levels were kept above 3-5 cm from the soil s during the whole growth period of the rice. Other intercultural operations such as ing and spraying pesticides were performed when necessary. At maturity, rice wa vested after 98 days of transplanting.

Estimation of Soil Moisture Content, the Volume of Irrigation Water and Water Use Efficiency
A soil moisture meter (FieldScout TDR 150, Aurora, IL, USA) was used to dete the moisture content during the drying cycles of alternate wetting and drying irriga

Estimation of Soil Moisture Content, the Volume of Irrigation Water and Water Use Efficiency
A soil moisture meter (FieldScout TDR 150, Aurora, IL, USA) was used to determine the moisture content during the drying cycles of alternate wetting and drying irrigation.
The total volume of water (L) consumed by the plants in a pot was calculated by subtracting the volume of water required for pot preparation before transplanting of seedlings from the total volume of water applied until crop harvesting. A volumetric jar was used to measure the amount of water applied during each irrigation. Water productivity (WP) of rice was calculated by dividing the grain yield by the total volume of water applied [47]: Grain yield (g) Total irrigated water (L)

Determination Yield Component and Yield
A SPAD meter (SPAD-502, Konica Minolta sensing Inc. Sakai Osaka, Japan) was used to determine the leaf chlorophyll content of rice at three major growth stages (tillering, flowering and grain filling). Different yield-contributing parameters such as plant height, tiller number per hill, panicle length and the number of grains per panicle were recorded during the harvest. Grain and straw were sorted and weighed after harvesting from each pot. The biological yield is the sum of total grain and straw production.

Analysis of Plant Sample
After harvesting the plants at maturity, grain and straw were collected, separated and dried in the oven at 70 • C for 3 days to get a stable weight. Furthermore, the dried samples were finely milled for total nitrogen and 15 N analysis in the samples. Total N content in grain and straw sample was measured by dry combustion using CNS analyzer (LECO, Corporation, Saint Joseph, MO, USA) and 15 N isotope in the milled grain and straw samples were determined by using a Europa EA-GSL sample preparation system connected to a Sercon 20-22 stable isotope ratio mass spectrometer running in continuous flow mode.
Per cent of N derived from fertilizer (%Ndff), N uptake, fertilizer N uptake, fertilizer N use efficiency and recovery were calculated using the following formulas: Partial factor fertilizer N productivity g g −1 = Grain yield obtained in 15 N fertilized pot (g) Quantity of N applied from urea in each pot(g) FertilizerNrecovery g g −1 = Total Ndff by plant (g) Quantity of N applied from urea in each pot (g)

Percent Relative Data
For each element, the relative data of the value were presented as percentages relative to control [48]. Relative data (%) = Treatment value − control value control value ×100 Sustainability 2022, 14, 7622 6 of 21

Statistical Analysis
The statistical software R (version 3.6.1, R Foundation for Statistical Computing, Vienna, Austria) was used to analyze the experimental data by analysis of variance (ANOVA) to evaluate the statistical difference among the treatments. Two-way ANOVA was performed to determine the effect of the treatments. Tukey's test was used to compare the significant difference between the mean values at a 0.05 level of significance. The relation between the parameters was determined by Pearson's correlation analysis. The figures of different impacted by treatments were prepared using Microsoft Excel.

Scanning Electron Microscopy (SEM) of Biochar
SEM visualization was performed to observe the internal structure of RHB and EFBB. The SEM micrograph showed porous structures in both biochars originating from the plant-derived cellular structure used as feedstocks ( Figure 2). The figure illustrates that the biochar originating from rice husk exhibited pores with cell wall composition varying from 0.5 to 10 micrometres (µm). Furthermore, the origins of the pore structure in EFBB was similar to that of rice husk, although pore size varied from 1 to 10 µm. A greater number of micropores (<1 µm) was observed in the case of RHB compared to EFBB.
For each element, the relative data of the value were presented as percentages relative to control [48]. Relative data (%) = Treatment value -control value control value ×100

Statistical Analysis
The statistical software R (version 3.6.1, R Foundation for Statistical Computing, Vienna, Austria) was used to analyze the experimental data by analysis of variance (ANOVA) to evaluate the statistical difference among the treatments. Two-way ANOVA was performed to determine the effect of the treatments. Tukey's test was used to compare the significant difference between the mean values at a 0.05 level of significance. The relation between the parameters was determined by Pearson's correlation analysis. The figures of different impacted by treatments were prepared using Microsoft Excel.

Scanning Electron Microscopy (SEM) of Biochar
SEM visualization was performed to observe the internal structure of RHB and EFBB. The SEM micrograph showed porous structures in both biochars originating from the plant-derived cellular structure used as feedstocks ( Figure 2). The figure illustrates that the biochar originating from rice husk exhibited pores with cell wall composition varying from 0.5 to 10 micrometres (µ m). Furthermore, the origins of the pore structure in EFBB was similar to that of rice husk, although pore size varied from 1 to 10 µ m. A greater number of micropores (<1 µ m) was observed in the case of RHB compared to EFBB. (a)

Leaf Chlorophyll (SPAD) Impacted by Various Biochar Treatments and Irrigation Regimes at Different Growth Stages of Rice
The main effect of the irrigation regime and biochar with 15 N urea combinations significantly (p < 0.05) impacted the leaf chlorophyll (SPAD value) at tillering stage of rice (Table 1), but their interaction was insignificant. The CF irrigation produced a significantly higher SPAD value over AWD irrigation (31.74 and 30.56, respectively). Moreover, biochar treatments B1 (RHB + 15 N urea) and B2 (EFBB + 15 N urea) exhibited similar SPAD values (33.45 and 33.45, respectively) over B3 ( 15 N urea alone), which were significantly lower than the values obtained by B0 (control). Moreover, at the flowering stage, identical SPAD values were exhibited by B1, B2 and B3 compared to B0, but no significant main effect was observed from the AWD and CF regimes. Concurrently, in the tillering stage, CF showed enhanced SPAD value over AWD at the grain filling stage. Furthermore, from the main effect of the biochar treatments, the B1 and B2 treatments showed similar SPAD values which were significantly greater than B3 and B0 in the AWD regime.

Leaf Chlorophyll (SPAD) Impacted by Various Biochar Treatments and Irrigation Regimes at Different Growth Stages of Rice
The main effect of the irrigation regime and biochar with 15 N urea combinations significantly (p < 0.05) impacted the leaf chlorophyll (SPAD value) at tillering stage of rice (Table 1), but their interaction was insignificant. The CF irrigation produced a significantly higher SPAD value over AWD irrigation (31.74 and 30.56, respectively). Moreover, biochar treatments B1 (RHB + 15 N urea) and B2 (EFBB + 15 N urea) exhibited similar SPAD values (33.45 and 33.45, respectively) over B3 ( 15 N urea alone), which were significantly lower than the values obtained by B0 (control). Moreover, at the flowering stage, identical SPAD values were exhibited by B1, B2 and B3 compared to B0, but no significant main effect was observed from the AWD and CF regimes. Concurrently, in the tillering stage, CF showed enhanced SPAD value over AWD at the grain filling stage. Furthermore, from the main effect of the biochar treatments, the B1 and B2 treatments showed similar SPAD values which were significantly greater than B3 and B0 in the AWD regime.

Impact of Biochar on Rice Yield, Irrigation Water Volume and Water Productivity under AWD and CF Irrigation
The grain and straw yield of rice was significantly (p < 0.05) influenced by the two irrigation and different biochar treatments but was unaffected by their interaction ( Table 2). The main effect showed that a significant grain yield increase was recorded by the B1 (202.57 g pot −1 ) compared to AWD (193.89 g pot −1 ). Moreover, B1 and B2 produced similar grain yields (226.73 and 229.83 g pot −1 , respectively), which were significantly higher thanB3 (211.20 g pot −1 ), and the lowest yield produced by B0 (125.16 g pot −1 ). Furthermore, the straw yield of rice exhibited a similar trend for the main effect of irrigation and biochar as such as grain yield. The AWD and CF regime produced 307.27 and 312.29 g pot −1 straw yield, respectively.  The main effect of the irrigation regimes and the biochar treatments significantly (p < 0.05) impacted the irrigation water volume and water productivity (WP), while no difference was observed from their interaction. The CF irrigation obtained maximum irrigation water consumption (116.25 L) in comparison to the AWD regime (94.63 L). From the effect of biochar and 15 N urea combination, the maximum water consumption was obtained by the B3 treatment (122.38 L) over B1 and B2 irrigation, which exhibited similar water uses (105.00 and 108.88 L, respectively), and the minimum water usage was obtained by B0 (85.50 L). Considering that the WP of rice by AWD irrigation showed profoundly greater results (2.04 g L −1 ) compared to CF (1.73 g L −1 ). The biochar treatments B1 and B2 showed identical but greater WP over B3 and B0. showed very near values (47.56%, 46.59%, 44.75% and 43.02%, respectively). Nevertheless, in the subsequent drying cycles of AWD, the biochar treatments (B1 and B2) presented significantly greater moisture contents over B3 and B0. The biochar treatments B1 and B2 showed adjacent value in most drying cycles except in D8. Similarly, B3 and B0 also exhibited somewhat similar soil moisture values from D1 to D8, but profound variation was only found in D7. Overall, the total number of drying cycles (10) were similar for B1, B2 and B3, while 8 drying cycles were found in B0. However, B1 and B2 remarkably boosted soil moisture content compared to B3 and control B0 at various drying cycles of AWD irrigation.

Impact of Biochar and 15 N Urea Combination on the %Soil Moisture Content at Various Drying Cycles of AWD Irrigation
The soil moisture percentage was significantly (p < 0.05) influenced by the inclusion of combined RHB and EFBB with 15 N urea treatments (Figure 3) at different drying cycles (D) of AWD. From the first drying cycle (D1), it was observed that B1, B2, B3 and B0 showed very near values (47.56%, 46.59%, 44.75% and 43.02%, respectively). Nevertheless, in the subsequent drying cycles of AWD, the biochar treatments (B1 and B2) presented significantly greater moisture contents over B3 and B0. The biochar treatments B1 and B2 showed adjacent value in most drying cycles except in D8. Similarly, B3 and B0 also exhibited somewhat similar soil moisture values from D1 to D8, but profound variation was only found in D7. Overall, the total number of drying cycles (10) were similar for B1, B2 and B3, while 8 drying cycles were found in B0. However, B1 and B2 remarkably boosted soil moisture content compared to B3 and control B0 at various drying cycles of AWD irrigation.

Impact of Biochar on N Uptake by Grain, Straw and Their Total under Two Irrigation Regimes
Different biochar treatments and irrigation regimes significantly (p < 0.05) impacted N content in grain, straw and their total uptake, but their interaction was unaffected (Table 3). In the main impact of irrigation, the AWD regime showed lower grain N content over CF (2.68 and 2.95 g pot −1 , respectively). Considering the main effect of biochar treatment, identical grain N was obtained by B1 and B2 (3.35 and 3.48 g pot −1 , respectively), followed by B3 (2.95 g pot −1 ), and reduced N was found from B0 (1.49 g pot −1 ). Meanwhile, similar to grain N content, similar treatment variations were observed in the straw and total N content for the main effect of the irrigation and biochar treatments. However, the straw and total N content were 1.81 and 4.50 g pot −1 , respectively, in the AWD while CF showed 2.04 and 4.98 g pot −1 . Nevertheless, for the main effect of biochar, straw and total N content varied from 0.84 to 2.43 g pot −1 and 2.33 to 5.92, respectively.

Impact of Biochar on N Uptake by Grain, Straw and Their Total under Two Irrigation Regimes
Different biochar treatments and irrigation regimes significantly (p < 0.05) impacted N content in grain, straw and their total uptake, but their interaction was unaffected (Table 3). In the main impact of irrigation, the AWD regime showed lower grain N content over CF (2.68 and 2.95 g pot −1 , respectively). Considering the main effect of biochar treatment, identical grain N was obtained by B1 and B2 (3.35 and 3.48 g pot −1 , respectively), followed by B3 (2.95 g pot −1 ), and reduced N was found from B0 (1.49 g pot −1 ). Meanwhile, similar to grain N content, similar treatment variations were observed in the straw and total N content for the main effect of the irrigation and biochar treatments. However, the straw and total N content were 1.81 and 4.50 g pot −1 , respectively, in the AWD while CF showed 2.04 and 4.98 g pot −1 . Nevertheless, for the main effect of biochar, straw and total N content varied from 0.84 to 2.43 g pot −1 and 2.33 to 5.92, respectively.

Impact of Biochar on Per Cent of N Derived from 15 N Urea Fertilizer and Soil in Grain and Straw under Two Irrigation Regimes
Different biochar and 15 N combination and irrigation regimes significantly (p < 0.05) impacted the %N content in grain and straw from fertilizer and soil sources, while their interaction did not show any significant result ( Table 4). The CF irrigation significantly enhanced the %N in grain and straw from urea (31.96% and 31.22%, respectively) and reduced from soil sources (68.04% and 68.78%, respectively) over the AWD regime. However, in the case of the main effect of biochar, B1 and B2 exhibited higher but similar %Nin grain from urea compared to B3. Furthermore, the biochar unamended treatment (B3) significantly increased the %N content in grain from the soil compared to the biochar treatments such as B1 and B2. Moreover, the %N derived from fertilizer and soil ranged from 29.67% to 31.63% and 68.37% to 70.33%, respectively, observed from straw.

Effect of Biocharon Grain, Straw, Total N Uptake from 15 N Urea and Fertilizer N Recovery (FNR) under Two Irrigation Regimes
Several biochar treatments and irrigation regimes significantly (p < 0.05) affected the N derived from 15 N urea (Ndff) in grain, straw, total uptake and (FNR), while no impact was found from their interaction ( Table 5). From the main effect of irrigation, it was observed that significantly increased value derived from 15 N urea (Ndff) in grain, straw, total and fertilizer N recovery (FRN) were obtained by CF irrigation (1.09 g pot −1 , 0.75 g pot −1 , 1.84 g pot −1 and 0.59 g g −1 , respectively) over the AWD irrigation. Furthermore, biochar treatments B1 and B2 showed similar results of N derived from 15 N urea (Ndff) in grain (1.09 and 1.11 g pot −1 , respectively), which was significantly higher than B3 (0.90 g pot −1 ). As in grain, B1, B2 and B3 also exhibited an identical trend of Ndff in straw in the AWD. Total Ndff also followed a similar pattern of the result by different biochar. However, the higher but similar values of FRN were obtained by B1 and B2 in comparison to B3. Table 5. Grain, straw, total N uptake from 15 N urea and fertilizer N recovery influenced by the significant main effect of irrigation and biochar treatment.

Effect of Biochar and Irrigation Regimes on Nitrogen Use Efficiency Parameters of Rice
The agronomic efficiency (AE), physiological efficiency (PE) and partial factor productivity (PFP) of nitrogen in rice were significantly (p < 0.05) influenced by various biochar and 15 N urea combination, but the effect of irrigation and its interaction with biochar treatments were insignificant ( Table 6). The biochar treatments B1 and B2 produced similar but significantly greater AE of nitrogen (32.77 and 33.67 g g −1 , respectively) over B3 (27.76 g g −1 ). For the PE of N, the maximum value was obtained by B3 (86.56 g g −1 ); which was significantly higher than B1 and B2 (80.05 and 74.39 g g −1 , respectively). Similar to AE, the maximum PFP of N was found from B2 (74.14 g g −1 ), which was similar to B1 (73.14 g g −1 ) but significantly greater than B3 (68.13 g g −1 ).

Effect of Biochar with 15 N Urea Combination on Soil Total C and N under AWD and CF Irrigation Regimes
Regarding the total carbon content in the soil (Figure 4), the biochar treatment B2 exhibited greater under both AWD and CF irrigation (6.92% and 7.04%, respectively) followed by B1 (6.55% and 6.60%, respectively). Whereas, profoundly decreased results were observed from B3 and B0 in both irrigations. Considering the same biochar treatment, B3 and B0 with AWD regime exhibited reduced soil carbon (4.38% and 4.23%, respectively) compared to CF irrigation (5.58 and 5.36, respectively).

Effect of Biochar with 15 N Urea Combination on Soil Total C and N under AWD and CF Irrigation Regimes
Regarding the total carbon content in the soil (Figure 4), the biochar treatment B2 exhibited greater under both AWD and CF irrigation (6.92% and 7.04%, respectively) followed by B1 (6.55% and 6.60%, respectively). Whereas, profoundly decreased results were observed from B3 and B0 in both irrigations. Considering the same biochar treatment, B3 and B0 with AWD regime exhibited reduced soil carbon (4.38% and 4.23%, respectively) compared to CF irrigation (5.58 and 5.36, respectively).
In the AWD irrigation, B1 and B2 showed similar but higher values of total soil N (0.34% and 0.37%, respectively) over B3 (0.22%) and the lowest soil N was found from B0 (0.12). Likewise, AWD regime, B1 and B2 showed a similar variation of soil N (0.36% and 0.38%, respectively) compared toB3 (0.30) and B0 (0.19) under the CF regime. However, in the same biochar treatment, B3 and B0 with CF irrigation significantly boosted soil C and N content values over AWD irrigation, though B1 and B2 produced similar results.  In the AWD irrigation, B1 and B2 showed similar but higher values of total soil N (0.34% and 0.37%, respectively) over B3 (0.22%) and the lowest soil N was found from B0 (0.12). Likewise, AWD regime, B1 and B2 showed a similar variation of soil N (0.36% and 0.38%, respectively) compared toB3 (0.30) and B0 (0.19) under the CF regime. However, in the same biochar treatment, B3 and B0 with CF irrigation significantly boosted soil C and N content values over AWD irrigation, though B1 and B2 produced similar results.

Effect of Biochar on pH and Available Nitrogen of Post-Harvest Soil
Different biochar and irrigation regimes significantly (p < 0.05) impacted the soil pH, but their interaction was insignificant (Table 7). However, the AWD irrigation profoundly reduced the soil pH value over the CF regime. Biochar treatment B2 obtained the maximum pH value (6.45) followed by B1 (6.28), which were markedly greater than B3 and B0, which produced identical values (5.36 and 5.27, respectively).
Various biochar and irrigation treatments significantly influenced the available N (NH 4 + -N and NO 3 − -N). The AWD irrigation significantly increased the NH 4 + -N of soil over the CF regime (49.02 and 37.41 mg kg −1 , respectively). Consecutively, AWD irrigation reduced the NO 3 -N in soil compared to CF. Biochar treatments such as B1 and B2 obtained identical values of NH 4 + -N (53.44 and 58.77 mg kg −1 , respectively) but greater than B3 (38.36 mg kg −1 ), and the lowest results were found from B0 (22.30 mg kg −1 ). Moreover, the maximum NO 3 -N in soil was observed from B2 (54.50 mg kg −1 ) followed by B1 (44.77 mg kg −1 ) and B3 (21.27 mg kg −1 ). In comparison, the minimum value was also observed from B0 (14.74 mg kg −1 ). Table 7. Main effect of irrigation and biochar with 15 N urea combination on pH, ammonium-N and nitrate-N of post-harvest soil.

Leaf Chlorophyll and Yield
Incorporating biochar such as RHB and EFBB remarkably boosted the SPAD value (up to 7.5%), water productivity (up to 25%) and rice yield (up to 7%). Compared to the sole application of 15 N urea, at the tillering and grain-filling stage, biochar combined with urea increased the leaf SPAD value up to 6.6% and 7.5%, respectively. A recent study by Win et al. [49] found that the application of RHB profoundly enhanced the SPAD value of rice. Moreover, Lai et al. [50] observed that rice straw biochar with 150 kg ha −1 significantly increased the SPAD value compared to a single application of N fertilizer. This increased leaf activity by the addition of biochar and 15 N fertilizer combination was also reported by a previous study by Ullah et al. [24].
Rice yield performance is to be considered as the key indicator to adopt a management practice. Different studies from the past obtained varieties of yield responses to the AWD irrigation. Among some of them reported the increase [9,51,52], decrease [11,53,54] or no prominent effect on the grain yield [18,55,56]. From the main effect of irrigation, the AWD regime sharply decreased the grain yield (4.2%) compared to the CF irrigation. In this experiment, safe-AWD was practiced, where water levels were allowed to drain up to 15 cm from the surface during the drying cycles and then irrigated with 5 cm ponding water depth. Thereby, no drastic yield loss because the plant roots always reached soil water during the drying cycles. Nevertheless, this little yield reduction was also reported by Haque et al. [57], where carbon isotope discrimination in the rice leaf revealed that rice faces a sharp moisture deficit condition to some extent under AWD, which can be recovered by applying biochar such as RHB and EFBB [57]. From a previous study, Bakar et al. [58] reported that the application of EFBB improved the soil quality by increasing soil cation exchange capacity (CEC) and soil carbon; in addition, it also alleviates soil pH, which ultimately increased the rice yield. Furthermore, this study also revealed that integrated RHB and EFBB with N fertilizer increased the grain yield up to 7-8% by the main effect of biochar. Many studies from the past also reported the positive impact of biochar on rice yield [24,57,59,60]. There are several factors through which biochar inclusion in the soil accelerates rice yield, including enhanced nutrient uptake [57], photosynthetic activity [61] and improved soil properties such as nutrient availability [62], CEC [58], porosity [28] and moisture retention [59].

Irrigation Water Usage and Productivity
The key component in AWD irrigation is reducing irrigation water usage and improved water productivity (WP). This research examined that AWD irrigation decreased irrigation water usage by 28.6% and increased water productivity by 17.9%. Furthermore, RHB and EFBB with 15 N urea reduced the water usage up to 14.2% and improved WP up to 25.3% compared to the 15 N urea(without biochar) application. The AWD irrigation can save up to 35% of irrigation water for rice production also found from previous studies [51,52,55,56]. Due to the RHB and EFBB addition with 15 N urea, the enhanced moisture retention at different drying cycles of AWD is presented in Figure 3. This improved moisture retention might be explained by SEM micrographs of RHB and EFBB (Figure 2). The figures of RHB and EFBB revealed the presence of different sized micropores, and these pores could be served as a capillary store of water [40]. However, RHB did not show porous properties as EFBB did, possibly due to the different feedstock and divergence in temperature during the biochar preparation. At a very high pyrolytic temperature (>350 • C), rice husk loses carbon (C), whereas oil-palm branch biochar produces higher C at temperatures from 400 to 500 • C [63]. This might be because their origins and cellulose/hemicellulose contents are different [64].
Moreover, biochar generated from agricultural residue is high in silicon, which reacts with water molecules and has characteristics similar to a silica hydrogel, preventing water loss by preserving it [65]. However, biochar can also influence soil moisture retention by changing several soil physical parameters such as bulk density, porosity, aggregate stability, cracking, etc. [66]. According to Chen et al. [67], the addition of rice straw and rice husk biochar improved the moisture content of sandy loam and silty loam soil up to 18.61% and 19.66%, respectively. In Vertisol clay soil, rice straw and woodchip biochar boosted the moisture content up to 18.4% and 6.8%, respectively, as observed by Sun and Liu [68]. Furthermore, the addition of bamboo and rice straw biochar combined with urea enhanced the soil moisture content by 9% and 15%, respectively [59].

Nitrogen Use Efficiency (NUE) and Recovery
The main effect of irrigation demonstrates that AWD remarkably reduced the nitrogen uptake, which is similar to the findings of previous studies [21,22]. Thereby, it also decreases the fertilizer N recovery. The inclusion of biochar with 15 N urea increased the total and fertilizer uptake up to 23.4% and 18.8%, respectively. A study by Huang et al. [34] reported that depending on the application rate, the addition of cassava stem biochar with 15 N boosted the fertilizer N uptake up to 27% in rice. Similarly, Ali et al. [69] documented that biochar application with urea increased the N uptake by rice. Another study using 15 N labelled urea with biochar also observed that biochar incorporation increased about 23.9% of leaf N in rice [24].
Moreover, the agronomic efficiency (AE) and partial factor productivity (PFP) of N also accelerated by combined application of 15 N urea with RHB (18% and 7%, respectively) and EFBB (up to 21% and 8%, respectively). These parameters are mainly correlated to the grain yield; in this study, biochar addition profoundly increased the rice yield, which ultimately resulted in improved AE and PFP of N. However, this study revealed that biochar addition reduced the physiological efficiency (PE) of N. The PE of N is the ratio between biological yield and N uptake. In this context, compared to biological yield response, higher N uptake obtained from biochar amended soil over lone 15 N urea might result in reduced PE. However, Oladele et al. [35] reported that the integrated use of biochar with N fertilizer increased the AE by 140% in rice over 2 years. Another study demonstrated that urea with chicken litter biochar significantly boosted the AE of N [70]. Nevertheless, Zheng et al. [71] found that incorporating rice straw biochar with urea fertilizer increased the AE of N by 4.0 kg −1 . Likewise, Chen et al. observed that biochar-based fertilizers enhanced about 33% to 74% of PFP of N [72]. This positive effect of biochar on NUE is also similar to the findings of several researchers [73][74][75].
In addition, RHB and EFBB increased the fertilizer N recovery (FNR) up to 24.5% over the sole application of urea since FNR is directly related to the fertilizer N uptake. From this study, 15 N isotopic analysis of grain and straw revealed that biochar incorporation markedly enhanced the N uptake from urea, which ultimately increased the FNR. Findings from a past study reported that biochar increased about 192% of grain N recovery in rice [35]. A similar positive impact of biochar on N recovery result of also observed from a previous study [76]. Liu et al. [77] demonstrated that the inclusion of biochar with urea accelerated fertilizer N retention and increased N mineralization in the soil, which possibly resulted in enhanced N uptake with the improved FNR of rice.

Soil pH, Carbon and Nitrogen Content of Post-Harvest Soil
A profound change in the pH of the post-harvest soil has been observed due to the incorporation of RHB and EFBB. Biochar shows a different mechanism to control soil pH due to its unique surface chemistry and the presence of various functional organic groups and compounds [78]. Biochar itself exhibits high pH due to carbonates and organic anions derived from acidic functional groups [79]. In addition, inorganic alkali such as silicate (SiO 4 4− ), phosphate (PO 4 3− ) and iron hydroxides (FeO 2− ) absorb proton by the combination of these anions with H + [80]. Thereby, biochar is used as a soil amendment to optimize soil pH for crop production. Most of the studies reported that biochar addition plays a significant role in altering the pH value of soil [81]. Xie et al. [82] reported that the application of urea in an acidic Ultisol increased soil acidity, and the addition of corn biochar reduced the soil pH and buffered the nitrogen acidification in paddy soil. In a recent study, rice husk biochar and rice straw biochar was applied in two types of paddy soil, and in both cases, the pH value increased 0.27 to 1.61 units, respectively [67]. The addition of rice straw biochar increased the pH value from 0.1-0.46 in a Ultisol in Southern China [83].
The addition of biochar with 15 N urea boosted the soil total carbon up to 57.9% and 26.3% in the AWD and CF irrigation, respectively, whereas the case of biochar-unamended soil with AWD irrigation lost an eminent percentage of soil carbon. Compared to CF irrigation, in the drying period of AWD irrigation, the soil prevails in aerobic conditions [84]. This oxidizing environment might hasten the degrading organic matter and diminishing organic carbon status in the soil, and this occurrence may lead to significant CO 2 emissions from the soil [85]. Furthermore, due to the aerobic environment, heterotrophic respiration occurs in soil, resulting in the accelerated mineralization of organic carbon during the drying cycles of AWD [17]. The increased soil C by biochar in this study due to the biochar enriched with the stable and recalcitrant form of carbon [86], through the application of this soil amendment act as both sink and source of carbon [25], resulting in improved soil fertility [87]. Findings from past studies revealed that biochar inclusion conspicuously enhanced the soil carbon content. Gamage et al. [28] found that the addition of 1% RHB increased the soil organic carbon (SOC) by 65% in sandy loam soil. Yang et al. [88] reported that biochar application increased the SOC up to 26.7% under water-saving irrigation. Moreover, in a clay loam paddy soil, the addition of bamboo chip and rice straw biochar with urea increased the SOC by 58.4% and 37.4%, respectively [59].
As with total carbon, the AWD irrigation without biochar inclusion significantly reduces the soil's total N content over CF irrigation. A theatrical transformation between the aerobic and anaerobic conditions in AWD affects the nitrogen mineralization, nitrification and denitrification due to the microbial activity in the soil [18]. In AWD irrigation, soil N is lost through the nitrification and denitrification process [22,59]. Nevertheless, AWD accelerates the N 2 O production by denitrification in the soil and declining the indigenous N [23]. However, in this study, the incorporation of RHB and EFBB with 15 N urea remarkably boosted the total N in soil under AWD (52.8% and 67.42%, respectively) and CF (21.2% and 28.0%, respectively) irrigation. Incorporation of biochar enhanced soil N was also reported from previous studies [40,58,59]. For example, Haefele et al. [62] observed that the application of RHB with fertilizer increased the N up to 15.6% in paddy soil. In another study, wheat straw biochar at 40 t ha −1 increased the soil N by about 22.7% in the subtropical paddy soil [89]. Likewise, the incorporation of sewage sludge biochar in acidic soil accelerated the N content up to 550% depending on application rates.
This study also revealed the enhancement of the mineral N (NH 4 + -N and NO 3 − -N) by biochar inclusion. Biochar application promotes NO 3 + -N in soil, possibly due to the absorption phenolic compound, which restricts the soil nitrification process [90], further increasing the activities of nitrifying bacteria in the soil [91]. Moreover, biochar has unique surface chemistry inclusive of acid functional groups and the presence of both cation and anion exchange sites [92]. This cation exchange site of biochar absorbs the NH 4 + ion and the acid functional groups react with NH 4 + and forms amine and amides, thereby decreasing the loss of NH 4 + from soil [93]. In addition, there are some limitations of this study such as this experiment being conducted in pots under controlled conditions, so there was no source of irrigation from precipitation and water loss by percolation and seepage. Nevertheless, this is a shortterm study, and the result may vary for long-term experiments under field conditions. A long-term field study should be carried out to validate the results.

Conclusions
The AWD irrigation noticeably curtails about 28% of the irrigation water usage with a 4% grain reduction over the CF regime. Concurrently, the application of RHB and EFBB with 15 N urea prominently boosted the rice yield compared to alone 15 N urea inclusion. Therefore, this minor yield penalty could be recovered by biochar application. Furthermore, RHB and EFBB incorporated soil enhanced the fertilizer N uptake and recovered more fertilizer N compared to a single 15 N urea application. Additionally, these biochars conspicuously accelerated the agronomic efficiency and partial factor productivity of N. RHB and EFBB also improved the soil pH and total carbon content of post-harvest. The biochar addition with 15 N urea further increased the total and available soil nitrogen. However, the biochar (RHB and EFBB) with 15 N urea improved the rice yield, N use efficiency and soil properties. Still, in the CF regime, excessive irrigation water is required to keep the soil inundated during the whole growing period of rice. Thereby, biochar utilizes more fertilizer N while reducing excessive N fertilizer use for better environmental quality. Therefore, the integrated use of biochar and urea with AWD irrigation might be a better adoption for sustainable rice production with better WP.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/ su14137622/s1. The non-significant interaction values of various parameters influenced by irrigation and biochar treatment are presented in Tables S1-S5. Table S1: Interaction effect of irrigation and biochar on leaf chlorophyll (SPAD) of rice at different growth stages of rice, Table S2: Interaction effect of irrigation and biochar on rice yield, irrigation water volume and water productivity, Table S3: Interaction effect of irrigation and biochar on N derived from 15 N urea fertilizer and soil in grain and straw under two irrigation regimes, Table S4: Interaction effect of irrigation and biochar on N content in grain, straw, their total grain, straw, total N uptake from 15 N urea and fertilizer N recovery, Table S5: Interaction effect of irrigation and biochar on different N use efficiency parameters and soil pH, total carbon, total and available nitrogen of post-harvest soil.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available because theyare a part of a PhD study.

Acknowledgments:
The authors are grateful to the National Agricultural Technology Program Phase-II Project, Bangladesh Agricultural Research Council, for the financial support and Universiti Putra Malaysia, Serdang, Selangor, Malaysia, for the research facilities. Last but not the least, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh 2202, Bangladesh for providing the 15 N labelled urea.

Conflicts of Interest:
The authors declare no conflict of interest.