Soil Phosphorus Fractionation and Bio-Availability in a Calcareous Soil as Affected by Conocarpus Waste Biochar and Its Acidified Derivative

Abstract

Biochar possesses more profound effects on the availability of soil P in acidic soil than in alkaline and/or calcareous soil, mainly due to P fixation. Therefore, biochar derived from Conocarpus waste (BC) was acidified with sulfuric acid to produce acidified biochar (ABC) and incorporated into a calcareous soil planted with alfalfa in order to investigate P availability and fractionation. Additionally, the changes in some other soil chemical properties were investigated. Both BC and ABC were applied at three rates (0%, 2.5%, and 5%) along with P fertilizer application at four rates (0, 75, 150 and 300 ppm). The results showed that acidification remarkably reduced the pH of ABC by 6.84 units. The application of ABC considerably lowered the soil pH; however, it did not significantly increase P availability in the studied soil. Furthermore, BC, especially at a higher application rate, increased the extractable soil K. Similarly, the amendments increased the soil cation exchangeable capacity (CEC) and soil organic matter (OM), where a profound increase was observed at a higher application rate in the case of soil OM. Similarly, soil-available micronutrients were increased over the control, where a more profound increase was observed in soils treated with ABC. The NaHCO₃⁻P (exchangeable) fraction increased with increasing fertilizer application rate while the residual–P decreased. Therefore, BC and ABC could be used to improve soil quality and enhance soil nutrient availability. However, further studies are required on how to significantly improve soil available P in calcareous soil.

1. Introduction

Phosphorus (P) is an essential macro-nutrient required by plants for optimum growth and development. Soils of many parts of the world are suffering from P deficiency mainly due to P fixation and precipitation [1,2]. About 15 million tons of P fertilizer is needed yearly all over the world in order to meet P demand for agricultural productivity [3]. The absence or less and less availability of P in soil cannot be compensated by any other nutrient. Plant growth and production are severely affected by P deficiency [4]. Crop production consumes a high percentage of the total P source. With the increasing demand for food and feed production, mineral fertilizers have been the major source of P input to crop production. Despite the addition of soil amendment to enhance soil available P, its availability, forms, and dynamics are dependent on soil characteristics. Phosphorus is available in soil between 6.5–7 pH, and its availability is relatively low in alkaline soils (pH of 7.5–8.5), mainly due to the formation of insoluble calcium phosphate compounds [5]. In this context, almost all the agricultural areas of arid regions are characterized by sandy soils with different levels of calcium carbonate (CaCO₃) and high pH, while the organic matter is low. Calcareous soil is characterized by high CaCO₃ mineral content, which fixes P through mineral precipitation [5,6]. In such soils, the amount of P used in fertilization is much higher than the actual plant requirement for this nutrient because most of the added P is fixed before it is used by the plant. This results in P being accumulated in agricultural soil. Therefore, there is a need for soil amendment like organic materials to enhance the soil fertility status in order to increase soil P availability and, thus, improve P use efficiency, reducing the amount of P fertilizer use and maximizing crop yield. In spite of the importance of organic fertilizers that are currently used to increase soil P availability, it may lead to negative and serious effects on the environment through the decomposition of organic matter that causes greenhouse gas emissions and global warming. Recently, recycling of organic waste in the form of biochar to reduce global warming and improve soil properties has received wide attention.

Biochar is a black, porous, carbon-based material produced by pyrolyzing biomass (plant and animal waste) in a limited or oxygen-free environment [7]. Adding biochar to the soil changes the soil environment and soil microbial communities, and this results in a no effect to positive effect on P availability [8]. Incorporating biochar into the soil raises the soil pH, soil water holding capacity, and soil cation exchange capacity (CEC). Biochars are characterized by high surface charge density with high CEC. Through cation exchange and internal porosity, biochar can retain cations on the surface. Likewise, the nutrients can be absorbed on the polar and non-polar surfaces of biochar [9]. Biochars have the ability to store and release vital nutrients needed by plants [9]. Inorganic nutrient elements like N, P, K, and Ca present in biochar may give plants vital macro- and micronutrients [10]. Due to its alkalinity, biochar application to alkaline soil could have a negative impact on P availability. To overcome the alkalinity problem derived from biochar, acidifying pyrolyzed materials could reduce the pH of biochar which is then hypothesized to reduce the pH of calcareous soil when added to the soil as a soil additive. The ability of acidified biochar to reduce the pH of alkaline soil to pH around neutral could be an effective method to lower P fixation and boost its availability in calcareous soils. The contradictory results from numerous studies show that it is still unclear how biochar affects the P-pools in various soils.

Conocarpus erectus L. is a widely planted tree in the kingdom of Saudi Arabia. The yearly pruning of its branches produces a lot of waste that needs to be disposed of. The transformation of these waste materials into biochar is a possible method of disposal of these materials and could serve as a soil additive that can be used to enhance the productivity of the soil. Therefore, the current study focus on using Conocarpus-derived acidified biochar as a soil ameliorant to evaluate changes in P availability and its fractionation in calcareous soil. According to Huang et al. [11], the modified biochar treatments could be either before or after pyrolysis. Modification of biochar by acid was used to increase the surface area and reduce the pH of biochar where the treatments were applied both before and after pyrolysis [12]. Furthermore, the acid treatment removes impurities and metallic precipitates from the surface and introduces carboxylic groups to the biochar, making it more active for cation sorption [13].

The major objectives of this research were to produce acidified biochar from Conocarpus waste as a soil amendment in order to investigate its impacts on the changes in P status in calcareous soil by (1) investigating the changes in chemical properties of calcareous soil resulting from acidified biochars addition, and (2) assessing the influences of acidified biochar addition on P availability to alfalfa plants and P fractionation in a calcareous soil.

2. Materials and Methods

2.1. Collection, Preparation and Characterization of Soil Samples

The calcareous sandy soil was gathered from the agricultural research station farm of King Saud University, Saudi Arabia, (Riyadh, Saudi Arabia), (24°24′43.0″ N latitude, 46°39′30.7″ E longitude). The collected soil was prepared by air-drying, after which it was passed through a sieve of 2 mm in size. Three replicates of this soil were analyzed for basic characterization following standard procedures, according to Sparks [14]. Soil pH was measured by pH meter (pH 523, Weilheim, Germany) with soil:water suspensions at a 1 to 2.5 ratio [15]. The soil electrical conductivity (EC) was measured with an EC meter (YSI Model 35, Yellow Springs, OH, USA). The hydrometer method was employed to analyze the soil texture [16]. The CaCO₃ content was analyzed by employing a calcimeter (Eijkelkamp Agrisearch Equipment, Giesbeek, The Netherlands). Following oxidation, K₂Cr₂O₇ was used to measure the amount of organic matter in the soil [17]. The available form of micronutrients (Fe, Mn, Zn, Cu) and phosphorus in the soil samples were extracted using ammonium bicarbonate diethylenetriaminepentaacetic acid (AB-DTPA). These extracts were analyzed by employing Inductively coupled plasma-optical emission spectrometry (ICP-OES) (Perkin Elmer Optima 4300 DV, Waltham, MA, USA) for measuring the amount of Fe, Mn, Zn, and Cu, while P was analyzed using the colorimetric method with the aid of a UV/VIS spectrophotometer (Lambda EZ 150, PerkinElmer, Waltham, MA, USA) [18]. In the colorimetric method, a color reagent was developed using a combination of ascorbic acid, ammonium molybdate, H₂SO₄ and antimony potassium tartrate. The absorbance of the sample was determined at 880 nm using a UV/VIS spectrophotometer after the sample pH had been adjusted and the color had been developed.

2.2. Biochar Production and Acidification

Biochar was derived from the pyrolysis of Conocarpus waste (CW) at 400 °C for 3 h after drying to a constant weight in an oven at a temperature of 65 °C [19]. The produced biochar was ground, sieved through a 53-μm mesh, stored in an airtight container and labeled as BC. Biochar was acidified with sulfuric acid. Sulfuric acid is selected due to its sulfate group (SO₄²⁻), which could compete with the phosphate group (PO₄³⁻, H₂PO₄⁻, and HPO₄²⁻) in forming a complex with Ca in the calcareous soil, thereby enhancing phosphorus availability. The biochar was acidified by shaking it in 0.5 N H₂SO₄ solution at a biochar-to-acid solution ratio of 1:10 for 45 min [20]. The suspension was allowed to stand for 24 h, after which it was filtered through a Whatman 42 filter paper. The biochar on the filter paper was washed about 5 times with deionized water. The collected biochar on the filter paper was oven-dried at a temperature of 65 °C for 48 h and then kept in an airtight container and referred to as acidified biochar (ABC).

2.3. Biochar Characterization

2.3.1. Proximate Analyses

The produced biochar was subjected to proximate analysis, which includes yield, moisture content, volatile matter, and ash contents, employing the standard method of ASTM E872-82 [21]. The produced yield of biochar was calculated as the fraction of the weight of biochar to the weight of biomass. Moisture content was examined by subjecting the biochar to heat at 105 °C for 24 h. The volatile matter was determined by subjecting the materials to heat (in covered crucibles) at 450 °C for 30 min, while the ash content was measured by heating the produced biochars (in open crucibles) at a temperature of 750 °C for 30 min. The difference between 100% and percentage moisture content, in addition to ash content and volatile matter, gives the resident matter (representing the fixed carbon).

2.3.2. Ultimate Analyses

With the aid of an elemental analyzer, the composition of carbon, hydrogen, nitrogen and oxygen of the biochar was analyzed (Euro EA 3000, Wegberg, Germany), and the atomic ratio was further calculated. The surface structure of biochar was analyzed through the images that were taken with the aid of a scanning electron microscope (SEM, EFI S50 Inspect, Amsterdam, The Netherlands). On the coated (with adhesive carbon tapes) aluminum stubs, samples were spread out (12 mm; PELCO, Wokingham, UK), which were then coated with nanogold particles for 60 s by employing 108 Auto/SE Sputter Coater (Ted Pella Inc., Redding, CA, USA). Images were captured at an acceleration voltage of 30 kV in a high vacuum with magnifications ranging from 2000 to 300. Using an X-ray diffractometer (MAXima_X XRD-7000, Shimadzu, Kyoto, Japan), the mineralogy of the materials was examined by adjusting the scan speed to 2-degree min⁻¹ with radiation of 30 mA Cu Kα in a continuous scan mode. The surface area was determined with the aid surface area analyzer (TriStar II 3020, Micromeritics, Norcross, GA, USA) following the Brunauer–Emmett–Teller (BET) method. Fourier transform infrared spectroscopy (FTIR) was employed to describe the different functional groups present in biochar. The EC and pH of the material were determined with the aid of EC and pH meters, respectively, in a soil-to-water ratio of 1:10. Cation exchange capacity (CEC) was measured according to Sparks [14]. Available P, K, Fe, Mn, Cu and Zn were determined in NH₄HCO₃-DTPA extract according to Soltanpour and Schwab [22]. The concentrations of P were analyzed using the color method by spectrophotometer. In order to measure the total nutrients (P, K, Fe, Mn, Cu and Zn), samples were digested following the Hossner method; Fe, Mn, Cu, and Zn were measured by ICP-OES, P was analyzed using a colorimetric method with a spectrophotometer and K was measured using a flame photometer.

2.4. Greenhouse Pot Experiment

A pot experiment was carried out in the greenhouse. The experimental setup was a completely randomized design (CRD). Plastic pots of 3 kg capacity with a diameter of 25 cm were filled with 3 kg finely ground, air-dried (after which it was sieved through a 2-mm mesh) collected soil samples. Amendments (BC and ABC) were mixed with the soil in the pots at rates of 0, 2.5, and 5% (w/w; approximately 0, 55, and 110 t/ha, respectively, to a depth of 15 cm). These were replicated 3 times. All treatments (both BC and ABC at 0, 2.5, and 5% w/w) were fertilized with 0.0, 75, 150, and 300 mg P kg⁻¹ in the form of triple superphosphate (Ca(H₂PO₄)₂·H₂O) solution.

Homogenization of the amendments with soil was ensured. The soil moisture content was sustained at about 80% of field capacity before planting and after planting until the end of the experiment by taking the daily weights of the pots. Ten seeds of alfalfa were sown in each plot. Alfalfa is among the most important fodder crops of Saudi Arabia and is sensitive to P and K availability. A week after plant emergence, plants were thinned to four per pot. The plants were watered on a daily basis with tap water to field capacity by taking the pot weight to know the quantity of water to be added. The growth condition was maintained at 14 h day, 10 h night and a temperature of 27 ± 2 °C. At 60 days after planting, soil samples were collected and prepared for soil chemical analysis. Moreover, plant biomass (shoot) samples were collected for dry matter, P and nutrients measurements.

2.5. Soil Chemical Properties and Nutrients Availability

Soil pH, EC, and soluble ions: pH was measured using a pH-meter, and the EC in the saturated soil paste extract (EC_e) was determined using an EC-meter. Concentrations of soluble cations in saturated paste extract, including Na⁺ and K⁺, were measured [14]. Cation exchange capacity (CEC) was measured according to Sparks [14]. Organic carbon: Soil organic carbon was determined by oxidization using potassium dichromate [17]. Furthermore, soil available P, K, Fe, Mn, and Zn were determined in NH₄HCO₃-DTPA extract according to Soltanpour and Schwab [22]. The concentrations of P were analyzed using the colorimetric method by spectrophotometer. K was measured by flame photometer, while Fe, Mn, Cu, and Zn were measured by ICP-OES.

2.6. Phosphorus Fractionation Analysis

At the end of the greenhouse pot experiment, the soil samples were sequentially extracted for P fractionation using a modified procedure of Hedley et al. [23] as described by Chen et al. [24]. Specifically, 0.5 g of air-dried soil was extracted using 30 mL of 1 mol L⁻¹ NH₄Cl, using a mechanical shaker to shake the mixture for 16 h, and this gave the Pi-NH₄Cl fraction. The residual soil then received 30 mL of 0.5 mol L⁻¹ NaHCO_3, and it was also shaken for 16 h, thereby resulting in the Pi-NaHCO₃ and Po-NaHCO₃ fractions. Furthermore, the remaining soil was extracted with 30 mL of 0.1 mol L⁻¹ NaOH and shaken for 16 h to give the exchangeable and soluble P fractions (Pi-NaOH I and Po-NaOH I). Also, the residue of the extract was further shaken with 30 mL of 1.0 mol L⁻¹ HCl for 16 h to get the Ca-bound P fraction (Pi-HCl). In continuation of the fractionation process, the remaining soil was again shaken with 30 mL of 0.1 mol L⁻¹ NaOH for 16 h to obtain the Pi-NaOH II and Po-NaOH II fractions. Finally, the residual soil was subjected to microwave digestion in nitric acid (HNO₃) and perchloric acid (HCLO₄) to get the P residual fraction. A centrifugation machine was used to separate the soil from the suspension after each extraction by setting the machine to 4000 r min⁻¹. The supernatant was then filtered through Whatman filter paper, which was then stored under a refrigerator before the determination of P by ascorbic acid [25].

2.7. Plant Height, Fresh Weight, and Dry Matter and Nutrients Contents of Plant Shoots

At 40 days after planting, fresh weight, plant height, and dry matter were measured. The shoots and roots of plants were removed from the soil by saturating the soil with water and carefully removing them from the soil. They were washed with distilled water and oven dried at a temperature of 65 °C to a steady weight. Oven-dried plant shoot samples were ground for P and nutrients (K, Fe, Mn, Cu, and Zn) measurements. According to Azcue and Mudroch [26], the dry ashing method was used to digest the plant samples. In order to determine the P concentrations, a spectrophotometer was used. A flame Photometer was used to analyze the K concentrations. Employing ICP-OES, the concentrations of Fe, Mn, Zn, and Cu were examined.

2.8. Statistical Analysis

The collected data were subjected to analysis of variance (ANOVA), and means were compared using the least significant difference (LSD) at a 5% probability level with the aid of the Statistica version 10 software package. Correlation and regression analyses were carried out to identify the relationship between P uptake by plants and characteristics of P availability, sorption, desorption, and quantity intensity.

3. Results and Discussion

3.1. Soil, Biochar and Acidified Biochar Characterization

The experimental soil was characterized for some selected physical and chemical properties (

Table 1. Properties of soil.

	Sand	Silt	Clay	Textural Class	pH (1:2.5)	EC (dS/m)	CEC (meq/100 g)	SOM (g/kg)	CaCO3 (%)	P	Cu	Fe	Mn	Zn
	%									AB-DTPA (mg/kg)
Soil	95.62	1.25	3.13	Sandy soil	8.20	0.15	5.32	1.26	42.50	15.28	0.52	2.38	3.23	nd

nd, not detectable.

). The soil was alkaline (with a pH of 8.20) sandy soil which is high in calcium carbonate content (42.50%). Biochar (BC) and acidified biochar (ABC) were characterized for proximate and ultimate analysis, and this is presented in

Table 2. Properties of Biochar (BC) and Acidified Biochar (ABC).

	Ph (1:10)	EC (dS/m)	CEC (meq/100 g)	Yield	Moisture	Volatile Matter	Ash	Fixed Carbon	Surface Area (m2/g)	Total P (mg/kg)	C	H	N
				%							%
BC	9.83	1.62	41.25	32.81	4.08	32.23	13.77	49.92	58.19	907.41	60.25	1.95	1.11
ABC	2.99	2.14	35.46	-	4.93	28.35	6.52	60.20	303.99	727.88	38.66	2.30	0.61

. From the result of this study, it was found that the pH of BC drastically reduced from 9.83 to 2.99 following acidification to produce ABC. There was little difference in the EC value. It increased from 1.62 to 2.14 dS m⁻¹. No significant difference was observed in the volatile matter and moisture contents, whereas, while ash content of ABC was approximately half of that of BC. The reduction in ash content following acidification could be a result of the washing away of the ash content during the process of acidification. Acidification of BC resulted in higher fixed carbon, which was 60.20%, while BC was 49.92%. Surface area increased multifold following acidification as it was 108.19 m² g⁻¹ in BC and 303.99 m² g⁻¹ in ABC.

The surface morphology of both BC and ABC was observed. Images produced from SEM are presented in Figure 1a,b. The images showed an abundance of both micro and macro pores on the surfaces of the materials. The abundance of pores present in these materials could be a result of the loss of volatile and organic compounds during pyrolysis [27]. Critically looking at the SEM images, there is little or no difference in the porosity of the biochar following acidification. Furthermore, the X-ray diffraction (XRD) pattern of BC and ABC is shown in Figure 2. Two different peaks of sulfite and quartz were seen in ABC while they were not present in the BC. This could be a result of the acidification with sulfuric acid.

3.2. Soil Chemical Properties

3.2.1. Effect of Biochar (BC) and Acidified Biochar (ABC) on the pH and EC

Figure 3 shows the influence of acidified and non-acidified Conocarpus waste biochar on the pH of soil grown with alfalfa following the application of P in the form of a triple superphosphate (Ca(H₂PO₄)₂·H₂O) solution at different rates (0, 75, 150, and 300 ppm P). At 0 ppm P application rate, the addition of BC5 and BC2.5 significantly increased the soil pH above the CK with a value of 8.47 and 8.36, respectively, while the CK had a pH of 8.12. On the other hand, the pH of soil treated with acidified biochar (applied at 2.5% and 5%) with different P application rates was significantly (p < 0.05) decreased when compared to the control. A more profound decrease was observed in soil treated with acidified biochar at a higher application rate (ABC5) which was even significantly lower than the pH of soil treated with ABC2.5. The soil pH of soil treated with ABC2.5 decreased by a unit of 0.28 while that of ABC5 decreased by a unit of 0.42 when compared with the control (CK). It could be deduced that the higher the application rate, the more the effect of the treated soil pH.

At the other P application rates (75, 150, and 300 ppm), the resulting changes in soil pH following the application of non-acidified and acidified biochars were also similar to that of 0 ppm P application rate though the increment or decrement was not significant in some cases. The increase in soil pH following the addition of non-acidified biochar could be a result of the alkalinity nature of biochar. Biochar is characterized by high pH, as shown in Table 2. The increase in soil pH is a result of the presence of a carboxylic functional group in biochar and oxidation of the biochar used for amendment as biochar has been reported severally to increase soil pH following its addition to soil [28,29]. The results of this study are similar to the result reported by Hartley et al. [30], where there was an increase in the pH of soils in which biochar produced from woody materials was added when compared to the untreated soil. Oo et al. [31] also recorded an increase in the pH of biochar-amended soil following a 71-day incubation period. In this study, soil pH increases with the increasing rate of BC application. This result is similar to that of Oladele et al. [32], where soil pH increased with biochar application dose in rain-fed rice fields amended with rice husk biochar. Likewise, biochar produced from Conocarpus was found to increase soil pH at all application rates (1%, 3%, and 5%), where a profound increase was reported at the highest application rate (5%), which increased by 0.16–0.17 unit [33]. The decrease in pH of the ABC-treated soil could be a result of the decrease in the pH of ABC following acidification, decreasing from 9.83 to 2.99 for the acidification of BC produce ABC (Table 2). Similarly, in our previous research, acidified biochar was found to reduce soil pH following its addition to alkaline soil in an incubation experiment [34].

In this study, it was observed that the soil pH decreases with increasing rate of P application under soil treated with non-acidified biochar and the control while treatment ABC2.5 and ABC5 does not follow a regular pattern. The decrease in soil pH following P application could be a result of the acidic groups (H₂PO₄) in the fertilizer (Ca(H₂PO₄)₂·H₂O) applied, which increased when more fertilizer was applied and thus had more effect on soil pH.

The impact of non-acidified and acidified Conocarpus waste biochar on soil EC growth with alfalfa with different applications of P fertilizer is shown in Figure 4. For treatment under 0 ppm P application rate, the soil EC is in the order ABC5 > BC5 > ABC2.5 > BC2.5 > CK with values of 2.1, 1.97, 1.80, 1.54, and 1.08 dS m⁻¹, respectively. Soil amended with ABC5 had a soil EC which was significantly (p < 0.05) higher than all other treatments, including the control, with the exemption of soil treated with BC5. Likewise, all the treatments showed significantly (p < 0.05) higher EC as compared to CK. Similar trends were followed at all other P application rates (75, 150 and 300 ppm P), where soils treated with ABC5 had the highest soil EC value and the CK had the lowest. The values range from 0.97–2.17, 0.96–2.13, and 0.97–1.89 dS m⁻¹ for 75, 150, and 300 ppm P application rates, respectively. Generally, it could be deduced from the study that soil EC increased with increasing biochar application rates irrespective of the biochar being acidified or non-acidified. This is a similar outcome to the result reported by Al-Wabel et al. [33], where there was an increase in soil EC following the addition of biochar. Usman et al. [35] also recorded an increase in soil EC following biochar application, and this increased with increasing biochar application though not significant in most cases. This could be a result of the accretion of soluble salts present in ashes [35]. From this study, it was observed that P application, irrespective of the rate, had little or no effect on soil EC.

3.2.2. Effect of Acidified and Non-Acidified Biochar on Soil Available P

Table 3. Impact of BC and ABC applied at different rates with different P fertilizer application rates on AB-DTPA extractable P of soil (mg kg⁻¹).

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	10.505 c	45.741 c	68.104 bc	89.904 b	8.324
BC2.5	16.707 b	69.514 a	99.018 a	86.333 b	11.717
BC5	37.848 a	57.956 b	65.661 bc	106.065 a	11.800
ABC2.5	8.062 c	51.097 bc	61.997 c	88.306 b	10.831
ABC5	8.344 c	50.533 bc	70.547 b	88.964 b	5.490
LSD	6.902	9.846	8.907	11.887
p-value	0.0000	0.0003	0.0000	0.0139
F-value	49.7413	14.9922	41.8533	5.4199

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

shows the AB-DTPA soil available P as affected by the addition of non-acidified and acidified biochar with different rates of P application grown with alfalfa. Under a 0 ppm P application rate, soil treated with BC5 had the highest available P with a value of 37.848 g kg⁻¹ when compared to other treatments under the same P application rate. Soil available P follows the order BC5 > BC2.5 > CK > ABC5 > ABC2.5 with values of 37.848, 16.707, 10.505, 8.344, and 8.062 g kg^−1, respectively. Despite the differences in the treatments notwithstanding, some treatments were not significantly (p < 0.05) different from the others. For example, CK, ABC2.5, and ABC5 were not significantly (p < 0.05) different from one another, while the available P of soil treated with BC2.5 was significantly higher than that of CK, ABC2.5, and ABC5. In the case of treatments under 75 ppm P application rate, soil treated with BC2.5 had the highest soil available P, which was significantly higher than all other treatments under the same P application rate. For soil available P under the influence of 150 ppm P application rate, P availability following the order BC2.5 > ABC5 > CK > BC5 > ABC2.5 with values of 99.018, 70.547, 68.104, 65.661, and 61.997 g kg⁻¹ respectively. This result was similar to that of the 75 ppm P application rate, where soil amended with BC2.5 exhibited the highest soil available P, which is also significant to all other treatments. Consequently, under the 300 ppm P application rate, the soil available P ranges from 86.333 to 106.065 g kg^−1, where the highest value was found in the soil treated with BC5, which is significantly higher than all other treatments. The other treatments (BC2.5, ABC2.5, ABC5, and CK) had similar values with no significant differences (p < 0.05). In general, soil treated with non-acidified biochar (BC), irrespective of application rates, had a profound impact in enhancing P availability in this study. The lower value of plant-available P in soil treated with acidified biochar when compared to that of ordinary biochar could be a result of the decrease in soil pH following acidified biochar addition. The reduction in soil pH could increase the solubility of CaCO_3, thereby releasing Ca ions into the soil, which could result in the bounding of Ca with P, thus, making P less available in the soil for plant uptake. The alteration in soil pH plays a significant role in the availability of nutrients (P, Ca²⁺, Mg²⁺ and K⁺) for plant uptake, the activity of microorganisms, and the mineralization of organic matter [36]. Generally, many researchers have reported an increase in plant-available P following biochar application [37,38,39]. Oladele et al. [32] reported an increase in available P after the addition of biochar produced from rice husk, and the increasing rate of biochar resulted in increasing available P. Also, in an experiment conducted to look into the influence of green wastes on the production of radish dry matter, biochar was observed to increase the concentration of P [40]. In another research by Sika [41], who investigated the impact of locally-produced biochar on the soil nutrients of sandy soil from the Western Cape in which the experimental soil was characterized to be of low nutrients, the ammonium bicarbonate-DTPA plant available P was observed to increase following biochar application. In addition to the results of past studies that are in agreement with the current study, there was a significant increase in plant available P following the addition of biochar to soils in an incubation experiment [42]. Likewise, some other researchers reported an increase in plant available P in biochar-amended soil [43,44,45,46]. The substantial increase in the contents of plant-available P in soil amended with biochar could be attributed to various mechanisms. Firstly, the biochar employed in this study contains a considerably higher amount of labile P, and this could directly release phosphate into the soil [32]. This is also confirmed by Xu et al. [47], who observed an increase in soil-available P following the addition of biochar produced from rice husk, suggesting that it could be a source of P bioavailability, where phosphate is being directly released to the aqueous soil solution. It was hypothesized by DeLuca et al. [8] that the thermal pyrolysis of organic biomass at a particular range of temperatures can significantly improve soil available P by C volatilization and the cleavage of organic P bonds resulting in soluble P salts residue allied with the products of the pyrolysis. Also, acidic and alkali metals (Fe³⁺, Al³⁺ and Ca²⁺) could be adsorbed directly by the surface of the biochar; this reduces the activities of these cations in solution, thereby enhancing P activity by causing a delay in the adsorption and precipitation of P in soil [47]. On the other hand, other researchers showed that there was no significant effect on soil-available P following the amendment of calcareous soil by biochar [48]. This could be due to some other soil properties like soil texture and the degree of CaCO₃ in the soil, among others. For example, biochar was found to reduce P leaching in sandy soil; hence, its addition to sandy soil is projected to enhance soil-available P [46].

Furthermore, within each treatment, the phosphorus fertilizer application rate influenced the plant-available P. Available P increases with increasing P application rates for all the treatments except BC2.5, where the application of 150 ppm P was found to be greater than 300 ppm P. The increasing plant available P with increasing P application rate is expected since the mineral P applied is the major source of soil available P.

3.2.3. Impact of Acidified and Non-Acidified Biochar on Soil Extractable K and Na

Table 4. Influence of BC and ABC applied at different rates with different P fertilizer application rates on soil Extractable K in mg/kg.

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	118.33 c	112.00 c	103.00 c	102.83 c	18.16
BC2.5	547.33 b	711.67 b	711.67 b	725.00 b	210.61
BC5	1247.50 a	1163.33 a	1231.67 a	1470.83 a	376.59
ABC2.5	75.50 c	78.83 c	86.83 c	83.17 c	21.56
ABC5	103.83 c	110.17 c	98.00 c	106.67 c	16.09
LSD	159.67	70.14	147.33	294.13
p-value	0.0000	0.0000	0.0000	0.0000
F-value	149.2737	722.3653	183.1290	64.3887

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

shows the impact of biochar and acidified biochar on extractable soil K under alfalfa cultivation. Soil treated with BC5 under 0 ppm P application rate had the highest extractable K, which is approximately 11 times folds greater than the control, and it was as well significantly higher than all other treatments (p < 0.05). Soil extractable K of BC5-treated soil maintained the highest value for all the P application rates. These values ranged from 1163.33 mg/kg to 1470.83 mg/kg. The soil extractable K is in the order BC5 > BC2.5 > CK > ABC5 > ABC2.5. The soil extractable K followed a similar trend throughout the experiment in order of its increment except for the treatments under 300 ppm P application rate, where ABC5 treated soil had a greater extractable K than the CK even though it was not significant (p < 0.05). On the other hand, P application rates had little or no effect on soil extractable K, indicating that there is no correlation between soil extractable K and soil P. Similar to the findings of this current study, Oladele et al. [32] also reported increased soil available K following biochar application which increased with increasing rate of biochar application. Also, Inal et al. [42] incorporated poultry manure and biochar into soils in an incubation study; exchangeable K was found to be increased significantly (p < 0.05) in the amended soil when compared to the control. Similarly, the addition of biochar produced from wood at an application rate of 10–20 t ha⁻¹ increased exchangeable soil K on soils classified as sandy and clay-textured soils [49,50]. Furthermore, there was a significant increase in the soil-available K of biochar-amended soil in research conducted by El-Naggar et al. [19]. Similarly, Sika [41] reported an increase in exchangeable basic cations, where K and Ca were found to be predominantly increased following biochar addition to soil. This result was also confirmed by Tryon [51], who found that mixing sand with hardwood charcoal increased the exchangeable bases and the effective cation exchange capacity (ECEC). Likewise, water-extractable cations, especially Na and K, were found to be increased in biochar-amended [41]. This could probably be a result of the fact that Na and K carbonates and salts are more soluble in water when compared to the salts and carbonates of Mg and Ca [52]. Also, Chan et al. [40] reported an increase in K concentration following biochar addition to soil for the production of radish dry matter though the increase was only significant at higher biochar application rates (greater than 50 t ha⁻¹) without N fertilizer application. The authors concluded that the increase was a result of the high exchangeable K concentration of the applied biochar. Likewise, the increase in the soil available K following biochar addition in this study could be attributed to the high K content in the Conocarpus biochar employed in this study. More so, the biochar had a relatively high amount of bioavailable K. In addition, results of research conducted by Yao et al. [53] showed that there was an increase in exchangeable K⁺ following biochar addition to a Typic Plinthudult soil when compared to the untreated soil; the authors suggested that this increase could be as a result of a kind of force known as the electrostatic force of attraction present in the matrix of soil-biochar mixture which aids K⁺ retention. Meanwhile, a number of studies reported enhanced or no influence of biochar addition on the availability of soil K [36,48,54], which is basically a result of different feedstocks employed in biochar production. Knowing that biochar is made from organic materials, fundamentally, the nutrients element present in the ash (or mineral) fractions are the inherent biochar nutrients. Therefore, amending soil with biochar supplies free exchangeable bases like Ca, K, and mg to occupy the exchange sites for soil, thereby leading to increasing soil pH and readily releasing nutrients for plant uptake [55].

The influence of biochar and acidified biochar on extractable Na of soil grown with alfalfa is shown in

Table 5. Effects of BC and ABC applied at different rates with different P fertilizer application rates on soil extractable Na in mg/kg.

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	227.00 b	222.17 c	246.50 c	224.67 c	56.43
BC2.5	461.17 a	327.83 b	357.00 b	289.83 b	30.70
BC5	440.67 a	441.00 a	408.17 a	405.67 a	92.52
ABC2.5	217.33 b	211.83 c	216.83 d	183.17 cd	34.95
ABC5	164.67 c	165.67 d	160.67 e	136.67 d	30.49
LSD	51.50	51.15	32.75	68.16
p-value	0.0512	0.0000	0.0000	0.0000
F-value	0.6321	70.6283	146.3657	35.4616

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

. The result obtained is similar to that of extractable K, though with few differences. Soil treated with BC5 had the highest extractable Na for all the P application rates with the exemption of the 0 ppm P application rate, where BC2.5-treated soil had the highest value though it was not significantly different from the BC5 treatment. The values for the extractable Na range from 164.67–461.17, 165.67–441.00, 160.67–408.17, and 136.67–405.67 mg kg⁻¹ for 0, 75, 150, and 300 ppm P application rates, respectively. For treatments under 0 ppm P, the extractable Na was in the order BC2.5 > BC5 > CK > ABC2.5 > ABC5, while the other P application rates (75, 150, and 30 ppm P) are in the order BC5 > BC2.5 > CK > ABC2.5 > ABC5. Similar to the result obtained for extractable soil K, P application had little or no effect on extractable soil Na. Similar to the result of this study, Saletnik et al. [56] studied the effect of biochar and ash produced from plant biomass on the yield of giant miscanthus and soil properties; it was observed that biochar and ash increased the Na concentration of soil. In addition, Xu et al. [57] presented that biochar is a sink of soluble cations like Na⁺, Mg²⁺, and Ca²⁺. High concentrations of these ions compete with K⁺ on the adsorption site of soil.

Biochar-amended soil having higher extractable cations in comparison with acidified biochar-amended soil could be attributed to the lower ash content of acidified biochar. This can be related to the research of Saletnik et al. [56], where the nutrient concentration of ash was greater than that of biochar in manifolds.

3.2.4. Soil Cation Exchangeable Capacity (CEC) as Affected by BC and ABC

The results for the influence of BC and ABC applied at different rates with different application rates of P fertilizer on CEC on soil cultivated with alfalfa are presented in

Table 6. Effects of BC and ABC applied at different rates with different P fertilizer application rates on soil CEC in meq/100 g soil.

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	5.246 c	4.681 c	4.894 c	5.372 c	0.774
BC2.5	6.280 b	6.976 ab	6.609 b	6.300 bc	0.590
BC5	7.285 a	7.681 a	7.662 a	7.913 a	0.590
ABC2.5	6.768 ab	6.502 b	6.894 b	6.560 b	1.529
ABC5	7.382 a	7.522 a	7.005 b	6.460 b	1.136
LSD	0.820	1.077	0.767	1.327
p-value	0.0002	0.0002	0.0000	0.0057
F-value	17.0671	18.9271	27.4386	7.0798

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

. Under the 0 ppm P application rate, soil treated with ABC5 had the highest CEC though it was not significantly different from ABC2.5 and BC5. The CEC of BC5 treated soil was 40.7%, 17.5%, 1.3%, 9.0% greater than CK, BC2.5, BC5, and ABC2.5, respectively. Meanwhile, under 75 ppm P application rate, BC5 treated soil had the highest CEC, which is significantly different from CK and ABC2.5 but not significantly different (p < 0.05) from BC2.5 and ABC2.5. Under this P application rate, the CEC values are in the order BC5 > ABC5 > BC2.5 > ABC2.5 > CK with values of 7.681, 7.522, 6.976, 6.502 and 4.681 meq/100 g soil, respectively. Similarly, BC5-treated soil exhibited the highest CEC among the treatments under 150 ppm P application rate. Also, under the 300 ppm P application rate, all treated soil had CEC values which were all significantly greater than the control (p < 0.05) with the exemption of BC2.5 treated soil. Application of biochar to soil has also been reported to strongly enhance soil nutrient availability, resulting in an increase in the pH and CEC of soil, which also improves soil physical properties and decreases the amount of soluble Al ions in the soil [54,58,59,60,61]. Similarly, Chan et al. [40] reported a significant increase in CEC at a higher biochar application rate (50 t ha⁻¹) when the influence of green waste biochar on the soil chemical properties of a highly acidic soil characterized with low organic matter content was investigated. Also, Yuan and Xu [62] reported an increase in CEC following the addition of biochar produced from 10 different crop residues to soil. Likewise, in an investigation of biochar produced from corn stover on the chemical properties of soil by Chintala et al. [63], CEC was reported to increase above the control. Similarly, El-Sharkawy et al. [64] reported an increase in CEC over the control and pristine biochar following the application of acidified cotton stalk biochar. In this current study, mostly, ABC-treated soil had lower CEC when compared with BC-treated soil, and this could be a result of the lower ash content of ABC since ash has been reported to contain more nutrients than biochar [56]. However, both ABC and BC can be used to improve the CEC of soil for better plant growth and development.

3.2.5. Influence of Acidified and Non-Acidified Biochar on Organic Matter (OM) of Soil

Organic matter, which is a function of organic carbon, is one of the most important soil contents that have to be sustained and managed in the soil for adequate functioning of the soil ecosystem, and its various soil organic carbon fractions are functions of the agro-technical management practices [65,66].

Table 7. Effects of BC and ABC applied at different rates with different P fertilizer application rates on soil OM in g kg⁻¹.

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	1.554 c	1.566 c	1.350 c	1.354 c	0.597
BC2.5	2.436 b	3.453 b	3.871 bc	3.627 b	0.843
BC5	6.146 a	6.750 a	7.948 a	7.539 a	2.761
ABC2.5	2.743 b	2.791 bc	2.843 c	3.093 bc	1.891
ABC5	5.240 a	6.688 a	5.172 b	6.246 a	3.303
LSD	1.121	2.617	1.842	2.379
p-value	0.0000	0.0007	0.0000	0.0002
F-value	46.2674	12.1833	27.8049	16.5331

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

shows the impact of acidified and non-acidified biochar on soil organic matter content. From the results of this study, BC5-treated soil had the highest soil OM at all levels of P application though not significantly different from some treatments in some cases. Under the 0 ppm P application rate, soil OM of BC5 treated soil was 3.95-, 5.52-, 2.24-, and 1.17-fold greater than CK, BC2.5, ABC2.5, and ABC5, respectively. For treatments under the influence of 75 ppm P application rate, BC5 treated soil was greater than CK, BC2.5, ABC2.5, and ABC5 by 4.31-, 1.95-, 2.42-, and 1.01-fold, respectively. Furthermore, under the 150 ppm P application rate, BC5-treated soil showed a greater soil OM than CK, BC2.5, ABC2.5, and ABC5 by 5.89-, 2.05-, 3.20-, and 1.54-fold, respectively. This increase in soil OM following the addition of BC5 is significant to all other treatments (p < 0.05). For the soil under the influence of 300 ppm P application rate, soil OM of BC5 treated soil were greater than CK, BC2.5, ABC2.5, and ABC5 by 5.57-, 2.08-, 2.44-, and 1.21-fold, respectively. These outcomes are consistent with those reported by [37,38,67]. Similar to the results of the current study, Chan et al. [40] reported a significant increase in organic carbon at a higher biochar application rate (50 t ha⁻¹) when the influence of green waste biochar on the soil chemical properties of a highly acidic soil with low content of OM was investigated. Similarly, in a previous study by Usman et al. [35], soil OM significantly (p < 0.05) increased following the application of plant waste biochar. Also, the addition of biochar at application rates of 20 and 40 t ha⁻¹ in combination with the addition of nitrogen-based fertilizer to a calcareous soil with low levels of organic C increased the SOC by 25% and 42.2%, respectively, when compared to the untreated soil [68]. Likewise, Cui et al. [69] reported a significant increase in SOM over the control when biochar produced from wheat straw was employed as an amendment in a 5-year experiment. The increase in soil organic matter following biochar addition could be a result of the high content of C in biochar which will directly add to the SOC. Based on C stability, soil OM can be improved by biochar by the decomposition of biochar by soil microorganisms or by the preservation of the OM that exists naturally in the soil. The high carbon content of biochar and the protection of native organic carbon from exploitation may be the causes of the increased SOC in soil modified with biochar [70]. Al-Wabel et al. [71] found that the biochar’s stable pool of fixed carbon rose while its unstable organic carbon decreased.

3.2.6. Impact of Acidified and Non-Acidified Biochar on Plant Available Micronutrients (Cu, Fe, Mn, and Zn)

Micronutrients are of great importance to the growth and development of crops; hence there is a need for their sustenance and enhancement soil to achieve good maximum crop growths and yields. Calcareous soils are known to be challenged with limiting the amount of micronutrients due to their fixation at high soil pH. The effects of the acidified and non-acidified biochar on Cu availability in a calcareous soil cultivated with alfalfa under the influence of different doses of P-fertilizer at different rates were presented in Figure 5. The results showed that, for all the P-fertilizer application rates, ABC5-treated soil had the highest soil available Cu though the differences were not significant (p < 0.05) in a few cases. Under O ppm P, available Cu ranged from 0.493–0.663 mg kg⁻¹ where the treatments were in the order ABC5 > BC5 > BC2.5 > CK > ABC2.5 with values 0.663, 0.610, 0.554, 0.509, and 0.493 mg kg⁻¹, respectively. In this case, the available Cu of ABC5-treated soil was significantly higher than all other treatments (p < 0.05). For the treatments under 75 ppm P application rate, the values of soil available Cu ranged from 0.731–0.906 mg kg⁻¹ in which the treatments are in the order ABC5 > BC2.5 > BC5 > ABC2.5 > CK with values of 0.906, 0.845, 0.791, 0.775, and 0.731 mg kg⁻¹, respectively. Furthermore, treatments under 150 ppm P application rates had available Cu values ranging from 0.503–0.643 mg kg⁻¹ where the treatments were in the order ABC5 > ABC2.5 > BC5 > BC2.5 > CK with values of 0.643, 0.577, 0.575, 0.509, and 0.503 mg kg⁻¹ respectively. Considering the effects of the different levels of P applied on the available Cu, there was no definite trend on Cu availability, and it showed little or no effect on its availability. From this current study, it could be deduced that there is no correlation between P availability and available Cu. Also, as reported by El-Naggar [19], biochar treatment significantly increased (p < 0.05) AB-DTPA-extractable Cu.

Figure 6 presented the influence of the applied treatments at different application rates on the availability of Fe in soils grown with alfalfa where different doses of P-fertilizer were applied. From the results of this study, there was no definite trend in Cu availability with respect to the treatments at different levels of P application. Under 0 ppm P dose of P-fertilization, soil amended with BC5 had the highest soil available Fe, which is significantly higher than all other treatments in this same P application dose (p < 0.05). Soil treated with BC5 was 27.9%, 16.4%, 25.5%, and 14.4% higher than the CK, BC2.5, ABC2.5, and ABC5 treatments, respectively. All the treatments in this category were significantly different from one another (p < 0.05). In the case of the treatments where 75 ppm P was applied in the form of P-fertilizer, ABC5-treated soil exhibited the highest value of soil available Fe with a value of 2.915 mg kg⁻¹. Soil amended with ABC5 had percentage increases of 23.9%, 8.8%, 7.0%, and 17.8% above CK, BC2.5, BC5, and ABC5, respectively. Furthermore, in treatments under the application of P-fertilizer at an application rate of 150 ppm, BC5 treated soil had the highest soil available Fe with a value of 2.728 mg kg⁻¹ while other treatments are in the order BC2.5 > ABC5 > ABC2.5 > CK having values of 2.700, 2.650, 2.493 and 2.465 mg kg⁻¹, respectively. Though there were differences in the soil available Fe of these treatments but these differences were not significant (p < 0.05). For the results of soil available Fe of treatments when 300 ppm P in the form of P-fertilizer was added, ABC5 treated soil had the highest amount of soil available Fe though not significantly higher than all the other four treatments (p < 0.05). Soil treated with ABC5 had percentage increases of 12.4%, 20.2%, 1.0%, and 5.9% over the CK, BC2.5, BC5, and ABC2.5, respectively. From the results of the current study, considering the relationship of P-fertilizer dose on soil available Fe, there was no specific trend in the relationship. The results showed that the application of P-fertilizer had little or no effect on the Fe availability of the studied soil. Similarly, in our previous studies, acidified biochar enhances micronutrient availability over the control after being added to alkaline sandy soil [34]. However, in previous studies, biochar reduced micronutrient availability with the exception of Cu [19]. This could be a result of the affinity of biochar to the sorption of trace elements, as explained by Kloss et al. [72].

There is variation in soil-available Mn, as affected by the addition of acidified and non-acidified biochar is soil cultivated with alfalfa where P-fertilizer was applied at different rates (Figure 7). The results of this study showed that there were mixed effects of the amendments on Mn availability at different P application rates. That is, there is no fixed trend in the order of increase or decrease in soil-available Mn with respect to the treatments used. In the case of the treatments under 0 ppm P application rate, BC5 treated soil had the highest amount of soil available Mn with percentage increases of 128.6%, 29.6%, 18.7%, and 2.9% over the CK, BC2.5, ABC2.5, and ABC5, respectively. Furthermore, under 75 ppm P application rate, ABC5 treated soil had the highest value of soil available Mn, which is significantly higher than all other treatments (p < 0.05). Soil treated with ABC5 had percentage increases of 137.6%, 31.7%, 22.6%, and 35.6% over CK, BC2.5, BC5, and ABC2.5, respectively. In the case of the treatments under 150 ppm P application rate, the amount of available Mn in the treated soil ranged from 4.199–7.268 mg kg^−1, where the CK had the minimum value. The treatments are in the order ABC2.5 > ABC5 > BC5 > BC2.5 > CK with values of 7.268, 6.979, 6.825, 6.479, and 4.199 mg kg⁻¹, respectively. In the case of the treatments under 300 ppm P application rate, ABC5 treated soil had the highest amount of soil available Mn, just like that of treatments under 150 ppm P with percentage increases of 93.4%, 21.1%, 0.9%, and 10.4% over the CK, BC2.5, BC5, and ABC5, respectively. However, there were significant differences among these other three treatments (p < 0.05). From the results of this study, considering Mn availability with respect to increasing P application rate, there was no definite trend in the availability of Mn. This indicates that available Mn is lowly or not affected by P availability. On the contrary, El-Naggar [19] reported a decrease in AB-DTPA extractable Mn when compared to the control and this was referenced to be probably due to the affinity of biochar to micronutrients sorption as described by Kloss et al. [72]. In a 67-day trial, biochar produced from pecan shells was found to increase Mn concentration in the soil. Due to its strong interaction with a variety of organic and inorganic forms in biomass, this proves that Mn was extensively maintained throughout biochar production [73].

From the results of this study, soil available Zn was under the detection limit of the machine used for the analysis (Perkin Elmer Optima 4300 DV ICP-OES). This indicates that the soil available Zn was very minimal in the treated soil and was not significantly enhanced by the applied treatments.

3.3. Plant Nutrients Uptake and Concentration

3.3.1. Influence of Acidified and Non-Acidified Biochar on Plant P Uptake in Shoot of Alfalfa Planted

Figure 8 shows the variation in the uptake of P by plant shoot of alfalfa as affected by the application of amendments. Under 0 ppm P fertilizer application rate, the shoots of plants treated with BC2.5 had the highest mean value of plant P uptake with a value of 601.69 µg/plant. Plant shoot P uptake was in the order BC2.5 > ABC2.5 > ABC5 > BC5 > CK. Also, BC2.5 treatment had the highest uptake of P in the shoot of the plant under 75 ppm P fertilizer application rate. The treatments followed the same order as in that of 0 ppm P fertilizer rate, and this trend was consistent for the other two P fertilizer application rates (150 and 300 ppm), except in the case of treatments under 300 ppm P fertilizer application rate were the highest mean value of shoot uptake of P was found in treatment ABC2.5 which is however not significantly different from treatment BC2.5. For that of treatments under 300 ppm P fertilizer rate, treatment ABC5 was not significantly different (p < 0.05) from treatment BC2.5 but significantly higher than all other treatments (BC5, ABC5 and CK). The amendment BC2.5 did not differ substantially (p 0.05) from the other treatments either.

Biochar treatments resulting in the higher uptake of P by plant shoot all through the different P fertilizer application rates could be a result of the higher P availability in soils treated with biochar, especially BC2.5 treatment. Biochar aids the uptake of plant nutrients by serving as a direct source of plant nutrients and also results in changes in some soil physical and chemical properties [74].

3.3.2. Effects of Acidified and Non-Acidified Biochar on Plant P Concentration

Table 8. Effects of BC and ABC applied at different rates with different P fertilizer application rates on plant P concentration (g kg⁻¹).

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	1.30 ab	1.39 a	1.51 a	1.44 ab	0.53
BC2.5	1.17 b	1.47 a	1.63 a	1.71 a	0.20
BC5	1.44 a	1.48 a	1.51 a	1.48 ab	0.11
ABC2.5	0.93 c	1.01 b	0.90 c	1.17 b	0.20
ABC5	1.12 bc	1.43 a	1.22 b	1.35 b	0.14
LSD	0.26	0.31	0.24	0.37
p-value	0.0032	0.0094	0.0000	0.0292
F-value	8.3242	6.1041	23.0720	4.2329

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

presents the impact of biochar and its acidified biochar derivative on plant P concentration measured in g kg⁻¹. Under 0 ppm P fertilizer application rate, treatment BC5 had the highest plant P concentration though not significantly higher (p < 0.05) than the control but significantly higher than all other treatments in this category. The control is as well significantly higher (p < 0.05) than the other treatments except for BC2.5, which are not significantly different (p < 0.05) from each other. In terms of the plant P concentration, the treatments are in the order BC5 > CK > BC2.5 > ABC5 > ABC2.5 with values of 1.44, 1.30, 1.17, 1.12, and 0.93 g kg⁻¹, respectively. Also, for the treatments under 75 ppm P fertilizer application rate, treatment BC5 had the highest plant P concentration though not significantly different (p < 0.05) from all other treatments except treatment ABC 2.5, which had the lowest value of plant P concentration. In order of decreasing value of plant P concentration under this fertilizer rate, BC5 > BC2.5 > ABC5 > CK > ABC2.5 with values of 1.48, 1.47, 1.43, 1.39, and 1.01 g kg⁻¹, respectively. For that of treatments under 150 ppm P fertilizer application rate, treatment BC2.5 had the highest plant P concentration though not significantly different (p < 0.05) from treatments BC5 and CK but significantly higher than treatments ABC2.5 and ABC5. In this category, in order of decreasing value of plant P concentration, BC2.5 > CK > BC5 > ABC5 > ABC2.5 with values of 1.63, 1.51, 1.51, 1.22, and 0.90 g kg⁻¹, respectively. Furthermore, treatments under 300 ppm P fertilizer application rate is similar to that of the previous category, i.e., treatments under 150 ppm P application rate. Here as well, treatment BC2.5 had the maximum plant P concentration with a value of 1.71 g kg^−1, while treatment ABC2.5 had the minimum concentration with a value of 1.17 g kg⁻¹. Despite the higher value of treatment BC2.5, it was not significantly different (p < 0.05) from treatment BC5 and CK, while the CK was also not significantly different from treatments ABC2.5 and ABC5. Concerning plant P concentration as affected by P application rate, there is no definite trend; however, plant P concentration tends to increase with increasing rate of P fertilizer.

In summary, biochar treatments both at higher and lower application rates exhibited the most significant influence on plant P concentration. The higher plant P concentration in plants treated with biochar could be a result of the ability of biochar to serve as a direct source of plant nutrients and supply directly to the plants for their uptake [74].

3.3.3. Uptake of K by Plant Shoots as Affected by the Amendments

Figure 9 shows the plant potassium uptake as affected by the amendments under different P fertilizer rates, which is measured in mg plant⁻¹. Under 0 ppm P fertilizer rate, ABC2.5 had the maximum K uptake by the shoots of alfalfa with a mean value of 6.77 mg plant⁻¹, while the minimum K uptake was observed in CK with a mean value of 6.21 mg plant⁻¹. In terms of their plant K uptake, the treatments are in the order ABC2.5 > ABC5 > BC5 > BC2.5 > CK. Meanwhile, for treatments under 75 ppm P fertilizer application rate, the mean values ranged from 4.52 to 14.27 mg plant⁻¹, where the maximum value was obtained by treatment BC2.5, and CK had the minimum value. Though the treatment BC2.5 had the highest mean value, it was not significantly higher than treatment ABC2.5, making this result similar to that of treatments under 0 ppm P fertilizer rate where treatment ABC2.5 had the highest plant K uptake. Similarly, under 150 ppm P fertilizer application rate, the maximum mean value of K uptake by plant shoots was observed in treatment ABC2.5 though the mean value was not significantly higher (p < 0.05) than in treatments BC2.5 and BC5. The mean values of the treatments ranged from 6.99 to 10.92 mg plant⁻¹. Considering the level of significant difference among the treatments mean, treatment BC5 was not significantly higher than (p < 0.05) the CK but significantly higher than ABC5. Likewise, treatment CK was significantly higher (p < 0.05) than treatment ABC5. Under the highest P fertilizer application rate, treatment ABC2.5 also had the highest mean value, while treatment ABC5 had the minimum mean value with values of 17.53 and 5.86 mg plant⁻¹, respectively. The treatments were in the order ABC2.5 > CK > BC2.5 > BC5 > ABC5 with mean values of 17.53, 11.27, 9.79, 9.02, and 5.86 mg plant⁻¹. Considering the impact of the P fertilizer rate on K uptake by plant shoots, there was a mixed result. Generally, it could be deduced that there is no correlation between P availability and K uptake by plant shoots. Similarly, in a previous study, biochar applied at higher rates was found to significantly enhance (p < 0.05) plant K uptake under non-saline irrigation [35]. Potassium (K) uptake has been demonstrated to increase when biochars, which are typically high in both total and accessible K, are used [28,40].

3.3.4. Plant K Concentration as Affected by the Amendments

Plant K concentration, as influenced by the treatments, is presented in

Table 9. Effects of BC and ABC applied at different rates with different P fertilizer application rates on Plant K concentration (g/kg).

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	10.80 c	11.07 b	18.22 a	18.30 a	2.70
BC2.5	10.43 c	12.67 b	12.73 b	15.28 a	2.24
BC5	16.92 a	21.37 a	18.59 a	16.77 a	3.25
ABC2.5	12.11 c	11.67 b	9.46 c	15.71 a	10.04
ABC5	14.00 b	12.89 b	12.78 b	10.37 a	4.43
LSD	2.16	4.43	3.05	8.51
p-value	0.0001	0.0005	0.0000	0.1957
F-value	22.8927	13.6901	25.0178	1.8517

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

. The mean values of plant K concentration of treatments under 0 ppm P fertilizer application rates range from 12.11 to 16.92 g kg^−1, where treatment BC5 had the maximum value and treatment ABC2.5 had the minimum. Treatment BC5 having the maximum value, was significantly higher (p < 0.05) than all other treatments. Furthermore, considering the treatments under 75 ppm P fertilizer application rates, treatment BC5 also had the highest plant K concentration among the treatments. The mean values of plant K concentration range from 11.07 to 21.37 g kg⁻¹. Treatment BC5, having the highest plant K concentration, was also significantly higher (p < 0.05) than all other treatments, where there were no significant differences (p < 0.05) among other treatments. Similarly, observing the plant K concentration of nutrients under 150 ppm P fertilizer rate, treatment BC5 also had the highest value, followed by CK, and then ABC5 and then BC2.5 and followed by ABC2.5 with values of 18.59, 18.22, 12.78, 12.73, and 9.46 g kg⁻¹. On the contrary, looking at treatments under 300 ppm P fertilizer rate, the control had the highest value of plant K concentration though not significantly different (p < 0.05) from the other treatments. The mean values for plant K concentration under this category range from 10.37 to 18.30 g kg⁻¹. Concerning the effects of P fertilizer rates on plant K concentration, it could be deduced from the results of this study that plant K concentration is not affected by the P application rate.

In summary, plants treated with biochar, especially at higher application rates, had the most positive influence on plant K concentration. This could be due to the higher availability of K by biochar which was readily available for the plants.

3.3.5. Impact of Acidified and Non-Acidified Biochar on Plant Shoot Uptake of Micronutrients (Cu, Fe, Mn, and Zn)

Micronutrient uptake by plant shoots measured in µg/plant is shown in Figure 10, Figure 11, Figure 12 and Figure 13. For Cu uptake by plant shoots under 0 ppm P fertilizer application rate, the mean values ranged from 6.77 to 23.23 µg plant^−1, where CK had the maximum mean value and treatment ABC2.5 had the minimum. It showed that biochar and acidified biochar-treated soil reduced the plant uptake of Cu as compared with the control. Treatments under 75, 150 and 300 ppm P fertilizer application rates also followed the same trend where biochar and acidified biochar treatments reduced plant uptake when compared with the control. For Fe uptake by plant shoots, mean values ranged from 244.74 to 800.53, 385.86 to 706.98, 387.05 to 709.53, and 208.75 to 839.59 µg plant⁻¹ under the 0, 75, 150, and 300 ppm P fertilizer rates, respectively. Similar to Cu uptake, CK had the highest Fe uptake by plant shoots for all P fertilizer application rates, while treatment ABC5 had the lowest all through. Although CK was observed to have the maximum mean value of plant Fe uptake, however, it is not significantly higher (p < 0.05) than all other treatments in some cases of P fertilizer application rates. For example, under 75 ppm P fertilizer application rate, CK was found not to be significantly higher (p < 0.05) than other treatments except treatment ABC5. Also, under treatment 150 ppm P fertilizer rate, CK was not significantly greater (p < 0.05) than all other treatments in their mean values, while CK was significantly higher (p < 0.05) than all other treatments under 0 and 300 ppm P fertilizer application rates. For Mn uptake by plant shoots, mean values ranged from 28.90 to 95.10, 31.88 to 95.85, 35.97 to 98.89, and 32.98 to 126.89 µg plant⁻¹ under 0, 75, 150 and 300 ppm P fertilizer rates, respectively. Similar to Cu and Fe uptake, CK had the highest Fe uptake by plant shoots for all P fertilizer application rates, while treatment ABC5 had the lowest all through. In this case, CK was significantly higher than all other treatments at all levels of P fertilizer application rate with the exception of 75 ppm P fertilizer application rate where CK was found not to be significantly different from BC2.5 and ABC2.5. Uptake of Zn by plant shoots followed the same trend as that of the other micronutrients, except that treatment BC2.5 had the highest Zn uptake under 75 ppm P fertilizer application rate. The mean values ranged from 16.47 to 53.67, 16.55 to 59.36, 16.60 to 74.70, and 13.99 to 75.67 µg plant⁻¹ under the 0, 75, 150, and 300 ppm P fertilizer rates, respectively.

Generally, in the current study, it could be deduced that both biochar and acidified biochar decreased the uptake of micronutrients by plant shoots. Similar to this, Moreno-Jiménez et al. [75] showed that adding oak-derived biochar had no impact on fortifying barley grain with Zn and Cu. They hypothesized that this might be because of the high biochar application rate, high CEC, and pH buffering. But it is also acknowledged that genetic differences between cultivars might impact Zn uptake [76]. In another previous study, wheat grain Fe and Cu contents decreased in all biochar-treated soils, while Zn concentrations considerably decreased in biochar treatments (p < 0.05) [30]. On the contrary, in another experiment, Mn and Zn uptake were significantly (p < 0.05) greater than the control. This could be due to some other factors like plant varieties and the type of biochar feedstock and its properties. The reason for the decrease in Fe uptake after the addition of biochar is that adding biochar to soil causes Fe to precipitate, rendering it inaccessible to plants and decreasing the movement of Fe into phloem cells for long-distance transport [77]. However, since Fe is chelated by organic matter and is largely present in soil solution as oxides and hydroxides and is impacted by soil pH [78], It is likely that biochar makes it possible for cations like calcium to form soluble complexes that are more readily available to plants [77]. A high pH above 7.5 and rising soil organic matter concentration is associated with a lack of Mn [78].

3.3.6. Influences of Acidified and Non-Acidified Biochar on Plant Micronutrients (Cu, Fe, Mn, and Zn) Concentration

Plant micronutrient (Cu, Fe, Mn, and Zn) concentrations as affected by biochar and its acidified biochar derivative are presented in

Table 10. Effects of BC and ABC applied at different rates with different P fertilizer application rates on Plant Cu concentration (mg/kg).

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	87.75 a	20.75 a	64.83 a	105.50 a	95.92
BC2.5	22.67 a	45.58 a	28.00 b	86.92 ab	39.77
BC5	74.58 a	28.50 a	9.83 b	39.75 abc	119.89
ABC2.5	12.08 a	47.75 a	11.50 b	28.42 b	82.30
ABC5	24.00 a	15.58 a	13.67 b	12.92 c	18.78
LSD	100.18	70.33	40.65	85.25
p-value	0.2110	0.6462	0.0194	0.0593
F-value	1.7721	0.6397	4.8655	3.2531

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

,

Table 11. Effects of BC and ABC applied at different rates with different P fertilizer application rates on Plant Fe concentration (mg/kg).

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	1629.75 a	517.25 a	1497.92 a	1363.17 a	1214.91
BC2.5	796.25 ab	943.17 a	1709.00 a	1176.83 a	1878.85
BC5	1596.75 a	1571.33 a	829.17 a	685.25 a	1522.86
ABC2.5	666.25 b	632.33 a	513.17 a	1797.00 a	1737.75
ABC5	588.25 b	749.00 a	717.08 a	369.00 a	800.27
LSD	1057.43	1078.99	1678.98	1741.41
p-value	0.0467	0.1375	0.2890	0.2559
F-value	3.5688	2.2381	1.4462	1.5707

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

,

Table 12. Effects of BC and ABC applied at different rates with different P fertilizer application rates on Plant Mn concentration (mg/kg).

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	107.33 a	65.83 a	112.58 a	158.67 a	154.70
BC2.5	74.92 a	65.83 a	76.50 ab	83.58 a	21.23
BC5	107.00 a	91.58 a	77.42 ab	74.17 a	49.83
ABC2.5	64.58 a	68.00 a	60.00 b	65.50 a	32.42
ABC5	65.00 a	61.83 b	65.67 b	58.33 a	20.07
LSD	47.13	32.42	54.36	121.42
p-value	0.0613	0.1645	0.1511	0.2295
F-value	3.2103	2.0392	2.1331	1.6834

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

and

Table 13. Effects of BC and ABC applied at different rates with different P fertilizer application rates on Plant Zn concentration (mg/kg).

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	101.45 a	82.08 a	236.00 a	345.58 a	76.07
BC2.5	80.08 a	87.88 a	255.58 a	42.50 c	67.14
BC5	66.00 ab	39.00 b	35.08 b	106.08 b	59.08
ABC2.5	30.00 b	34.42 b	18.75 b	149.75 b	27.12
ABC5	36.83 b	32.17 b	230.42 a	24.75 c	53.05
LSD	52.58	43.64	58.78	68.64
p-value	0.0197	0.0102	0.0000	0.0000
F-value	4.8392	5.9630	60.2873	52.8265

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

. For plant Cu concentration under 0 ppm P fertilizer application rate, the mean values range from 12.08 to 87.75 mg/kg, where the maximum value was observed in the untreated plant, and the minimum was observed in plants treated with ABC2.5. Though there are differences among the treatments but the differences are not significant (p < 0.05). Similarly, there were no significant differences (p < 0.05) among the treatments under 75 ppm P fertilizer application rate. Here, their mean values range from 15.58 to 47.78 mg/kg, where treatment ABC5 had the minimum and treatment ABC2.5 had the maximum mean value. Under the 150 ppm P fertilizer application rate, the CK had the maximum value, which is significantly higher (p < 0.05) than all other treatments, while the other treatments (BC2.5, BC5, ABC2.5 and ABC5) were not significantly different (p < 0.05) among themselves. Similarly, under 300 ppm P fertilizer application rate, the CK also had the highest value though not significantly different (p < 0.05) from treatment BC2.5 and BC5 but significantly higher (p < 0.05) than treatments ABC2.5 and ABC5. For plant Fe concentration under the 0 ppm P fertilizer application rate, the CK had the highest value with a mean value of 1629.75 mg/kg, while treatment ABC5 had the lowest value with a mean value of 588.25 mg/g. Despite the higher mean value of the CK, it was not significantly different (p < 0.05) from treatments BC2.5 and BC5 but significantly higher (p < 0.05) than treatments ABC2.5 and ABC5. In the case of treatments under the influence of 75 ppm P fertilizer application rate, treatment BC5 had the concentration of plant Fe while treatment ABC2.5 had the lowest value with values of 1571.33 and 632.33 mg/kg, respectively. However, treatment BC5 was not significantly different (p < 0.05) from all other treatments. Furthermore, plant Fe concentration of treatments under 150 and 300 ppm P fertilizer application rates range from 513.17 to 1709.00 mg/kg and 369.00 to 1797.00 mg/kg, respectively. In both cases, there were no significant differences (p < 0.05) among the treatments. For plant Mn concentration under 0, 75, 150, and 300 ppm P fertilizer application rates, the treatment mean values range from 64.58 to 107.33, 61.83 to 91.58, 60.00 to 112.58, and 58.33 to 158.67 mg/kg, respectively. Similar to the aforementioned micronutrients, the CK had the highest value in almost all P fertilizer application rates except treatments under 75 ppm P fertilizer application rate. Plant Zn concentration also followed a similar trend as in the case of Cu, Fe, and Mn, where the CK had the highest mean value under the influence of almost all fertilizer application rates. The treatments mean values of plant Fe concentration under 0, 75, 150, and 300 ppm P fertilizer application rates range from 30.00 to 101.45, 32.17 to 87.88, 18.75 to 236.00, and 24.75 to 345.58 mg/kg, respectively.

In summary, it could be deduced that both biochar and its acidified biochar derivative had little or no effect on plant micronutrient concentrations. According to Taiz and Zeiger [77], the reduction in Fe uptake following the addition of biochar is due to the precipitation of Fe caused by the addition of biochar to soil, which renders it physically unavailable to plants and reduces the movement of Fe into phloem cells for long-distance transport. Additionally, Moreno-Jiménez et al. [75] found that adding oak-derived biochar had little impact on fortifying barley grain with zinc and copper. They hypothesized that this might be because of the high application rate of biochar, high cation exchange capacity (CEC), and pH buffering.

3.4. Phosphorus Fractionation

Total phosphorus can be regarded as a basic soil property [79], even if over pedogenic timeframes, it can alter significantly [80]. The chemistry of P in soils is complicated. It is present in the soil in many forms. It could be present as available P or form complexes with Ca, Fe and Al. Phosphorus fractionation has been employed to investigate the forms of P in the soil, and the transformation of P applied to the soil. The phosphorus fraction in the studied soil as affected by the amendments was presented in Figure 14, Figure 15, Figure 16 and Figure 17. In most cases of the soil where 0 ppm P fertilizer was added, residual P had the highest fraction all through the amendments (CK, BC2.5, BC5, ABC2.5, ABC5). The percentage of residual P ranges from 58.63% to 76.55%, where ABC2.5 had the minimum and CK had the maximum. In order of decreasing P fractions, NaOH II–P was next to the residual P. This fraction is the Fe-bounded P. Fe/Al-bound P is widely believed to be moderately plant-available and has been found to act as a buffer for accessible P, particularly in heavily weathered [81] and sandy soils [82]. The percentage of NaOH II–P to the total P ranges from 0.65% to 1.48%, with BC2.5 and BC5 having the minimum and maximum values, respectively. In the same fertilizer rate, 0 ppm P, HCl–P, which is the Ca-bounded P, ranges from 13.38% to 28.23% in the fraction of the total P, where CK had the minimum value and ABC5 had the maximum value. Similarly, NaOH I–P, which is the Al- and Fe-bounded P, ranges from 3.35% to 9.54% in the fraction of the total P, where CK had the minimum value and ABC5 had the maximum value. From the results of the current study, the NaHCO₃–P fraction, which is the exchangeable fraction, increased with increasing fertilizer application rate, with BC2.5 relatively having the highest percentage and BC5 having the lowest in most cases. In percentage of the total P, the values ranged from 0.19% to 2.70%, 4.76% to 11.87%, 5.49% to 21.42%, and 12.25% to 26.19% for 0, 75, 150, and 300 pp P fertilizer rates, respectively. Similarly, NH₄Cl–P fraction, which represents the soluble and exchangeable fraction, decreased with increasing fertilizer application rate, with BC5 having the highest relative percentage and CK having the lowest in most cases. In percentage of the total P, the values ranged from 3.30% to 15.06%, 10.55% to 24.92%, 14.94% to 26.33%, and 14.39% to 31.39% for 0, 75, 150, and 300 pp P fertilizer rates, respectively.

P that is readily soluble and loosely bound is represented in the bicarbonate and water fractions [83,84]. Only slight chemical alterations are introduced by water and bicarbonate, simulating the impact of soil solution [84]. The Ca-bound P in minerals with low solubilities, like apatite, is believed to be represented by the HCl fraction [83,84]. It is believed that calcium-bound P is not easily accessible to plants [83], but in somewhat weathered soils, it might serve as a buffer for the available P [81]. According to Golterman [85], the extraction of phytate phosphate and organic-P occurs along with phosphate and is time- and concentration-dependent. Therefore, additional research will consider and delve more deeply into the application of this extraction phase.

Key: NH₄Cl–P, Soluble and exchangeable P; NaHCO₃–P, Exchangeable P; NaOH I–P, Al and Fe-bound P; NaOH II–P, Fe-bound P; HCl–P, Ca-bound P.

3.5. Growth Parameters

3.5.1. Effects of Acidified and Non-Acidified Biochar on Plant Height

The mean values of the heights of alfalfa plants as affected by the amendments were recorded in

Table 14. Effects of BC and ABC applied at different rates with different P fertilizer application rates on plant height in cm.

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	9.23 e	17.84 d	20.54 d	27.68 c	2.57
BC2.5	23.01 b	38.60 b	34.58 b	28.91 b	1.65
BC5	15.59 d	19.24 d	24.26 c	24.81 d	1.20
ABC2.5	25.56 a	46.16 a	38.51 a	38.27 a	1.81
ABC5	18.57 c	22.58 c	24.73 c	25.12 d	1.15
LSD	1.90	1.87	1.88	0.85
p-value	0.0000	0.0000	0.0000	0.0000
F-value	171.7498	695.1940	247.5158	631.5768

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

. All through the different P fertilizer application rates, treatment ABC2.5 had the highest plant height when compared to all other treatments together with the control. Under 0 ppm P fertilizer rate, the plant height ranges from 9.23 to 25.56 cm. The treatments were in the order ABC2.5 > BC2.5 > ABC5 > BC5 > CK with values of 25.56, 23.01, 18.57, 15.59, and 9.23 cm, respectively. In this case, all the treatments were significantly different (p < 0.05) from one another. For plant heights under that of 75 ppm P fertilizer rate, the treatments were in the same order as the previously mentioned P application rate with plant values of 46.16, 38.60, 22.58, 19.24, and 17.84 cm, respectively, but not all the differences in the treatments were significant (p < 0.05). Treatment BC5 was found not to be significantly different from CK. For the 150 ppm P fertilizer application rate, the height of alfalfa ranged from 20.54 to 38.51 cm. Also, the treatments were in the order of the previous P application rate, i.e., ABC2.5 > BC2.5 > ABC5 > BC5 > CK, with values of 38.51, 34.58, 24.73, 24.26, and 20.54 cm, respectively. Likewise, not all the differences are significant in this case. Treatments BC5 and ABC5 were not significantly different (p < 0.05) from each other. In the case of the 300 ppm P fertilizer application rate, the order of treatments in terms of plant height was slightly different from that of the three previous P fertilizer application rates. The treatments were in the order ABC2.5 > BC2.5 > CK> ABC5 > BC5 with corresponding values of 38.27, 28.91, 27.68, 25.12, and 24.81 cm, respectively.

Generally, in this current study, it could be deduced that biochar and acidified biochar treatments had greater plant heights than the untreated soil. This could be a result of the higher plant-available macro- and micronutrients in soil treated with biochar and acidified biochar which are readily available for plant uptake for proper growth. In a previous study, Usman et al. [35] also reported increased vegetative growth parameters following the addition of biochar as compared to the control under both saline and non-saline condition. Several other studies reported improved plant growth following the addition of biochar to soil having low quality and problem such as high salinity, high acidity, low fertility or water retention [42,60,68,86,87].

3.5.2. Effect of BC and ABC Applied at Different Rates with Different P Fertilizer Application Rates on Plant Dry Weight

Table 15. Effects of BC and ABC applied at different rates with different P fertilizer application rates on plant dry weight in g/pot.

Treatment	P Application Rate (ppm)
	0	75	150	300	LSD
CK	0.547 e	1.222 d	1.436 d	1.846 b	0.142
BC2.5	1.547 b	3.376 b	2.521 b	1.923 b	0.203
BC5	0.991 d	1.350 d	1.564 cd	1.615 c	0.114
ABC2.5	1.675 a	3.667 a	3.462 a	3.368 a	0.195
ABC5	1.333 c	1.547 c	1.632 c	1.701 c	0.137
LSD	0.101	0.201	0.164	0.140
p-value	0.0000	0.0000	0.0000	0.0000
F-value	309.5435	523.8641	417.3279	402.9944

According to the least significant difference (LSD) test at p < 0.05, means with different letters in each column imply significant differences. CK = Control, BC2.5 = Biochar added at 2.5% w/w, BC5 = Biochar added at 5% w/w, ABC2.5 = Acidified biochar added at 2.5% w/w, and ABC5 = Acidified biochar added at 5% w/w.

presented the influence of biochar and acidified biochar applied as amendments at different rates coupled with different rates of P fertilizer. According to this result, treatment ABC2.5 had the highest dry weight at all levels of P fertilizer application. Under 0 ppm P fertilizer application, ABC2.5 was found to increase over the CK, BC2.5, BC5 and ABC5 by 3.06-, 1.08-, 1.69-, and 1.26-fold, respectively. And all the differences among the treatments were significant (p < 0.05). For treatments under 75 ppm P fertilizer application, ABC2.5 was found to produce increases over CK, BC2.5, BC5, and ABC5 by 3.00-, 1.09-, 2.72-, and 2.37-fold, respectively. In this case, not all the differences among the treatments were significant. Treatments BC5 and CK had no significant differences (p < 0.05) in their mean values. In the case of 150 ppm P fertilizer application rate, ABC2.5 treatment was observed to increase over the CK, BC2.5, BC5, and ABC5 by 2.41-, 1.37-, 2.21-, and 2.12-fold, respectively. Also, not all the differences in the means of the treatments were significant. For example, treatments BC5 and ABC5 had no significant differences (p < 0.05) in their mean values, and also treatments BC and CK exhibited no significant differences (p < 0.05) in their mean values. Meanwhile, in the case of treatments under the application of 300 ppm P fertilizer, ABC2.5 treatment was recorded to increase over the CK, BC2.5, BC5, and ABC5 by 1.82-, 1.75-, 2.09-, and 1.98-fold, respectively.

In summary, it could be deduced that the application of biochar, either acidified or non-acidified, increased the dry weight of alfalfa over the control. Moreso, a profound increase in plant dry weight was observed in acidified biochar treatment applied at a lower rate. Similar to the results of this study, Abd El-Mageed [88] reported an increase in crop yield following acidified biochar application. Also, in a previous study by Usman et al. [35], increased plant yield was reported following the amendment of biochar to soil under both saline and non-saline condition. Similarly, in another study, biochar was reported to improve the yield of ryegrass [3]. The increase in the yield parameter following biochar addition could be a result of the higher plant-available macro- and micronutrients in soil treated with biochar and acidified biochar which are readily available for plant uptake for proper growth and thus result in higher yield. Numerous other studies hypothesized that the improvements in general soil properties, plant water and nutrient use efficiency, nutrient release and/or retention, direct nutrient additions, balanced nutrition of plants, and subsequently increased nutrient availability to plants are related to the increases in nutrient uptake and plant yield following the application of biochar [42,60,89]. Contrary to claims claiming that biochar always has a good impact on crop development and yield in acidic soil but not in calcareous soil [55,90], Inal et al. [42] reported that the growth of bean and maize plants was increased by biochar addition to a calcareous soil and this is similar to the result of the current study. Plant growth and yield response levels in calcareous soil might vary by species [91]. Van Zwieten et al. [92] discovered that whereas biochar lowered wheat and radish yield, it boosted soybean output. Additionally, a prior study indicated that the addition of biochar and chicken dung boosted the dry weight of lettuce [93]. An increase in plant production when amended with biochar can be associated with soil quality enhancement [94,95], the release of nutrients into the soil solution, an increase in chemicals and/or helpful organisms, as well as balanced plant nutrition [93].

4. Conclusions

Conocarpus waste biochar (BC) and its derived acidified biochar (ABC) with different rates of P fertilizer application were used as amendments in soils grown with alfalfa. The effects of these treatments on soil phosphorous (P) availability and soil quality were studied. The pH of the biochar was drastically reduced even up to a unit of 6.84 following acidification, and thus, reduced the pH of the studied soil. Therefore, acidified biochar has the potential to reduce the pH of alkaline soil. From the current study, biochar was capable of increasing soil available P, though the increment was minimal due to the calcareous nature of the soil. Moreover, biochar applied at higher application rates was able to increase soil extractable K and Na above the control soil. Likewise, soil cation exchangeable capacity and soil organic matter were increased following BC and ABC applications, especially at higher application rates. Similarly, the amendments were efficient in the enhancement of soil micronutrient availability. On the other hand, the uptake of micronutrients was decreased following the addition of the amendments, while the uptake of P and K was enhanced. Meanwhile, yields and growth of alfalfa were improved by both BC and ABC applications. Hence, both biochar and acidified biochar can be efficiently used by farmers to enhance soil available P and other soil chemical properties. Moreover, acidified biochar is recommended for enhancing soil micronutrient availability in calcareous soil having a deficiency of available micronutrients.