Potential short-term negative versus positive effects of olive mill-derived biochar on nutrient availability in a calcareous loamy sand soil.

In the present work, the olive mill solid waste (OMSW)-derived biochar (BC) was produced at various pyrolytic temperatures (300-700°C) and characterized to investigate its potential negative versus positive application effects on pH, electrical conductivity (EC), and nutrients (P, K, Na, Ca, Mg, Fe, Mn, Zn, and Cu) availability in a calcareous loamy sand soil. Therefore, a greenhouse pot experiment with maize (Zea mays L.) was conducted using treatments consisting of a control (CK), inorganic fertilizer of NPK (INF), and 1% and 3% (w/w) of OMSW-derived BCs. The results showed that BC yield, volatile matter, functional groups, and zeta potential decreased with pyrolytic temperature, whereas BC pH, EC, and its contents of ash and fixed carbon increased with pyrolytic temperature. The changes in the BC properties with increasing pyrolytic temperatures reflected on soil pH, EC and the performance of soil nutrients availability. The BC application, especially with increasing pyrolytic temperature and/or application rate, significantly increased soil pH, EC, NH4OAc-extractable K, Na, Ca, and Mg, and ammonium bicarbonate-diethylenetriaminepentaacetic acid (AB-DTPA)-extractable Fe and Zn, while AB-DTPA-extractable Mn decreased. The application of 1% and 3% BC, respectively, increased the NH4OAc-extractable K by 2.5 and 5.2-fold for BC300, by 3.2 and 8.0-fold for BC500, and by 3.3 and 8.9-fold for BC700 compared with that of untreated soil. The results also showed significant increase in shoot content of K, Na, and Zn, while there was significant decrease in shoot content of P, Ca, Mg, and Mn. Furthermore, no significant effects were observed for maize growth as a result of BC addition. In conclusion, OMSW-derived BC can potentially have positive effects on the enhancement of soil K availability and its plant content but it reduced shoot nutrients, especially for P, Ca, Mg, and Mn; therefore, application of OMSW-derived BC to calcareous soil might be restricted.

Introduction

Rapid expansion in agripan class="Chemical">culture to pan class="Chemical">feed the continuously growing world population has increased conventional intensification in farming systems, which consequently has resulted in soil nutrient depletion and various environmental concerns [1]. Intensive application of agro-chemicals has resulted in land degradation as well as declined soil health and quality [2, 3]. These problems are even more intensified in calcareous soils under arid and semi-arid climate due to lower organic matter and nutrient availability. Hence, a global transition towards modern farming systems with sustainable soil health, safe ecosystems, food security, and climate change mitigation is required. In this context, organic soil additives can potentially improve crop production, soil organic matter, rehabilitation of degraded land, and microbial activity with minimal environmental damage [4-7]. Biochar has been recently suggested as an emerging organic conditioner that can aid in overcoming soil problems and enhancing soil productivity [8-10].

Biochar is produced from organic materials (e.g., biomass) through pyrolytic processes under limited pan class="Chemical">oxygen supn>ply, and is generally characterized by its high content of fixed pan class="Chemical">carbon [11]. Biochar materials are largely employed as additives to overcome soil problems and limitations by enhancing soil properties in relation to chemical, biological, and hydro-physical parameters, as well as nutrient content and efficiency [8, 10, 12–14]. Owing to higher cation exchange capacity, sorption affinity, large surface area, higher porosity, and lower mobile matter, biochar could serve as an ideal candidate for improving physio-chemical properties of soils such as cation exchange capacity, pH, water holding capacity, and nutrient retention [15]. However, previous studies have demonstrated that application of biochar was more suitable for acidic soils than the alkaline soils, owing to liming effect induced by the alkaline nature of biochar [16, 17]. Nevertheless, some studies demonstrated that the application of biochar to calcareous sandy soil increases water holding capacity, organic matter, cation exchange capacity, and microbial activities, whereas, decreases the hydraulic conductivity [18-20]. Amin [21] reported that application of biochar to calcareous sandy soil improved phosphorus availability and barley production. Still, the effects of biochar application on the properties of alkaline soils with high pH and CaCO3 values are not well understood and have received much less attention.

Despite of positive impacts of biochar on soil quality, the performance of biochar is not always consistent. For instance, Van Zwieten [22] reported that paper mill waste derived biochar increased the production of pan class="Species">soybean, while reduced the production of wheat. Likewise, Jones [23] stated that hardwood derived biochar application significantly improved the soil fertility, while did not enhance the production of maize. Moreover, the performance of biochar varies with the characteristics of biochar, which are influenced by feedstock type, pyrolysis temperature, and resident time. For instance, biochar produced at lower temperature improved the cation exchange capacity and nutrient availability, whereas, decreased the salinity of the soil. Likewise, biochar produced above 500°C enhanced soil pH and decreased soil metal availability [24]. Therefore, selection of a biochar with distinct structural and chemical properties is a key factor in order to improve soil fertility and productivity.

The selection of suitable and low-cost pan class="Chemical">feedstock for biochar production is of critical importance in order to obtain maximum benefits. Recycling solid waste materials could serve as a potential low-cost strategy for biochar production. For instance, re-using solid pan class="Species">olive-derived waste might serve as a potential low-cost technology to produce biochar for soil application. The amount of olive mill solid waste (OMSW) produced globally accounted for 4 × 108 kg dry matter per year, comprising of 38–50% (w/w) cellulose, 23–32% hemi-cellulose, and 15–25% lignin. Therefore, converting OMSW into biochar could reduce surface pollution on one hand, and the produced biochar could serve as a soil conditioner for enhanced soil productivity on the other hand. Previous studies have demonstrated the enhancing effects of OMSW-derived biochar on soil microbial biomass C and N, and alteration of the structure of the bacterial community in soil [25, 26]. However, to date, there is no available information on the effects of OMSW-derived biochar on nutrient availability in alkaline sandy soils. Therefore, we investigated (i) the effects of pyrolysis temperature (300–700°C) on the properties of OMSW-derived biochar, and (ii) potential negative versus positive effects of OMSW-derived biochar on the chemical properties and nutrients availability in calcareous loamy sand soil and on maize (Zea mays L.) growth.

Material and methods

Production and characteristics of biochar

The solid waste from the pan class="Species">olive mill was collected from the Al-Jawf region, Saudi Arabia, and there is no specific permission was required from the company of pan class="Species">olive presses to collect the OMSW samples. The OMSW feedstock was dried at 60°C and then pyrolyzed in a closed container by furnaces under limited oxygen supply at temperatures 300, 400, 500, 600, and 700°C for 4 h at 5°C min-1. Biochar samples were collected, cooled in a desiccator, ground, sieved through a 2 mm sieve, labelled as BC300, BC400, BC500, BC600, or BC700, and stored for further analyses. The measurements of olive mill solid waste-derived biochar (OMSW-BCs) samples were carried out in duplicate.

In OMSW-derived pan class="Chemical">BC, the moisture content, and that of volatile and resident materials, and ash (proximate characteristics) were analyzed by the ASTM D1762-84 method [27]. The pH of OMSW-derived pan class="Chemical">BCs was measured in a mixture (1:25, w/v) of BC and deionized water using digital pH meter. After measuring biochar pH, the mixture of BC and water was extracted to determine the electrical conductivity (EC) using a digital EC meter. Zeta potential of the BC samples was determined by dynamic light scattering techniques, by measuring the electrophoretic mobility of 1 g L-1 BC suspensions in deionized water (Zetasizer Nano ZS, Malvern, UK). Additionally, BC samples were analyzed with a scanning electron microscope (SEM; FEI, Inspect S50), X-ray diffraction (XRD; XRD-7000; Shimadzu Corp, Kyoto, Japan), surface area analyzer (ASAP 2020, Micromeritics, USA), and the Fourier transformation infrared method (Nicolet 6700 FTIR).

Greenhouse pot experiment

The soil samples were collected from an agripan class="Chemical">cultural farm (24o21'33.1' N and 47o07'49.8' E, altitude 467 m), which is located in a dry land region at Al-Kharaj, Riyadh, Saudi Arabia. No specific permission was required from the farm owner to collect the composite soil samples. The collected composite soil samples were air-dried, sieved, and analyzed for their physico-chemical properties. To identify particle size distribution according to Bouyoucos [28], the hydrometer method was apn>plied. According to Spn>arks [29], and Nelson and Sommers [30], chemical soil properties, including pH, EC, pan class="Chemical">CaCO3, and organic matter were measured. The data for soil analyses showed that the soil samples, which were characterized as having a loamy sand texture (containing 80.89% sand, 12.07% silt, and 7.04% clay) and a low level of organic matter (0.12%), had an alkaline pH (measured in 1:1 suspension of soil to water) value of 7.8, EC (measured in 1:1 extracts of soil to water) value of 2.0 dS m-1, and high CaCO3 content (16.51%).

A greenhouse pot experiment with pan class="Species">maize plants (pan class="Species">Zea mays L.) was conducted. Specifically, 1 kg of soil treated with 1% and 3% (w/w) of unwashed OMSW-derived BC at various pyrolytic temperatures (300°C, BC300; 500°C, BC500; and 700°C, BC700). Additionally, treatments consisting of a control (CK) and inorganic fertilizer of NPK (INF) were applied in the study for comparison. Then, the treated and untreated soil samples were placed in pots, irrigated at the level of field capacity, and incubated for 21 d under laboratory conditions (at a temperature of 23 ± 2°C). After the incubation period, three replications of the treated and untreated pots were transferred to the greenhouse and placed in a randomized complete block design. Ten maize seeds were sown in each pot. After seedlings emerged, the plants in each pot were thinned to four. The planting period lasted 4 weeks. During the growth period, the plants were irrigated and maintained at 70% of field capacity by weight loss. After 4 weeks of cultivation, the shoots of maize plants and soil were collected from the pots. The collected soil samples were air-dried and analyzed for pH, EC, AB-DTPA-extractable nutrients (P, Fe, Mn, Zn, and Cu), and ammonium acetate-(NH4OAc)-extractable K, Na, Ca, and Mg [29]. Additionally, the shoots of maize plants were collected, dried at 70°C, and analyzed for P, Ca, Mg, K, Na, Fe, Mn, Zn, and Cu. The maize plant shoot samples were dried and digested using a dry ashing method [31]. In this study, inductively coupled plasma (ICP, Perkin Elmer Optima 4300 DV ICP-OES) was used to measure the concentrations of P, Ca, Mg, Fe, Mn, Zn, and Cu. The concentrations of K and Na were analyzed using a flame photometer.

Statistics

For the properties of biochar samples, the means and standard deviations (±SD) are computed. Moreover, to compare the efpan class="Chemical">fects of biochar treatments on soil and plant, the difpan class="Chemical">ferences of means were analysed statistically by a one-way analysis of variance (ANOVA) using the TIBCO Statistica 13.5.0 software [32]. In addition, the obtained data were evaluated by using the least significant difference (LSD) test for post hoc comparisons (at the 0.05 level of significance).

Results and discussions

Characteristics of OMSW-derived BC

The results showed that pan class="Chemical">BC yield declined with increasing thermal decomposition during the pyrolytic process (Table 1). The obtained yield of biochar produced at 300°C accounted for 40.3%, whereas it declined by 32.1%, 28.7%, 27.3%, and 26.7% as the pyrolysis temperature increased to 400, 500, 600, and 700°C, respectively. This reduction was mainly caused by the organic material decomposition and the pan class="Disease">dehydration of OH groups during the pyrolysis process. However, increasing thermal decomposition during the pyrolytic process increased the levels of fixed carbon and ash in the OMSW-derived BC (Table 1). Several other researchers have reported that the pyrolysis temperature is considered one of the main factors determining BC properties, and thus, the effects of its application in the environment [33-35]. In their studies, the levels of fixed carbon, ash, and pH increased with increasing temperature of the pyrolytic process, whereas yield and volatile matter of BCs tended to decrease. They suggested that high pyrolysis-temperature BCs possess more carbonaceous materials. Additionally, pyrolysis temperature could affect the thermal stability and chemistry of the BC surface [35].

Table 1

pH, Electrical Conductivity (EC), and approximate analysis of Olive Mill Solid Waste-derived Biochar (OMSW-BCs) samples as affected by pyrolysis temperature.

Parameters	Samples1
	FS	BC300	BC400	BC500	BC600	BC700
pH	5.97 ± 0.022	9.85 ± 0.08	10.12 ± 0.02	10.03 ± 0.02	10.11 ± 0.03	10.21 ± 0.04
EC, (dS m-1)	0.83 ± 0.01	1.06 ± 0.02	2.11 ± 0.02	2.09 ± 0.02	2.39 ± 0.00	2.54 ± 0.04
Yield, %	-	40.3 ± 0.47	32.1 ± 0.12	28.7 ± 0.82	27.3 ± 0.03	26.7 ± 0.49
Moisture, %	6.11 ± 0.49	1.16 ± 0.006	1.44 ± 0.007	1.72 ± 0.03	1.89 ± 0.02	0.95 ± 0.02
Volatile matter, %	74.51 ± 1.04	27.74 ± 1.33	17.07 ± 0.49	11.96 ± 0.44	11.00 ± 0.02	9.62 ± 0.02
Ash, %	2.02 ± 0.08	6.91 ± 0.15	8.92 ± 0.18	9.43 ±0 .30	9.59 ± 0.17	9.62 ± 0.03
Fixed C, %	17.36 ± 1.44	64.19 ± 1.19	72.57 ± 0.60	76.90 ± 0.11	77.51 ± 0.16	79.80 ± 0.03

1. FS: feedstock; BC300: biochar produced at 300°C; BC400: biochar produced at 400°C; BC500: biochar produced at 500°C; BC600: biochar produced at 600°C; BC700: biochar produced at 700°C.

2. Values represent the means ± standard deviations (±SD).

1. FS: pan class="Chemical">feedstock; pan class="Chemical">BC300: biochar produced at 300°C; BC400: biochar produced at 400°C; BC500: biochar produced at 500°C; BC600: biochar produced at 600°C; BC700: biochar produced at 700°C.

The FTIR results showed that OMSW-derived BC300 possessed a spectrum at 3430 cm-1 (S1 Fig), generally attributed to O–H. The spectra of O–H declined with pyrolysis temperature. Additionally, OMSW-derived BC300 exhibited broad bands in the region of 2850–2919 cm-1, mainly because of the C-H stretch caused by aliphatic compounds, waxes, and fatty acids. Additionally, the band at 2850 was attributed to symmetrical CH in–CH2, suggesting the presence of fatty acids and alkanes. These two peaks at 2850 and 2919 cm-1 declined in BC400 and completely disappeared with a temperature ≥ 500°C. The absorption at 1574–1652 cm-1 of biochar samples suggested the presence of–COOH, as well as C = O and C = C, especially in an aromatic form. However, the intensity of these peaks (1574–1652 cm-1) decreased in BC samples produced at higher pyrolysis temperatures (≥ 500°C). The absorption at approximately 1400 cm-1 (with an intense band for BC300 and BC400) suggested the presence of aliphatic and aromatic O-H groups, which decreased at higher pyrolysis temperatures.

In this study, XRD was used to identify the mineral composition of pan class="Chemical">BC samples (S2 Fig). In the samples of OMSW-derived pan class="Chemical">BC300, peaks at a spacing of 4.02, 2.06, 1.45, and 1.23 Å were identified, suggesting the presence of kalcinite [K(HCO3)], sylvite (KCl), and perclase (MgO), which were reduced or disappeared with increasing pyrolysis temperatures. Furthermore, a high pyrolysis temperature resulted in calcite minerals (CaCO3) at 3.04–3.14, 1.87, 1.81 Å. Additionally, the SEM analysis depicted a greater change in the surface structure of BCs compared to that of feedstock with temperature (S3 Fig).

Larger zeta potentials were observed at the lower pyrolysis temperatures of 300 and 400°C (Table 2), which was mainly attributed, as indicated by the FTIR results, to the increased number of O–H and–pan class="Chemical">COOH groupn>s observed in the pan class="Chemical">BC produced at these temperatures. As a result of the decline or the complete disappearance of the O–H and–COOH groups at higher pyrolysis temperatures, less negative charges remained on the surfaces of BC, and zeta potential were decreased for BCs produced at 500, 600, and 700°C. The surface area and pore characteristics of OMSW-derived BCs showed an improvement in the surface area and total and microporous pore volumes with pyrolysis temperature (Table 2). OMSW-derived BCs made at the lowest temperatures of 300°C and 400°C, respectively, possessed the lowest surface area of 0.35 m2 g-1 and 1.78 m2 g-1; however, OMSW-derived BC500, BC600, and BC700 had high values for the surface area of 108.4, 128.0, and 168.4 m2 g-1, respectively. In contrast, as pyrolysis temperature increased from 300°C to 400°C, 500°C, 600°C, and 700°C, the average pore width for OMSW-derived BCs decreased from to 170.8 to 46.1, 20.4, 19.8, and 19.7 nm, respectively. Generally, during the pyrolytic process, the loss of volatile materials and the functional groups H- and O- were the main reason for the improvement in surface area and pore characteristics of the resultant BCs. In this context, pyrolytic temperatures were positively correlated with BC characteristics, including EC (r = 0.885), fixed carbon (r = 0.9264), surface area (r = 0.9562), microporous surface area (r = 0.9691), total pore volume (r = 0.9532), and microporous pore volume (r = 0.9690) (Table 3). However, pyrolysis temperature showed a negative correlation with yield (r = -0.9054) and volatile matter (r = -9028). In general, the greatest changes were pronounced for high pyrolysis temperatures ≥500°C. When plotting the biochar characteristics (yield, volatile matter, fixed carbon, ash content, and surface area) in relation to different pyrolysis temperatures, the best model fitting was pronounced with a second-order polynomial (S4 Fig).

Table 2

Zeta potential, surface area and pore properties of Olive Mill Solid Waste-derived Biochars (OMSW-BCs).

Pyrolysis temperature (°C)	Zeta potential (mV)	SBET2 (m2 g-1)	Smicr3 (m2 g-1)	Vt4 (cm3 g-1)	Vmicro5 (cm3 g-1)	Dave6 (nm)
300	−43.0 ± 1.641	0.35 ± 0.01	0.35 ± 0.01	0.0015± 0.007	0.00018± 0.00	170.8 ± 6.4
400	−50.2 ± 1.96	1.78 ± 0.04	1.78 ± 0.03	0.0021± 0.00	0.00086± 0.00	46.1 ± 1.7
500	−29.3 ± 1.21	108.4 ± 1.9	80.5 ± 1.6	0.055± 0.00	0.037± 0.00	20.4 ± 0.7
600	−27.3 ± 1.53	128.0 ± 2.3	109.2 ± 2.2	0.063± 0.00	0.051± 0.00	19.84 ± 0.9
700	−24.7 ± 0.75	168.4 ± 3.2	143.8 ± 3.1	0.083± 0.00	0.066± 0.00	19.74 ± 0.6

1. Errors are ± SD.

2. SBET surface area.

3. Microporous surface area by the t-plot method.

4. Total pore volume

5. Microporous pore volume by the t-plot method.

6. Average pore width, estimated by 4Vt/SBET.

7. ±SD reported as zero have values ≤ 1.0 x 10−5

Table 3

Pearson correlation coefficient (r) among Olive Mill Solid Waste-derived Biochar (OMSW-BCs) properties.

BC properties	Pyrolysis temperature	pH	EC	Zeta potential	Yield	Volatile matter	Ash	Fixed carbon	SBET	Smic	Vt	Vmic	Dave
Pyrolysis temperature	1.0000
pH	0.8280	1.0000
EC	0.8850*	0.9590	1.0000
Zeta potential	-0.8475	-0.4195	-0.5800	1.0000
Yield	-0.9045*	-0.8730	-0.9726*	0.7127	1.0000
Volatile matter	-0.9028*	-0.8652	-0.9662*	0.7208	0.9990*	1.0000
Ash	0.8416	0.8797	0.9738*	-0.6175	-0.9903*	-0.9891*	1.0000
Fixed carbon	0.9264*	0.8751	0.9646*	-0.7422	-0.9947*	-0.9970*	0.9779*	1.0000
SBET	0.9562*	0.6468	0.7627	-0.9625*	-0.8476	-0.8534	0.7683	0.8771	1.0000
Smic	0.9691*	0.6680	0.7693	-0.9513*	-0.8398	-0.8429	0.7570	0.8687	0.9966*	1.0000
Vt	0.9532*	0.6426	0.7604	-0.9637*	-0.8475	-0.8540	0.7688	0.8776	0.9999*	0.9953*	1.0000
Vmic	0.9690*	0.6673	0.7702	-0.9517*	-0.8409	-0.8435	0.7581	0.8686	0.9963*	0.9999*	0.9949*	1.0000
Dave	-0.7926	-0.8689	-0.9611*	0.5578	0.9745*	0.9742*	-0.9960*	-0.9590*	-0.7147	-0.6997	-0.7159	-0.7006	1.0000

SBET: surface area; Smic: Microporous surface area; Vt: Total pore volume; Vmic: Microporous pore volume; Dave: Average pore width

Biochar effects on soil pH and EC

OMSW-derived pan class="Chemical">BC treatments increased soil pH from 7.85 to 8.05, 8.15, and 8.11 at an application rate of 1% for BC300, BC500 and BC700, respectively (Table 4). Meanwhile, at a high application rate of 3%, these corresponding values of soil pH increased to 8.28 (BC300), 8.31 (BC500) and 8.32 (BC700). The occurred increases in soil pH, due to BC application, are mainly because of the alkalinity induced by BCs as a result of the basic oxides and carbonates produced during the pyrolytic process. Previous studies have also indicated the application effects of BCs on increasing soil pH [36-38]. However, in contrast to our results, other studies have found that alkaline soil pH decreased with biochar addition [14]. The reductions in soil pH could be attributed to BC oxidation and the production of acidic materials. In the current study, the higher pH of produced BCs (9.85–10.21) compared to the control soil (7.80) could result in increasing soil pH. The increases in soil pH could have occurred because of the increase in the level of soil exchangeable base cations with BC addition. In a study conducted on calcareous soil, Cardelli et al. [36] suggested that the alkalizing effects of BC on soil pH could be explained by the poor buffering capacity of soil induced by the low levels of soil organic matter.

Table 4

The influences of the applied Olive Mill Solid Waste-derived Biochars (OMSW-BCs) on the soil pH and EC, and the soil concentrations of NH4OAc- and Ammonium Bicarbonate-DiethyleneTriaminePentaacetic Acid (AB-DTPA)-extractable nutrients.

Treatments1	pH	EC	NH4OAc-extractable nutrients				AB-DTPA-extractable nutrients
			K	Na	Ca	Mg	P	Fe	Mn	Cu	Zn
		dS m-1	. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . .. . .mg kg-1. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .. . . .
CK	7.85a2	2.03a	40.5a	26.0ab	8,140a	105a	10.09a	0.07a	0.66a	UDL3	0.31ab
INF	7.88a	2.08ab	65.0b	26.0ab	8,080a	99.3a	11.37a	0.07a	0.46bc	UDL	0.23a
BC300-1%	8.05b	2.04a	102c	24.8ac	8,077a	94.8a	10.83a	0.09a	0.57ab	UDL	0.30ab
BC300-3%	8.28c	2.15cd	209d	27.8bd	8,090a	93.9a	10.92a	0.06a	0.66a	UDL	0.34ab
BC500-1%	8.15d	2.16cd	130e	27.0bd	8,005a	98.4a	10.51a	0.06a	0.40c	UDL	0.35bc
BC500-3%	8.31c	2.21e	324f	32.5e	7,943a	91.1a	10.14a	0.05a	0.49bc	UDL	0.27ab
BC700-1%	8.11bd	2.10b	135e	26.8ab	8,003a	99.5a	10.73a	0.17b	0.42c	UDL	0.28ab
BC700-3%	8.32c	2.20cde	360j	30.8e	9,962b	134b	11.22a	0.22b	0.51bc	UDL	0.45c
Analysis of variance (ANOVA)
F-value	44.646	18.448	333	7.719	1.774	2.601	1.038	6.717	6.841	-	3.225
P-value	0.0000	0.0000	0.0000	0.0003	0.1620	0.0537	0.4434	0.0008	0.0007	-	0.0248

1. CK: control; INF: inorganic fertilizer (NPK); BC300-1%: biochar produced at 300°C and added at 1% application rate; BC300-3%: biochar produced at 300°C and added at 3% application rate; BC500-1%: biochar produced at 500°C and added at 1% application rate; BC500-3%: biochar produced at 500°C and added at 3% application rate; BC700-1%: biochar produced at 700°C and added at 1% application rate; BC700-3%: biochar produced at 700°C and added at 3% application rate.

2. Different letters indicate significant differences according to the LSD test (p = 0.05, n = 3).

3. UDL: under detection limit.

1. CK: control; INF: inorganic pan class="Chemical">fertilizer (NPK); BC300-1%: biochar produced at 300°C and added at 1% application rate; BC300-3%: biochar produced at 300°C and added at 3% application rate; BC500-1%: biochar produced at 500°C and added at 1% application rate; BC500-3%: biochar produced at 500°C and added at 3% application rate; BC700-1%: biochar produced at 700°C and added at 1% application rate; BC700-3%: biochar produced at 700°C and added at 3% application rate.

2. Difpan class="Chemical">ferent letters indicate significant difn>an class="Chemical">ferences according to the LSD test (p = 0.05, n = 3).

In this study, the EC values for control soils were 2.03 dS m-1 (CK) and 2.08 dS m-1 (CK+NPK) (Table 4). These EC values increased to 2.15 dS m-1 for 3% pan class="Chemical">BC300, to 2.16 (BC500 at 1%) and 2.21 dS m-1 (BC500 at 3%), and to 2.10 (BC700 at 1%) and 2.20 dS m-1 (BC700 at 3%). It has been previously reported that a high level of soil salinity following BC application was caused by the introduction of high levels of soluble ions derived from BC ash into the soil [39].

BC effects on soil nutrient availability

The incorporation of pan class="Chemical">BC into soils can affect soil chemistry, causing changes in the available fraction of soil P, mainly because of alterations in soil pH and cation exchange capacity (CEC). Although soil pH in the current study increased because of the application of OMSW-derived BC, soil AB-DTPA-extractable P concentrations exhibited some increases in the INF and BC treatments compared to that of the control soil, but they were not significant (Table 4). For example, inorganic fertilizer treatment increased soil available P by 1.28 mg kg-1, whereas the BC treatments increased its availability by 0.05–1.13 mg kg-1. In a meta-analysis study conducted by Glaser and Lehr [17] on the availability of soil P, the addition of BCs to acidic and neutral soils significantly enhanced the availability of soil P, whereas there was no significant effect in alkaline soils. Thus, contrary to the effects in acidic soils, it would be advisable to apply acidic biochar (not alkaline biochar) to alkaline soils with P constraints to improve the levels of plant-available P.

pan class="Chemical">NH4OAc-extractable K increased significantly by pan class="Chemical">BC application to the soil (Table 4). In INF-treated soil, the soil exchangeable content of K significantly increased from 40.5 to 65.0 mg kg-1. However, because of the application of BCs, the values of soil exchangeable K content greatly increased from 40.5 (CK) to 102 mg kg-1 (1% BC300), 209 mg kg-1 (3% BC300), 130 mg kg-1 (1% BC500), 324 mg kg-1 (3% BC500), 135 mg kg-1 (1% BC700), and 360 mg kg-1 (3% BC700). Notably, significant differences among BC treatments were found in the soil content of exchangeable K. Therefore, both pyrolysis temperature and application rates of BCs had a significant effect on the increase in the available form of soil K. Additionally, application of high rates of BCs pyrolyzed at high temperature (500°C and 700°C) exhibited greater significant increases in soil exchangeable Na than that in the control soil (Table 4), and exhibited increases from 26.0 mg kg-1 (CK) to 32.5 mg kg-1 (3% BC500) and 30.8 mg kg-1 (3% BC700). The experimental results of Jien and Wang [40] indicated that the levels of exchangeable K were significantly enhanced in BC-treated soil, suggesting that it improved the level in the soil. Our results suggested that OMSW-derived BC itself might be a K source, and thus, it enhanced its bioavailability in soils. It has also been reported that BC application could increase the concentrations of exchangeable Ca and Mg in the soil [40]. In the present study, among all BC treatments, only 3% BC700 resulted in significant increases in the concentrations of NH4OAc-extractable Ca and Mg by 22.4% and 27.9% compared to that of the control soil, respectively. This suggests that the availability of soil Ca and Mg in OMSW-derived BC could likely depend on both pyrolysis temperature and application rate. The high content of soil exchangeable basic cations in the high pyrolysis-temperature BC-treated soil could be explained by the increasing content of BC ash. Several other researchers have explained the improvements in the exchangeability of soil cations because of the presence of ashes in BC, which contain high levels of oxides and hydroxides of alkali cations [41].

Regarding micronutrient availability in soil, pan class="Chemical">BC significantly afpan class="Chemical">fected the soil concentrations of AB-DTPA-extractable micronutrients, depending upon pyrolysis temperature and application rates (Table 4). The concentrations of AB-DTPA-extractable Mn in INF, BC500, and BC700 treatments were lower than that in the CK. Although BC treatments caused significant increases in soil pH, application of BC pyrolyzed at the highest temperature (BC700) resulted in a significant improvement in the soil concentrations of AB-DTPA-extractable Fe (at application rates of 1% and 3%) and Zn (at an application rate of 3%). The soil concentrations of AB-DTPA-extractable Fe increased significantly from 0.07 mg kg-1 in the CK to 0.17 mg kg-1 with 1% BC700 and 0.22 mg kg-1 with 3% BC700, whereas AB-DTPA-extractable Zn increased significantly from 0.31 mg kg-1 in the CK to 0.45 mg kg-1 with 3% BC700. Among all micronutrients, soil concentrations of AB-DTPA-extractable Cu were undetectable by ICP-OES.

BC effects on dry matter and mineral content of maize plants

Statistically, pan class="Chemical">BC addition did not significantly affect plant growth parameters (fresh and dry weight and plant height) (Table 5). Similarly, Farrell et al. [42] found no improvement in wheat yield in calcareous soils because of BC addition. They suggested that the addition of BC to calcareous soil did not lead to better conditions for the uptake of plant nutrients. In this context, the results of the current study showed that the shoots of maize plants in BC-amended soils had significantly lower content of P, Ca, and Mg, especially with increasing application rate. In soil treated with high application rate (3%) of OMSW-derived BC, the shoot P content decreased from 2.24 g kg-1 (CK) to 1.68 g kg-1 (BC300), 1.82 g kg-1 (BC500), and 1.88 g kg-1 (BC700). The shoot content of Ca decreased from 13.06 g kg-1 in the CK to 10.50 g kg-1, 7.64 g kg-1, and 6.23 g kg-1 for BC300, BC500, and BC700 at 3% application rate, respectively. Furthermore, the shoot content of Mg decreased from 10.6 g kg-1 in the CK to 5.08, 3.88, and 3.71 g kg-1 for BC300, BC500, and BC700 at 3% application rate, respectively. In alkaline soils, calcareous substances can lead to the formation of insoluble compounds of Ca-Mg-P, decreasing the shoot content of these nutrients [43]. On the other hand, the great quantity of K obtained because of OMSW-derived BC could result in reduced plant uptake of Ca, Mg, and P by an antagonistic effect [44, 45]. Contrary to our results, several reports found increases in the plant content of nutrients [41, 46]. The discrepancy between our data and that of other studies could be explained because of varying soil characteristics and feedstock used to produce BCs. Our results suggested that the incorporation of OMSW-derived BCs into alkaline soils may limit the essential nutrient (such as Ca, Mg, and P) uptake by plants, and require the additional input of these nutrients (especially P fertilizer). Further research on OMSW-derived BCs is needed to clarify the mechanism responsible for governing uptake of these nutrients by plants.

Table 5

Olive Mill Solid Waste-derived Biochar (OMSW-BCs) effects on plant height, fresh and dry matter, and shoot mineral content of Zea mays.

Treatments1	Plant height (cm)	Plant fresh weight (g plant-1)	Plant dry weight (g plant-1)	K	Na	P	Ca	Mg	Fe	Mn	Zn	Cu
				g kg-1					mg kg-1
CK	54.5ab2	2.37a	0.21a	15.1a	0.14a	2.24a	13.06ab	10.6a	108a	49.5ab	64.5a	8.94a
INF	54.3ab	2.56a	0.22a	23.4ab	0.16ab	2.41ab	16.60c	9.51b	106a	53.9a	76.3ab	8.22a
BC300-1%	54.2ab	2.59a	0.20a	33.2bc	0.26ac	1.71c	12.37bd	6.45c	91.3a	43.1cd	74.3ab	7.39a
BC300-3%	50.2b	2.58a	0.18a	56.1de	0.30c	1.68c	10.50d	5.08d	105a	39.4d	71.9ab	9.67a
BC500-1%	53.6ab	2.76a	0.21a	49.9df	0.28bc	2.18ab	11.61bd	5.90ce	105a	47.1bc	81.8b	8.11a
BC500-3%	49.7b	2.53a	0.20a	65.3ej	0.31c	1.82c	7.64e	3.88f	92.4a	43.1cd	79.3b	7.56a
BC700-1%	59.4a	3.17a	0.24a	39.4cf	0.51d	2.17ab	11.35bd	6.33c	108a	46.2bc	77.4b	7.72a
BC700-3%	49.4b	2.62a	0.21a	73.7j	0.79e	1.88c	6.23e	3.71f	97.7a	40.2d	69.2ab	7.22a
Analysis of variance (ANOVA)
F-value	1.483	0.7809	0.3612	31.258	23.692	10.113	24.560	96.687	0.8794	7.672	1.790	0.8221
P-value	0.2421	0.6126	0.9117	0.0000	0.0000	0.0001	0.0000	0.0000	0.5436	0.0004	0.1584	0.5832

1. CK: control; INF: inorganic fertilizer (NPK); BC300-1%: biochar produced at 300°C and added at 1% application rate; BC300-3%: biochar produced at 300°C and added at 3% application rate; BC500-1%: biochar produced at 500°C and added at 1% application rate; BC500-3%: biochar produced at 500°C and added at 3% application rate; BC700-1%: biochar produced at 700°C and added at 1% application rate; BC700-3%: biochar produced at 700°C and added at 3% application rate.

2. Different letters indicate significant differences according to the LSD test (p = 0.05, n = 3).

1. CK: control; INF: inorganic pan class="Chemical">fertilizer (NPK); BC300-1%: biochar produced at 300°C and added at 1% application rate; BC300-3%: biochar produced at 300°C and added at 3% application rate; BC500-1%: biochar produced at 500°C and added at 1% application rate; BC500-3%: biochar produced at 500°C and added at 3% application rate; BC700-1%: biochar produced at 700°C and added at 1% application rate; BC700-3%: biochar produced at 700°C and added at 3% application rate.

2. Difpan class="Chemical">ferent letters indicate significant difn>an class="Chemical">ferences according to the LSD test (p = 0.05, n = 3).

Contrary to that of shoot P, Ca, and pan class="Chemical">Mg, OMSW-derived BCs treatments enhanced the levels of K and Na in plant shoots (Table 5). In soil treated with 1% and 3% OMSW-derived BC, respectively, the shoot K content increased from 15.1 g kg-1 (CK) and 23.4 g kg-1 (INF) to 33.2 and 56.1 g kg-1 (BC300), 49.9 and 65.3 g kg-1 (BC500), and 39.4 and 73.7 g kg-1 (BC700). This indicated that shoot K content increased with increasing pyrolysis temperature and application rate. In this study, soil exchangeable K was also significantly increased following BC application and it increased with both increasing pyrolysis temperature and application rate, reflecting enhanced K uptake by plant shoots. It has been reported that a high quantity of K in BCs could enhance the bioavailable pool of K in the soil [47]. Application of BCs might also improve soil mineral K release by enhancing the activity of K-dissolving bacteria and facilitating K uptake by crops [48]. Syuhada et al. [49] found that the K content in corn tissue was significantly higher but its tissue content of N, Ca, and Mg was lower in BC-amended plants than that of the control. They attributed the high tissue K content in BC treatments to increasing exchangeable soil K.

pan class="Chemical">BCs exhibited varying efpan class="Chemical">fects on the shoot content of micronutrients, depending upon the type of micronutrient. Statistically, BC addition did not have a significant effect on the shoot levels of Fe and Cu (Table 5). However, the addition of BCs (especially at the highest application rate) significantly decreased the shoot Mn levels in comparison with that of the CK. Conversely, application of 1% and 3% BC500 and 1% BC700 significantly enhanced the level of shoot Zn.

Recommendations and suggestions for future trials

This study was conducted to evaluate the effects of application of OMSW-derived BCs (in relation to pyrolysis temperature and application rates) on soil pH, EC, availability of soil nutrients to plants, and maize growth in arid alkaline soil. The results showed that the properties of BC were affected by increasing pyrolytic temperatures, reflecting on soil pH, EC, and the performance of soil nutrients availability to plants. The application of OMSW-derived BC decreased the levels of Ca, Mg, Mn, and P in plant shoots and enhanced the levels of shoots K, Na, and Zn. The high K content in BC treatments suggests high agronomic value in terms of replacing conventional K sources. However, despite the high soil available concentrations of K, the significant increase in soil pH values and decrease in P, Ca, Mg, and Mn of plant tissues because of the application of BC should not be ignored as these nutrients are of substantial importance in terms of agronomic potential. The quantity of K incorporated by OMSW-derived BC could negatively reflect the decreasing nutrient uptake by plants, as indicated in this study for the decrease in shoot content of P, Ca, Mg, and Mn. For OMSW-derived BC, lowering of shoot nutrient content may likely limit its use, and thus, care should be taken in its use under arid conditions. Collectively, our findings suggested that application of OMSW-derived BCs may not be able to provide sufficient nutrients to enhance plant growth; however, because of high alkalinity, OMSW-derived BC may be applied as a soil additive to acidic soils rather than alkaline ones. In the current study, OMSW-derived BCs were applied to a calcareous soil in unwashed form, which may have an opposite impact on the performance of soil properties and nutrients availability. Therefore, further studies on OMSW-derived BCs in washed form are needed to evaluate its effects on a wide range of alkaline and calcareous soils with varied properties in comparison with unwashed OMSW-derived BCs. Further studies are also required on the effects of OMSW-derived BC in combination with inorganic fertilizers on the antagonistic effect induced by the high quantity of K and the properties and fertility of arid-land soils.

FTIR spectra of the produced Olive Mill Solid Waste-derived Biochar (OMSW-BCs) (BC300: biochar produced at 300°C; BC400: biochar produced at 400°C; BC500: biochar produced at 500°C; BC600: biochar produced at 600°C; BC700: biochar produced at 700°C).

(DOCX)

Click here for additional data file.

XRD spectra of the produced Olive Mill Solid Waste-derived Biochar (OMSW-BCs) (BC300: biochar produced at 300°C; BC400: biochar produced at 400°C; BC500: biochar produced at 500°C; BC600: biochar produced at 600°C; BC700: biochar produced at 700°C).

(DOCX)

Click here for additional data file.

Scanning electron microscope (SEM) analyses of feedstock (FS) and olive mill solid waste-derived biochars (OMSW-BCs) pyrolyzed at different temperatures (a: FS: feedstock; b: BC300: biochar produced at 300°C; c: BC400: biochar produced at 400°C; d: BC500: biochar produced at 500°C; e: BC600: biochar produced at 600°C; f: BC700: biochar produced at 700°C).

(DOCX)

Click here for additional data file.

Model fitting for the produced Olive Mill Solid Waste-derived Biochar (OMSW-BCs) properties in relation with pyrolysis temperatures.

(DOCX)

Click here for additional data file.

1 Jun 2020

pan class="Chemical">PONE-D-20-11578

Potential short-term negative versus positive efpan class="Chemical">fects of pan class="Species">olive mill-derived biochar on nutrient availability in a calcareous loamy sandy soil

PLOS ONE

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Reviewer #1: This manuscript does 2 things: First it chemically characterized biochars produced by the pyrolysis of pan class="Species">olive mill waste and second, it compares pan class="Species">zea mays growth in soils with biochar (and controls). The title and abstract stress the greenhouse experiment, yet the results and discussion focus on the chemical analysis of biochar. It is overly long, but also seems lacking a lot of details. It is very repetitive. For it to be acceptable for publications the authors need to re-envision the paper and make it much more clear.

The introduction spends a lot of space talking about what biochar can do for the soil in a really general way. There is so many papers out there on this already you only need to reference them briefly and move on. Instead use the introduction to educate pan class="Species">people about what specifically you are actually doing- . What do we already know about how different production temperatures effect biochar – its nutrients, mass, etc? Then lay out predictions for how you would expect your materials to act. Similarly, this needs to be done for plants and soil nutrients – there are so many studies (and meta-analyses) that describe how biochar effects these things.

Yes, this paper is in alkaline soils and that is an interesting aspect, but you need to lay out what we know and then how do we expect that to change in alkaline soils.

THEN your results should only fopan class="Chemical">cus on those things laid out in the introduction. Did it do what you expn>ected? Why or why not?. For instance there is no need to reiterate your design or anything in the dispan class="Chemical">cussion, implications and conclusions. This should be simply laid out in the introduction (which is just a page or two away)

Minor things

I cannot speak to the chemical analysis of the biochar (XRD etc) I assume this was performed well.

The statistics section requires much more detail

Please indicate in the tables which values are difpan class="Chemical">ferent from each other

Recommendations and conclusions are redundant. The paper is not that long and the conclusions are just not needed.

Line 63 – mitigate climate– add change

Lines 72-75 These sentences are contradictory as one says there has not been many studies in alkaline soils and then the next sentence sites three. I would just delete the last sentence

Line 77 – delete “on the other hand” (there is no first hand)

Lines 82-87 should be tacked on to the paragraph above it. The last paragraph of the sentence should start “Previous studies…”

Line 93 - Replace “the aims of this study were to investigate” to “We investigated”. -this gets rid of redundant words and is in an active voice.

Delete line 143-144 – one can just put “Table 1” at the end of the next sentence

All tables need to be formatted similarly and to the journal’s standards.

Line 152- what is basicity (do you mean pH?)

Line 209 –This paragraph is very confusing and overly wordy – please simplify

Reviewer #2: The production of pan class="Chemical">BC from OMSW is an important area of research

In the section of Material and methods it is not clear if the pan class="Chemical">BC was washed before being mixed with the soil, otherwise some of contaminants were introduced into the soil

From the beginning you selected alkaline soil and the pan class="Chemical">BC is going to increase that. Did you try some acidic soil which is more suitable for your pan class="Chemical">BC?? Why not from the beginning you selected to compare acidic vs alkaline soil?? Your BC is better suit acidic soil

The Zeta potential was not measured and data should be supplied, otherwise it is diffipan class="Chemical">cult to expn>lain why for example the nutrients such as Ca, pan class="Chemical">Mg and P and others were limited to the plants and the BC was unable to adsorb them

The Tables 1 and 2 no mention how many replicates, SD and statistical analysis such as LSD are available. You are talking about significance, but I can't see that.

I don't see data concerning N content in all soils especially you used NPK in the INF treatment.

You see from the results (for example Table 5) that when you have increased the soil amendment from 1% to 3% you see increase in the negative efpan class="Chemical">fects on plant parameters which means you have contamination in your pan class="Chemical">BC which means you should wash it!!!

Might be that your supplied pan class="Chemical">BC have afpan class="Chemical">fected your soil properties and plant growth as well. Accordingly, your results are indicating that BC supplementation didn't improve plant growth parameters.

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Reviewer #2: No

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7 Jun 2020

Response to Reviewers

Dear Editor of PLOS ONE

On behalf of the authors of the manuscript entitled "Potential short-term negative versus positive efpan class="Chemical">fects of pan class="Species">olive mill-derived biochar on nutrient availability in a calcareous loamy sandy soil", I wish to express my respects for you and for the reviewers for reviewing this manuscript. The authors have modified the manuscript based on the valuable reviewer’s comments and responses to reviewers’ comments are addressed point by point and given below.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional information regarding the permits you obtained to collect samples for the present study. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

3. Please amend the manuscript submission data (via Edit Submission) to include author Hesham Ibrahim.

Response: All above requirements have been done and/or provided.

Reviewer #1:

Specific comments:

Comment 1: This manuscript does 2 things: First it chemically characterized biochars produced by the pyrolysis of pan class="Species">olive mill waste and second, it compares pan class="Species">zea mays growth in soils with biochar (and controls). The title and abstract stress the greenhouse experiment, yet the results and discussion focus on the chemical analysis of biochar. It is overly long, but also seems lacking a lot of details. It is very repetitive. For it to be acceptable for publications the authors need to re-envision the paper and make it much clearer.

Response: We are thankful to the reviewer for the critical comments of the reviewer. Based on your valuable comments, we revised our manuscript.

Comment 2: The introduction spends a lot of space talking about what biochar can do for the soil in a really general way. There is so many papers out there on this already you only need to reference them briefly and move on. Instead use the introduction to educate pan class="Species">people about what specifically you are actually doing- . What do we already know about how different production temperatures effect biochar – its nutrients, mass, etc? Then lay out predictions for how you would expect your materials to act. Similarly, this needs to be done for plants and soil nutrients – there are so many studies (and meta-analyses) that describe how biochar effects these things.

Yes, this paper is in alkaline soils and that is an interesting aspect, but you need to lay out what we know and then how do we expect that to change in alkaline soils. THEN your results should only fopan class="Chemical">cus on those things laid out in the introduction. Did it do what you expected? Why or why not?. For instance there is no need to reiterate your design or anything in the dispan class="Chemical">cussion, implications and conclusions. This should be simply laid out in the introduction (which is just a page or two away).

Response: We are highly thankful to the reviewer for valuable suggestions. The introduction has been revised extensively in the revised version of the manuscript. More fopan class="Chemical">cus has been drawn on the information related to the work performed in this study. The information about the impacts of biochar with difpan class="Chemical">ferent pyrolyzing temperature has been added. More information on expected performance of the biochar in calcareous alkaline soils has been added now. Moreover, the possible negative impacts of biochar are also mentioned as the performance of biochar is not always consistent. Thus, revised version is now possessing improved quality.

Minor things

Comment 1: I cannot speak to the chemical analysis of the biochar (XRD etc) I assume this was performed well.

Response: Thanks

Comment 2: The statistics section requires much more detail. Please indicate in the tables which values are difpan class="Chemical">ferent from each other.

Response: More detail information on statistical section were provided in the revised version.

Comment 3: Recommendations and conclusions are redundant. The paper is not that long and the conclusions are just not needed.

Response: Based on your suggestion, the conclusions section has been deleted and the section of recommendation has been improved in the revised version.

Comment 4: Line 63 – mitigate climate– add change

Response: Thanks, and it has been inserted.

Comment 5: Lines 72-75 These sentences are contradictory as one says there has not been many studies in alkaline soils and then the next sentence sites three. I would just delete the last sentence.

Response: The sentence has been deleted.

Comment 6: Line 77 – delete “on the other hand” (there is no first hand).

Response: it has been deleted.

Comment 7: Lines 82-87 should be tacked on to the paragraph above it. The last paragraph of the sentence should start “Previous studies…”

Response: Thanks for the reviewer comment and this paragraph has been improved in the revised version.

Comment 8: Line 93 - Replace “the aims of this study were to investigate” to “We investigated”. -this gets rid of redundant words and is in an active voice.

Response: It has been replaced.

Comment 9: Delete line 143-144 – one can just put “Table 1” at the end of the next sentence.

Response: Thanks for the reviewer comment and the mentioned sentence has been deleted.

Comment 10: All tables need to be formatted similarly and to the journal’s standards.

Response: All tables has been formatted according to the journal’s standards.

Comment 11: Line 152- what is basicity (do you mean pH?)

Response: Yes, the basicity reflects on increasing the value of pH. Therefore, word of basicity replaced by pH in the revived version.

Comment 12: Line 209 –This paragraph is very confusing and overly wordy – please simplify

Response: Thanks for the reviewer comment and the mentioned sentence has been rewritten and simplified.

Reviewer #2:

Comment 1: The production of pan class="Chemical">BC from OMSW is an important area of research

In the section of Material and methods it is not clear if the pan class="Chemical">BC was washed before being mixed with the soil, otherwise some of contaminants were introduced into the soil.

Response: Thanks for the reviewer comment and we agree with you that washed and unwashed biochar may have difpan class="Chemical">ferent behaviour and impacts on soil and plant. In the pan class="Chemical">current study, we used unwashed BC samples. We clarified it in the section of material and methods.

Comment 2: From the beginning you selected alkaline soil and the pan class="Chemical">BC is going to increase that. Did you try some acidic soil which is more suitable for your pan class="Chemical">BC?? Why not from the beginning you selected to compare acidic vs alkaline soil?? Your BC is better suit acidic soil

Response: Because this work was carried out in the arid region where soils have high pH value and high pan class="Chemical">CaCO3 content and there are no acidic soils under our condition. Therefore, our team of the biochar groupn> is interested in studying biochar as a soil amendment under alkaline soil conditions.

Comment 3: The Zeta potential was not measured and data should be supplied, otherwise it is diffipan class="Chemical">cult to expn>lain why for example the nutrients such as Ca, pan class="Chemical">Mg and P and others were limited to the plants.

Response: Based on your valuable comment, we measured the values of zetal potential for pan class="Chemical">BC samples and inserted in the revised version. However, we would like to clarify that there are other reasons could be responsible for nutrients limiting to plants (such as alkalinity) due to biochar apn>plication, especially with increasing pyrolysis temperature, as we found that the values of zeta potential tented to decrease with increasing pyrolysis temperature.

Comment 4: The Tables 1 and 2 no mention how many replicates, SD and statistical analysis such as LSD are available. You are talking about significance, but I can't see that.

Response: Thanks for the reviewer comment and the ±SD values are incorporated in Tables 1 and 2 because the part of biochar characterization carried out in duplicate. We clarified it in the section of material and methods.

Comment 5: I don't see data concerning N content in all soils especially you used NPK in the INF treatment.

Response: Unfortunately, the soil N availability and its content in plant shoots were not measured in the present work. However, we would like to clarify that the aim of this study was to fopan class="Chemical">cus on the efpan class="Chemical">fects of biochar on essential nutrients that mainly face problems (especially that subjected to fixation such as P and micronutrients) in alkaline soils and can change with changing soil pH. In addition, in the current study, the recommended dose of NPK was applied in this study because this is the common practice used by the farmers.

Comment 6: You see from the results (for example Table 5) that when you have increased the soil amendment from 1% to 3% you see increase in the negative efpan class="Chemical">fects on plant parameters which means you have contamination in your pan class="Chemical">BC which means you should wash it!!!

Might be that your supplied pan class="Chemical">BC have afpan class="Chemical">fected your soil properties and plant growth as well. Accordingly, your results are indicating that BC supplementation didn't improve plant growth parameters.

Response: Thanks for the reviewer comment and we agree with you that washed and unwashed biochar may have different behaviour and impacts on soil and plant. In the pan class="Chemical">current study, we used unwashed BC samples. In term of applying BC to agricultural soils, unwashed biochar would be mostly added because most of the fresh and unwashed biochars were a source of nutrients. And, our findings suggest that application of OMSW-derived BCs may not be able to provide sufficient nutrients to enhance plant growth. However, based in your comment, this point regarding washed BC will be taken into our account in the future work. Therefore, we referred to this point regarding washed biochar in section of recommendations and suggestions for future trials in the revised version of our manuscript. Thanks again.

Submitted filename: Response to reviewers comments.docx

Click here for additional data file.

15 Jun 2020

Potential short-term negative versus positive efpan class="Chemical">fects of pan class="Species">olive mill-derived biochar on nutrient availability in a calcareous loamy sand soil

pan class="Chemical">PONE-D-20-11578R1

Dear Dr. Al-wabel,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please repan class="Chemical">fer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supn>porting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. pan class="Species">participant privacy or use of data from a third party—those must be specified.

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Reviewer #1: They did a great job revising the manuscript. I am require to write 100 characters so I am just typing something.

Reviewer #2: AS I mentioned in my comments, it is important to wash the pan class="Chemical">BC before being used to pan class="Chemical">cultivate plants. I understood that you didn't wash the BC before planting in the pots which may cause negative effects on plants. The main points/comments I have raised were corrected which make the paper more suitable for publications.

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Reviewer #1: No

Reviewer #2: Yes: Prof. Hassan Azaizeh, Tel Hai Academic College, Upper Galilee 12210, Israel

18 Jun 2020

pan class="Chemical">PONE-D-20-11578R1

Potential short-term negative versus positive efpan class="Chemical">fects of pan class="Species">olive mill-derived biochar on nutrient availability in a calcareous loamy sand soil

Dear Dr. Al-Wabel:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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