Residual Effect of Finely-Ground Biochar Inoculated with Bio-Fertilization Impact on Productivity in a Lentil–Maize Cropping System

Abstract

Biochar fertilization improves soil fertility and carbon sequestration, implying agricultural and environmental advantages. The effect of different sized previously applied biochar and biofertilizer agents on succeeding crops remains poorly known for legume–cereal cropping cycles. This study compared different particle-sized biochar and biofertilizer strains applied to lentils for their residual impact on subsequent maize growth, nutrition, and soil fertility without further polluting the environment. Three particle sizes (<2, 2–5, 5–10 mm) of Babul tree (Acacia arabica) wood biochar was obtained through grinding and sieving and applied prior to the lentil (first) crop at a rate of 500 g m⁻². The commercial Rhizobium leguminosarum products Biozote-N and Rhizogold were inoculated to lentil seeds before sowing. The effect of biochar and biofertilizer agents on the succeeding maize (second) crops was evaluated for soil and crop performance. Findings revealed that particle sizes of <2 mm biochar and Biozote-N inoculation enhanced plant height, leaf area and leaf area index, biological yield, and thousand grain weight of the subsequent maize crop. Maize grain yield was enhanced by 2.5%, tissue N uptake by 15%, nitrogen uptake efficiency by 17%, grain protein content by 15%, extractable P by 17%, and soil bulk density by 3% with a residual biochar particle size of <2 mm and Biozote-N inoculation. It was concluded that the finely grounded (<2 mm) biochar particle combined with inoculation of Biozote-N was superior to larger particle sizes for enhancing crop growth and improving soil fertility status at the residual level, benefiting the subsequent crop in a legume–cereal rotation system.

1. Introduction

Biochar added to soil is beneficial due to carbon sequestration and long-term storage [1]. Biochar has several other environmental benefits, including improving soil properties, mitigating essential nutrients losses [2], soil biology [3], and heavy metal uptake [4]. These benefits lead to improved crop photosynthetic pigments and ultimately enhanced crop yield. Low fertility soils, especially, respond well to biochar application, since it can improve soil nutrient content for higher agronomic performance and soil health [5]. Biochar, as a thermo-chemically transformed biomass [6], possesses a variable rate of decomposition of different components under different conditions, whilst under natural conditions, it shows slow decomposition [7]. Therefore, its residual effects on plant growth parameters become more prominent after several months of its application. A one-time application may last for three cropping cycles and influences soil fertility and crop yield [8] and may fade down after multiple crop seasons [9].

In addition to structural improvements and the formation of soil aggregates, biochar addition improves soil organic C, CEC, and pH [8,10,11]; reduces leaching of N, P, and gaseous losses of N₂O and NH₃ [12]; and leads to enhanced plant growth and yield. Reduced-particle-size biochar improves nutrient availability and soil properties [13]. The reduced size of the applied biochar particle is associated with increased exposed surface area and improved reactivity in soil for adsorption of excess accessible nutrients from the soil solution and reducing their losses [14,15,16], resulting in a significant enhancement in the crop performance. The liming effects and nutrient exchange of biochar, as well as their ability to increase water storage capacity, have been suggested to be improved by decreasing the particle size of biochar [17]. Biochar with smaller particle sizes have a greater potential to boost water storage capacity while also improving liming effects due to more ash content and nutrient exchange [18]. Biochar with smaller particle sizes improves the sorption of nutrients and organic compounds [19]. Smaller particle size biochar may have more plant nutrient availability because of their increased micro-porosity, surface area, and exterior surface area [17].

Bio-fertilizers are promising replacements to reduce dependence on costly chemical fertilizers and maintain long-standing soil health [20]. These may contain a single type or a mixture of different types of useful micro-organisms that mobilize the plant’s nutrients and enhance their bio-availability for improved plant nutrition [21]. Therefore, selection of the potential right bio-fertilizer type concerning crop genotype and physiological state is very important [22]. The residual effect of bio-fertilizers on soil fertility status was also reported to be highly significant by Ali et al. [13]. Owing to the importance of biofertilizers on legumes to improve atmospheric nitrogen fixation, different research and teaching organizations have isolated rhizobium strains from lentils and introduced their biofertilizer products. Two such products are Biozote-N and Rhizogold released for commercial use on lentils.

The role of biochar and biofertilizers in agricultural sustainability can be effective and highly significant for future agricultural production, particularly in areas of poor soil fertility. However, very little information regarding the use of biochar of a suitable size is available, particularly its residual effects in combination with biofertilizer agents, and its legacy effects in the succeeding crops. Our work focused on comparing different particle sizes of biochar for effectiveness when applied to the soil at the same rate across two cropping cycles (lentil–maize). It was hypothesized that reducing the particle size of the biochar increases its long-term effectiveness for soil improvement and crop yield, and the reduced particle size has a higher residual effect on the next crop to improve its yield and soil properties. This study reports the residual effects of different biochar particle sizes and inoculation to preceding lentil crops in terms of improved growth and yield of the following maize crop, post-harvest soil properties, and soil fertility without polluting the soil environment.

2. Material and Methods

2.1. Cropping History of the Site

Lentil (Lens culinaris M.) was sown during winter in mid-November 2018 on the same field prior to the maize experiment and harvested at maturity (data not shown in this paper) during April 2019. The same treatment layout was retained for the succeeding maize (Zea mays L.) crop for assessing the residual effect of particle sizes of biochar and biofertilizer strains applied to previous lentils. Maize (cultivated variety: Iqbal) was sown and reared as per recommended agronomic management practices (75 and 20 cm row–row and plant–plant distance; 120, 90, 60 kg ha⁻¹ N, P₂O₅, K₂O application, respectively, to all treatment plots as basal dose with three splits of N and irrigation on an as needed basis). The details of the study site are given in

Table 1. (A) Characteristics of soil in the study site before experiment commencement. (B) Analysis of the biochar before the experiment.

(A)
Parameter	Unit	Value/Remarks
Texture	-	Silt loam
USDA Classification	Fine loamy, mixed, thermic, typic hapludalfs
Bulk density	g cm−3	1.3
Moisture percentage	%	8.3
Organic carbon	g kg−1	3.2
Total N	″	0.1
C/N ratio	-	32:1
pH1:5	-	7.78
EC1:5	dS m−1	0.53
AB-BTPA extracted
K	mg kg−1	72
P	″	4.32
Fe	″	0.9
Cu	″	0.5
Zn	″	0.45
Mn	″	1.52
(B)
Parameter	Unit	Value
Moisture	%	0.65
C	″	64.3
N	g kg−1	0.72
K	″	0.42
P	″	0.25
pH(1:5)	-	8.2
EC(1:5)	dS m−1	0.65

Source: Authors (soil and environmental sciences laboratory, Amir Muhammad Campus Mardan, Pakistan). “″” represents same unit as mentioned for the above trait.

A.

2.2. Biochar Particle Size and Rhizobia Strains

Acacia (Acacia Arabica L.) wood biochar was purchased from the market, analyzed for physical and chemical properties (Table 1B), ground, and separated into different sized particles using 2 mm, 5 mm, and 10 mm sieves and applied to previously sown lentil crops at a rate of 500 g m⁻². The seeds of lentil were treated with respective biofertilizers at a rate of 0.5%. Biofertilizer products (Biozote-N and Rhizogold) were received from respective research laboratories of the National Agriculture Research Council (NARC), Islamabad, and the University of Agriculture, Faisalabad, Pakistan. Biozote-N (Rhizobium inoculum-TAL 169) is a biofertilizer developed by the Pakistan National Agriculture Research Council (NARC) and consists of a formulation having diverse groups of microorganisms able to fix atmospheric nitrogen in soil [23,24]. “Rhizogold” (RG) is another biofertilizer developed by Islamabad, Soil Microbiology and Biochemistry group at University of Agriculture Faisalabad and contains a mix-culture of Rhizobium and ACC deaminase containing plant growth promoting rhizobacteria (PGPR) [25]. The biofertilizer-inoculated (Biozote-N and Rhizogold) seeds were dried under the shade and sown immediately in main plots with one biofertilizer control (Table 1B). The experiment was arranged in split-plot design replicated three times with sub-plot sizes of 3 m × 4 m.

2.3. Data Collection

Five plants per plot were randomly selected to record growth and yield data. The measuring tape was stretched from base to collar leaf to measure plant height. Leaf area (cm²) was measured at recently matured leaves as length × breadth × 0.75. Leaf area index was determined from total leaf area per plot (cm²) divided by ground area per plot. At harvest, the crop was sun-dried and weighed consecutively for 5 days until there was no considerable loss in weight. Cobs were threshed manually, and grains were air-dried again. For biological and grain yield (kg ha⁻¹), the whole plot was harvested. One hundred normal grains were counted randomly from grain yield, weighed, and multiplied by 10 to record 1000 grain weight.

2.4. Soil Sampling and Analysis

For determination of soil bulk density, total porosity, saturation water content, and particle size analysis, protocols described in Methods of Soil, Plant, and Water Analysis [26] were followed. In brief, soil core samples of known volume (Vt; 100 cm³) and disturbed samples through an auger from 0 to 20 cm depth were collected from each treatment plot. Core samples were dried in the oven at 105 °C for 72 h to estimate the mass of dry soil (Ms). Bulk density ($\mathsf{\rho }\mathrm{b}$) was calculated as $\frac{Ms}{Vt}$. The oven-dried cores were saturated with water and reweighed to calculate saturation percentage as $\mathsf{\omega }=\frac{Mt - Ms}{Ms}\times 100$, where Mt is saturated weight. Soil porosity (f) was determined as $\left(1-\frac{\mathsf{\rho }\mathrm{b}}{\mathsf{\rho }\mathrm{p}}\right)\times 10$, where $\mathsf{\rho }\mathrm{p}$ is particle density (2.65 Mg m⁻³). Samples collected through the auger were air-dried and sieved with a 2 mm sieve. For the determination of texture, the hydrometer method was followed. Ammonium bicarbonate di-ethylene tri-amine penta acetic acid (AB-DTPA) was used for determination of extractable nutrients [27]. Crop tissues, soil, and biochar analysis for N, P, K, micronutrients (Fe, Zn, Mn, Cu), organic C, pH, and EC were carried out in the filtrate as per procedures and instruments outlined in [26].

2.5. Statistical Analysis

Data were analyzed for significant variation among treatments through the ANOVA procedure [28] using Statistix version 8.1 for Windows (Tallahassee, FL, USA; https://www.statistix.com/ (accessed on 20 July 2022). Multiple pairwise comparisons of means were performed using the least significant difference (LSD) test [29] at a 0.05 significance level. The analysis of variance for main effects (biochar particle size and biofertilizer) and their interaction was also computed for each trait and given below mean values. The PCA analyses were performed using Statistica software (Dell™ Statistica™ 13.1). Correlation analysis was carried out using R Studio software.

3. Results

3.1. Plant Growth and Soil Bulk Density

Plant height, leaf area, leaf area index of maize, and soil bulk density were significantly influenced by different particle sizes of biochar and bio fertilizer inoculation previously applied to lentil crops. Taller plants were noted with <2 mm size biochar and Biozote-N followed by 2–5 mm biochar and Biozote-N; however, they were equal to the same biochar particle and Rhizogold, whereas the shorter plants were noted in the control where neither biochar nor bio fertilizer was added (

Table 2. Residual impact of biochar particle size grades and rhizobia applied to preceding lentils on maize plant height (cm), leaf area (cm²), leaf area index (LAI), and soil bulk density (BD).

Biofertilizer	Biochar	PH	LA	LAI	BD
	Less 2 mm	220a	483.7a	3.30a	1.04i
Biozote-N	2–5 mm	212ab	449.7b	2.92b	1.07hi
	5–10 mm	205b	443.7b	2.88b	1.19de
	Control	204b	442.3b	2.51efg	1.32ab
	Less 2 mm	208ab	448.3b	2.84bc	1.09ghi
Rhizogold	2–5 mm	207ab	427.0c	2.63cde	1.16def
	5–10 mm	207ab	441.0b	2.57def	1.21cd
	Control	203b	426.0c	2.41fg	1.33a
Control	Less 2 mm	207ab	449.7b	2.77bcd	1.11fgh
	2–5 mm	205b	442.3b	2.58def	1.14efg
	5–10 mm	202b	425.3c	2.48efg	1.18de
	Control	200b	408.3d	2.31g	1.26bc
+SOV	B	ns	**	**	**
	F	ns	**	**	*
	B×F	ns	**	ns	ns

SOV represents source of variance, B = biochar particle size, F = biofertilizer, * = significant at 5% probability, ** = significant at 1% probability, ns = non-significant at 5% probability. Means in columns followed by different lower-case letters are significantly different from each other at 5% level of probability.

). Leaf area plant⁻¹ and leaf area index of maize were enhanced with small particle size biochar (<2 mm) compared with 2–5 mm and 5–10 mm particle size treatments over the biochar control (425.56 cm²), respectively (Table 2). The <2 mm, 2–5 mm, and 5–10 mm particle size plots showed 23%, 12%, and 9% increases, respectively, in leaf area index of maize. The biochar particle sizes of 2–5 and 5–10 mm previously applied to lentils were statistically similar for leaf area and leaf area index (Table 2).

Across residual biochar applications, Bio-fertilizer strains applied to previous lentils did not show any residual effect; however, Biozote N showed significant improvement in maize leaf area plant⁻¹ (by 5% and 4%) and leaf area index (by 15% and 12%) over the control and Rhizogold strains, respectively.

3.2. Yield and Yield Components

The combined residual application of biochar and bio-fertilizer strains inoculated to previous lentils showed significantly increased biological yield, grain yield, 1000 grain weight, and grain protein content of the succeeding maize crop. The residual effect of Biozote-N and less than 2 mm particle size of biochar resulted in higher grain, biological yields, thousand-grain weight, and grain protein content of maize. The grain yield of maize was also at par with smaller particle biochar and Rhizogold (

Table 3. Residual impact of biochar particle size grades and rhizobia applied to preceding lentils on grain yield (kg ha⁻¹), biological yield (kg ha⁻¹), grain P concentration (%), and thousand grain weight (g) of maize.

Bio-Fertilizer	Biochar	GY	BY	TGW	GPC
	Less 2 mm	3558a	13,814a	250ab	15.8a
Biozote-N	2–5 mm	3424cde	13,031ab	236bc	15.9a
	5–10 mm	3468bcd	12,442abc	233bc	16.0a
	Control	3379ef	9926g	232ab	10.9e
	Less 2 mm	3504ab	12,008bcd	237bc	12.8cd
Rhizogold	2–5 mm	3472bc	11,767bcde	231bc	13.1bcd
	5–10 mm	3419cde	11,681bcdef	225c	13.4bc
	Control	3379ef	10,044fg	221b	12.1d
	Less 2 mm	3408def	108,66cdefg	235bc	12.7cd
Control	2–5 mm	3417cde)	105,17defg	227bc	13.2bcd
	5–10 mm	3383ef	102,96efg	225bc	13.9b
	Control	3354f	101,60efg	224bc	9.6f
+SOV	B	**	**	**	**
	F	**	**	**	**
	B × F	*	ns	ns	**

SOV represents source of variance, B = biochar particle size, F = biofertilizer, * = significant at 5% probability, ** = significant at 1% probability, ns = non-significant at 5% probability. Means in columns followed by different lowercase letters are significantly different from each other at 5% level of probability.

). There was an increase in grain yield with the smallest particles of biochar and Biozote-N as compared with no biochar and no biofertilizer, suggesting a positive role of the residual effect of biochar and bio fertilizer for enhancing maize yield. Statistically controlling the effect of biochar, the residual effect of Biozote-N and Rhizogold resulted in a 5% and 4% increase in biological yield, 2%, and 1.5% increase in grain yield, and 4.4% and 0.3% increase in 1000 grain weight over the bio-fertilizer control.

3.3. Crop Tissues N, N Uptake, and Uptake Use Efficiency

The concentration of N in grain and stover, N uptake, and nutrient use efficiency were significantly higher because of the residual impact of biochar amended at different size particles (<2, 2–5, 5–10 mm) to soil before the preceding lentil crop compared to no biochar treatment, bio-fertilizer, and interaction between biochar and biofertilizer (Figure 1A–F). The residual effect of Biozote-N and biochar at either particle size significantly enhanced the maize grain and stover N, N uptake, P concentration, organic matter, and NUE, compared to Rhizogold; the trend was, however, opposite in the no-biochar plot, where the residual impact of Rhizogold on the following maize grain N, N uptake, and NUE was incremented as compared to the Biozote-N (Figure 1). Across biochar particle sizes, the Biozote-N strain inoculated to preceding lentils revealed substantially higher grain and stover N, N uptake, and NUE over the Rhizogold and biofertilizer control and recorded an increase of 19%, 24%, 22%, 24%, and 19% over the biofertilizer control. Rhizogold did not show any residual impacts and was statistically similar in maize grain N and protein content to the control. However, its residual impact significantly increased the stover N, N uptake, and NUE of the succeeding maize as compared to the no-biofertilizer treatment. Overall, Rhizogold residual impact showed 4%, 10%, 7%, and 7% increases in the succeeding maize grain and stover N, N uptake, and NUE, respectively.

3.4. Principal Component Analysis (PCA) and Pearson’s Correlation Analysis

Principal component analysis (PCA) was performed for the studied traits in the current study and showed that different traits were strongly correlated, and different treatments were differentially distributed (Figure 2). Biplot revealed that the studied traits comprised of growth, yield-related traits, and quality traits accounted for 89.56% of the total variability of the data set. The first PC revealed 80.37% of the total variation, while PC2 accounted for 9.19%. The biplot revealed that PC1 was correlated with almost all studied traits excluding soil bulk density, which was negatively associated with P concentration and yield of the crop. The combined treatments of biochar and bio-fertilizer altered the values of the tested traits as explained by PCA1 and PC2 biplots (Figure 2); hence, substantial differences were observed between the biochar particle size and biofertilizer treatments for the studied traits in the maize crop. Moreover, a correlation among the studied traits revealed a significant and strong correlation of grain yield to grain protein content with grain N concentration, NUE, total nitrogen uptake, shoot nitrogen concentration, organic matter, biological yield, P concentration, leaf area, and leaf area index of maize (Figure 3). Soil bulk density was negatively associated with growth, yield, and yield attributing traits as well as grain and nutrient concentration and nitrogen use efficiency (Figure 3).

4. Discussion

Biochar as a soil amendment additive has gained significant support for soil fertility restoration and climate change mitigation worldwide [30]. Higher growth and improvement in yield parameters in the current study can be explained by enhanced nutrient sorption and reduced losses, resulting in higher crop nutrient availability. The effectiveness of smaller size biochar on improving growth may be due to the liming effects and nutrient exchange of biochar, as well as their ability to increase water storage capacity [17]. Biochar already reported by previous research demonstrated improved fertility and crop growth as well as higher grain yield and soil fertility [13,16,31]. However, our results further enhance our understanding of biochar′s increased potential regarding soil fertility and crop performance with its reduced particle size and vice versa.

A significant increase in nutrient content in soil might be the result of biochar directly releasing nutrients into soil during decomposition [32], or it may regulate plant nutrient concentration in soil solution after fertilizer application [33]. Reducing particle size showed increased N uptake over biochar control, which might be because of reduced N losses and its longer availability to the crop [14,15,34]. As the size of the amended biochar particle was lowered, higher N uptake could be associated with a larger exposed surface area and higher reactivity with free N in soil solution, mitigating gaseous losses or leaching [34,35]. Biochar with smaller particle sizes have a greater potential to boost water storage capacity while also improving liming effects due to more ash content and nutrient exchange [18]. Moreover, Xie et al. [19] also suggested that smaller particle sizes of biochar improve sorption of nutrients and organic compounds.

Results regarding the increased available fraction of N, P, and K in soil and the improved physical properties of soil with biochar application have been supported by different authors [13,31]. This can be explained through the adsorption of basic cations from the soil solution and preventing their fixation reaction with the applied P fertilizer to remain available for crop uptake. Reduced biochar particle size is tantamount to its enhanced reactivity for basic cations because of more exposed surfaces than larger particle sizes, resulting in higher P availability. Biochar with reduced particle size should uniformly dilute the soil matrix and efficiently block the applied P and K fertilizer to encounter soil particles or fix them therein. Higher P availability in biochar-amended soil has been reported by Glaser and Lehr [36]. Furthermore, uniform mixing of lower particle sizes of biochar with soil volume might be more helpful in soil aggregation compared to larger particle sizes, resulting in reduced bulk density and improved porosity and saturation percentage. In other words, the lowest size particle changes the nutrient retention more suitably than larger particles [37]. Likewise, Wenxi and Sean [17] also found that smaller particle size biochar enhanced plant nutrient availability due to its increased micro-porosity, surface area, and exterior surface area. Reduced bulk density with biochar application has been documented by previous researchers [31,38]. Improvements in soil macro-aggregates, structure, and water holding capacity have been ascribed to the application of biochar [39].

The higher growth and yield parameters with Biozote-N than Rhizogold over the bio-fertilizer control indicated the superiority of Biozote-N in N-fixation or its potential to add more nitrogen-rich organic matter to the soil after legume harvesting than the Rhizogold. The significant residual effect of the preceding legumes and the application of bio-fertilizer on soil fertility crop performance is evident from Ali et al. [13] and Billah et al. [40]. The interaction between the smallest size biochar particles and Biozote-N, showing significantly improved leaf area than biochar control and other particle size treatments, might jointly be attributed to improved plant-available N and P concentrations within these treatments. Rhizogold at the upper size of biochar particles significantly increased leaf area over the no-biofertilizer and the no-biochar plot that showed Rhizogold also contributes N to improve plant growth whilst biochar improves soil properties and nutrient status compared to the control. No interaction of either of the particle sizes of biochar and bio-fertilizer on leaf area index, biological yield, and 1000 grain weight indicated that each treatment’s individual effect was more able to affect these properties, whilst a significant influence on grain yield indicated their joint favorable effect. Nutrients as up-taken by the crop were finally translocated and accumulated in grains, and higher grain weight in the residual Biozote-N treated plot can be explained with higher N availability with Biozote than Rhizogold, and uptake that might better support Biozote-N than Rhizogold in terms of N economy.

Significant increases in N uptake, efficiency, and grain protein content with Biozote-N than the Rhizogold can be explained by Biozote-N appearing to have fixed the N throughout a longer period than Rhizogold and having added more N-rich organic material to the soil for the subsequent maize crop. Irisarri et al. [41] documented differences in the potential of different rhizobia strains on their symbiotic performance and N fixation rate based on their competitiveness and actual nodule occupancy in white clover. Significantly nutrient availability with Biozote-N over Rhizogold may be attributed to its greater potential to produce phytohormones that solubilize these nutrients [42]. Thus, the right candidate bio-fertilizer strain would perform better in the field based on its properties and technical background as well as its adoption to environmental conditions [43]. Biozote N in interaction with either of the particle sizes of biochar was significantly superior and indicated its improved performance. However, in the biochar control, the grain N, N uptake, and NUE in Biozote-N plots were lagging behind Rhizogold, indicating that biochar application creates a suitable environment for Biozote-N performance. Higher N availability by either strain interaction with the higher particle size of biochar or vice versa indicates the biochar smaller particle adsorption of N more than higher particle size. This is an important attribute of smaller particle sizes of biochar—that N availability is controlled better than higher particle sizes, and smaller particle sizes help to mitigate N losses.

Biozote-N associated increases in plant growth could have contributed more soil organic matter than Rhizogold and the biofertilizer control, supporting higher nutrient availability, reduction in bulk density, and increase in porosity and saturation in Biozote-N plots, since organic matter enhanced the soil aggregation [44]. This also explains the reduced soil bulk density and increased percent saturation for the interaction of Biozote-N with the smallest particle size of biochar compared to its interaction with larger particle sizes or the interaction of Rhizogold with either biochar particle size.

5. Conclusions

Biochar application preceding lentils crops and biofertilizer inoculation showed significant residual impacts on subsequent maize crop growth and yield, N nutrition, crop quality, and soil fertility were compared to the no-biochar plots. Particle sizes <2 mm and Biozote-N showed significantly higher residual beneficial impacts (legacy effects) for the subsequent maize crop. Biozote-N combined with <2 mm particle size also showed favorable residual impacts for growth, yield, N nutrition, grain protein, and soil properties and could be recommended for sustainable soil management in cereal–legume rotations. The experiment proved that the reasonable utilization of biochar can improve the growth of lentils and increase the yield of corn, which is also very beneficial for the improvement of soil. It should be developed and utilized to further promote the high-quality development of sustainable agriculture.