*2.2. Experimental Setup for Collection and Analysis of N2O*

Initially, air dried soil without any amendment was incubated in a plastic (polyethylene terephthalate) tub with 20% gravimetric water content (60% water filled pore space) at a temperature of 25 ± 1 ◦C for one week (Figure 1). After one week of initial incubation, incubated wet soil (100 g on dry basis) from the tub was placed in glass jars. Liming materials were added separately to the soil. The application dose of each liming material was 1 g kg−<sup>1</sup> dry soil with a particle size <sup>≤</sup>0.3 mm. Treatments for the present study were as follows: (i) Ca(OH)2, (ii) CaO, (iii) CaCO3, (iv) CaMg(CO3)2, and (v) control (soil without any amendment). Each treatment had three replicates. Treated soils in jars were placed in an electric-automated chamber (S-400-HP) and incubated at 25 ± 0.5 ◦C in the dark for four weeks (28 days). During incubation, a plastic sheet with pin holes (about 30) was used on the top of each jar to reduce water loss, but permit gas exchange. Soil water content in each jar was sustained at 20% throughout the study by weighing jars and refilling with distilled water on a daily basis.

**Figure 1.** Schematic diagram of the experimental setup.

Gas from the headspaces of jars, equipped with air-tight lids holding a 3-mm diameter pipe, was collected at days 1, 3, 5, 7, 10, 13, 16, 20, 24, and 28 using a special air-tight syringe made for sampling purposes. On gas sampling day, the tops of jars were uncovered prior to gas sampling and

soil in the jars was allowed to be exposed to ambient air for 30 to 40 min. After that, the jars were closed with air-tight lids and gas samples were taken immediately after closure to know the initial concentration of gas in the jars. Another gas sample from headspace was collected after 60 min to know the change in gas production. The gas samples were analyzed for N2O concentration using a gas chromatograph system (7890A, Agilent technology, Santa Clara, CA, USA). The concentration of N2O in the gas sample was calculated using the equation as given below [13].

$$\mathbf{F} = \mathbf{p} \times \mathbf{V}/\mathbf{W} \times \Delta \mathbf{c} / \Delta \mathbf{t} \times 273 / (\mathbf{T} + 273) \tag{4}$$

In Equation (1), F denotes the rate of N2O–N emission (μg kg−<sup>1</sup> h<sup>−</sup>1); ρ denotes the density (kg m<sup>−</sup>3) of N2O gas; V denotes the volume (m3) of headspace of jars; W denotes soil weight (kg); Δc denotes change in gas concentration during closure time of jars; Δt denotes the time period of closure (h) of the treatment jars; and T denotes the temperature at which the experiment was conducted (25 ◦C).

The cumulative emissions of N2O (μg kg<sup>−</sup>1) for the whole period of study were calculated based on the following formula [14].

$$\text{Cumulative N2O emission} = \sum\_{i=1}^{n} (\text{Ri} \times 24 \times \text{Di}) \tag{5}$$

where *Ri* is the N2O emission rate (μg kg−<sup>1</sup> h<sup>−</sup>1); *Di* are days between the sampling periods; and *n* is the number of samples.

### *2.3. Experimental Setup for Soil Analysis*

A separate experiment to that for gas analysis was concurrently performed for soil analysis. Treatments, pre-incubation period, temperature, and moisture conditions for the soil analysis study were identical as that for the gas analysis setup. After pre-incubation, a weight of 200 g soil was incubated after being placed in 1000 mL beakers. Soil sub-samples from jars were taken after one day of imposing treatments and then on a weekly basis over 28 days.

For pH determination of the soil-sub samples, a soil slurry was made by performing a 1:2.5 ratio suspension of soil:distilled water [12]. The slurry was shaken in an orbital shaker for 40 min, and the pH was tested using a pH-meter (2FPHS, Wincoms Co. Ltd., Shanghai, China) after 30 min of shaking. Soil was subjected to specific extraction for the subsequent determination of the mineral contents of soil nitrogen (NO3 <sup>−</sup>–N and NH4 <sup>+</sup>–N) by adding 1 M KCl (5 mL for 1 g soil), shaking for 60 min, and subsequently using a flow injector system analyzer (SEAL Co. Ltd., Henstedt-Ulzburg, Germany) [15]. Chloroform fumigation specific extraction method was adopted for testing microbial biomass C [16]. Dissolved organic C content in soil was determined by extracting the soil with distilled water (1:5, soil:distilled water) and using Elementar system analysis (Vario, Elementar-CN, Hanau, Germany).

### *2.4. Data Analysis*

Data pertinent to soil and gas parameters were analyzed using Analysis of Variance (ANOVA) one-way analysis of variance. Tukey's test was employed to identify significant differences for treatments of their mean results. The Kolmogorov–Smirnov test for the normality distribution of variables was performed before proceeding further for ANOVA [17]. All data were statistically evaluated using Windows-based software Statistical Package for the Social Sciences (SPSS) Statistics 23.

### **3. Results**

### *3.1. Soil pH*

Soil pH was statistically significantly (*p* ≤ 0.01) different among the treatments of liming materials. Soil pH before the immediate day of adding liming materials was 5.35, and liming of soil rapidly increased pH (Figure 2). On day 1, soil pH in all treatments was substantially higher than that of the control and thereafter continued to gradually increase up until the end of the study. The highest value of soil pH corresponded to Ca(OH)2 treatment on day 28. The pH values were 7.21, 6.99, 6.70, 6.43, and 5.30 for Ca(OH)2, CaO, CaCO3, CaMg(CO3)2, and the control, respectively, on day 28 of the experiment.

**Figure 2.** Dynamics of soil pH following the application of liming materials. Vertical bars denote the error bars of the mean of three replicates. Values at the same time followed by different letters indicate significant differences between different treatments (*p* < 0.05).

#### *3.2. Soil Mineral–N (NH4* <sup>+</sup>*–N and NO3* −*–N)*

Soil NH4 <sup>+</sup>–N concentrations were highly and significantly (*<sup>p</sup>* <sup>≤</sup> 0.01) influenced by the addition of liming materials. NH4 <sup>+</sup>–N concentration before the immediate day of adding liming materials was 35 mg kg<sup>−</sup>1, whereas the addition of liming materials caused diverse patterns of NH4 <sup>+</sup>–N concentrations (Figure 3). The CaCO3, CaMg(CO3)2 and control treatments showed continuous decline of NH4 <sup>+</sup>–N concentrations throughout the study period. However, NH4 <sup>+</sup>–N concentration in the Ca(OH)2 and CaO treatments declined on day 1 of the onset of the study, increased on day 2, and afterward speedily decreased throughout until the end of the experiment. The NH4 <sup>+</sup>–N concentrations were 9.1, 10.9, 6.8, 12.0, and 20.2 mg kg−<sup>1</sup> in the Ca(OH)2, CaO, CaCO3, CaMg(CO3)2 and control treatments, respectively, on day 28 of the study.

**Figure 3.** Variations in soil ammonium concentrations following the application of liming materials. Vertical bars denote the error bars of the mean of three replicates. Values at the same time followed by the different letters indicate significant differences between different treatments (*p* < 0.05).

The amendment of liming materials significantly (*p* ≤ 0.01) augmented soil NO3 −–N concentrations (Figure 4). The trend of increase of NO3 −–N concentration kept continuing in all treatments until the end of the study. The maximum concentration of NO3 <sup>−</sup>–N was observed in the CaCO3 treatment on day 28 of the study. The NO3 <sup>−</sup>–N concentrations were 60.2, 52.4, 75.1, 50.0, and 43.9 mg kg−<sup>1</sup> in the Ca(OH)2, CaO, CaCO3, CaMg(CO3)2, and control, respectively, on day 28 of the study.

**Figure 4.** Variations in soil nitrate concentrations following the application of liming materials. Vertical bars denote the error bars of the means of three replicates. Values at the same time followed by different letters indicate significant differences between different treatments (*p* < 0.05).

### *3.3. Dissolved Organic C and Microbial Biomass C*

Addition of liming materials significantly (*p* ≤ 0.01) impacted the microbial biomass C (MBC) as well as dissolved organic C (DOC) in soil. Before the addition of liming materials, the DOC content was 25 mg kg−<sup>1</sup> and instantly increased on day 1 in all treatments, except for the control (Figure 5). The DOC contents reached maximum values of 38.5, 33.2, 30.1, 28, 24.9 mg kg−<sup>1</sup> on day 7 in the Ca(OH)2, CaO, CaCO3, CaMg(CO3)2, and control treatments, respectively, and afterward declined until the end of the study.

In the case of MBC contents, all the liming treatments showed an increment on day 1, while a divergent trend was observed afterward (Figure 6). Only Ca(OH)2 treatment showed a rise in MBC content after day 1, reached the maximum at 59 mg kg−<sup>1</sup> on day 14, and after that gradually declined and reached 49.1 mg kg−<sup>1</sup> at the end of the experiment, whereas MBC contents decreased in all other treatments of CaO, CaCO3, and CaMg(CO3)2 as well as the control over the entire study period.

**Figure 5.** Fluctuations in soil dissolved organic carbon following the application of liming materials. Vertical bars denote error bars of mean of three replicates. Values at the same time followed by different letters indicate significant differences between different treatments (*p* < 0.05).

**Figure 6.** Fluctuations in soil microbial biomass carbon following the application of liming materials. Vertical bars denote the error bars of the means of three replicates. Values at the same time followed by different letters indicate significant differences between different treatments (*p* < 0.05).
