Biochar Aged for Five Years Altered Carbon Fractions and Enzyme Activities of Sandy Soil

Abstract

Biochar applied to soil has been considered as an effective tool for mitigation of atmospheric carbon dioxide emission and enhancement of carbon storage in soil, which may also enhance soil quality. However, the effect of biochar aged for 5 years on the different carbon fractions and enzyme activities as well as its changes, is not well understood in the cropland sandy soil of northern China. Therefore, a field trial was carried out in 2014 with biochar applied once at 0, 5.25, 10.50, 21.00 and 42.00 g·kg⁻¹ (BC0, BC1, BC2, BC3, and BC4, respectively). We evaluated the influence of biochar addition to the changes in soil organic carbon (SOC), labile carbon pools (readily oxidized carbon, dissolved organic carbon, and microbial biomass carbon) and enzyme activities (invertase, urease, and catalase). Biochar significantly increased SOC (122.00%) and altered the content of labile carbon (increased ROC, DOC and MBC by 71.29%, 10.35%, and 900.00%, respectively). Soil urease and invertase activities increased by 55.81% and 46.76%, respectively, with an increase in biochar application rate, but catalase activity significantly decreased by 31.79%. The values of the geometric means of labile carbon (0.88) and enzyme activities (2.39) for the BC3 treatment were higher than others, which indicated that the biochar application rate of 21.00 g·kg⁻¹ is suggested for the sandy soil. Our results suggest that the application of biochar in sandy soil for five years increased soil carbon sequestration, changed enzyme activities and ameliorated soil quality.

1. Introduction

Sandy soil is mainly distributed in the arid and semi-arid areas of northwest China, which is one of the most important cropland resources [1]. In addition, sandy soil is also the largest distribution area in Xinjiang, which is increasingly used for agricultural production [2]. However, low levels of soil organic carbon (SOC) in sandy soil can be the main cause of poor soil fertility. As far as sustainable development is concerned, it is necessary to utilize a suitable way to improve soil fertility. Biochar can achieve this goal because it contains condensed aromatic carbon to sequester more carbon and stabilization of soil organic carbon (SOC) by increasing C input to the soil [3]. Herein, biochar has attracted considerable attention in improving soil quality. Many scientists have shown concern about using biochar as a soil modifier [4,5]. Biochar is an emerging carbon-rich material derived from agricultural waste by high temperature pyrolysis under low oxygen or anaerobic conditions, which has a highly aromatic, carbon-rich porous solid granular material. It contains a large amount of carbon and plant nutrients, has a rich pore structure, a large specific surface area and a surface containing more oxygen-containing active groups, and is a multi-functional material [6,7]. It plays a vital role in facilitating soil chemical and biological properties. Therefore, biochar has been used for soil amendment, given that it affects soil bulk density, soil nutrients availability, water retention and soil pH [8,9,10], but it largely depends on the feedstock type, pyrolysis process, and soil type, especially biochar application rate [11].

Farmland soil organic carbon is one of the most important carbon pools in terrestrial ecosystems, which plays an important role in maintaining soil fertility and ensuring farmland productivity. It includes labile pools and recalcitrant pools [12], which slow responses to environmental changes [13]. Therefore, for the sake of better reflecting the effectiveness of SOC, the fractions of soil labile organic C include microbial biomass carbon (MBC), dissolved organic carbon (DOC), and readily oxidized carbon (ROC) as early prediction indicators of soil fertility and quality changes, have a rapid response to changes in soil properties caused by exogenous organic matter input [14,15,16]. The amount and composition of SOC would inevitably be affected by biochar that is widely incorporated in soil [17]. Biochar applied to soil can exert an essential influence on increasing soil organic C [18]. Gong et al. [19] recorded that biochar created a positive effect on soil quality by increasing the fractions of soil organic matter. Accordingly, biochar applied to soil can diversely influence individual SOM fractions. For instance, relative to unamended soil, biochar amendment improved the contents of DOC and MBC [20,21]. Moreover, biochar in the soil can improve soil habitat, affect microbial activity, promote the production of microbial assimilation stability carbon and continuous burial effect, and indirectly increase soil carbon sequestration [22]. Soil enzyme is one of the biologically active substances in soil. It has a high degree of catalysis. Organic matter is mainly decomposed by microorganisms through various enzymes, so the applied biochar to soil is an approach to ameliorating soil quality by increasing enzyme activity [20]. In general, biochar can influence soil microbial activity, change the proportion of soil bacteria to fungi, and improve soil enzyme activity [23]. Furthermore, with biochar application, enzyme activities, soil microbial biomass carbon, and nitrogen were also enhanced [24]. Taylor et al. [25] observed that soil organic matter content is a major influence factor on soil enzyme activities. Similarly, Li et al. [26] suggested that soil enzyme activity can influence the transformation direction of the soil carbon pool. Additionally, Bailey et al. [27] showed that biochar amendment might usually promote the activities of a range of enzymes while simultaneously decreasing the activities related to soil organic carbon mineralization [28].

To date, research on the application of biochar has mainly focused on agricultural and environmental applications such as soil improvement, greenhouse gas emission reduction, and treatment and remediation of polluted environments, but little attention has been given to the response of soil organic carbon fractions and enzyme activities to biochar application in sandy soil. Specifically, biochemical stability and alteration of biochar during aging in the soil environment are still unknown. The objective of this field research was: (i) to clarify the influence of biochar addition on SOC fractions and enzyme activities and the correlation among them and (ii) to investigate the change in soil labile carbon fractions and enzyme activities after 5 years of biochar application at different rates.

2. Materials and Methods

2.1. Study Site

Our field study site was laid on the PaoTai Experimental Station in Shihezi Reclamation Area, Xinjiang Province, China (84°58′–86°24′ E, 43°26′–45°20′ N). The area belongs to an inland arid semi-desert climate, and mean annual temperature and rainfall were 7.50 °C and 225 mm, respectively. Sunshine of 2525 h and frost-free period of 169 d. Corn is planted all year round in this area. The soil type is typical sandy soil, which is classified as arenosols, and its initial soil properties are provided in

Table 1. Basic properties of soil and fresh biochar in the study area in 2014.

Items	Soil	Biochar
Sand (>0.05 mm), %	53.2	-
Silt (0.05–0.002 mm), %	27.2	-
Clay (<0.002 mm), %	19.6	-
EC	66.13	-
pH	8.51	9.90
OC, g·kg−1	1.38	670
TN, g·kg−1	0.76	-
AP, mg·kg−1	4.6	82.2
AK, mg·kg−1	97	1590

Note: OC, organic matter; TN, total nitrogen; AP, available phosphorus; AK, available potassium.

. The biochar selected in this experiment was from Henan Sanli New Energy Technology Co., Ltd. (Luo’he, Henan Province, China), and the wheat straw was carbonized at 450 °C for 4 to 8 h under limited oxygen conditions. After crushing, it was sieved by a 2 mm sieve. The basic physical and chemical properties of biochar are shown in Table 1.

2.2. Experimental Design and Soil Sampling

The field experiment was organized in a randomized complete block design, which included five treatments and three replicates per treatment. Each plot measured 4.60 m × 7 m (32.20 m²). In order to prevent the interference of each plot, there was a spacing of 1 m wide protective band between plots. Similarly, in the previous study, considering the mixing level of biochar economically and efficiently applied to the soil under field conditions, the mixing ratio of biochar applied to soil was determined [29]. The five additional amounts of biochar included 0, 5.25, 10.50, 21.00 and 42.00 g·kg⁻¹, abbreviated as BC0, BC1, BC2, BC3 and BC4, respectively. The applied biochar rates are based on straw carbonization (2.625 t·hm⁻²·yr⁻¹). When the experiment began in 2014, biochar was added to 0–20 cm of soil at one time, and the biochar material and soil were fully mixed by ploughing the soil in the tillage layer through an agricultural tilling machine. The tested crops were cotton and corn; cotton was cultivated in March and reaped in September of the same year, corn was cultivated in May and reaped in September of the same year. Fertilizers were the same for all treatments, and fertilizer application rates followed local cultivation practices. Both cotton and corn were fertilized once at sowing. Basal fertilizer was applied with calcium superphosphate 225 kg·ha⁻², potassium sulfate 15 kg·ha⁻², Urea 660 kg·ha⁻², and potassium dihydrogen phosphate 165 kg·ha⁻² were applied with drip irrigation for no topdressing during the growth period.

We collected 0–20 cm soil samples in each plot by shovel before maize sowing in April 2019. Mixed the soil samples and removed other visible debris manually. Then the soil samples were separated into two parts; one was stored at 4 °C for DOC and MBC measurement, another part was air-dried at room temperature, passed through a 2 mm sieve, and then thoroughly homogenized for SOC, ROC, soil invertase, urease and catalase activities were measured.

2.3. Soil Biophysiochemical Properties Analysis

The SOC was determined using the K₂Cr₂O₇ external heating and FeSO₄ titration [30]. The ROC was oxidized using 333 mmol·L⁻¹ potassium permanganate, 565 nm wavelength colorimetric assay according to Blair et al. [31]. Soil DOC measurement was referred to the Jones et al. [32]. Soil MBC was analyzed by chloroform fumigation-extraction and was determined by extraction with 0.5 mol·L⁻¹ K₂SO₄ solution and determined by the colorimetric method [33]; the difference between the fumigated and un-fumigated extracts was used to calculate soil MBC and with a KEC factor of 0.45 [34].

The activities of one oxidative enzyme and two hydrolytic enzymes were measured [35,36,37]. Soil invertase activity (INV) was analyzed by the 3, 5-dinitrosalicylic acid colorimetry method, which was indicated in milligrams of glucose per gram of soil for 24 h. Soil urease (URE) activity was determined using urea as substrate and incubated for 2 h at 18 °C. Soil catalase activity (CAT) was determined via KMnO₄ as the substrate and incubated for 24 h at 37 °C.

2.4. Statistical Analysis

The geometric mean is the case of the continuous product of each unit mark value, which is suitable for the average level to reflect a phenomenon [38]. Therefore, to better investigate the change in soil properties, this study adopts the method to calculate the GMC and GME. The geometric mean of soil labile C is calculated as follows:

GMC = ∛(ROC × DOC × MBC)

(1)

where ROC, DOC and MBC are readily oxidizable labile C, dissolved organic C and microbial biomass C, respectively.

The geometric mean calculation formula of soil enzyme activity is as follows:

GME = ∛(CAT × INV × URE)

(2)

where CAT, INV and URE are the activities of catalase, invertase and urease, respectively.

To examine the effect of biochar addition on soil carbon fraction and enzyme activities, we used a one-way analysis of variance using SPSS 24.0 statistical software. Differences among BC0, BC1, BC2, BC3, and BC4 were evaluated by the least significant difference test (LSD) at 0.05 level. Correlation analysis used Pearson’s coefficient analysis.

3. Results

3.1. The Response of Soil Organic C and Soil Labile C to Biochar

The addition of biochar changed the SOC content, and the SOC content increased with the increase in biochar addition (Figure 1). The highest SOC concentration was added to the biochar treatment with 42.00 g·kg⁻¹ (BC4), and its content was 2.94 g·kg⁻¹. Compared with the addition of 0 g·kg⁻¹ treatment (BC0), BC4 treatment significantly increased SOC content by 122.00%. The SOC contents of the control treatment (BC0), 5.25 g·kg⁻¹ (BC1), 10.5 g·kg⁻¹ (BC2) and 21 g·kg⁻¹ (BC3) biochar treatments were 1.32, 2.01, 2.39 and 2.68 g·kg⁻¹, respectively, showing BC3 > BC2 > BC1 > CK.

Application of biochar affected soil labile C. Compared with BC0, the ROC content under BC4 treatment was significantly increased by 53.81% on average, while there was no significant difference between BC1, BC2 and BC3 treatments (

Table 2. The change of soil labile C content and its percentage to SOC under different biochar applications.

Soil Labile C	Treatments	Content of Labile C (g·kg−1soil)
ROC	BC0	0.944 ± 0.61 b
	BC1	1.044 ± 0.60 ab
	BC2	1.197 ± 0.75 ab
	BC3	1.331 ± 0.26 ab
	BC4	1.452 ± 0.12 a
DOC	BC0	0.137 ± 0.03 a
	BC1	0.138 ± 0.13 a
	BC2	0.141 ± 0.01 a
	BC3	0.140 ± 0.01 a
	BC4	0.139 ± 0.01 a
MBC	BC0	0.019 ± 1.2 b
	BC1	0.079 ± 34.05 b
	BC2	0.101 ± 31.04 ab
	BC3	0.190 ± 34.07 a
	BC4	0.088 ± 36.12 b

Note: DOC, dissolved organic carbon; ROC, readily organic carbon; MBC, microbial biomass carbon. The codes of BC0, BC1, BC2, BC3 and BC4 are the same as those in Figure 1; different letters in columns indicate significant differences (p < 0.05).

). The ROC accounted for 49.40% to 71.29% of SOC content under all treatments. No significant differences were found in DOC under all the treatments. Compared to the ROC, DOC only accounted for a small percentage of SOC, ranging from 4.73% to 10.35% under all treatments. Moreover, the percentage of the DOC accounting for the SOC was higher under the control (BC0) than the other treatments. In contrast to the other fractions, MBC content showed a clear trend with biochar application in Table 2, in which the BC3 was significantly higher than the rest of the treatments. The percentages of MBC to SOC were not significantly different among all treatments, whereas the value under the BC3 treatment was higher than others.

Biochar application exerted an obvious influence on the geometric mean of labile C (GMC), as provided in Figure 2. Compared with BC0, there were significant differences in GMC among treatments. The highest value of GMC was found in T3 at different biochar rates, which indicated that soil labile C was more sensitive than other treatments. As compared with BC0 and T0, the GMC values of T1, T2, T3 and T4 increased significantly by 35.34%, 45.81%, 48.78% and 46.16%, respectively.

3.2. The Response of Soil Enzyme Activities to Biochar

Biochar addition changed soil enzyme activity (Figure 3). INV increased under BC0, BC1, BC2, and BC3 treatments, yet it decreased in BC4, showing BC3 > BC2 > BC1 > BC4 > BC0. Compared with BC0, the increase in INV under BC3 treatment was the largest, increasing by 46.76%, while the increase in BC4 treatment was the smallest, increasing by 15.83% (Figure 3a). The trend of URE was similar to that of INV, showing BC3 > BC2 > BC1 > BC0 > BC4, and the URE of BC3 treatment was the highest, which was 3.42 mg·g⁻¹·d⁻¹. The URE of the BC4 treatment was the lowest, which was significantly different from that of the BC3 treatment (Figure 3b). Nevertheless, CAT showed a contradictory effect with INV (Figure 3c). With the increase in biochar application, CAT showed a downward trend. Compared with the BC0 treatment, there was no significant difference in BC1, BC2 and BC3, while BC4 was significantly reduced by 31.80%.

Biochar addition changed the geometric mean of soil enzyme activity. The average change trend of soil enzyme activity was basically consistent with INV and URE. Except for BC4, the GME values of biochar treatments were higher than BC0, showing BC1 > BC3 > BC2 > BC0. Compared with BC0, the GME value of BC1, BC2, and BC3 increased by 16.94%, 16.00%, and 23.96%, respectively (Figure 4), while the GME value of BC4 decreased by 9.33%.

3.3. Relationship between Organic Carbon and Enzyme Activities

The results of the correlation analysis showed that there was a significant positive correlation between SOC and ROC. There was a significant positive correlation between MBC, INV and URE. In contrast, we obtained an obvious negative correlation between ROC and CAT (

Table 3. Pearson correlation between organic carbon and enzyme activities.

	SOC	ROC	DOC	MBC	CAT	INV	URE
SOC	1
ROC	0.969 **	1
DOC	−0.121	−0.330	1
MBC	0.713	0.640	0.024	1
CAT	−0.863	−0.949 *	0.399	0.360	1
INV	0.576	0.410	0.251	0.879 *	−0.095	1
URE	0.321	0.200	0.177	0.879 *	0.129	0.895 *	1

Note: SOC, soil organic carbon; DOC, dissolved organic carbon; MBC, microbial biomass carbon; ROC, readily organic carbon; CAT, Catalase; INV, Invertase; URE, Urease activities. * and ** represent the significant difference levels of p < 0.05 and p < 0.01, respectively.

).

4. Discussion

4.1. Biochar Effects on Carbon Fractions

Our data revealed that under all treatments, SOC increased in contrast with the control as the amount of biochar increased, which was consistent with Liu et al. [39]. This was because biochar is recalcitrant and rich in aromatic carbon so as not to uneasily decompose, and it was easier to persist in soil [24]. Biochar, as an exogenous organic carbon, enters the soil, and its surface functional groups are oxidized over time, thereby affecting the carbon sequestration capacity of biochar [40]. Numerous results indicated that the effect of biochar on SOC was usually observed in a short period or under laboratory control, with a range of less than 2 years [41,42,43,44,45]. Singh et al. [46] noted that the application of biochar induced an initial positive priming impact on SOC by conducting an incubation experiment, but the effect attenuated after 5 years, which could be explained by the decomposition of soil labile C or stabilization of SOC resulted in biochar induced organic mineral interactions. Our research was based on a five-year field trial, the native SOC content under field conditions was relatively low, but biochar still increased SOC content in the sandy soil, which depended on the amount applied [47]. In this study, SOC increased with the increase in biochar application rate, showing BC4 > BC3 > BC2 > BC1 > CK.

The soil labile C is considerably vital to the turnover of SOC content [48]. In this study, The addition of different contents of biochar could change soil labile C. Several researchers have observed that the organic amendments applied to soil altered microbial biomass and community composition, which were associated with the SOC concentration [49,50] due to the enhanced the activities of soil microorganisms by the mineralization of SOC. Soil microbial biomass carbon is the most active part of organic matter, which can sensitively reflect the change in soil organic carbon. Our experiment found that MBC increased with the increase in biochar dosage. When the highest MBC in the biochar rate of 21.00 g·kg⁻¹ (BC3), which indicates that the activity of microorganisms is stronger, thereby decomposing more soil organic matter and labile organic C. The SOC and ROC are positively correlated with MBC support in this context. In addition, biochar could improve soil MBC content contrasted to the control, which was consistent with Kolb et al. [51]. On the one hand, biochar increased the input of SOC concertation; on the other hand, biochar provided a suitable living environment for microbial and accelerated the decomposition rate of microorganisms in the soil. In this study, the GMC combined ROC, DOC, and MBC responses to biochar application, it indicated that ROC, DOC, and MBC were excellent monitoring indicators for soil carbon quality change. We observed that the amount of biochar exerted a significant effect on GMC. GMC has a good quota of soil labile carbon, which was increased with biochar application. It is demonstrated that soil with biochar amendment was a potential carbon sink and that the biochar application rate of 21.00 g·kg⁻¹ (BC3) is a better choice to enhance SOC in sandy soil.

4.2. Biochar Effects on Enzyme Activities

Soil enzymes play an essential role in nutrient cycling [52]. Enzymes secreted by microorganisms can catalyze the decomposition and synthesis of SOC, as well as SOM can increase enzyme activity by stimulating carbon [20,26,53]. Biochar can decrease the soil enzyme activities related to ecological processes, especially soil C mineralization [53]. Our results illustrated that the response of soil UR activity on biochar is closely related to its application amount and was increased under biochar application treatments of BC1, BC2, and BC3, which was inconsistent with Bailey et al. [27]. Biochar can improve soil fertility on one side, and its porosity can also provide a suitable carrier for the survival of microorganisms, promote the reproduction of microorganisms, and stimulate the improvement of enzyme activity. On the other side, soil UR can promote urea, which is hydrolyzed to produce NH⁴⁺, and the biochar can promote the oxidation process of NH⁴⁺, which in turn accelerates the consumption of NH⁴⁺ [54,55], promotes the urease hydrolysis process and increases urease activity. In our study, UR was higher in BC3 and lower in BC4, which may be due to the higher microbial biomass under treatment, the more UR released and the higher amount of biochar application with a larger adsorption effect. This is because the long-term weathering of biochar caused a higher proportion of oxygen-containing functional groups in its surface functional groups, which may increase the adsorption of urease molecules on biochar, thus protecting the binding site of enzymatic reaction [56].

The enzyme activity increased as labile C content decreased [57]. Our data showed that the addition of different contents of biochar could change soil enzyme activity (INV, URE, CAT); the INV was significantly related to soil ROC. Yang et al. [45] observed that biochar can distinctly promote CAT activity and soil enzyme index, yet there was no significant influence on INV activity. Furthermore, soil INV activities are significantly related to the carbon process and reflect the carbon metabolism intensity [58]. In our study, biochar addition exerted an essential influence on increasing INV activity. This result may possibly be because of part of the accumulated soil DOM contents after biochar application, which was beneficial to improve the microbial structure and diversity. Indoor incubation experiments conducted by Yang et al. [45] indicated that soil CAT activity could be significantly affected by water, especially under flooding conditions, and such activity was stronger. In our study, soil CAT activity decreased with the increase of biochar application, which could be explained by soil environment changes, such as soil surface water status and temperature variability, which is not conducive to increasing CAT activity, the specific mechanism and mode of action need to be further studied. The GME combined diverse soil enzymes, which can illustrate the levels of soil enzyme activities and microbial activities [58]. Overall, the highest values of GME and GMC in the BC3 (21.00 g·kg⁻¹) treatment indicated that this biochar application rate was an optimum choice for soil enhancement.

5. Conclusions

Our results suggested that 21.00 g·kg⁻¹ biochar was beneficial to the soil carbon fractions and enzyme activities, although biochar aged in sandy soil for 5 years under field conditions. Biochar applied could ameliorate the sandy soil by altering the content of soil labile C and enzyme activities, as well as increasing the SOC content. However, the changes in microbial activity at different biochar rates may potentially be key, and the deeper mechanisms need to be further clarified. Moreover, in the perspective of improving soil quality, the effective amount of biochar applied to diverse soil types should be explored, which may contribute to the development of a robust assessment scheme on the long-term persistence of biochar in agroecosystems.