3.1 Biochar characterization
Selected physical and chemical properties of fresh biochar (BC) and aged biochars (OBC) are presented in Table 2. pH value decreased from 9.37 for BC to 8.18 for OBC. The CEC value increased from 12.20 cmol kg-1 for BC to 27.12 cmol kg-1 for OBC. CEC can be serve a way to measure biochar oxidation, with greater sensitivity than the O/C ratio (Hale et al., 2011). The increased CEC indicated the surface oxidation of OBC. The Brunauer–Emmett–Teller (BET) surface area decreased by 60.1% from BC (104.21 m2 g-1) to OBC (41.56 m2 g-1), indicating that aging decreased the specific surface area of biochar. This is consistent with Ghaffar et al. (2015) that the specific surface area of biochar reduced by 26–66% after aging by an oxidation treatment. The possible reasons include collapse of inner pores and the formation of oxygen-containing functional groups at pore entrances (Chang et al., 2018).
Aging also modified the elemental composition of biochars; the contents of C and H decreased while the N and O contents increased after aging (Table 2). As a result, the O/C ratio increased from 0.11 in BC to 0.15 in OBC, indicating that oxygen-containing functional groups have been introduced by aging (Uchimiya et al., 2011; Huff and Lee, 2016). As the (O+N)/C ratio is an indicator of polarity (Chen et al., 2005), its increase suggested that OBC had a greater polarity than BC.
The FTIR spectra of the BC and OBC were similar, suggesting they shared similar types of functional groups (Fig. 1a). For a close inspection of the intensity changes of functional groups after aging, we calculated the difference spectrum by subtracting the normalized spectrum of OBC from that of the BC (Fig. 1b). Three distinct IR transmittance peaks appeared in the difference spectrum at ca. 1400 cm-1, 1575 cm-1 and 1700 cm-1. Peaks at ca.1400 cm-1 and 1575 cm-1 corresponded to the asymmetric and symmetric modes of COO- functional group, respectively. The peak at 1700 cm−1 was assigned to the stretching mode of C=O (Ameur et al., 2018; Ren et al., 2018). These results indicated that O-containing functional groups were enriched after aging, which is in agreement with the observed increased in O/C ratio of the aged biochar (OBC). This statement is further supported by the results of XPS analysis. The XPS analysis showed that the OBC had higher O content than its fresh counterpart of BC (Fig. 2), in particular the proportion of O associated with C=O in OBC was much higher than that in BC (Fig. 2). In summary, aging increased the intensities of oxygen-containing functional groups, including carboxyl and carbonyl, on the biochar’s surface. Such changes may alter the interaction between biochar and ARGs.
3.2 Effects of biochar amendments on antibiotic resistance genes (ARGs) in soil
The relative abundances (in relative to the total 16S rRNA copy) of ARGs in chicken manure-fertilized soil as affected by fresh biochar and aged biochar amendments are shown in Fig. 3. intI1 had the highest relative abundance, with a magnitude of 10-2. The relative abundances of the other ARGs ranged at 10-3 to 10-4, following a decreasing order of sul2 > ermB > tetX > tetW. Fig.3 showed that biochar amendments may inhibit or promote the abundance of ARGs in the soil depending on the ARG species. Moreover, the abundance of ARGs in the rhizosphere soil was much different to that in the non-rhizosphere soil. To make a clear presentation, the ARGs abundance in the rhizosphere soil and in the non-rhizosphere soil was described separately as follows.
3.2.1 Comparative effects of fresh biochar and aged biochar on ARGs in rhizosphere soil
The relative abundances of tetW, sul2, and ermB genes in the rhizosphere soil generally decreased in both BC and OBC treatments as compared to the control (CK, without biochar), and the decreased amount in the rhizosphere soil was 15.0–24.3%, 27.6–74.7%, and 18.8–54.4% for tetW, sul2 and ermB, respectively (P < 0.05) (Fig. 3a, c, d). However, the abundances of tetX and intI1 increased significantly in both biochar treatments (P < 0.05, Fig. 3b, e). More specifically, the abundance of tetX and intI1 in the rhizosphere soils was 1.5–5.7 times and 2.3–3.5 times higher, respectively, than that of CK. intI1 is a proxy of antibiotic resistance and an indicator of mobile genetic elements (Amos et al., 2015; Duan et al., 2017). The increased abundance of intI1 indicated that both BC and OBC increased the risk of horizontal transfer of ARGs in soil. Moreover, the aged biochar OBC had a highly stronger increasing effect than that of fresh biochar BC (P < 0.05, Fig. 3e).
On the effects of BC and OBC on the ARGs abundance in the rhizosphere soil, there was no significant difference between the two types of biochars in decreasing the abundances of tetW, ermB and sul2 (P>0.05); while OBC showed a significant effect than BC on increasing the abundances of tetX and intI1 (P<0.05). These results suggested that the aging of biochar had negligible effect on the accumulations of tetW, ermB and sul2 but increased the potentials of tetX accumulation and the horizontal transfer of ARGs in the rhizosphere soil. Given that the occurrence of ARGs in soil is regulated by microorganisms which carry these ARGs (Dantas and Sommer, 2014), the decreases of tetW, ermB and sul2 suggest the downregulated producing of these genes, while the increased accumulations of tetX and intI1 indicate the upregulated producing of tetX and intI1 as influenced by biochar addition.
3.2.2 Comparative effects of fresh biochar and aged biochar on ARGs in the non-rhizosphere soil
Similarly, the abundance of tetW, sul2, and ermB genes was decreased by biochar addition, with a decreasing amount of 15.8–58.5% (Fig. 3a, c, d). In contrast, the abundance of tetX and intI1 increased by 6.5%–155.8% (Fig. 3b, e). Both biochars showed no significant difference in the abundance of tetW, sul2, and ermB (P>0.05), especially at a higher biochar application rate. However, the fresh biochar was superior to the aged biochars in increasing the abundance of tetX and intI1 in the non-rhizosphere soil (P<0.05). This result suggested that the aging of biochar had an opposite effect on the accumulation of tetX and intI1 in the non-rhizosphere soil versus the rhizosphere soil.
Rhizosphere soil has been a focused area in research regarding transmission of ARGs (Cui et al., 2018; Song et al., 2020). In the present study, the ARGs abundance in the rhizosphere was generally lower than that in the non-rhizosphere soil, especially in CK (Fig. 3), indicating that planting could significantly decrease the ARGs abundances in the rhizosphere soil, likely owing to the root exudates such as sugars, organic acids, and amino acids, which may create a low selective pressure of the antibiotics (Doornbos et al., 2012; Jechalke et al., 2013). In addition, the rhizosphere soil always creates conditions favorable for higher functional and microbial diversity than the non-rhizosphere soil which may also contributes to the decreased ARGs abundance (Nannipieri et al., 2008; Chen et al., 2018; Compant et al., 2019; Rivas et al., 2022).
The addition of biochar decreased the abundance of some genes while increased the abundance of others, both in the rhizosphere and non-rhizosphere soils. Some previous studies have shown that the application of biochar in soil reduced the dissemination of ARGs in soil (Ye et al., 2016; Liang et al., 2017; Jiao et al., 2018). This was attributed to the high specific surface area and microporous structure of biochar which could adsorb antibiotics and heavy metals, resulting in a decrease in their bioavailability. Moreover, the surface and porous structure could promote the hydrolysis and photolysis of antibiotics, providing a niche for antibiotic degrading bacteria as well, thus resulting the lower antibiotic concentrations in soil (Ye et al., 2016; Duan et al., 2017). These together attenuate the selective pressure on ARGs. On the other hand, there are also studies showing that the addition of biochar increased the abundance of some ARGs in soil. For example, Cui et al. (2018) found that the abundance of tet (tetX, tetG-01, tetG-02 tetM-01 tetM-02), sul (sul1 and sul2), and intI1 was increased in soil after 60 days of biochar amendment. The phenomenon was attributed to the pore structure and larger specific surface area of biochar which offer a niche for antibiotic-resistant bacteria (Ye et al., 2016). Apart from this, the bacterial biofilms formed within biochar and their proximity might contribute to an increase in the abundance of ARGs due to quorum sensing (Zheng et al., 2018).
To explore the effect of biochar type (BC/OBC), biochar addition rate (0%, 1%, and 2%), and planting (rhizosphere/non-rhizosphere soil) and their interactions on ARGs abundances in soil, multi-factor variance analysis was used and the results are presented in Table 3. Both biochar type and biochar addition rate had a remarkable effect on the abundance some ARGs. Specifically, biochar type significantly influenced genes of tetW, tetX, ermB and intI1 while biochar addition rate significantly affected genes of tetW, sul2 and ermB (P<0.05), indicating that the change of ARGs abundance in soil as affected by biochar was highly related to the ARGs species, the biochar addition level and its aging in soil. Table 3 shows that all tested genes were distinctively affected by planting, suggesting the distribution of ARGs abundance in rhizosphere soil was absolutely different from that in the non-rhizosphere soil (P<0.001). In addition, planting improved the performance of biochar (including its types and addition level) on ARGs abundance and significant interactive effects were observed among planting, biochar type, and biochar addition rate (Table 3).
3.3 ARGs enrichment in pakchoi
Shown in Fig. 4 are the relative abundance of ARGs in pakchoi after cultivation for 75 days. All selected genes (tetX, tetW, sul2, ermB and intI1) were detected in pakchoi tissues, suggesting that these genes were transferred from soil to the plant. Some root endophytes harboring ARGs might be responsible for the ARGs transfer. These endophytes inhabited in the interior of the roots via the stoma or mechanical injuries, allowing ARGs to reach the leaves (Hardoim et al., 2008; Bulgarelli et al., 2012).
The relative abundances of the abovementioned genes in pakchoi were significantly reduced by biochar addition, regardless of biochar type, with only one exception (that the fresh biochar applied at a lower rate increased the relative abundance of ermB in Fig. 4d). In comparison to CK (without biochar), the abundance of tetX, tetW, sul2, ermB, and intI1 in BC treatments decreased by 73.9-86.1%, 74.1-91.3%, 25.7-85.2%, 76.8-87.2%, and 58.7-93.4%, respectively. A similar pattern was observed for OBC treatments, where the corresponding genes decreased by 50.0-64.7%, 64.5-88.0%, 18.9-93.7%, 55.4-89.2%, and 71.7-95.4%, as compared with CK.
The reduction in ARGs accumulation in pakchoi could not be explained by the effects of biochar on the abundance of ARGs in either the rhizosphere or the non-rhizosphere soils. Although tetX and intI1 were less accumulated in the plant in the biochar treatments than CK, their abundances in soil was significantly increased by both fresh biochar and aged biochar additions. It suggested that the promoted abundance of ARGs in soil by biochar addition does not indicate an increasing risk of ARGs contamination in plants. The possible reason was due to that the antibiotic resistance bacteria which carry these genes could not be transferred to the other parts of the plant (Duan et al., 2017).
At 1% biochar application rate, BC showed significantly stronger effect than OBC (P < 0.05) on the abundance of some genes (tetW, tetX, and sul2), whereas an opposite trend was observed on the abundance of other genes (ermB and intI1). As biochar application rate increased to 2%, the difference between BC and OBC treatments diminished gradually and no significant difference between BC and OBC on the abundance of tetW, tetX and ermB (P > 0.05; Fig.4). The difference in ARGs accumulation in plants could be partly attributed to the changes of microbial community composition both in the soil and the plant (Forsberg et al., 2014; Jia et al., 2015; Sun et al., 2018; Awasthi et al., 2021). Future work with DNA sequencing should be performed in order to link the microbial community composition to the pattern of ARGs in plants, which could help better understand the biochar’s influence on ARGs transfer in the soil-plant system.
Although the process of biochar aging in the present study is somehow different to that occurs in the natural environment, changes in biochar properties tended to be similar. The surface area of biochar was reduced during the artificial aging process. In the natural environment, biochar undergoes slow aging process which also results in the decrease in its specific surface area. In addition, both aging processes caused the loss of liable organic fractions (e.g. extractable organic matter) and the formation of oxygen-containing functional groups. Therefore, our results obtained using an artificially aged biochar could provide insights into the effects of naturally aging on the biochar-mediated accumulation of ARGs in soils and plants.