Cow manure and its biochar characterization
Our results showed that C and N were greater in cow manure biochar than the cow manure, whereas the opposite trend was observed regarding oxygen (O), hydrogen (H), O/C and H/C ratios (Table 1). These results are in agreement with previous studies (Ahmad et al., 2012; Al-Wabel et al., 2013; Khadem and Raiesi, 2019). The O/C and H/C ratios can be used as an indicator of determining the stability, aromaticity and polarity of biochar. The lower the ratios indicate the higher the aromaticity or the lower the polarity (Al-Wabel et al., 2013; Khadem and Raiesi, 2019; Spokas, 2010). Therefore, cow manure biochar showed a higher stability than cow manure. Higher recalcitrant C and conversely, lower labile C in cow manure biochar than cow manure (Table 1) can also confirm the above-mentioned results.
Analyses of variance for studied characteristics
The results of two-way ANOVA showed that amendment additions had a significant effect on SOC, WSC, POXC, POC, MBC content and βG activity at both incubation times (P < 0.05). A significant influence of earthworm on SOC and MBC content and βG activity (at both incubation times), WSC (at 30-day incubation), POXC and POC (at 90-day incubation) content was also observed (P < 0.05; Table 2). But unexpectedly, the interaction between earthworms and amendment additions on the measured parameters was not significant (P > 0.05; Table 2) though we observed that the earthworms had a preference for cow manure over its biochar so that mortality and weight loss of E. fetida happened with biochar but not with manure applications (Nahidan and Ghasemzadeh, 2022). Therefore, our results revealed that the influence of earthworms on C fractions and βG activity was not dependent on amendment additions, inconsistent with our hypotheses.
Table 2
Summary of two-factor analysis of variance (ANOVA) results (mean square values) for different properties and effects considered.
Variables | Amendment (A) | Earthworm (E) | A*E |
| 30-day incubation |
SOC | 2.057*** | 0.020** | 0.002ns |
WSC | 95647*** | 4583* | 622ns |
POXC | 268209*** | 4008ns | 866ns |
POC | 0.897*** | 0.003ns | 0.022ns |
MBC | 320*** | 164*** | 3.47ns |
βG | 11944*** | 10052* | 43.1ns |
| 90-day incubation |
SOC | 1.84*** | 0.009** | 0.0001ns |
WSC | 34120*** | 1414ns | 358ns |
POXC | 249973*** | 17701** | 3445ns |
POC | 0.688*** | 0.023*** | 0.003ns |
MBC | 601*** | 1182*** | 11.53ns |
βG | 14590 *** | 30218*** | 261ns |
*, ** and *** denote significance at p < 0.05, p < 0.01 and p < 0.001, respectively. “ns” denotes not significant. SOC: Soil organic C; WSC: water-soluble C; POXC: permanganate oxidizable C; POC: particulate organic C; MBC: microbial biomass C; βG: β-glucosidase activity |
Effect of amendment additions on C fractions and βG activity
The addition of both amendments increased the SOC content compared to CK (44.7-245.1% at 30-day incubation and 67.8-291.7% at 90-day incubation) due to input of C. The increase was greater in CM than in CMB treatments (Fig. 1), which was consistent with the pattern of labile C content in organic amendments (Table 1). It can be concluded that the labile C component in manure and manure biochar is the initial source of increasing SOC content (Chagas et al., 2022; Chen et al., 2023). Chen et al. (2023) also stated that most of the C in biochar is stable and cannot be converted into SOC in a short time. Nevertheless, since manure biochar contained more recalcitrant C than manure (Table 1), we speculate that C is eventually sequestrated in biochar (compared to manure) amended soils when the labile C fractions of amendments in soil are decomposed. To investigate this hypothesis, a long-term SOC monitoring in amended soils is required.
The results also showed that WSC increased in 1% CMB (9.5 and 21% at 30- and 90-day incubation, respectively) but decreased in 2 and 5% CMB (42.9–71.4% and 26.3–63.2% at 30-and 90-day incubation) compared to CK. The addition of CM to soil increased WSC by 47.6-142.8% at 30-day incubation and by 42.1–78.9% at 90-day incubation (Fig. 1). A similar trend was also observed for POXC. The addition of CMB at the rate of 1% led to an increase in POXC compared to CK by 20.5 and 17.7% at 30- and 90-day incubation, respectively. There was no significant difference between CK and 2% CMB regarding POXC but the addition of 5% CMB decreased POXC (22.7 and 24% at 30- and 90-day incubation, respectively) compared to CK. The addition of CM increased POXC over the CK by 53.4–99.8% at 30-day incubation and by 37.6–97.7% at 90-day incubation (Fig. 1).
Both WSC and POXC are the labile substrates for microbial activity and have been shown to be sensitive to changes in management practices as well as environmental conditions (Tang et al., 2020; Weil et al., 2003). It has been widely accepted that application of manure markedly increases the content of WSC and POXC in soil (Du et al., 2022; Liu et al., 2013; Tang et al., 2020), which could be attributed to the decomposition of manure and its positive priming effect on the native SOC (Liang et al., 1996; Zhang et al., 2020). Previous studies have also shown that the application of biochar can decrease (Yang et al., 2018; Demisie et al., 2014; Qiu et al., 2023) or increase (Jiang et al., 2022; Li et al., 2018; Ouyang et al., 2014; Yang et al., 2017) WSC and POXC content in soil. In agreement with our results, Demisie et al. (2014) found that POXC decreased when a higher rate of biochar was applied. Zhao et al. (2018) concluded that the labile organic C generated during pyrolysis could result in an increase of the labile C fractions in soil. However, biochar may also adsorb them on their surface or within their pores and as a result decrease the content of the labile C fractions in soil. It should be noted that the positive or negative priming effect of biochar on C mineralization was also reported in the literature which could contribute to the content of labile C in soil (Luo et al., 2016; Zimmerman et al., 2011). These different responses of the labile C in the biochar-amended soils resulted from the complicated properties of biochar, application rate, time of exposure, soil characteristics and scale of the studies.
The POC fraction has been defined as a labile SOC mainly consisting of organic residues partially decomposed and not associated with soil minerals. POC serves as the core of macroaggregate that protects organic carbon against physical disruption (Cambardella and Elliott, 1992; Liu et al., 2013). Our results showed that both CM and CMB increased the POC content (44.6–223% at 30-day incubation and 53.1–203% at 90-day incubation), the increase was greater in CM than in CMB treatments (Fig. 1). Previous studies also reported that application of manure (Liu et al., 2013; Tang et al., 2020) and biochar (Jiang et al., 2022; Tian et al., 2016) can enhance the POC accumulation in soil, probably due to physical protection of POC by promoting aggregate formation. Therefore, an increase in the biochar and manure application rate can be conducive to C sequestration in soil.
Both amendment additions had an increasing effect (17-64.7% at 30-day incubation and 19.7-181.7% at 90-day incubation) on MBC compared to CK (Fig. 1), suggesting that the application of organic amendments has a beneficial effect on the activity of microorganisms. The increase was greater in CM than CMB treatments (Fig. 1) probably due to the presence of more labile C available for microbial utilization and growth in CM (Table 1). Our results agree with the results from Liang et al. (2016) and Khadem and Raeisi (2017) who demonstrated higher MBC in soils amended with unchared feedstocks than their biochars.
βG activity was increased by amendment additions from 230.9 and 216.5 µg pNP g− 1 h− 1 in CK to 364.4 and 369.9 µg pNP g− 1 h− 1 in CM treatment at 30- and 90-day incubation, respectively. BG activity was only significantly higher in CM5 than CMB5 at 30-day incubation but it was higher in all CM than those in CMB treatments at 90-day incubation (Fig. 1). The CMB compared to CM exhibits a recalcitrant and stable nature due to its high aromatic C content (i.e. lower atomic H/C and O/C ratios) (Table 1). These characteristics make biochar chemically resistant to microbial attack and degradation (Fang and Singh, 2014; Spokas, 2010). Indeed, the addition of the labile components of organic inputs stimulates microbial activity, which could tend to increase enzyme production and activity (Khadem and Raiesi., 2017; Wu et al., 2013). Khadem and Raiesi (2017) also stated that the presence of polycyclic aromatic hydrocarbons and other toxic compounds in biochar can potentially reduce enzyme production by the soil microbiota. Our findings correspond well with previous studies indicating a higher activity of various soil enzymes by addition of raw materials compared with charred materials (Khadem and Raiesi, 2017; Paz-Ferreiro et al., 2012; Wu et al., 2013).
Effect of earthworm additions on C fractions and βG activity
The results showed a significant slight increase of SOC content as affected by earthworm (3 and 2.2% at 30- and 90-day incubation, respectively). An increasing effect of earthworm was also observed on WSC (10.3%) at 30-day incubation, POXC (7%) and POC (5.2%) at 90-day incubation. Earthworm also increased MBC content (9.5 and 46% at 30- and 90-day incubation, respectively) and βG activity (10.2 and 19.7% at 30- and 90-day incubation, respectively (Fig. 2).
It was reported that earthworm casts contain higher SOC and POC content due to their ability to physically protectection of carbon against mineralization (Puche et al., 2022). Earthworms can also stabilize organic C by promoting the formation of macro and microaggregates in soil (Angst et al., 2019; Zhou et al., 2022). Nevertheless, Caravaca and Roldan (2003) stated that earthworms increase the degradation of organic residue, causing an increase in the WSC. It is also recognized that earthworm activities, such as feeding, digestion, mucus secretion, excretion, and burrowing may enhance microbial biomass by altering the microhabitat and nutrient status (Xu et al., 2023). The earthworm along with its associated microbial community produce a variety of digestive enzymes, incuding polysacharidases, glycosidases and peroxidases, and consequently stimulate mineralization of organic matter (De Menezes et al., 2018). Therefore, as mentioned above, earthworms not only may accelerate the decomposition of organic matter, but also they may stabilize organic matter by aggregate formation. Nevertheless, the net effect of these adverse processes (C mineralization and stabilization) induced by earthworms, changes the total amount of SOC and its forms (Xu et al., 2023).
Our results also indicated that the labile C fractions (especially MBC) are more sensitive to earthworm-induced changes than SOC and can be more informative regarding the C dynamic in earthworm-worked soils. This is in keeping with the results of other studies, which demonstrated that MBC responds much more quickly to changing environmental conditions and can provide an early indication of slow changes in organic C content (Brookes et al., 2008; Jiang-shan et al., 2005; Powlson et al., 1987).