3.1 Changes in elemental composition and DOC of the soil before and after the incubation experiment
The analysis of the elemental composition of the soil before and after the incubation are shown in Table 1, the carbon content of the topsoil was slightly higher than that of the deep soil. After the incubation, except for the addition of biochar (mainly because of the addition of biochar), their contents reduced to some extent, especially significantly in the presence of earthworms, while the N content did not decrease but even increased, which may be related to the secretion of nitrogenous compounds by microorganisms during the incubation process. Therefore, the C/N ratio after the incubation significantly decreased.
|
YB
|
B
|
B-BC
|
B-E
|
B-E-BC
|
YS
|
S
|
S-BC
|
S-E
|
S-E-BC
|
Table 1
Changes in soil element composition before and after the incubation
C%
|
1.33
|
1.27
|
1.61
|
0.73
|
1.42
|
1.22
|
1.18
|
1.63
|
0.84
|
1.36
|
N%
|
0.11
|
0.09
|
0.11
|
0.12
|
0.12
|
0.07
|
0.09
|
0.09
|
0.13
|
0.13
|
C/N
|
14.11
|
16.46
|
17.08
|
7.10
|
13.81
|
20.33
|
15.30
|
21.12
|
7.54
|
12.21
|
YB Original top soil, B topsoil, B-BC Top soil with biochar, B-E top soil added with earthworms, B-E-BC Top soil added with biochar and earthworms, YS Original deep soil, S deep soil, S-BC deep soil with biochar, S-E deep soil added with earthworms, S-E-BC deep soil with biochar and earthworms
The changes of pH and DOC content of soil before and after incubation are shown in Table 2. The soil pH ranged from 6.27–7.12, which basically showed a neutral bias. The addition of biochar increased the soil pH, while the presence of earthworms decreased the pH slightly. The pH of biochar was about 10.12, which is alkaline, so its addition in small amounts increased the soil pH to some extent. Most studies indicated that the presence of earthworms can stimulate microbial population and activity, and that they themselves and microorganisms may secrete some organic acids, leading to a decrease in soil pH (Valdez-Perez et al. 2011). This result was consistent with our findings.
Additionally, DOC content in soils decreased after the incubation and showed the following trend: B/SBC > B/S > B/S-E-BC > B/S-E. Obviously, the most significant decrease was observed in the presence of earthworms alone, earthworms accelerated the decomposition of DOC through the indirect or direct stimulation of microbial activity (Egert et al. 2004, Suthar 2008). However, the degree of decrease in DOC content with the addition of biochar was the lowest. On the one hand, this phenomenon may be due to the leaching of DOC from biochar itself; On the other hand, it was possible that the adsorption of DOC by biochar inhibited the degradation of DOC. Considering that the biochar was repeatedly washed before addition and added in a smaller amount, DOC released from biochar may contribute less to its DOC in short incubation time. Therefore, it was more likely that the small reduction in DOC content with biochar addition was due to its adsorption by biochar, which inhibited its degradation and thus retained more DOC content. This result was further confirmed by the release of CO2. Compared to top soils, the DOC content of deeper soils, generally with higher mineral fractions and relatively lower microbial activity, was reduced to a lesser extent after the incubation (Rukshana et al. 2013, Zhao et al. 2018). Moreover, the composition structure of DOC may further influence this phenomenon, which would be further elucidated from the 3D fluorescence map of DOC. Thus, this may led to a relatively slow decomposition of DOC in deep soils, which would be further evidenced based on the release of CO2.
Name
|
TOC (mg/g)
|
pH
|
Number of surviving earthworms (only)
|
Table 2
Changes in soil DOC and pH before and after the incubation
YB
|
0.2256
|
6.83
|
/
|
B
|
0.1551
|
6.98
|
/
|
B-BC
|
0.1724
|
7.12
|
/
|
B-E
|
0.1221
|
6.56
|
7
|
B-E-BC
|
0.1557
|
6.50
|
8
|
YS
|
0.2017
|
6.96
|
/
|
S
|
0.1594
|
6.79
|
/
|
S-BC
|
0.1737
|
7.11
|
/
|
S-E
|
0.1413
|
6.50
|
6
|
S-E-BC
|
0.1474
|
6.27
|
7
|
YB Original top soil, B topsoil, B-BC top soil with biochar, B-E top soil added with earthworms, B-E-BC Top soil added with biochar and earthworms, YS Original deep soil, S deep soil, S-BC deep soil with biochar, S-E deep soil added with earthworms, S-E-BC deep soil with biochar and earthworms
3.2 Analysis of three-dimensional fluorometry before and after the incubation experiment
To further investigate the difference in DOC composition, this study analyzed its composition with three-dimensional fluorescence spectroscopy (Fig. 1). Before the incubation, both DOC in top and deep soils were mainly dominated by tyrosine and tryptophan. Among them, the response degree of tyrosine and tryptophan was greater in the top soil. In general, the tyrosine and tryptophan are regarded as easily decomposed components (Tremblay et al. 2007). Thus, the top soil had more easily decomposed substances compared to the deep soil. In contrast, the deep soil DOC had a certain response at 420 nm, which belonged to the fulvic acid region, and its large molecular weight was more difficult to degrade compared with the easily degradable tyrosine and tryptophan, further indicating that the composition of DOC in the deep soil was more difficult to degrade than that in the top soil.
After the incubation, the DOC compositions of top and deep soils were mainly dominated by fulvic acids, which indicated that tyrosine and tryptophan were depleted during the incubation. Notably, the response intensity of the fulvic acid region of the topsoil DOC was higher than that of the deep soil (about three times higher than that of the deep soil), indicating that the top soil contained more fulvic acid fractions after the incubation. Specifically, it may be because the topsoil SOM was more easily decomposed into soluble fulvic acid by microorganisms. Furthermore, the fulvic acid response was more pronounced in the presence of earthworms, and the fulvic acid fraction of topsoil DOC was more significantly increased (about 5 times more than that of the deep soil) than that of the deep soil. This result further demonstrated that earthworms can promote the decomposition of SOM and conversion to soluble DOC fractions. In contrast, after the addition of biochar, the tryptophan, tyrosine and fulvic acid remained have a strong response in soil DOC. Combined with the earlier results showing that DOC content decreased less after biochar addition, this result could further indicate that the addition of biochar inhibited the decomposition of easily degradable fractions.
3.3 Effect of Earthworms/Biochar on Mineralization of SOM
3.3.1 CO2 release rate
According to the changes in soil respiration rate, it is clear that the soil CO2 respiration rate shows a decreasing trend with increasing incubation time, which is mainly for the rate of CO2 emission gradually decreases with time due to the decrease in microbial activity resulting from the reduction of food sources and the absence of the addition of foreign organic matter. There was a decrease in soil respiration rate after the addition of biochar, most DOC of which had been removed before the application, proving that SOM was absorbed and protected by the biochar, as was supported by the previous fluorometric measurements. In addition, as can be seen from Table 4, the respiration rate of the topsoil was slightly higher than that of the deeper soil after the addition of biochar. On the one hand, it is perhaps because the soil microorganisms decompose less organic matter in order to maintain a C/N ratio suitable for their survival under the circumstances that the C/N is greater than 20 with more pronounced nitrogen fixation (Sigua et al. 2014). On the other hand, it is clear from fluorescence that deep soils have a lower content of readily decomposable organic matter.
Regarding the CO2 respiration rate, it was much higher in the presence of earthworms than that of the earthworm-free treatment group. Particularly at the beginning of the incubation, the CO2 respiration rate could reach 100 g·g− 1·d− 1. On the one hand, the earthworm itself has a certain respiratory effect to emit CO2 (Binet et al. 1998); On the other hand, the addition of earthworms increased the number and activity of microorganisms in the culture group, resulting in the mineralization of most of the organic matter in the soil (Hoang et al. 2017). Moreover, considering the significant decrease of easily degradable DOC after the addition of earthworms as earlier reported, thus the addition of earthworms accelerated the mineralization of SOM especially for easily degradable DOC. It is worth noting that during the experimental period, the topsoil had a higher respiration rate than the deeper soil, which may be that the higher organic matter content of the topsoil and the higher number of microbial populations (Zhao et al. 2018) facilitate the induction of a dominant community by earthworm (Barois &Lavelle 1986) as well as the acceleration of SOM mineralization, while the deeper SOM is more stable and more difficult to be utilized. In contrast, it can be seen that the addition of biochar can inhibit the rate of earthworm CO2 release as the incubation time increases, mainly thanks to the action of the biochar as an agglomerate in the soil (Wang et al. 2017). In despite of the disruption of the formation of agglomerates from earthworm activity, it was observed that earthworm activity decreased with incubation time as the experiment proceeded, causing that the mineralization of the experimental group where earthworms were co-cultured with biochar was reduced relative to the experimental group where only earthworms were added.
3.3.2 Cumulative release of CO2
The cumulative release of CO2 after earthworms’ treatment was significantly higher than that without earthworms’ treatment, which was consistent with the release rate of CO2. In addition, in earthworm-free treatment, the addition of biochar did not change the cumulative release of soil CO2. However, under earthworms’ treatment, the addition of biochar had lower cumulative release of soil CO2 than earthworm activity alone and was more pronounced with increasing incubation time. Most studies suggested that biochar had insignificant effects on soil carbon sequestration in the short term or may even have positive priming effects (Major et al. 2010). However, our study found that biochar could inhibit the release of CO2 in the short term in the presence of earthworms, especially for deeper soils with lower organic matter content. For one thing, it may be because earthworms would promote the interaction between biochar and SOM, thus avoiding the further decomposition of SOM by adsorption and even formation of agglomerates, which complied with the composition of three-dimensional fluorescent DOC; For another, biochar may also have some toxicity to earthworms (Sohi et al. 2010), and in this experiment, it was found that earthworms had some tendency to avoid biochar at the beginning, and the earthworm activity was weaker in the group of earthworms with biochar added, which may also contribute to the reduction of cumulative soil respiration.