Optimizing biochar addition for vermicomposting: a comprehensive evaluation of earthworms’ activity, N2O emissions and compost quality

Biochar addition has been widely used in the field to mitigate soil nitrous oxide (N2O) emissions, and can be considered as a potential method to reduce N2O emissions during vermicomposting. However, excessive biochar addition may inhibit earthworms’ activity. Thus, it is crucial to clarify the optimum addition volumes of biochar during vermicomposting. This study evaluated the impact of addition of various amounts of biochar (0, 5, 10, 15, 20 and 25% of total amount of feedstock) on earthworms’ (Eiseniafetida) activity, N2O emission and compost quality during vermicomposting. Compared with the treatment without biochar added, 5% of biochar application significantly increased earthworm total biomass (from 177.5 to 202.2 g pot−1), and cumulative burrowing activity (from 47.0% to 52.2% pixel per terrarium). The increased earthworms activity stimulated the vermicomposting process and led to the best quality of compost, which showed the highest total nutrient content (5.38%) and a significantly higher germination percentage of seeds (88%). Although N2O emissions were slightly increased by 5% biochar addition, a non-significant difference was found between the treatment with 5% biochar and the treatment without biochar added. On the contrary, 20% and 25% biochar addition not only lowered N2O emissions, but also significantly decreased the quality of compost. The results suggest that 5% biochar application is an appropriate amount to improve the quality of compost without significant N2O emissions. Cattle dung can be effectively managed through vermicomposting Biochar showed both positive and negative effects on earthworms’ performance 5% biochar addition promoted compost quality by stimulating earthworms’ activity Higher doses of biochar were not suitable for vermicomposting Available N and functional genes drove compost quality and N2O emissions Biochar increased AOB but decreased nirS gene numbers in fresh cast of earthworms Cattle dung can be effectively managed through vermicomposting Biochar showed both positive and negative effects on earthworms’ performance 5% biochar addition promoted compost quality by stimulating earthworms’ activity Higher doses of biochar were not suitable for vermicomposting Available N and functional genes drove compost quality and N2O emissions Biochar increased AOB but decreased nirS gene numbers in fresh cast of earthworms


Introduction
Rapid increasing demand for livestock and dairy products has stimulated livestock manure production globally, estimating about 6252 million tons per year of production worldwide (Cao et al. 2021). Improper disposal or management of manures may cause serious environmental pollution (Zubair et al. 2020). However, nutrients and organic matter in livestock manures make them a potential organic fertilizer and ameliorating agent for cultivated land (Schlegel et al. 2017).
Vermicomposting is an economical and effective technique to turn organic wastes (e.g. livestock manure) into organic fertilizers under earthworms' activity (Garg et al. 2006). Earthworms increase air circulation to preserve an aerobic condition in the compost pile through continuous turning of substrate (Wu et al. 2015). By mixing and grinding the substrate, earthworms increase the surface area of the organic waste for microbial colonization and further decomposition (Lim et al. 2016). Moreover, earthworms' guts stimulate specific groups of microorganisms such as bacteria, which turn complex molecules into readily available molecules (Sapkota et al. 2020). Thus, the biological activities of earthworms are crucial factors that influence the decomposition process of solid waste and the quality of vermicompost for plant growth.
The biological activities of earthworms in vermicomposting may also increase nitrous oxide (N 2 O) emissions, thus leading to the losses of nitrogen (N), reducing the agronomic value of the vermicompost and environmental benefits from vermicomposting (Frederickson and Howell 2003;Lv et al. 2020). Previous meta-analysis indicated that earthworm presence increased soil N 2 O emissions by 42% (Lubbers et al. 2013). The gut of the earthworm comprises a mobile anoxic microzone and a large concentration of available nutrient. Thus, earthworms induce N 2 O emissions because denitrifying bacteria are stimulated by the unique micro-conditions of earthworm gut during the ingestion and excretion process ). In addition, enhanced organic matter mineralization by earthworms increases the substrates for N 2 O generation (Na et al. 2022).
Biochar is a fine-grained, degradation-resistant porous substance produced by slow pyrolysis of plant and animal biomass at low to medium temperatures under limited oxygen conditions (Shaaban et al. 2018a, b), and has been widely used in fields to mitigate soil N 2 O emissions (Zhang et al. 2021(Zhang et al. , 2022. The mechanisms of N 2 O reduction by biochar application include: (1) improvement of soil aeration, (2) suppression of denitrifiers in soil, and (3) adsorption of soil mineralized N (Wu et al. 2018;Gao et al. 2022;Wei et al. 2022). Biochar has also been proven to reduce earthworm-induced soil N 2 O emissions by changing the anoxic microzone of earthworms' gut and functional gene abundance of earthworms' casts during the short-term incubation experiment ). Thus, biochar addition was considered as a potential method to reduce N 2 O emissions during the vermicomposting. However, recalcitrant C in biochar is generally not palatable to earthworms, and high recalcitrant C content in materials may inhibit earthworm growth and slow down the vermicomposting process (Sharon and Kurt 2011). The application rate of nearly 60% biochar has been found to decrease earthworms' feeding activity in soil (Topoliantz and Ponge 2003). Therefore, an optimal biochar application amount is important for vermicomposting.
At present, however, little information is available on the literature for the comprehensive evaluation of earthworm performance, N 2 O emissions, and compost quality in response to various amounts of biochar addition. Thus, the objectives of this study were (1) to find the most optimal biochar application amount for vermicomposting by comparing the quality of composts and N 2 O emission, and (2) to investigate the main driving parameters that influence vermicomposting by the response of earthworms' activity and the key functional genes related to nitrification and denitrification.

Earthworm, cattle dung and biochar
Both earthworms (Eisenia fetida) and cattle dung were obtained from Hubei TianShengJia Biotechnology Co., Ltd. Adult earthworms with similar size and weight (about 0.37 ± 0.06 g individual −1 ) were hand collected and placed on a wet filter paper for 24 h to purge their guts. Cattle dung contained 29.5% total carbon (TC) and 1.23% total nitrogen (TN) on a dry weight basis with a C:N ratio of 24.0. The pH of cattle dung was 7.71.
Biochar used in the present study was produced from corn straw as it is cheap and easy to obtain from the field. Corn straw (Zea mays L.) was collected in the autumn, crushed, and put into a tube furnace to pyrolyze at 550 °C for 2 h under a continuous N 2 environment. Then, the prepared biochar was sieved (2 mm), washed with distilled water, and dried at 80 °C. The pH and ash content of biochar were 8.54 and 7.58%, respectively. The TC, TN and C/N value of biochar 66.6%, 1.14% and 58.4, respectively. The specific surface area (BET) and total pore volume (TPV) of biochar were 114 m 2 g −1 and 0.109 cm 3 g −1 , respectively.

Vermicomposting
The vermicomposting was designed as a single factor experiment, including six treatments: cattle dung without biochar addition (CD0), and cattle dung mixed with different doses of biochar [5% biochar (CD5), 10% biochar (CD10), 15% biochar (CD15), 20% biochar (CD20) and 25% biochar (CD25)]. Cattle dung was thoroughly mixed with different amounts of biochar, and then the distilled water was added to adjust the moisture content up to 65%. A total of 5 kg mixed material (dung, biochar and water) was added to plastic pots (40 cm × 25 cm × 25 cm), and then ~ 100 g (fresh weight) of earthworms were introduced for vermicomposting. There were six pots in each treatment: three for gas sampling, while three for compost sampling. Pots were covered with a thin black sheet with mesh to avoid earthworms escaping, and then incubated in a controlled greenhouse with temperatures ranging from 25 to 30 ℃. The moisture was maintained at 63-67% by spray-distilled water every 3 days. The duration of the experiment was 28 days, which was based on a preliminary experiment indicating more than 80% of material was ingested by earthworms within 28 days.
Closed chamber method was used for collecting gas samples to analyze N 2 O concentrations on a daily basis for the first fourteen days, and then every two days until the end of the experiment. The chamber was manually placed over the pots and sealed when gas sampling. Gas samples were collected from the headspace of each chamber at 0 and 60 min after the chamber closure (Shaaban et al. 2022) and analyzed immediately on a gas chromatograph (GC-7890A, Agilent Technologies, USA). The chamber was removed after sampling. Nitrous oxide fluxes were calculated assuming a linear increase of the N 2 O concentration over time after the chamber closed, and the cumulative gas emissions were calculated assuming linear changes between subsequent measurements.
Samples of compost were collected by a soil drill (2 cm diameter, 30 cm long) at three random locations in each pot on days 0, 7, 14 and 28. The TN and TC of samples were determined using a C/N elemental analyzer (Elementar Vario PYRO cube, Elementar, Germany). Available N (NH 4 + -N + NO 3 − -N) was analyzed using a continuous flow analyzer (SEAL Analytical GmbH, Norderstedt, Germany). The pH and electrical conductivity (EC) were measured in a 1:10 compost-deionized water mixture (Shaaban et al. 2013). After extraction with deionized water, the dissolved organic carbon (DOC) concentration of compost was measured using a total organic carbon analyzer (TOC 5000, Shimadzu, Kyoto, Japan). Germination assays (GI) for seeds of Lepidium sativum L. were conducted in a 1:1 extraction (shaken for 1 h at 25 ℃) of dry vermicompost and water according to Wu et al. (2019). The organic matter content of the final vermicompost (collected on day 28) was determined by dry combustion method. After H 2 SO 4 -H 2 O 2 digest, the total-N, total-P, and total-K contents of final vermicompost were determined by Kjeldahl method, spectrophotometry, and flame photometry, respectively.
At the end of the experiment, the earthworm was hand sorted, and then the total earthworm biomass (fresh weight basis) and density were measured.

Earthworm behavior
Burrowing is a normal behavior of earthworms. The changes in earthworms' burrowing behavior can be indicative of changes in the environment and the adverse effects of biochar on earthworms. Monitoring of earthworms' burrowing behavior in cattle dung mixed with different doses of biochar was carried out using a twodimensional terrarium (2D terrarium) according to Felten and Emmerling (2009). The experimental set-up was consistent with the vermicomposting experiment and included six treatments (CD0, CD5, CD10, CD15, CD20 and CD25). The terrarium was composed of two parallel glass plates with an external dimension of 40 × 50 cm. The distance between two glass plates was 6 mm. The mixed materials were prepared according to treatments, and then filled into the terrarium. Two individuals of earthworm were inoculated in each terrarium. Each treatment was replicated nine times. All terrariums were sealed with thin black sheets and stored in darkness at 25 °C for 120 h.
Pictures of both sides of the terrarium were captured using cameras at 12,24,36,48,60,72,84,96,108, 120 h after the commencement of the experiment. For each terrarium, data were summarized from both of the two glass plates. The pictures were imported into Adobe Photoshop 7 software package to analyze the burrowing activity of earthworms, which was shown as numbers of pixels.
At the end of the experiment, the terrarium was laid flat, and the upside of the glass plate was removed. Earthworms from each terrarium were collected and starved on wet filter papers for 12 h. Then the fresh casts left on filter paper were collected. Earthworms' cast from 4 to 6 replicates with the same treatment was composited to one sample to meet the quantity required for total DNA extraction and determination of functional genes including AOA, AOB, nirK, and nosZ.

Statistical analysis
Statistical analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). All data met the assumptions of normality. The effects of biochar on cumulative N 2 O emissions, earthworms' performance, functional genes and temporal dynamics of chemical properties in vermicompost were analyzed using oneway analysis of variance and considered statistically different at P < 0.05. Principal component analysis (PCA) was performed with CANOCO 4.5 (Microcomputer Power, USA) to determine the relationships of chemical and biota parameters in final compost.

Earthworms' biomass and density
Adult Eisenia fetida individuals with similar weight (about 0.37 g individual −1 ) were used for inoculation. Thus, there was no obvious difference on earthworm density between treatments at the beginning of vermicomposting (Table 1). However, the change in earthworms' biomass was significantly influenced by the application of biochar. Compared with CD0, 5% biochar addition significantly increased the total biomass of earthworms from 177.5 to 202.2 g pot −1 . On the contrary, a high amount of biochar addition (CD25) exhibited adverse effects on total earthworm biomass accumulation. Interestingly, there was no significant difference in earthworm density between CD0 and CD5, which indicated that 5% biochar addition enhanced earthworm performance by growth rate instead of reproduction. Nevertheless, 25% biochar addition led to not only weight loss for individuals but also the death or escaping of earthworms during vermicomposting.

Dynamic of chemical properties during vermicomposting
Addition of biochar at doses 0%, 5%, 10% and 15% resulted in a rapid pH decrease during the vermicomposting, while the pH in CD20 and CD25 treatments kept steady (Fig. 1a). Generally, the more biochar mixed in cattle dung, the higher the pH obtained both in raw materials and final compost. The final C/N ratio of compost varied within the 17.1-29.2 range, while only CD0, CD5 and CD10 fell into 20.0 (Fig. 1b). Obvious increases in available N contents were found in the treatment of CD0, CD5, and CD10 during the vermicomposting.
In contrast, a slight change in available N contents was detected in CD20 and CD25 (Fig. 1c). In the final compost, CD5 showed significantly higher available N contents than CD0, while CD20 and CD25 showed lower available N contents than other treatments. DOC content gradually decreased in all treatments during the whole vermicomposting process, and a non-significant difference was found between each treatment in the final compost (Fig. 1d). Compared with CD25 and CD20, the value of electrical conductivity (EC) in CD0, CD5, CD10, and CD15 treatments was stimulated at the initial period of vermicomposting, then decreased until the end of the study (Fig. 1e). The EC value of final compost varied within the 2.59-2.63 range for all treatments. Germination percentage (GI) gradually increased in all treatments with the vermicomposting process (Fig. 1f ), while the rate of increase tended to be quicker in CD0, CD5, CD10 and CD15 treatments, resulting in a significantly higher GI value (varied from 77.2 to 88.4%) at last than the treatments of CD20 and CD25 (varied from 39.1 to 43.2%). The nitrogen, phosphorus, potassium, and organic matter contents of the final compost were compared to Chinese national standards for agricultural organic fertilizer (NY525-2021) ( Table 2). For all treatments, the total nutrient (N + P 2 O 5 + K 2 O, dry basis) and organic matter content of the final compost met the requirements of national standard. However, the treatment of 5% biochar addition showed the highest total nutrient content.

N 2 O emissions and functional genes in the final compost
Compared with CD20 and CD25, a quick increase in N 2 O emissions was observed in CD0, CD5, CD10, and CD15 treatments, reaching maximum emissions on day 6 or 7, and then steadily declining with fluctuations of magnitudes until day 28 (Fig. 2a). The CD20 and CD25 treatments mitigated N 2 O emissions at the initial stage of vermicomposting, and resulted in significantly lower cumulative N 2 O emissions at last (8.32 and 7.72 mg N 2 O-N kg −1 dry for CD20 and CD25, respectively). The CD5 treatment showed the highest cumulative N 2 O emissions (15.09 mg N 2 O-N kg −1 dry vermicompost), but no significant difference was detected between the CD0, CD5, and CD10 treatments (Fig. 2b). The population size of the whole microbial community and key functional genes related to nitrification and denitrification in the final compost are shown in Table 3. Compared with CD0, the gene copy number of AOB was increased, while nirS decreased by biochar addition. However, significant differences were only observed between CD0, CD5, and CD10 treatments.

Earthworm behavior and functional genes in fresh cast
In CD0, CD5, CD10 and CD15 treatments, earthworms showed continuously increased cumulative burrowing activity in the terrarium environment for 120 h, albeit with a reduced burrowing rate at the later period of the study (Fig. 3a). Compared with CD0, 5% of biochar addition significantly stimulated the burrowing activity of earthworms, while 20% and 25% of biochar addition showed inhibiting effects on earthworms' burrowing behavior. As expected, burrowing activity of earthworms decreased with increasing materials depth (Fig. 3b). Especially for CD25, non-earthworms' activity was found below the depth of 10 cm.
The effect of biochar on earthworms' cast copy number of AOB, AOA, nirS and nosZ genes varied in different treatments (Table 4). Non-significant differences were found for AOA gene copy number between the treatments with and without biochar addition. The gene copy number of nosZ was slightly decreased by biochar addition, while the significant difference was only observed between CD0 and CD5 treatments. Compared with the treatment without biochar, the gene copy number of AOB was significantly increased by 1.83 to 2.07 times, and the gene copy number of nirS was significantly decreased by 1.31 to 1.76 times with biochar addition. However, nonobvious difference in AOB and nirS gene copy numbers was found between the treatments with different amount of biochar addition.
The functional genes and earthworms' performance in the final compost were used in the PCA analysis to identify the separation of biota parameters between each treatment (Fig. 4a). A noticeable isolated position was found between the treatments with low (0, 5, 10 and 15%) and high (20 and 25%) amounts of biochar addition. Treatments with 0-15% biochar addition were positivelycorrelated with maturity index and total nutrient content, while treatments with 20% and 25% biochar addition were negatively correlated.

Effects of biochar on earthworms' activity
Assuming that one earthworm consumes the feed up to half of its body weight per day (Maliå et al. 2017), the amount of the substrate (5.00 kg) was sufficient for   5.32 × 10 9 a 2.07 × 10 4 a 3.63 × 10 4 a 6.57 × 10 6 b 1.83 × 10 5 a CD10 4.87 × 10 9 a 1.84 × 10 4 a 3.41 × 10 4 a 7.17 × 10 6 ab 1.59 × 10 5 a CD15 5.81 × 10 9 a 1.98 × 10 4 a 3.27 × 10 4 ab 6.87 × 10 6 ab 1.69 × 10 5 a CD20 5.17 × 10 9 a 2.07 × 10 4 a 3.21 × 10 4 ab 6.67 × 10 6 ab 1.71 × 10 5 a CD25 4.98 × 10 9 a 2.14 × 10 4 a 3.14 × 10 4 ab 6.81 × 10 6 ab 1.69 × 10 5 a a 28-day period of vermicomposting. In addition, all the pots were incubated in a controlled greenhouse with optimum temperatures and moisture. Thus, the decreased biomass and density of earthworms in CD25 should be attributed to the high dose of biochar addition. This adverse effect of high doses of biochar can be further confirmed by burrowing behavior of earthworms, which was significantly lower in CD20 and CD25 treatments, and mainly restricted between 0 and 10 cm. Similar results have been reported in soil by Wu et al. (2021). Firstly, biochar has been documented to be resistant to both microbial and chemical degradation, and obviously, this is not an ideal food for earthworms (Sharon and Kurt 2011). Secondly, a high amount of biochar addition may have adsorbed the readily available nutrients in vermicompost, for example, lower available N contents were observed in CD25 treatment at the beginning of vermicomposting. Thirdly, the specific surface area and total pore volume of biochar in the present study were 114 m 2 g −1 and 0.109 cm 3 g −1 , respectively. Microbes colonized on biochar may have been protected by biochar pores (Sharon and Kurt 2011). Therefore, less food might Vertical burrowing activity of earthworms. Different letters among treatments indicate significant (P < 0.05) differences on determined properties at the end of behavior test be available to the earthworms in the treatment with a high amount of biochar addition, which then decreased the activity of earthworms. The results showed that 5% of biochar application increased substantial growth of earthworms. Similar positive effect has been reported by Zwieten et al. (2010) and Whalen et al. (2021), which may contribute to several factors including (1) immobilization of contaminants in materials (Tammeorga et al. 2014;Zhang et al. 2019), (2) increased material total porosity and water holding capacity (Gong et al. 2018) and (3) nutrient retention and enhancement of microbial activity (Maliå et al. 2017). Sharon and Kurt (2011) suggested that the response of earthworms to biochar may be the combined effect of characteristics of soil and biochar. While the present study further implied that the dose of biochar should also be considered to elucidate the effects of biochar on earthworm population. Table 4 Copy numbers of the 16S rRNA, AOA, AOB, nirS, and nosZ genes in fresh earthworm casts Values in the same column followed by the same lowercase letter are not significantly different (P > 0.05)

Effects of biochar on chemical properties of vermicomposting
Certainly, biochar's addition led to increased pH and C/N ratio of materials ( Fig. 1a and b). Nevertheless, the initial pH values and C/N ratios of all treatments were within the optimal range for the growth of E. fetida (Gong et al. 2018). The pH tended to decrease during the vermicomposting in all treatments, which might be attributed to the consumption of readily available nitrogen compounds, as well as the subsequent production of phenolic compounds (Hanc and Chadimova 2014). While the continuous decreased C/N ratio may be due to the rate of organic N mineralization being lower than that of organic C . It should be noted that both organic matter mineralization and readily available compound consumption were dominated by earthworms' activity. The inhibited earthworms' activity in CD25 may help to explain the little change in pH and C/N ratio during the experiment. The increased content of available N indicates the decomposition of organic matter. Earthworms increase N availability by enhancing microbial mineralization, and also by releasing their metabolic products (feces, urine, and mucus) (Buck et al. 1999;Blouin et al. 2013). Thus, it is not surprising that CD5 showed a quicker increase and a significant higher available N content than CD0 (Fig. 1c), as CD5 had a more active earthworm population. Since C is requisite during various metabolic activities for both earthworms and microorganisms (Devi and Khwairakpam 2020), DOC values gradually decreased in all treatments and a non-obvious difference was found between each treatment (Fig. 1d).
Electrical conductivity (EC) is a useful index to reflect salt concentration in the compost (He et al. 2016). For the treatments of CD0, CD5, CD10 and CD15, the increase in EC in the initial period of vermicomposting could be explained by the loss of organic matter by earthworm mineralization (Nayak et al. 2013). Then the volatilization of ammonia and precipitation of mineral salts may be attributed to the decrease of EC in the later period (Huang et al. 2004).
Germination tests are commonly used to assess compost phyto-toxicity (Das et al. 2011). The GI values of CD0, CD5, CD10 and CD15 gradually increased, suggesting that the inhibitors of seed germination were slowly eliminated. However, no obvious changes were found in EC and GI during vermicomposting of CD 20 and CD25, which may contribute to the limited effect of earthworms on organic matter mineralization.
In the determined indexes, C/N ratio, EC and GI are typically used in predicting the maturity of compost. The C/N ratio of compost should falls between 15 and 20 at the end of maturity (Devi and Khwairakpam 2020). Since salinity showed an adverse effect on seed germination and GI is a measure of phytotoxicity, He et al. (2016) and Das et al. (2011) recommended 4 ms cm −1 of EC and 80% of GI as the threshold for the field application of compost. In this view, only the treatments of CD0, CD5 and CD10 can meet the desired quality of compost, and 5% of biochar addition resulted in the highest total nutrient of products.

Effects of biochar on N 2 O emissions from vermicomposting
Existing research indicates that biochar addition is an effective way to reduce N 2 O emissions during thermophilic composting for example, Wang et al. (2013) (2) increasing the substrates for N 2 O generation by stimulated organic material mineralization (Wu et al. 2015Sun et al. 2020).
In the treatment of 5% biochar addition, it seems that stimulated earthworms' activity magnified the promoting effect of earthworms on N 2 O generation, and overlapped the mitigation effect of biochar on N 2 O emission. On the contrary, biochar influence N 2 O emission more in the treatments of CD20 and CD25. This view can be evidenced by (1) dynamic available N content during the vermicompost, as well as (2) the difference in functional gene numbers between final compost and fresh cast.
(1) Available N content: Available N is an important substance for N 2 O generation (Wu et al. 2015;Shaaban et al. 2019;Shaaban et al. 2018a, b). At day (2) Functional genes: Earthworm gut is ideally suited for N 2 O-producing bacteria, as it provides an anoxic microzone and a large amount of organic substrates (Horn et al. 2003;Wang et al. 2016). Obviously, biochar addition significantly changed the response of fresh cast bacteria, as shown in Table 3, a significant increased AOB and significantly decreased nirS gene copy numbers in all biochar-added treatments. This is because the specific surface and pore of biochar may serve as air (O 2 ) reservoirs when earthworms ingest biochar particles, which may slightly increase O 2 concentrations in earthworm guts ). On the contrary, the significant difference in AOB and nirS gene copy numbers in final compost was only detected between CD0 and CD5, instead of between treatments with and without biochar added. These results implied a negligible effect of gut-stimulated bacteria on vermicomposting microbe community with high amounts of biochar addition, which may explain the decreased earthworms' burrowing activity, since earthworms' activity is a crucial factor influencing the spreading of microorganisms. Figure 4b illustrates the potential driving parameters that influence compost quality and N 2 O emissions. Low amount of biochar addition stimulated earthworms' activity, improved the mature process of cattle dung, and thus resulted in a higher compost quality. Although available N content increased, the final N 2 O emission was mitigated by the change in compost microbial community, which was driven by earthworm gut associated microbes. No matter how much biochar was added, biochar showed the consistent effects on cast functional genes. However, the change in cast microbial community showed a limited effect on vermicomposting when a high amount of biochar mixed, which may be attributed to the inhabited earthworms' activity. Instead, the adsorption and dilution effect of high amount of biochar addition has sharply decreased the available N contents, which then significantly decreased N 2 O emissions.

Conclusion
Although slightly increased N 2 O emissions during the vermicomposting, 5% biochar addition clearly promoted the quality of final compost. On the contrary, 20% and 25% biochar additions lowered N 2 O emissions, but greatly decreased the quality of compost. The present study highlighted that low amount of biochar application is a potential method to improve the efficiency of vermicomposting during the practices. However, the parameters of biochar varied when different feedstocks were used for biochar production or biochar pyrolyzed under different conditions. Therefore, it is necessary to further investigate the role of different types of biochar on vermicomposting.