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The biological activities of earthworms in vermicom - 1 Introduction posting may also increase nitrous oxide (N O) emissions, Rapid increasing demand for livestock and dairy prod- thus leading to the losses of nitrogen (N), reducing the ucts has stimulated livestock manure production globally, agronomic value of the vermicompost and environmental estimating about 6252 million tons per year of produc- benefits from vermicomposting (Frederickson and How - tion worldwide (Cao et al. 2021). Improper disposal or ell 2003; Lv et al. 2020). Previous meta-analysis indicated management of manures may cause serious environmen- that earthworm presence increased soil N O emissions tal pollution (Zubair et al. 2020). However, nutrients and by 42% (Lubbers et al. 2013). The gut of the earthworm organic matter in livestock manures make them a poten- comprises a mobile anoxic microzone and a large con- tial organic fertilizer and ameliorating agent for culti- centration of available nutrient. Thus, earthworms induce vated land (Schlegel et al. 2017). N O emissions because denitrifying bacteria are stimu- Vermicomposting is an economical and effective tech - lated by the unique micro-conditions of earthworm gut nique to turn organic wastes (e.g. livestock manure) into during the ingestion and excretion process (Wu et al. organic fertilizers under earthworms’ activity (Garg et al. 2021). In addition, enhanced organic matter mineraliza- 2006). Earthworms increase air circulation to preserve tion by earthworms increases the substrates for N O gen- an aerobic condition in the compost pile through con- eration (Na et al. 2022). tinuous turning of substrate (Wu et al. 2015). By mixing Biochar is a fine-grained, degradation-resistant porous and grinding the substrate, earthworms increase the sur- substance produced by slow pyrolysis of plant and ani- face area of the organic waste for microbial colonization mal biomass at low to medium temperatures under lim- and further decomposition (Lim et al. 2016). Moreover, ited oxygen conditions (Shaaban et al. 2018a, b), and has earthworms’ guts stimulate specific groups of microor - been widely used in fields to mitigate soil N O emissions ganisms such as bacteria, which turn complex molecules (Zhang et al. 2021, 2022). The mechanisms of N O reduc- into readily available molecules (Sapkota et al. 2020). tion by biochar application include: (1) improvement of u Th s, the biological activities of earthworms are crucial soil aeration, (2) suppression of denitrifiers in soil, and factors that influence the decomposition process of solid (3) adsorption of soil mineralized N (Wu et al. 2018; Gao waste and the quality of vermicompost for plant growth. Wu et al. Biochar (2023) 5:4 Page 3 of 12 et al. 2022; Wei et al. 2022). Biochar has also been proven 2.2 Vermicomposting to reduce earthworm-induced soil N O emissions by The vermicomposting was designed as a single factor changing the anoxic microzone of earthworms’ gut and experiment, including six treatments: cattle dung without functional gene abundance of earthworms’ casts during biochar addition (CD0), and cattle dung mixed with dif- the short-term incubation experiment (Wu et al. 2021). ferent doses of biochar [5% biochar (CD5), 10% biochar uTh s, biochar addition was considered as a potential (CD10), 15% biochar (CD15), 20% biochar (CD20) and method to reduce N O emissions during the vermicom- 25% biochar (CD25)]. Cattle dung was thoroughly mixed posting. However, recalcitrant C in biochar is gener- with different amounts of biochar, and then the distilled ally not palatable to earthworms, and high recalcitrant water was added to adjust the moisture content up to 65%. C content in materials may inhibit earthworm growth A total of 5 kg mixed material (dung, biochar and water) and slow down the vermicomposting process (Sharon was added to plastic pots (40 cm × 25 cm × 25 cm), and and Kurt 2011). The application rate of nearly 60% bio - then ~ 100 g (fresh weight) of earthworms were intro- char has been found to decrease earthworms’ feeding duced for vermicomposting. There were six pots in activity in soil (Topoliantz and Ponge 2003). Therefore, each treatment: three for gas sampling, while three for an optimal biochar application amount is important for compost sampling. Pots were covered with a thin black vermicomposting. sheet with mesh to avoid earthworms escaping, and then At present, however, little information is available on incubated in a controlled greenhouse with temperatures the literature for the comprehensive evaluation of earth- ranging from 25 to 30 ℃. The moisture was maintained at worm performance, N O emissions, and compost quality 63–67% by spray-distilled water every 3 days. The dura - in response to various amounts of biochar addition. Thus, tion of the experiment was 28 days, which was based on the objectives of this study were (1) to find the most opti - a preliminary experiment indicating more than 80% of mal biochar application amount for vermicomposting by material was ingested by earthworms within 28 days. comparing the quality of composts and N O emission, Closed chamber method was used for collecting gas and (2) to investigate the main driving parameters that samples to analyze N O concentrations on a daily basis influence vermicomposting by the response of earth - for the first fourteen days, and then every two days until worms’ activity and the key functional genes related to the end of the experiment. The chamber was manu - nitrification and denitrification. ally 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 2 Materials and methods chromatograph (GC-7890A, Agilent Technologies, USA). 2.1 Ear thworm, cattle dung and biochar The chamber was removed after sampling. Nitrous oxide Both earthworms (Eisenia fetida) and cattle dung were fluxes were calculated assuming a linear increase of the obtained from Hubei TianShengJia Biotechnology Co., N O concentration over time after the chamber closed, Ltd. Adult earthworms with similar size and weight −1 and the cumulative gas emissions were calculated assum- (about 0.37 ± 0.06 g individual ) were hand collected ing linear changes between subsequent measurements. and placed on a wet filter paper for 24 h to purge their Samples of compost were collected by a soil drill guts. Cattle dung contained 29.5% total carbon (TC) and (2 cm diameter, 30 cm long) at three random locations 1.23% total nitrogen (TN) on a dry weight basis with a in each pot on days 0, 7, 14 and 28. The TN and TC of C:N ratio of 24.0. The pH of cattle dung was 7.71. samples were determined using a C/N elemental analyzer Biochar used in the present study was produced from (Elementar Vario PYRO cube, Elementar, Germany). corn straw as it is cheap and easy to obtain from the field. + − Available N (N H –N + NO –N) was analyzed using Corn straw (Zea mays L.) was collected in the autumn, 4 3 a continuous flow analyzer (SEAL Analytical GmbH, crushed, and put into a tube furnace to pyrolyze at 550 °C Norderstedt, Germany). The pH and electrical conduc - for 2 h under a continuous N environment. Then, the tivity (EC) were measured in a 1:10 compost-deionized prepared biochar was sieved (2 mm), washed with dis- water mixture (Shaaban et al. 2013). After extraction with tilled water, and dried at 80 °C. The pH and ash content deionized water, the dissolved organic carbon (DOC) of biochar were 8.54 and 7.58%, respectively. The TC, concentration of compost was measured using a total TN and C/N value of biochar 66.6%, 1.14% and 58.4, organic carbon analyzer (TOC 5000, Shimadzu, Kyoto, respectively. The specific surface area (BET) and total 2 −1 Japan). Germination assays (GI) for seeds of Lepidium pore volume (TPV) of biochar were 114 m g and 0.109 3 −1 sativum L. were conducted in a 1:1 extraction (shaken for cm g , respectively. Wu et al. Biochar (2023) 5:4 Page 4 of 12 1 h at 25 ℃) of dry vermicompost and water according to At the end of the experiment, the terrarium was laid Wu et al. (2019). The organic matter content of the final flat, and the upside of the glass plate was removed. Earth - vermicompost (collected on day 28) was determined by worms from each terrarium were collected and starved dry combustion method. After H SO -H O digest, the on wet filter papers for 12 h. Then the fresh casts left on 2 4 2 2 total-N, total-P, and total-K contents of final vermicom - filter paper were collected. Earthworms’ cast from 4 to post were determined by Kjeldahl method, spectropho- 6 replicates with the same treatment was composited to tometry, and flame photometry, respectively. one sample to meet the quantity required for total DNA Compost samples collected on day 28 were subjected to extraction and determination of functional genes includ- the total DNA extraction by FastDNA SPIN Kit (MP ing AOA, AOB, nirK, and nosZ. Biomedicals, Illkirch, France). Functional genes [Ammo- nia oxidizing archaea (AOA), ammonia oxidizing bac-2.4 Statistical analysis teria (AOB), nitrate reductase (nirS), and nitrous oxide Statistical analyses were performed using SPSS ver- reductase (nosZ)] that encode the key enzymes for nitri- sion 13.0 (SPSS Inc., Chicago, IL, USA). All data met fication and denitrification were determined by quanti - the assumptions of normality. The effects of biochar on tative real-time PCR (qPCR) in Majorbio Co., Ltd. The cumulative N O emissions, earthworms’ performance, pair of primers used for qPCR of AOA, AOB, nirS, and functional genes and temporal dynamics of chemical nosZ genes were Arch-amoAF/Arch-amoAR, amoA-1F/ properties in vermicompost were analyzed using one- amoA-2R, cd3aF/R3cd, and nosZ-F/nosZ-R, respectively. way analysis of variance and considered statistically dif- The gene copy number was determined by the standard ferent at P < 0.05. Principal component analysis (PCA) curve made with agarose gel-purified PCR products. was performed with CANOCO 4.5 (Microcomputer At the end of the experiment, the earthworm was hand Power, USA) to determine the relationships of chemical sorted, and then the total earthworm biomass (fresh and biota parameters in final compost. weight basis) and density were measured. 3 Results 3.1 Earthworms’ biomass and density 2.3 Earthworm behavior Adult Eisenia fetida individuals with similar weight Burrowing is a normal behavior of earthworms. The −1 (about 0.37 g individual ) were used for inoculation. changes in earthworms’ burrowing behavior can be u Th s, there was no obvious difference on earthworm den - indicative of changes in the environment and the adverse sity between treatments at the beginning of vermicom- effects of biochar on earthworms. Monitoring of earth - posting (Table 1). However, the change in earthworms’ worms’ burrowing behavior in cattle dung mixed with biomass was significantly influenced by the application of different doses of biochar was carried out using a two- biochar. Compared with CD0, 5% biochar addition signif- dimensional terrarium (2D terrarium) according to icantly increased the total biomass of earthworms from Felten and Emmerling (2009). The experimental set-up −1 177.5 to 202.2 g pot . On the contrary, a high amount was consistent with the vermicomposting experiment of biochar addition (CD25) exhibited adverse effects on and included six treatments (CD0, CD5, CD10, CD15, total earthworm biomass accumulation. Interestingly, CD20 and CD25). The terrarium was composed of there was no significant difference in earthworm den - two parallel glass plates with an external dimension of sity between CD0 and CD5, which indicated that 5% 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 indi - Table 1 Earthworms’ total biomass and density viduals of earthworm were inoculated in each terrarium. −1 Treatment Total biomass (g pot ) Earthworm density Each treatment was replicated nine times. All terrariums −1 (individual pot ) were sealed with thin black sheets and stored in darkness Beginning Ending Beginning Ending at 25 °C for 120 h. Pictures of both sides of the terrarium were captured CD0 100.3 a 177.5 b 273 a 362 ab using cameras at 12, 24, 36, 48, 60, 72, 84, 96, 108, 120 h CD5 100.9 a 202.2 a 270 a 374 a after the commencement of the experiment. For each CD10 100.2 a 188.3 ab 285 a 392 a terrarium, data were summarized from both of the two CD15 100.5 a 168.4 b 268 a 382 a glass plates. The pictures were imported into Adobe CD20 100.4 a 114.3 c 274 a 317 b Photoshop 7 software package to analyze the burrowing CD25 100.3 a 87.67 c 279 a 251 c activity of earthworms, which was shown as numbers of Values in the same column followed by the same lowercase letter are not pixels. significantly different (P > 0.05) Wu et al. Biochar (2023) 5:4 Page 5 of 12 biochar addition enhanced earthworm performance by In contrast, a slight change in available N contents was growth rate instead of reproduction. Nevertheless, 25% detected in CD20 and CD25 (Fig. 1c). In the final com - biochar addition led to not only weight loss for individu- post, CD5 showed significantly higher available N con - als but also the death or escaping of earthworms during tents than CD0, while CD20 and CD25 showed lower vermicomposting. available N contents than other treatments. DOC content gradually decreased in all treatments during the whole 3.2 D ynamic of chemical properties vermicomposting process, and a non-significant differ - during vermicomposting ence was found between each treatment in the final com - Addition of biochar at doses 0%, 5%, 10% and 15% post (Fig. 1d). Compared with CD25 and CD20, the value resulted in a rapid pH decrease during the vermicom- of electrical conductivity (EC) in CD0, CD5, CD10, and posting, while the pH in CD20 and CD25 treatments CD15 treatments was stimulated at the initial period of kept steady (Fig. 1a). Generally, the more biochar mixed vermicomposting, then decreased until the end of the in cattle dung, the higher the pH obtained both in raw study (Fig. 1e). The EC value of final compost varied materials and final compost. The final C/N ratio of com - within the 2.59–2.63 range for all treatments. Germina- post varied within the 17.1–29.2 range, while only CD0, tion percentage (GI) gradually increased in all treatments CD5 and CD10 fell into 20.0 (Fig. 1b). Obvious increases with the vermicomposting process (Fig. 1f), while the in available N contents were found in the treatment of rate of increase tended to be quicker in CD0, CD5, CD10 CD0, CD5, and CD10 during the vermicomposting. and CD15 treatments, resulting in a significantly higher Fig. 1 Dynamic of chemical properties during vermicomposting. a pH, b C/N, c available N contents, d DOC contents, e electrical conductivity (EC), f germination (GI). Different letters among treatments indicate significant (P < 0.05) differences in determined properties at the end of vermicomposting Wu et al. Biochar (2023) 5:4 Page 6 of 12 GI value (varied from 77.2 to 88.4%) at last than the treat- the study (Fig. 3a). Compared with CD0, 5% of biochar ments of CD20 and CD25 (varied from 39.1 to 43.2%). addition significantly stimulated the burrowing activ - The nitrogen, phosphorus, potassium, and organic ity of earthworms, while 20% and 25% of biochar addi- matter contents of the final compost were compared to tion showed inhibiting effects on earthworms’ burrowing Chinese national standards for agricultural organic ferti- behavior. As expected, burrowing activity of earthworms lizer (NY525-2021) (Table 2). For all treatments, the total decreased with increasing materials depth (Fig. 3b). nutrient (N + P O + K O, dry basis) and organic mat- Especially for CD25, non-earthworms’ activity was found 2 5 2 ter content of the final compost met the requirements of below the depth of 10 cm. national standard. However, the treatment of 5% biochar The effect of biochar on earthworms’ cast copy num - addition showed the highest total nutrient content. ber of AOB, AOA, nirS and nosZ genes varied in different treatments (Table 4). Non-significant differences were 3.3 N O emissions and functional genes in the final found for AOA gene copy number between the treat- compost ments with and without biochar addition. The gene copy Compared with CD20 and CD25, a quick increase in number of nosZ was slightly decreased by biochar addi- N O emissions was observed in CD0, CD5, CD10, and tion, while the significant difference was only observed CD15 treatments, reaching maximum emissions on day between CD0 and CD5 treatments. Compared with the 6 or 7, and then steadily declining with fluctuations of treatment without biochar, the gene copy number of AOB magnitudes until day 28 (Fig. 2a). The CD20 and CD25 was significantly increased by 1.83 to 2.07 times, and the treatments mitigated N O emissions at the initial stage gene copy number of nirS was significantly decreased by of vermicomposting, and resulted in significantly lower 1.31 to 1.76 times with biochar addition. However, non- cumulative N O emissions at last (8.32 and 7.72 mg obvious difference in AOB and nir S gene copy numbers −1 NO-N kg dry for CD20 and CD25, respectively). The was found between the treatments with different amount CD5 treatment showed the highest cumulative N O of biochar addition. −1 emissions (15.09 mg NO-N kg dry vermicompost), but The functional genes and earthworms’ performance no significant difference was detected between the CD0, in the final compost were used in the PCA analysis to CD5, and CD10 treatments (Fig. 2b). identify the separation of biota parameters between The population size of the whole microbial commu - each treatment (Fig. 4a). A noticeable isolated position nity and key functional genes related to nitrification and was found between the treatments with low (0, 5, 10 and denitrification in the final compost are shown in Table 3. 15%) and high (20 and 25%) amounts of biochar addition. Compared with CD0, the gene copy number of AOB Treatments with 0–15% biochar addition were positively- was increased, while nirS decreased by biochar addi- correlated with maturity index and total nutrient content, tion. However, significant differences were only observed while treatments with 20% and 25% biochar addition between CD0, CD5, and CD10 treatments. were negatively correlated. 3.4 Ear thworm behavior and functional genes in fresh cast4 Discussion In CD0, CD5, CD10 and CD15 treatments, earthworms 4.1 Eec ff ts of biochar on earthworms’ activity showed continuously increased cumulative burrowing Assuming that one earthworm consumes the feed up activity in the terrarium environment for 120 h, albeit to half of its body weight per day (Maliå et al. 2017), with a reduced burrowing rate at the later period of the amount of the substrate (5.00 kg) was sufficient for Table 2 Nutrient content in the final vermicompost Treatment National standard CD0 CD5 CD10 CD15 CD20 CD25 Total-N (%) 1.12 1.16 1.15 1.14 1.13 1.12 Total-P (%) 1.53 1.55 1.53 1.52 1.32 1.34 Total-K (%) 2.56 2.67 2.64 2.64 2.15 2.14 Organic matter (%) 33.7 c 34.2 c 39.1 bc 46.1 b 54.1 ab 56.4 a 30.0 Total nutrient (%) 5.21 a 5.38 a 5.32 a 5.30 a 4.60 b 4.60 b 4.00 National standard: the agricultural industry standard of the People’s Republic of China, Organic fertilizer, NY525-2021 Values in the organic matter and total nutrient content followed by the same lowercase letter are not significantly different (P > 0.05) Wu et al. Biochar (2023) 5:4 Page 7 of 12 Fig. 2 Dynamics of N O emission flux (a) and cumulative N O emission (b) from each treatment. In b, different letters among treatments indicate 2 2 significant (P < 0.05) differences on cumulative N O emissions at the end of vermicomposting Table 3 Copy numbers of the 16S rRNA, AOA, AOB, nirS, and nosZ genes in final compost from different treatments −1 Treatment No. of gene copies (g of vermicompost) 16S rRNA AOA AOB nirS nosZ 9 4 4 6 5 CD0 5.04 × 10 a 1.79 × 10 a 3.07 × 10 b 7.41 × 10 a 1.75 × 10 a 9 4 4 6 5 CD5 5.32 × 10 a 2.07 × 10 a 3.63 × 10 a 6.57 × 10 b 1.83 × 10 a 9 4 4 6 5 CD10 4.87 × 10 a 1.84 × 10 a 3.41 × 10 a 7.17 × 10 ab 1.59 × 10 a 9 4 4 6 5 CD15 5.81 × 10 a 1.98 × 10 a 3.27 × 10 ab 6.87 × 10 ab 1.69 × 10 a 9 4 4 6 5 CD20 5.17 × 10 a 2.07 × 10 a 3.21 × 10 ab 6.67 × 10 ab 1.71 × 10 a 9 4 4 6 5 CD25 4.98 × 10 a 2.14 × 10 a 3.14 × 10 ab 6.81 × 10 ab 1.69 × 10 a Values in the same column followed by the same lowercase letter are not significantly different (P > 0.05) Wu et al. Biochar (2023) 5:4 Page 8 of 12 Fig. 3 Burrowing behavior of earthworms in cattle dung mixed with different amount of biochar. a Cumulative burrowing activity of earthworms. b Vertical burrowing activity of earthworms. Different letters among treatments indicate significant (P < 0.05) differences on determined properties at the end of behavior test a 28-day period of vermicomposting. In addition, all both microbial and chemical degradation, and obviously, the pots were incubated in a controlled greenhouse this is not an ideal food for earthworms (Sharon and with optimum temperatures and moisture. Thus, the Kurt 2011). Secondly, a high amount of biochar addi- decreased biomass and density of earthworms in CD25 tion may have adsorbed the readily available nutrients in should be attributed to the high dose of biochar addition. vermicompost, for example, lower available N contents This adverse effect of high doses of biochar can be fur - were observed in CD25 treatment at the beginning of ther confirmed by burrowing behavior of earthworms, vermicomposting. Thirdly, the specific surface area and which was significantly lower in CD20 and CD25 treat - total pore volume of biochar in the present study were 2 −1 3 −1 ments, and mainly restricted between 0 and 10 cm. Simi-114 m g and 0.109 cm g , respectively. Microbes col- lar results have been reported in soil by Wu et al. (2021). onized on biochar may have been protected by biochar Firstly, biochar has been documented to be resistant to pores (Sharon and Kurt 2011). Therefore, less food might Wu et al. Biochar (2023) 5:4 Page 9 of 12 Table 4 Copy numbers of the 16S rRNA, AOA, AOB, nirS, and nosZ genes in fresh earthworm casts −1 Treatment No. of gene copies (g of cast) 16S rRNA AOA AOB nirS nosZ 10 5 6 7 6 CD0 1.85 × 10 b 0.53 × 10 ab 1.17 × 10 b 3.24 × 10 a 3.27 × 10 a 10 5 6 7 6 CD5 2.37 × 10 a 0.57 × 10 ab 2.14 × 10 a 2.48 × 10 b 1.76 × 10 b 10 5 6 7 6 CD10 2.14 × 10 ab 0.58 × 10 ab 2.26 × 10 a 1.84 × 10 b 2.27 × 10 ab 10 5 6 7 6 CD15 2.13 × 10 ab 0.62 × 10 a 2.31 × 10 a 2.28 × 10 b 2.57 × 10 ab 10 5 6 7 6 CD20 2.05 × 10 ab 0.53 × 10 ab 2.27 × 10 a 2.08 × 10 b 2.76 × 10 a 10 5 6 7 6 CD25 1.86 × 10 b 0.48 × 10 b 2.42 × 10 a 1.96 × 10 b 2.64 × 10 a Values in the same column followed by the same lowercase letter are not significantly different (P > 0.05) Fig. 4 Relationships of chemical and biota parameters in final compost (a) and the schematic diagram (b) illustrating the potential driving parameters that influence compost quality and N O emissions. In b, the red or blue color indicates low (5%, 10% and 15%) or high (20% and 25%) amount of biochar addition. Circle with color indicates the positive (shown as “ + ”) or negative (shown as “−”) effects between two determined characters, and the round size of circle indicates the intensity interactions be available to the earthworms in the treatment with a (2) increased material total porosity and water holding high amount of biochar addition, which then decreased capacity (Gong et al. 2018) and (3) nutrient retention and the activity of earthworms. enhancement of microbial activity (Maliå et al. 2017). The results showed that 5% of biochar application Sharon and Kurt (2011) suggested that the response of increased substantial growth of earthworms. Similar earthworms to biochar may be the combined effect of positive effect has been reported by Zwieten et al. (2010) characteristics of soil and biochar. While the present and Whalen et al. (2021), which may contribute to sev- study further implied that the dose of biochar should also eral factors including (1) immobilization of contaminants be considered to elucidate the effects of biochar on earth - in materials (Tammeorga et al. 2014; Zhang et al. 2019), worm population. Wu et al. Biochar (2023) 5:4 Page 10 of 12 4.2 E ec ff ts of biochar on chemical properties the end of maturity (Devi and Khwairakpam 2020). Since of vermicomposting salinity showed an adverse effect on seed germination Certainly, biochar’s addition led to increased pH and C/N and GI is a measure of phytotoxicity, He et al. (2016) and −1 ratio of materials (Fig. 1a and b). Nevertheless, the initial Das et al. (2011) recommended 4 ms cm of EC and 80% pH values and C/N ratios of all treatments were within of GI as the threshold for the field application of com - the optimal range for the growth of E. fetida (Gong et al. post. In this view, only the treatments of CD0, CD5 and 2018). The pH tended to decrease during the vermicom - CD10 can meet the desired quality of compost, and 5% posting in all treatments, which might be attributed of biochar addition resulted in the highest total nutrient to the consumption of readily available nitrogen com- of products. pounds, as well as the subsequent production of phe- nolic compounds (Hanc and Chadimova 2014). While 4.3 Eecffts of biochar on N O emissions the continuous decreased C/N ratio may be due to the from vermicomposting rate of organic N mineralization being lower than that of Existing research indicates that biochar addition is an organic C (Wu et al. 2019). It should be noted that both effective way to reduce N O emissions during thermo- organic matter mineralization and readily available com- philic composting for example, Wang et al. (2013) found pound consumption were dominated by earthworms’ that total N O emissions from compost were lower by activity. The inhibited earthworms’ activity in CD25 may 25.9% with biochar addition. Those phenomenons may help to explain the little change in pH and C/N ratio dur- result from: (1) capture of nitrates by the biochar; (2) ing the experiment. increasing pH; (3) improving aeration, and (4) changing The increased content of available N indicates the the denitrifier community composition (Awasthi et al. decomposition of organic matter. Earthworms increase 2016; Wang et al. 2021; Ravindran et al. 2022). How- N availability by enhancing microbial mineralization, and ever, it should be noted that earthworms may increase also by releasing their metabolic products (feces, urine, N O emissions by a similar pathway: (1) stimulating the and mucus) (Buck et al. 1999; Blouin et al. 2013). u Th s, activity of denitrifiers by inoculating gut-derived micro - it is not surprising that CD5 showed a quicker increase organisms through casting and (2) increasing the sub- and a significant higher available N content than CD0 strates for N O generation by stimulated organic material (Fig. 1c), as CD5 had a more active earthworm popula- mineralization (Wu et al. 2015, 2021; Sun et al. 2020). tion. Since C is requisite during various metabolic activi- In the treatment of 5% biochar addition, it seems that ties for both earthworms and microorganisms (Devi and stimulated earthworms’ activity magnified the promot - Khwairakpam 2020), DOC values gradually decreased in ing effect of earthworms on N O generation, and over- all treatments and a non-obvious difference was found lapped the mitigation effect of biochar on N O emission. between each treatment (Fig. 1d). On the contrary, biochar influence N O emission more Electrical conductivity (EC) is a useful index to reflect in the treatments of CD20 and CD25. This view can be salt concentration in the compost (He et al. 2016). For evidenced by (1) dynamic available N content during the the treatments of CD0, CD5, CD10 and CD15, the vermicompost, as well as (2) the difference in functional increase in EC in the initial period of vermicompost- gene numbers between final compost and fresh cast. ing could be explained by the loss of organic matter by earthworm mineralization (Nayak et al. 2013). Then the (1) Available N content: Available N is an important volatilization of ammonia and precipitation of mineral substance for N O generation (Wu et al. 2015; salts may be attributed to the decrease of EC in the later Shaaban et al. 2019; Shaaban et al. 2018a, b). At day period (Huang et al. 2004). 0 of vermicomposting, available N contents in mate- Germination tests are commonly used to assess com- rials were gradually decreased with the increasing post phyto-toxicity (Das et al. 2011). The GI values of amount of biochar addition, indicating the nitrate CD0, CD5, CD10 and CD15 gradually increased, suggest- capture effects of biochar. However, the available N ing that the inhibitors of seed germination were slowly content in CD5 increased quickly during the ver- eliminated. However, no obvious changes were found in micomposting and exceeded CD0 on day 14, indi- EC and GI during vermicomposting of CD 20 and CD25, cating that earthworms stimulated more N release which may contribute to the limited effect of earthworms than biochar capture. On the contrary, 20% and 25% on organic matter mineralization. of biochar addition limited the activity of earth- In the determined indexes, C/N ratio, EC and GI are worms, and showed the lowest available N content typically used in predicting the maturity of compost. The during the whole vermicomposting process. C/N ratio of compost should falls between 15 and 20 at Wu et al. Biochar (2023) 5:4 Page 11 of 12 (2) Functional genes: Earthworm gut is ideally suited for under different conditions. Therefore, it is necessary to N O-producing bacteria, as it provides an anoxic further investigate the role of different types of biochar microzone and a large amount of organic substrates on vermicomposting. (Horn et al. 2003; Wang et al. 2016). Obviously, bio- Acknowledgements char addition significantly changed the response of Not applicable. fresh cast bacteria, as shown in Table 3, a significant Author contributions increased AOB and significantly decreased nirS The first draft was written by YW. Supervision and project administration gene copy numbers in all biochar-added treatments. were addressed by MS and RH. Experiment was conducted by YW, YZ and This is because the specific surface and pore of bio - XX. Review was performed by QL, YC, MS and RH. All authors commented on previous versions of the manuscript. All authors read and approved the final char may serve as air (O ) reservoirs when earth- manuscript. worms ingest biochar particles, which may slightly increase O concentrations in earthworm guts (Wu Funding This work was supported by Major Science and Technology Program of et al. 2021). On the contrary, the significant differ - Hainan Province (ZDKJ2021009), National Natural Science Foundation of ence in AOB and nirS gene copy numbers in final China (32171638), and Yunan Science and Technology Talents and Platform compost was only detected between CD0 and CD5, Program (202205AF150004). instead of between treatments with and without Availability of data and materials biochar added. These results implied a negligible All data generated or analyzed during this study are available from the cor- effect of gut-stimulated bacteria on vermicom - responding author on reasonable request. posting microbe community with high amounts of biochar addition, which may explain the decreased Declarations earthworms’ burrowing activity, since earthworms’ Competing interests activity is a crucial factor influencing the spreading The authors declare that they have no known competing financial interests of microorganisms. or personal relationships that could have appeared to influence the work reported in this paper. Figure 4b illustrates the potential driving parameters Author details that influence compost quality and N O emissions. Low College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China. Institute of Environment and Plant Protection, Chinese amount of biochar addition stimulated earthworms’ Academy of Tropical Agricultural Sciences, Haikou 571158, China. Power activity, improved the mature process of cattle dung, China Kunming Engineering Corporation Limited, Kunming 650051, China. and thus resulted in a higher compost quality. Although Institute of Plant Protection and Soil Fertilizer, Hubei Academy of Agricultural Sciences, Wuhan 430064, China. Department of Soil Science, Bahauddin available N content increased, the final N O emission Zakariya University, Multan 60800, Pakistan. was mitigated by the change in compost microbial com- munity, which was driven by earthworm gut associated Received: 14 October 2022 Revised: 23 December 2022 Accepted: 30 December 2022 microbes. No matter how much biochar was added, biochar showed the consistent effects on cast functional genes. 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Biochar – Springer Journals
Published: Jan 17, 2023
Keywords: Earthworm; Biochar addition; Nitrous oxide; Functional genes; Burrowing activity
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