3.1 Physiochemical characteristics
Similar variations in composting temperature was observed for all groups (Fig. 2a). The temperature of all experiment groups increased significantly to their summits (about 70°C) on day 3–4 due to rapid decomposition of organic matter and then decreased gradually. A secondary increase in temperature was observed in all experiment groups on day 10, probably because the organics in anaerobic zone was decomposed for heat production after turning on day 9. In the later stage of composting, the temperature of all groups was maintained at 40°C or below. After composting, the thermophilic phase (≥ 50°C) lasted for all groups was all over 7 days and SB2 had the longest duration. Compared with the CK, the B1 and B2 warmed up faster and had higher peaks, reaching 71.7°C and 68.8°C on day 4, respectively, which might be due to the promoted microbial activity from abundant, exogenous microorganisms in the mature compost. However, the temperature in S was obviously lower than CK in the thermophilic phase. This was mainly related to the lower pH due to the addition of acidic additive SP, which might inhibit the microbial activity, thus delaying the heating rate of the pile in the early stage.
During composting, microorganisms consumed O2 to biodegrade organic substances to enhance the substrate temperature (Li et al. 2018). A sharp decline and then gradual increase to the ambient level in O2 content was observed for all experiment groups due to the intensive biodegradation and then depletion of easily organic matter, respectively (Xu et al. 2020) (Fig. 2b). On day 3–4 of composting, the O2 content of each group dropped to the lowest value. The O2 content of B2 and SB2 reached the lowest values of 7.5% and 8.0% on day 3, respectively, and then remained at a lower level, suggesting higher strength of microbial aerobic metabolism due to the introduce of 2B.
The trend of pH changes remained basically the same for all groups, with a trend of rapidly rising tin the early stage then leveling off (Fig. 2c). The initial pH of each group ranged from 4.69 to 5.21. Microorganisms multiplied and produced heat under sufficient nutrient conditions, which was favorable to ammonification. With the volatilization of NH3 and the enhancement of nitrification, the pH of each group slightly fluctuated and finally stabilized. The pH of S raised slowly in the early stage, which was mainly due to the acidic SP. The EC values reflect the salt content in the pile. The changes of EC values were similar for each group, with an overall increasing trend (Fig. 2d). In the early stage of composting, organic macromolecules decomposed and produced NH4+ and small molecule fatty acids, leading to the gradually increased soluble ions (Pan et al. 2019). By the end of composting, the EC values of all groups ranged from 2.35 to 4.12 mS/cm, and the EC values of S, SB1 and SB2 were significantly higher than that of CK, probably because the addition of SP inhibited the conversion of NH4+ to NH3 and thus increased the EC values. The GI value of each group ranged from 43.2–127.8%, and the final GI value of B2 showed significantly higher than others (p < 0.05) (Fig. 2e). In the composting process, GI increased and tended to steady, as the toxic substances in the piles were gradually degraded (Pan et al. 2019). GI in the pile with adding SP raised slowly, and the lowest value at the end, suggesting that SP had an inhibitory effect on GI.
The variation of MC for different groups was shown in Fig. 2f, and the initial MC of each group was 60–65%. The overall MC of each group showed a continuous decreasing trend in the composting process. The dewatering effect of each group was obvious on day 3–12, reaching 22.4%-38.9%. After 15 d of composting, the MC of each group ranged from 12.8–35.5%, and the MC removal rates ranged from 81.4–93.3%. Compared with CK, SP and mature compost amendments improved the MC removal rate, with the best dewatering effect in SB2. After composting, the VS degradation rates of CK, B1, B2, S, SB1, and SB2 were 38.9%, 42.7%, 44.1%, 35.6%, 37.9%, and 40.5%, respectively (Fig. 2g). VS degradation rates of B2 and SB2 were higher than other groups, indicating that the addition of 2B significantly promoted the organic matter degradation.
3.2 Nitrogenous gases emissions and nitrogen conversion
3.2.1 Emission of nitrogenous gases
The NH3 emission could be ascribed to the intensive mineralization of organic nitrogen to NH4+, which was further transformed into NH3 under high temperature and alkaline conditions (Xu et al. 2021). The NH3 emission rate of each experimental group raised obviously since day 3–4, reaching the peak emission on day 5–6 (Fig. 3a), and then decreased as the organic matter degradation rate became slower and the pile temperature started to decrease. The peaks of NH3 emission rate in B1 and B2 were earlier than CK, and that of S was delayed, which was related to the increased temperature as mature compost added and the decreased pH as the introduce of SP. The accumulative NH3 emission decreased in the order: CK > B1 > B2 > S > SB2, SB1 (Fig. 3b). NH3 emission was more intense in the thermophilic stage of composting, accounting for 82.0%-88.1% of the total NH3 emission. Compared with CK, all the piles with additives had a suppressive effect on ammonia emissions, and the accumulative NH3 emissions of B1, B2, S, SB1, and SB2 were reduced by 7.05%, 18.62%, 32.73%, 34.69%, and 39.86%, respectively. It was reported that mature compost had fluffy structure and high adsorption capacity (Song et al. 2021), which might benefit the reduction of NH3 emission. The decreased pH due to SP addition had more effect on reducing NH3 volatilization compared to adding mature compost. The combined application of SP and mature compost could significantly reduce NH3 emissions, with the best reduction of 39.86% for SB2 based on the physical and chemical reactions. Furthermore, the application of additives might promote the action of functional microorganisms, which had a key role in the conversion of NH3. (Jurado et al. 2014; Zhang et al. 2018)
The N2O emission mainly occurred in the initial and late stages of composting for all groups (Fig. 3c). There were 85.9%-93.0% of N2O emissions in the first week of composting. This was mainly due to the rapid increase in temperature at the beginning of composting, and the oxidation of organic nitrogen may promote the emission of N2O. It was reported that the nitrification gradually increases in the later stages of composting due to the drier substrate, good aeration conditions and decreasing pile temperature (Guerra-Gorostegi et al. 2021), resulting in smaller N2O emissions. The accumulative N2O emission was decreased as S > CK > SB2, B2 > SB1 > B1 (Fig. 3d). All the additives except S could suppress N2O emission, which was reduced by 36.9%, 17.0%, 23.6%, and 15.7% for B1, B2, SB1, and SB2, respectively, compared to CK, and the addition of mature compost had the most significant inhibitory effect on N2O emission.
3.2.2 Nitrogen conversion and loss
An increase and then decrease of NH4+-N content was observed for all groups during composting and the initial NH4+-N content of each group ranged from 0.20 to 0.42 g/kg (Fig. 4a). In the early stage of composting, the NH4+-N content of each group started to rise and reached the peak on day 3–9 because nitrogenous organic matter was decomposed by microbial assimilation to produce a large amount of NH4+-N (Wang et al. 2017). Then, NH4+-N content decreased as the ammonification effect gradually weakened and a large amount of NH4+-N was converted into NH3 and escaped (Moenne-Loccoz and Fee 2010). By the end of composting, the peak of NH4+-N content was found in S, which was mainly due to the small NH3 emission in the early stage of and the rate of NH4+-N production was greater than the conversion rate.
The initial NO3−-N content of each group ranged from 16.55 to 20.65 mg/kg (Fig. 4b). In the first 3 days, the NO3−-N content of each group showed a decreasing trend, which was mainly due to the significant denitrification of the pile in the early stage, and part of NO3−-N was converted to N2O and escaped. On day 3–9, NO3−-N content in each group showed a slight increase, but remained at a low level, which was mainly due to the inhibition of temperature-sensitive nitrifying bacterial activity during the thermophilic stage. Then the pile temperature started to decrease and nitrification gradually increased, and the NO3−-N content showed an increasing trend on day 12–15. By the end of composting, NO3−-N content of CK, B1, B2, S, SB1, and SB2 was significantly different (p < 0.01), which increased by 81.6%, 98.3%, 87.8%, 58.4%, 127.5% and 139.9%, respectively, compared with the initial NO3−-N content.
The initial TN concentration ranged from 31.10-33.67 g/kg in each group (Fig. 4c). In the early stage, TN concentration of each group showed a decreasing trend due to the loss of nitrogen caused by the volatilization of large amount of NH3. As the pile entered the cooling stage, nitrification was enhanced and TN concentration slightly rebounded. The nitrogen loss rates of each treatment ranged from 29.9–49.3% and SB2 had the lowest nitrogen loss rate (Table 3). Overall, CK had the largest nitrogen loss rate, which was mainly due to the high NH3 emission and inhibited nitrification as the pile was maintained at a high temperature by the external heating method. All types of additives could reduce the nitrogen loss to different degrees. The ratio of N2O emission to nitrogen loss for each group ranged from 1.5–3.1% and the ratio of NH3 emission to nitrogen loss for each group ranged from 32.8–49.2%, meaning that NH3 emission was the main factor causing nitrogen loss in the composting.
Table 3
Nitrogen loss and nitrogen balance for different experiment groups
Experiment groups | TN | NH3 | N2O |
Initial content (g/kg) | Final content (g/kg) | Nitrogen loss rate (%) | NH3 emission (g) | Ratio of NH3 emission to nitrogen loss (%) | N2O emission (g) | Ratio of N2O emission to nitrogen loss (%) |
CK | 33.30 | 26.17 | 49.3 | 33.81 | 49.2 | 2.38 | 2.7 |
B1 | 31.73 | 27.93 | 46.2 | 31.43 | 40.7 | 1.50 | 1.5 |
B2 | 31.37 | 27.53 | 44.1 | 27.82 | 34.3 | 1.97 | 2.0 |
S | 33.67 | 28.17 | 41.5 | 22.75 | 34.5 | 2.65 | 3.1 |
SB1 | 31.40 | 29.20 | 40.0 | 22.09 | 32.8 | 1.82 | 2.1 |
SB2 | 31.10 | 31.70 | 29.9 | 20.33 | 39.1 | 2.00 | 3.0 |
Person correlation analysis showed that during the composting, temperature integration (TI) index had a significant positive correlation with NH3 emission (r = 0.943, p < 0.01) and nitrogen loss (r = 0.961, p < 0.01) (Fig. 5). This result suggested that the increase of the temperature facilitated the release of NH3 and caused a greater nitrogen loss. Nitrogen loss was significantly and positively correlated with pH (r = 0.810, p < 0.01), NH3 emission (r = 0.957, p < 0.01), N2O emission (r = 0.816, p < 0.01), and negatively correlated to VS (r=- 0.738, p < 0.01), and MC (r=-0.805, p < 0.01), indicating that the temperature, the dewatering and capacity reduction effect of the pile significant affected, the transfoemation of nitrogen fractions during composting.
3.3 Dynamics of bacterial community and its potential functions
Dynamics of bacterial community from each experiment group were sequenced and analyzed, and 36 samples were screened to obtain 1081089 valid sequences, including 38 phyla, 82 orders, 146 species, 305 families and 925 genera.
An increase and then decrease in all α-diversity indices (Chao 1 and Shannon index) occurred during the composting (Fig. 6a and b). The highest Chao1 and Shannon index were found for B2 and SB2 on day 0 of composting, which was mainly related to the abundant microorganisms in 2B. The Shannon index of each additive group increased in the mesophilic stage of composting (days 0–3), which was due to the sufficient nutrients at the early stage of composting that were favorable to the growth and metabolism of microorganisms. When the pile entered the thermophilic stage, the Shannon index started to decrease, which was mainly due to the fact that heat-resistant bacteria started to dominate during the thermophilic stage, while other species died due to intolerance of high temperature. By the 15th day of composting, the Shannon index of each group increased again, because the thermophilic microorganisms in the pile began to recover. By the end of composting, the Chao1 index of S was the largest, which may be be related to the postponed microbial decomposition process in the early stage of composting due to SP addition. The variation of beta diversity of bacterial community during composting were shown in Fig. 6c. Principal component analysis (PCA) also showed the pattern of microbial community variation with time. The results showed that the variation due to different experiment groups and composting time variation explained 59.5% and 16.8% of the total variation of bacterial community, respectively, indicating that the effect of composting time on bacterial community structure was greater than that of treatments.
To clarify the effects of different additives on nitrogen loss in the composting, a hierarchical cluster analysis was conducted based on different nitrogen indicators (NH4+-N, NO3−-N, NH3 emission, N2O emission, TN, and nitrogen loss) (Fig. 7), indicating that there were two major classes for six experiment groups, with SB2 as the first major class (A) and the remaining groups as the second major class (B). B could be divided into two subcategories, where the first subcategory was SB1 and the second subcategory were the remaining groups. The results of the sample hierarchical clustering analysis based on OTUs at different time points in the six experiment groups of test samples were shown in Fig. 7b-g. In the six sampling periods of day 0, 3, 6, 9, 12, and 15 of composting, the bacterial communities of different experiment groups formed different clustering branches. It could be seen that B2 and SB2 clustered into one group on day 0 of composting, and the rest of the groups clustered into one group, with S, B1, and SB1 clustered into one subgroup at a closer distance. A clear branch was formed between the CK and the rest of the additive groups at the middle 4 sampling times (day 3, 6, 9, and 12). By the 15th d of composting, the CK formed a separate cluster, B1 and B2 formed a branch, S and SB1 clustered into one category, and SB2 formed a separate cluster, which also corresponded to the cluster analysis of the nitrogen fraction content. The above results indicated that each additive changed the community structure of the flora in the pile, and all types of additives changed the living environment of microorganisms after adding to the pile, which in turn significantly affected the nitrogen loss and transformation of the pile, with SB2 showing the most obvious performance.
The succession of bacterial community during composting was examined by taxonomic analysis at the phylum level (Fig. 8). The dominant bacterial phyla mainly consisted of Firmicutes, Actinobacteria, and Proteobacteria. Previous studies suggested that these phyla were closely responsible for the biodegradation of organic substances and were detected ubiquitously in composting processes (Liu et al. 2018b; Wang et al. 2018a). In the mesophilic and thermophilic stages of composting, Firmicutes of each group was absolutely dominant, which is more related to the high heat tolerance of Firmicutes, and can grow rapidly under nutrient-rich conditions (Liu et al. 2018a; Wang et al. 2018a; Mao et al. 2019). As the pile temperature gradually decreased, the relative abundance of Firmicutes gradually decreased, while the relative abundance of Actinobacteria and Proteobacteria gradually increased and became dominant. Actinobacteria and Proteobacteria have been shown to be important phyla for the degradation of lignin, cellulose and proteins, which can effectively degrade organic matter in the pile and have a major contribution to the degradation and decay of the pile (de Gannzs et al. 2013). Thus, it is clear that Firmicutes play a major influence on pile warming, while Actinobacteria and Proteobacteria play a major influence on decomposition during composting.
The succession of bacterial community during composting was shown at the genus level (Fig. 9). Among the top 20 genera in terms of relative abundance in all experiment groups, 13 genera belonged to Firmicutes, 5 genera belonged to Actinobacteria. The remaining 2 genera belonged to Proteobacteria and Cyanobacteria. At the beginning of composting, the dominant species in all groups were Lactobacillus in Firmicutes, with relative abundance ranging from 26.7–58.7%. Lactobacillus showed the highest relative abundance in CK on day 0. As the temperature increased, the relative abundance of Lactobacillus began to decrease. On day 3–6, the pile temperature increased to above 50°C, and the relative abundance of Thermobifida of Actinobacteria and Ureibacillus of Firmicutes gradually increased as the dominant species. It is reported that Thermobifida and Ureibacillus, as two thermophilic bacteria, are the main microbial taxa that degrade organic matter at high temperature and have been detected in most of the high temperature periods of composting tests (Li et al. 2020). The relative abundance of Ureibacillus and Thermobifida in S was low (< 2%) in the early stage and increased substantially in the later stage of composting, which corresponded to the low degradation rate in the early stage and the substantial increase of fermentation rate in the later stage (Fig. 10). In contrast, Ureibacillus and Thermobifida in B2 and SB2 contained higher abundance on day 0 compared to other experiment groups, which might be related to the thermophilic bacteria retained by the 2B after multiple fermentation screening. Combined with the efficient dewatering capacity of B2 and SB2 and the changes of degradation rate of S, it was speculated that Ureibacillus and Thermobifida were the key strains for the high dewatering and decomposition capacity of 2B.
The relative abundance of nitrogen-fixing bacteria such as Azotobacter, Azospirillum, and paenibacillus in CK was low (0.1%-1.2%). In contrast, the relative abundance of nitrogen-fixing bacteria were higher in composting process with different additives (0.3%-2.4%), with the highest in SB2. The average relative abundance of Bacillus and Pseudomonas in composting process with additives were lower than that of CK (Fig. 10). These two genera of predominantly ammonifying and denitrifying bacteria are important for nitrogenous gas emissions as important drivers of ammonification and denitrification, and it was reported that changes in NH3 and N2O were correlated with the number and activity of this genus (Otawa et al. 2006). Towards the end of the composting, the temperature gradually decreased and the community diversity of the groups gradually recovered, with Saccharomonospora becoming the new dominant species in the experiment groups, while Lactobacillus almost completely disappeared.
In order to study the relationship between each nitrogen fractions and bacterial community, the association between nitrogen indicators and bacterial community was analyzed by redundancy analysis (RDA), as a way to clarify the main bacterial species affected by the use of additives on the transformation of nitrogen fractions. RDA showed that Firmicutes and Cyanobacteria were positively correlated with TN and negatively correlated with NH4+-N, nitrogenous gases (NH3, N2O), and nitrogen loss (Fig. 11a). While Actinobacteria and Proteobacteria were negatively correlated with TN, and positively correlated with inorganic nitrogen (NH4+-N, NO3−-N), nitrogenous gases (NH3, N2O), and nitrogen loss. These results indicated that the addition of additives had an important driving role for the conversion of nitrogen fractions in the composting process and could significantly affect the nitrogen loss and conversion process. As shown in Fig. 11b, the response of microorganisms to environmental factors in the composting process indicated that key microbes affecting different nitrogen fractions were significantly regulated by MC, which had the highest contribution to the change of bacterial community composition (p < 0.01).