3.1. Physicochemical properties of compost
Table 2 shows the changes in the physicochemical properties of the compost during different stages. The temperatures in all treatments increased to their highest levels (> 60°C) on day 2. The reason for this result might be that microorganisms prioritized to use the easily degradable organic components. The thermophilic phase in O2 treatment had the highest temperature, which was consistent with Zhu (2020b) on the impact of sugarcane molasses on the composting temperature, indicating that nutrient-regulating substances could effectively increase the temperature in thermophilic phase. However, the duration of high temperature for all treatments in this study was relatively short, which could be related to the size of the compost (Meng et al., 2020). The pH increased at the beginning of the composting and then decreased. The increase in the pH might have related to the mineralization of nitrogen-containing organic matter. And a large amount of nitrogen was released from the compost in the form of ammonium nitrogen (Yang et al., 2019). As the composting process continued, ammonia volatilization and nitrification increased, and inorganic acids and organic acids were produced, and thus the presence of H+ made the pH decrease gradually before it then tended to stabilize (Zhang et al., 2017). At the end of the composting process, the pH value decreased most significantly in the O2 treatment compared with the other treatments (P < 0.05).
Table 2
Changes in physicochemical properties (temperature, pH, C/N ratio (total organic carbon/total nitrogen), and seed germination index (GI)) under different treatments during composting (n = 3).
Treatment
|
Temperature (°C)
|
pH
|
C/N
|
GI (%)
|
Day 0
|
CK
|
30.0 ± 1.2a
|
7.79 ± 0.01a
|
24.7
|
23.3 ± 3.1a
|
Day 1
|
CK
|
43.7 ± 1.5c
|
8.52 ± 0.00c
|
24.4
|
31.0 ± 3.9bc
|
O1
|
52.0 ± 2.0b
|
8.66 ± 0.02a
|
24.7
|
35.0 ± 0.3b
|
O2
|
51.5 ± 0.9b
|
8.56 ± 0.01b
|
24.4
|
40.0 ± 2.2a
|
O3
|
57.8 ± 1.9a
|
8.32 ± 0.02d
|
24.5
|
33.5 ± 12.1bc
|
Day 2
|
CK
|
57.4 ± 1.2b
|
8.72 ± 0.01a
|
24.5
|
41.6 ± 2.5ab
|
O1
|
62.8 ± 1.3a
|
8.55 ± 0.02b
|
24.2
|
39.1 ± 0.7b
|
O2
|
63.2 ± 1.2a
|
8.41 ± 0.01c
|
23.8
|
44.8 ± 7.9a
|
O3
|
62.0 ± 1.2a
|
8.39 ± 0.00c
|
23.8
|
42.7 ± 1.4ab
|
Day 6
|
CK
|
42.7 ± 0.6c
|
8.39 ± 0.01a
|
23.1
|
57.3 ± 2.6b
|
O1
|
46.7 ± 0.5a
|
8.18 ± 0.00b
|
22.8
|
66.7 ± 4.4a
|
O2
|
46.3 ± 1.2a
|
7.97 ± 0.00c
|
23.3
|
64.4 ± 4.7ab
|
O3
|
44.7 ± 1.0b
|
7.85 ± 0.01d
|
23.1
|
62.0 ± 1.7ab
|
Day 13
|
CK
|
43.5 ± 1.3a
|
8.28 ± 0.01a
|
21.9
|
79.4 ± 4.1b
|
O1
|
43.3 ± 0.5a
|
8.00 ± 0.01b
|
20.2
|
82.4 ± 6.0ab
|
O2
|
42.7 ± 0.6a
|
7.57 ± 0.00d
|
20.1
|
84.7 ± 6.8a
|
O3
|
39.2 ± 0.5b
|
7.74 ± 0.04c
|
21.3
|
69.2 ± 2.0c
|
Day 25
|
CK
|
34.0 ± 0.9a
|
8.14 ± 0.01a
|
20.8
|
93.9 ± 21.1c
|
O1
|
32.0 ± 0.5ab
|
8.06 ± 0.03b
|
20.0
|
91.6 ± 19.3c
|
O2
|
31.0 ± 0.6ab
|
7.68 ± 0.02d
|
19.5
|
109.4 ± 34.7a
|
O3
|
30.5 ± 0.5b
|
7.76 ± 0.02c
|
21.6
|
100.8 ± 11.2b
|
Note: The C/N values were calculated based on the average total carbon and total nitrogen contents, and they did not differ significantly. The a, b, c, d means difference between groups, and the same superscripts denoted not significant difference (P > 0.05), difference superscripts denoted significant difference (P < 0.05). |
The GI value in the four treatment increased in general trend. The initial GI, 23%, increased to 93% in CK, 92% in O1, 109% in O2, and 101% in O3 in the end compliance with the compost maturity standard proposed by Bernal (2009) of GI > 80%. The GI value was relatively high with the O2 treatment, thereby indicating that adding 0.5% MO had an important effect on reducing the toxicity of the compost. Thus, adding MO at a specific concentration may have effectively inhibited the growth of harmful bacteria and promoted the reproduction of beneficial bacteria to reduce the toxicity of the compost and increase the GI value to some extent (Ismail et al., 2019). Similar to the findings obtained in other studies, the C/N ratio decline in trend with the composting time (Zhao et al., 2017), and the C/N ratio decreased significantly with the O2 treatment, possibly due to the carbon loss caused by mineralization.
3.2. Lignocellulose contents
Plant cell walls mainly comprise cellulose, hemicellulose, and lignin. Figure 1a shows that the hemicellulose contents stabilized in the four treatments, after the initial decreased rapidly in mesophilic phase with the composting time, as also found by Wang et al. (2017). Because hemicellulose is an active component of lignocellulose and it can be utilized as a carbon and energy source by the microorganisms in compost (Xu et al., 2019). Microorganisms require large amounts of energy to reproduce and grow during the mesophilic and thermophilic phases of composting which made hemicellulose contents decreased rapidly. The hemicellulose contents descend with the time and tend to be stable because of the consumed by microorganisms. At the end of the composting process, the hemicellulose contents were 3.12% in CK, 2.95% in O1, 2.25% in O2, and 2.65% in O3. Figure 1b shows that the changes in the cellulose contents were similar to those in the hemicellulose contents, but the hysteresis was observed clearly, possibly due to the enzymatic degradation of cellulose being inhibited by the formation of the lignin–hemicellulose complex (Xu et al., 2019). At the end of composting process, the cellulose contents were 13.99% in CK, 12.76% in O1, 11.25% in O2, and 11.83% in O3. Thus, the final proportions of hemicellulose and cellulose was lowest in O2, thereby indicating that inoculation with 0.5% manno-oligosaccharide effectively improved the enzymatic reaction with lignocellulose to reduce the hemicellulose and cellulose contents.
Because of its special structure, lignin is not easy to be degraded by microorganisms (Kluczek-Turpeinen et al., 2003). The lignin contents decreased slowly in initial (Fig. 1c), it was due to the preferential use of simple organic matter (polysaccharides, proteins, hemicellulose, etc.) by microorganisms. After the cooling period, the microorganisms associated with lignin degradation gradually became the dominant population, and thus the lignin content decreased more rapidly in the middle and late composting periods. At the end of the composting process, the final lignin contents were 7.76% in CK, 7.50% in O1, 7.07% in O2, and 7.95% in O3. The lignin content decreased significantly in O2 in the later stage of composting, thereby indicating that adding 0.5% MO could effectively adjust the microbial community structure in the compost and reduce the lignin content in this stage.
Composting usually takes a long time. Cellulase can promote the biotransformation of organic matter and it has an important role in the degradation of lignocellulose and fasten compost maturation process. Previous studies have shown that the activity of cellulase depends on the abundances and structural composition of microorganisms involved in cellulose decomposition (Goyal et al., 2005). Figure 1d shows that the cellulase activity increased initially, and peaked in the cooling stage because microorganisms could store sufficient energy to release cellulase and degrade cellulose (Du et al., 2019). Compared with the other treatments, the cellulase activity of O2 treatment was significantly higher in maturation phase, indicating that adding 0.5% MO enhanced the microbial activity in the late stage to promote the secretion of cellulose-degrading enzymes and accelerate the degradation of cellulose.
3.3. Carbon contents
It is noteworthy that Fig. 2a shows TOC contents first decreased and gradually stabilized because microorganisms can degrade organic matter and the carbon will be emitted as the form of CO2 and CH4 (Pisa et al., 2020). However, with the humification strengthened and mineralization weakened during composting process, the rate of carbon loss decreased, and thus the TOC contents gradually stabilized. The carbon degradation rates of CK, O1, O2 and O3 in the thermophilic phases (day 25) were 9.7%, 10.8%, 11.2% and 9.0%, respectively. The total TOC content in each treatment on day 26 was ranked as follows: O3 (255.4 mg/g) > CK (248.9 mg/g) > O1 (241.5 mg/g) > O2 (238 mg/g). The results indicated that a small number of MO could effectively improve the carbon degradation of compost, and excessive MO had an inhibitory effect. The carbon degradation rate in O3 treatment was the lowest, which could be explained by two reasons. The first reason was related to the high content of MO, the existence of MO resulted in the high TOC content. The second reason was that microorganisms preferentially use MO, which led to the organic matter in O3 treatment could not be fully degraded in the initial stage of compost.
WSC comprises active carbon components and the WSC content can reflect the maturity and stability of compost (Bai et al., 2020). As shown in Fig. 2b, the WSC contents inside composting gradually decreased due to the presence of abundant degradable substances with high water solubility in the raw materials. As the degree of humification increased, large amounts of stable substances such as humus were formed and the water solubility of the materials decreased. Therefore, the WSC contents were low in the mature compost (Zhu et al., 2020a; Duan et al., 2020). After 25 days, the WSC contents were ranked in the order: CK (mg/g) > O1 (9.07 mg/g) > O3 (6.37 mg/g) > O2 (5.53 mg/g), and all were less than the compost maturity index (WSC < 17 mg/g) proposed by Bernai (1998), thereby indicating that the compost products were fully mature.
3.4. Changes in the bacterial community
According to a previous study, compared with O2, O3 treatments, the values and contents of pH, GI, hemicellulose, cellulose and WSC in O1 treatment were closer to CK treatment. So, the Illumina HiSeq PE250 platform was used to conduct high-throughput sequencing of the variable V4 region of the 16S rRNA gene for CK, O2, and O3. The sequencing results are shown in Fig. 3. The four bacterial phyla with the highest abundances in all treatments were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes, which accounted for more than 80% of the total, and the similar results was obtained in previous studies of compost (Wei et al., 2018; Akyol et al., 2019). In all of the composting stages, the highest relative bacterial abundances occurred in the thermophilic phase. The dominant phylum in this stage were Firmicutes and Actinobacteria. The addition of MO significantly changed the bacterial composition during composting, where the relative abundance of Firmicutes increased most in O2 and this phylum accounted for more than 55% of the total bacteria. The similar results were obtained in a previous study (Zhang et al., 2016). The high simple organic matter content (hemicellulose and cellulose) in the early stage could have been utilized directly by Firmicutes to facilitate its rapid reproduction. The simple organic matter content decreased in the cooling and maturation phase, and the relative abundance of Firmicutes decreased. The dominant phylum in the maturation phase was Proteobacteria. Correlation analysis also showed that the abundance of Proteobacteria had a strong negative correlation with the lignocellulose content (Table 3), possibly because the members of Proteobacteria remained in a dormant state during the thermophilic phase before reactivating during the cooling phase (Tkachuk et al., 2014), and the lignocellulose content decreased gradually with the composting time. In addition, Actinobacteria was the main phylum involved in cellulose degradation (DeAngelis et al., 2011) and this phylum was mainly detected in the maturation phase. Actinobacteria can regulate the activities of key enzymes to degrade lignocellulose (Wei et al., 2019). The abundance of Actinobacteria had strong positive correlations with the cellulose and lignin contents, thereby indicating that Actinobacteria made an important contribution to the degradation of lignocellulose during the composting process. Figure 3a shows that the relative abundance of Actinobacteria was highest in O2, which explains the low cellulose and lignin contents in this treatment.
Table 3
Correlations between lignocellulose contents and bacterial phyla.
|
Proteobacteria
|
Firmicutes
|
Actinobacteria
|
Bacteroidetes
|
Cellulose
|
–0.736**
|
0.859**
|
0.679*
|
–0.715**
|
Hemicellulose
|
–0.690**
|
0.883**
|
0.830**
|
–0.754**
|
Lignin
|
–0.647*
|
0.817**
|
0.766**
|
–0.683*
|
Note: * represents a significant correlation (P < 0.05), and ** represents a highly significant correlation (P < 0.01). |
At the genus level, Thermobifida (Actinobacteria) was the most abundant in all treatments, followed by Thermopolyspora (Actinobacteria), Geobacillus (Firmicutes), Planifilum (Firmicutes), and Streptomyces (Actinobacteria). The dominant bacteria differed among the various periods and treatments. Streptomyces, Romboutsia, and Planococcus were the main genera in the early stage of composting, and Thermobifida, Bacillus, Thermopolyspora, and Planifilum were highly abundant in the thermophilic phase. Previous studies have shown that members of the genus Thermobifida in Actinobacteria can degrade cellulose (Zhang et al., 2020), which might explain the rapid decline in the cellulose contents during the thermophilic phase. The abundance of Thermobifida decreased during the composting process under all treatments, but a high abundance of Thermobifida was observed in O2 during the maturation phase, and especially in the early maturation phase. Bacillus was an abundant genus in the phylum Firmicutes. According to Gannes (2013), members of Bacillus can tolerate high temperatures and degrade lignocellulose. The abundances of Luteimonas (Proteobacteria) and Chelativorans (Proteobacteria) were high in the cooling stage, and previous studies have shown that these bacteria can degrade lignocellulose and xylan (Zhang et al., 2011). Chryseolinea, Altererythrobacter, Taibaiella, and Cellvibrio were the dominant bacterial genera in the maturation phase. Chryseolinea is present in the soil rhizosphere and it can improve the soil structure and crop growth (Visioli et al., 2018).
3.5. Microbial functional genes
During composting, the transcription and expression of the corresponding functional genes were giving the microbial functions and the secreted enzyme. The classification information for the microbial community is usually insufficient to assess its impact on the composting process (Manoharan et al., 2017; Chen et al., 2020). Therefore, it is more appropriate to analyze the effects of microorganisms during the composting process by quantifying the diversity of the microbial functional genes. In this study, PICRUSt was used to investigate the microbial functional diversity and to determine the effects of MO on the microbial functions. The level 1 KEGG prediction results (Fig. 4a) identified seven functions, which “Metabolism” accounted for 49.7–51.7% and the largest proportion of all the functional genes, the following 15.3–16.2%, 12.6–15.6%, 12.8–13.8%, 2.9–4%, 0.8–1.2%, 0.8–0.9% were in genetic information processing, environmental information processing, unclassified, cellular processes, human diseases, and others, respectively. The abundances of functional genes increased in all treatments with the composting time.
The relative abundances of the first 30 functional genes among the level 3 KEGG prediction results are shown in Fig. 4b. There were some differences in the efficiencies of the bacterial functions during composting. The relative abundances of pyruvate metabolism, glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism genes related to carbohydrate metabolism increased during the thermophilic phase, especially O2 treatment. Similarly, Wang (2018) found that the relative abundances of genes related to the biosynthesis and metabolism of sugars and ketones increased during the thermophilic stage, and the carbon transformation was positively correlated with carbohydrate degradation genes (Hartman et al., 2017). It suggested that the rich carbohydrate metabolism genes at this stage were the main reason for the rapid degradation of lignocellulose and carbon. Membrane transcription and nucleotide metabolism also accounted for a large proportion in thermophilic stage, which was related to the rapid reproduction of microorganisms. Obviously, the relative abundance of these functional genes in O2 treatment was higher. The relative abundances of genes associated with arginine and proline metabolism, glycine, serine and threonine metabolism, and valine, leucine, and isoleucine degradation enhanced gradually in the cooling and maturation phase. This was because the carbohydrate degrading enzymes secreted by bacteria decomposed the proteins, cellulose and hemicellulose in thermophilic phases (Goh et al., 2013). A previous study also found that more sequences were annotated with roles in amino acid metabolism in this stage to significantly enhance the synthesis of humus (Wu et al., 2017). Replication, transcription, and translation are the main pathways related to bacterial enzyme secretion, and the abundances of genes associated with ribosome, chromosome, and ribosome biogenesis increased with the composting time, thereby indicating that bacteria continued to produce secretions to participate in lignocellulose metabolism. In addition, some functions were processed by genes associated with “Environmental information,” such as “Secretion system,” which also demonstrates that the secretion of related enzymes increased with the composting time. The propanoate and butanoate contents are closely related to the pH, and the increased of butanoate metabolism and propanoate metabolism mainly explained the decrease and stabilization in the pH during the composting process (Duan et al., 2020). Figure 4b shows that the relative abundance of tuberculosis (human diseases) in O2 treatment was lowest in all treatments, which indicated that 0.5% of MO had a certain inhibitory effect on pathogenic bacteria.
3.6. Correlations between physicochemical properties, microbial community composition, functional genes, and lignocellulose contents
The bacterial community and its functions were affected by the physicochemical properties of compost, which affecting lignocellulose content. Thus, RDA was conducted to assess the correlations between the composting environment, microbes, and changes in the lignocellulose contents. Figure 5 shows the effects of the temperature, pH, C/N, microbial community, and metabolic functions on the lignocellulose, TOC, and WSC contents. RDA1 and RDA2 accounted for 98.8% and 0.2% of the variation, respectively (Fig. 5a). Among the eight factors were considered, C/N explained 84.5% of the total variation in the lignocellulose, TOC, and WSC contents (P = 0.002), Streptomyces explained 5.5% of the total variation (P = 0.012), temperature explained 4.9% of the total variation (P = 0.008), Luteimonas explained 1.7% of the total variation (P = 0.094), pH explained 1.5% of the total variation (P = 0.206), and Chryseolinea explained 0.8% of the total variation (P = 0.202). Thus, the C/N ratio had the greatest influence on the lignocellulose content because lignocellulose is rich in carbon. Carbon loss was inevitable due to the continuous degradation of lignocellulose, and thus the C/N ratio explained much of the total variance. The temperature was also positively correlated with the lignocellulose and carbon contents because the temperature decreased gradually with the compost matured, and the lignocellulose and carbon contents decreased due to microbial degradation. Streptomyces was the dominant genus in the early stage and it could tolerate the weak alkali environment. Studies have shown that Streptomyces can utilize lignocellulose as a sole carbon source to support growth, as well as effectively degrading hemicellulose, cellulose, and lignin (Feng et al., 2021). Luteimonas and Chryseolinea were mainly detected in the maturation phase and their abundances were negatively correlated with temperature, and thus these two bacterias were not resistant to high temperature but they were involved with the degradation of organic matter in the later stage.
As shown in Fig. 5b, RDA1 and RDA2 explained 99.1% and 0.3% of the variation in the cellulose contents, respectively. Among the eight factors were considered, C/N still explained most of the changes in the cellulose contents during composting, where it explained 83.6% of the total variation (P = 0.002), while the secretion system, pH, amino sugar and nucleotide sugar metabolism, purine metabolism, and temperature explained 4.6% (P = 0.084), 3.3% (P = 0.116), 1.5% (P = 0.43), 2.3% (P = 0.188), and 1.6% (P = 0.166), respectively. All of the functional genes were mainly distributed in the maturation stage and they had no significant negative correlations with the lignocellulose and carbon contents, mainly because the degradation of lignocellulose was more rapid in the early stage and bacterial functional genes were more abundant in the maturation stage. Compared with the other treatments, the O2 treatment had a stronger correlation with functional genes in the maturation phase. The results showed that adding 0.5% MO could effectively improve the abundance of functional genes and degrade lignocellulose. Furthermore, the pH was positively correlated with the lignocellulose and carbon contents, but negatively correlated with butanoate metabolism. It is well known that the pH is mainly affected by the organic acids produced via the degradation of complex organic matter (e.g., lignocellulose) (Duan et al., 2020). Therefore, organic acids accumulated and the pH decreased as the lignocellulose contents decreased. In addition, butanoate metabolism increased and butanoate was consumed in large quantities to inhibit the accumulation of organic acids.