Microbial communities have a central role in driving composting performance by mediating organic solid waste component transformation, but their roles are poorly understood [1, 2, 7, 34]. Determining the keystone microbial factors related to the superior performance of hTC (improved composting temperature, NC, N2O-N mitigation and GI) [7, 12, 14–16] is useful for better understanding the microbially driven mechanisms. In this study, we surveyed the variation in microbial functions and communities and their linkages with composting performance in factory-scale experiments in both hTC and cTC. We identified keystone genes involved in C degradation and N cycling that potentially play a pivotal role in the improved performance of hTC (enhanced composting temperature, NC, N2O mitigation and GI). Moreover, the keystone genes are more influential on performance indices than taxonomy is in hTC but not in cTC. These findings highlighted the importance of keystone gene abundances in mediating composting temperature, NC, N2O emissions and GI, providing the possibility to improve performance by regulating microbial functions in composting ecosystems.
Previous work has demonstrated that hTC resulted in a distinct bacterial community composition compared with that resulting from cTC [7, 15]. Our results go further, showing that hTC markedly shifted both the function and phylogenetic structures of microbial communities, as indicated by NMDS-based ordination for GeoChip and pyrosequencing data, respectively (Fig. 1, Fig. 3, Additional file 1, Figures S2, S3 and S7). Importantly, C-degrading and N-cycling genes experienced the greatest shifts between the two treatments (Fig. 1) and predicted selected individual performance indices accurately in both treatments (for hTC, adjusted R2 = 0.89–0.96; for cTC, adjusted R2 = 0.95–0.97; Fig. 2). This supports previous studies of functional genes such as those involved in the N cycle that have been targeted to obtain insights into various aspects of performance such as the N content, N2O-N emissions and N conservation [15, 16]. Notably, our results showed that hTC not only stimulates C-degrading genes but also suppresses genes involved in N cycling aspects such as nitrification, denitrification and N mineralization (Fig. 1). A potential explanation for the increased number of C-degrading genes in hTC may be that microbes have great potential to decompose C substrates under ultrahigh temperatures [35–37]. For instance, the amyA gene was significantly enriched in hTC; this gene encodes α-amylase, which exists widely in hyperthermophiles and is the most studied hyperthermostable enzyme [20, 21]. The enriched amyA gene might catalyze starch degradation to release more monomeric or oligomeric sugars in hyperthermic conditions [38, 39], which could enhance heat production by microbial metabolism and facilitate humic substance formation by the Maillard reaction (condensation between amides and reducing sugars) . All N-cycling genes exhibited lower relative abundance in the hTC treatment, suggesting a shift to hinder some microbial functional groups that can utilize N-containing substrates or losses in certain organisms that thrive at elevated composting temperatures [9, 41–43]. These results were in accordance with our finding that the relative abundance of the genus Bacillus, a strongly ammonifying taxon , was lower in hTC (Additional file 1, Figure S7). This shift may reflect the adaptation of microbial communities in hTC to hyperthermophilic conditions over time [45, 46]. Consistently, both inorganic N content (NO2−, NO3− and NH4+) and gaseous N emission (NH3 and N2O) were decreased in hTC (Additional file 1, Table S2), and NC was increased (Additional file 1, Figure S1).
We further detected the variations in bacterial abundance and community composition by applying qPCR and high-throughput sequencing of bacterial 16S rRNA genes, respectively. The results showed that the unique hyperthermophilic stage (composting temperature above 80°C) can be sustained for at least 5 days (Additional file 1, Figure S1a), leading to lower bacterial abundance and diversity by selection and environmental adaptation in hTC than in cTC (Additional file 1, Figure S5). Consistently, the phyla Proteobacteria, Firmicutes, and Bacteroidetes dominated in cTC and were replaced with hyperthermophilic taxa (Deinococcus-Thermus and Actinobacteria) during the hyperthermophilic stage (Fig. 3a, Additional file 1, Figure S7). Hyperthermophiles have many properties making them suitable for organic waste treatment, as they typically have higher growth rates and tolerances to wide ranges of environmental conditions such as temperature, salt, pH and low nutritional requirements [35, 36, 47]. For example, the high temperatures at which thermostable C-degrading enzymes in hyperthermophiles can operate at allow more substrate to dissolve, which can increase diffusion and mass transfer rates and thus shift the equilibrium [39, 48]. Additionally, although a wide variety of hyperthermophiles catalyze exergonic redox reactions involving nitrogenous compounds, nitrification, denitrification and dissimilatory nitrate reduction were strongly inhibited at high temperatures during composting [9, 42, 49]. The correlations between the microbial community composition (Bray-Curtis distance) and functional structure were further confirmed by Procrustes tests (for hTC, P < 0.05, M2 = 0.8026, R = 0.3111, 9999 permutations; for cTC, P < 0.01, M2 = 0.70544, R = 0.5428, 9999 permutations; Additional file 1, Figure S8) and pairwise similarity with linear regressions (P < 0.001, similarity was calculated by Bray-Curtis distance, Additional file 1, Figure S9). Such features might explain the distinct functional patterns in hTC compared with those in cTC.
Microbial co-occurrence relationships associated with composting performance indices were resolved in greater detail by constructing correlation networks among microbial taxonomy, functions and selected performance indices for both treatments (Fig. 3b) [50, 51]. Our results indicate that the selected composting performance has a much closer link with keystone genes than with taxonomy in hTC, while these associations in cTC were comparable. This is not particularly surprising since the microbial community displayed drastic variations in composition, while the function maintained a relatively similar structure throughout hTC (Fig. 3a, Additional file 1, Figure S7) [8, 13]. This functional similarity within changing communities may be attributed to strong stoichiometric balancing between multiple metabolic pathways, the majority of which serve to decompose complex organic molecules into simpler molecules . These metabolic pathways have been discovered in nearly every form of microbial life, resulting in metabolic functional structures that are relatively conserved across divergent communities . Furthermore, we found that the complexity of taxonomy-function-performance networks and the numbers of highly connective taxa and genes were significantly decreased in hTC. These results are consistent with those of previous studies showing that the correlations between species and the complexity of the microbial cooperation network of a community are usually simplified under harsh environments such as oil-, mercury- and alkaline tailing-contaminated ecosystems [18, 53, 54]. More importantly, given that more than half of the edges were identified between microbial taxonomy and function in both treatments, the numbers of connected genes in hTC were lower than those in cTC, while the contribution of function-performance edges to cooccurrence patterns was higher in hTC. This highlighted the important role of keystone genes in influencing performance indices in hTC, suggesting that functional redundancy might be reduced in hyperthermophilic communities [55, 56]. The results of PLS-PM further indicated that the dominant effects of keystone genes on hTC performance indices were maintained after multiple biotic drivers (community composition, other genes such as those involving organic remediation, metal homeostasis, and phosphorus cycling) were simultaneously considered. Previous studies also indicated that the community composition of the macroalga Ulva australis was best explained in terms of functional information rather than taxonomic information, and the human gut microbiota exhibits a core set of genes despite high taxonomic differences between individuals [57, 58]. Similarly, microbial functional attributes were the predominant biotic factors driving soil processes, including soil respiration, denitrification and nitrification . However, although the GeoChip method can provide comprehensive information about microbial functions in C degradation and N cycling, it cannot determine the active portions of communities during composting [20, 55, 56]. Therefore, determining the transcription related to functions might indicate stronger relationships between composting performance and microbial functions [22, 59].
In conclusion, this is the first comprehensive study to demonstrate that functional gene abundances were the most influential biotic factors in composting performance in hTC. More importantly, our study provides a list of keystone genes involved in C degradation and N cycling that potentially play a pivotal role in dominating composting performance, showing that hTC altered bacterial communities and their functions, which led to improved composting temperature, NC, N2O mitigation and GI. Understanding the functional contributions of microbial communities to composting performance may also allow us to better predict how composts will function in further fertilization. Overall, our study provides evidence for a strong relationship between microbial functions and composting performance and indicates that functional genes rather than taxonomic information were more predominant drivers of the improved composting temperature, N2O emissions, NC and GI in hTC.