4.3 Changes in soil bacterial and fungal community composition and structure
The restoration of microbial diversity is a key issue in reclaimed soil systems (Lucas et al ., 2014). After a 90-day incubation, bacterial co-ordinated alpha and beta diversity were affected by the amendments. The Chao1 estimator of alpha diversity revealed a higher bacterial than fungal species richness in mine soils. Meanwhile, Chao1 estimator of bacterial was significantly higher in N2 level, indicating that proper N addition based on biochar (corresponding to C/N ratio of 25:1) promoted the restoration of species richness of soil bacterial community. However, Chao1 estimator of fungal was significantly higher in M2 level, indicating that higher M addition promoted the restoration of species richness of soil fungal community. We also observed that bacterial alpha-diversity (represented by Simpson indices) in all amended treatments (especially in N2 level) increased significantly compared with CK treatment, while no significant increase was recorded for in soil fungal community, indicating that amended treatments promoted the restoration of species diversity of soil bacterial community, but were not sufficient for increasing that species diversity of soil fungal community.
Beta diversity further indicated that the bacterial community composition formed three separate clusters based on N level, while the fungal community composition was mainly separated by M addition. This indicated that the N level may be a key driving factor behind the positive influence of biochar on bacterial community composition. A possible reason is that the composition of soil bacterial community may be regulated by C/N ratio in the combined N level and biochar. However, composition of the microbial agent itself may also affect clusters of fungal communities, indicating that fungal community composition was not regulated by biochar and N but by microbial agent in this study. This finding may attribute to fungi have higher soil nutrient level requirements than we provided by biochar and N fertilizer compared with bacteria (Niu et al ., 2015). The addition of microbial agent affected the composition of soil fungal community, but further studies are needed to confirm this result. Meanwhile, our study also revealed that soil physicochemical properties and microbial biomass together explained a larger proportion of variation in bacterial communities (74.73%) than in fungal communities (35%). This result further confirmed that soil bacteria are highly sensitive to the changes in soil nutrients (Yao et al ., 2014).
In this study, high-throughput sequencing revealed significant changes in soil bacterial community structure due to the application of amendments at the end of the incubation. The Proteobacteria phyla dominated soil bacterial communities across all soil samples, which was consistent with predominant microbiota found in the mining area in a previous study (Kolton et al ., 2011; Narendrula-Kotha and Nkongolo, 2016). This may be related to the extensive degradation properties of Proteobacteria and their ability to inhabit a wide range of habitats (Hanna et al ., 2013). At the same time, in our study, the increase over control in the abundances of Proteobacteria phyla after addition of amendments may be due to fast growth rates when levels of available substrates are high (Zhang et al ., 2016; Su et al ., 2017; Wang et al ., 2017). Moreover, Fig.5 a also showed that the accumulation of soil nutrients provided resources for the survival of Proteobacteria (Fiereret al ., 2007). We also found that the relative abundance of Bacteroidetes (P <0.001) and Firmicutes (P <0.01) increased significantly in C-M-N treatments, especially in N2 and N1 treatments, respectively. Bactericide have fast growth rates and are more likely to grow in eutrophic conditions (Willet al ., 2010), which explains the increase in Bacteroidetes in C-M-N treatments. Firmicutes have the ability to secrete enzymes that are key to the nitrogen fixation pathway and are directly involved in various other nitrogen metabolism functions such as nitrate reduction, dissimilatory nitrate reduction, and denitrification (Ren, 2018); thus, Firmicutes are considered to have the potential to promote nitrogen cycling after addition proper amounts of N fertilizer like N1 level in our study. In addition, the relative abundance of Acidobacteria decreased significantly with addition of N fertilizer. The result of RDA also confirmed that the abundance of Acidobacteria was negatively related to TN (Fig.6a). Acidobacteria are generally classified as slow-growing oligotrophs (Fierer et al ., 2007; Wang et al ., 2020), and their abundances usually decrease with N fertilizer application (Francioli et al ., 2016).
Overall, the relative abundance of Bacteroidetes (P <0.001), Firmicutes (P <0.01) and Acidobacteria (P <0.001), changed significantly after proper N addition especially N1 and N2 level (corresponding to C/N ratio of 35 and 25:1), indicating that proper C/N ratio (35 and 25:1) has a significant effect on the relative abundance of these three bacteria.
The dominant fungal phylum in this study was Ascomycota, corresponding to findings of previous studies in mining soils. Also, the relative abundance of Ascomycota phyla decreased with the addition of N fertilizer but not significantly. This decrease was likely due to preferred habitat of Ascomycota, which are particularly important under conditions of low N availability, and decline with increased N availability (Beimforde et al ., 2014; Yuet al ., 2020) in agreement with the negative correlation betweenAscomycota and TN (Fig.5b). Notably, the relative abundance of Chytridiomycota (p<0.05) decreased significantly at all amended treatments, which may be due to a more sensitive response of Chytridiomycota to changes in soil acidity and nutrient availability. However, the pH was still alkaline in our experiment although it was neutralized after amendments addition. The relative abundance of Basidiomycota, a decomposer of glucose and cellulose, increased after M addition, and decreased in C and C+N treatment. This may be related to the addition of M which can promote the metabolism of recalcitrant organic carbon by Basidiomycota (Yanget al ., 2019). In summary, soil properties (especially related to N), played an important role in shaping fungal community composition.
Conclusions
This work is one of the first attempts to determine the effects of three amendments (biochar, N fertilizer, microbial agent) based on C/N ratio( regulated by biochar and three N level) on soil quality and soil microbial structure and diversity. Our results showed relatively higher contents of N and M addition based on biochar in the short-term can improve soil quality by neutralizing soil pH and increasing soil nutrient content. N2-treated soil (corresponding to C/N ratio of 25:1) could contribute to the increase of microbial biomass and effectively improve the utilization rate of soil N.
N2-treated soil combined with biochar and the microbial agent could significantly promote the restoration of species richness and diversity of soil bacterial community; meanwhile, PCoA further indicated that the N level (corresponding to C/N ratio) may be a key driving factor behind the positive influence of biochar on bacterial community composition. N2 and N1-treated soil (C/N ratio of 25 and 12.5:1) has a significant effect on the relative abundance of Bacteroidetes, Firmicutes and Acidobacteria. M2-treated soil could promote the restoration of species richness of soil fungal community, PCoA further indicated that fungal community composition was regulated by M addition in this study. In addition, RDA analysis indicated that soil bacteria are highly sensitive to the changes in soil nutrients than fungal. Overall, our study provided a new idea for changing soil microbial community by regulating C/N ratio by amendments to achieve restoration of damaged habitats, which provided a basis for field application to land managers at this coal mine in Qilian mountains.