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.