4 Discussion
The results showed that land reclamation had a positive effect on the
provision of soil ecosystem functions, which were measured in the light
of the enzymatic activities (Figs. 2G-J). All the investigated plots
subjected to land reclamation showed higher enzymatic activities than
the control plots. The results suggested that URA, DHG and FDA increased
rapidly after reclamation (Figs. 2G, 2I and 2J), indicating that
long-time reclamation may change soil functions such as the conversion
of soil nitrogen, or the soil respiratory metabolism (Delgado-Baquerizo,
et al., 2016). Yet, an increase in the activity of PPO was only
noticeable after 11 years from reclamation, increasing steadily
subsequently (Fig. 2H), which might be related with the microbial
activity of Chloroflexi . In the Fig. S5 (see supplemental
material), the PPO showed the positive relation with phylumChloroflexi , whereas had negative relationships withProteobacteria and Bacteroidetes . In the Fig. 3A, the
abundance of increased Chloroflexi and declinedProteobacteria and Bacteroidetes along the reclamation
time. In addition, the activities of DHG and FDA presented some
fluctuations over time, suggesting that continuous land management (such
as crop planting, irrigation, or fertilization) is needed to maintain
their activities levels high (Xiao et al., 2019; Hu et al., 2016). All
the variations of soil enzyme activities in the study might suggest that
the reclaimed soil became healthier, which might be related with the
continuous agricultural production activities, as well as the natural
successional processes (Dick, 1994; Sarathambal et al., 2016).
The effect of land reclamation on the studied key soil attributes was
uncertain. Only the concentration of AK in the soil seemed to increase
following reclamation (Fig. 2D), while the levels of soil N and P were
lower in the reclaimed than in the control plots (Figs. 2E and 2F). The
increasing AK in this study (Fig. 2D) might be related with coal gauges
which contain potassium, and those coal gauges partially dissolving over
time (Hu et al., 2015; Spargo and Doley, 2016). This observation could
be also related to the fertilization schemes implemented in the studied
plots by the local farmers as well as to the depletion of soil nutrients
by the crops rotated in the studied plots (Yu et al., 2012; Wang et al.,
2016). In addition, SOM showed a decreasing trend along the reclamation
timeline and only 17 years after reclamation an increase in SOM was
noticeable (Fig. 2A). The latter observation could be related to the
accumulation of crop litter, leaves and roots over time, facilitating
the formation of humus in the soil (Ezeokoli et al., 2019; Mukhopadhyay
et al., 2016). It is also worth noting that the observed decreasing
trend in SOM could be related to higher mineralization levels of SOM
provided by a changing soil microbial community following reclamation,
as we found herein (Zhong et al., 2015; Duval et al., 2016). The
reclaimed soil composition has influenced the soil organic matter, which
thus might affect the change of total N and Olsen-P contents, which
indicated that the soil phosphorus content might be positively
correlated with soil organic matter (Fig. S5, see supplemental
material).
Contrariwise, the soil pH and EC levels increased over the reclamation
timeline, indicating that soil alkalinity increased after reclamation.
These observations contradicted earlier studies (Mukhopadhyay et al.,
2014, Adeli et al., 2013) and were likely produced by the presence of a
water table near the ground surface and with high concentration in salt
(Qu et al., 2018) resulting from the release of cations (e.g.
Ca2+, Mg2+) present in the coal
gangue from past mining activities (Hu et al., 2015). Another possible
source for the increase in soil pH and EC following land reclamation
could be the application of inorganic fertilizers by the local farmers
(Liu et al., 2018), or the soil materials used for reclaiming the land.
The latter stresses that careful monitoring of the attributes and origin
of the soil materials used in land reclamation should be considered to
avoid worsening the attributes of the soil following restoration
(Mukhopadhyay et al., 2016; Bai, et al., 2018).
In general, our results seemed to differ from previous studies
indicating that land reclamation led to decrease the soil pH and to
increase SOM and TN (Shrestha and Raj, 2010; Spargo and Doley, 2016; Li
et al., 2014a; Sheng et al., 2015). However, as the plots under study
have been subjected to intensive farming activities since they were
reclaimed, it was hard to acknowledge which changes in the key soil
attributes studied herein were influenced by land reclamation or by the
existing farming practices. Future research will aim at disentangling
the effects of farming and land reclamation on key soil attribute by
identifying if key soil attributes played an important role in driving
the multifunctionality in soil ecosystem (Delgado-Baquerizo, et al.,
2016).
The results from analyzing the taxonomic composition of the SMCs (Fig.
3) indicated that the distributions at the phylum level differed among
the reclamation plots (Fig. 3A), which evidenced the effect of land
reclamation on the SMCs composition (Li, et al., 2014b). The relative
abundance of phyla such as Bacteroidetes , Proteobacteriaand Verrucomicrobia declined along the reclamation timeline,
whereas the relative abundance of the phyla Acidobacteria ,Actinobacteria , Chloroflexi , Planctomycetes andThaumarchaeota increased (Fig. 3A). These findings are somehow in
disagreement with the results gathered in Li et al. (2014c), who
suggested a decrease in the abundance of Actinobacteria andChloroflexi in old, restored sites, and who also reported a
negative effect of land reclamation on the abundance ofActinobacteria . However, the increasing abundance ofAcidobacteria along the reclamation timeline was interpreted
herein as a good indicator of soil health, provided the role that this
phylum plays in relevant soil ecosystem processes (e.g. soil
acidification; Tringe et al., 2005; Liu et al., 2016). In fact, we
observed that Acidobacteria became the dominant phylum in all the
reclaimed plots (Fig. 3A and Fig. 5), which is in agreement with
previous studies evaluating the abundance of SMCs after land restoration
(Li et al., 2014bc). Nonetheless, we detected a positive correlation
between the abundance of Acidobacteria and soil pH (Fig. S5, see
supplemental material), which differs from the findings reported before,
that the phylum Acidobacteria decreased in relative abundance as
soil pH increased (Chu et al., 2010; Griffiths et al., 2011). On other
hand, the increasing levels of alkalinity found in the reclaimed plots
(Fig. 2B) likely explained the increasing abundance ofActinobacteria (Fig. 3A), which has a great affinity for
alkaline, arid soils. Yet this phylum was the seventh most abundant of
the detected phyla, which is in opposition with previous studies
reporting Actinobacteria as the most abundant phylum in reclaimed
mine soil in semiarid climates (Bastida et al., 2013). Our finding may
thus be related to the high soil moisture encountered during sampling
(i.e. 35%, there was a five-day thunder and heavy rain prior to
sampling;). In fact, the observed increase in abundance for the phylumChloroflexi was indicative of water tables present near the
ground surface (Chen et al., 2017), and it correlates with the high soil
moisture levels found at sampling time and with the relatively high
abundance of the phylum Planctomycetes - i.e. aquatic bacteria
commonly found in brackish water (Lindsay et al., 2001). Still, the
steady increase in abundance of Chloroflexi along the reclamation
timeline (Fig. 3A) may be related to the increase in the water retention
capacity of the soil resulting from the observed increase in SOM in the
plot assessed 17 years after reclamation - i.e. r17 (Fig. 2A).
Organic manure has been used as soil fertilizer in Eastern China since a
long time ago. This popular farming practice could have distinctly
affected the composition of the SMC found herein (Geng et al., 2008).
The long-term application of organic fertilizer in the plots subjected
to reclamation may have affected the soil physicochemical properties
(Liang et al., 2014), which in turn may have affected the microbial
microenvironments harboring different microbial communities (Jangid et
al, 2008). For example, the increasing trend observed for the phylumThaumarchaea (Fig. 3A) could be related to the fertilization
schemes implemented by the local farmers in the studied plots. It is
worth noting that Thaumarchaea is an important ammonia oxidizer
in aquatic and terrestrial environments, and that ammonia is a common
form of nitrogen in organic manure (Brochier-Armanet et al., 2008). In
fact, Thaumarchaea is the first archaea identified as being
involved in
the nitrification
process (Brochier-Armanet et al., 2012). Hence, the observed abundance
trend for Thaumarchaea may explain why the levels of soil TN
decreased over the reclamation timeline, as discussed above (Fig. 2F)
-i.e. more nitrification (Nitrification, the two-step oxidation of
ammonia to nitrate via nitrite – is a critical component of the global
nitrogen cycle. Biller et al., 2012) triggered by Thaumarchaeaderived into lower levels of soil N. Contrariwise, the decreasing trend
observed for the phyla Proteobacteria can be related to the
reduction in mine-based, soil pollution in the reclaimed plots over time
(Banning et al., 2011; Lewis et al., 2012; Ma et al., 2017), stressing
additional benefits of land reclamation on soil health. To clarify
further the latter, we will investigate the levels of soil pollution
(e.g. Polycyclic Aromatic Hydrocarbon (PAHs)), as well as the
relationship between PAHs and phyla Proteobacteria in the study
site in the future. As well known that, Protobacteria are the
best bacteria to PAH degradation, containing PAH-degrading genes (e.g.
naphthalene 1,2-dioxygenase, extradiol dioxygenase, Martin et al., 2012;
Guarino et al. 2019). Guarino et al. (2019) have also reported thatProteobacteria was a phylum with higher proportions (82%),
containing known species of PAH degraders (e.g. Pseudomonas ,Burkholderiales ) in the contaminated soil of Bagnoli brownfield
site (Southern Italy). Other phyla, however, showed a relatively stable
abundance trend along the reclamation timeline (Fig. S2, see
supplemental material), as indicated in Li et al. (2014b). This can be
due to having detected those SMCs at a stable stage in their succession
at the time sampling was conducted (Novianti et al., 2018; Sun et al.,
2019). Anyhow, the array of phyla for the SMCs detected in this study
agrees with previous studies focusing on land reclamation (e.g. Li et
al., 2014b; Chen et al., 2016; Luo et al., 2019), reflecting the eight
major bacterial phyla commonly encountered in the soil (Fig. 3A, Fig.
S2, see supplemental material; Chen et al., 2017). Moreover, the most
abundant genera of SMCs found herein (i.e. RB41 and UTCFX1; Fig. 3B)
were attributed to the high abundance of Acidobacteria andChloroflexi , given the demonstrated affinity between these phyla
and the most abundant genera (Ngugi et al., 2018). Flavobacteriumand Pedobacter classified to Bacteroidetes , as well asSphingomonas and Luteimonas belonged toProteobacteria presented the similar phenomenon (Fig. 3B).
In this study, we identified strongly significant or significant
correlations between the modules of the microbial ecological networks
(MEN) and the studied key soil attributes, such as soil pH, OP, or DHG
(Fig. 6), supporting that land reclamation had an effect on the SMC
provided through changes in the key soil attributes. In this regard,
Ngugi et al. (2018) have investigated the soil bacterial characteristics
of 21 coal-mining sites, and found that soil bacterial structure was
significantly correlated with soil OP value, which was similar to the
conditions identified in the r8 network (Figs. 2 and 6). Phosphorus can
be easily fixed in the soil, but its utilization rate is slow, making
the concentration of available phosphorus (OP) in the soil small (Ngugi
et al.,2018). Yet, some soil microbes, such as Acidobacteriamight produce acidic substances through metabolism, which then dissolve
some insoluble phosphates and utilize them for their own metabolism (Chu
et al., 2010), reducing the concentration of soil P. In the network
identified for the plot sampled 11 years after reclamation (r11; Fig.
6B), we observed that the differences in terms of SMC were mostly
affected by AK. Sun et al. (2019) pointed out similar findings, in which
the distribution of bacteria was primarily affected by AK in coal-mining
areas, although SOM and OP were also identified as key drivers of SMC
change. We also identified a significant correlation between soil pH and
the structure of SMC (Fig. 6), which is in agreement with earlier
studies (e.g. Li et al., 2018; Hartman et al., 2008; Lauber et al.,
2009), and which was mainly attributed to the role thatAcidobacteria may play on the soil pH (Table 2; Fig. 6). Although
our result suggested that the key soil attributes present a complex
relationship with soil microbial communities, which need further
research to be disentangled, our results also confirmed that network
analysis was an effective and feasible tool to analyze the relationship
between environmental factors and microbial community structures.
The features of the microbial ecological networks (MEN) established
between the detected SMCs varied across the five study sites
(Tables 1-2; Fig. 4; Fig. S4, see
supplemental material), suggesting that the time after reclamation
substantially affected SMC stability and MEN complexity. MEN were more
complex in the control plots (CK) than in the plots undergoing
reclamation (Fig. 4). The latter may be related to the presence of more
stable SMCs in the CK plots due to the absence of operations provoking
soil disturbance as opposed to the plots that were reclaimed (Hunt and
Ward, 2015; Helingerová et al., 2010). This is further supported by the
higher MEN complexity found in r17 -i.e. higher SMC stability, and thus
MEN complexity, is expected along the reclamation timeline (Luo et al.,
2020; Ma et al., 2020). These findings may be directly related to the
effect of time after reclamation on soil ecosystem health and stability,
as more stable and complex SMCs are more resilient to environmental
stress and ecosystem disturbance (Hunt and Ward, 2015; Dimitriu et al.,
2010). Furthermore, the microbial species involved in each network was
different, suggesting shifts in the dominant phyla along the reclamation
timeline (Figs. 4 and 5). For example, the
phyla Acidobacteria , Planctomycetes andProteobacteria were always present while the keystone phyla
changed from plot to plot (Figs. 4 and 5, File S1-S5, see supplemental
material). The variation here may cause changes in soil ecological
functioning (Delgado-Baquerizo et al., 2018). Therefore, the observed
changes in the composition of the SMCs and their interaction through
MENs in relation to the time after reclamation is another indicator of
the evolution of the soil ecosystem towards a more complex but stable
system (Odum, 1969; Gonzalez-Ollauri and Mickovski, 2017) as time from
reclamation (i.e. disturbance) progresses in spite of the soil
disturbance triggered by past mining activities.