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Effects of agronomic treatments on functional diversity of soil microbial community and microbial activity in a revegetated coal mine spoil
Geoderma 338 (2019) 40–47
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Effects of agronomic treatments on functional diversity of soil microbial community and microbial activity in a revegetated coal mine spoil
T
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Pramod Shrestha , Resham Gautam, Nanjappa Ashwath Central Queensland University, School of Health, Medical and Applied Sciences, Rockhampton, QLD 4702, Australia
A R T I C LE I N FO
A B S T R A C T
Handling Editor: I. Kögel-Knabner
Successful restoration of coal mine spoil requires functional microbial community for soil development and biogeochemical cycling to ensure long term sustainability of established plants. The current study investigated functional diversity of the soil microbial community, microbial activities, and physico-chemical properties of a revegetated coal mine spoil that was exposed to different agronomic practices. Treatments included Control (conventional revegetation practice), selective herbicide (SH), mulching (M), succession planting (SP), and green manure (GM) crops. Fluorescein diacetate (FDA) hydrolysis revealed significantly (P < 0.05) higher microbial activity in the GM treatment compared to the Control and all other treatments. Patterns of microbial biomass C (MBC), determined by the chloroform fumigation extraction method, also showed significant (P < 0.05) differences between treatments, with the SH treatment showing the lowest MBC. Basal respiration (BR) and substrate induced respiration (SIR) did not differ amongst the treatments. Principal component analysis of community level physiological profiles data based on Biolog® Ecoplate data revealed differing bacterial functional (metabolic) diversity amongst the treatments. Overall, the study demonstrated significantly (P < 0.05) higher average well color development (AWCD), indicating high metabolic activity, and substrate richness (R) and Shannon's index (H) showed greater functional diversity of the bacterial community in the SP treatment, as compared to other treatments. These results demonstrate that the agronomic treatments such as SP will not only boost microbial activity, but they also play a constructive role in increasing functional diversity of soil microbial communities. Based on these results, we recommend that agronomic practices such as SP should be used as an important component in mine site revegetation programs.
Keywords: Microbial community Microbial activity Microbial biomass C FDA hydrolysis Revegetation Coal mine spoil
1. Introduction Extraction of coal via open-cut mining alters topography, hydrology, soil chemistry and plant-soil-microbial interactions (Ngugi et al., 2017; Sheoran et al., 2010). Soil quality is an important component in the restoration of ecosystems due to its physical, chemical and biological (nutrient) support for plant recolonization and establishment (Zhang and Chu, 2013). Soil microbial communities play a vital role in ecosystem function as they are important for establishing sustainable plant communities that rely upon healthy soil development and biological interactions (Lopez-Lozano et al., 2016). Similarly, microbial communities support revegetation processes that can further help stabilize soils to prevent the loss of soil and nutrients due to erosion (Sheoran et al., 2010). Therefore, successful restoration of postmining landscapes relies not only on the establishment of plant cover, but also on regeneration of soil microbial communities (Ohsowski et al., 2012); thus, soil microbial assessment can help obtain an insight into
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the processes that occur within a restored ecosystem. Rhizosphere microbial communities and root exudates promote plant growth by mobilization of nutrients, transformation of organic matter in the soil, protection of plants from phytopathogens and the production of growth-promoting substances (Azcón-Aguilar and Barea, 2015; Prashar et al., 2014; Sheoran et al., 2010). Despite the importance of the role played by the soil microbial communities in rehabilitated post-mining landscapes, little is known about the role of organic amendments in carbon usage profiles of microbial communities on coal mine spoils. There is global interest in the use of soil amendments during mine reclamation or restoration processes (Hu et al., 2015; Showalter et al., 2010; Wilson-Kokes et al., 2013; Young et al., 2015), however, most of these studies have focused mainly on re-establishment of plants. An understanding of ecological processes in ecosystem restoration is critical during the first 5–10 years of the reclamation (Wortley et al., 2013). Kumar et al. (2015) suggested that reclamation age had a significant influence on soil properties, due to
Corresponding author. E-mail address: [email protected] (P. Shrestha).
https://doi.org/10.1016/j.geoderma.2018.11.038 Received 7 December 2017; Received in revised form 18 November 2018; Accepted 20 November 2018 0016-7061/ © 2018 Elsevier B.V. All rights reserved.
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Perennial nitrogen fixing plants were used, as plants such as Acacia holosericea were found to be responsible for changes in soil microbiota in depauperate soils (Bilgo et al., 2012). Furthermore, this species is widely used in mine site rehabilitation in tropical Australia (Corbett, 1999). The aim was to test if the introduction of such plants would enhance microbial activity in mine spoils and assist in the establishment of a wide range of native species on mine spoils. After one year of establishing the GM and SP treatments, the entire experimental site (including SP and GM treatments) was deep ripped and sown (broadcasted) with the seeds of 76 native species. This seed mix represented acacias (16 species), eucalypts (20), casuarinas (3), melaleucas (2), grasses (6) and other species (29), and they represented trees, shrubs, legumes and climbers. Immediately after sowing, the remaining treatments, such as SH and M were introduced, and Controls were set up. The SH treatment was imposed to reduce weed (wild sorghum) growth in the experimental plot, as the top soils used in the revegetation program contained a high density of weed seeds, and these weeds are known to compete with the establishment and survival of native species. The M treatment was introduced to conserve soil moisture as previous studies had demonstrated improved plant establishment and growth in mulched treatments (Ashwath and Rank, 2009; Radloff, 2003). The SH treatment was introduced by spraying Verdict 520 (haloxyfop, 1 L/ha) twice, the first spray at three months after sowing and the second at nine months after sowing. In the M treatment, a layer of locally available hay mulch was spread immediately after the seeds were sown. In the Control, the experimental plot was left as is, and hence it consisted of deep ripping and sowing of native species only, common to all the treatments used in this study. After one and a half years from sowing the native species (or two and half years of establishing the SP and GM treatments), soil samples (0–20 cm) were collected from each of the three replicated plots using a 10 cm diameter soil core. The sampling occurred in early summer (December). From each replicated treatment plot, 27 soil cores were taken to a depth of 10–20 cm and were bulked on the site into three samples per plot. The samples were brought to the laboratory in an icecooled Esky. On the following day, the samples from each plot were passed through a 2 mm sieve and the three samples were mixed in equal proportion to produce one sample per plot. These composite samples were stored at 4 °C until used in microbial- and physico-chemical analyses.
changes in functional and structural changes in vegetation. However, soil microbial communities can assist in this process as an early indicator of successful restoration (Emmerling et al., 2000; Izquierdo et al., 2005; Sheoran et al., 2010). Soil microbial communities can provide more precise representation of the immediate recovery of degraded land, whereas plant species diversity assessments may mislead in the short-term because they may simply reflect the artificial planting schedules, rather than genuine re-establishment (Harris, 2003; Sheoran et al., 2010). Soil microbial communities in reclaimed soils can be assessed using various methods (Gil-Sotres et al., 2005; Sheoran et al., 2010). Soil microbial properties such as microbial biomass carbon (MBC), soil respiration, and activities of different soil enzymes have been studied (Anderson et al., 2008; Baldrian et al., 2008; Helingerová et al., 2010; Izquierdo et al., 2005). One of the important functions of the microbial community in revegetation programs is its ability to metabolize diverse carbon sources as this feature is essential for organic matter turnover. Thus, measurements of microbial diversity may give additional information on the function of microbial communities in revegetated mine spoils (Chodak et al., 2009). The microtiter plate (Biolog®, Inc., Hayward, CA) technique is one of the methods employed for the analysis of physiological profiles of microbial communities, based on carbon utilization patterns. In spite of several limitations, such as the difficulty in culturing all microbes (Ros et al., 2008), the Biolog® assay can be a valuable tool for rapid and convenient screening of microbial functional abilities in rehabilitated mining landscapes. This method is sensitive and reproducible, and yields information on important functional attributes of microbial communities (Gomez et al., 2004). Furthermore, canonical variate analysis (CVA) data of community level physiological profiles (CLPP) have been shown to match the results of the culture-independent phospholipid fatty acid (PLFA) method (Grayston et al., 2004). A number of agronomic treatments were tested for their effects on microbial properties of coal mine spoil, with the view to identifying suitable treatments that would help promote healthy soils that are crucial for establishment and long term sustainability of native vegetation. Weed management, moisture conservation and erosion loss are the major challenges for native plant establishment in Central Queensland. Because the topsoils used in mine site rehabilitation are stockpiled for various periods, the loss of microbial activity in these soils is another challenging issue for revegetation. We hypothesized that the soil microbial functional diversity and microbial activities on coal mine spoils would respond to introduced agronomic treatments. The outcome of this study would therefore be beneficial for the choice of suitable agronomic treatments that would help improve the restoration process and provide an early indication of restoration success.
2.2. Physico-chemical analyses
2. Materials and methods
The pH and electrical conductivity in 1:5 H2O extract of soil samples were measured according to Rayment and Higginson (1992). Moisture content was calculated after drying the soil at 105 °C for 72 h. Chemical compositions of soils were determined at the CSBP Soil and Plant Analysis Laboratory, Perth, Australia using standard CSBP methods.
2.1. Study sites and soil sampling
2.3. Fluorescein diacetate (FDA) hydrolysis and microbial biomass carbon
A coal mine spoil with a five-hectare experimental site was selected for the study in Central Queensland, Australia. This site contained excavated mine spoil and was covered with a layer (20–30 cm) of stored topsoil (black cracking clay). The experimental site was exposed to various agronomic treatments such as Succession Planting (SP), Green Manure (GM) crops, Selective Herbicide (SH) and Mulching (M) with hay, along with a Control. Each treatment contained three replication plots with a completely randomized design. The GM and SP treatments were imposed one year prior to the introduction of other treatments. In the GM treatment, nitrogen fixing exotic species such as Clitoria ternatea, Vigna unguiculata and Dolichos lablab were hand sown using a seed drill. In the SP treatment, perennial nitrogen fixing native species such as Acacia holosericea, Acacia simsii and Acacia harpophylla were sown in rows using a seed drill. GM crops were introduced to increase soil organic matter (soil carbon) content.
A modified method of fluorescein diacetate (FDA) hydrolysis was used to measure microbial activity (Adam and Duncan, 2001). The soil sample (equivalent to 1 g dry weight) was added to 30 mL of phosphate buffer (60 mM, pH = 7.6), followed by addition of FDA, and incubated at 37 °C for 1 h. The reaction was quenched with 10 mL of acetone, and the developed color was measured spectrophotometrically at 490 nm. The concentrations of fluorescein released from the soil samples were calculated using a standard curve produced from the standards containing 0 to 10 μg/mL fluorescein. Microbial biomass carbon (MBC) was determined by the chloroform fumigation-extraction method (Vance et al., 1987). Briefly, the air-dried samples (equivalent to 10 g dry weight) were weighed in a 100 mL beaker, placed in a desiccator, and fumigated in a vacuum with 20 mL alcohol-free chloroform. After incubation in the dark for 5 days, the fumigation was stopped, and 50 mL of 0.5 M K2SO4 was added and 41
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Table 1 Characteristics of the coal mine spoil exposed to different agronomic treatments. Parameters ⁎
pH (1:20) EC⁎ (μS/cm) (1:20) Organic C (%) NH4+-N (mg kg−1 d wt) NO3-N (mg kg−1 d wt) Phosphorus (mg kg−1 d wt) Potassium (mg kg−1 d wt) Sulphur (mg kg−1 d wt)
Control b
7.8 187.7a 1.1 ± 0.1a 14.5 ± 5.0abc 3.3 ± 0.9a 4.3 ± 1.3ab 172.4 ± 59.5a 2.2 ± 0.2a
GM
M
a
SH ab
7.4 196.5a 1.4 ± 0.2a 24.0 ± 4.6c 8.9 ± 2.8b 4.7 ± 0.9ab 177.3 ± 16.6a 3.5 ± 1.2b
7.6 180.9a 1.0 ± 0.2a 12.7 ± 0.3a 2.7 ± 0.3a 3.0 ± 0.0a 184.3 ± 56.9ab 3.6 ± 0.5b
SP b
7.8 187.9a 1.1 ± 0.3a 15.3 ± 1.2ab 7.8 ± 2.7b 2.5 ± 0.9a 172.9 ± 61.0a 2.7 ± 0.2ab
7.7ab 175.8a 1.8 ± 0.2a 21.7 ± 3.7bc 5.0 ± 1.0a 5.7 ± 1.2b 196.7 ± 14.5b 2.9 ± 0.4ab
The data shown are the means and standard errors (n = 3). Values in the same row followed by different letters are significantly (P < 0.05) different. ⁎ pH and soil electrical conductivity (1:5 soil: water extract w/v) of a mine spoil exposed to different agronomic treatments.
AWCD = ∑(X − X0) / 31, where X was the mean of the same three wells per plate and X0 was the mean of control (water blanks) per plate. Functional diversity was measured as substrate richness (R) and Shannon's index (H). R was the number of different carbon substrates that were oxidized by the microbial community. H encompassed both R and the intensity of substrate use, which was calculated as H = ∑Pi (ln Pi) where Pi was proportional color development of the ith well over total color development of all wells (Schutter and Dick, 2001; Zak et al., 1994). GENSTAT (v 13) was used to perform ANOVA of AWCD, R and H data, and the means were compared using Student's t-test at 5% significance. Principal component analysis (PCA) was used to characterize the bacterial community level profile and was performed by using XLSTAT software.
shaken for 1 h at 200 rpm. The resulting mixture was then filtered through a Whatman No. 42 filter paper. Another set of non-fumigated samples was also extracted as per the fumigated samples. The filtered solution was stored in a freezer at −20 °C. The concentrations of C in both fumigated and non-fumigated extracts were determined spectrophotometrically using dichromate digestion (Walkley and Black, 1934). The MBC was calculated using a KEC factor of 0.33 (Sparling and West, 1988) as follows:
Microbial Biomass C (C from fumigated soil − C from non‐fumigated soil) = 0.33
2.4. Microbial respiration 3. Results and discussion
Soil microbial respiration was determined after wetting the soil samples to 40–50% water holding capacity and incubating at room temperature for 7 days. Basal respiration (BR) was estimated by measuring CO2 evolved from the soil (equivalent to 10 g dry weight) that was incubated in a closed vessel for 24 h at 25 °C in the dark. The CO2 produced was absorbed in 0.5 M NaOH and was quantified by titration with 0.5 N HCl after the addition of saturated BaCl2, using phenolphthalein as the indicator. Substrate induced respiration (SIR) was estimated using the same method as for the BR, except that 1 mL of glucose (0.5 g/mL) was added to each sample before incubation at 25 °C for 24 h in the dark (Anderson, 1982).
3.1. Physico-chemical properties Soil pH was significantly lower (P < 0.05) in the GM treatment compared to SH and the Control (Table 1). However, no differences in pH were observed amongst the other treatments. The highest pH (7.8) was observed in the Control and SH, and the lowest in the GM treated plots (7.4). The GM treatment included nitrogen-fixing plants such as Clitoria ternatea, Vigna unguiculata and Dolichos lablab. These plants accumulate large quantities of biomass and release protons during the nitrogen fixing process (Yan et al., 1996). This nitrogen fixation process would have contributed to reduce pH in the GM treatment. Studies have demonstrated an increase in soil organic matter and acidification of the soil during nitrogen cycling in agricultural soils (Bolan et al., 1991) and mined sandy soils (Chodak et al., 2009; Graham and Haynes, 2006) that supported leguminous plants. Soil EC varied from 175 μS/cm to 197 μS/ cm (Table 1) and remained similar amongst all the treatments. Soil organic C did not differ amongst the treatments, as the Organic C was highly variable within the site as well as between the sites, ranging from 1.1% to 1.8% (Table 1). However, the GM (1.4 ± 0.2%) and SP (1.8 ± 0.2%) treatments had elevated levels of organic C. The NH4+-N was significantly (P < 0.05) higher (24.0 ± 4.6 mg kg−1 dw) in the GM treatment and was the lowest (12.7 ± 0.3 mg kg−1 dw) in the M treatment. No marked differences were observed in the other treatments. Interestingly, the NO3-N levels were significantly (P < 0.05) higher in GM (8.9 ± 2.8 mg kg−1 dw) and SH (7.8 ± 2.7 mg kg−1 dw) treated plots compared to all other treatments. A distinct effect of GM and SP treatments was expected on N content, as the plants used in these treatments are able to fix atmospheric N2 through symbiotic bacteria (Brockwell et al., 2005; Peoples and Herridge, 1990). As Johnson and Curtis (2001) observed in a similar study, soil N and organic C content of the GM and SP treatments were consistently higher than those in other treatments, although some of these differences were not statistically significant. This suggests the beneficial use of N fixing plants. The higher NO3-N level in the SH
2.5. Community level physiological profiles The Ecoplate (Biolog® Inc., USA) was used to identify microbial functional diversity, wherein the rates of utilization of 31 different C substrates (see the table in the Supplementary file) were assessed (Insam, 1997). Soil samples (equivalent to 1 g dry weight) were serially diluted to 10−3 in 9 mL of sterile saline solution in triplicate. Solutions (200 μL) were then inoculated onto each well of the Biolog® Ecoplates. The microplates were monitored periodically at 0, 24, 48, 72, 96, 120 and 144 h by an ELISA reader at 570 nm. The absorbance readings that were negative or below 0.06 were set to zero. The average well color development (AWCD) of all absorbance data for different C substrates was individually calculated prior to statistical analysis (Konopka et al., 1998). 2.6. Statistical analysis All measurements, including pH, electrical conductivity (EC) and respiration, were calculated from triplicates of each treatment (3 replications). The data was subjected to one-way analysis of variance (ANOVA) using GENSTAT (v13) after testing of the data for outliers, and normality and homogeneity of error variances. All Biolog data were based on 96 h incubation. Microbial activity in each micro-plate was expressed as AWCD, and was determined using the formula: 42
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treatment can be attributed to a higher availability of NH4+-N, as the weed plant density was low in this treatment. These observations concur with those reported by Damin and Trivelin (2011), who revealed an increase in NO3-N content of soil after plants were sprayed with herbicide. Significantly higher (P < 0.05) phosphorus content was recorded in SP (5.7 mg kg−1 dw) compared to the M and SH treated plots; however, the phosphorus content was similar in the rest of the treatment plots (Table 1). Except in the M treatment plot, a significantly higher level of K was observed in the SP treatment plot (196.7 mg kg−1 dw) and no differences were found in other treatments (Table 1). Similarly, sulphur concentration varied from 2.2 to 3.6 mg kg−1 and was highest in M and GM compared to all other treatments. Significant changes in soil nutrients in the SP treatment could result from increased amounts of organic matter added by Acacia species that were included in the succession planting (Sparling and West, 1988). According to Souza-Alonso et al. (2015), invasion of Acacia dealbata not only contributed to higher C and N levels, but also enhanced the level of other soil nutrients, including P and K. Similarly, Forrester (2004) observed a significant increase in the rate of N and P recycling when Acacia mearnsii was grown with eucalypts.
(Fig. 1A). The high microbial activity observed in GM and SP treatments presumably reflected the presence of N fixing bacteria, whereas the M treatment contributed via enhanced moisture availability in the soil. Soil microorganisms and their activities play an important role in soil formation, decomposition of organic matter, N fixation and nutrient cycling (Mummey et al., 2002; Samuel et al., 2008), and hence they can provide information about the degree of change in biochemical processes in the soil. The FDA hydrolysis was used as an indicator of the changes in biochemical processes occurring in the soil (Adam and Duncan, 2001). Previous studies have shown alteration in FDA hydrolysis activity by exotic tree legumes (Bilgo et al., 2012) and pesticides (Chowdhury et al., 2008) in different conditions. The MBC, determined via the chloroform fumigation extraction method, was the lowest (0.3 g/kg) in SH treatment compared to the other treatments, which had similar levels (~1 g/kg) of C (Fig. 1B). Comparison between various treatments for both microbial activity and MBC clearly demonstrated the effect of these treatments in improving microbial activities in mined spoil following rehabilitation. The marked reduction in microbial activity and MBC in SH treatments suggests that the application of herbicide has led to the extinction of soil microbial activity in the mine spoil. Earlier study has revealed that an extensive use of herbicide impacted on soil microbial communities by altering their number, microbial activity, enzyme activity and even microbial diversity (Sebiomo et al., 2011). Furthermore, the presence of plants is critical to the build-up of microbial activity in mine spoil dumps. The presence of any plant, and even weeds, could enhance microbial processes in such soils (Melo et al., 2014; Sheoran et al., 2010). It is also recommended that numerous native ground cover species can be selected and established as soon as the spoil dumps are reshaped to increase microbial processes in the soil. The established native species will not only protect the soil but they will also help build microbial activity, whilst minimizing weed growth. Several studies have shown considerable effects on soil microbial activity by establishing plant communities and providing favorable conditions to soil microflora (Anderson and Ineson, 1984; Dinesh et al., 2003). Building up of soil organic C is crucial for long term sustainability of native plant communities on mine spoil dumps. The current study demonstrates that the use of appropriate organic treatments can enhance microbial activities in such soils.
3.2. Fluorescein diacetate (FDA) hydrolysis and microbial biomass C The current results revealed significantly (P < 0.05) higher microbial activity in GM treatments, followed by SP and M treatments c
µg Fluorescien release/g dw soil/h
140
A
120 100
b
80 60
b a a
40 20
3.3. Soil respiration
0 1.2 bc 1.0
c bc
Soil respiration is one of the most important components of the C cycle in forest ecosystems (Adachi et al., 2006). Generally, soil respiration varies with time and space (Darenova et al., 2016; Fang et al., 1998), and soil temperature and soil moisture are the key factors responsible for variations in soil respiration (Inoue and Koizumi, 2012; Shi et al., 2011). Soil respiration for a specific ecosystem can be characterized by its magnitude and temporal and spatial variability (Fang et al., 1998). Results of this study showed that BR did not differ amongst the treatments, although the values were very low in the SH treatment (Fig. 2A). Similarly, fewer differences were observed between the treatments for SIR except in the Control (Fig. 2B). Interestingly, the differences between the treatments for SIR were smaller than those observed for BR, due to the additional supply of C. In the SH treatment, the SIR increased substantially, as compared to the BR, indicating that the low microbial activity in this treatment could be due to low C supply (lack of plants). These results emphasize the need to maintain vegetation in the spoils to allow a healthy soil microbial environment, as the plants add C to the soil. An increase in plant cover leads to increased root density, resulting in enhanced soil microbial activity and respiration; both of which in turn contribute to soil organic matter formation (Lehmann and Kleber, 2015) and the build-up of organic C in the soil (Brohon et al., 2001; Liu et al., 2016).
B
b
g C\Kg d soil
0.8 a 0.6
0.4
0.2
0.0 Control
GM
M
SH
SP
Treatments Fig. 1. A: Microbial activity in variously treated mine spoils, as measured by the FDA hydrolysis test (n = 3). B: Microbial biomass C, as measured by the chloroform fumigation method. Bars indicate standard errors of means (n = 3). Columns with different letters are significantly (P < 0.05) different according to the Duncan multiple range test. 43
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a
35
µg CO2/g DW/h
30
A
a
a
25 a 20
a
15 10 5 0 50
a
a
µg CO2/g DW/h
40
30
B a
a
a
20
10
0 Control
GM
M
SH
SP
Treatments Fig. 2. A: Basal respiration (BR), and B: substrate-induced respiration (SIR) in variously revegetated mine spoils (n = 3). Bars indicate standard errors of means (n = 3). Columns with different letters are significantly (P < 0.05) different according to the Duncan multiple range test.
3.4. Community level physiological profiles The functional diversity of culturable soil microorganisms in different treatments was differentiated on the basis of bacterial kinetics and substrate utilization patterns (Konopka et al., 1998). The treatments showed differences in overall activity, as expressed by AWCD, across time. Based on AWCD kinetics (data not shown), measurements from 96 h incubation were chosen for data analysis (Garland, 1996). The AWCD values after 96 h of incubation were significantly (P < 0.05) higher in the SP and GM treatments, compared to SH and Control, showing variations amongst the treatments in functional diversity of the microbial community (Fig. 3A). Although higher total microbial activity (Fig. 1A) and MBC (Fig. 1B) were recorded in the M treatment compared to SH, no difference was observed between M and SH treatments for substrate utilization. This could be due to antagonistic interactions between the microbiota or toxicity caused to microorganisms by the redox dyes used in the Biolog wells (Ullrich et al., 1996). The R data showed the following order: SP, M, GM, C, SH (Fig. 3B) indicating three groups, viz. SP in one group, M, GM and C in the second group, and SH in the third group. The H followed the order: SP, M , GM , C, SH (Fig. 3C), and the treatments fell again into three groups. = Group 1 contained SP, M and GM, Group 2 contained M, GM and C, and Group 3 contained SH. Both the R and H analysis consistently separated the SH treatment from the rest of the treatments (Fig. 3B & C). These results could be taken as evidence for the positive effect of some
Fig. 3. A: Average well-color development (AWCD), B: richness (R), C: Shannon's index (H) of carbon substrate utilization on the Biolog® Ecoplate by soil bacterial communities in a mine spoil exposed to different agronomic treatments in a revegetated coal mine. Error bars indicate standard errors of means (n = 3). Columns with different letters are significantly (P < 0.05) different according to the Duncan multiple range test.
treatments and negative effect of SH treatment on soil microbial activity and the microbial community. According to average utilization of specific substrate data analyzed by the corresponding functional guilds, SP treated plots averaged higher utilization of all functional guilds of C substrates than the other treatments (Table 2). Similarly, compared to the Control, M and SH, the GM treatment had higher averaged carbohydrate utilization. Although significant differences were noted between the M and SH treatments for microbial activity (as measured by FDA) and MBC (Fig. 1), no difference was observed between these two
=
44
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Table 2 Average carbon substrate utilization in different substrates of the Biolog® Ecoplate by the mine spoils of a revegetated coal mine exposed to various agronomic treatments. The samples were collected from 96 h incubations of Biolog® Ecoplate, and the substrate utilization was measured as the average optical density across all substrates within each guild, which included seven types of carbohydrates, nine carboxylic acids, four polymers, six amino acids, two amines/amides and three miscellaneous substrates. Carbon sources
Control
Carbohydrates Carboxylic acids Polymers Amino acids Amines/amides Miscellaneous
0.89 0.98 1.25 0.92 0.53 0.85
± ± ± ± ± ±
GM 0.06 0.06 0.12 0.03 0.04 0.15
a
M
1.68 1.09 1.69 1.10 0.41 1.09
ab a a a ab
± ± ± ± ± ±
0.16 0.06 0.05 0.16 0.06 0.11
b
1.07 1.09 1.57 1.34 0.73 0.82
ab ab ab a b
SH ± ± ± ± ± ±
0.07 0.10 0.02 0.11 0.03 0.08
a
SP
1.05 0.88 1.16 0.98 0.55 0.54
ab ab ab a ab
± ± ± ± ± ±
0.09 0.10 0.20 0.09 0.08 0.15
a a a a a a
2.05 1.22 1.88 1.48 1.35 1.81
One way ANOVA ± ± ± ± ± ±
0.29 0.14 0.24 0.32 0.19 0.07
b b b b b c
P = 0.002 P = 0.122 P < 0.071 P = 0.081 P = 0.026 P < 0.001
The data shown are the means and standard errors of means (n = 3). Values in the same column followed by different letters are significantly (P < 0.05) different.
6
SH 4
PC2 (13.28 %)
SH
2
SP
SH GM 0
Control
SP GM M
Control -2
Control
GM SP
M
M -4 -4
-2
0
2
4
6
8
10
PC1 (35.51 %) Fig. 4. Principal component analysis (PCA) based on the carbon utilization profile of bacterial communities of a revegetated coal mine spoil exposed to different agronomic treatments (n = 3).
the conclusions of other researchers such as Remigi et al. (2008) and Lupwayi et al. (1998), who claimed that legume species may be responsible for changes in soil microbiota, affecting the structure and functions of microbial communities.
treatments in terms of substrate utilization according to Ecoplate data (Table 2). This suggests that larger microbial communities may be functionally more diverse, and that smaller differences between microbial communities could be difficult to describe from the Biolog® Ecoplate data (Lynch et al., 2004). In this study, the PCA of Biolog® Ecoplate data showed significant differences between treatments for functional diversity of the microbial community. The PC1 and PC2 explained 35.5% and 13.3% of the total variance, respectively (Fig. 4). The PC1 separated the Control, SH and M from the GM and SP, probably due to the rhizosphere bacterial community, as a result of introduction of different legume species. This provides strong support for
4. Conclusions The results of the current study clearly showed that agronomic treatments such as GM, SP and M increased microbial activity and MBC, whereas the SH treatment decreased both microbial activity and MBC. Additionally, the Biolog® Ecoplate results showed that application of 45
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various agronomic treatments had a positive effect on bacterial community structure, with the highest functional diversity present in the SP treatment. These data demonstrate that there were beneficial effects of agronomic treatments to soil microbial activity, and C usage profile of soil microbial communities. Further studies should evaluate if the molecular techniques and microbial functions such as C and N cycling could provide a better indication of the partial or total recovery of the functional diversity of the microbial community in disturbed mine spoils. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.geoderma.2018.11.038. Acknowledgments The authors are grateful to Professor David Midmore, Tom Hayes and Larry Hantler for their encouragement and support during this study. Special thanks to Dr Leonie Barnett for her constructive criticism of the manuscript. We also acknowledge the funding support of Anglo Coal Australia. 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Geoderma journal homepage: www.elsevier.com/locate/geoderma
Effects of agronomic treatments on functional diversity of soil microbial community and microbial activity in a revegetated coal mine spoil
T
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Pramod Shrestha , Resham Gautam, Nanjappa Ashwath Central Queensland University, School of Health, Medical and Applied Sciences, Rockhampton, QLD 4702, Australia
A R T I C LE I N FO
A B S T R A C T
Handling Editor: I. Kögel-Knabner
Successful restoration of coal mine spoil requires functional microbial community for soil development and biogeochemical cycling to ensure long term sustainability of established plants. The current study investigated functional diversity of the soil microbial community, microbial activities, and physico-chemical properties of a revegetated coal mine spoil that was exposed to different agronomic practices. Treatments included Control (conventional revegetation practice), selective herbicide (SH), mulching (M), succession planting (SP), and green manure (GM) crops. Fluorescein diacetate (FDA) hydrolysis revealed significantly (P < 0.05) higher microbial activity in the GM treatment compared to the Control and all other treatments. Patterns of microbial biomass C (MBC), determined by the chloroform fumigation extraction method, also showed significant (P < 0.05) differences between treatments, with the SH treatment showing the lowest MBC. Basal respiration (BR) and substrate induced respiration (SIR) did not differ amongst the treatments. Principal component analysis of community level physiological profiles data based on Biolog® Ecoplate data revealed differing bacterial functional (metabolic) diversity amongst the treatments. Overall, the study demonstrated significantly (P < 0.05) higher average well color development (AWCD), indicating high metabolic activity, and substrate richness (R) and Shannon's index (H) showed greater functional diversity of the bacterial community in the SP treatment, as compared to other treatments. These results demonstrate that the agronomic treatments such as SP will not only boost microbial activity, but they also play a constructive role in increasing functional diversity of soil microbial communities. Based on these results, we recommend that agronomic practices such as SP should be used as an important component in mine site revegetation programs.
Keywords: Microbial community Microbial activity Microbial biomass C FDA hydrolysis Revegetation Coal mine spoil
1. Introduction Extraction of coal via open-cut mining alters topography, hydrology, soil chemistry and plant-soil-microbial interactions (Ngugi et al., 2017; Sheoran et al., 2010). Soil quality is an important component in the restoration of ecosystems due to its physical, chemical and biological (nutrient) support for plant recolonization and establishment (Zhang and Chu, 2013). Soil microbial communities play a vital role in ecosystem function as they are important for establishing sustainable plant communities that rely upon healthy soil development and biological interactions (Lopez-Lozano et al., 2016). Similarly, microbial communities support revegetation processes that can further help stabilize soils to prevent the loss of soil and nutrients due to erosion (Sheoran et al., 2010). Therefore, successful restoration of postmining landscapes relies not only on the establishment of plant cover, but also on regeneration of soil microbial communities (Ohsowski et al., 2012); thus, soil microbial assessment can help obtain an insight into
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the processes that occur within a restored ecosystem. Rhizosphere microbial communities and root exudates promote plant growth by mobilization of nutrients, transformation of organic matter in the soil, protection of plants from phytopathogens and the production of growth-promoting substances (Azcón-Aguilar and Barea, 2015; Prashar et al., 2014; Sheoran et al., 2010). Despite the importance of the role played by the soil microbial communities in rehabilitated post-mining landscapes, little is known about the role of organic amendments in carbon usage profiles of microbial communities on coal mine spoils. There is global interest in the use of soil amendments during mine reclamation or restoration processes (Hu et al., 2015; Showalter et al., 2010; Wilson-Kokes et al., 2013; Young et al., 2015), however, most of these studies have focused mainly on re-establishment of plants. An understanding of ecological processes in ecosystem restoration is critical during the first 5–10 years of the reclamation (Wortley et al., 2013). Kumar et al. (2015) suggested that reclamation age had a significant influence on soil properties, due to
Corresponding author. E-mail address: [email protected] (P. Shrestha).
https://doi.org/10.1016/j.geoderma.2018.11.038 Received 7 December 2017; Received in revised form 18 November 2018; Accepted 20 November 2018 0016-7061/ © 2018 Elsevier B.V. All rights reserved.
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Perennial nitrogen fixing plants were used, as plants such as Acacia holosericea were found to be responsible for changes in soil microbiota in depauperate soils (Bilgo et al., 2012). Furthermore, this species is widely used in mine site rehabilitation in tropical Australia (Corbett, 1999). The aim was to test if the introduction of such plants would enhance microbial activity in mine spoils and assist in the establishment of a wide range of native species on mine spoils. After one year of establishing the GM and SP treatments, the entire experimental site (including SP and GM treatments) was deep ripped and sown (broadcasted) with the seeds of 76 native species. This seed mix represented acacias (16 species), eucalypts (20), casuarinas (3), melaleucas (2), grasses (6) and other species (29), and they represented trees, shrubs, legumes and climbers. Immediately after sowing, the remaining treatments, such as SH and M were introduced, and Controls were set up. The SH treatment was imposed to reduce weed (wild sorghum) growth in the experimental plot, as the top soils used in the revegetation program contained a high density of weed seeds, and these weeds are known to compete with the establishment and survival of native species. The M treatment was introduced to conserve soil moisture as previous studies had demonstrated improved plant establishment and growth in mulched treatments (Ashwath and Rank, 2009; Radloff, 2003). The SH treatment was introduced by spraying Verdict 520 (haloxyfop, 1 L/ha) twice, the first spray at three months after sowing and the second at nine months after sowing. In the M treatment, a layer of locally available hay mulch was spread immediately after the seeds were sown. In the Control, the experimental plot was left as is, and hence it consisted of deep ripping and sowing of native species only, common to all the treatments used in this study. After one and a half years from sowing the native species (or two and half years of establishing the SP and GM treatments), soil samples (0–20 cm) were collected from each of the three replicated plots using a 10 cm diameter soil core. The sampling occurred in early summer (December). From each replicated treatment plot, 27 soil cores were taken to a depth of 10–20 cm and were bulked on the site into three samples per plot. The samples were brought to the laboratory in an icecooled Esky. On the following day, the samples from each plot were passed through a 2 mm sieve and the three samples were mixed in equal proportion to produce one sample per plot. These composite samples were stored at 4 °C until used in microbial- and physico-chemical analyses.
changes in functional and structural changes in vegetation. However, soil microbial communities can assist in this process as an early indicator of successful restoration (Emmerling et al., 2000; Izquierdo et al., 2005; Sheoran et al., 2010). Soil microbial communities can provide more precise representation of the immediate recovery of degraded land, whereas plant species diversity assessments may mislead in the short-term because they may simply reflect the artificial planting schedules, rather than genuine re-establishment (Harris, 2003; Sheoran et al., 2010). Soil microbial communities in reclaimed soils can be assessed using various methods (Gil-Sotres et al., 2005; Sheoran et al., 2010). Soil microbial properties such as microbial biomass carbon (MBC), soil respiration, and activities of different soil enzymes have been studied (Anderson et al., 2008; Baldrian et al., 2008; Helingerová et al., 2010; Izquierdo et al., 2005). One of the important functions of the microbial community in revegetation programs is its ability to metabolize diverse carbon sources as this feature is essential for organic matter turnover. Thus, measurements of microbial diversity may give additional information on the function of microbial communities in revegetated mine spoils (Chodak et al., 2009). The microtiter plate (Biolog®, Inc., Hayward, CA) technique is one of the methods employed for the analysis of physiological profiles of microbial communities, based on carbon utilization patterns. In spite of several limitations, such as the difficulty in culturing all microbes (Ros et al., 2008), the Biolog® assay can be a valuable tool for rapid and convenient screening of microbial functional abilities in rehabilitated mining landscapes. This method is sensitive and reproducible, and yields information on important functional attributes of microbial communities (Gomez et al., 2004). Furthermore, canonical variate analysis (CVA) data of community level physiological profiles (CLPP) have been shown to match the results of the culture-independent phospholipid fatty acid (PLFA) method (Grayston et al., 2004). A number of agronomic treatments were tested for their effects on microbial properties of coal mine spoil, with the view to identifying suitable treatments that would help promote healthy soils that are crucial for establishment and long term sustainability of native vegetation. Weed management, moisture conservation and erosion loss are the major challenges for native plant establishment in Central Queensland. Because the topsoils used in mine site rehabilitation are stockpiled for various periods, the loss of microbial activity in these soils is another challenging issue for revegetation. We hypothesized that the soil microbial functional diversity and microbial activities on coal mine spoils would respond to introduced agronomic treatments. The outcome of this study would therefore be beneficial for the choice of suitable agronomic treatments that would help improve the restoration process and provide an early indication of restoration success.
2.2. Physico-chemical analyses
2. Materials and methods
The pH and electrical conductivity in 1:5 H2O extract of soil samples were measured according to Rayment and Higginson (1992). Moisture content was calculated after drying the soil at 105 °C for 72 h. Chemical compositions of soils were determined at the CSBP Soil and Plant Analysis Laboratory, Perth, Australia using standard CSBP methods.
2.1. Study sites and soil sampling
2.3. Fluorescein diacetate (FDA) hydrolysis and microbial biomass carbon
A coal mine spoil with a five-hectare experimental site was selected for the study in Central Queensland, Australia. This site contained excavated mine spoil and was covered with a layer (20–30 cm) of stored topsoil (black cracking clay). The experimental site was exposed to various agronomic treatments such as Succession Planting (SP), Green Manure (GM) crops, Selective Herbicide (SH) and Mulching (M) with hay, along with a Control. Each treatment contained three replication plots with a completely randomized design. The GM and SP treatments were imposed one year prior to the introduction of other treatments. In the GM treatment, nitrogen fixing exotic species such as Clitoria ternatea, Vigna unguiculata and Dolichos lablab were hand sown using a seed drill. In the SP treatment, perennial nitrogen fixing native species such as Acacia holosericea, Acacia simsii and Acacia harpophylla were sown in rows using a seed drill. GM crops were introduced to increase soil organic matter (soil carbon) content.
A modified method of fluorescein diacetate (FDA) hydrolysis was used to measure microbial activity (Adam and Duncan, 2001). The soil sample (equivalent to 1 g dry weight) was added to 30 mL of phosphate buffer (60 mM, pH = 7.6), followed by addition of FDA, and incubated at 37 °C for 1 h. The reaction was quenched with 10 mL of acetone, and the developed color was measured spectrophotometrically at 490 nm. The concentrations of fluorescein released from the soil samples were calculated using a standard curve produced from the standards containing 0 to 10 μg/mL fluorescein. Microbial biomass carbon (MBC) was determined by the chloroform fumigation-extraction method (Vance et al., 1987). Briefly, the air-dried samples (equivalent to 10 g dry weight) were weighed in a 100 mL beaker, placed in a desiccator, and fumigated in a vacuum with 20 mL alcohol-free chloroform. After incubation in the dark for 5 days, the fumigation was stopped, and 50 mL of 0.5 M K2SO4 was added and 41
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Table 1 Characteristics of the coal mine spoil exposed to different agronomic treatments. Parameters ⁎
pH (1:20) EC⁎ (μS/cm) (1:20) Organic C (%) NH4+-N (mg kg−1 d wt) NO3-N (mg kg−1 d wt) Phosphorus (mg kg−1 d wt) Potassium (mg kg−1 d wt) Sulphur (mg kg−1 d wt)
Control b
7.8 187.7a 1.1 ± 0.1a 14.5 ± 5.0abc 3.3 ± 0.9a 4.3 ± 1.3ab 172.4 ± 59.5a 2.2 ± 0.2a
GM
M
a
SH ab
7.4 196.5a 1.4 ± 0.2a 24.0 ± 4.6c 8.9 ± 2.8b 4.7 ± 0.9ab 177.3 ± 16.6a 3.5 ± 1.2b
7.6 180.9a 1.0 ± 0.2a 12.7 ± 0.3a 2.7 ± 0.3a 3.0 ± 0.0a 184.3 ± 56.9ab 3.6 ± 0.5b
SP b
7.8 187.9a 1.1 ± 0.3a 15.3 ± 1.2ab 7.8 ± 2.7b 2.5 ± 0.9a 172.9 ± 61.0a 2.7 ± 0.2ab
7.7ab 175.8a 1.8 ± 0.2a 21.7 ± 3.7bc 5.0 ± 1.0a 5.7 ± 1.2b 196.7 ± 14.5b 2.9 ± 0.4ab
The data shown are the means and standard errors (n = 3). Values in the same row followed by different letters are significantly (P < 0.05) different. ⁎ pH and soil electrical conductivity (1:5 soil: water extract w/v) of a mine spoil exposed to different agronomic treatments.
AWCD = ∑(X − X0) / 31, where X was the mean of the same three wells per plate and X0 was the mean of control (water blanks) per plate. Functional diversity was measured as substrate richness (R) and Shannon's index (H). R was the number of different carbon substrates that were oxidized by the microbial community. H encompassed both R and the intensity of substrate use, which was calculated as H = ∑Pi (ln Pi) where Pi was proportional color development of the ith well over total color development of all wells (Schutter and Dick, 2001; Zak et al., 1994). GENSTAT (v 13) was used to perform ANOVA of AWCD, R and H data, and the means were compared using Student's t-test at 5% significance. Principal component analysis (PCA) was used to characterize the bacterial community level profile and was performed by using XLSTAT software.
shaken for 1 h at 200 rpm. The resulting mixture was then filtered through a Whatman No. 42 filter paper. Another set of non-fumigated samples was also extracted as per the fumigated samples. The filtered solution was stored in a freezer at −20 °C. The concentrations of C in both fumigated and non-fumigated extracts were determined spectrophotometrically using dichromate digestion (Walkley and Black, 1934). The MBC was calculated using a KEC factor of 0.33 (Sparling and West, 1988) as follows:
Microbial Biomass C (C from fumigated soil − C from non‐fumigated soil) = 0.33
2.4. Microbial respiration 3. Results and discussion
Soil microbial respiration was determined after wetting the soil samples to 40–50% water holding capacity and incubating at room temperature for 7 days. Basal respiration (BR) was estimated by measuring CO2 evolved from the soil (equivalent to 10 g dry weight) that was incubated in a closed vessel for 24 h at 25 °C in the dark. The CO2 produced was absorbed in 0.5 M NaOH and was quantified by titration with 0.5 N HCl after the addition of saturated BaCl2, using phenolphthalein as the indicator. Substrate induced respiration (SIR) was estimated using the same method as for the BR, except that 1 mL of glucose (0.5 g/mL) was added to each sample before incubation at 25 °C for 24 h in the dark (Anderson, 1982).
3.1. Physico-chemical properties Soil pH was significantly lower (P < 0.05) in the GM treatment compared to SH and the Control (Table 1). However, no differences in pH were observed amongst the other treatments. The highest pH (7.8) was observed in the Control and SH, and the lowest in the GM treated plots (7.4). The GM treatment included nitrogen-fixing plants such as Clitoria ternatea, Vigna unguiculata and Dolichos lablab. These plants accumulate large quantities of biomass and release protons during the nitrogen fixing process (Yan et al., 1996). This nitrogen fixation process would have contributed to reduce pH in the GM treatment. Studies have demonstrated an increase in soil organic matter and acidification of the soil during nitrogen cycling in agricultural soils (Bolan et al., 1991) and mined sandy soils (Chodak et al., 2009; Graham and Haynes, 2006) that supported leguminous plants. Soil EC varied from 175 μS/cm to 197 μS/ cm (Table 1) and remained similar amongst all the treatments. Soil organic C did not differ amongst the treatments, as the Organic C was highly variable within the site as well as between the sites, ranging from 1.1% to 1.8% (Table 1). However, the GM (1.4 ± 0.2%) and SP (1.8 ± 0.2%) treatments had elevated levels of organic C. The NH4+-N was significantly (P < 0.05) higher (24.0 ± 4.6 mg kg−1 dw) in the GM treatment and was the lowest (12.7 ± 0.3 mg kg−1 dw) in the M treatment. No marked differences were observed in the other treatments. Interestingly, the NO3-N levels were significantly (P < 0.05) higher in GM (8.9 ± 2.8 mg kg−1 dw) and SH (7.8 ± 2.7 mg kg−1 dw) treated plots compared to all other treatments. A distinct effect of GM and SP treatments was expected on N content, as the plants used in these treatments are able to fix atmospheric N2 through symbiotic bacteria (Brockwell et al., 2005; Peoples and Herridge, 1990). As Johnson and Curtis (2001) observed in a similar study, soil N and organic C content of the GM and SP treatments were consistently higher than those in other treatments, although some of these differences were not statistically significant. This suggests the beneficial use of N fixing plants. The higher NO3-N level in the SH
2.5. Community level physiological profiles The Ecoplate (Biolog® Inc., USA) was used to identify microbial functional diversity, wherein the rates of utilization of 31 different C substrates (see the table in the Supplementary file) were assessed (Insam, 1997). Soil samples (equivalent to 1 g dry weight) were serially diluted to 10−3 in 9 mL of sterile saline solution in triplicate. Solutions (200 μL) were then inoculated onto each well of the Biolog® Ecoplates. The microplates were monitored periodically at 0, 24, 48, 72, 96, 120 and 144 h by an ELISA reader at 570 nm. The absorbance readings that were negative or below 0.06 were set to zero. The average well color development (AWCD) of all absorbance data for different C substrates was individually calculated prior to statistical analysis (Konopka et al., 1998). 2.6. Statistical analysis All measurements, including pH, electrical conductivity (EC) and respiration, were calculated from triplicates of each treatment (3 replications). The data was subjected to one-way analysis of variance (ANOVA) using GENSTAT (v13) after testing of the data for outliers, and normality and homogeneity of error variances. All Biolog data were based on 96 h incubation. Microbial activity in each micro-plate was expressed as AWCD, and was determined using the formula: 42
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treatment can be attributed to a higher availability of NH4+-N, as the weed plant density was low in this treatment. These observations concur with those reported by Damin and Trivelin (2011), who revealed an increase in NO3-N content of soil after plants were sprayed with herbicide. Significantly higher (P < 0.05) phosphorus content was recorded in SP (5.7 mg kg−1 dw) compared to the M and SH treated plots; however, the phosphorus content was similar in the rest of the treatment plots (Table 1). Except in the M treatment plot, a significantly higher level of K was observed in the SP treatment plot (196.7 mg kg−1 dw) and no differences were found in other treatments (Table 1). Similarly, sulphur concentration varied from 2.2 to 3.6 mg kg−1 and was highest in M and GM compared to all other treatments. Significant changes in soil nutrients in the SP treatment could result from increased amounts of organic matter added by Acacia species that were included in the succession planting (Sparling and West, 1988). According to Souza-Alonso et al. (2015), invasion of Acacia dealbata not only contributed to higher C and N levels, but also enhanced the level of other soil nutrients, including P and K. Similarly, Forrester (2004) observed a significant increase in the rate of N and P recycling when Acacia mearnsii was grown with eucalypts.
(Fig. 1A). The high microbial activity observed in GM and SP treatments presumably reflected the presence of N fixing bacteria, whereas the M treatment contributed via enhanced moisture availability in the soil. Soil microorganisms and their activities play an important role in soil formation, decomposition of organic matter, N fixation and nutrient cycling (Mummey et al., 2002; Samuel et al., 2008), and hence they can provide information about the degree of change in biochemical processes in the soil. The FDA hydrolysis was used as an indicator of the changes in biochemical processes occurring in the soil (Adam and Duncan, 2001). Previous studies have shown alteration in FDA hydrolysis activity by exotic tree legumes (Bilgo et al., 2012) and pesticides (Chowdhury et al., 2008) in different conditions. The MBC, determined via the chloroform fumigation extraction method, was the lowest (0.3 g/kg) in SH treatment compared to the other treatments, which had similar levels (~1 g/kg) of C (Fig. 1B). Comparison between various treatments for both microbial activity and MBC clearly demonstrated the effect of these treatments in improving microbial activities in mined spoil following rehabilitation. The marked reduction in microbial activity and MBC in SH treatments suggests that the application of herbicide has led to the extinction of soil microbial activity in the mine spoil. Earlier study has revealed that an extensive use of herbicide impacted on soil microbial communities by altering their number, microbial activity, enzyme activity and even microbial diversity (Sebiomo et al., 2011). Furthermore, the presence of plants is critical to the build-up of microbial activity in mine spoil dumps. The presence of any plant, and even weeds, could enhance microbial processes in such soils (Melo et al., 2014; Sheoran et al., 2010). It is also recommended that numerous native ground cover species can be selected and established as soon as the spoil dumps are reshaped to increase microbial processes in the soil. The established native species will not only protect the soil but they will also help build microbial activity, whilst minimizing weed growth. Several studies have shown considerable effects on soil microbial activity by establishing plant communities and providing favorable conditions to soil microflora (Anderson and Ineson, 1984; Dinesh et al., 2003). Building up of soil organic C is crucial for long term sustainability of native plant communities on mine spoil dumps. The current study demonstrates that the use of appropriate organic treatments can enhance microbial activities in such soils.
3.2. Fluorescein diacetate (FDA) hydrolysis and microbial biomass C The current results revealed significantly (P < 0.05) higher microbial activity in GM treatments, followed by SP and M treatments c
µg Fluorescien release/g dw soil/h
140
A
120 100
b
80 60
b a a
40 20
3.3. Soil respiration
0 1.2 bc 1.0
c bc
Soil respiration is one of the most important components of the C cycle in forest ecosystems (Adachi et al., 2006). Generally, soil respiration varies with time and space (Darenova et al., 2016; Fang et al., 1998), and soil temperature and soil moisture are the key factors responsible for variations in soil respiration (Inoue and Koizumi, 2012; Shi et al., 2011). Soil respiration for a specific ecosystem can be characterized by its magnitude and temporal and spatial variability (Fang et al., 1998). Results of this study showed that BR did not differ amongst the treatments, although the values were very low in the SH treatment (Fig. 2A). Similarly, fewer differences were observed between the treatments for SIR except in the Control (Fig. 2B). Interestingly, the differences between the treatments for SIR were smaller than those observed for BR, due to the additional supply of C. In the SH treatment, the SIR increased substantially, as compared to the BR, indicating that the low microbial activity in this treatment could be due to low C supply (lack of plants). These results emphasize the need to maintain vegetation in the spoils to allow a healthy soil microbial environment, as the plants add C to the soil. An increase in plant cover leads to increased root density, resulting in enhanced soil microbial activity and respiration; both of which in turn contribute to soil organic matter formation (Lehmann and Kleber, 2015) and the build-up of organic C in the soil (Brohon et al., 2001; Liu et al., 2016).
B
b
g C\Kg d soil
0.8 a 0.6
0.4
0.2
0.0 Control
GM
M
SH
SP
Treatments Fig. 1. A: Microbial activity in variously treated mine spoils, as measured by the FDA hydrolysis test (n = 3). B: Microbial biomass C, as measured by the chloroform fumigation method. Bars indicate standard errors of means (n = 3). Columns with different letters are significantly (P < 0.05) different according to the Duncan multiple range test. 43
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a
35
µg CO2/g DW/h
30
A
a
a
25 a 20
a
15 10 5 0 50
a
a
µg CO2/g DW/h
40
30
B a
a
a
20
10
0 Control
GM
M
SH
SP
Treatments Fig. 2. A: Basal respiration (BR), and B: substrate-induced respiration (SIR) in variously revegetated mine spoils (n = 3). Bars indicate standard errors of means (n = 3). Columns with different letters are significantly (P < 0.05) different according to the Duncan multiple range test.
3.4. Community level physiological profiles The functional diversity of culturable soil microorganisms in different treatments was differentiated on the basis of bacterial kinetics and substrate utilization patterns (Konopka et al., 1998). The treatments showed differences in overall activity, as expressed by AWCD, across time. Based on AWCD kinetics (data not shown), measurements from 96 h incubation were chosen for data analysis (Garland, 1996). The AWCD values after 96 h of incubation were significantly (P < 0.05) higher in the SP and GM treatments, compared to SH and Control, showing variations amongst the treatments in functional diversity of the microbial community (Fig. 3A). Although higher total microbial activity (Fig. 1A) and MBC (Fig. 1B) were recorded in the M treatment compared to SH, no difference was observed between M and SH treatments for substrate utilization. This could be due to antagonistic interactions between the microbiota or toxicity caused to microorganisms by the redox dyes used in the Biolog wells (Ullrich et al., 1996). The R data showed the following order: SP, M, GM, C, SH (Fig. 3B) indicating three groups, viz. SP in one group, M, GM and C in the second group, and SH in the third group. The H followed the order: SP, M , GM , C, SH (Fig. 3C), and the treatments fell again into three groups. = Group 1 contained SP, M and GM, Group 2 contained M, GM and C, and Group 3 contained SH. Both the R and H analysis consistently separated the SH treatment from the rest of the treatments (Fig. 3B & C). These results could be taken as evidence for the positive effect of some
Fig. 3. A: Average well-color development (AWCD), B: richness (R), C: Shannon's index (H) of carbon substrate utilization on the Biolog® Ecoplate by soil bacterial communities in a mine spoil exposed to different agronomic treatments in a revegetated coal mine. Error bars indicate standard errors of means (n = 3). Columns with different letters are significantly (P < 0.05) different according to the Duncan multiple range test.
treatments and negative effect of SH treatment on soil microbial activity and the microbial community. According to average utilization of specific substrate data analyzed by the corresponding functional guilds, SP treated plots averaged higher utilization of all functional guilds of C substrates than the other treatments (Table 2). Similarly, compared to the Control, M and SH, the GM treatment had higher averaged carbohydrate utilization. Although significant differences were noted between the M and SH treatments for microbial activity (as measured by FDA) and MBC (Fig. 1), no difference was observed between these two
=
44
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Table 2 Average carbon substrate utilization in different substrates of the Biolog® Ecoplate by the mine spoils of a revegetated coal mine exposed to various agronomic treatments. The samples were collected from 96 h incubations of Biolog® Ecoplate, and the substrate utilization was measured as the average optical density across all substrates within each guild, which included seven types of carbohydrates, nine carboxylic acids, four polymers, six amino acids, two amines/amides and three miscellaneous substrates. Carbon sources
Control
Carbohydrates Carboxylic acids Polymers Amino acids Amines/amides Miscellaneous
0.89 0.98 1.25 0.92 0.53 0.85
± ± ± ± ± ±
GM 0.06 0.06 0.12 0.03 0.04 0.15
a
M
1.68 1.09 1.69 1.10 0.41 1.09
ab a a a ab
± ± ± ± ± ±
0.16 0.06 0.05 0.16 0.06 0.11
b
1.07 1.09 1.57 1.34 0.73 0.82
ab ab ab a b
SH ± ± ± ± ± ±
0.07 0.10 0.02 0.11 0.03 0.08
a
SP
1.05 0.88 1.16 0.98 0.55 0.54
ab ab ab a ab
± ± ± ± ± ±
0.09 0.10 0.20 0.09 0.08 0.15
a a a a a a
2.05 1.22 1.88 1.48 1.35 1.81
One way ANOVA ± ± ± ± ± ±
0.29 0.14 0.24 0.32 0.19 0.07
b b b b b c
P = 0.002 P = 0.122 P < 0.071 P = 0.081 P = 0.026 P < 0.001
The data shown are the means and standard errors of means (n = 3). Values in the same column followed by different letters are significantly (P < 0.05) different.
6
SH 4
PC2 (13.28 %)
SH
2
SP
SH GM 0
Control
SP GM M
Control -2
Control
GM SP
M
M -4 -4
-2
0
2
4
6
8
10
PC1 (35.51 %) Fig. 4. Principal component analysis (PCA) based on the carbon utilization profile of bacterial communities of a revegetated coal mine spoil exposed to different agronomic treatments (n = 3).
the conclusions of other researchers such as Remigi et al. (2008) and Lupwayi et al. (1998), who claimed that legume species may be responsible for changes in soil microbiota, affecting the structure and functions of microbial communities.
treatments in terms of substrate utilization according to Ecoplate data (Table 2). This suggests that larger microbial communities may be functionally more diverse, and that smaller differences between microbial communities could be difficult to describe from the Biolog® Ecoplate data (Lynch et al., 2004). In this study, the PCA of Biolog® Ecoplate data showed significant differences between treatments for functional diversity of the microbial community. The PC1 and PC2 explained 35.5% and 13.3% of the total variance, respectively (Fig. 4). The PC1 separated the Control, SH and M from the GM and SP, probably due to the rhizosphere bacterial community, as a result of introduction of different legume species. This provides strong support for
4. Conclusions The results of the current study clearly showed that agronomic treatments such as GM, SP and M increased microbial activity and MBC, whereas the SH treatment decreased both microbial activity and MBC. Additionally, the Biolog® Ecoplate results showed that application of 45
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various agronomic treatments had a positive effect on bacterial community structure, with the highest functional diversity present in the SP treatment. These data demonstrate that there were beneficial effects of agronomic treatments to soil microbial activity, and C usage profile of soil microbial communities. Further studies should evaluate if the molecular techniques and microbial functions such as C and N cycling could provide a better indication of the partial or total recovery of the functional diversity of the microbial community in disturbed mine spoils. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.geoderma.2018.11.038. Acknowledgments The authors are grateful to Professor David Midmore, Tom Hayes and Larry Hantler for their encouragement and support during this study. Special thanks to Dr Leonie Barnett for her constructive criticism of the manuscript. We also acknowledge the funding support of Anglo Coal Australia. 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