Analysis of mining waste
The common test used to assess the presence of soluble acid salts in mining wastes is paste pH. The mining waste used in this study has a paste pH of 3.23 and a water-soluble concentration of 1.94 g/l.
Concentration and relative distributions of heavy metals and As in mining waste are shown in Table 1 and Fig. 1.
Table 1
Concentration of heavy metals and As in mining waste
Fraction
|
Cu
|
Zn
|
Fe
|
Mn
|
Cd
|
Al
|
As
|
mg/kg
|
Exchangeable
|
221.3
|
55.6
|
21.75
|
161.25
|
< 0.1
|
222.15
|
< 0.1
|
Carbonates
|
25
|
0.4
|
6.55
|
8.55
|
< 0.1
|
10.8
|
< 0.1
|
Fe and Mn oxides
|
249.8
|
18.4
|
5350
|
98.3
|
< 0.1
|
2506
|
57.45
|
Organic matter and secondary sulfides
|
220
|
44.4
|
638.5
|
5
|
< 0.1
|
1501
|
18.85
|
Residual
|
242.2
|
332.0
|
36925
|
206.95
|
< 0.1
|
15155
|
175.6
|
Total
|
958.3
|
450.8
|
42941.8
|
480.05
|
< 0.5
|
19394.95
|
252.1
|
The total concentration of Cu in the mining waste was 958.3 mg/kg. Distribution of copper between exchangeable, Fe - Mn oxyhydroxides, organic matter and residual fraction was approximately the same (ranging from 23–26%). The results obtained can be interpreted according to a sequential extraction procedure adapted for geochemical studies of copper sulfide mine waste (Dold 2003), and it can be concluded that 23.1% of copper is in a water-soluble fraction the form of water-soluble fraction (e.g., chalcanthite (CuSO4.5H2O)) and as Cu, which may be released in the exchangeable fraction from vermiculite-type mixed-layer mineral in the mining sample. Twenty-five percent of copper was incorporated in iron-phases. In oxidizing conditions performed by a H2O2 leach, one quarter of the total copper was dissolved. It can be concluded that part of the copper in the mining waste is in the form of supergene Cu-sulfides such as covellite and chalcocite–digenite and twenty-five percent of copper is in the residual fraction.
The content of total zinc in the mining waste was 451 mg/kg. Distribution of zinc between exchangeable and organic matter fractions was 16.7% and 13.4 %, respectively. The major part of Zn was found in residual fraction as sphalerite accounting for 73.6% of total Zn. Schaider et al., 2007, found that relatively labile and bioaccessible mineral phases of Zn increased with decreasing particle size of mine waste.
The mining waste in this study contained pyrite at 4%. Iron was mainly distributed in two fractions, residual (86% of iron content) or Fe and Mn oxides (dissolved schwertmannite, ferrihydrite, Mn-hydroxides, secondary jarosite, as well as goethite formed acid mine drainage) which contained 12.5% of the iron content. High pyrite content in mining waste and low paste pH values are a prerequisite for the growth and activity of iron-oxidizing hemolithotrophic bacteria such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans.
Unlike iron, manganese was present in high concentrations in exchangeable fraction (33.6%). Distribution of manganese in Fe and Mn oxides and residual fractions was 20.4 and 43% respectively. A number of authors have found that Mn had the higher mobility potential to be released from the mining waste, since its content in the exchangeable fraction in much of the research is high (Chotpantarat et al. 2015; Soltani et al. 2017).
The presence of minerals such as albite, microcline, muscovite, clinochlore in the studied mining waste is a premise for the significant fraction of total aluminum to be in the residual fraction (78%). 13% of Al was in the composition of the Fe and Mn oxides fraction, and only 1% was distributed in exchangeable fraction.
The total concentration of As in the mining waste was 252.1 mg/kg. Sequential fractionation of As showed that 25% of total arsenic was distributed in the Fe and Mn oxides fraction. The arsenate and arsenite ions had an affinity to be sorbed from formed ferrihydroxides having a highly developed specific surface. In oxidizing conditions, 5% of the arsenic was dissolved. Most of the toxic elements (69.7%) were found in the residual fraction.
Analysis of the effluents
Data on the measured pH, redox potential, electrical conductivity (EC), sulfate and heavy metals concentrations in the effluents are presented in Table 2. Treatment with PGPR or/and humic substances led to a slight increase in pH of the effluents. The Eh values in all five cases ranged from 301 to 388 mV, with the highest values being established in the control (364±24 mV). The highest EC values were found in the first effluents in all variants. Over time, the electrical conductivity of the effluents decreased. From the obtained results, it can be concluded that the applied treatment approaches reduce the activity of the hemolytotrophic microflora. These findings are also supported by data on the concentration of sulfate in the effluents. The highest concentrations of sulfate in the leachates were established in the control (1.76±0.22 g/l). The concentrations of sulfates in cases of separate treatments of PGPR and humic acids were 1.49±0.14 and 1.27±0.20 g/l, respectively. The suppression of oxidation of sulfide minerals in mining waste was more significant by the combination treatment with bacteria and humic acids, where the sulfate concentration is lowest.
The highest concentrations of Cu, Fe and Zn were determined in effluents of control treatment yielding values of 14.3-15 (mean 14.65 mg/l), 0.52-0.76 (mean 0.64 mg/l) and 1.84-3 (mean 2.57 mg/l), respectively. The concentration of manganese in effluent of control treatment was 5.79±1.21 mg/l. The high concentrations of copper, manganese and zinc in the leachate were due to the high proportion of these heavy metals in the easy-soluble exchangeable fraction (Fig. 1) and microbial oxidation of copper and zinc minerals, distributed in sulfide fractions. In studies on long-term acid generation and heavy metal leaching from the tailings, Khoeurn et al. (2019) proved the involvement of these two mechanisms in different phases of contaminated water generation from mining waste.
We found that the application of both humic substances and PGPR resulted in a decrease in Cu, Fe and Zn concentrations in leachates. An increase in the concentration of manganese 7.28±0.34 mg/l was found only in the case of separate humic acid treatment (Table 2). In combination treatment with PGPR and humic acids, the lowest concentrations of all heavy metals in effluents were recorded.
Table 2 General parameters measured at the effluents from the in vivo pot tests
Parameters
|
Control
|
PGPR
|
HA
|
PGPR + HA
|
2xPGPR + 2xHA
|
pH
|
3.58±0.26
|
3.65±0.31
|
3.78±0.31
|
4.02±0.22
|
4.13±0.25
|
Eh, mV
|
364±24
|
319±17
|
335±18
|
337±21
|
325±24
|
EC, mS/cm
|
2.457±0.39
|
2.050±0.24
|
2.032±0.23
|
1.411±0.36
|
1.375±0.31
|
SO42-, g/l
|
1.76±0.22
|
1.49±0.14
|
1.27±0.20
|
0.63±0.32
|
0.55±0.29
|
As, mg/l
|
<0.01
|
<0.01
|
<0.01
|
<0.01
|
<0.01
|
Cu, mg/l
|
14.65±0.35
|
4.02±0.13
|
2.17±0.09
|
1.19±0.11
|
1.59±0.07
|
Fe, mg/l
|
0.64±0.12
|
0.29±0.05
|
0.24±0.14
|
0.05±0.04
|
0.09±0.03
|
Mn, mg/l
|
5.79±1.21
|
2.11±0.75
|
7.28±0.34
|
0.64±0.14
|
0.58±0.18
|
Zn, mg/l
|
2.57±0.73
|
0.64±0.21
|
1.08±0.32
|
0.61±0.29
|
0.56±0.17
|
The application of humic substances to mine tailings significantly decreased Cu leaching, due to the formation of organomineral complexes (Tapia et al. 2019). According to Chotpantarat et al. (2015), the addition of humic acid strongly inhibited the bioavailability of Cu and Pb, whilst slightly decreasing the mobility factor of Co, Cr, and Zn but slightly increasing that for Mn and Ni, depending on the dose. However, humic substances can form both soluble and insoluble complexes with heavy metals which can increase or decrease metal mobility (Violante et al. 2010). Wang and Mulligan (2009) reported that humic acid could enhance the mobilization of arsenic and heavy metals from the mine tailings under alkaline conditions (pH 11). The origin and composition of humic substances, the dose of application, pH, metal concentration and speciation in mining waste are important factors that need to be taken into account in reclamation technologies.
Metal–mineral–microbe interactions have also been the subject of a number of studies (Gadd 2010; Kong and Glick 2017). Microorganisms have a variety of properties that lead to the mobilization or immobilization of metals due to the changes in metal speciation, toxicity and mobility. Solubilization mechanisms are related to the production of siderophores, excreted metabolites (amino acids, phenolic compounds and organic acids) with metal-complexing properties, chemical oxidation or reduction. On the other hand, processes such as biosorption to cell walls, formation of biopolymers such as proteins, nucleic acids and extracellular polysaccharides, intracellular accumulation, or precipitation of metal in and/or around microbial cells are involved in the immobilization of metals. The variety of mechanisms that mobilize or immobilize metals makes a number of soil microorganisms attractive for bioremediation of contaminated mining sites (Karthiga and Natarajan 2015; Kong and Glick 2017; Li et al. 2017; Ashraf et al. 2017).
Analysis of plants
Data on fresh and dry weights of the aboveground biomass are presented in Table 3. This data suggests that the application of PGPR and humic acid improves plant growth on poor soil used for mine waste reclamation. The results of statistical analysis of fresh and dry weights of the aboveground biomass are presented graphically in Fig. 2 and Fig. 3 respectively.
Table 3 Fesh and dry weights of the aboveground biomass
Variant
|
07.June
|
13. August
|
26.October
|
Fresh
weight, g
|
Dry
weight, g
|
Fresh
weight, g
|
Dry
weight, g
|
Fresh
weight, g
|
Dry
weight, g
|
Control
|
40.801
|
2.781
|
16.902
|
3.330
|
28.195
|
4.302
|
PGPR
|
45.502
|
3.228
|
18.076
|
3.637
|
34.304
|
5.044
|
HA
|
48.913
|
3.899
|
22.305
|
4.435
|
38.105
|
5.392
|
PGPR + HA
|
49.812
|
3.930
|
24.112
|
4.850
|
38.611
|
5.623
|
2xPGPR + 2xHA
|
47.113
|
3.458
|
21.092
|
4.362
|
33.413
|
5.071
|
Treatment with only PGPR increased fresh aboveground biomass yield by 12%, 7% and 22% for different months of determination. Fresh biomass was 20 - 35 % above controls in the case of separate humic acid treatment. The highest results (22%, 43% and 37% above controls for different months) were obtained by treating the plants with a combination of PGPR and humic acid. applied at a dose of 2 ml/l. The fresh biomass of plants was lower in the combined treatment in a double dose. respectively 15%, 25% and 19%. Since the P-value of the F-test is less than 0.05, there is a statistically significant difference between the means of the 5 variables at the 5% significance level.
The application of PGPR separately has the effect of increasing the dry biomass by 16%, 9% and 17% respectively (Fig. 3). Treatment with only humic acid increased dry biomass yield by 40%, 33% and 25% over specific months. Dry biomass data also demonstrate that the combination of PGPR and humic acid at a dose of 2 ml/l has the greatest positive effect on plant growth (41%, 46% and 31% above controls for different months). Dry aboveground biomass was lower in the combined treatment in a double dose. Khaled and Fawy (2011) studied the effect of different levels of humic acids on plant growth and nutrient content. and reported that the dry weight and nutrients uptake were negatively affected by the application of higher dosage humic acids (4 g humus/kg). Since the P-value of the F-test is less than 0.05, there is a statistically significant difference between the means of the 5 variables at the 5% significance level.
The applications of PGPR and humic acid had a significant effect on the uptake of biogenic and macroelements in plants growing on poor soil (content of the humus and Kjeldahl-are respectively 0.98% and 0.196%). In all cases of treatment. the nitrogen content was higher than the control (Table 4), as the highest nitrogen uptake (10.3% above control) was obtained with combined treatment in a double dose.
The application of PGPR impacted phosphorus uptake having the effect of increasing the phosphorus content by 2.2%. The application of humic acids affected the uptake of phosphorus with 12.9%. The highest assimilation of phosphorus from the grass (30.3% above control) is observed with combined treatment in a double dose.
This study found that treatment with PGPR and humic acids significantly increased the uptake of potassium by plants. Treatment with only PGPR increased K uptake to 49.5% and in the case of separate humic acid treatment K uptake was 81.3% above control. Combination of both treatment variants showed an increase in potassium assimilation of more than 100% compared to the control.
According to the analysis of results, the application of PGPR and humic acids also increased the uptake of Ca and Mg (Table 4). The highest uptake of Ca and Mg by plants was obtained in the case of humic acid treatment, respectively 31.4% and 21.3% above the control.
The application of PGPR and humic acids increased the uptake of Fe from 31.8% to 76.9% for the different variants of treatment. An increase in Mn and Zn uptake (54.7 % and 54.6%) was found only in the case of humic acid treatment. The application of PGPR (both alone and in combination with humic acids) had as effect the decrease the Mn and Zn content. In all cases of treatment, the uptake of copper from the plants was reduced as accumulation was undetectable (Table 4).
Table 4 Effect of application of PGPR and humic acid on plant nutrients and pollutants uptake
Treatments
|
N.
%
|
P. mg/kg
|
K. mg/kg
|
Ca. mg/kg
|
Mg. mg/kg
|
Fe. mg/kg
|
Mn. mg/kg
|
Cu. mg/kg
|
Zn. mg/kg
|
As. mg/kg
|
Control
|
1.84
|
1870
|
10403
|
5937
|
2397
|
402
|
349
|
79.5
|
53.10
|
<5
|
PGPR
|
1.98
|
1911
|
15550
|
7643
|
2435
|
530
|
341
|
33.5
|
43.9
|
<5
|
HA
|
1.94
|
2111
|
18870
|
7801
|
2908
|
711
|
540
|
35.6
|
82.1
|
<5
|
PGPR + HA
|
1.99
|
2112
|
20898
|
6767
|
2496
|
588
|
275
|
25.7
|
34.9
|
<5
|
2xPGPR + 2xHA
|
2.03
|
2436
|
20957
|
6402
|
2461
|
623
|
252
|
34.5
|
53.2
|
<5
|
The results obtained for the effect of applied treatments on the aboveground biomass of grass and and mineral elements uptake show the applicability of a combination of PGPR and humic acids in the reclamation of mining waste. Selected strains of bacteria belonging to the genera Bacillus and Pseudomonas have a number of properties that classify them as plant growth-promoting bacteria. B. subtilis CI R1 and B. amyloliquefaciens CI R2 are very effective for increasing plant available phosphorus (Bratkova et al. 2015). Also, these strains produce Indole-3-acetic acid. Ps. chlororaphis 1S4 completely inhibits the growth of three molds (Aspergillus flavus, Penicillium claviforme and Rhizopus arrhizus) (Georgieva et al. 2018). B. megaterium АМ1, B. simplex АМ3, Ps. fluorescens АМ2 and Ps. arsenicoxydans АМ4 have diverse lytic enzyme activities – esterase, esterase lipase, alkaline phosphatase, acid phosphatase, protease, amylase (data not published). The combination of these strains, applied in poor soil for the reclamation of acid-generated mining waste has the effect of not only improving the mineral nutrition of the grass, but also reducing the uptake of Cu and Zn. The similar effects on Lupinus luteus inoculated with metal resistant PGPR have been reported by Dary et al. 2010. According to Tripathi et al. (2005) inoculation with a Pb- and Cd-tolerant Pseudomonas putida KNP9 strain increased plant growth but reduced the Pb and Cd uptake by Phaseolus vulgaris. However, it should be noted that PGPB may also alter metal bioavailability and increase plant metal uptake (Li and Ramakrishna 2011; Ashraf et al. 2017; Ren et al. 2019). The large number of studies on the effects of PGPR on different plants for the reclamation of mining sites indicate the possibility of their application in both phytoextraction and phytostabilization (Kong and Glick 2017). The combination of PGPB with humic substances has an even greater effect on plant growth and improving their mineral nutrition.