Feldspar Mine Spoil Rehabilitation: Use of Legumes and Bio Inoculants to Establish Soil Fertility on Agro–Silviculture Basis

Feldspar is one of the most abundant rock forming minerals in the earth’s crust. Feldspar deposition in India is 132 million tones which is 7% of the global availability. India stands fourth, in its production, whereas, Turkey (30%) is leading followed by Italy (19%) and China (8%) (Anonymous, 2015). It is one of the important minor minerals and is valuable for its use in ceramic and glass industries as a source of alumina and alkali, which accounts for more than 90% consumption (Anonymous, 2018). In India more than 80 per cent of the mineral production comes from open cast mines (Pandey et al., 2005) and feldspar is one of them. Open cast mining includes removal of vegetation, disturbance of soil profile, filling of undesirable waste materials and compaction, thus resulting in environmental degradation of the site and makes the soil unfertile (Piha et al., 1995). Similarly with severe land deterioration, feldspar mining often has detrimental effects on ecosystems. The gaseous waste of feldspar mining reduces microbial activities in the soil. Feldspar is a major source of potassium (K), sodium (Na) and calcium (Ca) (Vapur et al., 2015) but high sodium content commonly limits the use of water for irrigation. Feldspar ores often associated with the impurities like iron (Fe) (Vapur et al., 2015) which slowly leaches out in the water through chemical reactions and change the pH of water body towards acidic nature (Adhikary, 2015). Feldspar sludge is found to be rich in arsenic (Smedley and David, 2001) which is hazardous and needs special treatment before its disposal (Xue et al., 2017). Thus to neutralize these ill effects of feldspar mining some rehabilitation measures should be implemented for sustainable soil development. So far the aim of restoration has been achieved by the addition of top soil with organic amendments (Juwarkar et al., 2009), by the application of microorganisms (Rani et al., 2018) and by site-specific plantation (Singh et al., 2002).  But these methods are found to be very slow (Jha and Singh, 1992), time-consuming and technically and economically challenging (Botin, 2009). Today, microbial bio-inoculants are considered as key agricultural components that improve crop productivity and contribute in sustainable agro-ecosystems, by enhanced their bioactivities that stimulate and improve plant-microbe-soil consortium (Joshi et al., 2019). Moreover, site-specific tree plantation also enhances soil fertility (Nair, 2012) and agro-silviculture system is believed to have a great potential to reclaim mine contaminated site (Tewari and Singh, 2006). This two-tier vegetation enhances the mine spoil biological diversity and fertility besides contributing in socio-economic upliftment (Singh et al., 1998). In this context, microbial interventions in combination with agro-silviculture can be opted for enhancing soil fertility and this synergistic approach may deal with soil and agro-ecosystem simultaneously, thus it may play a major role in restoring soil health management (Dubey et al., 2017). Under agro-silvilculture system legume trees and crops can be opted, as being nitrogen fixing species, they have dramatic effects on soil fertility through the production of readily decomposable nutrient rich litter and also have capability to develop self-sustaining environment (Zhang et al., 2001). The present paper studies the response of rehabilitation process of feldspar mine spoil by using legumes and bioinoculants under agro-silviculture technique.

Feldspar mine at Sarana village is one of the major sites of feldspar mining, situated at Ajmer, India (25o38² and 26o58² north latitudes and 73°54² and 75°22² east longitudes). The field experiments were conducted in greenhouse at Govindgarh, Pushkar (Ajmer) and laboratory analysis has been done at Amity University Rajasthan, Jaipur. Total duration of the experiment has been eighteen months for field work (May 2017-October 2018) and seven months for laboratory work (October 2018-April 2019). Three trees species Prosopis cineraria (L.), Acacia nilotica (L.) and Acacia senegal (L.), two agricultural crops Cyamopsis tetragonoloba (L.) and Cassia angustifolia (Lam), two bacterial species Azospirillum brasilense and Azotobacter chrococcum and one symbiotic fungi Glomus mosseae (AMF) were identified for the study. Total 16 combinations (including one control) for each plant species were fixed in triplicate using randomised block design. The treatments were: (1) mine spoil only (control); (2) mine spoil + Azotobacter  (3) mine spoil + Azotobacter+ organic manure (OM); (4) mine spoil + Azotobacter + AMF; (5) mine spoil + Azotobacter+ NPK (50%) (6)  mine spoil + Azotobacter+ OM + NPK (50%); (7) mine spoil + Azospirillum (8) mine spoil + Azospirillum + OM; (9) mine spoil + Azospirillum + AMF; (10) mine spoil + Azospirillum + NPK (50%) (11) mine spoil + Azospirillum +OM + NPK (50%) (12) mine spoil+ AMF; (13) mine spoil + OM+ AMF; (14) mine spoil +AMF + NPK (50%) (15) mine spoil + AMF + OM + NPK (50%) (16) full dose of NPK (100%). Total 480 plants and their rhizosphere soil sample have been tested from initial analysis to eighteen months.
       
One ton of feldspar mine spoil from the core area of feldspar mine at Sarana village has been transported to green house and initial analysis was carried out in the laboratory. Soil texture was analyzed by sieving method. While in greenhouse, nursery pot experiment has been performed.  9 kg mine spoil was filled in earthen pots (12-15 kg capacity). Azotobacter, Azospirillum and Glomus mosseae (AMF) were isolated from soil samples by using Jensen Nitrogen Free media, semi solid malate media and wet – sieving and decanting technique respectively. These three cultures were mass produced on the same and inoculated as per the treatments. The pots with control (T₁), of respective plants, were also inoculated with their respective microbial media after the sterilization. The full dose (100%) of NPK comprised nitrogen (urea) = 10.5 gm, super phosphate = 15.5 gm, potash = 4.25 gm and half dose (50 % of the same), were given on 30^th day after sowing. All pots were watered up to field capacity; alternate days in first month and weekly onwards. The plant height, shoot diameter and canopy were recorded at the interval of three months. Plants were harvested after three months for crops and eighteen months for trees and their roots, shoots and leaves were oven dried and weighed for the further analysis.
       
The plant samples were processed in the laboratory for the estimation of chlorophyll a and chlorophyll b at 645 and 663 nm respectively (Arnon, 1949) and reducing sugar at 540 nm (Nelson, 1944). Dried powder was digested using Triacid (Nitric + Sulphuric + Perchloric acid) digestion method given by Piper (1942). Total nitrogen was determined by using Kjeldahl method (Saez-Plaza et al., 2013), phosphorus (P) determined by vanadomolybdate yellow colour method by Jackson, (1967), potassium (K), calcium (Ca), magnesium (Mg) and micronutrients (Cu, Mn, Zn and Fe) were determined through Atomic Absorption Spectroscopy (AAS) method. Rhizosphere soil was taken for the estimation of physico-chemical and biological properties of soil pH, EC, available phosphorus by using method given by Olsen (1954), dehydrogenase activity was measured by using 2,3,5-triphenylotetrazolium chloride (TTC), spectrophotometrically given by Tabatabai (1982), phosphatase by Tabatabai and Bremner (1969) and organic carbon (OC) method given by Walkley and Black (1934).
 
Statistical analysis
 
The data was subjected to analysis of variance and mean of inoculated and control treatments were compared by the Scheffe’s test for planned comparison up to LSD (Least Significant Difference).

Initial analysis
 
Average values of the physico-chemical properties of feldspar mine spoil were shown in (Fig 1). The texture of the investigated mine spoil was dominated by gravel (72%), with varying shares of sand (11.5%), silt (10.8) and clay (5.5%). The pH of the mine spoil was found highly alkaline in nature and seasonally provide cohesive environment for water holding and microbial mineral solubilization. Organic C and EC of feldspar mine spoil were also found to be low.  Extremely low contents of macro and micronutrients (N, P, K, Zn, Cu and Mn) were recorded. Whereas, the values of K, Ca and Fe elements were recorded slightly high as feldspar is a major source of these elements (Vapur et al., 2017). The spoil had a very low dehydrogenase activity. Thus biological activity of the studied mine spoil was also found to be low.
 


Growth parameters
 
Feldspar mine spoil has given good response in all the five plant species upon inoculation with various microorganisms. Improvement in plant height, shoot diameter, canopy and dry weight were observed, shown in (Table 1). In the present study microorganisms contributed maximum in the growth of Acacia nilotica with the treatment of Azotobacter+ OM+ NPK 50%. In Cyamopsis tetragonoloba and Cassia angustifolia, treatment of Azospirillum+ OM+ NPK 50% gave the maximum increment in growth. Plant wise maximum height is attended by A. nilotica (130 cm) and C. tetragonoloba (121 cm), which is higher in comparison with C. angustifolia (67), A. senegal (55 cm) and P. cineraria (92 cm) as in Fig 2.
 

 

       
Microorganisms have enhanced 30-35% of the plant growth over the control and observed equivalent/higher, when it gives in combination with 50% NPK. Azotobacter releases growth promoting hormones, which are responsible for the enhancement in plant growth (Jnawali et al., 2015) this is evident from the results by Azotobacter + NPK 50% than chemical fertilizer alone. Wu et al., (2006) supports our findings. Whereas, in Acacia senegal and Prosopis cineraria treatment of AMF + Om + NPK 50% have shown highest growth stimulatory effect, followed by Azotobacter + OM+ NPK 50%.  Muthukumar and Udaiyan, (2006), found similar results in significant growth of Prosopis cineraria. Organic manure has been commonly used in the ecological restoration of waste mine land (Peng et al., 2016) and in our study also it is responsible for improving the quality of soil. Also organic manure acts as a slow-release fertilizer, along with microbial inoculums in different combinations (Xu et al., 2016). Added to the our findings Mere et al., (2013) observed that organic manures provide nutrients, substrate for the growth of microorganisms, reduce the soluble and exchangeable aluminum (one of the main components of feldspar) temporarily by forming complexes or chelates in acidic soils and providing favorable environment for plant growth. Thus, in the present study too, OM might have supported the bio-inoculants in establishment of various plant species in feldspar spoil.
 
Enzymatic activities
 
Dehydrogenase (DHA) and alkaline phosphatase in rhizosphere were significantly increased after the introduction of bio-inoculants in comparison with uninoculated control shown in (Table 2). Feldspar mine spoil inoculated with Azospirillum showed enhanced DHA activity followed by Azotobacter and AMF, but maximum DHA activities were observed in the treatment of Azospirillum + OM+ NPK 50%. Highest dehydrogenase was may be due to the presence higher organic matter that supports microbial biomass and consequently affects the concentration of dehydrogenase. Our findings are supported by Prasanthi et al., (2019). DHA is well responded with AMF also as, Qian et al., (2012) found in maize plant on coal mine spoil. Whereas, alkaline phosphatase was found maximum in treatment with AMF+ OM+ NPK 50% and similar trend was seen in all plant species. This finding is supported by Gucwa-Przepiora et al., (2016). In our study along with the amendments legumes are also playing a critical role in the recovery of microbial activity and N accumulation that results in the improvement and enhancement of enzymatic activity in the mine spoil. 
 

 
Olsen P and organic carbon
 
Availability of Olsen P and organic-C was found maximum with AMF+ Om+ NPK 50% which is higher than NPK 100% alone, as AMF have been found to mineralise maximum soil phosphorous in this treatment as shown in (Table 3). The enhancement is may be due to the accumulation of organic matter in the mine soil, which has accelerated organic carbon production as stated by Ahirwal et al., (2017). We also found that organic carbon is directly proportional to the soil organic matter therefore organic matter in the treatments also played equally important role along with the bioinoculants in enhancing organic carbon. Our findings are confirmed by Rath et al., (2010). Legumes are also known to increase soil organic carbon and we have also seen the same increment of organic carbon in our plants grown on feldspar spoil. This is supported by Kumar et al., (2018), who stated that legumes have the capacity to store 30% higher soil organic carbon than other species, due to nitrogen fixing capability.
 

 
Metabolites
 
Total chlorophyll and reducing sugar were significantly enhanced in all the plant species upon microbial inoculation in various combinations, in the range of 40-45 % and 45-51% respectively over the control as shown in (Fig 3 and 4).  The chlorophyll content in the leaves of Acacia nilotica increased by 44.82% and 41.08% after inoculation in Azotobacter+ OM+ NPK 50% treatment and AMF+ OM+ NPK 50% respectively over the control, whereas the corresponding increase in the reducing sugar by 50.6% and 49.3%, respectively for both the treatments. Chlorophyll content (41.8%) and reducing sugar (49.1%) also increased in the leaves of Cyamopsis tetragonoloba after inoculation with Azotobacter+ OM+ NPK 50%. These results agree with Dhawi et al., (2015).  The increase in chlorophyll content may be due to microbial solubilization and mobilization of macro and micronutrients in the rhizosphere by the organic acids produced by microbial inoculants as supported by Subba-Rao, (1981). Kaur et al., (2015) in a study on chickpea stated that Rhizobium inoculation significantly increased chlorophyll content as compared to control thus supported the role of AMF in our study. Jin et al., (2015) reported that the level of sugar content increased in the leaves of maize plant as level of nitrogen increased. Similarly, in our studies percentage of reducing sugar enhanced significantly with the increase of nitrogen, may be due to the nitrogen fixation by the microbial inoculants and legumes.
 
Macronutrients
 
Microorganisms are getting acquainted in new environment and significantly contributing in nutrient solubilization, as evident by the enhanced uptake of nitrogen (N), phosphorous (P), potassium (K), calcium (Ca) and magnesium (Mg) in the range of 30-60% over the uninoculated control shown in (Table 4). Azospirillum+ OM+ NPK 50% (T-12) recorded maximum nitrogen uptake in Acacia nilotica, Acacia senegal and Prosopis cineraria, followed by Azotobacter + OM+ NPK 50% (T-7) and AMF+ OM+ NPK 50% (T-16) while Azotobacter + OM+ NPK 50% has been observed to support high nitrogen uptake in Cyamopsis tetragonoloba and Cassia angustifolia. Nitrogen fixing microorganisms along with 50% NPK have shown at par nitrogen uptake as of with 100% NPK inoculation. In phosphorus uptake, AMF showed maximum phosphorus influx in all five plants with feldspar mine spoil and have given better results in the presence of 50% NPK and higher in AMF+ OM+ NPK 50%. AMF easily absorb elements such as phosphorus and nitrogen which are essential for plant growth as stated by Maiti and Ahirwal, (2019). This result is in the line with Yang et al., (2016), who observed that legume- rhizobia have always provided a synergistic approach on nitrogen management. Ahirwal et al., (2017) has also confirmed the results and found increment in nitrogen upon introducing leguminous trees with mine spoils. We have observed highest N uptake with Azospirillum and significant intake with AMF. Similarly T-7 is responsible for the highest uptake of K. Comparatively crops had maximum uptake of K than trees. Feldspar has insoluble form of potassium (8-10%) and therefore, this K could be effectively utilized by native potassium solubilizing microorganisms (Kasana et al., 2017). This could be the reason for the highest accumulation of K in the plants.
 

       
Overall both bacteria and fungi have increased the uptake of Ca and Mg in all the five plants. Comparatively Ca uptake has been higher than Mg as feldspar already contains Ca in excess (Vapur et al., 2017) as shown in (Fig 5). Crops showed best results with T-12 and T-16 whereas, trees were best with T-7 in the uptake of Ca and Mg. Other than bioinoculants legumes also contributed in the uptake of these nutrients. As supported by Nyoki and Ndakidemi, (2014) that rhizobial inoculation of legume crops supplemented with P fertilizer improves the uptake of Ca and Mg.
 
Micronutrients
 
The uptake of micronutrients copper (Cu), manganese (Mn), zinc (Zn) and iron (Fe) increased over the control by 80% maximum in (Table 5). There is no uniformity in the absorption of micronutrients among all the introduced bio-inoculants along with various combinations, but AMF have shown consistently highest uptake in all the plants with feldspar spoil. Since feldspar and mica are most likely responsible for the release of these metal elements (Singh and Schulze, 2015) may be therefore, significant higher availability of micronutrients in feldspar mine spoil, is the reason for the high concentration of these elements in the plants, as Rao et al., (1996) found the same results with gypsum mine spoil. The mycorrhiza-assisted plants have also reported for the uptake of nutrients such as phosphorus (P) and zinc (Zn) that normally have low diffusion rates in the soil (Lendenmann et al., 2011).
 

       
In our studies bio-inoculants might have developed microclimate and initiate nutrient solubilization which has readily taken up by the plants and used in the metabolism and resulted in higher growth over or among the treatments. The microbial inoculants performed well in disturbed feldspar soil in the presence of organic manure and inorganic NPK. Legume species have also supported vice-versa. Application of organic manure and bioinoculants supported legumes to increase yield and soil fertility. Therefore, plantation of legume species for improving the fertility status of feldspar mine spoil could be a good option because of their N-fixing ability and consequently for restoring the soil-nutrient cycling in the degraded spoil. Thus the combination of legumes under agrosilviculture technique and used treatments proved to be a satisfactory trial for the future rehabilitation processes.

In conclusion, we recommend that the eco-friendly approach of integrated use of potential microbial inoculants like Azotobacter, Azospirillum, AMF for enhancing soil fertility along with the suggested legume species A. nilotica, A. senegal, P. cineraria, C. tetragonoloba and C. angustifolia under agro-silviculture practices with organic and inorganic amendments, enhances soil health and fertility, thus paving the path for feldspar mine spoil rehabilitation.