Influence of arbuscular mycorrhizal fungi on the growth and nutrient status of bermudagrass grown in alkaline bauxite processing residue

Influence of arbuscular mycorrhizal fungi on the growth and nutrient status of bermudagrass grown in alkaline bauxite processing residue

Environmental Pollution 159 (2011) 25e29 Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate...

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Environmental Pollution 159 (2011) 25e29

Contents lists available at ScienceDirect

Environmental Pollution journal homepage: www.elsevier.com/locate/envpol

Influence of arbuscular mycorrhizal fungi on the growth and nutrient status of bermudagrass grown in alkaline bauxite processing residue A. Giridhar Babu, M. Sudhakara Reddy* Department of Biotechnology, Thapar University, Patiala 147 004, India

Inoculation of red mud tolerant AM fungi enhanced the growth and nutrient status of bermudagrass and the physico-chemical properties of the bauxite residues amended with gypsum or sewage sludge.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 May 2010 Received in revised form 20 September 2010 Accepted 23 September 2010

A nursery experiment was conducted to evaluate the potential role of arbuscular mycorrhizal (AM) fungi in encouraging the vegetation cover on bauxite residue (red mud) sites. An alkali tolerant bermudagrass (Cynodon dactylon) adapted to local conditions were grown in red mud with different amendments with and without AM fungi to assess mycorrhizal effects on plant growth, mineral nutrition, metal uptake and neutralization of bauxite residue. Inoculation of AM fungi significantly increased the plant growth, nutrient uptake and reduced Fe, Al accumulation in plant tissue and also improved the soil physicochemical and biochemical properties. Gypsum and sludge amended treatments inoculated with AM fungi had maximum biomass, nutrient uptake and reduced accumulation of metals. The neutralization of red mud was significant in presence of AM fungi than control. The experiment provided evidence for the potential use of bermudagrass in combination with AM fungi for ecological restoration of bauxite residue sites. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Bermudagrass Glomus spp. Gypsum Fly ash Sewage sludge

1. Introduction Alumina extraction from bauxite ore with caustic soda (NaOH) at elevated temperatures produces waste bauxite refinery residue consisting of residue sand and residue mud (red mud). Red mud (RM) is characterized by high pH (pH > 10), high electrical conductivity (EC > 30 dS m1), high exchangeable sodium percentage (>70) (Friesl et al., 2004; Snars et al., 2004) and rich in iron (Fe) (typically 25e40%) and Al oxides (15e20%) (Gray et al., 2006). World production of this waste has been estimated at 30 million Mg per year (Menzies et al., 2004), the majority of which is disposed on land. These deposits also pose a potential risk of ground water contamination and also to human health. Establishing a sustainable vegetation cover on residue storage areas represents a challenge to alumina producers worldwide because of its inherent hostile properties (Chen et al., 2009; Courtney et al., 2009; Wehr et al., 2006). Such unfavorable conditions for plant growth require applying methods such as different amendments or microbial inoculation for improving nutrient balance, microbial activity and soil quality.

* Corresponding author. E-mail addresses: [email protected] (A. Giridhar Babu), [email protected] (M. Sudhakara Reddy). 0269-7491/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2010.09.032

The main aim of any restoration process is to create sustainable plant communities representative of the composition and diversity of the surrounding natural plant communities (Jefferson, 2004). Available published work on establishment of plant cover on RM has been demonstrated by amending with different organic and inorganic substrates (Benjamin et al., 2010; Courtney and Timpson, 2005; Eastham et al., 2006; Wehr et al., 2006). However, successful rehabilitation must involve the development of microbially driven organic matter turnover and mineral nutrient cycling for the longterm provision of plant nutrients (Banning et al., 2010). Studies of primary successional ecosystems have suggested that microorganisms play a critical role to establish early ecosystem development, due to functional abilities such as nitrogen fixation, organic matter turnover, mycorrhizal symbiosis, and potential facilitation of plant establishment (Hodkinson et al., 2002; Walker et al., 2003). Among soil microorganisms, arbuscular mycorrhizal fungi (AMF) play relevant roles for establishment, survival of plant species and improved soil properties in stressed environments (OrtegaLarrocea et al., 2010; Vivas et al., 2005; Zarei et al., 2008). AM fungi also alter the soil microbial communities in rhizosphere directly or indirectly through changes in root exudation patterns (Barea et al., 2005) and enhance the soil enzyme activities (Wang et al., 2006). Reports dealing with remediation of bauxite residue sites using microorganisms are scarce or not available. In the present study, an attempt has been made to remediate the bauxite

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residue sites using AM fungi which play an important role in establishment of vegetation in disturbed sites. The AM fungi associated with plants growing in the adjoining areas of RM pond were isolated and multiplied in RM to adapt AM fungi to these extreme conditions.

water to remove surface adhered RM and cleared with 10% KOH for 15 min at 90  C, placed in 10% HCl for 2 min and then stained with 0.05% lactic-glycerol-Trypan Blue for 20 min at 90  C by modified Phillips and Hayman (1970) method. The percentage of root length colonized by AM fungi was calculated by the gridline intersect method (Giovannetti and Mosse, 1980). 2.5. Plant analysis

2. Material and methods 2.1. AM fungal collection and inoculum production Root samples along with the RM flooded rhizospheric soil samples were collected from plants such as Acacia pennata (Mimosaceae), Lantana camara (Verbenaceae) growing adjoining to RM pond due to absence of vegetation in RM pond. Soil samples were passed through a 2 mm sieve and AM fungal spores were isolated from these samples by wet sieving and decanting technique (Gerdemann and Nicholson, 1963). Spores were multiplied on the roots of three trap plants like maize, wheat and sorghum separately in earthen pots containing RM and sandy loam soil in ratio of 40:60 for two successive cycles, each cycle of 3 months. No organic/manuring amendment was added while multiplying the AM fungi. The inoculum was prepared with density of 90e100 spores per 10 g of soil and RM along with the root pieces from pot culture. Prior to inoculation, isolated spores were identified as Glomus heterogama, Glomus mosseae, Glomus delhinious and Glomus facundisporum following the current taxonomic criteria (Schenck and Perez, 1990) and also from the INVAM (http://www.invam.caf.wdu.edu). 2.2. Soil collection and preparation RM was collected from the impoundments of National Aluminum Company Limited (NALCO), Damanjodi, Orissa, India. Fly ash, local nursery sludge and top soil were used to amend with RM. Sludge, top soil, fly ash and gypsum were added at a final concentration of 10% (v/v) to the RM. The physico-chemical properties of RM and different amendments were analyzed and presented in Table 1.

2.6. Soil sampling and analysis After harvest, the soil samples were collected from each plot and kept in sealed plastic bags at 4  C until brought to laboratory. The pH and electric conductivity (EC) was determined in 1:2.5 (w/v) water extract using Deluxe Water and Soil Analysis Kit (Model 191 E). Organic carbon was estimated by Walkley and Black (1934) method. Total N was determined by total organic carbon analyzer (TOC). Available P was estimated by Olsen method (Olsen et al., 1954). Urease activity was determined by the method of McGarity and Myers (1967) and the assay of acid and alkaline phosphatase activity was determined by measuring the p-nitrophenol (PNP) released by phosphatase activity followed by method of Tabatabai and Bremner (1969). The soil analysis was performed in triplicates by collecting the soils sample randomly and mixing them together for each treatment. 2.7. Statistical analysis The data were analyzed by analysis of variance (ANOVA) and the means were compared using Turkey’s tests at P < 0.05. The statistical analyses were performed using GraphPad prism software v.4.03.

2.3. Nursery experiment Nursery experiment was conducted in NALCO nursery at Damanjodi, Orissa, India. Randomized block design was prepared and the treatment plots were designed in such a way that every treatment had five replicates such as RM amended with gypsum contained control (without any inoculation) and AM fungal inoculation. Same pattern was followed for sewage sludge, top soil, fly ash and unamended RM. The plot size of each treatment was 72 cm length and 72 cm width. All the amendments added to RM were mixed in the range of 15 cm from the upper layer to reduce the dilution of amendments. An alkali tolerant bermudagrass (Cynodon dactylon) maintained at NALCO nursery was collected washed with water prior to planting. The indigenous AM mycorrhizal colonization was determined (about 3.0%) prior to planting. About 125 seedlings having similar length (5 cm) were planted in each plot. While planting, AM fungal inoculum (50 g/plot having 90e100 spores/ 10 g of soil) was applied near the roots. The grass was irrigated without adding any fertilizer. The grass was replanted after 2 months of initial plantation in unamended RM due to failure of the growth. After six and twelve months of plantation, the above ground grass was harvested to evaluate the growth and nutritional status of grass. The physico-chemical and biochemical properties of rhizosphere soils were also analyzed. 2.4. AM root colonization To determine the AM fungal colonization, the roots were dug out (about 30 plants) randomly from each plot both at 6 and 12 months. The dug out plants were replanted with grass again. Roots were washed in 0.1% ‘Teepol’ followed by distilled

Table 1 Physico-chemical properties of RM and its amendments. Parameters RM

The above ground biomass of grass was harvested both at 6 and 12 months by clipping to a height of 3 cm to determine the biomass. The grass was washed with de-ionized water to remove the soil particles and dried at 70  C for 48 h or till the constant weight was obtained. Following biomass determination, sub-samples were digested with conc. HNO3 and perchloric acid (3:1) and analyzed for K, Ca Na, Mg, Al and Fe with Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). Total P content in the grass was determined by colorimetry as described in Kitson and Mellon (1944).

RM þ G

RM þ TS

RM þ S

RM þ FA

pH 11.45  0.11 10.22  0.09 10.47  0.06 10.57  0.06 10.41  0.41 EC 1.86  0.01 2.09  0.04 0.92  0.01 1.23  0.02 1.35  0.01 (mS/cm) Organic 0.34  0.01 0.47  0.02 0.64  0.00 1.05  0.02 0.71  0.02 carbon (%) Avi. P 0.26  0.03 0.88  0.06 0.47  0.06 1.48  0.06 1.03  0.02 (mg/kg) Total N 4.00  0.01 9.00  0.00 19.00  7.00 26.0  0.00 11.0  7.00 (mg/kg) RM, red mud; RM þ G, red mud with gypsum; RM þ TS, red mud with top soil; RM þ S, red mud with sludge and RM þ FA, red mud with fly ash.

3. Results Though the data was collected after 6 and 12 months of nursery trial, we are presenting the data of only 12 months in this study as data of 6 and 12 months showed same pattern of results. The colonization of AM fungi was significantly increased in all the treatments inoculated with AM fungi compared to non-inoculated soils. Maximum colonization was observed in gypsum amended treatment followed by sludge (Fig. 1). The survival and the growth of grass in all amended treatments were significantly increased than controls and it was higher in AM inoculated treatment. Maximum biomass was observed in AM inoculated gypsum followed by sludge amended treatment (Fig. 1). Inoculation of AM fungi significantly increased the nutrient uptake of P, K and Ca compared to their respective controls. Maximum uptake of P, K and Ca were observed in AM fungi inoculated gypsum amended treatment followed by sludge amendment (Table 2). As compared to other treatments, the uptake of Mg was very low in gypsum amended treatments. However AM inoculation enhanced the uptake of Mg compared to the respective controls. The uptake of Ca in AM fungal inoculation significantly enhanced compared to their respective controls and the maximum Ca content was observed in gypsum followed by sludge amendment. Inoculation of AM fungi significantly reduced uptake of Fe and Al content of grass in all treatments. Highest accumulation of Fe and Al was observed in top soil amended RM and lowest in gypsum amended RM. However uptake of Na levels increased with AM inoculation compared to the respective controls (Table 2). The properties of unamended and amended RM significantly improved in various degrees in all inoculated treatments compared to control treatments. The pH was significantly reduced and the maximum pH reduction was observed in gypsum amended treatment. The electrical conductivity was higher in gypsum amended treatment compared to other amendments. The EC values were

A. Giridhar Babu, M. Sudhakara Reddy / Environmental Pollution 159 (2011) 25e29

AM colonization (%)

a

higher than acid phosphatase activity in all the treatments. Urease activity increased in presence of AM fungi compared to the respective controls and the maximum activity was recorded in gypsum amended treatment followed by sludge (Table 3).

120 100 80 60

4. Discussion

40

Establishment of vegetation on residues produced from the bauxite refining process is a beneficial part of their environment management. However, bauxite residue rehabilitation represents a significant challenge as this material is highly alkaline, saline, sodic, has poor water retention and containing virtually no organic matter and has numerous nutrient deficiencies (Gherardi and Rengel, 2001; Eastham and Morald, 2006). Till date, rehabilitation performance has been variable and very little information available on the effect of microorganisms on the vegetation cover. Remediation of contaminated sites may be facilitated by selection of tolerant plant species as well as soil microbes, such as AM fungi to promote the growth of plants in contaminated soils/substrates (Ortega-Larrocea et al., 2010). In the present study, we have selected an alkali tolerant bermudagrass for vegetation cover because of its multi metal tolerance and adaptability to local conditions and AM fungi isolated from the adjoining area of RM pond. The inoculum of AM fungi was produced in pot culture in presence of RM to make AM fungi tolerance to these conditions. Oliveira et al. (2010) reported that AM fungi quickly lose their symbiotic efficacy when cultivated without edaphic stresses of the environment from where they are originally isolated. They also recommended that the inoculum should be produced with original edaphic stresses especially for AM isolates from extreme environments. In order to promote plant establishment in contaminated sites, it is essential to improve the physical and chemical properties of the substrate (Chen et al., 2007). We have amended gypsum, fly ash, sewage sludge and top soil to RM to improve the properties of the RM. The bermudagrass fail to grow initially in RM without any amendments, which might be due to the inherent hostile properties of RM (Chen et al., 2009; Courtney et al., 2009; Pagano et al., 2002). Fortin and Karam (1998) and Pagano et al. (2002) reported the failure of vegetation in unamended RM. Bermudagrass replanted after 2 months showed the growth which might be due to leaching of some of the toxic components of the RM. Inoculation of AM fungi increased the growth of bermudagrass grown in RM with different amendments compared to their respective controls. AM fungi are reported to reduce the detrimental effects of soil associated plant stresses such as lack of nutrients, organic matter, high salinity or high pH (Entry et al., 2002; Oliveira et al., 2010). Among the different amendments to RM, AM fungi inoculated in gypsum amended treatment showed

20 0

RM

RM+G

RM+TS

RM+S

RM+FA

Treatments

Dry Biomass (gm/plot)

b

27

300

200

100

0

RM

RM+G

RM+TS

RM+S

RM+FA

Treatments Fig. 1. (a) Arbuscular mycorrhizal fungal colonization and (b) grass biomass of different treatments inoculated with AM fungi (empty bars) and respective controls (dotted bars) (RM, red mud; RM þ G, red mud with gypsum; RM þ TS, red mud with top soil; RM þ S, red mud with sludge and RM þ FA, red mud with fly ash).

significantly reduced during the growth of grass in both inoculated and non-inoculated treatments. Inoculation of AM fungi significantly decreased the pH compared to their respective controls in fly ash and sludge amendments. In AM fungi inoculated samples, organic carbon and available P was significantly enhanced in all treatments compared to their respective controls. Total N increased in all treatments inoculated with AM fungi and high N content was observed in sludge followed by gypsum amended treatments (Table 3). Acid phosphatase and alkaline phosphatase enzyme activities were increased in presence of AM fungi compared to controls. The maximum acid phosphatase and alkaline phosphatase activities were observed in gypsum amended treatment followed by sludge amended treatment. However, alkaline phosphatase activity was

Table 2 Uptake of different nutrients and heavy metals (mg/kg) in aerial portion of bermudagrass in different treatments after 12 months. Treatments

Fe

Al

Na

P

K

Ca

Mg

RM þ C RM þ AM GþC G þ AM TS þ C TS þ AM SþC S þ AM FA þ C FA þ AM

2652  8a 1752  3c 1358  4e 1126  4f 1972  6b 1188  5f 1483  5d 776  4 g 1852  6bc 1089  4f

2187  4a 1580  7b 1063  5c 879  3de 776  5f 669  6 h 891  6d 633  4i 861  1e 747  5 g

225  3.7d 364  5.0b 146  1.5e 218  .0d 242  7.5 cd 343  4.0b 255  1.0c 396  6.0a 238  3.0 cd 366  6.0b

319  8 g 419  8e 688  2c 1068  6a 237  5 h 364  6f 376  3f 887  3b 374  5f 537  2d

785  5e 1031  4b 829  3de 1366  4a 826  4de 934  4c 940  3 cd 1112  2b 842  7de 1068  7b

869  5e 997  8c 1154  8b 1329  7a 810  5f 930  6d 760  4 g 1129  11b 559  3 h 1003  3c

250  2ef 308  5c 271  1de 205  8 h 219  3gh 351  3b 287  9 cd 395  3a 229  6fg 332  6b

RM, red mud; S, sludge; TS top soil, FA, fly ash; G, gypsum; C, non-inoculated treatment; AM, arbuscular mychorrhizal fungi. Values in columns sharing same letter are not significant at P < 0.05.

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Table 3 Physico-chemical and biochemical properties of soil of different treatments after 12 months. Treatmentsa

pH

EC (mS/cm)

Organic carbon (%)

Avail. P (mg/kg)

Total N (mg/kg)

Acid. P.ase (mM/g/h)

Alk. P.ase (mM/g/h)

Urease (mM/g/h)

RM þ C RM þ AM GþC G þ AM TS þ C TS þ AM SþC S þ AM FA þ C FA þ AM

9.3  0.1a 8.8  0.7b 7.7  0.07e 7.5  0.2f 8.7  0.05bc 8.5  0.06c 8.5  0.01c 8.2  0.1d 8.2  0.2d 8.1  0.03d

0.49  0.02c 0.67  0.02b 1.70  0.02a 1.62  0.03a 0.70  0.01b 0.71  0.02b 0.48  0.02 cd 0.52  0.06c 0.43  0.02d 0.43  0.03d

0.51  0.3f 1.07  0.3bc 0.60  0.0ef 1.29  0.0b 0.57  0.2f 0.88  0.1 cd 1.09  0.8bc 1.90  0.3a 0.79  0.0de 1.2  0.5b

0.86  0.00 h 1.31  0.07 g 4.47  0.46c 7.92  0.11a 1.39  0.05 g 2.00  0.04f 2.85  0.08e 5.84  0.06b 2.11  0.09f 3.70  0.06d

57.0  1j 100  1i 389  2d 485  2b 267  1 h 342  5f 352  2e 690  1a 322  3 g 448  2c

171  2j 233  6 h 293  4f 394  4b 209  1i 262  3 g 362  2d 413  1a 335  2e 382  2c

274  2 h 385  5f 416  1d 515  4b 315  1 g 400  1e 417  2d 592  1a 415  1d 472  1c

8.1  0.3 g 19.4  0.1e 47.9  0.5b 67.5  0.6a 28.8  0.7d 69.3  0.7a 28.9  0.6d 41.4  0.8c 13.8  0.6f 30.4  0.7 g

a RM, red mud; S, sludge; TS, top soil, FA, fly ash; G, gypsum; C, non-inoculated treatment; AM, arbuscular mychorrhizal fungi. Values in columns sharing same letter are not significant at P < 0.05.

maximum growth followed by sludge treatment. Addition of gypsum or sludge might have increased the nutrition level and lowered the pH of the residue which might have promoted the maximum growth of grass. Courtney and Timpson (2005) reported the plant growth in treatments that had received gypsum amendment, with higher plant biomass, Mn nutrition and lower Al and Fe concentration. The higher percent root length colonization of AM fungi observed in gypsum and sludge amended treatments might also have contributed for the maximum growth. The AM fungi may act indirectly, by enhancing the plant mineral nutrition and increasing plant growth with a resulting dilution effect of the metal in the host plant or directly, by binding of the metal to the fungal mycelium and immobilization in the rhizosphere or the roots (Chen et al., 2001). In RM, the main alkaline anions buffering the solution are HCO 3/ 2  CO3 , Al(OH) 4 and OH . Gypsum addition effectively lowers the pH which is related to the ability of gypsum to dissolve and release 2 Ca2þ into the solution to react with OH, Al(OH) 4 and CO3 (Oster et al., 1999). Xendis et al. (2005) observed that gypsum solubility limited the extent of the pH reduction and only upon activation with H2SO4 did the desired pH reductions takes place. This limitation might have occurred due to the precipitation of CaCO3 on gypsum particles as has been observed by Kopittke et al. (2004). RM neutralization by microbial means has been investigated by a small number of researchers despite early results showing significant promise (Hamdy and Williams, 2001; Krishna et al., 2005; Vachon et al., 1994). In the present study, the pH of the RM was significantly reduced compared to the initial levels and inoculation of AM fungi reduced further compared to their respective controls. Though the exact mechanism of neutralization reaction is not fully understood, it is assumed to be a combination of organic acids released by the microbes and root exudates and the diffusion of the respiratory gases into the environment (Hamdy and Williams, 2001; Krishna et al., 2005). The neutralization of RM using microorganisms is significant because it is continuously controlled by a biological entity rather than the application of acid, the pH is buffered by microbes as long as they are provided with nutrients and microbes also improves drainage, nutrient exchange and chances for establishing a plant cover (Grafe et al., 2009). Addition of different amendments to RM have improved the residue properties by lowering the pH, increasing the organic carbon, available P, total N and the soil enzyme activities such as acid phosphatase, alkaline phosphatase and urease compared to unamended RM. Benjamin et al. (2010) reported that organic waste amendment improves the physical and microbial properties of the bauxite residue sand. Phosphorus nutrition is limited in RM due to high P adsorption of sesquioxides (Snars et al., 2004). The maximum P increase was observed in gypsum amended treatment which might be due to the reduction of pH and fixation of Al and Fe.

In the present study, high content of Fe and Al was observed in tissues of grass grown in top soil amended treatment due to presence of these metals at higher concentration in top soil. The top soil used in this study is mined out soil of bauxite ore, generally rich in Al and Fe. However in AM inoculated treatments the physicochemical and biochemical properties were improved by various degrees compared to controls. AM fungi can interact with other rhizospheric microorganisms and can affect rhizodeposition and thus the quantity and quality of organic C delivered to soil via fugal hyphae (Barea et al., 2002). Enzymes are potential indicators of the extent to which soil disturbance by a given activity may affect the immediate environment (Pascual et al., 2000). Among the soil enzymes, phosphatases and urease play an important role in P, N and C mineralization respectively. AM fungi inoculated treatments enhanced the activity of enzymes in all treatments. The increase of enzymatic phosphatase and urease activities of inoculated plants could be due to the effect of nutrient leakage from roots (qualitative and quantitative changes in root exudates) (Vivas et al., 2005). 5. Conclusion In conclusion, RM with different amendments positively affected the growth of bermudagrass especially in gypsum and sludge. Further, inoculation of AM fungi improved the survival and growth of bermudagrass by increasing the nutrient uptake and reducing metal translocation to aerial portion of grass, tolerance to alkaline, saline, sodic and metal stress conditions. We are reporting for the first time the influence of AM fungi on the growth of bermudagrass in RM. Inoculation of RM tolerant AM fungi with gypsum and sludge amendments may be promising approach to promote the vegetation on RM ponds. Acknowledgments The authors are thankful to TIFAC-CORE, Thapar University, Patiala, Punjab, India for provided lab facilities for research. We would also like to thank Department of Biotechnology (DBT) for their financial support to this project (BT/PR-4697/BCE/08/327/ 2004). We also thank National Aluminum Company Limited (NALCO), Damanjodi, Orissa, India for support in setting up and managing this experiment in NALCO nursery. References Banning, N.C., Phillips, I.R., Jones, D.L., Murphy, D.V., 2010. Development of microbial diversity and functional potential in bauxite residue sand under rehabilitation. Restoration Ecology 17, 350e358. Barea, J.M., Azcón, R., Azcón-Aguilar, C., 2002. Mycorrhizosphere interactions to improve plant fitness and soil quality. Antonie van Leeuwenhoek International Journal of General and Molecular Microbiology 81, 343e351.

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