Environ Sci Pollut Res (2014) 21:7938–7944 DOI 10.1007/s11356-014-2719-9
RESEARCH ARTICLE
Chromium phytoextraction from tannery effluent-contaminated soil by Crotalaria juncea infested with Pseudomonas fluorescens Anamika Agarwal & Harminder Pal Singh & J. P. N. Rai
Received: 8 October 2013 / Accepted: 28 February 2014 / Published online: 22 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The aim of present study was to remediate chromium (Cr)-contaminated soil by Crotalaria juncea in the presence of Pseudomonas fluorescens. Inoculation of P. fluorescens in pot soil grown with C. juncea significantly increased (~2-fold) the water-soluble (Ws) and exchangeable (Ex) Cr contents in contaminated soil under greenhouse condition. It also enhanced the chlorophyll content by 92 % and plant biomass by 99 % as compared to the uninoculated C. juncea plant. The analysis showed that root and shoot uptake of Cr in C. juncea inoculated by P. fluorescens was 3.08- and 2.82-fold, respectively. This research showed that the association of C. juncea and P. fluorescens could be a promising technology for increasing the soil Cr bioavailability and plant growth for successful phytoextraction of Cr from the contaminated soil. Keywords Tannery effluents . Cr-phytoextraction . Cr-bioavailabilty . Cr-uptake . Biomass accumulation
Introduction Tannery industry is one of the major sources of pollution in India, as it releases large quantity of chromium (Cr)- and salt-
Responsible editor: Elena Maestri A. Agarwal : H. P. Singh (*) Department of Environmental Studies, Panjab University, Chandigarh 160014, India e-mail: [email protected] H. P. Singh e-mail: [email protected] J. P. N. Rai Department of Environmental Science, G.B. Pant University of Agriculture and Technology, Pantnagar, India
rich effluents (Mahimairaja and Shenbagavalli 2010). Chromium occurs in the aqueous system as both trivalent and hexavalent forms. The latter is more toxic, carcinogenic, mutagenic and terratogenic to the living beings (Singh et al. 2013). The traditional technologies used for the remediation of a Cr(VI)-contaminated environment are based on physical and chemical approaches, which are expensive, require large amount of energy, and also generate hazardous byproducts (Gonzalez et al. 2003). Thus, biological approaches employing microbes have been considered as an alternative bioremediation strategy for reduction of toxic Cr(VI) contamination in groundwater and soil (Tripathi and Garg 2010). Bioreduction of Cr(VI) can occur directly by microbial metabolism (enzymatic) or indirectly when mediated by bacterial metabolite such as (H2S) (Sultan and Husnain 2000). A number of Cr(VI)-reducing microorganisms, including Pseudomonas, have been reported (Iftikhar et al. 2007). Although phytoremediation involving phytoextraction, phytovolatilisation and/or phytostabilisation (Suresh and Ravishankar 2004) is time consuming (Nascimento and Xing 2006), it requires acceleration by exploiting rhizospheric microflora and their modifications. This calls for microbial consortium approach having selection and engineering of microorganisms with capabilities for pollutant degradation, beneficial effects on the phytoremediator crops or modifying effects on pollutant bioavailability (Wenzel 2009). Microorganisms including bacteria, algae, fungi and yeast are found to be capable of efficiently accumulating heavy metal ions (Gadd 2010). Microbial populations in metalpolluted environments adapt to toxic concentrations of heavy metals and become metal resistant (Prasenjit and Sumathi 2005) and bioaccumulate the metals. For example, species of Aspergillus (Congeevaram et al. 2007), Pseudomonas (Durga Devi et al. 2012), Bacillus, and Staphylococcus (Sharma and Adholeya 2012; Mythili and Karthikeyan 2011) have been reported as efficient Cr reducers. Such
Environ Sci Pollut Res (2014) 21:7938–7944
organisms prevent metal entry in to the cell by mobilizing/ immobilizing heavy metals. Plant growth-promoting bacteria have the ability to promote plant growth and biomass and increase tolerance to toxic heavy metals by nitrogen fixation, phosphate solubilisation, sulphate oxidation and synthesis of phytohormones (Zhuang et al. 2007) and induced systemic resistance (ISR) mechanism in the plant (Pal and Rai 2010, Saraswat and Rai 2011). Rhizospheric microflora also assists the phytoremediation process by increasing the availability and mobility of heavy metals to plants through acidification, redox changes and releasing of chelating agents such as siderophores (Khan 2005, Wani et al. 2008). Moreover, members of the family Brassicaceae along with mycorrhizal fungi and rhizospheric bacteria have been found to bioremediate a variety of industrial effluents by sequestration of several heavy metals (Chauhan and Rai 2009, Fuloria et al. 2009). Likewise, legume species have been demonstrated to possess high metal tolerance and grow well in metalcontaminated soil without any negative effect on symbiotic N2 fixation (Chen et al. 2003). Crotalaria juncea, a leguminous plant, has been found to grow well in tannery effluentcontaminated soil. The present study was conducted to assess the Cr-phytoremediation potential of C. juncea, a legume, by augmenting it with Pseudomonas fluorescens in controlled conditions with a view to its possible application in metal decontamination of nitrogen poor soil.
Materials and methods
7939
Isolation and identification of Cr-resistant bacteria For isolation of Cr-resistant bacteria, soil samples were serially diluted in sterile phosphate buffer (pH 7.2) and spread on to King’s B medium, a selective one (Kings et al. 1954) amended with 50 μg/ml of Cr(VI). A filter-sterilized solution of K2Cr2O7 was used as the source of Cr(VI), which was added to the sterile molten nutrient agar to prevent problems associated with autoclaving chromate containing solutions. The inoculated plates were incubated at 37 °C for 48 h and afterwards observed for the growth of microorganisms. After incubation, well-separated individual colonies with yellow-green and white pigments were detected by viewing under UV light. The most dominant strain was processed for its identification by Gram staining (Holt et al. 1994), fluorescence, microscopic, and biochemical tests, i.e. catalase, oxidase, MR-VP, indole, citrate, urease, nitrate reduction and fermentation of various sugars, were applied (Table 1) to the isolate according to Bergey’s Manual of Systematic Bacteriology (Holt et al. 1994). Based on the observation, the genus to which the isolates belong was similar as P. fluorescens (Mythili and Karthikeyan 2011) with strain pf 27. Identification and phylogenetic characterization was done by 16S rRNA partial sequence analysis to identify the bacterial isolate pf 27 and deposited in gene bank database, and the accession number is pf 295578. Homology search using BLAST revealed 97 % similarity of sequence with that of gene sequence of P. fluorescens (pf 27) have given final genetic relationship of this bacterial isolate with several other Pseudomonas species and thus the isolate was named P. fluorescens (AA 12).
Soil sampling and analysis Cr extraction from bacterial supernatant containing soil The soil samples were collected from the tannery waste including effluent disposal site at the longitudinal distance of about 10 and 1,000 m of a functional leather processing factory at the Unnao district of the Uttar Pradesh (India) and designated as polluted soil (PS) and non-polluted soil (NPS), respectively. The samples were air-dried for 2 weeks and sieved through a 2-mm wire mesh. Physicochemical properties of soil samples such as pH (using digital pH meter), EC (using electrical conductivity meter), and organic matter (Walkley and Black 1934) were analyzed. Chromium content was determined using an atomic absorption spectrophotometer, AAS (Model: Sens AA dual from GBC Scientific Equipment PTY LTD, Australia) after digesting 0.5 g of dried samples with 15 ml of HNO3, H2SO4 and HClO4 in a 5:1:1 ratio at 80 °C (Page et al. 1982) and filtered through Whatman #42 filter paper, followed by dilution up to 50 ml with triply microfiltered water. The water-soluble (Ws) metal content was assessed after adding distilled water in a ratio of 1:1 (w/v) and the exchangeable (Ex) fraction by extraction with 0.01 mol/l CaCl2 (1:10, w/v, soil: CaCl2), followed by acidification with HNO3 and using AAS (McGrath and Cunliffe 1985).
The ability of P. fluorescens AA 12 to enhance water-soluble (Ws) and exchangeable (Ex) fractions of Cr in polluted soil (PS) was tested under in vitro conditions, as these fractions are most readily available for plant uptake (Datta and Sarkar 2004). For this, the strain was grown overnight in 500-ml Erlenmeyer flasks containing 300 ml of sterilized nutrient broth in a shaker at 150 rpm at 30 °C. Another flask containing sterile nutrient broth was uninoculated as a sterile (axenic) control. From each flask, 50 ml of medium was centrifuged at 8,000g for 15 min, the supernatant was decanted and vacuum-filtered through a sterile filter (0.22 μm pore size). The ability of the filtrate to extract metal from the soil was determined after 0, 12, 24, 36, 48 and 60 h in triplicate. The soil suspensions were centrifuged at 4,000g for 15 min, filtered and acidified with HNO3 to determine Ws and Ex metal fractions using AAS. Pot experiment The soil of PS and NPS were separately mixed with fertilizers (0.44 g of urea and 0.88 g KH2PO4 kg−1 of soil) and filled in
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Environ Sci Pollut Res (2014) 21:7938–7944
20-cm diameter plastic pots. Seeds of C. juncea obtained from the Crop Research Centre of G.B. Pant University of Agriculture and Technology, Pantnagar (India), were first sterilized with a mixture of ethanol and 30 % H2O2 (1:1, v/v) for 10 min and then washed with sterile water to remove surface contaminations. Ten seeds per pot (each having 4 kg of soil) were allowed to germinate for 15 days, after that, the seedlings were thinned to three per pot. Pot soil moisture was maintained as 70 % WHC. Each experiment (inoculated with Pseudomonas and uninoculated) was replicated three times, and pots were maintained in a greenhouse at 25±4 °C. The plant harvesting was done at different times, i.e. 30, 60 and 90 days after planting. P. fluorescens inoculation in soil P. fluorescens was grown in sterilized nutrient broth. Cells in exponential phase were collected after 16 h, followed by centrifugation at 16,099g for 10 min, and the pellets were then washed twice with sterile-distilled water. Bacterial inoculum was prepared by re-suspending pelleted cells in steriledistilled water to get an inoculum density of ca. 108 cfu/ml using dilution plate technique. The soil was inoculated with live bacterial suspension (10 ml/pot) after 10 days of seedling emergence, whereas in uninoculated pots, an equal amount of sterile distilled water was sprayed.
Table 2 Chemical characteristics of polluted soil (PS) and non-polluted soil (NPS) as affected by tannery industry effluent Parameters
PS
pH EC (ds/m) Soil moisture (%) Organic carbon (%)
8.65±0.02 0.48±0.025 28.07±0.07 1.82±0.01
7.05±0.035 0.31±0.015 15.5±0.171 3.10±0.061
Total Cr (mg/kg) Ws Cr (mg/kg) Ex Cr (mg/kg)
92.77±1.243 2.48±0.03 2.70±0.025
21.38±0.055 0.48±0.015 0.36±0.02
Values are presented as mean±SE of three replicates
and dried at 105 °C for biomass determination. Total leaf chlorophyll was estimated by the Arnon method (Arnon 1949). To assess the nitrogen-fixing ability of C. juncea, the number of root nodules and dry weight of nodules were estimated after 90 days of seedling emergence. To estimate nodulation, the roots were carefully washed and nodules were detached, counted, oven-dried at 80 °C, and weighed. The milled plant material (0.5 g per treatment) was digested with a mixture of concentrated HCl: HNO3 (4:1, v/v) (Allen et al. 1986) and analyzed for Cr using AAS. After each harvest, pot soil was also analyzed for Ws and Ex fractions of Cr.
a
Plant and soil analyses
P. fluorescens uninocluated P. fluorescens inocluated
Isolated Bacterial Strain
Catalase activity Oxidase activity Indole test Methyl red test Voges–Proskauer test Citrate utilization Glucose fermentation Lactose fermentation Sucrose fermentation Mannitol fermentation Urease activity Nitrite reduction Gram staining Fluorescence Identified species
+ + − − − + A − − − − + − + Pseudomonas fluorescens
+ positive, − negative, A acid production
(mg kg-1)
4 3 2 1 0
0
12
24
36
48
60
48
60
Culture time (h)
b
8
6
(mg kg-1)
Biochemical Test
Exchangeable (Ex) Cr
Table 1 Biochemical characterization of bacterial isolate
Water soluble (Ws) Cr
5
Three plants per treatment were harvested after 30, 60 and 90 days of seedling emergence. After removing the plants, the roots and shoots were separated, washed with deionised water,
NPS
4
2
0 0
12
24
36
Culture time (h)
Fig. 1 Extraction of water-soluble (a) and exchangeable (b) Cr from metal-contaminated soil inoculated by Pseudomonas fluorescens under in vitro conditions (vertical bars represent SE of means, n=3)
Environ Sci Pollut Res (2014) 21:7938–7944
a
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2
Bacterial efficiency for metal solubilisation
1
Fig. 2 Chlorophyll content (a) and biomass content (b) of Crotalaria juncea grown in polluted soil (PS) and non-polluted soil (NPS) under Pseudomonas fluorescens-inoculated and Pseudomonas fluorescens-uninoculated conditions (vertical bars represent SE of means, n=3)
P. fluorescens inoculation for 24 h resulted in a significant (P≤ 0.05) increase in soil Ws and Ex metal fractions (Fig. 1). Maximum Ws Cr (3.86 mg kg−1) was recorded after 60 h of bacterial growth. The values increased by 66 % as compared to the control. However, the maximum Ex Cr (6.94 mg kg−1) increased by 45 % over the uninoculated control. In general, Ex Cr content was higher than the Ws fraction in both P. fluorescens-inoculated as well as P. fluorescens-uninoculated soils. This depends on the texture and other physicochemical properties such as organic matter, pH and cation exchange capacity of soil (Imperato et al. 2003). Microorganisms can increase solubility and change speciation of metals/metalloids through the production of organic ligands via microbial decomposition of soil organic matter and exudation of metabolites (e.g. organic acids) and microbial siderophores that can complex cationic metals or desorb anionic species by ligand exchange (Gadd 2004). Our results are corroborated by an earlier study reporting conspicuous increase in Cr solubility in soil inoculated with P. putida (Huang et al. 2005).
Statistical analyses
Influence of P. fluorescens inoculation on plant growth
Experimental data were presented as mean values±standard error (SE). To verify the statistical significance of difference among treatments, the data were analyzed using Student’s t test as available in the SPSS statistical package (ver. 11) and were expressed at P≤0.05 and P≤0.01.
Cr-toxicity in plants is observed at multiple levels from reduced yield to effects on leaf and root growth, through the inhibition of enzymatic activities and mutagenesis (Singh et al. 2013). Plants grown in PS for 90 days showed toxicity symptoms in terms of a reduction of biomass and chlorophyll contents by 50 and 75 %, respectively, compared to the plant grown in NPS (Fig. 2 a, b). Cr(VI) toxicity causes brown coloration of roots, stunts root growth, impairs root hair formation and enhances lateral root growth (Mallick et al. 2010). Decrease in leaf size and area, chlorosis, necrosis and wilting were observed in Brassica oleracea (Chatterjee and Chatterjee 2000) and Brassica juncea (Pandey et al. 2005) under Cr-stress. Various studies have reported a decrease in total chlorophyll,
Total Chlorophyll (mg g-1 FW)
3
soil, NPS (Table 2). Percent organic carbon and Cr contents were significantly greater (P≤0.01) in PS than in NPS. It was largely due to the use of large amount of salts in tanning process (Mondal et al. 2005). Further, the water-soluble Cr in PS was ~5.2-fold higher than that in NPS, whilst the corresponding increase in Ex fraction was ~7.5-fold (Table 2).
PS uninoculated PS inoculated NPS uninoculated NPS inoculated
5 4
0 30
60
90
Duration (Days)
Biomass (g plant-1)
b
6 5 4 3 2 1 0 30
60
90
Duration (Days)
Results and discussion Soil characteristics Cr-contaminated soil (PS) was found to have higher pH, higher EC values and higher moisture content than the non-polluted
Table 3 Number of root nodules (plant−1) and nodules dry weight (mg plant−1) in Pseudomonas fluorescens-inoculated/Pseudomonas fluorescensuninoculated Crotalaria juncea plants in polluted soil (PS) and non-polluted soil (NPS) after 90 days NPS
PS
Treatment
Nodule number. plant−1
Nodule dry weight (mg plant−1)
Nodule number. plant−1
Nodule dry weight (mg plant−1)
Uninoculated Inoculated
23.90±1.60 26.50±1.77
18.70±1.23 21.80±1.61
20.30±1.32 21.89±1.47
15.70±1.02 16.12±1.26
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Environ Sci Pollut Res (2014) 21:7938–7944
Cr content (mg kg-1 DW)
500
Root uninoculated Root inoculated Shoot uninoculated Shoot inoculated
to enhance plant growth through various mechanisms such as reduction of ethylene production, (thus allowing plants to develop longer roots and establish better during early stages of growth, Glick et al. 1998), producing specific enzyme activity (Khan 2005), and supply of bioavailable phosphorus and other trace element for plant uptake and production of phytohormones such as auxins, cytokinins and gibberellins (Glick et al. 1995). Substantial increase in the bioavailability of metals in soil and plant biomass led to increased Craccumulation in the plant under P. fluorescens-inoculated conditions, signifying the synergistic role of plant and bacteria in Cr-uptake, as has also been reported by Saraswat and Rai (2011).
400 300 200 100 0
30
60
90
Duration (Days)
Fig. 3 Amount of Cr in roots and shoots of Crotalaria juncea grown in polluted soil under Pseudomonas fluorescens-inoculated and Pseudomonas fluorescens-uninoculated conditions (vertical bars represent SE of means, n=3)
Bioconcentration factor (BCF) and translocation factor (TF) BCF and TF (ratio of metal concentration in roots to soil and shoots to roots, respectively) are an index of phytoremediation capacity of plants. BCF values of >1 for metals indicate a better phytoextraction potential of a plant, whereas TF values of 1, which indicated the good phytoextraction potential of C. juncea for Cr. In uninoculated conditions, a maximum BCF was found for Cr (9.62), which increased significantly (P≤0.05) due to bacterial inoculation by 42 % through increasing metal bioavailability to plants (Saraswat and Rai 2011). Further, TF values of
RESEARCH ARTICLE
Chromium phytoextraction from tannery effluent-contaminated soil by Crotalaria juncea infested with Pseudomonas fluorescens Anamika Agarwal & Harminder Pal Singh & J. P. N. Rai
Received: 8 October 2013 / Accepted: 28 February 2014 / Published online: 22 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The aim of present study was to remediate chromium (Cr)-contaminated soil by Crotalaria juncea in the presence of Pseudomonas fluorescens. Inoculation of P. fluorescens in pot soil grown with C. juncea significantly increased (~2-fold) the water-soluble (Ws) and exchangeable (Ex) Cr contents in contaminated soil under greenhouse condition. It also enhanced the chlorophyll content by 92 % and plant biomass by 99 % as compared to the uninoculated C. juncea plant. The analysis showed that root and shoot uptake of Cr in C. juncea inoculated by P. fluorescens was 3.08- and 2.82-fold, respectively. This research showed that the association of C. juncea and P. fluorescens could be a promising technology for increasing the soil Cr bioavailability and plant growth for successful phytoextraction of Cr from the contaminated soil. Keywords Tannery effluents . Cr-phytoextraction . Cr-bioavailabilty . Cr-uptake . Biomass accumulation
Introduction Tannery industry is one of the major sources of pollution in India, as it releases large quantity of chromium (Cr)- and salt-
Responsible editor: Elena Maestri A. Agarwal : H. P. Singh (*) Department of Environmental Studies, Panjab University, Chandigarh 160014, India e-mail: [email protected] H. P. Singh e-mail: [email protected] J. P. N. Rai Department of Environmental Science, G.B. Pant University of Agriculture and Technology, Pantnagar, India
rich effluents (Mahimairaja and Shenbagavalli 2010). Chromium occurs in the aqueous system as both trivalent and hexavalent forms. The latter is more toxic, carcinogenic, mutagenic and terratogenic to the living beings (Singh et al. 2013). The traditional technologies used for the remediation of a Cr(VI)-contaminated environment are based on physical and chemical approaches, which are expensive, require large amount of energy, and also generate hazardous byproducts (Gonzalez et al. 2003). Thus, biological approaches employing microbes have been considered as an alternative bioremediation strategy for reduction of toxic Cr(VI) contamination in groundwater and soil (Tripathi and Garg 2010). Bioreduction of Cr(VI) can occur directly by microbial metabolism (enzymatic) or indirectly when mediated by bacterial metabolite such as (H2S) (Sultan and Husnain 2000). A number of Cr(VI)-reducing microorganisms, including Pseudomonas, have been reported (Iftikhar et al. 2007). Although phytoremediation involving phytoextraction, phytovolatilisation and/or phytostabilisation (Suresh and Ravishankar 2004) is time consuming (Nascimento and Xing 2006), it requires acceleration by exploiting rhizospheric microflora and their modifications. This calls for microbial consortium approach having selection and engineering of microorganisms with capabilities for pollutant degradation, beneficial effects on the phytoremediator crops or modifying effects on pollutant bioavailability (Wenzel 2009). Microorganisms including bacteria, algae, fungi and yeast are found to be capable of efficiently accumulating heavy metal ions (Gadd 2010). Microbial populations in metalpolluted environments adapt to toxic concentrations of heavy metals and become metal resistant (Prasenjit and Sumathi 2005) and bioaccumulate the metals. For example, species of Aspergillus (Congeevaram et al. 2007), Pseudomonas (Durga Devi et al. 2012), Bacillus, and Staphylococcus (Sharma and Adholeya 2012; Mythili and Karthikeyan 2011) have been reported as efficient Cr reducers. Such
Environ Sci Pollut Res (2014) 21:7938–7944
organisms prevent metal entry in to the cell by mobilizing/ immobilizing heavy metals. Plant growth-promoting bacteria have the ability to promote plant growth and biomass and increase tolerance to toxic heavy metals by nitrogen fixation, phosphate solubilisation, sulphate oxidation and synthesis of phytohormones (Zhuang et al. 2007) and induced systemic resistance (ISR) mechanism in the plant (Pal and Rai 2010, Saraswat and Rai 2011). Rhizospheric microflora also assists the phytoremediation process by increasing the availability and mobility of heavy metals to plants through acidification, redox changes and releasing of chelating agents such as siderophores (Khan 2005, Wani et al. 2008). Moreover, members of the family Brassicaceae along with mycorrhizal fungi and rhizospheric bacteria have been found to bioremediate a variety of industrial effluents by sequestration of several heavy metals (Chauhan and Rai 2009, Fuloria et al. 2009). Likewise, legume species have been demonstrated to possess high metal tolerance and grow well in metalcontaminated soil without any negative effect on symbiotic N2 fixation (Chen et al. 2003). Crotalaria juncea, a leguminous plant, has been found to grow well in tannery effluentcontaminated soil. The present study was conducted to assess the Cr-phytoremediation potential of C. juncea, a legume, by augmenting it with Pseudomonas fluorescens in controlled conditions with a view to its possible application in metal decontamination of nitrogen poor soil.
Materials and methods
7939
Isolation and identification of Cr-resistant bacteria For isolation of Cr-resistant bacteria, soil samples were serially diluted in sterile phosphate buffer (pH 7.2) and spread on to King’s B medium, a selective one (Kings et al. 1954) amended with 50 μg/ml of Cr(VI). A filter-sterilized solution of K2Cr2O7 was used as the source of Cr(VI), which was added to the sterile molten nutrient agar to prevent problems associated with autoclaving chromate containing solutions. The inoculated plates were incubated at 37 °C for 48 h and afterwards observed for the growth of microorganisms. After incubation, well-separated individual colonies with yellow-green and white pigments were detected by viewing under UV light. The most dominant strain was processed for its identification by Gram staining (Holt et al. 1994), fluorescence, microscopic, and biochemical tests, i.e. catalase, oxidase, MR-VP, indole, citrate, urease, nitrate reduction and fermentation of various sugars, were applied (Table 1) to the isolate according to Bergey’s Manual of Systematic Bacteriology (Holt et al. 1994). Based on the observation, the genus to which the isolates belong was similar as P. fluorescens (Mythili and Karthikeyan 2011) with strain pf 27. Identification and phylogenetic characterization was done by 16S rRNA partial sequence analysis to identify the bacterial isolate pf 27 and deposited in gene bank database, and the accession number is pf 295578. Homology search using BLAST revealed 97 % similarity of sequence with that of gene sequence of P. fluorescens (pf 27) have given final genetic relationship of this bacterial isolate with several other Pseudomonas species and thus the isolate was named P. fluorescens (AA 12).
Soil sampling and analysis Cr extraction from bacterial supernatant containing soil The soil samples were collected from the tannery waste including effluent disposal site at the longitudinal distance of about 10 and 1,000 m of a functional leather processing factory at the Unnao district of the Uttar Pradesh (India) and designated as polluted soil (PS) and non-polluted soil (NPS), respectively. The samples were air-dried for 2 weeks and sieved through a 2-mm wire mesh. Physicochemical properties of soil samples such as pH (using digital pH meter), EC (using electrical conductivity meter), and organic matter (Walkley and Black 1934) were analyzed. Chromium content was determined using an atomic absorption spectrophotometer, AAS (Model: Sens AA dual from GBC Scientific Equipment PTY LTD, Australia) after digesting 0.5 g of dried samples with 15 ml of HNO3, H2SO4 and HClO4 in a 5:1:1 ratio at 80 °C (Page et al. 1982) and filtered through Whatman #42 filter paper, followed by dilution up to 50 ml with triply microfiltered water. The water-soluble (Ws) metal content was assessed after adding distilled water in a ratio of 1:1 (w/v) and the exchangeable (Ex) fraction by extraction with 0.01 mol/l CaCl2 (1:10, w/v, soil: CaCl2), followed by acidification with HNO3 and using AAS (McGrath and Cunliffe 1985).
The ability of P. fluorescens AA 12 to enhance water-soluble (Ws) and exchangeable (Ex) fractions of Cr in polluted soil (PS) was tested under in vitro conditions, as these fractions are most readily available for plant uptake (Datta and Sarkar 2004). For this, the strain was grown overnight in 500-ml Erlenmeyer flasks containing 300 ml of sterilized nutrient broth in a shaker at 150 rpm at 30 °C. Another flask containing sterile nutrient broth was uninoculated as a sterile (axenic) control. From each flask, 50 ml of medium was centrifuged at 8,000g for 15 min, the supernatant was decanted and vacuum-filtered through a sterile filter (0.22 μm pore size). The ability of the filtrate to extract metal from the soil was determined after 0, 12, 24, 36, 48 and 60 h in triplicate. The soil suspensions were centrifuged at 4,000g for 15 min, filtered and acidified with HNO3 to determine Ws and Ex metal fractions using AAS. Pot experiment The soil of PS and NPS were separately mixed with fertilizers (0.44 g of urea and 0.88 g KH2PO4 kg−1 of soil) and filled in
7940
Environ Sci Pollut Res (2014) 21:7938–7944
20-cm diameter plastic pots. Seeds of C. juncea obtained from the Crop Research Centre of G.B. Pant University of Agriculture and Technology, Pantnagar (India), were first sterilized with a mixture of ethanol and 30 % H2O2 (1:1, v/v) for 10 min and then washed with sterile water to remove surface contaminations. Ten seeds per pot (each having 4 kg of soil) were allowed to germinate for 15 days, after that, the seedlings were thinned to three per pot. Pot soil moisture was maintained as 70 % WHC. Each experiment (inoculated with Pseudomonas and uninoculated) was replicated three times, and pots were maintained in a greenhouse at 25±4 °C. The plant harvesting was done at different times, i.e. 30, 60 and 90 days after planting. P. fluorescens inoculation in soil P. fluorescens was grown in sterilized nutrient broth. Cells in exponential phase were collected after 16 h, followed by centrifugation at 16,099g for 10 min, and the pellets were then washed twice with sterile-distilled water. Bacterial inoculum was prepared by re-suspending pelleted cells in steriledistilled water to get an inoculum density of ca. 108 cfu/ml using dilution plate technique. The soil was inoculated with live bacterial suspension (10 ml/pot) after 10 days of seedling emergence, whereas in uninoculated pots, an equal amount of sterile distilled water was sprayed.
Table 2 Chemical characteristics of polluted soil (PS) and non-polluted soil (NPS) as affected by tannery industry effluent Parameters
PS
pH EC (ds/m) Soil moisture (%) Organic carbon (%)
8.65±0.02 0.48±0.025 28.07±0.07 1.82±0.01
7.05±0.035 0.31±0.015 15.5±0.171 3.10±0.061
Total Cr (mg/kg) Ws Cr (mg/kg) Ex Cr (mg/kg)
92.77±1.243 2.48±0.03 2.70±0.025
21.38±0.055 0.48±0.015 0.36±0.02
Values are presented as mean±SE of three replicates
and dried at 105 °C for biomass determination. Total leaf chlorophyll was estimated by the Arnon method (Arnon 1949). To assess the nitrogen-fixing ability of C. juncea, the number of root nodules and dry weight of nodules were estimated after 90 days of seedling emergence. To estimate nodulation, the roots were carefully washed and nodules were detached, counted, oven-dried at 80 °C, and weighed. The milled plant material (0.5 g per treatment) was digested with a mixture of concentrated HCl: HNO3 (4:1, v/v) (Allen et al. 1986) and analyzed for Cr using AAS. After each harvest, pot soil was also analyzed for Ws and Ex fractions of Cr.
a
Plant and soil analyses
P. fluorescens uninocluated P. fluorescens inocluated
Isolated Bacterial Strain
Catalase activity Oxidase activity Indole test Methyl red test Voges–Proskauer test Citrate utilization Glucose fermentation Lactose fermentation Sucrose fermentation Mannitol fermentation Urease activity Nitrite reduction Gram staining Fluorescence Identified species
+ + − − − + A − − − − + − + Pseudomonas fluorescens
+ positive, − negative, A acid production
(mg kg-1)
4 3 2 1 0
0
12
24
36
48
60
48
60
Culture time (h)
b
8
6
(mg kg-1)
Biochemical Test
Exchangeable (Ex) Cr
Table 1 Biochemical characterization of bacterial isolate
Water soluble (Ws) Cr
5
Three plants per treatment were harvested after 30, 60 and 90 days of seedling emergence. After removing the plants, the roots and shoots were separated, washed with deionised water,
NPS
4
2
0 0
12
24
36
Culture time (h)
Fig. 1 Extraction of water-soluble (a) and exchangeable (b) Cr from metal-contaminated soil inoculated by Pseudomonas fluorescens under in vitro conditions (vertical bars represent SE of means, n=3)
Environ Sci Pollut Res (2014) 21:7938–7944
a
7941
2
Bacterial efficiency for metal solubilisation
1
Fig. 2 Chlorophyll content (a) and biomass content (b) of Crotalaria juncea grown in polluted soil (PS) and non-polluted soil (NPS) under Pseudomonas fluorescens-inoculated and Pseudomonas fluorescens-uninoculated conditions (vertical bars represent SE of means, n=3)
P. fluorescens inoculation for 24 h resulted in a significant (P≤ 0.05) increase in soil Ws and Ex metal fractions (Fig. 1). Maximum Ws Cr (3.86 mg kg−1) was recorded after 60 h of bacterial growth. The values increased by 66 % as compared to the control. However, the maximum Ex Cr (6.94 mg kg−1) increased by 45 % over the uninoculated control. In general, Ex Cr content was higher than the Ws fraction in both P. fluorescens-inoculated as well as P. fluorescens-uninoculated soils. This depends on the texture and other physicochemical properties such as organic matter, pH and cation exchange capacity of soil (Imperato et al. 2003). Microorganisms can increase solubility and change speciation of metals/metalloids through the production of organic ligands via microbial decomposition of soil organic matter and exudation of metabolites (e.g. organic acids) and microbial siderophores that can complex cationic metals or desorb anionic species by ligand exchange (Gadd 2004). Our results are corroborated by an earlier study reporting conspicuous increase in Cr solubility in soil inoculated with P. putida (Huang et al. 2005).
Statistical analyses
Influence of P. fluorescens inoculation on plant growth
Experimental data were presented as mean values±standard error (SE). To verify the statistical significance of difference among treatments, the data were analyzed using Student’s t test as available in the SPSS statistical package (ver. 11) and were expressed at P≤0.05 and P≤0.01.
Cr-toxicity in plants is observed at multiple levels from reduced yield to effects on leaf and root growth, through the inhibition of enzymatic activities and mutagenesis (Singh et al. 2013). Plants grown in PS for 90 days showed toxicity symptoms in terms of a reduction of biomass and chlorophyll contents by 50 and 75 %, respectively, compared to the plant grown in NPS (Fig. 2 a, b). Cr(VI) toxicity causes brown coloration of roots, stunts root growth, impairs root hair formation and enhances lateral root growth (Mallick et al. 2010). Decrease in leaf size and area, chlorosis, necrosis and wilting were observed in Brassica oleracea (Chatterjee and Chatterjee 2000) and Brassica juncea (Pandey et al. 2005) under Cr-stress. Various studies have reported a decrease in total chlorophyll,
Total Chlorophyll (mg g-1 FW)
3
soil, NPS (Table 2). Percent organic carbon and Cr contents were significantly greater (P≤0.01) in PS than in NPS. It was largely due to the use of large amount of salts in tanning process (Mondal et al. 2005). Further, the water-soluble Cr in PS was ~5.2-fold higher than that in NPS, whilst the corresponding increase in Ex fraction was ~7.5-fold (Table 2).
PS uninoculated PS inoculated NPS uninoculated NPS inoculated
5 4
0 30
60
90
Duration (Days)
Biomass (g plant-1)
b
6 5 4 3 2 1 0 30
60
90
Duration (Days)
Results and discussion Soil characteristics Cr-contaminated soil (PS) was found to have higher pH, higher EC values and higher moisture content than the non-polluted
Table 3 Number of root nodules (plant−1) and nodules dry weight (mg plant−1) in Pseudomonas fluorescens-inoculated/Pseudomonas fluorescensuninoculated Crotalaria juncea plants in polluted soil (PS) and non-polluted soil (NPS) after 90 days NPS
PS
Treatment
Nodule number. plant−1
Nodule dry weight (mg plant−1)
Nodule number. plant−1
Nodule dry weight (mg plant−1)
Uninoculated Inoculated
23.90±1.60 26.50±1.77
18.70±1.23 21.80±1.61
20.30±1.32 21.89±1.47
15.70±1.02 16.12±1.26
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Environ Sci Pollut Res (2014) 21:7938–7944
Cr content (mg kg-1 DW)
500
Root uninoculated Root inoculated Shoot uninoculated Shoot inoculated
to enhance plant growth through various mechanisms such as reduction of ethylene production, (thus allowing plants to develop longer roots and establish better during early stages of growth, Glick et al. 1998), producing specific enzyme activity (Khan 2005), and supply of bioavailable phosphorus and other trace element for plant uptake and production of phytohormones such as auxins, cytokinins and gibberellins (Glick et al. 1995). Substantial increase in the bioavailability of metals in soil and plant biomass led to increased Craccumulation in the plant under P. fluorescens-inoculated conditions, signifying the synergistic role of plant and bacteria in Cr-uptake, as has also been reported by Saraswat and Rai (2011).
400 300 200 100 0
30
60
90
Duration (Days)
Fig. 3 Amount of Cr in roots and shoots of Crotalaria juncea grown in polluted soil under Pseudomonas fluorescens-inoculated and Pseudomonas fluorescens-uninoculated conditions (vertical bars represent SE of means, n=3)
Bioconcentration factor (BCF) and translocation factor (TF) BCF and TF (ratio of metal concentration in roots to soil and shoots to roots, respectively) are an index of phytoremediation capacity of plants. BCF values of >1 for metals indicate a better phytoextraction potential of a plant, whereas TF values of 1, which indicated the good phytoextraction potential of C. juncea for Cr. In uninoculated conditions, a maximum BCF was found for Cr (9.62), which increased significantly (P≤0.05) due to bacterial inoculation by 42 % through increasing metal bioavailability to plants (Saraswat and Rai 2011). Further, TF values of
