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Abstract

Fluoride (F) contamination is a worldwide problem which has severe consequences on human and animal health. Physiochemical characteristics of F-polluted soil in different sites (Banasthali, Rajasthan, India) were tested. Soil pH (7 - 9.3), E.C (2.1 - 6.1 dSm^-1), O.C (0.30% - 0.60%), N (11.4 - 53.4), P (3.3 - 5.8), K (106 - 117), S (1.3 - 8.3), Ca (11.7 - 14.2), Mg (1.8 - 11.7 kgha^-1) and total Fe (3.6 - 5.6), Mn (1 - 2.4), Zn (0.30 - 2.6), Cu (0.70 - 4.1) and total F contents (9.6 - 11.0 mgkg^-1) were observed. Prosopis juliflora is a hyperaccumulator plant, known and suitable species for phytoremediation of F polluted soil. A repeat experiment for confirmation using F contaminated soil with amendments of Pseudomonas fluorescence (P.F) and Pseudomonas aeruginosa (P.A) were applied to soil in small scale for 120 days. Moreover, total plant biomass increase (67.73% - 74.33%), chlorophyll reduced (50% - 34.67%) and mineral contents enhanced (46.38% - 58.68%) at 100 mgkg^-1 were found as compared to the control. These two strains, increased bioaccumulation factor (BF) 0.22 - 2.89 and translocation factor (TF) 0.63 - 1.08 of plant, accumulated high amount of F in root. Comparative results (biomass, chlorophyll, mineral content, TF and BF) between P.F and P.A indicated that applications of Pseudomonas fluorescence significantly (P ≤ 0.05) impacted on the study of P. juliflora tolerance capacity. Results showed P. juliflora has good tolerance capacity when soil F concentration at 100 mg kg^-1 with P.F - 108 cfu ml^-1kg^-1 of soil. In conclusion, Pseudomonas fluorescence identified in our study may be potentially used for phytoremediation purpose in field scale.

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Keywords

Fluoride; Phytoremediation; Prosopis juliflora; Pseudomonas fluorescence; Pseudomonas aeruginosa

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Introduction

Soil pollution by Fluoride (F) non-metal is one of the main worldwide problems. Several reports suggest that Fluoride (F) is an essential element for the normal growth of plants and in higher concentration, it is toxic for plants [1]. Seed germination and early seedling growth are important phases for the successful growth and survival of plants and these physiological parameters of plants are affected by F stress [2]. Several physiological and biochemical processes are known to be affected by F such as chlorosis and necrosis of leaf, low nutrient uptake, reduction of plant biomass, and enzymatic activities [3]. Higher soil salinity increases the catalase, peroxidase and superoxidase activity among tolerant and sensitive varieties of plant [4]. The relationship between antioxidants and salinity indicate that O_-2 radical and H₂O₂ could play an important role in the mechanisms system [5].

Fluoride pollution is spreading all over the world causing India to severely suffer from its effects [6]. According to survey, high concentration of F in drinking water has been found in South and North American countries (Italy, Holland, Mexico and Spain) [7]. In India, 17 out of 32 states are critically affected by high F concentration [8]. A state named Rajasthan in India, having arid to semi-arid environment, is severely affected from high F concentration. In India, about 20% of F concentration were found in the household water supply; out of these 10% was found in Rajasthan [9]. According to Saini et al. total F content in soil was higher level than normal ones Krashi Vigyan Kendra farm (127.56 μgg^-1) and Banasthali (679.63 μgg^-1) Rajasthan, India [10].

The conventional technologies (adsorption & biosorption, ionexchange, electrocoagulation, flotation and reverse osmosis) are too expensive so it is important to develop eco-friendly and more effective method to decontaminate soils. Recently plants using for F contamination has gained importance. Phytoremediation is low cost and effective technology used for degradation of contaminants [11]. The cost for conventional technology expected is “US$ 100,000 and 10,00000 per ha” [12], whereas the cost of phytoremediation was “US$ 60,000 and 100,000 per ha” [13]. Microorganisms are able to survive in extreme cold, heat and desert, salt conditions due to their potentially produced enzymatic activities, which allow to converting hazardous compounds into harmless compounds [14,15]. Different microorganism evolved mechanism for resistance to metal toxicity and adapted to live in the presence of soil pollutants [16,17]. Soil bacteria that were resistant to metals (Zn, Cu, and Pb) and found that a P-solubilizer (Bacillus megaterium HKP^-1) and a K-solublizer (Bacillus mucilaginosus HKK^-1) had a high resistance to heavy metal toxicity to Zn (250 mgl^-1), Cu (100 mgl^-1), and Pb (300 mgl^-1) due to formation of endospores which are capable to lived in extreme condition [18]. Pseudomonas aeruginosa strain Wl-1 could tolerate up to 0.6 mM lead nitrate and also accumulated lead at 26.5 mgg^-1 of dry cell biomass [19]. Bioremediation technology which is generally accepted and carried out on contaminated sites without affecting soil fertility metabolic pathway in microbes and is consequently a potential threats to the human health and also prevent environment from different variety of contaminants. Successful phytoremediation depends not only on the interaction of plants root with microbes but also on the bioavailability of heavy metals in soils.

In the present study of leguminous species, Prosopis juliflora hyperaccumulator plant promoted us to assess the potential use of this plant in the reclamation of F contaminated soils [20]. This is also naturally grown in Rajasthan (India) without showing any morphological distortions against the F stress. It is tolerant to very high temperature (45 °C). This tree can grow in different areas and the roots can enter to large depths in the soil. The objective was to investigate (a) soil characteristics of Banasthali, Tonk, Rajasthan, India (b) effect of microbial consortium on plant biomass, chlorophyll, mineral content of plant organs (c) check efficacy of phytoremediation in small scale of pot experiment under given microbial treatment.

Random soil samples were collected from a depth of 0-10 cm from nearby areas of Braham Mandir, Botanical Garden and Krashi Vigyan Kendra regions of Banasthali University (26°60’ N 75°54’ E) Tonk, Rajasthan, India. Samples were stored in cold temperature at 4 °C and brought to a soil testing laboratory. The soil sample were analyzed for pH, electrical conductivity (E.C), organic carbon (C), nitrogen (N), phosphorus (P), potassium (K), available DTPA (diethylenetriaminepentaacetic acid) extractable iron (Fe), manganese (Mn), zinc (Zn), copper (Cu) and fluoride (F). The organic carbon was estimated by Walkley method [21]. Available N was estimated by alkaline permanganate method [22]. Available phosphorus was extracted by using Olsen’s reagent and estimated through spectrophotometer after developing the blue color by ascorbic method. Available K was extracted with neutral ammonium acetate and estimated by flame photometer [23]. The DTPA extractable Fe, Mn, Zn and Cu were estimated by using atomic absorption spectrophotometer (AAS). The total F content was estimated by alkali fusion-ion selective method [24].

Prosopis juliflora seeds were collected from Central Arid Zone Research Institute (CAZRI), Jodhpur (Rajasthan) India. Seeds were surface sterilized with 10% H₂SO₄ for 15 min and rinsed with millipore water. Seeds were germinated in plastic pots with 1 kg of sterilized soils. Each pot received six seeds which were placed at 1 cm depth. Pots were rearranged in the greenhouse chamber. The F contaminated soil of pot experiment was performed artificially given NaF of different concentration 25, 50, and 75 and 100 mgkg^-1 in soil. Other set of experiment was subjected to a different type of inoculation: PF 8904 (Pseudomonas fluorescence), and PA 1934 (Pseudomonas aeruginosa) strains obtained from Microbial Tissue Culture Collection (MTCC) Chandigarh together with F concentration. Three replicates were used for each F level inoculation type treatment and without microbial treatment. Bacterial strain suspension (108 cfu ml^-1) in nutrient broth was used for the inoculation, by spraying them on soil surfaces, after 10 days of germination [25]. To the control pots, 10 ml of sterile distilled millipore water was added (Table 1). Plants were harvested after 120 days, washed with tap water and deionized sterile water for further analysis. Plant biomass (roots and shoot length) was determined using measuring scale.Photosynthesis pigments

Fresh experimental plant leaves were taken for the determination of photosynthetic pigments e.g. Total Chlorophyll (total Chl), Chlorophyll a (Chl a) and Chlorophyll b (Chl b). The leaves were cut into tiny pieces; and homogenized 100 mg material in 5 ml of chilled 100% acetone by grinding the leaves of control and stressed seedlings in a pre-cooled mortar and pestle until the powdered materials becomes completely non-green. The homogenate was centrifuged for 5 min at 3000 rpm at 4 °C in a cooling centrifuge. The pellet was discarded and the supernatant was re-adjusted to 5 ml acetone. To 1.6 ml of the supernatant, 0.4 ml double distilled water was added. The chlorophyll content absorbance of the resulting supernatant was recorded at 645 and 663 nm using a double beam UV-Vis spectrophotometer as given below [26]:

Chl a (mg/l) = 12.7 (A663) - 2.69 (A645)

Chl b (mg/l) = 22.9 (A645) - 4.68 (A663)

Total Chl (a and b) (mg/l) = a + b

The above formula expressed the pigments content in mgg^-1 fw of sample.

The plants were harvested after 120 days of inoculation and analyzed for further experiments. The whole plants were washed with distilled water, separate out into root, shoot and leaf than ovendried for 72 h at 70 °C to determine the contents of plant organs. The plant organs (root, shoot and leaf) were analyzed for Nitrogen - N, Phosphorus - P, Potassium - K, Iron - Fe, Manganese - Mn, Zinc - Zn and Cooper - Cu. The samples were analyzed (except N) by taking 1 g materials digesting in di-acid mixture Zn, and (9:4 ratio of nitric and perchloric acids) using standard analytical methods. N was estimated by Kjeldahl method [27], whereas available P was extracted by using Olsen’s reagent and estimated through spectrophotometer after developing the blue color by ascorbic method and K was analyzed by flame photometer [28]. The micronutrients (Fe, Mn, Zn and Cu) were analyzed by using atomic absorption spectrophotometer [28].

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Determination of F

The bioaccumulation and translocation factor were measured according to Niu et al. [29]. A total F content in plant organs and remaining soil was calculated by alkali fusion-ion selective technique [30].

(BF = {F concentration in shoot} / {F concentration in soil})

(TF = {F concentration in shoot} / {F concentration in root})

Statistical analysis were determined according to analysis of variance (ANOVA) test and significant T-test was computed using Standard Statistical Package (SPSS 17.0) to examine difference between each treatment at P ≤ 0.05 and all graphs was prepared by OriginPro 8.5 software.

This paper incorporates the results of present studies on preliminary investigation of physiochemical characteristics of the soil and samples sites areas of Braham Mandir, Botanical Garden and Krashi Vigyan Kendra regions of Banasthali University (26°60’ N 75°54’ E) Tonk Rajasthan, India (Table 2 and Figure 1). The concentrations of macronutrients was higher as per the Banasthali standard for F in soils: pH were (7 - 9.3), E.C (2.1 - 6.1 dSm^-1), O.C (0.30% - 0.60%), total N (11.4 - 53.4), P (3.3 - 5.8), K (106 - 117), S (1.3 - 8.3), Ca (11.7 -4.2) and Mg (1.8 - 11.7) kgha^-1 that were highly alkaline soil in both summer and winter season. Meanwhile the concentrations of micronutrients were of moderate levels as follows: Fe (3.6 - 5.6 mgkg^-1), Mn (1 - 2.4 mgkg^-1), Zn (0.3 - 2.6 mgkg^-1), Cu (0.7 - 4.1 mgkg^-1) and F (9.6 - 11.00 mgkg^-1). F-polluted soil is just because of use phosphorus fertilizers, which contain more than 1.5% Fluorine. Polluted soil generally influences human well being by direct contact with soil or vegetation through inhalation of soil contaminants which have vaporized and move into the food chain. Rajasthan soil is generally highly alkaline, have poor soil structure and low infiltration capacity so they have a hard calcareous layer and nfavorable for agriculture land in desert region. Alkaline soil have trouble with nutrient mobility in soil and total 16 elements should be available within the soil for good plant growth (nitrogen, phosphorus and potassium are the most important elements for plant growth). Growth parameters

The effect of microbes on plant growth was observed under different amendments (P.F and P.A) (Figure 2). Plant did not showed promoting effects on root, shoot length (Figure 2- 1,2) and biomass production (Figure 2- 3,4,5,6) of P. juliflora under given microbes treatment (Figure 2). Although the length and biomass of plant tissues were significantly (p ≤ 0.05) affected at 100 mgkg^-1 F. P. juliflora did not show any toxic symptom when plant under normal stress condition but if we were to go above more than 100 mgkg^-1 than it showed chlorosis and necrosis effect on plant due to more accumulated in tissue part and no translocation of F. Then the P.F and P.A significantly (p ≤ 0.05) increased both root length (12.76 - 23.96 cm) and the root biomass (fresh weight from 0.247 to 0.288 g and dry weight from 0.203 to 0.293 g). P.F performed better as compared to P.A in case of root parameters and P.F promoted shoot length (10.43 to 18.53 cm) and the shoot biomass (fresh weight from 0.269 to 0.349 g and dry weight from 0.195 to 0.322 g) of plants under F stress condition as compared to P.A. However, P.A increased both the root length (17.26 to 19.36 cm) and the root biomass (fresh weight from 0.248 to 0.284 g and dry weight from 0.204 to 0.283 g) in comparison to control. Meanwhile, P.A increased both the shoot length (10.60 to 16.03 cm) and the shoot biomass (fresh weight from 0.259 to 0.346 g and dry weight from 0.260 to 0.291 g) was significantly increased but did not as P.F. However, F stress was decreased root length, shoot length and also affected plant biomass under 100 mgkg^-1 F stresses. However, microbial consortium treatment has increased plant biomass as compared to control due to the bioavailability of mineral content in soil. P.F have tendency to increase F mobility in soil therefore, its formed complex with cation (P, Al, Fe and Cu) because of F have more electronegativity power to interact with other metal. However, these phenomenon’s have important phases for enhanced plant biomass.Chlorophyll contents

The chlorophyll content of P. juliflora leaves were reduced under F stress as compare to control but increased when microbes treatment has given as Chl a from (3.329 mgg^-1 fw), Chl b from (1.101 mgg^-1 fw), total chl a + b (4.431 mgg^-1 fw) and Chl a/b (3.021 mgg^-1 fw) (Figure 3). Heavy metal stresses are known to be interfering in chlorophyll contents through the direct inhibition of enzyme activity which effects on the plant nutrients. The chlorophyll substance was reduced under copper contaminated soil in Triticum aestivum cv. Brassica oleracea [31,32]. In previous research, it was found out that chlorophyll biosynthesis reduced and inhibit chlorophyll. P.F affect on chlorophyll content was highly reduced at the transient phase (stationary state) approximately 90 days. After that crucial period, plant was not affected under given P.F treatment as compared with P.A and F stress at 120 days.Mineral content

The analysis of N, P and K in root, shoot and leaves of P. juliflora were given (Figure 4). The N, P and K amount significantly increased (p = 0.98) when microbial consortium level was increased with enhanced F concentration but decreased when only with F concentration (Figure 4). The N, P and K concentration in root under P.F treatment was found to be (19.78%, 21% and 78%) higher in comparison to the treatment with P.A in Prosopis juliflora. However the N, P and K levels was affected by different F treatments (25, 50, 75 and 100 mgkg^-1), but has only enhanced when the microbes treatment has given to the plant due to the biosynthesis of enzymes which has helpful under stress. Thus Cd can inhibit the activity of enzymes related with phosphorus metabolism. The Increasing chromium concentrations from 10-40 mgkg^-1 in the soil decreased the N, P, K, and Fe contents of Brassica juncea shoot system [33]. Significant decreases in shoot K contents of white lupine (Lupinus albus) with 18 and 45 μM Cd treatments were also reported [34]. However, Fe, Mn, Zn and Cu concentration in root significantly increased (p = 1.00) with microbial treatment. The mean value of micronutrients Fe, Mn, Zn and Cu were analyzed separately (41.98%, 81.76%, 47.30% and 51.48%).The nutrient (N, P and K) present in shoot under P.F treatment was 68.04%, 20.39% and 87.25% which significantly increased value (p = 0.98). The available micronutrients ranges were also enhanced in case of Fe, Mn, Zn and Cu (94.94%, 92.5%, 68.88% and 74.37%). Meanwhile, in leave N, P and K was low ranged (45.86%, 39.06% and 55.55%) as compare to root and shoot due to negative effects of F. The available micronutrient ranges in leave of Fe, Mn, Zn and Cu was 63.33%, 51.97%, 78.12% and 52.59%. Different kinds of nutrients were involved including calcium, zinc, magnesium, phosphorus, potassium, iron and cooper [35]. Heavy metals such as Cu, Zn, Al, Cd, Ni, Hg and As has negative effects on macronutrient and micronutrients [9]. Heavy metals bind to all calcium binding sites on the cell surface at low pH (4.5). It works with calcium absorption and uptake calcium by roots [36]. High amount of heavy metals were exposed to the inhibition of calcium. Inhibit the influx of calcium ion at 100 mM aluminium (Al) [37]. Aluminium ions interfere with the action of guanosine 5’ triphosphate (GTP) binding protein as well as the inhibit calcium ion uptake by binding verapamil-specific channel [38]. The uptake of calcium ions was decreased in beech plants due to the combination of nitrogen and aluminium at high concentration [9]. Under aluminum treatment the concentration of calcium is unaffected in shoots because aluminium enters the plant cells through calcium channel [39]. Potassium channel influx inhibit due to the toxicity of heavy metals like Aluminium. Active pathway involvement of potassium uptake is also inhibited by high concentration of aluminium [40]. Durum wheat is more tolerant against aluminium toxicity but potassium ion concentration was decreased in wheat under aluminium concentration [41]. Concentration decreased of magnesium ions in roots and shoots by increasing the concentration of heavy metals [42]. Iron concentration decreased at a pH of 4.5 when they were treated with different concentration of heavy metals and also significantly affected root growth [43]. Therefore, it may be calcium channel and active pathway interferes with the action of F with P.F. to increase mineral content with specific channel like GTP and Verapamil. Therefore, P.F has positive effect on mineral content. It seems that F has combative interactions especially at high concentrations with mineral content in plant, which this led to decreased mineral level.

The bioaccumulation factor (BF) values were 0.12 - 3.30 at different microbial consortium amendments (P.F and P.A) with various F concentrations. The P. juliflora showed a translocation factor (T.F) which was 0.51 - 1.08 in (Figure 5). The ratio of translocation factor was greater than 1 mean hyperaccumulator plant [44]. The result of the present investigation showed that microbial consortium had high ability and increased the efficiency of F accumulation. Plant growth promoting bacteria deserve a special attention because they can directly improve the phytoremediation process by increasing the metal solubility through altering soil pH, release of chelators (e.g. organic acids, siderphores), and they also help to increased metal mobility for high accumulation in plant organ [45]. 

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Conclusion

Present study demonstrated that Pseudomonas fluorescence improve abilities of P. juliflora phytoremediation efficiency, mineral content and increase plant biomass as compare to the control. Results suggested that F had negative impact on chlorophyll pigments and mineral content of P. juliflora. Fluoride has highly interactions especially at high concentrations with mineral content in plant especially with P, Fe and Cu, which led to decrease the mineral levels in P. juliflora. Based on the results soil nutrients contents are sensitive to F concentration and it should be considered F contaminated soil. P. juliflora need the microbial treatment in soil to decline F toxicity. However, this work could be applied in the field scale and can be commercialized using microbial treatment with F. Pseudomonas fluorescence (P.F) for the use for remediation, counter balance nutrients content and could contribute to sustainable agriculture especially in areas where F contaminated problem in soil and water.

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Acknowledgement

Authors are deeply thankful to Prof. Aditiya Shastri for generous financial support to complete this research work and Bioinformatics Centre of Banasthali University, Newai (Rajasthan) India for computional work and also funding support from “MHRD project on center for excellence on Water and Energy” under training and research in frontiers areas of science and technology (FAST 5-5/2014 TS VII). We also acknowledge the Ms Swati Agarwal and Ms Sami Husain for language corrections.