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Wednesday 8 June 2016
Effect of applying rhizobacteria and fertilizer on the growth of Ludwigia octovalvis for arsenic uptake and accumulation in phytoremediation
Published Date September 2013, Vol.58:303–313,doi:10.1016/j.ecoleng.2013.07.018
Open Access, Creative Commons license
Title
Effect of applying rhizobacteria and fertilizer on the growth ofLudwigia octovalvisfor arsenic uptake and accumulation in phytoremediation☆
Author
Harmin Sulistiyaning Titah a,b,d,,,,
Siti Rozaimah Sheikh Abdullah a,
Idris Mushrifah c
Nurina Anuar a
Hassan Basri b
Muhammad Mukhlisin b
aDepartment of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
bDepartment of Civil and Structural Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
cTasik Chini Research Centre, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
dDepartment of Environmental Engineering, Faculty of Civil Engineering and Planning, Institut Teknologi Sepuluh Nopember (ITS), 60111 Keputih, Sukolilo, Surabaya, Indonesia
Received 28 January 2013. Revised 10 June 2013. Accepted 5 July 2013. Available online 30 July 2013.
Highlights
•
The addition of six rhizobacteria consortium at 2% (v/v) could alleviate the toxic effect of As in Ludwigia octovalvis.
•
The addition of NPK fertilizer at 0.02% (w/w) also lightens the toxic effect.
•
Those additions could increase the biomass weight of L. octovalvis.
•
The effectiveness of phytoremediation in treatment with NPK fertilizer addition was the highest compared with As only and As with rhizobacteria at 2%.
•
The NPK fertilizer addition showed the best results in As phytoremediation using L. octovalvis.
Abstract
The aim of this study was to investigate the effect of applying a six-rhizobacterial consortium and nitrogen phosphate potassium (NPK) fertilizer in inorganic arsenic (arsenate) phytoremediation using Ludwigia octovalvis (Jacq.) Raven plants. The experiment included control L. octovalvis plants and three phytoremediation treatments with L. octovalvis plants, namely an arsenic (As) concentration of 39 mg kg−1 only, the addition of a six-rhizobacterial consortium at 2% (v/v) with an As concentration of 39 mg kg−1 and the addition NPK fertilizer at 0.02% (w/w) with an As concentration of 39 mg kg−1. In the As phytoremediation treatment with the presence of As only, plants showed signs of phytotoxicity such as wilting and senescent leaves. L. octovalvis grew well until the end of exposure (Day 42) in the phytoremediation treatment with 2% rhizobacteria consortium or with 0.02% NPK fertilizer addition. The As phytoremediation treatment with 2% rhizobacteria showed the highest removal percentages of bioavailable As (89%), total extractable As (78%) and As in the leachate of the reed beds (98%) resulting in lower As uptake by L. octovalvis than in the other two phytoremediation treatments. This indicates that the bioremediation process played a role in the treatment with 2% rhizobacteria. The fresh weight of L. octovalvis biomass in treatment with 0.02% NPK fertilizer increased by almost five-fold compared with the As only treatment. The effectiveness of phytoremediation in terms of As uptake at Day 42 reached 49.8% in the phytoremediation treatment with 0.02% NPK fertilizer addition. It was the highest than in the other two phytoremediation treatments. In conclusion, NPK fertilizer addition gave the best results in As uptake using L. octovalvis plants in terms of the effectiveness of phytoremediation.
Keywords
Rhizobacteria
Fertilizer
Arsenic
Phytoremediation
Ludwigia octovalvis
Uptake
1 Introduction
Arsenic contaminated soils, sediments, and sludge were the major sources of arsenic contamination of the food chain, surface water, groundwater, and drinking water (Gonzaga et al., 2006). Contamination of As due to industrial activity was estimated reached 4.53 million tons at worldwide (Han et al., 2003). Arsenic species are bioactive and toxic (Zhang et al., 2002) and were toxic to most plant species (Quaghebeur and Rengel, 2005). In soils and groundwater, inorganic arsenic presents mainly as arsenate [As(V)] and arsenite [As(III)] (Wang and Zhao, 2009). As species have different physical and chemical characteristics, resulting in various degrees of mobility, bioavailability and toxicity (Quaghebeur and Rengel, 2005). According to Glick (2010), the Agency for Toxic Substances and Disease Registry (ATSDR) has made a list of chemicals that are often found in polluted areas, including hazardous materials. According to the list released by the ATSDR, there are 275 hazardous and toxic materials that have a negative impact on human health. Based on this list, As is ranked as the first chemical in terms of hazardous and toxic materials, followed by lead (Pb) and mercury (Hg) in second and third place, respectively.
Currently, there are many different types of technologies to remediate contaminated areas. One such technology is the remediation of contaminated soil and groundwater, including heavy metal contamination, through phytotechnology. The concept of using plants to restore areas contaminated with heavy metals is an effective approach and is categorized as a green technology. Phytoremediation is a technology that uses green plants to remediate various media (soil, water or sediment) that are contaminated with different types of contaminants (organic and inorganic) and interact with microorganisms (ITRC, 2001, Ghosh and Singh, 2005, Cho-Ruk et al., 2006 and Sao et al., 2007). Phytoremediation can be conducted using ex situ or in situ methods. Soil or contaminated areas can be directly planted using suitable plants and non-dredged soil when the in situ method is used (USEPA, 2002). Phytoremediation is a cost effective, environmentally friendly, engineering economical, safe, alternative technology to remediate As-contaminated soils and is suitable for use in developing countries (Yang et al., 2012, Ghosh and Singh, 2005and Lasat, 2002). Moreover, phytoremediation based on the ecological principles (Mirza et al., 2011). In addition to treating heavy metal contaminated areas, an additional benefit of phytoremediation is the potential for bioenergy production from the plants used in the phytoremediation process (Ginneken et al., 2007).
In addition to plants, rhizosphere bacteria or rhizobacteria, also known as plant growth promoting rhizobacteria (PGPR), also play an important role in phytoremediation. They are capable of aggressively colonizing plant roots and promoting plant growth (Khan et al., 2009). Some research has been conducted to evaluate the interactions of rhizobacteria and plants to remediate As. Yang et al. (2012) reported that the addition of As-reducing bacteria could promote the growth of Petris vittata and increase As accumulation. Other research has been conducted to evaluate the application of fertilizer on phytoremediation. According to Fayiga and Ma (2006), phosphate rock can be applied as an effective amendment for As phytoremediation since it significantly increased As uptake in P. vittata.
Ludwigia octovalvis was chosen for this study since it was one of the plants that could survive at a contaminated site in Malaysia (Rahman et al., 2009). L. octovalvis is a suffruticose shrub or perennial woody herb growing to 2 m in height with a stem diameter of 1 cm. L. octovalvis plants are supported by sinker roots, one of which may be the taproot, and long white, lateral roots that grow just under the soil surface. According to Chauhan and Abugho (2012), longfruited primrose-willow (L. octovalvis) is a major weed of rice in South and South East Asia. Its other common names include false primrose, primrose willow, swamp primrose, water primrose, willow primrose and yellow willow herb, while in Malaysia it is locally known as “buyang samalam”, “lakom ayer” and “pujang malam”.
The aims of this study were to determine the effects of applying rhizobacteria as a PGPR and fertilizer on the growth of L. octovalvis as well as to assess the effects of these treatments on As uptake and accumulation in L. octovalvis during phytoremediation at optimized conditions of some factors (As loading, retention time and aeration). Based on our previous study (data not published), the optimized conditions were obtained at 39 mg kg−1 As, 42 days and 0.22 L min−1 for aeration.
2 Materials and methods
2.1 Propagation of plant species
L. octovalvis plants were propagated from seeds gathered from a parent plant growing at a contaminated site in Malaysia. The seeds were grown in the greenhouse using garden soil with ratio of top soil:organic material:sand of 3:2:1 until the next generation plants produced seeds in large quantities. Only plants from this generation were used to run the phytoremediation study. The seeds were planted in plastic crates (37 cm × 27 cm × 10 cm). After 3 weeks, individual seedlings were selected from the nursery and transferred to polybags, with two plants per polybag. All of the plants used in the experiment were 8 weeks old with an average fresh weight of 5.9 g per plant at the beginning of the study.
2.2 Treatment with As-spiked sand in the pilot reed bed
The pilot reed beds were constructed of fiberglass tanks, the walls of which were 0.5 cm thick and black in color with dimensions of 92 (L) cm × 92 (W) cm × 60 (H) cm. A layer of medium gravel (2 cm in diameter) was placed at the bottom of the reed bed, and another layer of fine gravel (1 cm in diameter) was placed at the top. The depth of both the medium and fine gravel layers was 10 cm. As-spiked sand was placed in the reed bed at a depth of 10 cm. Each reed bed had a freeboard of approximately 10 cm with a pipe at the bottom for sampling the leachate. The pilot reed beds were supplied with aeration of 0.22 L min−1. The aeration system was run using an air compressor model HP2 (Orimas, Malaysia), and the air flow rate was measured using a flow meter (Cole-Parmer, USA). The aeration system was placed on a layer of medium-sized gravel and run continuously throughout the experiment.
The sand was then spiked with As salt as sodium arsenate dibasic heptahydrate (AsHNa2O4·7H2O) (FlukaChemika, Switzerland). The As concentration was selected based on an arsenate range finding test against L. octovalvis (Titah et al., 2012) and the previous study on optimization (data not published). The As concentrations were 0 mg kg−1 as the control and 39 mg kg−1 in the phytoremediation plants. Control L. octovalvis plants were used in addition to three phytoremediation treatments in L. octovalvis plants, namely an As concentration of 39 mg kg−1, the addition of a six-rhizobacterial consortium at 2% (v/v) with an As concentration of 39 mg kg−1 and the addition of NPK fertilizer at 0.02% (w/w) with an As concentration of 39 mg kg−1. Each pilot scale reed bed for phytoremediation treatment was planted with 100 healthy 8-week-old L. octovalvis plants with a total fresh weight of 590 g. Fig. 1 shows the schematic of the pilot reed beds.
Fig. 1. Schematic diagram of pilot reed beds.
Macronutrients (N, P, K, S, Mg, Ca, K) and a micronutrient (Cl) were analyzed by a Compact IC plus 882 ion chromatography (IC) (Metrohm, Switzerland), while other micronutrients (Fe, Zn, Mn) and trace elements (Pb, As) were determined using an Optima 7300DV inductively coupled plasma-optical emission spectrometer (ICP-OES) (Perkin Elmer, USA). Based on this analysis, the sand contents of the macronutrients were 29.2 mg kg−1 N (nitrate), 1.2 mg kg−1 K, 13.0 mg kg−1 SO42−, 86.5 mg kg−1 Ca, 7.4 mg kg−1 Mg whereas the micronutrient contents were 6.4 mg kg−1 Cl−, 5.5 mg kg−1 Fe, 0.04 mg kg−1 Zn and 1.62 mg kg−1 Mn. The trace elements were not detected.
During the test, plants were watered alternate days since the sand had a low moisture-holding capacity. Moisture was monitored using a moisture-meter model ECH2O (Decagon, USA), while pH and temperature of the spiked sand were monitored using a pH-meter model pH 300 (Cyberscan, Singapore).
2.3 Addition of the rhizobacteria consortium
Based on our previous study, ten rhizobacteria isolated from the roots of L. octovalviscan biosorb As (data not published). However, only six selected rhizobacteria were used in the study. The selected rhizobacteria were Sphingomonas paucimobilis, Arthtobacter globiformis, Bacillus cereus, Bacillus pumilus, Rhizobium rhizogenesand Rhizobium radiobacter. All rhizobacteria were identified using the Biolog GEN III System (USA) and Vitek2 System (France). The calculation of the amount of rhizobacteria added to the reed bed pilot scale was as follows: the bulk density of sand was 26 mL per 100 g of sand, so there was 34.1 L in 131 kg of sand in each pilot reed bed. An amount of 2% (v/v) rhizobacteria was added to each pilot scale reed.
2.4 Addition of fertilizer
The fertilizer used in this study was NPK (15%:15%:15%). Fertilizer addition was based on a study by Huang et al. (2012), with basic nutrient availability of nitrogen (N), phosphorus (P) and potassium (K) in the soil of 200 mg kg−1 each. The mass of sand in the pilot scale reed bed was 131 kg, which was spiked 26.2 g of NPK fertilizer or 0.02% of the total weight of the sand.
2.5 Sampling and analysis of sand and plants
Sampling of the sand was carried out on Day 0, Day 14, Day 28 and Day 42. About 10 g of sand taken from around the plant roots were sampled in three replicates using the grab sampling technique. Meanwhile, after the designated exposure time, three plants were sampled by digging up the plant together with the roots using a plastic scoop and pulled up slowly to prevent any plant parts from being left behind in the tank. Harvesting of the plants was conducted on Day 14, Day 28 and Day 42. The sand and plant samples were then placed in labeled plastic bags and brought to the laboratory for further analysis.
2.6 Laboratory analysis
2.6.1 Sand extraction
Sand extraction was carried out to determine bioavailable As using methods described by Quevauviller (1998). A 5 g sample of sand from each of the sand samples spiked with various concentrations of As was taken using a composite sampling method for extraction. Each sand sample was placed in a plastic tube. After the addition of 50 mL of a solution of ethylene diamine tetraacetic acid disodium (EDTANa2) (Merck, Germany), samples were agitated using an orbital shaker multi RS-60 (Biosan, Lutvia) at 30 rpm for 1 h at room temperature. Then, each sample was centrifuged at about 3000 rpm for 10 min using an 5810R centrifuge (Eppendorf, USA). The samples were filtered through filter paper (Whatman, UK) with a porosity of 0.2–1.1 μm using a vacuum filter model DOA–P504–BN (GAST, USA) The supernatant was collected in polyethylene bottles and stored at 4 °C before analyzing the As content using an ICP-OES Optima 7300DV (Perkin Elmer, USA).
The total extractable As concentration in the spiked sand was determined using the wet digestion method (USEPA, 1996). Approximately 2 g of the wet sand sample was added to 5 mL of 69% HNO3 in 100 mL Erlenmeyer flasks, and the samples were incubated overnight. All samples were placed in hot block tubes, and the hot blocks were placed in a block digester (AIM 600 Digestion System, Australia). Total As was analyzed using an Optima 7300DV ICP-OES apparatus (Perkin Elmer, USA).
2.6.2 Plant extraction
Plant parts (roots, stems and leaves) were dried prior to the extraction procedure. In this experiment, As extraction from the plants was performed using a modified wet digestion method (Plank, 1992, Kalra, 1998 and Temminghoff and Hauba, 2004). Approximately 0.1–0.5 g of the dried plant material was added to 10 mL of 69% HNO3in 100 mL Erlenmeyer flasks, and the samples were incubated overnight. All of the samples were placed in hot block tubes, and the hot blocks were placed in a block digester (AIM 600 Digestion System, Australia). All of the samples were subsequently heated at 95 °C for 1 h and 30 min. After cooling to 80 °C, 8 mL of 30% H2O2 was added. The heating process was then continued at 95 °C for 2 h (the residue should be translucent or white when the digestion is complete). An aliquot of 2.5 mL of dilute aqua regia (HNO3:HCl = 1:3) was then added, and the digested solution was transferred to a 50 mL plastic tube; deionized water was added to bring the volume of the solution to 50 mL. Lastly, the solution was filtered using a 0.45 μm membrane filter paper (Sastec, Malaysia) to further remove any particulates. The As content was then analyzed using the Optima 7300DV ICP-OES instrument (Perkin Elmer, USA).
2.7 Sampling and analysis of leachate
The leachate was sampled on Days 0, 14, 28 and 42 of exposure. About 60 mL of the leachate were collected from the pipe at the bottom of the reed bed. Samples of the leachate were placed in a plastic tube (10 mL) and stored at 4 °C for further As analysis using ICP-OES. All samplings were conducted in three replicates.
2.8 SEM analysis
The scanning electron microscopy (SEM) analysis objective is to find the impact of As on tissues of roots, stems and leaves of L. octovalvis. SEM analysis was conducted using FESEM Model Supra 55VP (Zeiss, Germany). SEM analysis was conducted after 42 days of exposure. Sample preparation was carried out using a method from Pathan et al. (2008).
2.9 Determination of the rhizobacterial populations
The method to determine the rhizobacterial content from the roots of L. octovalvis, sand near plants and the leachate were based on several previously published reports (Harley and Prescott, 2002, Abou-Shanab et al., 2005, Cakmakci et al., 2007and Mittal and Johri, 2007). The determination of the rhizobacterial number using the standard plate count method was performed on Day 1, Day 14, Day 21, Day 28 and the final day of exposure (Day 42).
2.10 Statistical analysis
All statistical tests were performed using SPSS Version 17.0 (IBM, USA). Two-way analysis of variance (ANOVA) at the 95% confidence level (p ≤ 0.05) was used to evaluate significant changes in the fresh-dry weight of L. octovalvis biomass, length of roots and stems, concentration of As in the spiked sand and leachate and the concentration of As in whole L. octovalvis plants.
3 Results and discussion
3.1 Observation of the physical growth of L. octovalvis
According to Greenberg et al. (2012), aeration is a one factor in the multi-process phytoremediation system (MPPS) on farmland which aims to accelerate and complete the removal of heavy metals or organic pollutants in the soil. Based on the observation of physical growth in this study, symptoms of As toxicity, such as wilting and senescent leaves, occurred in the phytoremediation treatment with As only. The toxic effects of As on L. octovalvis were alleviated at the As concentration of 39 mg kg−1 with the addition of the selected six-rhizobacterial consortium. All L. octovalvisplants grew well until the end of exposure. The biomass of L. octovalvis increased in the As phytoremediation treatment with the addition of rhizobacteria. The fresh weight biomass of L. octovalvis at the end of the exposure period with the addition of rhizobacteria reached 12.1 ± 1.7 g and was almost double the fresh weight biomass of L. octovalvis with As only (7.3 ± 3.3 g) (Fig. 2). This situation has occurred due to the addition of the six-rhizobacterial consortium that acted as PGPR and enhanced the growth of L. octovalvis. According to Khan et al. (2009), rhizobacteria as PGPR can facilitate plant growth.
Fig. 2. Fresh-dry biomass weight of L. octovalvis (WW, fresh weight; DW, dry weight).
Several studies have been conducted showing similar results. As reported by Nie et al. (2002), there was significant growth of canola plants in As-spiked medium after inoculation with the bacteria Enterobacter cloacae. According Shilev et al. (2006), an increase in biomass of Heliathnus annuss (sunflower) plants on land containing As occurred after adding the bacteria Pseudomonas fluorescens. Wang et al. (2011)reported that the addition of R. radiobacter (Agrobacterium tumefaciens) to increase resistance against Populus deitoides improved As uptake efficiency and increased As translocation.
According to Jetiyanon and Kloepper (2002), the application of microorganisms could reduce or increase the uptake and accumulation of heavy metals by plants. According to Lebeau et al. (2008), PGPR could enhance plant growth and biomass plants in two ways: first indirectly by producing antibiotics that can inhibit pathogenic bacteria in the soil and second in a direct way through improved nutrient and water uptake.
Based on the observation of the physical growth of L. octovalvis, the toxic effects of As in L. octovalvis could be reduced with the addition of NPK fertilizer. Toxic effects were not observed until the end of exposure. All L. octovalvis plants were fresh, healthy and grew well until the average height of the plants reached 102.7 ± 18.2 cm. According to Lihong and Guilan (2009), increased concentrations of phosphate in the medium could reduce the toxic effects of arsenate due to a decrease in As uptake by plants. The addition of phosphate nutrients could reduce the toxic effects of As on plants and could alleviate plant growth inhibition (Gulz, 2002). Cao et al. (2009)reported that the use of additional phosphates may reduce the toxic effects As in Scutellaria baicalensis Georgi plants. Fig. 2 shows that the addition of NPK fertilizer increased the biomass of L. octovalvis. The fresh weight biomass of L. octovalvis at the end of exposure with the addition of NPK fertilizer reached 34.1 ± 6.1 g, which was higher than the fresh weight biomass of L. octovalvis in As phytoremediation with As only (7.3 ± 3.3 g), with nearly a five-fold increase compared to the biomass in phytoremediation with As only.
The ANOVA results of the fresh weight biomass of L. octovalvis showed a significant difference (p < 0.05) in the fresh weight biomass of L. octovalvis in all phytoremediation treatments. Meanwhile, the ANOVA results on the dry weight biomass of L. octovalvis showed a significant difference (p < 0.05) in the phytoremediation treatment with the addition of 0.02% NPK fertilizer against the control and the other two treatments.
L. octovalvis were analyzed for SEM after 42 days of exposure. According to Bercu (2007), the cross section of roots can be subdivided into epidermis, cortex, endodermis and stele, the stele contains the xylem and phloem. Ions were translocated to shoot through the xylem and photosynthate was supplied from shoot to the root through the phloem. Results of SEM analysis for roots at the control (Fig. 3a-1), in treatment with the addition of 2% rhizobacteria (Fig. 3c-1) and in treatment with the addition of 0.02%NPK fertilizer showed the cross sections of the root such as epidermis, cortex, endodermis and stele could be seen clearly (Fig. 3d-1). While the root sections seems less visible and also the toxic effects of As in root tissue of L. octovalvis were detected in the treatment with As only (Fig. 3b-1). SEM analysis results on stems showed the SEM at the control (Fig. 3a-2) and in the treatment with the addition of 0.02% NPK fertilizer looks like the outside part of stems (Fig. 3d-2). SEM of stem in the treatment with the addition of 2% rhizobacteria showed more clear the parts of the stem (Fig. 3c-2), while the sample in the treatment with As only shows the damage of stems tissues and there was a hole inside the stem (Fig. 3b-2).
Fig. 3. SEM micrograph of L. octovavis plant (a-1 to a-4) at control, plant growing at 39 mg kg−1As, (b-1 to b-4) only As, (c-1 to c-4) with addition of 2% rhizobacteria, (d-1 to d-4) with addition of 0.02% NPK fertilizer.
Trichomes were hair cells or leaf hair derived from specialized epidermal cells on leaf or stems surface (Dennis, 2002). Results of SEM analysis on the leaves of L. octovalvis in the treatment with As only showed the damages on tissue leaves were detected and trichomes were not visible (Fig. 3b-3). Meanwhile, trichomes were detected clearly at the control (Fig. 3a-3), treatment with the addition of 2% rhizobacteria (Fig. 3c-3) and treatment with the addition of 0.02% NPK fertilizer (Fig. 3d-3). The size of stomata on the leaves in the treatment with As only (Fig. 3b-4) was even smaller than the stomata on the leaves at the control (Fig. 3a-4), in the treatment with the addition of 2% rhizobacteria (Fig. 3c-4) and in the treatment with addition of 0.02% NPK fertilizer (Fig. 3d-4).
Fig. 4a shows the root length of L. octovalvis. The root length in the phytoremediation treatment with the addition of NPK fertilizer was highest (34.3 ± 10.8 cm) at Day 42. The ANOVA results on the root length of L. octovalvis showed that there was a significant difference (p < 0.05) in the phytoremediation treatment with the addition of 0.02% NPK fertilizer against the control and the other two phytoremediation treatments. The retention time at 14 days was significantly different (p < 0.05) than at 28 and 42 days in phytoremediation with NPK fertilizer addition.
Fig. 4. (a) Root length of L. octovalvis and (b) stem length of L. octovalvis.
Fig. 4b shows the stem length of L. octovalvis. The length of L. octovalvis stems in the phytoremediation treatment with the addition of NPK fertilizer was highest (102.7 ± 18.2 cm) at Day 42. The ANOVA results show that the stem length of L. octovalvisplants was significantly different (p < 0.05) in all phytoremediation treatments. All the retention periods showed a significant difference (p < 0.05).
3.2 Concentrations of bioavailable As, total extractable As in spiked sand and the concentration As in the leachate
The bioavailable concentrations of As in the sand are shown in Fig. 5. The concentration of bioavailable As in the As phytoremediation treatment with the addition of 2% rhizobacteria was less when compared with the other two phytoremediation treatments. According to Khan et al. (2009), rhizobacteria as PGPR could reduce the concentration of bioavailable heavy metals.
Fig. 5. Concentration of bioavailable As and total extractable As in As-spiked sand.
The concentration of bioavailable As in the phytoremediation treatment with the addition of 0.02% NPK fertilizer was lower than the concentration of bioavailable As in phytoremediation treatment with As only. The presence of phosphate could decrease the sorption of As by soil particles and lead to increased concentrations of bioavailable As (Alvares-Benedí et al., 2005). According to Tu and Ma (2003), arsenate and phosphate will compete with each other for soil sorption sites, resulting in a reduction in their sorption by soil and an increase in solution concentrations. Phosphate can replace the sorption of As by soil and lead to increased concentrations of bioavailable As but this depends on the type of soil as the addition of phosphate does not affect the concentration of bioavailable As in volcanic soil. Phosphate addition to arsenic-contaminated soils would enhance arsenic release from soil through competitive exchange (Smith et al., 2002) and thereby increase soil As bioavailability. The content of iron oxide (Fe2O3) and aluminum oxide (Al2O3) in sand used in this study was low. It may be cause phosphate sorption of arsenate low such that the concentration of bioavailable As did not increase. Additionally, bioavailable As was taken up by the roots of L. octovalvis. The ANOVA results show a significant difference (p < 0.05) in the concentrations of bioavailable As in all three phytoremediation treatments compared to the control.
Table 1 shows the removal of bioavailable As in spiked sand. The highest removal percentages (70%) at Day 14 occurred in the phytoremediation treatment with the addition of 2% rhizobacteria. This situation may have occurred due to the addition of As-resistant rhizobacteria assisting in the removal of As. It has been suggested that the bioremediation process plays a greater role in the early phase of the treatment. Aeration in the bioremediation process could support rhizobacteria survival since the six rhizobacteria added in the As phytoremediation treatment were aerobic bacteria. Meanwhile, the percentage of bioavailable As removal in the phytoremediation treatment with As only reached 22%, 53% and 86% at 14, 28 and 42 days of exposure, respectively. This situation occurred because most of the As was taken up and accumulated by L. octovalvis plants, which could be determined from the concentration of As in whole plants of L. octovalvis; this value reached 1103.7 ± 273.3 mg kg−1 at 42 days of exposure. The same of percentage (86%) at Day 42 occurred in As phytoremediation treatment with the addition of NPK fertilizer. Although the concentration of As taken up by L. octovalvis was lower compared with the As only treatment, the dry weight of L. octovalvis was higher than in the As only treatment, resulting in a higher percentage of total As uptake in As phytoremediation with the addition of NPK fertilizer compared with the other two treatments.
Table 1. Percentage of bioavailable As removal in spiked sand.
Phytoremediation treatment
Removal of As bioavailable in spiked sand (%)
Day 14
Day 28
Day 42
39 mg kg−1 As only
22
53
86
39 mg kg−1 + with addition of 2% rhizobacteria
70
58
89
39 mg kg−1 + with addition of 0.02% NPK fertilizer
51
54
86
In order to give a true assessment of phytoremediation performance, we tried to convert the uptake concentration by L. octovalvis to total As uptake to indicate the effectiveness of the phytoremediation by using the equation below:
equation1
where,
C As in plant, concentration of As uptake per plant (mg kg−1)
DW plant, dry weight of plant (kg)
N plant, number of plants
C bioavailable As, concentration of As bioavailable in sand (mg kg−1)
W sand, total weight of sand (kg)
These results are depicted in Fig. 6. The effectiveness of the phytoremediation at 42 day of exposure were 17.5, 8.8 and 49.8% in treatment with As only, treatment with the addition of rhizobacteria and treatment with the addition of NPK fertilizer, respectively. Based on the results, it indicated that the best As phytoremediation application was As phytoremediation with NPK fertilizer since total As uptake by L. octovalvis had the highest effectiveness of the phytoremediation at 42 days.
Fig. 6. Percentages of total As uptake.
A comparison of the concentration of bioavailable As and the total concentration of extractable As in the sand treatments shows in Fig. 5. The average ratio of the concentration of bioavailable As to the total concentration of extractable As in the spiked sand for the three phytoremediation treatments was in the range of 0.1–0.5. The total concentration of extractable As at the end of the retention time (42 days) in the three phytoremediation treatments was below the quality standard (16 mg kg−1) of Malaysia (Department of Environment Malaysia, 2009). Phytoremediation using L. octovalvis plants could reduce As concentrations in accordance with the current quality standard.
Fig. 7 shows the concentration of As in the leachates of the reed beds. The concentration of As in the leachate in the phytoremediation treatment with the addition of 2% rhizobacteria showed lower concentrations compared to the other two phytoremediation treatments. According to Yang et al. (2012), the addition of bacteria as PGPR in P. vittata plants decreases the concentration of As in the leachate compared to treatment without the addition of bacteria. The ANOVA results show significant differences in the As concentrations in the leachates (p < 0.05) from the three phytoremediation treatments (As only, As with the addition of 2% rhizobacteria and As with the addition of 0.02% NPK fertilizer).
Fig. 7. Concentration of As in the leachate.
Table 2 shows the percentages of As removal in the leachate. The highest percentage removal of As in the leachate (98%) occurred in the phytoremediation treatment with the addition of 2% rhizobacteria at a retention time of 14 days. At the end of exposure (Day 42), the highest percentage removal of As in the leachate (94%) was still seen in the phytoremediation treatment with the addition of 2% rhizobacteria. The consortia of rhizobacterium may have assisted in removing As in the leachate. While the addition of NPK fertilizer showed an increased percentage removal of As in the leachate from the beginning to the end of exposure, this may have been due to the increasing biomass of L. octovalvis since more As could accumulate in the plants.
Table 2. Percentage of As removal in the leachate.
Phytoremediation treatment
Removal of As in leachate (%)
Day 14
Day 28
Day 42
39 mg kg−1 As only
88
91
91
39 mg kg−1 + with addition of 2% rhizobacteria
98
95
94
39 mg kg−1 + with addition of 0.02% NPK fertilizer
90
92
93
3.3 Uptake and accumulation of As
L. octovalvis plants were harvested at 14, 28 and 42 days. Fig. 8 shows the As concentrations in L. octovalvis plants at every harvesting time point. The highest concentration of As in L. octovalvis plants was 1103.7 ± 273.3 mg kg−1 in the As phytoremediation treatment with As only. The concentration of As in L. octovalvis in As phytoremediation with the addition of 2% rhizobacteria was 198.2 ± 30.7 mg kg−1, while the concentration of As in phytoremediation with the addition of 0.02% NPK fertilizer reached 352.4 ± 77.8 mg kg−1. The ANOVA results on As concentrations in L. octovalvis showed that the As concentration was significantly different (p < 0.05) in the phytoremediation treatment with As only compared to the other two treatments.
Fig. 8. Concentration of As in whole L. octovalvis plants.
The concentration of As in L. octovalvis plants in phytoremediation with the addition of 2% rhizobacteria was lower than the other two treatments, resulting in the highest removal percentages of bioavailable As (89%), total As extractable removal (78%) and As in the leachate (94%). The consortium of six rhizobacteria was resistant to As and were able to biosorb As, so this consortium of six rhizobacteria could assist in the process of As phytoremediation. According to Khan et al. (2009), rhizobacteria as PGPR make the system more effective in minimizing phytobacteria and in mitigating the biotoxicity of bioavailable heavy metals.
In phytoremediation with the addition of 0.02% NPK fertilizer, As concentrations in L. octovalvis plants were lower when compared to phytoremediation with As only. Arsenate [AsO4−3] (the inorganic from of As) and phosphate (PO42−) have analogous forms (Yang et al., 2012). However, plants prefer to take up phosphate over arsenate if the two anions exist at the same time since phosphate has a high affinity uptake system (Tu and Ma, 2003). According to Cao and Ma (2004), phosphate competes with arsenate for plant uptake from the external solution but phosphate more readily enters the plant, so it can prevent arsenate uptake. Low levels of phosphates displace arsenic from soil particles to increase uptake and phytotoxicity, while larger amounts of phosphates compete with arsenic at root surfaces to decrease uptake and phytotoxicity (Patra et al., 2004). The addition of phosphate can reduce the uptake and accumulation of arsenate by plants (Mahimairaja and Bolan, 2011). Pigna et al. (2010) reported the addition of phosphate fertilizer application could prevent the uptake and translocation of As on wheat plants, increasing plant biomass and alleviate the toxic effects of As.
According to Tu and Ma (2003), uptake of arsenate and phosphate by plants is generally competitive. Also, the presence of phosphate in contaminated soils has a role in the phytoextraction process (phytoremediation) (Gonzaga et al., 2006). According to Tu and Ma (2003), the presence of phosphate could affect plant growth, plant biomass weight and biosorption of As by plants. The impact could be an increase in plant growth and biomass weight, a reduction in the toxic effects of As in plants or a reduction or increase in As uptake and biosorption by plants. The effects of the presence of phosphate are predicted to depend on the P/As molar ratio in the soil (Tu and Ma, 2003).
The addition of NPK fertilizer could also directly increase the concentration of nitrogen in the spiked sand, hence reducing As uptake by L. octovalvis plants. According to Chen et al. (2008), the addition of nitrate can alleviate As uptake by rice plants.
The phytoremediation ability of L. octovalvis was assessed by calculating both the bioaccumulation factor (BF) and translocation factor (TF) (Melo et al., 2009) as well as the percentage of translocation from roots to stems after As exposure, as described by Poynton et al. (2004):
equation2
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Fig. 9 shows the values of BF, TF and the percentage of translocation. The determination of BF showed that the highest BF value was 36.6 in the As phytoremediation treatment with the addition of 0.02% NPK fertilizer on Day 42. This result supports the findings of total As uptake in the previous paragraph.
Fig. 9. Average BF, TF and percentage of translocation of L. octovalvis in As spiked sand.
In terms of TF and the percentage of translocation, phytoremediation with As only provided the highest value at the end of exposure (Day 42). This result also shows that the concentration of As in the plants in this treatment was high (1103.7 ± 273.3 mg kg−1). As a result, the plants showed signs of phytotoxicity, as explained above.
The highest TF (6.3) and percentage of translocation (69.5%) occurred in the As phytoremediation treatment with the addition of 2% rhizobacteria at Day 14. This result shows that the bioremediation process plays a greater role in the early stages of treatment, as explained before.
3.4 Bacterial population
The rhizobacteria population on plant roots of L. octovalvis, in the sand and in the leachate is shown in Fig. 10. Total populations of rhizobacteria on the roots of L. octovalvis were higher than in the sand and leachate. The total population on the roots of L. octovalvis was greater in the phytoremediation treatments with the addition of 2% rhizobacteria and 0.02% NPK fertilizer compared to the total population of rhizobacteria in phytoremediation treatment with As only. Rhizosphere soil with high concentrations of nutrients released by the roots attracts more bacteria (Penrose and Glick, 2001). According to Cakmakci et al. (2007), the addition of fertilizer containing nitrogen and phosphorus leads to a significant increase in the number of bacteria. In the present study, the indigenous rhizobacterial population was reduced in control sand. This situation occurred since the experiment was not carried out with the addition of fertilizer in control sand. The ANOVA results on the bacterial population showed significant differences (p < 0.05) in the phytoremediation treatments with the addition of 2% rhizobacteria and 0.02% NPK fertilizer compared to the control and phytoremediation treatment with As only.
Fig. 10. Bacterial population in the roots, As-spiked sand and leachate.
4 Conclusions
We conclude that the effect of applying the six-rhizobacterial consortium at 2% and NPK fertilizer at 0.02% could alleviate the toxic effects of As in L. octovalvis and increase the biomass weight of L. octovalvis. NPK fertilizer addition at 0.02% during As phytoremediation treatment increased the effectiveness of the phytoremediation by L. octovalvis due to increased L. octovalvis biomass. It showed the highest effectiveness of the phytoremediation (49.8%) compared with in other two phytoremediation treatments. Among the three As phytoremediation treatments (As only, As with rhizobacteria at 2% and As with NPK fertilizer at 0.02%), As phytoremediation with NPK fertilizer addition at 0.02% showed the best results.
Acknowledgments
The authors would like to thank Tasik Chini Research, Universiti Kebangsaan Malaysia (UKM) and the Ministry of Higher Education, Malaysia (grant no. UKM-KK-03-FRGS 0119-2010) for funding this research, and the Ministry of National Education of the Republic of Indonesia for providing a doctoral scholarship to the first author.
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Corresponding author at: Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia. Tel.: +60 149341284.
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