Phytotoxicity test of Scirpus grossus on diesel-contaminated water using a subsurface flow system

Ecological Engineering 54 (2013) 49–56

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Phytotoxicity test of Scirpus grossus on diesel-contaminated water using a subsurface flow system Israa Abdulwahab Al-Baldawi a,b,∗ , Siti Rozaimah Sheikh Abdullah c , Fatihah Suja’ a , Nurina Anuar c , Mushrifah Idris d a

Department of Civil and Structural Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, Selangor, Malaysia Department of Biochemical Engineering, Al-khwarizmi College of Engineering, University of Baghdad, Baghdad, Iraq Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, Selangor, Malaysia d Tasik Chini Research Centre, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Selangor, Malaysia b c

a r t i c l e

i n f o

Article history: Received 18 September 2012 Received in revised form 28 December 2012 Accepted 16 January 2013 Available online 19 February 2013 Keywords: Phytotoxicity Scirpus grosuss Hydrocarbon Sub-surface flow system

a b s t r a c t A phytotoxicity experiment with diesel as a model hydrocarbon pollutant at different concentrations (0, 8700, 17,400 and 26,100 mg/L) was performed on the emergent wetland bulrush of Scirpus grossus in a subsurface flow system (SSF). After 72 days of exposure, maximum removal occurred at the diesel concentration of 17,400 mg/L at 91.5%; in the corresponding control without plants, the removal was only 54.1%. Furthermore, the removal efficiency of hydrocarbons from sand was determined to be in the range of 67.2–69.9% for all treatments. According to the plant growth parameters, it was shown that S. grossus could effectively promote the degradation of total petroleum hydrocarbons (TPH) when the concentration of diesel in water was up to17,400 mg/L. The population of living microorganisms in the planted aquariums could also adapt to ≤17,400 mg/L diesel contaminated water. This study showed that S. grossus and rhizobacteria in a subsurface flow system has potential in reclaiming hydrocarboncontaminated water. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The application of phytoremediation in recent years has improved the environment using efficient and inexpensive in situ methods (Huesemann et al., 2009). Constructed wetlands (CWs) are an excellent technology for treating wastewater effectively in phytoremediation systems, due to their low cost, simple operation and maintenance and favourable appearance. The use of emergent plants for the phytodegradation of hydrocarbons in constructed wetlands is widely applied nowadays as a new alternative treatment for wastewater. CWs are used for a wide variety of pollutants, including agricultural, petroleum and petrochemical industry wastewaters, various runoff waters and landfill leachates (Vymazal, 2009). Diesel is a widely used fuel, especially in industry, and with the increase technological development, it has become one of the most common organic pollutants in the environment. Moreover, it is toxic to many organisms and detrimental to human

∗ Corresponding author at: Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, Selangor, Malaysia. Tel.: +60 3 89216407; fax: +60 3 89216148. E-mail addresses: [email protected], [email protected] (I.A. Al-Baldawi), [email protected] (S.R.S. Abdullah). 0925-8574/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ecoleng.2013.01.016

health (Moreira et al., 2011). Generally, diesel is more toxic to plants than crude oil because it contains higher concentrations of light hydrocarbon components than crude oil. Thus, the treatment of wastewater contaminated with diesel appears to be more of a challenge compared to crude oil (Lin and Mendelssohn, 2009). In this study, the emergent wetland plant Scirpus grossus was used. This plant is commonly found in tropical and temperate regions. Emergent plants are conspicuous plants that dominate wetlands, shallow lakes and streams. S. grossus is a native plant in Malaysia, and is locally known as “Rumputmusiang” (Anonymous, 2011). S. grossus is an aquatic species with a high growth rate and has the ability to degrade contaminants. According to Stottmeister et al. (2003), the bulrush S. grossus is suitable and frequently used in CWs. It is an annual aquatic bulrush, often found growing in large colonies in water. It has sharp to soft stems, triangular leaves, obvious leaf blades, inflorescences always on the stem tips, seen as tight clusters or spreading open with the leaves resembling stems (UF/IFAS, 2007). To date, no research has been done to explore the potential of S. grossus in treating diesel-contaminated water. According to Vymazal (2009), constructed wetlands with horizontal sub-surface flow (SSF) for wastewater treatment was started in Germany based on the research by Seidel beginning in the 1960s and by Kickuth in the 1970s. The SSF wetland consists of a channel with a bed filled with porous media, such as rock and gravel as a

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Fig. 1. (a) Diagram of an aquarium for the phytotoxicity test, (b) experimental set-up of the phytotoxicity experiment.

substrate for the growth of rooted emergent wetland plants. The media supports the vegetation root structure (Haberl et al., 2003). Plants can transfer oxygen to the rhizosphere and release soluble exudates that feed microorganisms and enhance biodegradation. Thus, plants support microbial growth and thereby enhance the associated contaminant-degradation processes (Fernandez et al., 2011). The rhizosphere encompasses the interactions between roots or rhizomes and the soil matrix, as the main supporting material for plant growth and microbial films (Stottmeister et al., 2003). In SSF constructed wetlands, the interactions between water, the granular media, macrophytes and microorganisms are very complex (Zhang et al., 2011b). SSF wetlands are designed to keep the water level below the top of the soil media. The flow path through operational SSF systems is usually horizontal, but may be vertical (USEPA, 2000). In SSF systems, wastewater is supplied through an inlet and flows slowly through the porous medium under the surface of the bed in a more or less horizontal path until it reaches the outlet zone where it is collected before leaving through a level control arrangement at the outlet. The advantage of SSF system wetlands is that they are less expensive to construct, operate and maintain compared to mechanical treatment processes designed to produce the same quality of effluent. The removal of BOD, COD, TSS, metals and organics in municipal wastewaters is very effective and reliable. The water level is maintained below the top layer of soil so colonisation by mosquitoes and similar insect vectors is avoided (Kadlec, 2009; USEPA, 2000). The objectives of this study were (1) to determine the tolerance limits of S. grossus to diesel fuel at different concentrations

(8700, 17,400 and 26,100 mg/L) in a phytotoxicity test and (2) to assess microbe–plant interactions in the biodegradation of diesel fuel from wastewater using a subsurface flow system.

2. Materials and methods 2.1. Experimental set-up for the phytotoxicity test with the SSF This study was conducted in an open natural environment in a greenhouse at Universiti Kebangsaan Malaysia (UKM), and the phytotoxicity experiment employed a subsurface flow system (SSF). In the study, 13 aquariums made of glass were used to minimise any sticky oils on the walls. All the aquariums were operated batchwise in single exposure. Each aquarium with dimensions of L 30 cm × W 30 cm × D 30 cm was filled from the bottom layer to the upper layer with: (1) 8 cm of gravel with a size of  10–20 mm, (2) 3 cm of gravel with a size of  1–5 mm and (3) 10 cm of sieved fine sand of  2 mm (Fig. 1a). For each concentration, there were three replicates (R1, R2 and R3) and an aquarium for the control contaminant without plants (CC), as well as another aquarium without the diesel contaminant as a plant control (PC), as shown in Fig. 1b. Fourteen healthy bulrush plants of S. grossus (one month old) were planted into each aquarium containing 7 L of synthetic wastewater prepared by mixing water with standard diesel obtained from a local Petronas petrol station at different concentrations (8700, 17,400 and 26,100 mg/L). The water level was maintained within the sand layer surface (up to 21 cm height of the aquarium) to simulate a

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subsurface flow system as normally used in a constructed wetland (USEPA, 2000).

2.2. Determination of the physicochemical properties of water The experimental work was done during 72 days of exposure with sampling performed on days 0, 7, 14, 28, 42 and 72. The parameters of temperature (T, ◦ C), dissolved oxygen (DO, mg/L), oxidation reduction potential (ORP, mV) and pH were recorded to observe the physicochemical changes in water using a multi-probe of IQ 150 (IQ Scientific Instruments, UK) for pH, ORP and temperature measurements, and a dissolved oxygen sensor (GLI International, Model 63, USA).

2.3. Water sampling Water samples from the growth medium (100 mL each) were collected periodically in clean containers from each aquarium on sampling days for all treatments to extract total petroleum hydrocarbons (TPH) as an indicator of diesel contamination. The samples were taken 5 cm from the bottom of the aquarium using two pipes as depicted in Fig. 1a. The TPH concentration in the synthetic wastewater was determined using a liquid–liquid extraction method and gas chromatography (Lohi et al., 2008). The method followed the Environmental Protection Agency (EPA) Method 3510C (USEPA, 2011). Dichloromethane (Merck, Germany) was used as the solvent. A 100 mL wastewater sample was transferred to a 1 L separatory funnel and shaken for 2 min after adding 25 mL of dichloromethane. The lower organic layer was withdrawn from the bottom of the flask and then dried over sodium sulphate (Merck, Germany). The residuals were put in a 10 mL vial inside an overhead fume hood to evaporate the remaining water for 3–4 days.

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2.6. Plant root-associated bacteria The microbial population was obtained from the roots and the sand attached to the rhizosphere zone. The count of live bacteria in the rhizosphere of plants irrigated with diesel contaminated water was determined by the serial dilution method. At first, 10 g of plant roots were harvested and then put into 100 mL of sterile distilled water to obtain a 10−2 dilution (Prescott and Harley, 2002). This was shaken at 150 rpm for 1 h to release adhered microorganisms. After transferring 1–9 mL of sterile saline water, subsequent dilutions up to 10−4 were made. Next, 0.1 mL of the three (10−2 , 10−3 and 10−4 ) dilutions was spread onto sterile plates containing a nutrient agar medium (tryptic soya agar, TSA). The plates were incubated inverted at 37 ◦ C for 24 h. Colonies of more than 20 and less than 300 cells were counted. The number of colonies counted was multiplied by the reciprocal of the dilution and the amount plated, the results are expressed as colony forming units, CFU per mL (CFU/mL) (Peng et al., 2009; Moreira et al., 2011). 2.7. Analysis and removal percentage of TPH The extracts from water and sand were concentrated to 2 mL in GC vials and analysed by GC-FID using capillary column gas chromatography (Agilent Technologies, Model 7890A, GC System, UK), with a HP-5 5% phenyl methyl siloxane column (30 m × 0.32 mm i.d. × 0.25 ␮m) and helium as the carrier gas. The column temperature was held at 50 ◦ C for 1 min, and then ramped at 15 ◦ C per min to 320 ◦ C for 10 min. The percentage of TPH removal on each sampling day was determined using Eq. (1): % Removal =

(TPH0 − TPHt ) × 100 TPH0

(1)

where TPH0 = total petroleum hydrocarbon on sampling day 0 and TPHt = total petroleum hydrocarbon on each sampling day.

2.4. Sand sampling 2.8. Statistical analysis To obtain more information on diesel absorption by sand, the sand was extracted based on the US EPA 3550C method (USEPA, 2007). To analyse the sand, 10 g of sand was collected at 5 cm depth from the top surface of sand layer periodically in clean containers from each aquarium on the same sampling days for all treatments to extract total petroleum hydrocarbon (TPH). TPH was detected ultrasonically using a solvent extraction method (Liu et al., 2011). First, soil samples were dried by mixing with sodium sulphate (Na2 SO4 ) then put in a 100 mL Schott bottle with 50 mL of dichloromethane used as the solvent. Afterward, the Schott bottle was put in an ultrasonic cleaner (Kwun Wah International Ltd., China) for 30 min at a temperature of 50 ◦ C. Then, the samples were filtered through glass wool and the extracted solution was poured into a 15 mL vial and left in the fume hood for 3–4 days to evaporate any traces of water and dichloromethane.

The experimental results were statistically evaluated using SPSS version 16 (SPSS Inc., USA). All the experiments were performed in triplicate to compensate for experimental errors and are reported as mean ± standard deviation (SD). The removal efficiency, plant growth and bacterial populations (dependent variables) according to day, concentration and treatment (independent factors) were analysed using the general linear model test with Duncan’s multiple range tests to separate means. Statistical significance was defined as p < 0.05. The correlations between TPH degradation, total rhizosphere bacteria and root length were analysed by Pearson correlations. 3. Results and discussion 3.1. Monitoring of physicochemical parameters

2.5. Plant growth The growth of S. grossus with different diesel concentrations (0, 8700, 17,400 and 26,100 mg/L) was observed for 72 days. One plant was harvested on each sampling day (0, 7, 14, 28, 42 and 72) from the three replicates. The plant was fully rinsed with tap water and then the water was absorbed using tissue to record the stem height, root length and wet weight. Then, all plant samples were dried in an oven (Memmert, Germany) until a constant weight was achieved at 70 ◦ C for 72 h to determine the dry weight (Peng et al., 2009).

Physical parameters (T, pH, DO and ORP) were recorded throughout the phytotoxicity test as depicted in Fig. 2 for treatments with plants and without plants at diesel concentrations of 8700, 17,400 and 26,100 mg/L. In general, the results show that the temperature mean values ranged between 24 ◦ C and 28 ◦ C throughout the 72 days; this is normal for a tropical region. The average pH value of the aquariums ranged from 5.9 to 7.6, indicating that pH did not differ significantly among the treatments. Regarding DO, average values ranged between 3.3 and 7.1 mg/L. According to Ong et al. (2010), the conditions of the phytotoxicity test can be distinguished whether it is aerobic or anaerobic through DO and

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Without plants 29

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Fig. 2. Physical parameter variations in the phytotoxicity test with Scirpus grossus using diesel as the contaminant.

ORP measurements (Ong et al., 2010). The results of this study showed the treatment environment was between aerobic and anoxic. Hydrocarbons are effectively degraded through microbial degradation under anoxic/anaerobic conditions as the concentration of dissolved oxygen in the filtration beds is very limited (Vymazal, 2010). ORP oscillated between −33.3 and +64.83 mV, indicating that all diesel concentrations and plant controls were in the range between aerobic and anoxic conditions. Diesel affected the environment of treatments around the rhizosphere and caused a decrease in the ORP readings, indicating that the environment was becoming more anaerobic with increasing diesel concentration (Lin and Mendelssohn, 2009). Generally, anaerobic processes play a major role in SSF CWs (Langergrabera et al., 2009). 3.2. Degradation and removal of TPH from water The degradation percentage of total petroleum hydrocarbons (TPH) by S. grossus was recorded from the extraction of synthetic wastewater of different diesel concentrations (8700, 17,400

and 26,100 mg/L) with plants and the corresponding control contaminant without plants during the 72 day treatment period; the results are depicted in Fig. 3. It was clearly seen that significant differences in TPH degradation by S. grossus were found between the treatments with plants and without plants. In other words, S. grossus enhanced the removal of diesel from contaminated water during growth. The removal of TPH by S. grossus in the three treatments of 8700, 17,400 and 26,100 mg/L was statistically significant compared with the natural degradation in the control treatment without plants, as depicted in Fig. 4. The results for the percentage of TPH degradation in wastewater at the end of 72 days for the three treatments (8700, 17,400 and 26,100 mg/L) were 30.1–91.5%, while degradation in the control aquariums without plants was only 16.4–54.1%. This demonstrates that all the three treatments accelerated the removal of TPH from diesel-contaminated water. Phytoremediation by S. grossus had a positive influence on the removal of diesel from water. TPH removal during the 72-day period achieved a maximum value of 91.5% in the treatment with 17,400 mg/L of diesel with an average removal in its corresponding control of only 54.1%. The average degradation rate in the

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Fig. 3. Degradation percentage by Scirpus grossus exposed to synthetic wastewater with diesel concentrations of 8700, 17,400 and 26,100 mg/L. Bars indicate the standard error of three replicates (n = 3). Letter A: statistically significant diesel removal from water between two treatments with plants and without plants was represented (p < 0.05).

treatment with 26,100 mg/L of diesel was 81.1% compared with 51.6% in its corresponding control. These results show that the degradation of TPH by S. grossus with diesel concentrations of 17,400 and 26,100 mg/L was greater than in the control treatment without plants. Diesel degradation with plants was higher than the corresponding control, possibly due to the action of microorganisms in the rhizosphere (Zhang et al., 2011a). Similarly, the degradation rates with a diesel concentration of 8700 mg/L and the corresponding control were 77.0 and 42.6%, respectively. In other words, the degradation rate with a diesel concentration of 8700 mg/L was still higher than that in its corresponding control. Generally, in the subsurface flow system (SSF) with variable concentrations of diesel, S. grossus could survive and enhance degradation of hydrocarbons in all treatments. The submerged substrate includes the plant roots growing in the media of large gravel, small gravel and fine sand which enhance hydrocarbon degradation throughout the plant rhizosphere (Agamuthu et al., 2010). Indeed, all the mechanisms of volatilisation, photochemical oxidation, sedimentation, sorption and biological degradation have taken part in the major processes promoting organic contaminants removal in wetlands (Imfeld et al., 2009).

3.3. Degradation of TPH in sand To obtain more information about diesel degradation in the substrate, sand extraction was conducted. Fig. 4 shows degradation and removal over 72 days for the three different diesel concentrations treatments with plants and without plants (8700, 17,400 and 26,100 mg/L). The removal efficiency of diesel pollutants in most treatments was significantly different between the three concentrations and sampling days (7, 14, 28, 42 and 72). The maximum TPH degradation removal in sand of 69.9% occurred with a diesel concentration of 17,400 mg/L after 72 days of treatment, while the average removal in its corresponding control treatment was only 51.9%. Similarly, the degradation rates with diesel concentrations of 8700 and 26,100 mg/L were 67.2 and 67.6%, respectively, while the average removal in the corresponding control treatments was 44.3 and 58.1%, respectively. The convergence of results indicates the ability of S. grossus to enhance degradation of TPH and survive in these three diesel concentrations in a subsurface flow system. Statistical analysis was performed between treatments with plants and without plants at each sampling time for all diesel concentrations as illustrated in Fig. 4. Due to the interaction between

Fig. 4. Degradation percentage in sand extraction by Scirpus grossus exposed to diesel contamination at 8700, 17,400 and 26,100 mg/L. Bars indicate the standard error of three replicates (n = 3). Letter A: statistically significant diesel removal from water between two treatments with plant and without plant was represented (p < 0.05).

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Fig. 5. Plant (DW/WW) ratio percentage of the three diesel concentrations with diesel-free (plant control).

the plants themselves and the rhizobacteria, the hydrocarbons were metabolised. In the unplanted sands, TPH was degraded by volatilisation, eluviation and photolysis, as reported by Peng et al. (2009). In addition to these factors, the decrease in the diesel concentration in control sand may also be the consequence of biodegradation by indigenous microorganisms (Peng et al., 2009). As what being claimed by Lu et al. (2010), the mechanisms of TPH phytoremediation in the sand include volatilisation, leaching, photo-degradation (contaminated at the surface), plant uptake, biodegradation, and other abiotic losses. However, the main mechanism of TPH phytoremediation in contaminated soils is assumed to be rhizodegradation. The rhizodegradation is the stimulation of bacteria in the rhizosphere zone to degrade and enhance removal of TPH (Cai et al., 2010). There have been many studies on the phytoremediation of TPH, especially in soil. According to Liu et al. (2011), the removal of diesel from soil contaminated with 15,000 mg/kg diesel by Scirpus triqueter was 67.41 and 72.62% with plants and without plants, respectively. Moreira et al. (2011) attempted to use Rhizophora mangle L. to phytoremediate TPH with 87% removal from a TPH concentration of 33.2–4.5 mg/g, whereas in the corresponding control without plants, the removal was only 70% of TPH, from a concentration of 33.2–9.2 mg/g. Huesemann et al. (2009) studied in situ phytoremediation of PAH and PCB-contaminated marine sediments with eelgrass (Zostera marina). They obtained 73 and 60% removal, respectively, while in the corresponding control treatment without plants; the efficiency of removal was only 25 and 0%, respectively. In addition, Peng et al. (2009) studied the degradation of TPH by Mirabilis jalapa and showed that the average efficiency of removing TPH over a 127-day culture period was high, up to 41.6–63.2%, while the removal rate by natural attenuation was only 19.7–37.9%. The maximum reduction occurred in the saturated hydrocarbon fraction compared with other components of petroleum contaminants. It was indicated that M. jalapa had a peculiar tolerance to petroleum contamination and could effectively promote the degradation of TPH when the concentration of petroleum hydrocarbons in soil was equal to and lower than 10,000 mg/kg (Peng et al., 2009). 3.4. Plant responses to the diesel contaminant Throughout 72 days of culture, none of the plants growing in sand irrigated with diesel-contaminated water showed obvious differences in appearance compared with those in the

corresponding controls (S. grossus without the contaminant). No plant death was recorded with diesel concentrations of 8700 and 17,400 mg/L. However, the growth of S. grossus was badly affected at a diesel concentration of 26,100 mg/L, indicating that there was inhibition of plant growth compared with the corresponding control. Obviously, for 17,400 and 26,100 mg/L diesel concentrations, some of the plants showed signs of phytotoxicity, such as yellowing leaves and impaired growth compared with the control. These signs are in agreement with the results of Agamuthu et al. (2010). Water content in the plant tissue was affected under stress conditions of contaminated media. To evaluate this parameter, the effect of diesel on the dry weight to wet weight (DW/WW) ratios of the plants was depicted in Fig. 5. The ratio ranges between 15.7 and 15.6% for plant control during 72 days of exposure. It was found that for 8700, 17,400 and 26,100 mg/L diesel concentrations, the ratio of DW/WW decreased in the first 14 days, then start to increase indicating some signs of phytotoxicity effect to diesel contamination until the end of 72 days. The ratio (DW/WW) increased as the diesel concentration increases. For 8700, 17,400 and 26,100 mg/L diesel concentrations, the ratio increased until 72 days to 18.0, 19.3 and 28.3% respectively relative to the diesel-free (plant control) which is only 15.6% (Fig. 5). Statistically analysis showed no significant difference between the (DW/WW) ratio for 8700 and 17,400 mg/L diesel concentrations with corresponding diesel-free (plant control). While there is a significant difference with the 26,100 mg/L diesel concentration due to the inhibit plants after day 14. Fig. 6 depicts the tolerance of S. grossus based on the growth parameters recorded after 72 days of diesel exposure. In the experiment, the wet and dry weights of S. grossus growing in sand irrigated with 8700, 17,400 and 26,100 mg/L of diesel were significantly (p < 0.05) less than those of S. grossus in the corresponding control. However, there was no significant difference between the concentrations of 8700 and 17,400 mg/L in terms of the wet and dry weights due to phytoremediation efficiency of S. grossus up to 17,400 mg/L of diesel. According to Fig. 6c, the stem length of S. grossus growing in sand irrigated with 8700 and 17,400 mg/L of diesel was insignificantly (p > 0.05) shorter than that of S. grossus planted in the corresponding control. However, the stem length of S. grossus with 26,100 mg/L of diesel was significantly (p < 0.05) shorter than that of S. grossus planted in the corresponding control. The root length of plants in sand irrigated with 8700 mg/L diesel was insignificantly (p > 0.05) reduced when compared with the corresponding control and in sand irrigated with 17,400 mg/L

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Fig. 6. Growth response parameters: (a) wet weight, (b) dry weight, (c) stem height and (d) root length in the phytotoxicity test of Scirpus grossus with diesel as the contaminant. Error bars indicate the standard deviation (n = 3). The means among different diesel concentrations followed by the same letter (A–C) were not significantly different at p < 0.05.

diesel. Visibly, extremely high diesel concentrations might inhibit the growth of S. grossus. 3.5. Rhizosphere microbial count The microbial population in S. grossus rhizosphere zone was evaluated at different diesel concentrations (0, 8700, 17,400 and 26,100 mg/L), as shown in Fig. 7. It was found that the diesel pollutant inhibited the microbial population and reduced its diversity. The removal of hydrocarbons through degradation mainly depends on the capabilities of the microorganisms in the surrounding rhizosphere (Liu et al., 2011). During the experiment, microbial populations in the control aquarium were significantly

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Fig. 7. Total count of the bacterial population during the 72 days of the experiment. Comparison of the microbial populations in the control and with different diesel concentrations of 8700, 17,400, and 26,100 mg/L.

lower than those in the aquaria with diesel concentrations of 8700, 17,400 and 26,100 mg/L. Liu et al. (2011) inferred that, during their experiment on diesel degradation, microbial populations in the control soils were significantly lower than those in the indigenous hydrocarbon-degrading microorganisms (HDMs) and S. triqueter L. (STL). The population of rhizobacteria in the plant control aquarium without contamination (0 mg/L) was 4.6 × 105 CFU mL−1 water at day 0, and was obviously lower than that in the treatments with different diesel concentrations. In other words, the population of bacteria in aquaria irrigated with diesel-contaminated water was clearly higher than in the control aquarium. Similarly, the population of bacteria in treatments with the highest diesel concentration of 26,100 mg/L amounted to 1.43 × 106 CFU mL−1 water at day 0, which was similar with a diesel concentration of 8700 and 17,400 mg/L. The bacterial population in the treatment with lowest diesel concentration of 8700 mg/L amounted from 5.5 × 105 to 1.88 × 105 CFU mL−1 water during 72 days of treatment, which was lower than with the other diesel concentrations of 17,400 and 26,100 mg/L. Generally, the bacterial population in the treatments with diesel concentrations of 8700, 17,400 and 26,100 mg/L increased until 7 days, and then started to decrease to the end of the 72 day period of exposure. The growth of bacteria with a diesel concentration of 17,400 mg/L for 72 days was stable within a range of 9.5 × 105 –6.5 × 105 CFU mL−1 water. Thus, the growth of bacteria with 17,400 mg/L diesel was not highly affected which may promote the degradation of petroleum hydrocarbons. In other words, the bacterial population was suitable for the bioremediation of ≤17,400 mg/L diesel-contaminated water. A significant difference was shown in the microbial populations between the control aquaria (0 mg/L) and those with different diesel concentrations (8700, 17,400 and 26,100 mg/L) during the exposure period (p < 0.05). The populations noticeably increased in the rhizosphere

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Table 1 Correlation coefficients for TPH degradation, rhizosphere bacterial counts and root length in Scirpus grossus planted in sand irrigated with diesel-contaminated water. TPH degradation TPH degradation 1 Total rhizosphere bacteria −0.685a Root length 0.685a a

Total rhizosphere bacteria

Root length

−0.658a 1 −0.691a

0.685a −0.691a 1

Correlation is significant at the 0.01 level.

of S. grossus irrigated with water contaminated with diesel compared with the control rhizosphere of S. grossus irrigated with water. This provided evidence that the growth of microorganisms in the S. grossus rhizosphere zone was enhanced when irrigated with water contaminated by diesel. The correlation coefficients between S. grossus root–microbial interactions and TPH degradation are presented in Table 1. Degradation of TPH showed a negative correlation (r = −0.658, p < 0.01) with the total rhizosphere bacteria, while it was significantly (r = 0.685, p < 0.01) related to the root length of S. grossus. It is suggested that the population of bacteria decreased gradually throughout the 72 day experimental period while TPH removal increased. The degradation of TPH may have weakened the bacterial populations (Xiao et al., 2010). A significant (r = −0.691, p < 0.01) negative correlation was also found between the total rhizosphere bacterial count and the root length of S. grossus. These results indicate that the total rhizosphere microbes had an influence with high diesel concentrations; however, the growth of S. grossus corresponded well with TPH degradation. 4. Conclusions After 72 days of wastewater treatment in a subsurface flow system, it was demonstrated that S. grossus has the capability to survive and provide good conditions for rhizobacteria to degrade hydrocarbon at all investigated diesel concentrations (8700, 17,400 and 26,100 mg/L). Based on wastewater and sand extraction, the highest TPH removal of 91.5 and 69.9% was obtained at a diesel concentration of 17,400 mg/L respectively. Results indicated that SSF system could be implementing for future pilot scale study with S. grossus to remediate contaminated water with diesel concentration up to 17,400 mg/L. Acknowledgments The authors would like to thank Universiti Kebangsaan Malaysia (UKM-KK-03-FRGS0119-2010) and the Tasik Chini Research Centre for supporting this research project. They also acknowledge with thanks to the Iraqi Ministry of Higher Education and Scientific Research for providing a doctoral scholarship for the first author. References Agamuthu, P., Abioye, O., Abdul Aziz, A., 2010. Phytoremediation of soil contaminated with used lubricating oil using Jatropha curcas. J. Hazard. Mater. 179, 891–894. Anonymous, 2011. http://www.cabicompendium.org/NamesLists/CPC/Full/SCIRPS. htm (27.07.11).

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