Enhanced remediation of sewage effluent by ...

Ecological Engineering 84 (2015) 58–66

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Enhanced remediation of sewage effluent by endophyte-assisted floating treatment wetlands Amna Ijaz, Ghulam Shabir, Qaiser M. Khan, Muhammad Afzal ∗ Environmental Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan

a r t i c l e

i n f o

Article history: Received 2 May 2015 Received in revised form 22 June 2015 Accepted 28 July 2015 Keywords: Floating treatment wetlands Sewage effluent Phytoremediation Endophytic bacteria Wastewater treatment Plant–bacteria partnerships

a b s t r a c t The use of floating treatment wetlands (FTWs) is a promising approach for the remediation of sewage effluent. The efficiency of FTWs can be improved by the combined use of plants and pollutant-degrading bacteria. The aim of this study was to evaluate the influence of inoculation of endophytic bacteria on the detoxification of sewage effluent in FTWs. A terrestrial plant, Brachiaria mutica, was vegetated on a floating mat and inoculated with three endophytic bacterial strains, Acinetobacter sp. strain BRSI56, Bacillus cereus strain BRSI57, and Bacillus licheniformis strain BRSI58, to develop FTWs for the remediation of sewage effluent of Faisalabad city (Pakistan). Results indicated that B. mutica has the potential to remove both organic and inorganic contaminants from sewage effluent. However, endophytic inoculation in FTWs further enhanced the removal efficiency. Maximum reduction in chemical oxygen demand (COD), biochemical oxygen demand (BOD5 ), total nitrogen (N), and phosphate (PO4 ) was achieved by the combined use of plants and bacteria. Moreover, the inoculated bacteria showed persistence in water as well as colonization in the root and shoot of the plant. Treated effluent met the national wastewater discharge standards of Pakistan and can be discharged in the environment without any environmental risks. This study provides useful evidence of endophyte-assisted FTWs to be the most sustainable and affordable approach for in situ remediation of sewage effluent. © 2015 Published by Elsevier B.V.

1. Introduction Worldwide urbanization has increased the release of sewage effluent in surface water resources. Sewage effluent contains different types of organic and inorganic contaminants, which deteriorate the quality of water resources (Bueno et al., 2012). The use of FTWs is a relatively new approach whereby plants are vegetated on soilless buoyant mats in a manner that underground biomass hangs freely in the water column flowing underneath the mat (De Stefani et al., 2011; Hwang and LePage, 2011). Despite the simple setup and ease of establishment of FTWs, researchers have reported them to be a highly effective approach toward improving the quality of sewage effluent (Borne et al., 2013; Lynch et al., 2015; Zhang et al., 2014). In FTWs, plants, in combination with microbial partners, employ natural physical, chemical, and biological processes to remove pollutants from contaminated water (Chang et al., 2013; Zhang et al., 2014). In plant–bacteria association, bacteria are capable of

∗ Corresponding author at: Environmental Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad 38000, Pakistan. E-mail addresses: [email protected], [email protected] (M. Afzal). http://dx.doi.org/10.1016/j.ecoleng.2015.07.025 0925-8574/© 2015 Published by Elsevier B.V.

contributing toward overall pollutant removal by degrading complex organic pollutants and assimilating N and phosphorous (P) (Afzal et al., 2014a; Khan et al., 2013a; Li et al., 2010). Bacteria are also reported to reduce concentration of nitrate (NO3 ) (Nagadomi et al., 2000), PO4 , and heavy metals in wastewater (El-Sheekh et al., 2005), through metabolism-dependent and independent methods (da Costa and de Franc¸a, 2003). Although microorganisms play an important role in the mineralization of organic pollutants and the biogeochemical transformation of nutrients in wetlands, nothing is known about the persistence and activity of the inoculated bacteria in different components (water, root and shoot) of FTWs. Several microorganisms from water can colonize on the root or rhizome surface and establish the so-called biofilm through a repeating proliferation process (Zhang et al., 2014). Some of the root colonizing bacteria penetrate the root, colonize within it, and/or migrate to the aerial parts; these are known as endophytes (Afzal et al., 2014b; Compant et al., 2010). Microbial population on the root surface and inside the plant tissues enhances the removal of pollutants from water (Newman and Reynolds, 2005; Weyens et al., 2013; Shehzadi et al., 2014). Different aquatic plants, such as Pontederia cordata, Schoenoplectus tabernaemontani (Wang et al., 2015), Cyperus ustulatus, Juncus edgariae (Tanner and Headley, 2011), Juncus effuses, Pontederia cordata (Chang et al., 2013), Typha angustifolia (Keizer-Vlek

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Fig. 1. Schematic diagram showing the development of floating treatment wetlands for the treatment of sewage effluent.

et al., 2014), Oenanthe javanica (Zhou and Wang, 2010), Gardenia jasminoides (Zhu et al., 2011), Canna indica (Sun et al., 2009) and Carex virgate (Borne et al., 2013) have been used to develop FTWs. However, Brachiaria mutica, commonly known as para grass, has not been tested to develop FTWs to date. Moreover, the combined use of plants and endophytic bacteria has not been evaluated in FTWs for the remediation of sewage effluent. Therefore, the aim of this study is to evaluate the potential of B. mutica on its own and in combination with endophytic bacteria in FTWs for the remediation of sewage effluent. Removal of organic and inorganic pollutants as well as toxicity reduction of the treated effluent has been observed. Moreover, the persistence of the inoculated bacteria has been determined in different components (root, shoot and water) of the FTWs as well.

2. Methodology 2.1. Sewage effluent collection and characterization Faisalabad city is the industrial hub of Pakistan with a population of more than 2.7 million and sewage effluent generation of nearly 435 million gallons per day that is discharged in the Chenab and Ravi rivers through Paharang and Madhuana drains, respectively (Kahlown et al., 2006). Many small and large scale industries are located in the city area and discharge their wastewater in these sewage effluent drains. Sewage effluent was collected in September and October 2014 from Paharang and Madhuana drains, respectively, and analyzed for various physicochemical parameters such as pH, electrical conductivity (EC), COD, BOD5 , total dissolved solids

Fig. 2. Different components of floating treatment wetland microcosm.

Batch microcosm experiments were performed in September and October 2014 under ambient conditions of temperature and light at National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad. Experiments were performed in twelve 30 l polyethylene tanks. Floating mats (Diamond JumbolonBoard, manufactured by Diamond Foam Company Private Limited,

6.6b (0.3) 6.5b (0.2) 3.6b (0.2) 3.5b (0.2) 2.7c (0.3) 2.9c (0.2) 1.5ab (0.2) 1.5ab (0.3) 156ab (10) 146b (6) 6.8b (0.3) 3.8ab (0.3) 2.6c (0.4) 1.6ab (0.3) 168ab (12) 6.1bc (0.2) 7.6a (0.1) 7.2b (0.2) 1.6cd (0.3) 4.7a (0.1) 4.2a (0.2) 7.1a (0.4) 0.5d (0.1) 1.5cd (0.3) 0.5d (0.2) 1.9a (0.1) 1.8a (0.2) 76d (5) 196a (5) 170ab (8) 7.2a (0.2) 3.8ab (0.2) 1.5cc (0.2) 1.5ab (0.2) 136b (7) 6.8b (0.2) 7.6a (0.1) 2.0c (0.2) 4.7a (0.1) 5.9b (0.5) 0.5d (0.1) 0.8c (0.2) 1.9a (0.1) 90cd (4) 196a (6) 7.0ab (0.3) 3.9ab (0.1) 2.8c (0.4) 1.2b (0.1) 125bc (6)

7.2c (0.3) 7.0cd (0.6) 3.1ab (0.2) 2.9ab (0.3) 1.5cd (0.3) 1.6cd (0.3) 1.66b (0.2) 1.58b (0.3) 58ab (4.8) 55b (3.1) 7.5bc (0.6) 3.4a (0.3) 0.5d (0.1) 1.75ab (0.2) 61ab (5.5) 7.2c (0.5) 6.1e (0.3) 7.7a (0.4) 7.5bc (0.5) 3.0ab (0.4) 1.2d (0.2) 3.5a (0.2) 3.5a (0.2) 3.5bc (0.7) 6.8ab (0.8) 0.5d (0.1) 0.5d (0.1) 1.63b (0.2) 1.01d (0.2) 2.05a (0.1) 1.84ab (0.1) 62ab (4.9) 34c (2.3) 79a (4.8) 68a (4.2) 7.7a (0.4) 3.5a (0.2) 0.5d (0.1) 2.05a (0.1) 79a (3.8) 7.2c (0.5) 6.5d (0.4) 2.6b (0.3) 1.7c (0.2) 4.5b (0.7) 8.0a (1.2) 1.65b (0.1) 1.25c (0.2) 63ab (3.8) 49b (3.4)

48 h 24 h 0h

T4

72 h 48 h 24 h 0h

T3

Units: EC, mS cm−1 ; DO, mg l−1 ; Na, g l−1 ; and K, mg l−1 . Each value is a mean of three replicates, standard deviations are presented in parentheses. Comparisons between treatments were carried out by one-way analysis of variance (ANOVA).

2.4. Experimental setup

7.8a (0.2) 7.6a (0.1) 7.3a (0.2) 4.5a (0.2) 4.7a (0.1) 4.2a (0.2) 0.3d (0.1) 0.5d (0.1) 1.5cd (0.3) 1.6ab (0.2) 1.9a (0.2) 1.5ab (0.2) 173ab (8) 196ab (4) 144b (7)

Bacterial strains, Acinetobacter sp. strain BRSI56, Bacillus cereus strain BRSI57, and Bacillus licheniformis strain BRSI58, previously isolated from the shoot of B. mutica (Fatima et al., 2015), were used in this study. These bacteria were chosen on the basis of their potential to reduce COD and BOD in sewage effluent (data not shown). Moreover, these bacterial species possessed plant growth promoting activities such as production of 1-aminocyclopropane1-carboxylate (ACC) deaminase, phosphorous solubilization, and siderophore production (Fatima et al., 2015). These strains were cultivated in Luria-Bertani (LB) broth. Cells were harvested by centrifugation and re-suspended in 0.9% (w/v) sodium chloride (NaCl) solution and the optical density (OD) of culture was adjusted to 0.7 at 600 nm.

Madhuana drain 7.6a (0.1) 7.7a (0.2) 7.7a (0.1) pH 4.7a (0.1) EC 4.7a (0.1) 4.6a (0.2) DO 0.5d (0.1) 0.5d (0.1) 0.4d (0.1) 1.9a (0.1) 1.8a (0.2) 1.7a (0.2) Na 196a (4) 192a (5) 188a (6) K

2.3. Endophytic bacterial strains

8.2a (0.8) 7.7b (0.4) 7.5bc (0.3) 3.4a (0.3) 3.5a (0.2) 3.1ab (0.4) 0.5d (0.1) 0.5d (0.1) 2.5c (0.4) 2.05a (0.1) 1.91a (0.2) 1.68b (0.2) 65ab (5.2) 79a (4.8) 72a (5.3)

B. mutica is a terrestrial plant chosen from among those that can survive and grow in the harsh environmental conditions of Pakistan. It is a perennial grass with long, coarse stolons up to 5 m. Its reproduction is usually by vegetative means. It is a fastgrowing plant and has been used widely around the world for the phytoremediation of contaminated soils (Mohanty and Patra, 2012).

Paharang drain 7.7b (0.2) 7.6b (0.8) 7.8a (0.7) pH 3.5a (0.2) 3.5a (0.2) 3.5a (0.2) EC 0.5d (0.1) 0.5d (0.1) 0.5d (0.1) DO 2.05a (0.1) Na 2.02a (0.1) 1.98a (0.3) K 79a (4.8) 76a (6.4) 71a (5.2)

2.2. B. mutica (Forssk.) Stapf

72 h

(TDS), total solids (TS), N, P, and different metals as described earlier (Eaton et al., 2005).

48 h

Each value is a mean of three replicates, standard deviations are presented in parentheses, NG = not given in NEQS list, NEQS = National Environmental Quality Standards for wastewater discharge, set by Government of Pakistan.

24 h

6–10 NG 150 80 NG NG 3500 150 NG NG 1000 600 NG NG 0.1 NG 1 NG 2 NG 1 NG 10

0h

7.6 (0.85) 5.1 (0.28) 508 (83) 213 (65) 0.5 (0.04) 4354 (620) 4113 (450) 180 (24) 2040 (260) 165 (23) 1150 (108) 505 (78) 34.3 (3.6) 11.4 (2.4) 0.78 (0.08) 0.70 (0.09) 8.7 (1.33) 0.82 (0.12) 13.7 (1.60) 1.3 (0.28) 6.5 (0.62) 0.62 (0.08) 15.7 (2.7)

T2

7.7 (1.3) 3.9 (0.6) 460 (105) 189 (58) 0.5 (0.1) 3444 (580) 3279 (460) 164 (12) 1926 (320) 79.9 (17) 939 (82) 328 (45) 41.5 (4.6) 16.3 (2.9) 0.5 (0.07) 0.65 (0.08) 2.3 (0.36) 0.56 (0.04) 11.6 (1.2) 0.74 (0.28) 3.8 (0.48) 0.65 (0.73) 8.5 (1.3)

72 h

Madhuana drain

48 h

NEQS

Paharang drain

24 h

pH EC (mS cm−1 ) COD (mg l−1 ) BOD (mg l−1 ) DO (mg l−1 ) TS (mg l−1 ) TDS (mg l−1 ) TSS (mg l−1 ) Na (mg l−1 ) K (mg l−1 ) Cl (mg l−1 ) SO4 (mg l−1 ) Total N (mg l−1 ) PO4 (mg l−1 ) Cd (mg l−1 ) Co (mg l−1 ) Cr (mg l−1 ) Cu (mg l−1 ) Fe (mg l−1 ) Mn (mg l−1 ) Ni (mg l−1 ) Pb (mg l−1 ) Oil and grease (mg l−1 )

Sources

0h

Parameter

T1

Table 1 Characteristics of sewage effluent collected from Paharang and Madhuana drains, Faisalabad, Pakistan.

72 h

A. Ijaz et al. / Ecological Engineering 84 (2015) 58–66 Table 2 Remediation of sewage effluent without vegetation and endophytes inoculation (T1), with vegetation of B. mutica (T2), with vegetation of B. mutica and endophytes inoculation (T3), and with endophytes inoculation only (T4) at different time intervals.

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Fig. 3. Percentage removal of COD, BOD, sulphates, chlorides and oil and grease from sewage effluent of Paharang drain by FTWs after 96 h. Different treatments were: T1: sewage wastewater without vegetation and bacterial inoculation; T2: sewage wastewater with vegetation only; T3: sewage wastewater with vegetation and bacterial inoculation; T4: sewage wastewater with bacterial inoculation only. Each vale is mean of three replicates and labels (a)–(f) indicate statistically significant differences between treatments at a 5% level of significance.

Pakistan) with dimension of 50.8 cm length, 38.1 cm width, and 7.6 cm thickness were used. Five holes (5 cm diameter of each) were made in each floating mat (Figs. 1 and 2). Five cuttings of B. mutica were planted in each hole of the floating mat. The floating mats were placed in polyethylene tank filled with 25 l tap water. The plants were allowed to establish their roots in the tap water

for a month after which sewage effluent was introduced in the tanks. To evaluate the effect of plant, bacteria, and plant–bacteria partnership, plants and bacteria were applied in combination as well as individually in the tanks containing the sewage effluent. For bacterial inoculation, 100 ml culture (OD 0.7) of the three strains, Acinetobacter sp. strain BRSI56, B. cereus strain BRSI57, and

Fig. 4. Percentage removal of COD, BOD, sulphates, chlorides and oil and grease from sewage effluent of Madhuana drain by FTWs after 192 h. Different treatments were: T1: sewage wastewater without vegetation and bacterial inoculation; T2: sewage wastewater with vegetation only; T3: sewage wastewater with vegetation and bacterial inoculation; T4: sewage wastewater with bacterial inoculation only. Each vale is mean of three replicates and bars indicate standard deviation among them. Labels (a)–(f) indicate statistically significant differences between treatments at a 5% level of significance.

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Table 3 Nutrients (N and PO4 ) removal from sewage effluent without vegetation and bacterial inoculation (T1), with vegetation of B. mutica (T2), with vegetation of B. mutica and endophytes inoculation (T3), and with endophytes inoculation only (T4) at different time intervals. Treatments

Paharang drain 0h −1

Total N (mg l T1 T2 T3 T4 PO4 (mg l−1 ) T1 T1 T2 T3

Madhuana drain 24 h

48 h

72 h

0h

24 h

48 h

72 h

) 40.8a 40.8a 40.8a 40.8a

(1.2) (1.2) (1.2) (1.2)

39.9a (3.2) 36.1a (2.4) 32.4b (1.5) 36.4a (2.6)

38.4a (3.7) 31.1b (1.5) 27.6bc (1.8) 32.8b (3.8)

37.4a (2.9) 17.9d (2.8) 13.4e (1.7) 26.7b (4.2)

37.8a 37.8a 37.8a 37.8a

(1.5) (1.4) (1.1) (1.2)

35.3a (3.1) 27.3b (2.5) 26.3b (1.8) 31.9ab (3.8)

33.4a (2.8) 25.2b (1.5) 13.4c (2.6) 28.2ab (1.8)

31.9a (1.9) 14.6c (1.2) 6.9d (1.8) 25.2b (1.3)

15.3a 15.3a 15.3a 15.3a

(0.7) (0.7) (0.7) (0.7)

15.1a (1.1) 10.8b (1.4) 8.4c (0.7) 14.3a (0.9)

14.7a (0.9) 7.8b (0.8) 5.1c (0.9) 13.6a (0.8)

13.6a (0.8) 3.8b (0.4) 2.9c (0.3) 12.2a (0.5)

10.3a 10.3a 10.3a 10.3a

(0.5) (0.3) (0.3) (0.3)

10.2a (0.8) 7.1b (0.3) 5.1c (0.5) 9.7a (0.3)

10.1a (0.5) 6.4b (0.7) 3.2c (0.6) 9.2a (0.8)

9.7a (1.2) 3.7b (0.2) 2.1c (0.2) 7.4b (0.6)

Each value is a mean of three replicates, standard deviations are presented in parentheses. Comparisons between treatments were carried out by one-way analysis of variance (ANOVA).

B. licheniformis strain BRSI58, was used to inoculate the wastewater. Different treatments (in triplicates) were: T1: sewage wastewater without vegetation and bacterial inoculation T2: sewage wastewater with vegetation only T3: sewage wastewater with vegetation and bacterial inoculation T4: sewage wastewater with bacterial inoculation only

2.8. Statistical analysis SPSS software package version 17.0 (SPSS, Inc., Chicago, IL) was used for analyzing data. The data (three replicates of each treatment) were subjected to analysis of variance (ANOVA) and the means [±standard deviation (SD)] were compared using Duncan’s multiple range test. 3. Results and discussion

2.5. Sewage effluent remediation Treated sewage effluent samples were collected after every 24 h to determine the effect of plants, bacteria, and plant–bacteria partnerships on the remediation of sewage effluent. pH and EC were measured using bench top pH and EC meter, respectively. COD, BOD5 , TS, TDS, total suspended solids (TSS), dissolve oxygen (DO), oil and grease, sulphate (SO4 ), and chloride (Cl) were measured as described earlier (Eaton et al., 2005). Total N was estimated using Millipore nitrogen (total) cell test kit by Merck & Co., while PO4 were determined using Millipore phosphate test kit by Merck & Co., and sodium (Na) and potassium (K) were measured by flame photometery (manufacture: FP 20, SEAC, Italy). Heavy metals including Cd, Co, Cr, Cu, Fe, Mn, Ni, and Pb were determined quantitatively using atomic absorption spectroscopy (manufacture: Varian SpectrAA.200, Varian Australia, Australia).

2.6. Enumeration of inoculated bacteria At the end of the experiment, the roots and shoots of T3 treatment were collected and surface sterilized using freshly prepared 70% ethanol and 2% sodium hypochlorite solutions. Surface sterilized roots and shoots were homogenized in 0.9% NaCl solution and plated on LB agar plates. Moreover, the treated sewage effluent samples of T3 and T4 treatments were also plated on LB agar plates. The plates were incubated at 37 ◦ C overnight followed by randomly selecting 100 isolates that were subjected to restriction fragment length polymorphism (RFLP) analysis whereby their identity was compared with the inoculated strains.

2.7. Estimation of toxicity using fish toxicity assay At the end of the experiment, treated sewage effluent was collected from each container and its toxicity was determined as described earlier (Afzal et al., 2008). Briefly, 10 healthy fish (Labeo rohita) of uniform weight were exposed to treated effluent. The number of fish that died were counted every 24 h for 4 days.

3.1. Effluent characteristics Physicochemical analysis of sewage effluent from Paharang and Madhuana drains revealed that both drains contained high values of COD, BOD5 , TSS, Cd, Cr, Fe, and Ni than those allowed by the wastewater discharge standards [National Environmental Quality Standards (NEQS, 1999)] of Pakistan (Table 1). Among these two drains, Madhuana drain was found to be more polluted as it contained higher values of TDS, Cl, and oil and grease. Our analyses indicate that sewage effluent from drains of Faisalabad city should be treated before discharge in the environment. 3.2. Remediation of sewage effluent The floating mats vegetated with B. mutica efficiently removed organic and inorganic pollutants from the sewage effluent of both drains. Plants are naturally capable of utilizing their metabolic and hydraulic processes to remove a wide range of toxic and harmful chemicals like chlorinated solvents, pesticides, metals, crude oil, explosives, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons, landfill leachates etc. (Schwitzguébel et al., 2009; Afzal et al., 2014a; Vymazal, 2014). However, a significantly better performance of the same plant species in association with endophytes was observed. This can be attributed to the fact that inoculated bacteria can degrade the recalcitrant organic pollutants present in effluent (Afzal et al., 2014b; Khandare et al., 2013; Shehzadi et al., 2014). Moreover, plants provide a microenvironment around their roots, which is optimal for microbes to thrive in and remove pollutants of nearly all types (Van de Moortel, 2008). The amount of DO in the effluent of both drains was very low at the start of experiment, and it steadily increased in all treatments (Table 2). However, contrary to previous research (Iamchaturapatr et al., 2007; Sooknah and Wilkie, 2004), the amount of DO was significantly higher in the vegetated treatments as compared to the unvegetated treatments. This might be due to the fact that in our study, unvegetated treatment was also provided cover with the floating mat that blocked sunlight in it as well and hence ruled out

Units: mg l−1 , each value is a mean of three replicates, standard deviations are presented in parentheses. Comparisons between treatments were carried out by one-way analysis of variance (ANOVA).

0.70a (0.15) 0.62ab (0.07) 7.6a (0.4) 0.78a (0.09) 12.5a (0.8) 1.07a (0.1) 5.8a (0.4) 0.52a (0.05) 0.72a (0.08) 0.65a (0.17) 8.1a (0.3) 0.80a (0.05) 12.8a (0.4) 1.1a (0.1) 6.2a (0.2) 0.55a (0.06) 0.75a (0.13) 0.68a (0.06) 8.3a (0.5) 0.82a (0.11) 13.5a (0.5) 1.1a (0.2) 6.5a (0.4) 0.58a (0.04) 0.78a (0.06) 0.72a (0.07) 8.7a (0.4) 0.82a (0.05) 13.7a (0.4) 1.3a (0.1) 6.5a (0.3) 0.62a (0.02) 0.24d (0.03) 0.25d (0.02) 1.2de (0.1) 0.18e (0.02) 2.16e (0.2) 0.21d (0.01) 0.54de (0.08) 0.15d (0.02) 0.47bc (0.05) 0.36c (0.03) 4.5bc (0.3) 0.34cd (0.02) 5.3cd (0.4) 0.30c (0.04) 2.5bc (0.1) 0.25c (0.03) 0.63ab (0.08) 0.48bc (0.14) 5.2b (0.7) 0.54bc (0.04) 8.6bc (0.3) 0.62b (0.04) 3.2b (0.2) 0.34b (0.04) 0.78a (0.06) 0.72a (0.07) 8.7a (0.4) 0.82a (0.06) 13.7a (0.4) 1.3a (0.1) 6.5a (0.3) 0.62a (0.02) 0.28d (0.02) 0.25d (0.03) 1.5d (0.2) 0.20d (0.04) 3.1e (0.4) 0.20d (0.01) 0.84d (0.07) 0.22c (0.02) 0.38c (0.06) 0.38c (0.05) 3.2c (0.6) 0.38c (0.06) 6.5c (0.2) 0.33c (0.02) 2.8bc (0.1) 0.35b (0.03) 0.57b (0.04) 0.58b (0.08) 4.6bc (0.4) 0.65b (0.06) 10.4b (0.6) 0.68b (0.06) 3.5b (0.2) 0.38b (0.04) 0.78a (0.06) 0.72a (0.06) 8.7a (0.4) 0.82a (0.05) 13.7a (0.2) 1.3a (0.1) 6.5a (0.6) 0.62a (0.2) 0.74a (0.07) 0.65a (0.10) 8.4a (0.5) 0.74a (0.07) 12.8a (0.4) 1.1a (0.1) 6.1a (0.3) 0.58a (0.06) 0.76a (0.04) 0.72a (0.08 8.6a (0.6) 0.80a (0.04) 13.7a (0.3) 1.3a (0.1) 6.4a (0.5) 0.62a (0.04) Madhuana drain Cd 0.78a (0.06) Co 0.72a (0.07) Cr 8.7a (0.4) Cu 0.82a (0.05) 13.7a (0.4) Fe Mn 1.3a (0.1) 6.5a (0.3) Ni Pb 0.62a (0.02)

0.75a (0.06) 0.68a (0.13) 8.5a (1.1) 0.75a (0.08) 13.5a (0.5) 1.2a (0.2) 6.2a (0.2) 0.60a (0.03)

0.5a (0.02) 0.58a (0.02) 1.57b (0.2) 0.45ab (0.03) 10.3a (1.1) 0.64ab (0.07) 3.3a (0.2) 0.60a (0.08) 0.5a (0.02) 0.62a (0.02) 1.74ab (0.2) 0.48a (0.06) 10.8a (0.2) 0.68a (0.06) 3.3a (0.5) 0.62a (0.13) 0.5a (0.02) 0.65a (0.01) 1.90a (0.1) 0.55a (0.04) 11.4a (0.3) 0.73a (0.03) 3.7a (0.3) 0.63a (0.07) 0.5a (0.02) 0.65a (0.01) 2.06a (0.2) 0.56a (0.04) 11.6a (0.7) 0.74a (0.04) 3.8a (0.4) 0.65a (0.05) 0.24c (0.03) 0.24e (0.02) 0.46e (0.03) 0.22c (0.08) 1.7f (0.4) 0.18d (0.03) 0.92d (0.2) 0.28c (0.05) 0.35b (0.04) 0.45c (0.04) 1.07c (0.1) 0.28b (0.03) 4.7c (0.3) 0.29c (0.03) 1.6c (0.1) 0.37bc (0.08) 0.41ab (0.07) 0.52bc (0.03) 1.54b (0.1) 0.32b (0.06) 6.4b (0.8) 0.51b (0.06) 2.4b (0.2) 0.42b (0.06) 0.5a (0.02) 0.65a (0.05) 2.06a (0.2) 0.56a (0.04) 11.6a (0.7) 0.74a (0.04) 3.8a (0.4) 0.65 (0.05) 0.30c (0.06) 0.25d (0.01) 0.58e (0.04) 0.24c (0.01) 2.4e (0.3) 0.25c (0.04) 1.1cd (0.1) 0.32c (0.04) 0.38b (0.05) 0.38c (0.05) 1.18c (0.1) 0.30b (0.02) 4.8c (0.4) 0.38bc (0.6) 1.5c (0.3) 0.38bc (0.04) 0.43a (0.06) 0.53bc (0.03) 1.64b (0.2) 0.34b (0.05) 6.2b (0.5) 0.47b (0.03) 2.7b (0.6) 0.45b (0.09) 0.5a (0.02) 0.65a (0.01) 2.06a (0.2) 0.56a (0.04) 11.6a (0.7) 0.74a (0.11) 3.8a (0.4) 0.65a (0.05) 0.47a (0.05) 0.58b (0.02) 1.98a (0.1) 0.50a (0.04) 10.5a (0.9) 0.68a (0.04) 3.4a (0.3) 0.54a (0.04) 0.48a (0.01) 0.60a (0.02) 1.98a (0.1) 0.54a (0.03) 11.0a (0.8) 0.70a (0.2) 3.6a (0.3) 0.56a (0.06) 0.5a (0.02) 0.63a (0.02) 2.04a (0.2) 0.54a (0.03) 11.3a (0.6) 0.72a (0.05) 3.6a (0.4) 0.58a (0.08)

72 h 48 h 24 h 0h 48 h 24 h

Paharang drain Cd 0.5a (0.02) Co 0.65a (0.01) Cr 2.06a (0.2) Cu 0.56a (0.04) Fe 11.6a (0.7) Mn 0.74a (0.04) 3.8a (0.4) Ni Pb 0.65a (0.05)

T4

72 h 48 h 24 h 0h

T3

72 h 48 h 0h

24 h T2

0h

72 h T1

Table 4 Heavy metals removal from sewage effluent without vegetation and endophytes inoculation (T1), with vegetation of B. mutica (T2), with vegetation of B. mutica and endophytes inoculation (T3), and with endophytes inoculation only (T4) at different time intervals.

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the possibility of growth of algal systems for photosynthetic oxygen (O2 ) production in the unvegetated treatment. The pH of the effluent was observed to be lesser in the vegetated treatments as compared to the unvegetated treatments (Table 2), which might be due to the release of acidic root exudates and carbon dioxide (CO2 ) production by root respiration (Bezbaruah and Zhang, 2004; Iamchaturapatr et al., 2007; Lynch et al., 2015; Sooknah and Wilkie, 2004). Moreover, the release of organic acids during microbial degradation of organic matter can also decrease the pH of the effluent. Similarly more EC reduction (13–20%) was observed in the vegetated treatments as compared to the unvegetated treatments (Table 2), these results are corroborated by previous findings (Lynch et al., 2015; Van de Moortel, 2008). The concentration of Na and K were reduced substantially in the same proportions as EC in all the treatments indicating the uptake of both ions by the plant. Natural Resources Conservation Service (NRCS) stated that water with EC 2 ms cm−1 is safe for all crops, while 4 ms cm−1 is safe for salt tolerant crops only (NRCS, 1999). Sewage effluent from Paharang drain and Madhuana drains had initial EC of 3.5 and 4.7 ms cm−1 , respectively, while the EC of the treated effluents by treatment T3 was 1.2 and 1.6 ms cm−1 , respectively, which renders the treated effluent safe for use in irrigation. The reduction in BOD5 and COD of aquatic system mainly depends upon the oxygen concentration in effluent as decomposition of pollutants occurs through oxidation reactions (Vymazal, 2010). Vegetation of B. mutica increased the concentration of oxygen in the effluent of both drains and consequently the degradation of organic pollutants (Figs. 3 and 4). Maximum COD and BOD5 reductions were observed in the treatments applied with B. mutica in combination with endophytic bacteria. It might be due to bacterial transformation and decomposition of organic matter (Vymazal, 2010). Similar results were reported previously with bacterially assisted Canna-carrying FTWs (Sun et al., 2009). Moreover, the removal of organic compound, 2,4-dichlorophenoxyacetic acid was observed to be greatly enhanced in two different studies with Hordeum vulgare (Jacobsen, 1997) and Triticum aestivum (Kingsley et al., 1994), when used in combination with endophytes, Burkholderia cepacia and Pseudomonas strains, respectively. Similarly, Ronchel and Ramos performed successful removal of 3-methylbenzoate using Zea mays inoculated with Pseudomonas putida (Ronchel and Ramos, 2001). This validates the use of plants in combination with endopytic bacteria as a promising means of COD and BOD5 reduction. The amounts of SO4 , Cl, and oil and grease were also significantly decreased in the sewage effluent in the presence of vegetation and their removal was further increased by endophytic bacterial inoculation. The vegetated treatments showed significantly better removal of nutrients (N and PO4 ) than the unvegetated treatments. Treatment T3 showed maximum removal efficiency of total N and PO4 (Table 3) hence revealing that plant–endophyte synergism is the most efficient approach for the purpose of eliminating eutrophication from wastewaters. Sun et al. (2009) experimented to reveal that nitrogen was removed up to 72.1% using Canna indica in combination with bacteria in 5 days while only 50.4% was removed in the same time by Canna only. In our study, the removal efficiency of total N by vegetated FTWs was 56.2% (Paharang drain) and increased to 67.4% on inoculation of endophytes. In the case of Madhuana drain, the removal of total N increased from 61% in T2 to 82% in T3. Similarly, more PO4 reduction was observed by bacterial inoculation in the effluent of both drains. The removal of N, which is present as NO3 or NH4 in effluent, occurs through natural metabolic processes of plants and associated microbes including ammonium oxidation, nitrification, and denitrification (Tao and Wang, 2009). Furthermore, plants take up these two entities as nutrients (Vymazal, 2010). Phosphorous is an essential nutrient for

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Table 5 Survival and colonization of the inoculated endophytes in the water, root and shoot of Brachiaria mutica. Treatments

T3 T4

Paharang drain

Madhuana drain

CFU ml−1 water × 103

CFU g−1 root × 103

CFU g−1 shoot × 103

CFU ml−1 water × 103

CFU g−1 root × 103

CFU g−1 shoot × 103

8.7 (2.6) 4.6 (1.4)

17.5 (3.1) –

10.2 (3.6) –

8.1 (1.5) 3.8 (1.8)

15.5 (3.7) –

21.6 (2.4) –

T3: sewage wastewater with vegetation and endophytes inoculation, T4: sewage wastewater with endophytes inoculation only. Each value is a mean of three replicates, standard deviations are presented in parentheses.

both plants and bacteria, although the portion of P in biomass is significantly lower than N (Brix, 1994). 3.3. Reduction in heavy metals Vegetation with B. mutica removed all the heavy metals (Cd, Co, Cu, Cr, Mn, Ni, Fe and Pb) present in the sewage effluent of both drains (Table 4). Cr, a dangerous heavy metal incorporated from textile industries, was effectively removed from the effluent in T2 and T3 (Table 4). Bacterial inoculation again improved the phytoremediation efficiency of B. mutica, which can be attributed to bacterial capacity of sorbing metallic ions on cell walls (Mullen et al., 1989). Moreover, bacterial inoculation enhances the bioavailability and uptake of heavy metals by plants (Sessitsch et al., 2013; Khan et al., 2014). Similarly, there was promising reduction in Fe content in the effluent of Paharang drain: it was reduced down to 79% in T2 and 85% in T3. In case of Madhuana drain, the Fe removal efficiency was increased from 77.4% to 85% by bacterial inoculation. Previously, research has indicated the usefulness of plant–endophyte interaction to lower metal contamination validating our research with B. mutica; for instance, Astragalus sinicus in combination with Mesorhizobium huakii was observed to lower Cd in contaminated substrate (Sriprang et al., 2002). 3.4. Detoxification Fish toxicity analysis revealed that B. mutica partially detoxified the sewage effluent. Partial detoxification is indicated by the survival of 8 and 9 fish (from initial number of 10 living fish) in T2 effluent of Paharang drain and Madhuana drain, respectively, in comparison to only 3 and 2 fish surviving in T1 effluent of Paharang drain and Madhuana drain, respectively (data not shown). However, no fish expired in the treated effluent of T3 of both experiments where bacterial inoculation had been provided with vegetation. This can be attributed to better reduction in salinity, heavy metal concentration, and toxic organic pollutants degradation by the combined use of B. mutica and endophytic bacteria (Shehzadi et al., 2014). 3.5. Persistence of the inoculated endophytes In phytoremediation, organic pollutants are mineralized mostly by plant-associated microbial populations and it has been proposed that the remediation potential of plants correlates with the number of bacteria in their environment (Yousaf et al., 2011; Afzal et al., 2013a; Shehzadi et al., 2014). In this study, the inoculated endophytes showed high level of colonization in the root, shoot, and wastewater (Table 5). This might be due to the fact that these endophytes were isolated from shoot of B. mutica and they were already adapted to proliferate in different compartments of the plant vegetated in sewage water. In unvegetated treatment (T4), comparatively lower numbers of the inoculated bacteria were observed in wastewater than that in the vegetated treatment (T3). This might be due to the use of nutrients in wastewater by the inoculated and indigenous microbes and lack of a symbiotic

partner resulted in declined numbers of inoculated endophytes in T4. Plants provide nutrients and residency to their associated microbial populations (Weyens et al., 2009; Afzal et al., 2012; Khan et al., 2013b) and higher numbers of the inoculated bacteria have been observed in vegetated soil and water than in unvegetated soil and water (Afzal et al., 2013b; Shehzadi et al., 2014). Moreover, inoculation of endophytic bacteria isolated from a specific plant exhibited more efficient colonization within that plant due to better adaptation to conditions of that specific plant (Yousaf et al., 2011). Although bacteria were inoculated in wastewater, larger numbers of the inoculated bacteria were found in the root and shoot than wastewater. This might be due to the fact that these bacteria were endophyes and could colonize more efficiently in the plant endosphere than the wastewater. Previous studies also demonstrated that endophytes exhibited better colonization in the root and shoot than in soil and water (Afzal et al., 2011; Shehzadi et al., 2014). 3.6. Durability of the mat In this research, we have employed the commercially available Diamond Jumbolon-Board composed of non-cross linked polyethylene as a floating mat. The actual purpose of the board is insulating surfaces of buildings, but it was considered ideal for establishment of FTWs due to its inherent strength, moisture barrier, temperature tolerance between −50 ◦ C to 75 ◦ C, weight tolerance of 52 N cm−2 , and resistance to rotting (http://www.jumbolon.com/ board technical properties.htm). Previously, floating structures of plastic pipes filled with foam (Van de Moortel, 2008), coconut fibers (Nakamura and Mueller, 2008), polyvinyl chloride/polypropylene pipes, bamboo reed net, etc. (Hubbard et al., 2004) have been used. However, Jumbolon-Board is an optimal choice as it does not require any special preparation but only drilling of holes. Moreover, the board showed no decomposition, bending, or breakage due to high temperature, sunlight, or moisture as long as even one year. It is a durable and affordable product, especially for the establishment of small artificial wetlands. 3.7. Possible advantages and problems of FTWs FTW is an affordable method of remediating wastewater as it does not require specifically designed experimental field or instrumentation, which may save up to 80% of process energy and 50% of material input (Luederitz et al., 2001). Being based on the natural remediating power of plants and their associated microbes harbored on floating mats, this technology is sustainable and environment-friendly as it can provide long-term remediation and pollutant degradation with minimal environmental intervention. Other advantages of FTWs are their capability to cope with variations in water levels, their esthetic value, their provision of habitat for invertebrates, fish, and birds (Keizer-Vlek et al., 2014). However, in field certain efficient water purification plants can be dangerous to the innate ecosystem when introduced to the areas outside their native distribution areas (Vera et al., 2010). Improperly chosen and managed FTWs could cause undesirable influence on the local agriculture, aquaculture, and biodiversity

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due to invasive species. The decayed, long soaked floating materials can also become a source of pollution (Yeh et al., 2015). 4. Conclusions The study conducted here reinforces the applicability of FTWs for feasible and cost-effective restoration of sewage effluent quality and presents the prospects of evaluating the effectiveness of FTWs at pilot and field-scale studies. Most importantly, we have concluded that B. mutica can not only survive well in extremely contaminated effluent but decontaminate it as well. In this way, an entirely new addition has been made in the plant species that are well-known to be useable for developing floating wetlands. Experimentation has revealed that B. mutica can effectively support bacterial endophytes in its roots and shoots and manipulate this interaction for the better removal of organic and inorganic pollutants, heavy metals, salinity, excess nutrients, and toxicity. The fact that plants are harbored on buoyant mats makes FTWs a suitable and sustainable ecological technology as the mats can be retrofitted as per the water body in which they need to be used. They are particularly appropriate in locations, where water depths and pressures vary with time. Conflict of interest All authors of this manuscript have no conflict of interest. It is declared that the research was not involved human participation and/or animals. The manuscript is neither published nor is under consideration to publish, elsewhere. Moreover, the content and authorship of the submitted manuscript has been approved by all the authors as well as by responsible authorities. Acknowledgements This study was financially supported by Higher Education Commission (HEC, research grant number 1997), Pakistan, and International Foundation of Science (IFS) Sweden and Organization for the Prohibition of Chemical Weapons (OPCW) (research grant number W/5104-2) to Muhammad Afzal. References Afzal, M., Khan, Q.M., Sessitsch, A., 2014b. Endophytic bacteria: prospects and applications for the phytoremediation of organic pollutants. Chemosphere 117, 232–242. Afzal, M., Khan, S., Iqbal, S., Mirza, M.S., Khan, Q.M., 2013a. Inoculation method affects colonization and activity of Burkholderia phytofirmans PsJN during phytoremediation of diesel-contaminated soil. Int. Biodeterior. Biodegrad. 85, 331–336. Afzal, M., Shabir, G., Hussain, I., Khalid, Z.M., 2008. Paper and board mill effluent treatment with the combined biological-coagulation-filtration pilot scale reactor. Bioresour. Technol. 99, 7383–7387. Afzal, M., Shabir, G., Tahseen, R., Ejazul, I., Iqbal, S., Khan, Q.M., Khalid, Z.M., 2014a. Endophytic Burkholderia sp. strain PsJN improves plant growth and phytoremediation of soil irrigated with textile effluent. Clean Soil Air Water 42, 1304–1310. Afzal, M., Yousaf, S., Reichenauer, T.G., Kuffner, M., Sessitsch, A., 2011. Soil type affects plant colonization, activity and catabolic gene expression of inoculated bacterial strains during phytoremediation of diesel. J. Hazard. Mater. 186, 1568–1575. Afzal, M., Yousaf, S., Reichenauer, T.G., Sessitsch, A., 2012. The inoculation method affects colonization and performance of bacterial inoculant strains in the phytoremediation of soil contaminated with diesel oil. Int. J. Phytoremediation 14, 35–47. Afzal, M., Yousaf, S., Reichenauer, T.G., Sessitsch, A., 2013b. Ecology of alkanedegrading bacteria and their interaction with the plant. In: de Bruijn, F.A. (Ed.), Molecular Microbial Ecology of the Rhizosphere. John Wiley & Sons, Inc., Hoboken, NJ, USA, pp. 975–989. Bezbaruah, A.N., Zhang, T.C., 2004. pH, redox, and oxygen microprofiles in rhizosphere of bulrush (Scirpus validus) in a constructed wetland treating municipal wastewater. Biotechnol. Bioeng. 88, 60–70. Borne, K.E., Fassman, E.A., Tanner, C.C., 2013. Floating treatment wetland retrofit to improve stormwater pond performance for suspended solids, copper and zinc. Ecol. Eng. 54, 173–182.

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