Bioremediation of Soil Irrigated with Sewage Effluent ...

Vol. 72 | No. 3 | Mar 2016

International Scientific Researches Journal

Bioremediation of Soil Irrigated with Sewage Effluent Benefitting New Kinetic Tactics M. Saber 1, E. Hoballah 1, H.F. Abouziena 2*, Soad El-Ashry 3, Wafaa M. Haggag 4 and A. M. Zaghloul 3 1

Agricultural Microbiology Department, National Research Centre, Cairo, Egypt, 12622 2 Botany Department, National Research Centre, Cairo, Egypt, 12622 3 Soil and Water Use Department, National Research Centre, Cairo, Egypt, 12622 4 Plant Pathology Department, National Research Centre, Cairo, Egypt, 12622 Corresponding author: [email protected] Abstract

Interdisciplinary approach using microbial enhancers [Acidithiobacillus thiooxidans, arbuscular mycorrhiza (AM)], chemical stabilizer (probentonite) and phytoremediation with Brassica napus and Brassica juncea Czern was evaluated at Abou-Rawash farm in soil ecosystem irrigated with sewage effluent for 30 years for the sake of lowering Zn equivalent from 630 to a save level (less than 250). The experiment was conducted for four months in a complete randomize plot design with three replicate for each treatment. Results confirmed that canola hyper-accumulator plant was more effective than Mustard plant particularly on the uptake of Ni compared to either copper or zinc. Inoculation with A. thiooxidans and AM significantly enhanced the ability of trailed hyper-accumulator plants to uptake the studied PTE's. Nevertheless, according to the kinetic parameters, the mixture of all remediative amendments was the best treatment in minimizing Zn equivalent value to a save level. Different mechanisms took place between trailed remediative amendments in the soil ecosystem were discussed. Keywords: Zn equivalent, Kinetic models, PTEs, Soil ecosystem, Sewage farming. 2. Introduction Sewage effluent had been extensively manipulated in farming in many sites in Egypt since several decades [1]. It is well known that long-term application of sewage effluent in farming might result in the accumulation of PTEs in the soil ecosystem [2, 3]. The rate at which PTEs are accumulated in the soil ecosystem depends on the physiochemical properties of the soil ecosystem and the relative efficiency of grown plants to uptake PTEs from the soil ecosystem. PTEs accumulated in cultivated soils can be transferred to humans through various exposure pathways causing adverse effects on human health [3]. One major exposure

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pathway of heavy metals to humans is through the consumption of crops grown in the soils which are contaminated by heavy metals from wastewater irrigation [4]. Contaminated crops by heavy metals have been documented in many soils irrigated by wastewater [5, 6, 7]. Sewagewater irrigation is one of the main causes for vegetable contamination by heavy metals. The use of treated and untreated wastewater for irrigation increased the contamination of Cd, Pb, and Ni in the edible portion of vegetables, thus posing potential health risks for humans [8]. The excessive accumulation of dietary heavy metals such as Cd, Cr, and Pb in the human body can result in serious systemic health problems [9]. Therefore, the Food and Agriculture Organization and World Health Organization (WHO), US Environmental Protection Agency (US-EPA), and other regulatory bodies of various countries have established the maximum permitted concentrations of heavy metals in foodstuffs or soils [3, 10]. The level of contamination risks posed by wastewater with heavy metals was determined using different indices, including the available forms in soils system, for human bean by transfer factor (TF), daily intake of metals (DIM), and health risk index (HRI) or health quotient (HQ) [2,4,10]. In this work the hazard of PTE's is represented by Zn equivalent value ZEV [1], the value of this constant must be less than 200 in sewaged soils to get save food from contaminated land. In this work we aimed to examine integrated management technique to decrease ZEV constant from about 633 to the save level. 3. Materials and Methods 3.1 Soil used A field experiment was conducted in Abo-Rawash farm and was cultivated by artichoke; this sandy soil was irrigated with untreated sewaged water from the 70th of last century and cultivated with different kind of trees and vegetables. The chemical characterization [11] of the used soil with pH 8.67, EC 0.2 dS m-1, 2.6% OM (organic matter), and the texture is sandy soil (typic psamments). The experiment consisted of 24 plots irrigated by sewage effluent using furrow irrigation. Plot area was 10.5 m2 (3.5 m width by 3 m length= 1/400 fed.), containing five ridges spaced 70 cm apart. 3.2 Microbiological Methods: To develop a microbial decontamination process in sewage soils, a steady supply of efficient microorganisms was ensured in a microbial culture collection containing highly active isolated pure cultures.

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3.2.1 Acidithiobacillus thiooxidans was obtained from Agricultural Microbiology Department, National Research Centre, Cairo, Egypt [1]. It was grown on modified Waksman medium [12]. 3.2.2 Mycorrhizal (AM [arbuscular mycorrhiza]) Conidia: Arbuscular mycorrhiza (AM) fungi spores were extracted from soil by wet sieving and sucrose density gradient centrifugation [13]. Soil sample was placed in a plastic bag and airdried (with the tops of the bags rolled 2mm mesh and stored at 4°C. The procedure included passage of 25 g of air-dried soil or 30 cm of harvested trap culture substrate through 1,000 µm, 500 µm, 125 and 45 µm sieves. The contents of the 125 µm and 45 µm sieves were layered onto a water-sucrose solution (70% wt/v) gradient and centrifuged at 900 g for 2 min. The resulting supernatant was passed through the 45 µm sieves, washed with tap water and used to inoculate the sewage soil. Mycorrhiza is not specific in terms of the partner plant they choose, which means that the same fungus could be grown on a down) for 24 hours under cover, then brushed through a large number of plant species. AM inoculums were prepared by mixing the spores in tap water (about 200 spore 10ml-1), and the soil at the rate of 20 ml pot-1. The detection of AM in infected plant roots (Clearing and staining) was done according to [7]. Microbial cultivation and fortification: A. thiooxidans microorganism used in the bioremediation trails was grown on the last mentioned specific medium in Bioflo and Celligen fermentor/bioreactor, A. thiooxidans suspension was impregnated on a proper mordant at the rate of 20 ml microbial suspension per 100 gm mordant oven dried soil. Sewage soils were solely inoculated with the prepared microorganism at the rate of 100 gm impregnated mordant/400 gm soil [14, 15]. 3.3 Chemical Methods Soil pH: The pH value in the soil was measured using the glass electrode method in a 1: 2.5 soil water suspension. 3.3.1 Instrumentation and analysis of PTEs Flame atomic absorption spectrometry (FAAS) is a simple and well available technique frequently used in determining PTEs in natural aquatic samples. A Perkin–Elmer ﬂame atomic absorption spectrometer (FAAS) and HACH DR890 colorimeter was used in this study. Atomic absorption measurements were carried out using air: acetylene ﬂame while HACH colorimeter measurement with the provided test kits. The operating parameters for working elements were set of as recommended by the manufacturer. 3.3.2 Soil Quality Criterion Zn Equivalent Model was numerically expressed for the levels of PTEs toxicity according to the following equation (ppm): 1* Zn concentration + 2*Cu concentration + 8 *Ni

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concentration .A quality criterion index over 250 units indicated a risky situation necessitating remediation for sustainable Farming management [1]. 3.4 Soil treatments In randomly complete plot design, the selected farm treated with probentonite 1.25 ton/ feddan (4200 m2), A. thiooxidans, AM and mixture of these treatments. The treatments were well mixed with the treated sandy soil and three replicates were took place for each treatments. 3.5 Hyperaccumulator Plants used 3.5.1 Indian Mustard (Brassica juncea Czern.) Indian mustard has been shown to be effective in accumulating high tissue concentrations of lead when grown in contaminated soil [1]. 3.5.2 Canola (Brassica napus, L.) Canola grows well in dry environments and can tolerate-moderately saline soil conditions. Also, it is an essential-oil crop of the Cruciferae family. It has a tap root system and profuse root hairs that allow the plant to be well equipped for hyper accumulation of PTE's. 3.5.3 Black nightshade (Solanum nigrum L.) Black nightshade is a pioneer species growing in a polluted site in Egypt. This plant has an opportunity to absorb high concentration of Zn [16]. Black nightshade was also found to proliferate in other metal polluted sites. Black nightshade (S. nigrum) which growing in a steel works waste site in Australia, accumulating up to 82 mg Zn, 15 mg Pb, 3 mg Cu and 17 mg Cd per kg dry tissue [17]. Worth to mention that the authors found this plant normally grown in polluted area selected for this study. 3.6 Statistical Analysis All data were processed by Microsoft Excel. Regression of linear and other statistical analyses were conducted using the programs of Costate version 6.400, a statistical analysis software package published by CoHort Software [18].

4. Results 4.1 PTE's uptake by Indian Mustard

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Figure (1) represents Ni Cu and Zn uptake by Indian mustard plant in sewage soil previously treated with modified bentonite, Acidithiobacillus, Mycorrhiza and combination between these materials. Before delving into explaining the data it should be mention that all these material individually showed priority in minimizing the hazards of pollution in soil system. In the beginning, it should be mention that a variation in pollutants uptake was observed according to the remediation material, type of pollutant and plant used as a hyperacumulator. As shown in Fig. (1) Ni uptake by Mustard plant was differed according to remediation material applied. The higher uptake of Ni was significantly observed in sewage soils treated with bentonite and mixture of all treatments compared to control treatment, The uptake of these treatments reached to about 26 ppm, meanwhile the lowest uptake was observed in A. thiooxidans treatment (13 ppm). In contrast, the obtained results showed that the highest uptake of Cu by Mustard was observed in A. thiooxidans soil treatment compared to other treatments applied. The numerical values of Cu uptake in different treatments were 6.31,11.5 ppm for control, A. thiooxidans, meanwhile it were 7.75 ppm for AM and bentonite and the lowest value was observed in mixture between all treatments. According to SD statistical analysis, it could be seen that the A. thiooxidans treatment significantly varied compared to other treatments applied, and the significance between different pollutants will be take into consideration in selectivity uptake in Mustard individually and the comparison between different plants tested. Compared to other pollutants, Zn uptake was the highest pollutants found in Mustard, this result could be related to the presence of Zn in high concentration in sewage soils. The results indicated that after control, cultivation of Mustard in bentonite treatments take the highest value of Zn compared to other treatments applied. In this treatment, about 92 ppm Zn was found in Mustard plant, while the lowest value was observed in mixture treatment, the other treatments values applied in sewage soils were in-between. However, it should be mention that the standard division SD analysis indicated that no significant was observed between different treatments applied. 4.2 PTE's uptake by Canola Figure (2) represents the uptake of Ni, Cu and Zn by canola as affected by different treatments applied in sewage soils. In contrast with Mustard, data indicated that control treatment (untreated soil) was the highest treatment in Ni uptake followed by AM and A. thiooxidans and the lowest one was observed in bentonite with significant statistical observation between different treatments compared to control. The uptake of Cu, illustrated in the same figure, indicated that the trend of uptake of Cu was almost the same like Ni in having the lowest value observed in bentonite. 71

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Numerically, data indicated that canola planted in sewage soil treated with A. thiooxidans absorb about 7 ppm Cu, the same value in control treatment, however, was about 9 ppm and the same value was observed in bentonite, the highest absorption of Cu was observed in soil treated with AM. Worth to mention that no significant observed between control and bentonite treatment. 45 40 Ni Concentration (ppm)

35 30

control

25

Bentonite

20

Thio

15

AM

10

all

5 0 Treatments

14

Cu concentration (ppm)

12 10 control 8

Bentonite Thio

6

AM 4 all 2 0 Treatments

120

Zn concentration (ppm)

100

80 control Bentonite

60

Thio AM

40

all 20

0 Treatments

Fig. 1. PTE's concentrations in Mustard plant at maturity stage as affected by different treatments applied in high contaminated sewage soil (Field experiment).

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The uptake of Zn by canola illustrated in the same figure indicated that again like Ni, control treatment was the highest treatment in Zn uptake by canola, meanwhile, the canola planted in mixture treatment was the lowest treatment. 45 40

Ni concentration (ppm)

35 30 control Bentonite

25

Thio 20

AM all

15 10 5 0 Treatments

14

Cu concentration (ppm)

12 10 control Bentonite

8

Thio 6

AM all

4 2 0 Treatments

120

Zn concentration 9ppm)

100

80 control Bentonite 60

Thio AM all

40

20

0 Treatments

Fig. 2. PTE's concentrations in Canola plant at maturity stage as affected by different treatments applied in high contaminated sewage soil (Field experiment).

Data showed that canola uptake about 101 ppm against 48 ppm in sewage soil treated with mixture of bentonite, AM and A. thiooxidans.

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Other canola plants cultivated under individual abovementioned treatments gave inbetween values such as 68 ppm for A. thiooxidans, 51 ppm for AM and 57 ppm in bentonite treatment. It should be mention that according to SD statistical analysis, a significant variation observed between control and all other treatments, in other words, the uptake of PTE's was decreased according to these treatments. 4.3 PTE's uptake Black Nightshade Data in Fig. (3) represents the phytoextraction of Ni, Cu and Zn by black Nightshade used in phytoremediation of contaminated soil. Generally, significant variations were observed in applied treatments in their ability to absorb of pollutants studied. In addition, as shown in the figure, high amounts of PTE's were absorbed by plant used with variations inside different pollutants studied. In Ni pollutants, data showed that without remediation treatments applied only about 1.6 ppm was absorbed by the plant used, however, application of remediation materials except bentonite individually, led to increase the absorption to about 47 ppm in A. thiooxidans treatment and 50 ppm by application of AM treatment, however application mixture of all abovementioned treatments in sewage soil led to increase the absorb Ni to about 56 ppm, worth to mention that according to statistical analysis, significant variations were observed between different treatments applied. The absorption of copper (Cu) from contaminated soil take another trend in both control treatment and remediation treated soil. Data showed that about 34 ppm of total cu was absorbed by black nightshade against 1.6 ppm Ni absorbed by the same plant, which represents the plant specific absorption for PTE's. Concerning the other treatments, data showed that about 37 ppm Cu was absorbed in the presence of bentonite, this value increased to 71 ppm in the sewage soil treated with AM, meanwhile absorption of Cu by black nightshade decreased to 17 and 34 ppm by treated the sewage soils with Acidithiobacillus and mixed treatments. Zinc assumed to be the highest pollutant absorbed by black nightshade compared to other pollutants studied in both control and treated soils. According to the data depicted in the same figure, plant cultivated in the sewage soil without any treatment absorb about 134 ppm Zn without any significant variation within sewage soil treated with mixture of different remediation material (127 ppm). In contrast, data showed that black nightshade plants cultivated in bentonite treated soil absorb about 235 ppm from total Zn increased to about 360 ppm in AM treatment, however, on reverse with Ni plants cultivated in A. thiooxidans treated soil gave the lowest value 166 ppm.

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Ni concentraion (mg/kg plant)

70 60 50

control Bentonite Thio AM all

40 30 20 10 0

Cu concentration (mg/kg plant)

90 80 70 60


50 40 30 20 10 0

400

Zn concentration (mg/kg plant)

350 300


250 200 150 100 50 0

Treatments

Fig. 3. PTE's concentrations in Nightshade plant at maturity stage as affected by different treatments applied in high contaminated sewage soil (Field experiment).

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The comparison between different plants used and according to roles of selection specifically in control treatments, black nightshade was the best followed by canola in absorb the PTE's tested from sewage soil under our experimental conditions especially in Cu and Zn. However, in some cases, application of remediation treatments applied in this study changed the preferability of using black nightshade and it could be replaced by canola. In more details concerning this point, in Cu uptake, all soil treatments applied especially AM, increased the ability of Black nightshade in absorb this pollutant reached to about 72 ppm against 33 ppm in control, other treatments also enhanced the pollutant uptake with lesser values compared to Am. Also, we can see that application of A. thiooxidans in soil system led to increase Cu uptake by mustard with about more than 80% over control, which indicates the importance of this material in enhancing phytoextraction process. In Ni, the arrangement of used plant in phytoextraction of PTE's was as follow: Black Nightshade > Mustard > Canola In other words, with respect to Black Nightshade we can see that mustard has advantage in absorb Ni than canola especially in the presence of bentonite and all mixture treatments. The comparison between different plant species in Zn absorption emphasized the preferability of using Black nightshade in sewage soils individually or with other treatments applied for minimizing the hazards of PTE's. In control treatment, the absorption of Zn was about 133 ppm against 101 and 96 ppm in canola and mustard respectively. Application of AM, increased the uptake of Zn by the same plant to about 360 ppm and the respective value for the abovementioned plants were 50 and 86 ppm, means that application of AM decreased the uptake of pollutants by these plants. 4.4 Rate process of PTE's desorption from sewage soils as affected by phytoextracted plants and remediation materials applied The rate process of PTE's desorption from soils media become the matter of concern since the increasing of industrial activities in Egypt. The Elovich equation has general application to chemisorption kinetics. This equation has been applied satisfactorily to some chemisorption data and has been found to cover a large range of slow adsorption. Taylor [19] studied the kinetics of zinc ion sorption by soils and concluded that the Elovich equation is the best fitting equation. Elovich’s equation has been widely used to describe the adsorption of gas onto solid systems [20, 21].

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Recently it has also been applied to describe the adsorption process of pollutants from aqueous solutions, such as cadmium removal from effluents using bone char [22], and Cr (VI) and Cu (II) adsorption by chitin, chitosan, and Rhizopus arrhizus [23]. The Elovich equation is often valid for systems in which the adsorbing surface is heterogeneous. The equation is formulated as: dqt / dt =  exp(-qt) or in integrated form as qt = 1/ l na + 1/ ln t The rate constants of PTE's desorbed from treated sewage soils described by Elovich model are presented in Table (1). The obtained results indicated that Elovich equation was the best of all tested models through having high and significant R2 compared to other used model with low Standard error (SE). Numerically, for all pollutants studied, the calculated R2 values of Zn desorption from the soil cultivated with Canola were ranged between 0.92**0.97**, the respective values after Mustard were ranged between 0.89** – 0.94**, for black nightshade, however, the coefficient of determination values were ranged between 0.94**0.99**. In the work of trace elements reactions with soil systems, again Elovich model was convenient to describe PTE's release from different soil systems such as Pb desorption [24], Cobalt desorption [25] Fluorin [26] and Iron [27]. The  values of Zn desorption after Canola plantation presented in the table indicated that highest value was observed in control treatment reached to about 64 mg kg-1 min-1 decreased to 39, 36, 14 and o 4.5 mg kg-1 min-1 in sewaged soils treated with AM, A. thiooxidans, mixture of all treatments and the lowest value was observed in Bentonite treated soil. The same pollutant desorbed after Mustard plant showed that again highest value 57 mg kg-1 min-1 was observed in control treatment, in other treatments applied treatments with variation in values the same trend of Canola was observed. In Black nightshade, Data indicated that again the highest rate was observed in control compared to other treatments applied in sewage soils. The lowest value of Zn release was observed in Bentonite treated soil 1.5 mg kg-1 min-1, increased to about 10, 17, and 24 mg kg-1 min-1 in AM, mixed, and A. thiooxidans treated soils respectively. The capacity factor represented by ἀ, irrespective of control which showed highest values of Zn desorption in all cultivated soil, data in the same table showed that application of Bentonite led to increase this factor in Canola and Black nightshade plants used and a reverse trend was observed in mustard. The comparison between different plants showed that capacity factor was 102 ppm after canola, decreased to 52 ppm in Black nightshade and although it was 77 ppm in Mustard, data indicated that it was the lowest value in Bentonite compared to other treatments applied in Mustard cultivated soils. 77

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The rate of Cu desorption as described by Elovich equation represented in the same table showed increasing in  values in control for any plant type used. According to the observed data, the rate of Cu release after Canola was about 11.5 mg kg-1 min-1 in untreated soil, mean while it were 7.5, 5.7, 6.9 and 1.2 mg kg-1 min-1 in the soil treated with Bentonite, A. thiooxidans, AM and the mixture of all abovmentioned treatments, data indicated that again the same trend was observed in rate of Cu release after cultivation of other plant species tested. Regarding the capacity factor of Cu release from sewage soils, data indicated that like Zn, control soil was the highest compared to other treatments applied regardless the type of cultivated plants used with priority to Black nightshade in having lowest capacity factor followed by canola and to less extent Mustard. The presence of Bentonite in soil system regardless the type of plant used always increased the capacity factor and this result could be important indication for the important of such materials to be applied in contaminated sewage soils; meanwhile the application of mixture treatment gave the lowest value. Table 1. Rate constants of Elovich model used to describe the kinetics of PTE's desorption from sewage soils as affected by remediation materials applied and cultivation of Canola, Mustard and Nightshade as a hyper accumulated plants. Canola Treatments

β

α

Mustard 2

R

SE

β

Black Nightshade 2

α

R

SE

β

α

R2

SE

Zn Control

56.2

98.0

0.94**

4.32

66.9

264.8 0.93**

4.2

49.6

181.4 0.95**

2.21

Bentonite

4.5

102.0 0.82**

2.66

5.5

77.0

0.81**

1.1

1.5

52.0

0.94**

1.14

Thio

36.0

49.0

0.80**

6.35

38.7

109.9 0.80**

3.1

24.1

90.2

0.98**

1.75

AM

39.4

41.5

0.79**

4.1

42.5

138.9 0.84**

3.7

9.92

65.5

0.99**

1.66

All

13.7

36.4

0.94**

2.8

25.6

94.5

1.4

16.7

86.4

0.98**

1.45

8.98

22.6

0.97**

2.75

0.83** Cu

Control

11.48

58.15 0.97**

Bentonite

7.46

Thio

8.2

4.62

73.4

62.46 0.97** 13.33

1.18

22.37 0.87**

1.84

1.54

20.87 0.99**

1.63

5.65

47.89 0.96**

8.26

1.38

35.23 0.86**

2.44

0.88

11.63 0.99**

2.41

AM

6.94

60.77 0.98** 14.45

3.42

20.9

0.85**

2.25

2.56

10.49 0.98**

1.51

All

1.2

15.59 0.96**

1.21

21.3

0.93**

1.55

0.98

16.02 0.94**

1.1

2.11

0.97** 13.47

Ni Control

4.26

3.3

0.92**

0.98

5.3

4.2

0.96**

1.98

2.56

3.75

0.98**

0.73

Bentonite

1.34

1.3

0.83**

1.21

1.07

3.35

0.79**

0.74

1.01

1.09

0.97**

0.22

Thio

2.81

5.66

0.80**

1.87

2.49

0.88

0.85**

0.96

1.97

2.51

0.99**

0.87

AM

1.67

1.88

0.96**

0.57

2.22

2.14

0.75*

0.76

1.41

2.01

0.95**

0.55

All

0.72

0.89

0.80*

0.37

1.71

2.57

0.80*

0.81

0.51

1.73

0.90**

0.53

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The rate of Ni release take the same trend of other pollutants studied with respect to low values observed according to decrease of concentration of this pollutant compared to others studied. Worth to mention that soil cultivated with Black nightshade was the lowest soil in release of these pollutants like other pollutants studied. 4.5 Effect of integrated managements applied in sewaged soils in sewage soils on Zn equivalent values The results of this part as shown in Fig. (4) could be divided into two main sections, the 1 indicated that all treatments applied (involving hyper accumulator plants applied individually or T2 as shown in the figure) significantly reducing the concentrations of PTE's in soil system to the save level of this constant (