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Science and Technology Food, Agriculture & Environment Vol.2 (2) : 381-390. 2004

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Using wheat-mungbean plant system and arbuscular mycorrhiza to enhance in-situ bioremediation
Gamal H. Rabie
Botany Department, Faculty of Science, Zagazig University, Egypt. Present address: Science Department, Teachers College, El-Hassa, Hufuf city,P.O.Box, 2313, Saudi Arabia. e-mail: rabiegam@hotmail.com
Received 7 January 2004, accepted 15 April 2004.

Abstract
Mixed wheat-mungbean systems together with mycorrhizal inoculants (Glomus mosseae) were used to remediate a polycyclic aromatic hydrocarbons (PAHs) spiked agricultural soil in a pot experiment. The soil was spiked with 500 mg kg-1 dry soil of each anthracene, pyrene and chrysene and 50 mg kg-1 dry soil of benz(a,h) anthracene, representing common PAH compounds with three, four and five aromatic rings, respectively and then aged for 5 weeks. After three months from sowing date, PAH residues in the soil, soil enzymatic activities, total organic carbon, pH, biochemical potential fertility indicator, microbiological analysis, mycorrhizal infection % and growth parameters of wheat and mungbean plants were determined. All of these measurements were significantly reduced in spiked soil with PAHs pollutant. Enhanced losses of PAHs were seen in planted soil and PAH degradation reached 60% of the initial PAHs 1550 mg kg-1 soil. Wheat-mungbean systems considerably decreases the negative influence of PAHs during this study. Vesicular-arbuscular mycorrhiza inoculated to wheat-mungbean system may significantly increase the efficiency of this system to minimize the negative effects of PAHs pollutants as well as enhance the dissipation of these pollutants in the soil. We conclude that VAM improves remediation in wheat-mungbean system by encouraging plant vigour, root growth and stimulating rhizospheric microbial activities to degrade PAHs in the soil. Key words: Hydrocarbons, phytoremediation, rhizosphere, pollutants, mycorrhiza, soil enzymes, fertility index.

Introduction In Saudi Arabia and other countries soil pollution by polycyclic aromatic hydrocarbons (PHAs) is a serious problem, especially in the vicinity of industrial plants29, petroleum refineries 9, motorways and petrol stations 1, air-fields11 and wastewater treatment plants22. These pollutants (PAHs) represent a group of persistent and toxic soil pollutants that is a major public concern due to their mutagenic and carcinogenic capacity. Phytoremediation is a technology that combines low costs with efficient erosion control and biodegradation of a wide range of organic pollutants, thus reducing the risk that these substances represent hazard for human health18. A wide range of parameters that influences the efficiency of phytoremediation still remains to be identified. However the main potential obstacles that need to be overcome to improve phytoremediation system are low bioavailablity of the pollutants due to adsorption to soil particles23 and lack of adequate microbial activity7. The use of plants in PAH bioremediation schemes is still in its infancy, but positive effects of plants on the degradation of PAH have been observed 45, 47. Phytoremediation of organics has been attempted using grasses 6 or legumes alone 19. The role of mycorrhiza during the remediation of PAH in mixed sward of clover and ryegrass was examined in a recent study and found enhanced losses of chrysene and dibenz(a,h)anthracene in planted soil containing a mycorrhizal inoculum 26. Different mechanisms have been proposed to explain the effect of the plant rhizosphere on PAH dissipation such as an increase in microbial numbers, an improvement in the physical and chemical soil conditions, increased humification and adsorption of
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pollutant in rhizoshere. However, the relative contribution of each presses has not been clearly elucidated 57 . Leyval and Binet 34 showed that VAM colonization of some plant species was negatively affected by increasing additions of an industrial soil polluted with PAH, but not by similar concentrations of a single PAH (anthracene) added to an agricultural soil, still VAM enhanced survival and plant growth in PAH-contaminated soils. However, to the authors view points, few studies have examined the impact of VAM inoculum during remediation of PAHs using different cropping systems. VAMs are likely to play an important role in these systems, due to their ability to alleviate nutrient limitations and thus increase plant and root growth. It is known that VAM can increase exudation from host roots which may in turn support the growth of microbial degraders or influence pollutant availability. However, the ability of VAM within the plant root or direct role of VAM to degrade PHAs is still unknown. This study is designed to (i) examine whether the dissipation of PAHs is enhanced in a soil artificially contaminated with PAHs and planted with a mixed wheat and mungbean sward and (ii) to examine whether a vesicular arbuscular mycorrhizal inoculum singly or in combination with biogas amendment further enhances the dissipation. Materials and Methods Contaminated soil: An agricultural topsoil (Table 1) was sieved to <2 mm sterilized by (25 kGy gamma rays) and left un-spiked or 381

Table 1. Properties of experimental soil.

Parameter Sand Silt Clay Texture class Total organic carbon Total nitrogen Total phosphorus Total potassium pH

74% 12% 14% Sandy loam 10.2 g kg-1 3.6 g kg-1 29 mg kg-1 32 mg kg-1 7.9

spiked with 500 mg kg-1 dry soil of each anthracene, pyrene and chrysene and 50 mg kg-1 dry soil of benz(a,h) anthracene. Soil microorganisms were reintroduced by adding 10 ml per pot of soil suspension (10 g of the corresponding soil in 200 ml water filtered through a 20 m filter and mist-sprayed on to soil during mixing). The spiked soil was then stored for 5 weeks for stabilization and ageing of the PAH amendment. Experimental: The experiment included factorial combinations of 1) two soil types a) non-spiked (non-polluted) soil and b) spiked (polluted) soil, 2) two soil states a) unplanted and b) planted, 3) two mycorrhizal treatments a) non-inoculated and b) inoculated with Glomus mosseae (Nicol.&Gerd.) and 4) two biogas manure amendments a) unamended soil b) amended soil. Five replications per each treatment were used to give a total 80 pots. Seeds of wheat (Triticum aestivum cv. Sakha 8) and mungbean (Vigna radiata V 2010) plants were disinfected by soaking in 30% H2O2 for 20 min, washed three times in distilled water, grown in black plastic pots containing 200 g of the spiked soil. Mycorrhized planted pots received 10 g of a mycorrhizal inoculum (10 spores g-1 Glomus mosseae Nicol.&Gerd.). Half pots received biogas manure before sowing at a rate of 6 ton fed-1. Phosphorus fertilizer at a rate of 31 kg P2O5 in form of calcium super phosphate (15.5%) during preparation of the soil for all treatments. Nitrogen and potassium fertilizers were added at rates of N 90 kg fed-1 and K2O 96 kg fed-1 in forms of ammonium sulphate (20.5% N) and potassium sulphate (48% K2O) in three equal doses at 15 , 30 and 60 days after emergence. Pots were sown with two pre-germinated seeds of wheat and mungbean and thinned to one plant of each species after 10 days. The soil was covered with a layer of coarse sand in an attempt to minimize PAH volatilization. The pots were arranged in growth chamber at 25/20oC day/night, 11 h day, 60-70% relative humidity and watered to 75% of WHC 2-7 times week-1. The plants were harvested 90 days after sowing date. PAH analysis: The root density in the planted pots was very high and all the soil was considered as rhizospheric soil. The soil from planted (rhizospheric soil) and non-planted pots (nonrhizospheric soil) was carefully collected, homogenized and crushed. Soil samples were dried in the dark at room temperature under a fumehood. PAHs were Soxhlet extracted from the soil (50 g dry soil with 200 ml chloroform for 4 h). Soil extracts were filtered through cellulose before analysis. The extracts were concentrated to 0.5 ml and analyzed on HPLC (Hewlett Packard 1050 fitted with a 250 mm C-18 vydac column, using 3D fluorescence detection (HP 1100) as described by Szolar et al. 51.

Determination of soil properties and enzymatic activity: Physicochemical soil properties were determined using methods generally applied in soil chemical laboratories. Soil pH was determined by a potentiometric method in a suspension 1 mole KCl. Total organic carbon (TOC) content was determined by the Tiurins method as modified by Simakow 8. The activity of the following enzymes was determined: dehydrogenase 54, phosphatases 52, protease 32 and urease 59. Fertility indicator (index) (Mw) was determined according to Kucharski et al. 31 as follows: Mw = [(UP/10) + DH + APa + APac] TOC, where DH= dehydrogenase activity [cm3 H2 kg-1 24 h-1], APal=alkaline phosphatase activity (p-nitro phenyl phosphate (PNP) mmol kg-1 h-1), APac=acid phosphatase activity (mmol PNP kg 1 h-1), UP=urease activity (mg NH4-N kg-1 h-1 ), TOC = total organic carbon (%). Statistical analysis: Statistical analysis of the results were subjected to ANOVA and Pearson correlation coefficient. Significance was set at *P <0.05 and ** P < 0.01. Results The data in Table 2 revealed that dehydrogenase activity had highly significant inverse correlation with soil pollutants PAHs. Dehydrogenase enzyme showed in non-polluted soil higher activity (P< 0.01) than in polluted soil (Fig 1). On the other hand, dehydrogenase activity was decreased by nearly 42% in the presence of PAHs in polluted soil and the maximum decrease was observed in non-planted non-mycorrhized soil amended with biogas. Dehydrogenase activity was significantly affected by biogas application and exhibited significant correlation with biogas amendment to the soil (Table 2). It was increased over other treatments especially in presence of VA-mycorrhiza. In polluted soil, dehydrogenase activity was significantly increased in planted soil treatments as compared with non-planted one (Fig. 1). Phosphatase (acid and alkaline) activities in the soil followed a similar pattern to dehydrogenase activity with soil type (Table 2 and Fig. 2). In soil spiked with PAHs pollutants acid and alkaline phosphatase showed approximate 55% and 64% reduction in their activities. Moreover, acid and alkaline phosphatases were completely inhibited in polluted non-planted non-mycorrhized soil without biogas amendments (Fig. 2). Phosphatase activities had a significant effect and correlation with mycorrhizal associations (Table 2). In polluted soil, acid and alkaline phosphatase activities were significantly higher in planted soil than in unplanted one especially in presence of VAM (Fig. 2) . The results of Table 2 showed that urease activity had a highly significant effect and inverse correlation with the presence of PAH pollutants in soil. The reduction in urease activity was about 53% in soil spiked with PAHs pollutants. Planting the soil (soil state) had a significant effect and correlation on urease activity in the soil (Table 2). Urease activity was increased by planting the soil either in presence or absence of PAHs pollutants and still showed higher activity in nonpolluted soil than that polluted one with increasing tendency for biogas treatments during this study (Fig. 1). The results also revealed that urease activity was decreased by nearly 52% in polluted soil compared to non-polluted one (Fig.1). Protease activity showed a highly significant effect and
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Table 2. Oneway ANOVA (signicant F) and correlation coefficient (r) for the effects of different treatments on soil enzymes activities.
Statistics Soil type Soil state VAmycorrhiza Biogas Sig. F r Sig. F r Sig. F r Sig. F r Dehydrogenase 0.009** - 0.630** 0.575 0.152 0.198 0.340 0.025* 0.557* Acid phoshatase 0.01** -0.586* 0.243 0.310 0.019* 0.577* 0.668 0.116 Alkaline phosphatase 0.01** -0.616* 0.212 0.330 0.041* 0.578* 0.430 0.212 Urease 0.002** -0.704** 0.026* 0.555* 0.504 0.180 0.560 0.158 Protease 0.191 -0.345 0.000** 0.917** 0.595 0.144 0.769 0.080

* significant difference (P<0.05) * * highly significant difference (P<0.01)

Table 3. Oneway ANOVA (signicant F) and correlation coefficient (r) for the effects of different treatments on pH, total organic carbon (TOC) and polycyclic aromatic carbon (PAH).
Treatment Soil type Soil state VAmycorrhiza Biogas Statistics Sig. F r Sig. F r Sig. F r Sig. F r pH 0.001** -0.737** 0.476 0.192 0.954 -0.016 0.297 0.278 T OC 0.001** -0.351 0.327 0.262 0.478 0.191 0.297 -0.084 Fertility index 0.899 0.034 0.124 0.400 0.025* 0.556* 0.043 0.512* PAHs residue a a 0.003** -0.890** 0.484 -0.292 0.560 -0.244

* significant difference (P<0.05) * * highly significant difference (P<0.01)

Table 4. Mycorrhizal infection % of wheat and mungbean plants and bacterial and fungal counts in soil with and without PAH and biogas.
Treatment VAM state Biogas state Mycorrhizal infection % Wheat Mungbean Bacterial count colonies g-1 soil Unplanted Planted soil soil 4 .93x10-4 6.03 x 10-5 6.57x10-4 7.1 x 10-5 6.43x10-4 8.4 x 10-5 8.22x10-4 9.7 x 10-5 0.52x10-4 6.57 x 10-4 2.6x10-4 7.07 x 10-4 0.31x10-4 7.53 x 10-4 2.53x10-4 7.9 x 10-4 Fungal count spores g1 soil Unplanted Planted soil soil 2.23x10-4 3.9 x 10-4 3.83x10-4 4.77 x 10-4 3.33x10-4 6.43 x 10-4 4.03x10-4 7.6 x 10-4 2.1x10-3 6.9 x 10-3 4.1x10-3 7.43 x 10-3 3.07x10-3 8.03 x 10-3 4.83x10-3 8.73 x10-3

Non-polluted soil

- ve +ve

Polluted soil

- ve + ve

- ve + ve - ve + ve - ve + ve - ve + ve

74.5 77.3

71.8 75.3

51.7 53.9

37.7 39.1

Table 5. One-way ANOVA (F) and correlation coefficient (r) for the effect of different treatments on mycorrhizal infection and bacterial and fungal counts in the soil.
Treatment Soil type Soil state VAmycorrhiza Biogas Statistics Sig. F r Sig. F r Sig. F r Sig. F r Bacteria 0.294 -0.280 0.405 -0.224 0.320 0.266 0.317 0.267 Fungi 0.345 0.253 0.000** 0.792** 0.247 0.307 0.382 0.261 Mycorrhizal infection 0.007 * * -0.742* *

0.002** 0.703** 0.620 0.134

correlation with soil state (Table 2). However, planted soil had a higher protease activity in absence and presence of PAHs pollutants in the soil (Fig. 1). In addition, unpolluted soil still exhibited protease activity higher than that in polluted soil. The results also revealed that PAHs pollutants in the soil reduced protease activity by about 26% compared to the absence of these pollutants. Table 3 summarizes the results of some important characteristics done on model soils at the end of the experiment (after 90 days)
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in this study. The polycyclic aromatic hydrocarbon sequestration in the soil showed highly significant effects by soil state and had inverse high significant correlation with planting the soil. The soils were found to encompass a wide range of PAHs residues with the lowest level (480 mg kg-1) found in polluted planted mycorrhized soil with biogas amendment and the highest one (950 mg kg-1) in polluted non-planted non-mycorrhized soil with biogas treatments (Fig. 3). Soil pH exhibited highly significant effects by soil type and had inverse highly significant correlation with the presence of pollutants (PAHs) in the soil (Table 3). Soil pH in polluted soil was lower than in non-polluted one (Fig. 4). Soil type showed a highly significant effect on total organic carbon of the soil in the present study (Table 3). Fig. 4 displays the extents to which total organic carbon was affected by soil pollutants after the first experiment. Total organic carbon was markedly lower in non-polluted soil. Polluted and planted soil was highly enriched in total organic carbon when compared to non-planted soil with the highest value in planted mycorrhizal soil with biogas amendments (Fig. 4). The presence of mycorrhiza in the soil had a significant effect on soil fertility index with maximal effect in polluted planted 383

Table 6. Height, root length, root to shoot ratio and dry weight of non-mycorrhizal and mycorrhizal plants grown in soil with or without polycyclic aromatic hydrocarbons (PAH) and biogas. Each value represents the mean of 5 replica.
VAM state Biogas state Height (cm) Wheat Nonpolluted soil Polluted soil - ve +ve - ve + ve - ve + ve - ve + ve - ve + ve - ve + ve 45.6 56.6 69.4 71.3 21.3 29.6 43.3 46.2 Mungbean 73.2 77.6 95.3 98.6 32.2 33.6 45.3 48.6 Root length (cm) Wheat 16.8 17.1 26.3 26.3 15.0 15.5 26.0 28.0 Mungbean 18.9 19.0 27.3 28.0 14.3 14.7 23.3 25.1 Root to shoot ratio Wheat Mungbean 0.27 0.27 0.30 0.27 0.33 0.30 0.38 0.32 0.25 0.21 0.27 0.22 0.32 0.26 0.38 0.29 Dry weight g plant-1 Wheat Mungbean 19.6 28.6 21.3 30.1 25.3 36.3 31.6 38.9 10.6 11.2 12.3 14.6 21.6 24.6 27.3 26.6

* significant difference (P<0.05) * * highly significant difference (P<0.01)

mycorrhizal soil with biogas treatments (Table 3, Fig. 4). The fertility index was in significant correlation with mycorrhiza and biogas treatment in the soil with higher values in planted soil. Polycyclic aromatic hydrocarbons had inhibitory effects on microbial numbers in the soil where microbial counts (bacteria and fungi) were significantly lowered in polluted soil (Table 4-5). Bacterial numbers decreased nearly 94% in polluted soil and still showed in planted soil higher count than in unplanted one. The bacterial number in polluted and planted soil was four folds that of unplanted one, while in non-polluted soil it increased 63% in planted soil. On the other hand, fungal spores decreased about 88% in polluted soil (Table 4). The count of fungi in soil displayed a highly significant effect and higher significant correlation with soil state (Table 5). In planted soil, fungal spores increased about 120% and 57% in polluted and non-polluted soil respectively. In addition, soil type (the presence of PAHs pollutants) had a highly significant effect and highly significant inverse correlation with % of mycorrhizal infection in the plants (Table 5). The % of mycorrhizal infection showed approximately 30% and 48% reduction in polluted soil compared to non-polluted one for wheat and mungbean plants respectively when grown in mixed swards (Table 4). Also % of mycorrhizal infection was reduced by nearly 23% and 54% in presence of PAHs for wheat and mungbean respectively when grown separately. The results in Tables 6-7 reveal the growth responses of wheat and mungbean plants in mixed swards. The height of wheat and mungbean plants was significantly affected by soil type and negatively correlated with the presence of PAHs in the soil (Table 6). The height of plants grown in the polluted soil was significantly smaller than that of plants grown in the non-polluted soil (Table 7). PAHs pollutants in the soil decreased height of wheat by 35-53.3 % and that of mungbean by 50.7-56.7% of the plant height in non-polluted soil. On the other hand, VAM and biogas treatment showed tendencies to increase plant height in

soil treated with VAM and biogas in the soil. The root length showed weak inverse insignificant correlation with polycyclic aromatic hydrocarbons in the soil (Table 6). The root length of wheat plants showed 1-11% reduction in polluted soil but an unexpected result was found for wheat plants (root length increased nearly by 6.5% in polluted soil amended with biogas in the presence of mycorrhiza). On the other hand, mungbean plants showed 10.424% reduction in polluted soil. VAM symbiosis showed significant effects and significant positive correlation with root length. The presence of VAM caused an increase in root length in comparison to absence of VAM in the soil during this experiment (Table 7). The root length of mycorrhized wheat plants increased 73% and 81% of that in non-mycorrhized one and for mycorrhized mungbean plants the increases were 63% and 71% in the absence and presence of biogas amendment respectively. Although soil type had insignificant effect on root to shoot dry weight ratios of the two plants (Table 6), these ratios were lowered in polluted soil in comparison with non-polluted one (Table 7). The presence of PAHs pollutants in the soil decreased root to shoot ratio by 0-10% for wheat and 928.5% for mungbean plants (Table 7). The results of Table 6 also indicate that the presence of VAM fungi in the soil had a significant effect and exhibited positive correlation with root to shoot ratios of studied plants. In the presence of mycorrhiza, root to shoot ratios were higher by nearly 28% and 41% for wheat and for mungbean 24% and 32% in the absence and presence of biogas respectively (Table 7). Wheat and mungbean dry weights showed inverse significant correlation with the presence of PAHs pollutants in the soil (Table 6). The dry weight of wheat and mungbean plants decreased about 29% and 44% in polluted soil respectively. VAM fungi had a significant effect on wheat and mungbean dry

Table 7. One-way ANOVA for height, root length, root to shoot ratio and dry weight of non-mycorrhizal and mycorrhizal plants grown in soil with or without polycyclic aromatic hydrocarbons (PAH) and biogas.
Statistics Wheat Soil type VA-mycorrhiza Biogas Sig. F Sig. r Sig. F Sig. r Sig. F Sig. r 0.962 -0.013 0.974 0.974 0.993 0.002 Height Mungbean 0.006** -0.858** 0.216 0.491 0.810 0.102 Root length Wheat Mungbean 0.406 0.150 -0.317 -0.559 0.405 0.017* 0.318 0.802* 0.407 0.724 0.316 0.149 R : S ratio Wheat Mungbean 0.788 0.354 -0.114 -0.379 0.008** 0.349 0.848** 0.783* 0.742 0.357 0.139 0.337 Dry weight Wheat Mungbean 0.221 0.033* -0.487 -0.748* 0.021* 0.135 0.786* 0.698* 0.489 0.720 0.288 0.152

* significant difference (P<0.05) * * highly significant difference (P<0.01)

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Dehydrogenase activity H2 cm3/kg/24 h, Urease activity NH4-N mg/kg/h, Preotease activity tyrosine mg/kg/h

dehyd Urease Protease 12

10

Figure 1. Dehydrogenase, urease and protease activities in unpolluted and polluted soil (up=unpolluted, np=non-planted, nm= nonmycorrhized, nb=non-biogass amendment,po=polluted, p=planted, m= mycorrhized, b= biogas amendment).
Alk phos Acid phos 1.2

p. .n up

p. .n up

p. .n up

b .n .m .p po .b m .n .p b po .n m .n .p po .b m p. .n b po .n m p. .n b po .n m p. .n .b po nm p. .n b .n po nm p. .n po .b .m .p up b .n .m .p up .b m .n .p b up .n m .n .p up .b m p. .n b up .n m

.b nm b .n nm

1
Phosphatase activity PNP mmol/kg/h

0.8

0.6

0.4

0.2

0
up .n po po po up po po po up up po po up up up up .p .n .p .n .p .n np .p .p .n np .n .n .p .p .n p. p. .m .n p. .n .m p. p. p p . . . . n m m m m .n m m .m nm m m nm nm .n .b m .n .b .n m .b .n .n .b .n b . . n b b . .n b . . b b b b n b b b b

Figure 2. Acid and alkaline phosphatase activities in unpolluted and polluted soil (up=unpolluted, np=non-planted, nm=non-mycorrhized, nb=non-biogass amendment,po=polluted, p=planted, m=mycorrhized, b= biogas amendment).

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0.9

0.8

0.7

PAH mg/kg soil

0.6

0.5

0.4

0.3

0.2

0.1

0 po.np.nm.nb po.np.nm.b po.np.m.nb po.np.m.b po.p.nm.nb po.p.nm.b po.p.m.nb po.p.m.b

Figure 3. PAHs residues in polluted soil at the end of the experiment (np=non-planted, nm= non-mycorrhized, nb=non-biogass amendment, po=polluted, p=planted, m=mycorrhized, b=biogas amendment).

TOC Fert. indix PH 30

25

TOC (g/kg soil), fertility index, pH

20

15

10

.p up

Figure 4. Total organic carbon, fertility index (Mw, biochemical potential of soil fertility) and pH in unpolluted and polluted soil at the end of the experiment (up=unpolluted, np=non-planted, nm=non-mycorrhized, nb=non-biogass amendment, po=polluted, p=planted, m= mycorrhized, b=biogas amendment).

.n up n p.

.p po

.p po

.n up

.n po

.n up n p. p .n m

.n up

.p po

.p po

.p up

.p up

.p up

.n po

.n po

.n po

.m

.m

.m

.n m p. b

.b m p.

.b m p.

.n m .n

.b m .n

.n m p.

.n m .n

.b m .n

.m b .n

n p.

n p.

.b

b .n

. .b

.b m

b .n m

.b m

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weight (Table 6). The % of plant dry weight reduction by the presence of PAHs in soil was lowered in mycorrhizal plants specially in presence of biogas amendments (Table 7). Discussion The main findings of this study indicate that polycycling aromatic hydrocarbon reduced soil enzymes activities, microbial density in the soil and growth of wheat and mubgbean plants grown in mixed swards of spiked soil with these organic pollutants. The observed effects in this study are in accordance with the findings of Sverdrup et al. 50, who tested the toxicity of eight polycyclic aromatic compounds to red clover, ryegrass and mustard plants, and found that the EC 20values for the PAHs were from 37 to >1000 mg kg-1 which are in the same range as EC 20values reported by Maliszewska-Kordybach and Smerczak 36 . Another interesting observation was that polluted and planted soil had significantly lower concentrations of extractable polycyclic aromatic hydrocarbon compared to unplanted soil. PAH degradation reached 60% of the initial 1550 mg kg-1 in planted soil. This suggested that the rhizosphere of wheatmungbean system significantly enhanced the dissipation of PAHs. The results are in accordance with those of Reilley et al. 45 , who showed an increase in the dissipation of anthracene and pyrene with fescue and alfa alfa. Also Binet et al.12 found that the concentration of anthracene was lower in the planted soil. Anderson et al.5 listed plant species that can facilitate the dissipation of hazardous organic compounds; gramineae appeared to increase the disappearance of PAHs. The degree of xenobiotic dissipation in the rhizosphere appears to be greater with monocots, particularly grass plants, than the dicots; the difference has been attributed to different root cell wall constituent or root exudates 48. However, it has been pointed out 47, 48 that grasses have been used extensively due to their fibrous root system that provides a high root surface area that interacts with soil microorganisms to enhance degradation. The correlations obtained from studied soil confirm inverse relations between PAHs content and dehydrogenase, acid and alkaline phosphatase, urease and protease activities. These results are in harmony with those of Kanaly and Harayama 30 and Loehr et al. 35 and contrary to data obtained by Boopathy 13 and Margesin et al. 38, 39. From the present study it can be seen that in polluted soil the highest activity of soil enzymes was in planted soil (wheat -mungbean rhizosphere) as compared to unplanted one. Dehydrogenase, acid and alkaline phosphatases, urease and protease activities were increased approximate by 34, 68 and 72, 58 and 58% respectively as compared to those in unplanted soil. These results confirm a stimulating influence of plant rhizosphere on the activity of the enzymes and consequently on exhaustive dissipation of PAHs in the soil. In this connection, faster and more exhaustive dissipation of PAHs pollutants in soil rhizosphere of certain plants was proved also by other authors 3, 28, 42. Biochemical potential of soil fertility (fertility index) gives information on the intensity of the biochemical processes taking place in the soil while considering the content of total organic carbon, which regulates the transfer of organic pollutants in the soils 44, 50. The results of the present study revealed that fertility index in polluted soil was increased by increasing PAHs
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degradation especially in planted soil. Fertility index of planted soil was increased by 95% of unplanted one in polluted soil. These findings in accordance with others 31, 58 suggest the beneficial role of plants in PAHs degradation in the soil. Studies by Maliszewska-Kordyback et al.37 and Baran et al.10 showed that soil enrichment in organic matter considerably decreases the negative influence of PAHs on enzymatic activity. Our results confirmed these previous studies, and also the total organic carbon of soil increased nearly 86% and 103% in planted soil and with mycorrhized plants compared with unplanted soil and with non-mycorrhized plants respectively. Hence, it can be concluded that the important role of plants and mycorrhizal fungi for increasing total organic carbon in polluted soil and consequently enhanced dissipation of PAHs in these soils. Numerous studies pointed out to the role of organic matter for sorption processes of organic pollutants 21,40, 50 and agreed that soil sorption of organic chemicals increases with increasing organic carbon content in soil. Pollutants introduced into the soil exert an influence on the microbiota, which manifests itself in changes in enzyme activity, soil respiration, biomass and microbial counts. These parameters are most often used for the description of the general condition of soil microorganisms 38, 46, 56. Our results confirm those of the previous studies 30, 35, 58 that various chemical organic pollutants have inhibitory effects on different microbiota including bacteria, fungi and actinomycetes, as well as other soil microorganisms. It is interesting that PAHs pollutants in the soil significantly reduces the % of mycorrhizal infection and other mycorrhizal parameters (VAM data will be presented elsewhere). In spite of that biodegradation of PAHs by soil microorganisms (bacteria and fungi) has been identified as a major mechanism for bioremediation of contaminated soils 14, 16. Recent studies have shown the ability of plants to stimulate the dissipation of PAHs in contaminated soil 45, 47. It has been found that plants may support a microflora in the rhizosphere with much greater adaptability for growth on different carbon sources (including pollutants) than non-rhizosphere microflora 49. In this connection, numbers of bacteria and fungi were significantly higher in soil rhizosphere of wheat/mungbean mixed swards compared to unplanted soil. Based on these data we suggest that plants may enhance microbial degradation by providing specific microenvironments for pollutant-degrading commensal (such as Pseudomonas) or symbiotic (e.g. mycorrhizae, rhizobia) microorganisms. The use of plants to enhance the bioremediation of persistent organic pollutants in soils may be an economically and environmentally feasible way of remediation in large areas of surface-contamination or of residual contamination after pre-treatment. Our results indicate that the rhizosphere of wheat and mungbean plants significantly enhanced the dissipation of PAHs pollutants. These results are in accordance with those of Reilley et al. 45 who showed an increase in the dissipation of anthracene and pyrene with fescue and alfalfa. Binet et al.12 pointed that the rhizosphere of ryegrass enhanced the dissipation of anthracene. Johnson et al. 25 proved that the dissipation of chrysene is enhanced in a soil planted with mixed clover/ryegrass sward. Based on these data together with our results we can partially support the hypothesis that the dissipation of PAHs pollutants is enhanced in a soil planted 387

with mixed wheat/mungbean sward. Different mechanisms have been proposed to explain the effect of the plant rhizosphere on PAH dissipation in the polluted soil. Certain lower molecular weight organic compounds may be translocated by plants and are either lost by transpiration from the vegetative plant parts 2 or are degraded within plant tissue 41 or increasing plant root growth and exudates stimulating the microbial community 49. A recent study by Leigh et al.33 went one step further and demonstrated that seasonal fine root death releases several flavones which act as substrates for degrading bacteria. Thus, upon death, fine roots may serve not only as injectors of bacterial substrates but also may facilitate soil aeration through the formation of air channels left after root senescence. In the present study interesting observation was that the negative effect of PAHs pollutants on both root length and R/S ratio of wheat and mungbean plants lowered more than that on other growth measurements. These findings in accordance with others 28, 47, 49 suggest that the mechanism by which plant enhance dissipation of PAHs in contaminated soil is via stimulating root growth and root exudates as well as soil microbial populations. Therefore, it follows that any improvement of root growth will lead to enhanced PAHs dissipation in contaminated soil. The other main results in this study were that significantly lower concentrations of PAHs were observed in the planted treatment that had received a mycorrhizal inoculum. Moreover, all measurements including soil condition (total organic carbon, fertility index and soil enzymes), microbial numbers (bacteria and fungi) as well as plant growth (height, root length, R:S ratio and dry weight) were significantly higher in mycorrhizosphere than non-mycorrhizosphere in PAH-contaminated soil. In addition, surprising result was that root length of mycorrhized wheat plants in polluted soil was higher than in nonpolluted one. These results which confirm previous studies 12, 16, 27, 34 suggest that mycorrhizal symbionts in the plants considerably decrease the negative influence of PAHs on plant growth. It is conceivable to conclude that VAM fungi may have a direct effect on PAH dissipation and an indirect effect on PAH bioavailability by modifying root exudation. VAM fungi, especially the root external mycelium extending the root system, may influence the maintenance and activity of microbial communities in the rhizosphere, including PAH-degrading micro-organisms. It is interesting to claim that our results indicated on the growth of wheat plants were better than that of mungbean plants in polluted soil. This difference may significantly correlated with the % of mycorrhizal infection (frequency of root segments) and intensity of mycorrhizal colonization in the root tissue as well as the rate of arbuscular formation in root segments (mycorrhizal data will be presented elsewhere). While planting and mycorrhizal symbiosis have frequently been proved as an efficient means of enhancing degradation of PAHs in the present study, the addition of biogas manure has given contradictory and astringent results. These results are in agreement with previous studies 15, 23, 28, 42 where it was found that coupling of planting and fertilization only had transitory positive effects, which were limited to three-ring PAHs. PAHs in the present study includes mixture of three-, four- and fivering compounds. Therefore biogas amendments showed inconstant results suggesting that the type of PAHs limited the dissipation 388

of these pollutants during phytoremediation in the soil. However, careful examination of results reveals an significant increase in dehydrogenase activity and fertility index in the presence of biogas amendments. In this connection, an increase in dehydrogenase activity has been observed after adding horticulture compost 37, straw 31 and manure 17 to soil polluted with PAH. Hence, it can be concluded that the differences in enzymatic activities and fertility index between presence and absence of biogas amendment, result from different contents of total organic carbon flourished from biogas treatments. The present study pointed out that integrated approaches, taking into account plants and their root-associated microorganisms including VAM fungi, may be an important key to understanding and successful use of phytoremediation. However, it still remains to be resolved to assess the feasibility of phytoremediation versus other bioremediation treatments in pilot scale experiments prior to large-scale efforts under field conditions. References
1

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