Chemosphere 91 (2013) 1236–1242

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Improved sustainability of feedstock production with sludge and interacting mycorrhiza

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.02.004

⇑ Corresponding author. Tel.: +358 4 587 46881; fax: +358 9 191 58463.E-mail address: mahmoud.seleiman@helsinki.fi (M.F. Seleiman).

Mahmoud F. Seleiman ⇑, Arja Santanen, Jouko Kleemola, Frederick L. Stoddard, Pirjo S.A. MäkeläDepartment of Agricultural Sciences, University of Helsinki, P.O. Box 27, FIN-00014, Helsinki, Finland

h i g h l i g h t s

" Sludge N and P are readily available for feedstock crops." Mycorrh iza improved nutrient availability of sludge." Sludge increased biomass accumulation in compariso n to synthetic fertilizer." Sludge increased number of mycorrhizal spores in soil and root colonization." Sludge with mycorrhizal improve sustainability of bioenergy feedstocks.

a r t i c l e i n f o

Article history:Received 7 December 2012 Received in revised form 5 February 2013 Accepted 7 February 2013 Available online 5 March 2013

Keywords:BiomassFeedstock crops Heavy metals Mycorrhizal fungi N and P availability Sewage and digested sludge

a b s t r a c t

Recycling nutrients saves energy and improves agricultural sustainability. Sewage sludge contains 2.6% Pand 3.1% N, so the availability of these nutrients was investigated using four crops grown in either soil orsand. Further attention was paid to the role of mycorrhiza in improveme nt of nutrient availability. The content of heavy metals and metalloids in the feedstock was analyzed. Sewage sludge application resulted in greater biomass accumulation in ryegrass than comparable single applications of either syn- thetic fertilizer or digested sludge. Sewage sludge application resulted in more numerous mycorrhizal spores in soil and increased root colonization in comparison to synthetic fertilizer. All plants studied had mycorrhizal colonized roots, with the highest colonization rate in maize, followed by hemp. Sewage sludge application resulted in the highest P uptake in all soil-grown plants. In conclusion, sewage sludge application increased feedstock yield, provided beneficial use for organic wastes, and contributed to the sustainability of bioenergy feedstock production systems. It also improves the soil conditions and plant nutrition through colonization by mycorrhizal fungi as well as reducing leaching and need of synthetic fertilize rs.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Production of synthetic fertilizers increased from 33 to 146 Mtbetween 1961 and 2002 (FAO, 2012 ). Increasin g use of synthetic fer- tilizers not only causes adverse environmental effects due toleaching, but also increases the global warming potential by 2%,which could be decreased markedly with nutrient recycling (Gilbert et al., 2011 ). Recycling nutrients from organic wastes, such as sludge, provides an opportunity to save energy and improve sustainabili ty in agriculture. The total energy required for the production of ammoniu m nitrate is 79.6 MJ kg�1 N and the carbon requiremen t is 3.9 kg CO2 eq kg�1 N (Elsayed and Mortime r, 2001 ).In comparison, the total energy required for the production of

digested liquid sewage sludge is 1.14 MJ kg�1 N and the carbon re- quired is 0.20 kg CO2 eq kg�1 N (Water UK Report, 2006 ).

The total amount of sludge produced annually in the European Union is approximat ely 11.6 Mt dry matter (DM), of which 45% isrecycled in agriculture (EUROSTAT, 2010 ). The N content of sewage sludge is about 4% of DM, and is in ammonium , nitrate and organic forms (EPA, 1994 ). The total P content is about 2% of DM, depend- ing on the product quality. From 15% to 85% of sludge N is plant- available , whereas the share of plant-ava ilable P is usually around 50% (European Communitie s, 2006; Gilbert et al., 2011 ). Up to 90%of P in the treated waste water is precipitated as Al, Ca and Fe salts,so it is generally unavailable to plants (Coker et al., 1987 ). Sewage sludge and other materials such as manure, green waste and en- ergy crops can be used as feed-stocks for anaerobic digestion toproduce methane (Rulkens, 2008 ). The remaining solid matter inmethane production is digested sludge, which can also be used as a soil conditioner.

M.F. Seleiman et al. / Chemosphere 91 (2013) 1236–1242 1237

Some rhizospher ic mycorrhizal fungi and bacteria can solubilize P from the organic and inorganic P pools of soil. Bacteria are usu- ally more effective at solubilization than fungi (Chen et al.,2006). Arbuscular mycorrhi zal (AM) fungi are the most common and widespre ad symbioses occurring in terrestria l plant (Smithand Smith, 2011 ). Around 90% of land plants distribut ed in all ma- jor biomes are potential ly mycorrhizal (Smith and Read, 2008;Smith et al., 2010; Smith and Smith, 2011 ). The remaining 10% ofplants, including Chenopodiacea e and Brassicaceae, are considered non-mycorrhi zal. AM fungi can improve the nutrition al status ofthe host plant through uptake of mineral nutrients, especially P,based on extensive mycelium in the soil. The mycelium accumu- lates and translocates nutrients and water to the plant roots, where the fungus grows between and within cortical cells forming arbus- cules or intracellular coils and vesicles involved in nutrient transfer and storing (Lambert et al., 1979; Smith and Read, 2008; Smith et al., 2010; Smith and Smith, 2011 ). The contribution of AM fungi to total N uptake is not clear (Smith and Smith, 2011 ). Even though NO�3 and NHþ4 are both taken up, NHþ4 is preferred by AM fungi (Govindaraju lu et al., 2005; Jin et al., 2005 ). The mineral N ismainly assimilated into arginine, transported via the hyphae, and then transferred in the form of NHþ4 to the plant (Govindarajuluet al., 2005 ). Use of synthetic fertilizers, particularly P, decreases the colonization of mycorrhi zal fungi, so it may limit P availability (Ortas, 2012 ).

The aims of this work were to investiga te the germination and growth of a set of crops followed by sewage and digested sludge applications , and N and P availability of sludge. We also investi- gated the potential interactions between sludge use and mycorrhi- zal activity. In addition, the content of heavy metal and metalloid in the plant biomass was analyzed.

2. Material and methods

2.1. Growing conditions

The experiments were conducted in the environmental ly con- trolled glasshouses of the Universit y of Helsinki. The day and night temperature s were 21 and 19 �C respectivel y, and the relative humidity was 60%. High pressure sodium lamps provided 18 hphotoperiod with photosyn thetic photon flux density of400 lmol m�2 s�1 throughout the day at the top of the canopy.Water was supplied daily.

2.2. Ryegrass biomass and nutrient availability

In two experiments , Italian ryegrass (Lolium multiflorum L. ssp.italicum, cv. Barmultra) seeds (200) were planted in 5-L pots con- taining 4 kg of fine sandy soil (Supplementary Material (SM),Table SM-1 ). Before seeding, 60 kg N ha�1 was added either in form of synthetic fertilizer (N–P–K: 17–4–25, 4-Supere x, Kekkilä Oy,Eurajoki, Finland, and N–Mg: 11–16, Mult-Magnis al, Haifa Chemi- cals Ltd., Israel), sewage sludge (Lahti Aqua, Lahti, Finland), or di- gested sludge (Envor Biotech Oy, Forssa, Finland), and mixed into the top 5 cm of soil (Table SM-1 ). After each harvest, a further 60 kg N ha�1 was applied on half of the pots fertilized with synthetic fertilizer. Plants were cut 4 cm above soil surface every 20 d from seeding until senescence, dried at 70 �C for 48 h, and weighed. Rela- tive growth rate has been calculated from the obtained dry weight asfollows: Relative growth rate = (lnW2 � lnW1)/(t2 � t1), where W1and W2 = dry weight at time t1 and t2, respectively . The experiments were arranged in a randomized complete block design with six replicates.

2.3. Feedstock s and their quality

In a further two experiments , maize (Zea mays L., cv. Ronaldino),fiber hemp (Cannabis sativa L., cv. Uso 31) and oilseed rape [Brassicanapus L. ssp. oleifera (Moench.) Metzg., cv. Wildcat] seeds (100)were planted in 5-L pots containing either 5 kg of fine sandy soil (Table SM-1 ) or 4.5 kg sand (SP Minerals Oy, Virkkala, Finland)mixed with unfertilized peat (Luonnonturve P2-4, Kekkilä Oy, Lap- inneva, Finland) in volumes of 1:1. Before seeding, either synthetic fertilizer (N–P–K: 28–3–5, Cemagro Oy, Lohja, Finland), sewage sludge or digested sludge were mixed on the top 5 cm, equal to120 kg N ha�1 for maize, 60 kg N ha�1 for hemp, and 90 kg N ha�1

for oilseed rape (Table SM-1 ). The experiment was arranged in arandomized complete block design with three replicates .

Germina tion was recorded at 7 and 14 d after sowing (DAS). At14 DAS, 5 plants were sampled randomly. Shoots and roots were separated to measure root and shoot length, dried at 70 �C for 48 h, and weighed.

In the second experiment, the dried samples were ground into fine powder (0.5 mm size). The content of key elements (As, Cd,Cr, Cu, Mn, Ni, Pb, Zn, K, Mg, Na, P, S and Si) was determined on300 mg ground plant samples as described previousl y (Seleimanet al., 2012 ) with Inductively Coupled Plasma-Opt ical Emission Spectrom etry (iCAP 6200, Thermo Fisher Scientific, Cambridge,UK).

The total N and C contents were analyzed from ground plant samples (200 mg) by the Dumas combustion method using a Vario MAX CN (Elementar Analysensysteme GmbH, Hanau, Germany).

2.4. Mycorrhiz a analyses

Mycorrh izal spore density in soil and sand samples was counted accordin g to the modified method of Allen et al. (1979). Soil sam- ples were collected at 15 DAS from each pot using a borer (£5 cm).Samples were carefully mixed and sieved (1 and 63 lm). A sub- sample of 10 g was weighed into a centrifuge tube containing 15 mL of distilled water. Samples were allowed to hydrate for 15 min, and centrifuged for 10 min at 2000 rpm at 10 �C to remove organic matter. Samples were resuspended in 20 mL of 2 M sucrose solution and centrifuged. The supernatant, containing the spores,was poured into a separato ry funnel and allowed to settle for 10 min. The liquid was then slowly drained out (10 mL min �1).Spores were washed carefully from the funnel walls with 2 mL ofdistilled water into a Petri dish (Tissue Culture Plate 6-Well Flat Bottom, Sarstedt, USA). The number of spores was counted imme- diately under a stereo microscop e (Leica MZ FL III, Fluorescent Ste- reo Microscope , Heerbrugg, Germany ).

For determination of mycorrhi zal root colonizati on, lateral roots were randomly sampled at 15 DAS and washed under running tap water. 1 g of fresh root was cut into 2 cm pieces, placed in bottles containing 40 mL of 10% KOH (Merck KGaA, Darmstad t, Germany),and kept in a water bath at 60–90 �C for 2–4 h (Phillips and Hay- man, 1970 ). Samples were rinsed with 10 mL of 10% HCl (ACS re- agent, Sigma–Aldrich, Germany), followed by distilled water,then stained with cotton blue (Riedel-de Haën AG, Seelze, Ger- many) according to Grace and Stribley (1991). A light microscope (Leitz, Wild Leitz GmbH, Wetzlar, Germany) with an attached CCD camera was used for viewing the AM mycelium and vesicles in the roots.

2.5. Statistica l analyses

Data were subjected to analysis of variance using PASW statis- tics 20.0 (SPSS, Chicago, IL, USA). Where the ANOVA showed signif- icant differenc es, the means were compare d using Tukey’s multiple range test. Simple correlation coefficient analysis was performed to

Table 1Growth parameters of soil- and sand-grown maize, hemp and oilseed rape fertilized with synthetic fertilizer (N), sewage sludge (SS) and digested sludge (DS). Data shown are the average of the two experiments, n = 6.

Root length (cm)

Shoot height (cm)

Dry mass

Root (mg)

Shoot (mg)

1238 M.F. Seleiman et al. / Chemosphere 91 (2013) 1236–1242

determine the relationshi p of the number of mycorrhizal spores insoil with the content of P and N in the plant biomass. Growth of the ryegrass in the nutrition experiment was fitted to Gompertz curves [Biomass = A + C � Exp(�Exp(�B � ((DAS/20) �M))) where A is the lower asymptote, C the upper asymptote, B the rate of increase,DAS the days after sowing and M the point of inflection] using PASW.

MaizeSoil + N 7.76 35.12 111.95 168.36 Soil + SS 7.75 34.71 112.46 164.65 Soil + DS 7.63 31.88 108.50 159.46 Sand + N 8.01 31.81 78.06 128.15 Sand + SS 9.02 27.83 84.56 93.81 S.E.M. (df = 16) 0.50 0.75 2.64 6.17 Hemp Soil + N 5.20 17.78 7.90 85.30 Soil + SS 5.66 18.08 7.88 91.00 Soil + DS 4.48 16.33 5.39 71.25 Sand + N 5.18 6.68 3.22 11.90 Sand + SS 5.61 10.68 4.60 48.13 S.E.M. (df = 16) 0.26 0.39 0.18 1.21 Oilseed rape Soil + N 4.06 10.70 4.39 55.46 Soil + SS 5.48 11.05 4.76 56.65 Soil + DS 5.18 11.30 4.43 51.03 Sand + N 5.42 4.93 3.22 29.12 Sand + SS 5.78 5.11 3.44 32.66 S.E.M. (df = 16) 0.26 0.24 0.06 0.45

3. Results

3.1. Biomass accumulation of ryegrass and availability of nutrients from organic sources

The highest biomass yield of ryegrass was obtained with syn- thetic fertilizer applied after each harvest (Fig. 1). In contrast, the yield was lowest when the synthetic fertilizer was applied only at sowing (Fig. 1). Sewage sludge application resulted in higher accumulate d biomass than digested sludge or singly applied syn- thetic fertilizer (Fig. 1). At 140 DAS, the accumulated biomass ofryegrass fertilized with sewage sludge was 67% higher than that treated once with the synthetic fertilizer (Fig. 1).

The Gompertz curves showed a very good fit to the data (r2 = 0.999, for all four nutrient sources) (Table SM-2 ). The point of inflection on the curve, M, was at 39 DAS for sewage sludge application and 36 DAS for synthetic fertilizer applied at sowing or digested sludge application, a non-sign ificant difference, and the correspond ing point in the multiple application of synthetic fertilizer was significantly later (91 DAS). The lower asymptote val- ues, A, were all not significantly different from zero. The upper asymptote values, C, were all significantly different from each other. The upper asymptote was particularly high for sewage sludge than singly applied synthetic fertilizer or digested sludge,confirming that it would ultimately produce the greatest biomass among these three treatments (Table SM-2 ).

Table 2Macronutrient content in soil- and sand-grown maize, hemp and oilseed rape fertilized with synthetic fertilizer (N), sewage sludge (SS), and digested sludge (DS).Data shown are means, n = 3.

Treatment Macronutrient content (g kg–1 DM)

3.2. Feedstock and mycorrhiza with different nutrient sources

Although minor differenc es in germination percentage were seen in each crop at 7 DAS, the differences were no longer signifi-cant by 14 DAS (Table SM-3 ).

Root length, root biomass, and shoot biomass were greater insewage sludge-ferti lized, sand-grown plants that in those treated

0

25

50

75

100

125

150

0 20 40 60 80 100 120 140

Biom

ass

(g)

Days after sowing

Fig. 1. Accumulated biomass yield of ryegrass treated once with synthetic fertilizer (N; j), sewage sludge (SS; d) and digested sludge (DS; N), or treated sequentially with synthetic fertilizer (Nm; �). Data shown are means from two experiments and error bars show S.E.M., n = 12.

with synthetic fertilizer (Table 1), but in soil-grow n plants, the dif- ferences in shoot biomass were not significant (Table 1).

In all three species, the N content was highest in sand-grown plants fertilized with synthetic fertilizer (Table 2). However, insoil-grow n maize and hemp, the N content was higher in sewage sludge-fer tilized plants than in those treated with synthetic fertil- izer (Table 2). Sewage sludge applicati on resulted in significantlyhigher P content than synthetic fertilizer in all three species when grown in sand (Table 2). In soil-grown oilseed rape treated with sewage sludge, the P content was 1.4 g kg�1 DM higher than with

N K P Mg Na S Si

MaizeSoil + N 21.6 20.5 2.67 1.79 0.026 1.68 0.120 Soil + SS 28.0 21.2 2.70 2.35 0.026 2.51 0.116 Soil + DS 27.5 21.1 2.68 2.35 0.033 2.28 0.096 Sand + N 46.7 8.1 2.85 4.54 0.033 2.44 0.126 Sand + SS 37.1 4.6 3.35 9.80 0.030 4.89 0.076 S.E.M. (df = 8) 0.41 0.92 0.046 0.117 0.006 0.050 0.005

HempSoil + N 30.9 17.9 1.79 3.27 0.030 2.58 0.070 Soil + SS 33.9 20.2 1.91 3.23 0.026 2.10 0.073 Soil + DS 31.1 23.2 1.99 2.82 0.040 2.73 0.106 Sand + N 45.3 7.1 1.73 6.96 0.030 2.71 0.090 Sand + SS 34.3 12.1 1.98 5.31 0.050 3.07 0.103 S.E.M. (df = 8) 0.11 1.02 0.085 0.140 0.002 0.140 0.008

Oilseed rape Soil + N 29.7 17.6 2.45 3.51 0.786 6.70 0.106 Soil + SS 29.2 25.5 3.03 2.43 0.863 7.94 0.120 Soil + DS 27.8 24.1 1.61 2.12 0.213 8.52 0.096 Sand + N 46.6 13.4 2.41 4.19 1.080 7.95 0.083 Sand + SS 40.9 4.1 2.86 5.65 2.386 9.71 0.103 S.E.M. (df = 8) 0.15 0.73 0.050 0.293 0.087 0.347 0.006

S.E.M., Standard error of mean; df, Degrees of freedom.

Table 3Heavy metal and metalloid content in sand- and soil-grown maize, hemp and oilseed rape fertilized with synthetic fertilizer (N), and sewage sludge (SS), and digested sludge (DS).Data shown are means, n = 3.

Treatment Heavy metal and metalloid content (mg kg–1 DM)

As Cd Cr Cu Mn Ni Pb Zn

MaizeSoil + N 0.013 0.013 2.89 4.29 2.00 1.44 0.34 21.9 Soil + SS 0.173 0.023 4.35 4.59 2.96 2.14 0.90 30.2 Soil + DS 0.107 0.020 2.47 3.86 2.80 1.08 0.49 30.0 Sand + N 0.013 0.013 2.64 3.08 22.82 1.14 0.14 37.4 Sand + SS 0.150 0.020 3.47 4.55 29.63 1.62 0.63 46.2 S.E.M. (df = 8) 0.025 0.006 0.144 0.228 2.708 0.057 0.025 0.98

HempSoil + N 0.176 Nd 0.96 3.19 3.77 0.62 Nd 26.8 Soil + SS 0.236 Nd 1.07 3.56 4.98 1.40 0.26 26.7 Soil + DS 0.040 Nd 0.73 3.07 3.32 0.31 Nd 22.5 Sand + N 0.013 Nd 0.54 3.16 22.86 0.22 Nd 28.9 Sand + SS 0.250 Nd 3.13 4.33 29.31 0.59 0.05 36.5 S.E.M. (df = 8) 0.046 – 0.288 0.154 0.292 0.081 0.016 1.14

Oilseed rape Soil + N Nd 0.012 3.15 2.72 3.02 1.46 Nd 36.9 Soil + SS Nd 0.077 3.31 2.90 4.29 2.27 Nd 32.5 Soil + DS Nd 0.033 0.93 2.14 2.47 0.48 Nd 28.5 Sand + N Nd 0.043 1.26 2.31 12.92 0.58 Nd 32.3 Sand + SS Nd 0.063 1.41 2.66 22.07 0.55 Nd 35.0 S.E.M. (df = 8) – 0.012 0.170 0.127 0.513 0.151 – 1.57

S.E.M., Standard error of mean; Nd, Not detected; df, Degrees of freedom.

M.F. Seleiman et al. / Chemosphere 91 (2013) 1236–1242 1239

digested sludge and 0.6 g kg�1 DM higher than with synthetic fer- tilizer (Table 2). Similarly, the P content was 0.5 mg kg�1 DM high- er in sand-grown maize and 0.3 g kg�1 DM higher in sand-grown hemp fertilized with sewage sludge than in the correspond ing syn- thetic fertilizer treatments (Table 2). Sewage sludge applicati on de- creased the K content of sand-grown maize and oilseed rape, and increased the Mg and S content in all three sand-grown crops (Table 2). In soil-grow n plants, the nutrient sources did not signif- icantly affect the Mg, S, and Si content.

Highest P uptake of all crops studied was obtained when sew- age sludge was applied into soil. The highest N uptake of hemp (310 mg) and oilseed rape (170 mg) was obtained when sewage sludge was applied into soil. The N uptake of maize was highest (585 mg), however, with synthetic fertilizer applied in sand, fol- lowed by sewage sludge applied in soil.

Heavy metal and metalloid content increased both in sand- and soil-grown plants only following sewage sludge application (Ta-ble 3). The greatest increases were in Cr (33%) and Pb (62%) in sew- age sludge-ferti lized, soil-grown maize in comparison to the correspondi ng synthetic fertilizer treatment. Ni was higher inmaize (32%) and hemp (60%) grown with sewage sludge in compar- ison to synthetic fertilizer in both soil and sand, and was 64% higher in sewage sludge-ferti lized than in synthetic fertilized, soil-grown oilseed rape. Moreover, Zn increased markedly (20%) in maize and hemp fertilized with sewage sludge mixed with sand (Table 3).

The number of mycorrhizal spores was outstandingl y high in di- gested sludge-fertilized , soil-grow n hemp (Fig. 2). In all three crops, and on both potting media, the sewage sludge treatment was associated with significantly more (30–40%) mycorrhizal spores than the synthetic fertilizer treatment, with high repeatabi l-ity between experiments . In maize, the spore numbers in the two sludge types were not significantly different, but in oilseed rape,there were fewer spores in the digested sludge treatment than inthe sewage sludge treatment.

Full mycorrhi zal colonization was found in roots of sewage sludge-ferti lized, sand-grown maize and digested sludge-fertilized ,soil-grown hemp grown. Colonization was not detectable in the roots of any plants treated with synthetic fertilizer (Fig. 3). Fungal

hyphae and arbuscul es were observed in roots of sewage sludge- fertilized , soil-grown maize, whereas vesicles and hyphae were ob- served in sewage sludge-fer tilized, sand-grown and digested -fer- tilized, soil-grown hemp roots. Only vesicles were seen in roots of sludge-ferti lized oilseed rape grown in either soil or sand (Fig. 3).

The number of mycorrhizal spores was positivel y correlate dwith P content in maize and hemp, but this correlation was not sig- nificant in oilseed rape (Fig. 4). Similarly, the correlation between the number of mycorrhizal spores and N content in maize and oil- seed rape was positive, but not significant.

4. Discussion

These experime nts showed that digested and sewage sludge are suitable nutrient sources for various crops, with the added benefitof increasing mycorrhizal colonization of plant roots. Furthermore,there were differences between crop species, with hemp being par- ticularly responsive to digested sludge.

The sludge applicati ons resulted in a higher biomass yield in the ryegrass experiments , even though the treatments were balanced to deliver the same amount of N (Fig. 1). The similarity of the rates of biomass accumulati on in the three treatments implies that nutri- ents were exhausted from all sources at similar, but not identical,rates (Fig. 1 and Table SM-2 ). This was also observed as differences in relative growth rate, which was highest (0.0025 g g�1 d�1) at 100 DAS following sewage sludge application in comparis on to single applicati on of synthetic fertilizer (0.0016 g g�1 d�1) or digested sludge (0.0014 g g�1 d�1). Relative growth rate also remained until the end of the experiment higher in sewage sludge than single applicati on of synthetic fertilizer and digested sludge. However,in dry conditions there was no difference in biomass yield of rye- grass due to organic or synthetic fertilizer (Pascual et al., 2004 ).

Although there appeared to be a small advantage to germina- tion in the presence of sewage sludge instead of synthetic fertilizer at 7 DAS, this disappea red by 14 DAS (Table SM-3 ). In earlier exper- iments, there have been some difficulties in achieving good germi- nation in the presence of sludge, attributabl e to its sticky nature

Fig. 2. Number of mycorrhizal spores in soil and sand treated with synthetic fertilizer (N), sewage sludge (SS) and digested sludge (DS) in (A) maize, (B) hemp,and (C) oilseed rape. Data shown are means from two experiments and error bars show S.E.M., n = 6.

1240 M.F. Seleiman et al. / Chemosphere 91 (2013) 1236–1242

that inhibits thorough mixing into the growing medium, and the high doses that are often used (Seleiman et al., 2012 ). Furthermore,a seed germination bioassay is relatively insensitiv e to many toxic substances in soil for two reasons: first, seeds are quite self-suffi-cient, and thus many elements might not be absorbed from soil,and secondly, the embryo is effectively isolated from the environ- ment (Kapustka, 1997 ).

Both in sand and soil, the increase in number of mycorrhizal spores following the sewage sludge application indicates anenhancement of growth and sporulation of the fungus (Fig. 2).Moreover, sewage sludge application increased the mycorrhizal colonization, particularly of maize and also of hemp (Fig. 3). This interspecific difference may have been attributabl e to the more extensive nature of the maize root system, or to its greater exuda- tion of sugars and other attractants and promoters of fungus growth. The reduction of mycorrhi zal colonization in plant roots following the use of synthetic fertilizer could be associated with the established reduction in hyphae and arbuscul es when syn- thetic fertilizers have been used (Johnson, 1993; Ortas, 2012 ).Moreover, it has also been observed that a plentiful N and P supply is deleterious to mycorrhiza (Lin et al., 2012 ).

The greater biomass accumulation of sewage sludge-fertilized ,sand-grown hemp and oilseed rape in comparison to plants treated

with synthetic fertilizer may also be associated with mycorrhi za- tion, which has been shown to increase the productivity of many crops including maize (Sylvia et al., 1993 ). The positive effect ofAM fungi on host-plant growth and developmen t is clear in soils of low fertility (Jeffries, 1987 ). AM fungi also improve plant P up- take more when the P is from a poorly soluble organic source than when it is in a soluble form in conventional fertilizer (Feng et al.,2003). When the potting medium was soil rather than sand, the differenc es in biomass between sludges and fertilizer were much smaller and seldom significant (Table 1), and it is well known that in fertile soils, plants can take up both N and P sufficiently without mycorrhi za (Lin et al., 2012 ).

Increases in N and P uptake and thus, also in the N content ofmaize and hemp and the P content of hemp and oilseed rape were observed when sewage sludge was applied in soil (Table 2). Soil aggregat ion and microbiota in plant rhizosphere affect symbiotic nutrient uptake (Andrade et al., 1998 ). AM fungi have been shown to increase the transfer of the mineralized N from organic sources to their associate d host but this role is considered to be seldom important (Hodge et al., 2001; Smith and Smith, 2011 ). Similarly,the P content of all three sand-grown crops was much higher inthe presence of sewage sludge than in the synthetic fertilizer treat- ment (Table 2). AM fungi can increase directly the uptake of slowly diffusing ions, such as PO3�

4 , through production of organic acids orproton extrusion (Lambert et al., 1979; Jacobsen et al., 1992; Leyval et al., 1997 ). In the present experiment, the results are consistent with increased availability of P from sludge due to the action ofmycorrhi zal fungi.

Application of sewage sludge in soil and sand resulted in an in- crease in Cu, Cr, Mn, Ni and Zn content in all crops, and in As content in maize and hemp (Table 3). The uptake of trace elements depends not only on their availability in the soil solution, but also on the absorption efficiency of the root system (Liu et al., 2000 ) which isaffected by the presence of AM fungi that can affect the availabili tyof sparingly soluble ions with narrow diffusion zones (Lambert et al.1979; Liu et al., 2000 ). Mycorrhiza l fungi can solubilize different minerals , including metal-co ntaining rock phosphates , by produc- ing organic acids and releasing protons (Leyval et al., 1997 ), thus potential ly increasing the availability of heavy metals and metal- loids in the rhizosphere. Once these ions have entered the hyphae,they can be sequestered there or transferred to the roots and trans- ported to the plant shoot (Leyval et al., 1997 ). In addition, the absorption of elements such as Cu and Zn is further increased byexternal hyphae (Li et al., 1991; Bürkert and Robson, 1994 ) due tothe increased surface area and decreased distance for diffusion,thus enhancing the absorption of immobile elements (Jacobsen,1992). In contrast, Bi et al. (2003) showed that mycorrhizal plants accumulate d less Na+ in shoots than non-mycorrhi zal plants, sothe mychorriza provided some protection against excessive accu- mulation of this highly soluble cation. In the present experiment,Na accumulation was lower in shoots of mycorrhi zal maize and hemp than in non-myc orrhizal oilseed rape (Table 2). Higher Mncontent in sand-grown plants fertilized with sewage sludge than in plants grown with other treatments could be due to the increase in nitrification process of NHþ4 , which decreases the pH in the grow- ing media. Subseque ntly, acidic pH causes a reduction in content ofNO�3 as an alternativ e electron acceptor and results in a high con- tent of Mn in plant material (Mukhopadhy ay and Sharma, 1991 ).

5. Conclusion s

In conclusion, there was no difference in N availabili ty and thus mineraliz ation of organic nitrogen whether originating from organic or synthetic sources. Nevertheles s, the growth of maize,hemp, oilseed rape and ryegrass was better with the sludge

Fig. 3. AM fungi, hyphae and vesicles in roots of (A, B) maize, (C, D) hemp, and (E, F) oilseed rape treated with (A, C, E) synthetic fertilizer and (B, D, F) sewage sludge.a = arbuscular mycorrhizal fungi, h = hyphae, v = vesicle.

Fig. 4. Simple correlation coefficient of the relationship between the number of mycorrhizal spore and (a) P and (b) N content of maize, (c) P and (d) N content in hemp, and (e) P and (f) N content in oilseed rape in the second experiment; j, soil + synthetic fertilizer; d, soil + sewage sludge; N, soil + digested sludge; h, sand + synthetic fertilizer;s, sand + sewage sludge. (a) y = 0.016 x + 1.671, r = 0.731 ⁄⁄, (b) y = 0.190 x + 18.44, r = 0.256 ns, (c) y = 0.002 x + 1.742, r = 0.666 ⁄⁄, (d) y = �0.056x + 38.78, r = �0.358ns, (e)y = 0.034 x + 1.214, r = 0.427 ns, and (f) y = 0.083 x + 31.74, r = 0.068 ns.

M.F. Seleiman et al. / Chemosphere 91 (2013) 1236–1242 1241

treatments, probably due partly to mycorrhi za colonization linked to sewage sludge application and partly to slow release of the nutrients from their organical ly bound forms. In addition, the occurrence of mycorrhiza in soil across the different treatments correlated well with plant P content.

Acknowled gments

Finnish Cultural Foundation, Niemi-Säätiö and August Johannes ja Aino Tiuran maatalouden tutkimussäätiö are gratefully acknowl- edged for their financial support. Helsingin Seudun Ympäristöpal-

velut–kuntayhtymä HSY, Jätevedenpuhdistusosasto, Lahti Aqua,and Envor Biotech Oy are acknowledged for providing the sludge.Also, we wish to thank Markku Tykkyläinen and Sanna Peltola for technical assistance.

Appendi x A. Supplementar y material

Supplement ary data associate d with this article can be found, inthe online version, at http://dx.doi .org/10.1016/j.chemospher e.2013.02.0 04.

1242 M.F. Seleiman et al. / Chemosphere 91 (2013) 1236–1242

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