Szarvasi-1 energy grass efficiently utilizes high doses of sewage sludge in hydroponics

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1 Szrvsi-1 energy grss efficiently utilizes high doses of sewge sludge in hydroponics Amrus Rév, Brigitt Tóth, Ádám Solti c, Gyul Sipos d, Ferenc Fodor c,* Deprtment of Ecology, Fculty of Science nd Technology, University of Derecen, 1 Egyetem Sq., H-4010 Derecen, Hungry. Deprtment of Botny, Crop Physiology nd Biotechnology, Institute of Plnt Sciences, Fculty of Agriculturl nd Food Sciences nd Environmentl Mngement, University of Derecen, 138 Böszörményi Str., H-4032 Derecen, Hungry. c Deprtment of Plnt Physiology nd Moleculr Plnt Biology, ELTE Eötvös Loránd University, Budpest, 1/C Pázmány P. lne, Budpest, H-1117, Hungry d Agriculturl Reserch nd Development Institute, 30 Szdság Str., H-5540, Szrvs, Hungry. * corresponding uthor E-mil: Amrus Rév, revmrus@gmil.com Brigitt Tóth, toth@gr.unide.hu Ádám Solti, dm.solti@ttk.elte.hu Gyul Sipos, gyul.sipos@sonic.co.hu Ferenc Fodor, ferenc.fodor@ttk.elte.hu Key words Elymus elongtus; Elytrigi elongt; hevy metl; nutrition; tll whetgrss; toxicity Astrct Sewge sludge (SS) originting from communl wstewter is hzrdous mteril ut hve potentilly gret nutritive vlue. Its disposl fter tretment in griculturl lnds cn e very economicl nd sfe wy of utiliztion once fst growing, high iomss, perennil plnts of renewle energy production re cultivted. Szrvsi-1 energy grss (Elymus elongtus susp. ponticus cv. Szrvsi-1), good cndidte for this ppliction, ws grown in hydroponics in order to ssess its metl ccumultion nd tolernce under incresing SS mendments. The pplied SS hd composition chrcteristic to SS from communl wstes nd did not contin ny toxic hevy metl contmintion from industril sludge in high concentrtion. Toxic effects ws ssessed in qurter strength Hoglnd nutrient solution nd only the two highest doses ( g dm -3 ) cused decreses in root growth, shoot wter content nd length nd stomtl conductnce wheres shoot growth, root wter content, chlorophyll concentrtion nd the mximl quntum efficiency of photosystem II ws unffected. Shoot K, C, Mg, Mn, Zn nd Cu content decresed ut N nd Ni incresed in the shoot compred to the unmended control. The nutritive effect ws tested in 1/40 strength Hoglnd solution nd only the highest dose (12.5 g dm -3 ) decresed root growth nd stomtl conductnce significntly while lower doses ( g dm -3 ) hd stimultive effect. Shoot K, N, Fe nd Ni incresed nd C, Mg, Mn, Zn nd Cu decresed in this tretment. It ws concluded tht SS with low hevy metl content cn e potentilly good fertilizer for high iomss non-food crops such s Szrvsi-1 energy grss. Introduction Sewge sludge (SS) from communl wstewter-tretment plnt is toxic nd iologiclly hzrdous product, which requires creful hndling efore deposition. With the development of

2 industriliztion nd urniztion, lrge mount of sewge is generted. Thus, the mngement of SS is n issue of growing importnce. It usully contins pthogenic microorgnisms s well s orgnic constituents nd hevy metls in different composition nd concentrtion. Therefore, in ll countries of the Europen Union, there re directives for storge, stiliztion nd sfe recycling. Before recycling, SS tretment usully involves disinfection, digestion, composting, nd het drying (Epstein 2003, Fytili nd Zniotou 2008, Yng et l. 2015, Cieślik et l. 2015). During the disinfection process, the microflor of SS is chnged. However, these tretments generlly do not modify the hevy metl content. Storge, deposition or utiliztion of the SS depends on the concentrtion nd iovilility of hevy metls (Uri nd Simon 2008). Nevertheless, dilution nd composting mkes it possile to utilize its fertilizer potentil due to its high N, P nd K content (Epstein 2003). The ppliction of SS compost on degrded lnds rted not suitle for griculturl food production my indeed led to lnd utiliztion in renewle energy production. Fst growing, lrge iomss perennil plnts, such s Szrvsi-1 energy grss (Elymus elongtus ssp. ponticus cv. Szrvsi-1) (Csete et l. 2011, Mrtynk et l. 2017) require incresing nutrient resupply to the soil which cn e provided y SS compost. The SS is good source of plnt nutrients such s N, P, K, C or Mg (Mrtinez et l. 2003). Hevy metls such s Cu nd Zn re often undnt in SS ut these re lso micronutrients for plnts. Wheres non-essentil ones, like Cd nd P re often toxic nd my hve deleterious effect on plnts (Mrschner, 1995). In order to void decresed yield it is importnt to test potentil plnt vrieties for sensitivity or tolernce to the hevy metls. Szrvsi-1 energy grss hs recently een tested for tolernce to Cd, Cu, Ni, P nd Zn in nutrient solution nd it proved to e tolernt to Ni nd P while sensitive to Cd nd Cu. Furthermore, this plnt not only ccumultes Zn ut its growth is lso stimulted y 10 µm ZnSO 4 in the nutrient solution (Sipos et l. 2013). In the present study we tested the Szrvsi-1 energy grss with exposure to communl SS in nutrient solution in order to ssess the potentil toxic effect of such tretment y pplying incresing doses of the SS over the norml element composition of the nutrient solution nd, on the other hnd, to ssess the fertilizer effect of the SS smple using diluted nutrient solution in similr experiment. Mterils nd Methods Plnt mteril nd tretments Seeds of the tll whetgrss cultivr Szrvsi-1 energy grss (E. elongtus susp. ponticus [Podp.] Melderis cv. Szrvsi-1, syn. Agropyron elongtum, Elytrigi elongte) (Csete et l. 2011) were germinted for seven dys on wet filter ppers in Petri dishes t room temperture nd sunlight. Three seedlings with 2 5 cm long roots were plced on 2 cm wide strip of sponge-ruer, rolled up nd fstened in polystyrene ring. The seedlings were trnsferred to plstic continers filled up with 10 dm 3 modified, continuously erted, unuffered, 1/4 strength Hoglnd nutrient solution () of the following composition: 1.25 mm KNO 3 ; 1.25 mm C(NO 3 ) 2 ; 0.5 mm MgSO 4 ; 0.25 mm KH 2 PO 4 ; 11.6 μm H 3 BO 3 ; 4.5 μm MnCl 2 4H 2 O; 0.19 μm ZnSO 4 7H 2 O; 0.12 μm N 2 MoO 4 2H 2 O; 0.08 μm CuSO 4 5H 2 O nd 10 μm Fe(III) citrte-hydrte. The plnts were grown in climte controlled growth chmer t 20/25 C, 75% reltive humidity nd 150 μmol m 2 s 1 photosynthetic photon flux density (PPFD) with 10/14 h drk/light period. The nutrient solution ws continuously erted nd replced with fresh solution once week. After 30 dys of growth the plnts were trnsferred to individul pots contining 0.8 dm 3 erted nutrient solution in two tretment groups. One group ws supplied with the sme nutrient solution s during the pregrowth period () ut supplied lso with 0, 1, 5, 10, 15 g dried SS directly dded to the solution in ech pot nd left unfiltered throughout the tretment period. The other group ws supplied with diluted nutrient solution of 1/40 strength () nd with 0, 1, 5, 10 g dried SS similrly s the other group. The SS ws derived from pilot-scle microerophilic, thermophilic digester, where the

3 temperture ws 63 C. The originl SS used in this experiment hd n originl dry mtter content of 6.8%. The orgnic mtter content ws 6.5-7% on dry mtter sis (dt were kindly provided y the compny). Element composition of the dried SS, deionized wter nd nutrient solution mended with SS nd filtered to remove solid prticles is shown in Tle 1. Ech pot ws supplemented with 0.1 dm 3 deionized wter fter 7 nd 14 dys of the tretment period in order to refill the trnspired/evported wter nd to void further supply of nutrients. In one experiment 3 prllel pots ws used for the sme tretment nd the experiment ws done in triplicte. At the end of the 3-week tretment period the plnts were hrvested for mss mesurements nd element nlysis. Mss mesurements The roots were thoroughly clened in deionized wter to remove SS prticles, then centrifuged etween filter ppers t 300 g nd weighed to determine fresh mss. Dry mss of ll tissues (shoots nd roots seprtely) ws determined fter drying t 80 C. Chlorophyll concentrtion The mesurements were mde fter 8 dys of tretment with the first fully developed leves. The chlorophyll (Chl) concentrtion ws determined spectrophotometriclly (Shimdzu UV-2101PC) from 80% cetone extrcts using the equtions of Porr et l. (1989). Ech mesurement ws crried out on three individul plnts in ech tretment group. Stomtl conductnce Stomtl conductnce ws mesured with porometer (DELTA-T Devices Ltd.) on the dxil epidermis of the middle sections of the youngest, fully developed leves fter 8 dys of tretment. Trnspirtion ws clculted s mmol H 2 O m 2 s 1. Ech mesurement ws crried out three times on three individul plnts in ech tretment group. Fluorescence induction of chlorophyll Fluorescence induction mesurements were crried out with intct leves using PAM Chlorophyll Fluorometer (Wlz, Effeltrich, Germny). Leves were drk-dpted for 15 min. The F 0 level of fluorescence ws determined y switching on the mesuring light (modultion frequency of 1.6 khz nd PPFD less thn 1 µmol m -2 s -1 ) fter 3 s illumintion with fr-red light in order to eliminte reduced electron crriers (Belkhodj et l. 1998). The mximum fluorescence yields, F m in the drk-dpted stte ws mesured y pplying 0.7 s pulse of white light (PPFD of 3500 µmol m -2 s -1, light source: KL 1500 electronic, Schott, Minz, Germny). The mximl quntum efficiency of photosystem (PS) II centres were determined s F v /F m = (F m F 0 )/F m. Element concentrtions Element content of the SS, the SS-contining medi (mended deionized wter nd nutrient solution) nd the shoots hve een mesured. Roots hve not een mesured s it ws not possile to fully remove the SS mteril. Mesurement of solution smples were mde fter filtrtion through MN 640 W filter pper. Mesurements of the SS nd plnt smples were mde fter cidic digestion ml cc. HNO 3 ws dded to ech grm of the smples for overnight incution. Then the smples were predigested for 30 min t 60 o C. Finlly, 2-3 ml H 2 O 2 (30 m/m %) ws dded for 90 min oiling t 120 C. The solutions were filled up to ml, homogenised nd filtered through MN 640 W filter pper. The element content of the filtrte ws determined y ICP-MS. All smples were prepred in triplicte. Sttistics Bsic sttisticl nlysis ws crried out with one-wy ANOVA nd Tukey-Krmer multiple comprisons post-hoc test (P < 0.05) using InStt 3.0 (GrphPd) softwre.

4 Results Anlysis of the SS The SS pplied in this experiment hs een nlysed s dried mteril nd s dissolved dditive in deionized wter nd (10 g dm -3 dried SS in wter or nutrient solution) (Tle 1). The dried SS is rich in K, N nd Fe ut otherwise contins low concentrtion of hevy metls such s Cu, Zn, Cd, Co, P. Deionized wter dissolves nutrients nd nonessentil elements from the dried SS mteril. The elementl composition of the resulting solution is chrcterised y concentrtions exceeding tht of unmended, for most mcro nd micronutrients mesured except for C nd Mn which re lower nd N which is much higher. Some nonessentil elements (Al, Cr, Li, Sr, Ti) were lso detected in the solution in low concentrtion. The mended with SS contins ll the mesured elements in concentrtions elevted in vrile extent s compred to the originl composition except for Mn which ws slightly lower. Tle 1. Element composition of the dried SS, deionized wter nd contining 10 g dm -3 dried SS. For reference, the theoreticl composition of the used for control plnts is lso shown. (n=3, SD 5%, DL = detection limit.) Element dried SS deionized wter + dried SS + dried SS control mg kg -1 μmol dm -3 μmol dm -3 μmol dm -3 C K Mg P S B Cu Fe Mn N Zn Al Cd 4.95 < DL < DL 0 Co 3.31 < DL < DL 0 Cr Li Ni < DL < DL 0 P < DL < DL 0 Sr Ti Growth prmeters The root dry mss of control plnts in ws lmost hlf of tht in wheres the shoot dry mss ws the sme. The growth of plnts hs only een inhiited t the highest SS doses. Root dry weight

5 ws significntly decresed y 10 nd 15 g dried SS while shoot dry weight ws significntly chnged only t the 15 g SS dose (Fig. 1). root dry mss (g -1 ) c c sewge sludge (g pot -1 ) shoot dry mss (g -1 ) c d sewge sludge (g pot -1 ) Figure 1. Root nd shoot dry mss of Szrvsi-1 energy grss grown in nd mended with 0, 1, 5, 10 nd 15 g dried SS pplied in 0.8 dm 3 pots. (men±sd, n=3; vlues mrked with different letters re significntly different t P<0.05) The wter content of oth roots nd shoots ws mrkedly lower in compred to control plnts. Root wter content decresed only t the highest SS dose in while in it continuously incresed with the incresing SS dose wheres shoot wter content continuously decresed with the incresing SS doses in ut in it continuously incresed until the ppliction of 5 g dried SS mteril (Fig. 2). root wter content (g H 2 O g -1 dw) c c c sewge sludge (g pot -1 ) shoot wter content (g H 2 O g -1 dw) c sewge sludge (g pot -1 ) Figure 2. Root nd shoot wter content of Szrvsi-1 energy grss grown in nd mended with 0, 1, 5, 10 nd 15 g dried SS pplied in 0.8 dm 3 pots. (dw=dry weight; men±sd, n=3; vlues mrked with different letters re significntly different t P<0.05) Shoot growth ws lower compred to the control t 5, 10 nd 15 g SS mendments in ut 1 nd 5 g tretments resulted in incresed shoot length in (Fig. 3).

6 shoot length (cm -1 ) c d 0 sewge sludge (g pot -1 ) Figure 3. Shoot length of Szrvsi-1 energy grss grown in nd mended with 0, 1, 5, 10 nd 15 g dried SS pplied in 0.8 dm 3 pots. (men±sd, n=3; vlues mrked with different letters re significntly different t P<0.05) Physiologicl prmeters The ph of the nutrient solution during the tretment period hs incresed in ll cses s compred to the initil vlue in the fresh control nd mended solutions (Fig. 4). The highest ph (8.5) ws detected in t 10 nd 15 g SS doses while the lowest one (6.9) ws in t 1 g dose. The highest increse of lmost 3 ph units ws oserved in the control dy 0 dy 21 c d ph 7 c sewge sludge (g pot -1 ) Figure 4. ph of the nutrient solution ( nd ) t the time of tretment with different doses (0, 1, 5, 10 nd 15 g dw in 0.8 dm 3 pots) of SS (dy 0) nd t hrvest (dy 21). The nutrient solution ws continuously erted ut not replced during the tretment. Evported/trnspired wter ws resupplied twice during the tretment. (men±sd, n=3; vlues mrked with different letters re significntly different t P<0.05) The stomtl conductnce ws the sme in nd controls (Fig. 5). The SS tretments hve negtive effect on the stomtl conductnce showing shrply decresing trend with incresing doses.

7 mmol H 2 O m -2 s c d c 0 sewge sludge (g pot -1 ) Figure 5. Stomtl conductnce in the youngest fully developed leves of Szrvsi-1 energy grss grown in nd mended with 0, 1, 5, 10 nd 15 g dried SS in 0.8 dm 3 pots. (men±sd, n=3; vlues mrked with different letters re significntly different t P<0.05) The control plnts grown in nd hd similr chlorophyll concentrtion in the youngest fully developed leves, 1.79 nd 1.83 mg Chl g -1 DW with 2.9 nd 3.0 Chl / rtio, respectively. There ws no considerle effect of ny SS tretments on Chl concentrtion nd / rtio ( ). 3.0 Chl (mg g -1 dw) c 3.1 d 2.9 cd c 3.0 c sewge sludge (g pot-1) Figure 6. Chlorophyll concentrtion nd Chl / rtio (indicted in ech column) in the youngest fully developed leves of Szrvsi-1 energy grss grown in nd mended with 0, 1, 5, 10 nd 15 g dried SS in 0.8 dm 3 pots. (dw=dry weight; men±sd, n=3; vlues mrked with different letters re significntly different t P<0.05) Mximl quntum efficiency of PSII The mximl quntum efficiency of PSII ws not ffected y the incresing SS tretment compred to the controls ut 10 g SS in significntly decresed it compred to the highest vlues in oth nd (Fig. 7).

8 0.90 F v /F m sewge sludge (g pot -1 ) Figure 7. Mximl quntum efficiency of PS II in the youngest fully developed leves of Szrvsi-1 energy grss grown in nd mended with 0, 1, 5, 10 nd 15 g dried SS in 0.8 dm 3 pots. (men±sd, n=3; vlues mrked with different letters re significntly different t P<0.05). Metl uptke Most of the elements hd similr concentrtion in the shoots of control plnts grown in nd except for K nd Zn which were higher in (Tle 2). Appliction of SS in the nutrient solution hd well-defined effect on the metl uptke of Szrvsi-1 energy grss. The mjority of the elements shows decresing ccumultion in the shoot s the SS doses incresed in either the or. These re the C, Mg, Mn, Zn, Cu, Cd. Some elements incresed in the shoot: N, Fe, Ni. Potssium decresed in the shoots of plnts grown in ut incresed in. Discussion SS dded to nutrient solutions The SS contins significnt quntities of orgnic mtter nd inorgnic elements tht provides the possiility to use it s orgnic mendment to reverse the degrdtion of soil nd to restore soil orgnic mtter s well s supplying redily ville nutrients to plnts (Shheen et l. 2014). For this reson, its ppliction to griculturl soils is n economiclly ttrctive mngement strtegy, ut its use hs een limited ecuse SS often contins significnt mounts of potentilly toxic metls (Singh nd Agrwl 2010). The totl elementl composition of the dried SS pplied in this study is typicl of communl wstes (Fytili nd Zniotou 2008, Singh nd Agrwl 2008, Usmn et l. 2012). It contins low concentrtion of hevy metls (Cu, Fe, Mn, Zn, Cr, Ni, Co, P) except for Cd which is little higher s compred to typicl metl content in SSs (Tle 1)(Fytili nd Zniotou, 2008). However, the orgniclly ound metls in SS re less ville thn more moile metl slts found in commercil fertilizers (Frost nd Ketchum Jr. 2000). This ws found when the dried SS ws dded to deionized wter or nutrient solution. There ws moderte (1-5x) increse in the solule concentrtion of most of the elements mesured (C, K, Mg, S, P, B, Cu, Fe, Zn) nd gret increse in tht of N. Moreover, the SS mended nutrient solution contined solule Al, Cr, Li, Sr nd Ti in low concentrtion. Mn concentrtion decresed compred to the unmended control solution while Cd, Co, Ni nd P levels remined elow detection limit lthough the dried SS contined them in considerle concentrtion. These chnges cn e explined y the inding of metl ctions to the orgnic mtter in the studied SS (Amir et l. 2005). The initil ph of the unmended nutrient solutions

9 in this study ws moving from the slightly cidic level towrds neutrl level y the ddition of incresing mounts of SS. The incresingly lkline ph my lso contriute to the decresing vilility of some metl ions especilly Fe (Mrschner 1995). Tle 2. Metl concentrtions in the shoots of Szrvsi-1 energy grss grown in nd mended with 0, 1, 5, 10 nd 15 g dried SS in 0.8 dm 3 pots. (men±sd, n=3; vlues mrked with different letters re significntly different t P<0.05) SS (g pot -1 ) K ( mol g-1 dw) 1315± ±86 949±79 c 857± 40 d 802±29 e K ( mol g-1 dw) 668± ±88 844±83 828±132 C ( mol g-1 dw) 130± ± 4 68±18 c 60± 1 d 54± 2 e C ( mol g-1 dw) 129± 8 118±21 51± 7 c 62± 20 c Mg ( mol g-1 dw) 114± 8 126±12 87± 20 72± 4 c 60± 0 d Mg ( mol g-1 dw) 91± ±21 77± 4 69± 16 c N ( mol g-1 dw) 13± 3 20± 4 27± 6 45± 8 c 49±1 c N ( mol g-1 dw) 26± 19 39±22 30± 8 45± 13 Fe ( mol g-1 dw) 1.0± ± ± ± ±0.3 Fe ( mol g-1 dw) 0.7± ± ± ±0.6 Mn ( mol g-1 dw) 2.3± ± ±0.4 d 0.8±0.1 cd 0.8±0.1 c Mn ( mol g-1 dw) 1.8± ± ±0.1 c 1.2±0.3 c Zn ( mol g-1 dw) 1.6± ± ± ± ±0.0 c Zn ( mol g-1 dw) 1.0± ± ± ±0.1 Cu ( mol g-1 dw) 0.14± ± ± ± ±0.01 Cu ( mol g-1 dw) 0.11± ± ± ±0.01 Cd (nmol g-1 dw) 2.5± ± ± ±0.3 c 0.6±0.2 c Cd (nmol g-1 dw) 2.1± ± ±1.0 <DL Ni (nmol g-1 dw) 4.1± ± ± * 9.6±0.9 Ni (nmol g-1 dw) 1.2± ± ± ±0.4 c DL, detection limit *, only one dtum ville Toxic effects of the SS tretments The root nd shoot growth ws oppositely ffected y the SS tretments in (Figs. 1-3). While root iomss decresed, shoot iomss ws lrgely unffected ut the shoot wter content nd shoot length decresed nd root wter content ws not influenced. The toxic effects ws exerted mostly y the two lrgest doses of the SS tretment while t the lowest dose root nd shoot iomss nd shoot length ws

10 even incresed slightly. The mjority of studies hve reported positive effect of SS on the crop yield even in cse of high dose or long term ppliction (Singh nd Agrwl, 2008). However, Qsim et l. (2001) found tht t the highest mendment doses in field ppliction the germintion nd yield hd declined. The wter content of the root tissues my hve correlted with the vilility of osmoticlly ctive solutes in the nutrient solution. Nevertheless, the prllel decrese in the root iomss nd the shoot wter content nd length implicte tht dmges my hve occurred in the roots tht lso inhiited the wter uptke somewht. Thus, the toxic effects influence the iomss production t the level of the physiology of roots. Similr chnges were oserved on the root nd shoot iomss production of willow, where the nutrient supply ws high enough to mke willows decrese the root growth proportionlly to the oveground prts (Jeri et l. 2015). The ph of the nutrient solutions incresed significntly during the 3 week growth period fter mendment. Being Pontic txon tht origintes from cronte-rich soils round the Blck Se region, Szrvsi-1 energy grss prefers lkline ph (Csete et l. 2011) nd this ws shown in cse of the control plnts nd the two lower tretment doses in which the ph reched 7.8. However, in the two higher tretment doses the ph further incresed up to 8.5 which my reflect tht the roots struggled with mintining the required cytoplsmic homeostsis (Fig. 4). Chnges in the Chl concentrtion nd Chl / rtio of the plnts s well s the mximl quntum efficiency of PSII were not influenced y ny doses of the SS tretments significntly (Figs. 6 nd 7). This mens tht neither the composition nor the photochemicl functioning of the photosynthetic pprtus suffered from ny toxic or inhiitory effects. Nevertheless, the stomtl conductnce (Fig. 5) decresed in prllel with the incresing SS concentrtion tht could hve inhiited the cron ssimiltion nd thus led to decresed iomss production. Since disturnces in the cron ssimiltion do not ffect the PSII quntum efficiency in generl, this effect ws not mesured in the photochemicl ctivity. Nevertheless no severe dmges, such s incresed ROS production occurred y the decresed stomtl conductnce which could hve led to the increse in the functioning of PSII rection centres. The dt re in greement with the work of Mt-Gonzlez et l. (2002) who studied gs exchnge nd photosynthesis rte in desert grsses. In nodulted lflf plnts sujected to cyclic drought, SS tretment lso improved photosynthetic ctivity (Antolin et l. 2010). Toxic elements ccumulting in the shoot my e the reson for the toxic effects of high doses of SS. However, in our study, most of the toxic elements decresed with incresing SS doses such s Cd nd Cu tht hve een proved to cuse toxicity in Szrvsi-1 energy grss (Tle 2) (Sipos et l. 2013). Cd is known to decrese the stomtl conductnce y intercting with the C metolism nd thus disrupting the stomtl opening (Perfus-Breoch et l. 2002). Cd ws lso shown to lter the cell wll thickening, leding to stomtl dysfunctions (Vitóri et l. 2003). The elements tht were incresing with SS doses were N, Fe nd Ni. Even though plnts hve different sensitivity ginst high iron concentrtions, iron toxicity my pper in plnts t severl orders of mgnitude higher concentrtion thn tht mesured in our plnts (2 µmol g -1 dw). In rice plnts iron toxicity mnifested in the decrese of the grin yield ws found over mg Fe kg -1, equl to round mmol Fe kg -1 (Audeert nd Shrvt, 2000). In sensitive species, sodium my cuse decline in yield nd disturnces in the wter regime ut lso in much higher concentrtion thn mesured here (50 µmol g -1 dw)(munns 2002). Furthermore it ws reported tht Szrvsi-1 energy grss is well dpted to slinity (Csete et l. 2011). Ni my e nother toxic metl once high concentrtions rech the sensitive tissues. For Szrvsi-1 energy grss the shoot concentrtion resulting in decline in iomss nd physiologicl prmeters ws 0.5 µmol g -1 dw (Sipos et l. 2013) wheres in this study its highest shoot concentrtion ws 0.01 µmol g -1 dw. As there ws no cler symptom of metl toxicity even t the highest SS tretments such s decresed Chl concentrtion nd photosynthetic ctivity we concluded tht the min reson for decreses in the mesured indices ws i. the nchoring of essentil metls in the orgnic components of the SS resulting in nutrient inlnce, ii. the cumultive nd synergistic effect of non-essentil elements, iii. the effect of toxic orgnic components tht we did not mesure.

11 Nutritive effects of the SS tretments In the root dry mss of control plnts ws lmost doule compred to tht in wheres the shoot dry mss ws lmost the sme (Fig. 1). This my refer to the root growth stimultion y the nutrient deficiency (Hermns et l. 2006). Decresing root iomss ws oserved with incresing rtes of SS ppliction until the control level of ws reched nd simultneously the shoot iomss ws incresing showing the stimulting effect of incresing SS doses. Only the highest, 10 g per pot SS ppliction resulted in significnt drop in root nd shoot iomss indicting the toxicity of high-dose of the pplied SS y metolic disturnces in plnts. Shoot length showed similr chnges (Fig. 3). But the wter content of the root nd shoot showed cler incresing tendency with incresing SS ppliction rtes (Fig. 2). Prmeters showing physiologicl chnges lso reflected stimultion or no effect up to 5 g SS ppliction to (Figs. 4-7). The highest pplied dose, 10 g per pot resulted in extreme ph increse nd decline in the PSII mximl quntum efficiency. Only stomtl conductnce showed decresing tendency with incresing SS doses ove 1 g per pot. This ltter, however, corresponds to the incresing root nd shoot wter content. Similr effect ws oserved on Lrix decidu seedlings, where SS ppliction improved the net photosynthesis, elevted the chlorophyll content nd enhnced the dry mtter ccumultion rte (Bourioug et l., 2015) Bsed on the ove oservtions the moderte doses of SS pper to hve good nutritive vlue. This is further confirmed y the shoot element concentrtions. Toxic metls were detected in lower concentrtion compred to the SS tretments in while essentil ones were t similr level in spite of the one tenth dilution of the originl () solution. Potssium nd Fe showed increse s the SS dose incresed tht could highly contriute to the positive effect of the tretments. Nutrient solution s model system for testing SS In conclusion, pplying SS in nutrient solution hs comined effect: the concentrtion of plnt ville nutrients nd other ions my increse with the SS element content ut lso decrese due to introduction of orgnic components with C.E.C. This my represent quick model system for testing how the SS my lter the nutrient lnce in the medium. In this study we hve experienced the pplied SS with low hevy metl content hs moderte toxic effect on Szrvsi-1 energy grss t the highest ppliction rtes. Stomtl conductnce ppered to e the most sensitive prmeter. While pplying the SS in solution with decresed nutrient content clerly showed the nutritive its vlue. Bsed on the present findings SS mended soil cultures will e further tested to improve degrded lnds with SS mendments tht would e utilized in renewle crop production for energy production. This ppliction would void humn nd niml helth prolems nd further reduce expenses emerging due to environmentl issues. Acknowledgements Á. Solti ws lso supported y the Bolyi János Reserch Scholrship of the Hungrin Acdemy of Sciences (BO/00207/15/4). References Antolín M C, Muro I, Sánchez-Díz M (2010) Appliction of sewge sludge improves growth, photosynthesis nd ntioxidnt ctivities of nodulted lflf plnts under drought conditions. Environmentl nd Experimentl Botny 68:

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