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1 Chemosphere 73 (2008) Contents lists vilble t ScienceDirect Chemosphere journl homepge: Crbon deposition in soil rhizosphere following mendments with compost nd its soluble frctions, s evluted by combined soil plnt rhizobox nd reporter gene systems Edordo Puglisi, George Frgoulis, Attilio A.M. Del Re, Riccrdo Spccini b, Alessndro Piccolo b, Giovnni Gigliotti c, Dniel Sid-Pullicino c, Mrco Trevisn, * Is ti tu to di Chi mi c Ag rr i ed Am bi en tle, Uni ver sità Ctto li c del Scro Cu or e, Vi Emil i Pr mense 84, Pi cen z, Itly b Di pr ti men to di Sci en ze del Su ol o, dell Pint, dell Am bi ente e delle Prod uzi on i Ani mli, Uni ver sità di Npoli Fede ric o II, Vi Uni ver sità 100, Por tic i, Itly c Di pr ti men to di Sci en ze Agr rie e Am bi en tl i, Uni ver sitá di Pe ru gi, Borgo XX Giu gno 72, Pe ru gi, Itly rticle info Article history: Received 11 April 2008 Received in revised form 6 July 2008 Accepted 8 July 2008 Avilble online 2 September 2008 Key words: Com post DOM Rhi zo box Pseu do mo ns flu o res cens pucd607 NMR bstrct We deter mined the orgnic cr bon relesed by roots of mize plnts (Ze mys L.) when grown in soils mended with com post nd its sol u ble frc tions. In rhi zo box sys tems, soil nd roots re sep rted from the soil of lower com prt ment by nylon mem brne. Tret ments re pplied to the upper com prtment, while in the lower com prt ment lumi nes cent bio sen sors me sure the bio vil ble orgnic cr bon relesed by roots (rhi zo de po si tion). The rhi zo box plnts sys tems were mended with com post (COM), its wter extrct (TEA), the hydro pho bic (HoDOM) nd hydro philic (Hi DOM) frc tions of the dis solved orgnic mt ter (DOM) extrcted from the com post. After root devel op ment, the lower untreted comprt ments were sm pled nd sliced into thin ly ers. The bio vil ble orgnic cr bon in ech lyer ws ssessed with the lux-mrked bio sen sor Pseu do mo ns flu o res cens pucd607, nd com pred with totl orgnic cr bon (TOC) nl y ses. The TOC vl ues rnged between 8.4 nd 9.6 g kg 1 nd did not show ny sig nifi cnt dif fer ences between bulk nd rhi zo sphere soil sm ples in ny tret ment. Con versely, the bio sen sor detected sig nifi cnt dif fer ences in vil ble C com pounds for rhi zo sphere soils mended with vr i ous orgnic mte ri ls. Con cen tr tions of vil ble orgnic com pounds in the first 2 mm of soil rhizo sphere were 1.69 (con trol), 1.09 (COM), 2.87 (Hi DOM), 4.73 (HoDOM) nd 2.14 (TEA) lmol C g 1 soil g 1 roots. The pplied rhi zo box bio sen sor inte grted method ws suc cess ful in detect ing nd qun tify ing effects of orgnic mend ments on orgnic cr bon relesed by mize plnt roots. This pproch my become impor tnt in ssess ing the cr bon cycle in gri cul turl soils nd soil tmo sphere com prtments Else vier Ltd. All rights reserved. 1. Intro duc tion The rhi zo sphere is bio log i clly ctive zone in soil where com plex inter c tions occur mong plnt roots, soil pr ti cles nd microbes. Inves ti g tions on the rhi zo sphere help to gin bet ter under stnd ing on vri ety of pro cesses such s nutri ent cycling, eco sys tem func tion ing, cr bon dynm ics, nd to evl u te the impcts of nthro po genic ctiv i ties on gri cul turl nd nt u rl ecosys tems. An impor tnt rhi zo sphere pro cess is rhi zo de po si tion, tht involves the trns fer of C from plnt roots to soil in the form of wter-sol u ble exu dtes (low molec u lr weight mol e cules such s orgnic cids, mino cids nd sug rs), secre tions of higher molecu lr weight sub stnces, lystes from cells fter utol y sis, gses * Cor re spond ing uthor. Tel.: ; fx: E-mil ddress: mrco.tre vi sn@unic tt.it (M. Tre vi sn). such s eth yl ene, cr bon diox ide, nd hydro gen cy nide (Gry ston et l., 1996). Through rhi zo de po si tion, plnts sup ply soil microbes with red ily vil ble orgnic C, which increses micro bil bio mss round roots nd ffects ctiv ity nd com po si tion of micro bil com mu ni ties. On the other hnd, microbes mobi lize nutri ents, primr ily nitro gen, nd ren der them vil ble to plnts, s described by the micro bil loop model (Pt er son, 2003; Bon kow ski, 2004). Rhi zo sphere nutri ent flow lso plys n impor tnt role in orgnic C mobi li z tion nd immo bi li z tion pro cesses, with impor tnt conse quences on cr bon diox ide fluxes involved in the green house effect (Cheng nd John son, 1998). In pr tic u lr, it hs been estimted tht bout 15 25% of cr bon fixed below-ground is exuded from roots into soil, includ ing root turn over (Ku zy kov, 2002). Sev erl envi ron men tl fc tors influ enc ing plnt growth nd physi ol ogy hve been reported to ffect rhi zo de po si tion, includ ing light inten sity, tmo spheric CO 2 con cen tr tion, pres ence of specific micro or gn isms, soil texture, nd orgnic mt ter com posi tion (Me hrg nd Kill hm, 1995; Pt er son et l., 1997). Knowl /$ - see front mtter 2008 Else vier Ltd. All rights reserved. doi: /j.chemosphere

2 E. Pugl is i et l. / Chemosphere 73 (2008) edge of rhi zo de po si tion pro cesses is still incom plete, pr tic u lrly due to the lim ited meth ods vil ble for study ing rhi zo sphere C depo si tion nd flow under rel is tic con di tions. More over, meth ods with com ple men try, cross-dis ci plin ry (phys ics, chem is try nd biol ogy) strengths hve not yet been effec tively com bined. Rhi zo box sys tems were intro duced to study root-induced chnges in soil prop er ties (Ku chen buch nd Jungk, 1982; You ssef nd Chino, 1989; Wen zel et l., 2001). They re bsed on soil root com prt ment nd one or more dj cent soil com prt ments sep rted by porous mem brnes, thus llow ing the mon i tor ing of gr di ents of rhi zo sphere chr c ter is tics t high sp til res o lu tion. Rhi zo box sys tems re com posed of two phys i clly sep rted comprt ments: n upper plnt roots soil com prt ment, nd lower rhi zo sphere com prt ment, where the soil is sep rted from roots by nylon mem brne. This enbles tret ment ppli c tions to the upper com prt ment nd evl u tion of con se quent effects in the untreted lower com prt ment. The devel op ment of reporter gene tech nol o gies such s biosen sors hs improved reserch not only on rhi zo sphere C depo sitions, but lso on the fte of other sso ci ted nutri ents (Kill hm nd Yeo mns, 2001). The most com mon bio sen sors re micro orgn isms into which reporter genes (e.g., lux, encod ing lucif er se; lcz, encod ing b-glc to si dse nd luc, encod ing fire fly lucif er se) re intro duced nd even tu lly con trolled by pro mot ers cti vted by spe cific com pounds. Pseu do mo ns spp. re ubiq ui tous in mny gri cul turl soils nd, s such, serve s use ful recipient orgn isms for bi or e port ing genes. Pseu do mo ns flu o res cens pucd607 (Hm in-hn jn i et l., 1993) is lumi nes cence-bsed bio sen sor con structed by genetic trns for m tion involv ing the inser tion of lux genes from the nt u rl mrine bc te rium Vib rio fisc her i. These genes re inserted upstrem of gen erl met bolic pro moter nd thus revel the orgn ism s gen erl metb o lism by lumi nes cence. If the bio sen sor is plced under strv ing con di tions, the emit ted light inten sity is reduced to min i mum. How ever, the emit ted light cn be restored qun ti t tively when C sources re vil ble. This fe ture hs been suc cess fully exploited for revel ing orgnic C depo si tion under dif fer ent con di tions, by using pro to col developed by Yeo mns et l. (1999), nd sub se quently pplied to nonster ile soils by Stnd ing et l. (2005). The bio sen sor my be directly pplied in situ on soil sm ples nd the light emis sion becomes func tion of the con cen tr tion of red ily bio vil ble orgnic compounds tht re com monly pres ent in root exu dtes. In recent yers, recy cled orgnic wstes hve been widely used to increse soil fer til ity nd s n eco nom i cl wste dis posl strtegy. An impor tnt eco log i cl role my be plyed by the sol u ble or red ily vil ble frc tions from com post mte ri ls. The hydro phobic orgnic frc tion (HoDOM) of com post ws shown to increse the dsorp tion nd hydro ly sis of pes ti cides (Sid-Pulli cin o et l., 2004) nd to be involved in the st bil iz tion of orgnic cr bon in soils by hydro pho bic pro tec tion (Pic colo et l., 2004). Aer ted compost te (TEA) is becom ing widely used com mer cil fer til izer nd it ws shown to pos i tively ffect the pro duc tion of fruits such s rsp ber ries (Hr greves et l., 2008). While mny stud ies focused on the inter c tions between xeno bi ot ics nd com post, humic cids, nd dis solved orgnic mt ter (DOM) (Br ri uso et l., 1995; Tot sche et l., 1997; Pic colo et l., 1998), little is known bout the effects of these mte ri ls on plnt rhi zo de po si tion pro cesses. Swin nen et l. (1995) dem on strted tht more C tended to be trns ferred to br ley (Hord e um vulg re L.) roots under con ven tionl mn ge ment with high fer til izer ddi tions thn in inte grted mn ge ment with reduced inputs. Addi tion of com posted mte ril incresed wtersol u ble C nd enzy mtic ctiv i ties in rhi zo sphere of Ole eu ro pe, thus sug gest ing n increse micro bil ctiv ity induced by plnt roots (Cr vc et l., 2002). Con versely, syn thetic nitro gen fer tiliz tion ws shown to cuse sig nifi cnt decrese in the mount of orgnic C trns ferred to roots (Ngu yen, 2003). The min objec tive of this study ws to evl u te the rhi zo de po si tion of mize plnts (Ze mys L.) mended with dif fer ent well-chr c ter ized orgnic mte ri ls by com bined ppli c tion of rhi zo box nd reporter gene sys tems. The rhi zo sphere com prt ment ws sliced into thin ly ers using micro tome tech niques nd the vil ble frc tion of orgnic C in ech lyer ws ssessed with the lux-mrked P. flu o res cens pucd607, nd com pred to totl orgnic C (TOC) nl y ses. 2. Mte ri ls nd meth ods 2.1. Chr c ter iz tion of orgnic mte ri ls The urbn wste com post used in this work ws pro duced mechn i clly on n indus tril scle t the Ges enu SpA com post ing fcil ity in Pie trm e li n (Pe ru gi, Itly). The feed stock ws com posed of source-sep rted municipl solid wste (55% w/w), yrd trim mings from prun ing ctiv i ties (30%) nd folige res i dues from tobcco gro-indus try (15%). Com post ing ws cr ried out under er o bic con di tions nd involved ther mo philic phse of pprox i mtely 28 d dur ing which the feed stock ws dily turned, fol lowed by cur ing phse of pprox i mtely 3 ddi tionl months in piles. The chem i cl com po si tion of the com post used is reported in Tble 1. Dis solved orgnic mt ter (DOM) ws extrcted from compost s described by Gig liotti et l. (2002). Briefly, com post ws plced in con tct with deion ized degssed wter in n extrc tion rtio of 1:10 w/v for 24 h t room tem per ture nd sub se quently cen tri fuged t 2500g. The super n tnt ws col lected nd fil tered through 0.7 lm glss mi cro fi bre fil ter nd 0.45 lm mem brne fil ter to obtin the totl DOM extrct. ph of the DOM extrct ws round 8.2. This extrct ws divided into two frc tions by mens of Am berlite XAD-8 nd XAD-4 res ins. The DOM extrct ws cid i fied to ph 2 with 0.1 N HCl nd pssed through the XAD-8 nd XAD-4 res ins. The orgnic frc tion retined by the XAD-8 resin ws eluted with 0.1 N NOH, pssed through AG MP-50 ct ion exchnge resin nd sub se quently freeze-dried. This frc tion ws defined s the hydropho bic frc tion (HoDOM). The orgnic frc tion retined by the XAD-4 resin ws eluted with wter:ce to ni trile (1:3) mix ture. Ace to ni trile ws removed from the elu te by rotry evp o r tion t 35 C nd the remin ing que ous solu tion freeze-dried. This frction ws defined s the hydro philic frc tion (Hi DOM). The HoDOM nd Hi DOM frc tions hd n orgnic C con tent of 499 mg g 1 nd 523 mg g 1, respec tively. The er ted com post te (TEA) frc tion ws obtined by suspend ing com post in deion ized wter (1:5 w/v rtio) nd lev ing it under stir ring for 10 d t 20 C. In order to ensure er o bic con- Tble 1 Chr c ter is tics of municipl wste com post Mois ture con tent (g kg 1 ) 239 ±17 Ash con tent (g kg 1 ) 446 ±10 ph 8.0 ±0.1 Totl orgnic cr bon (g OC kg 1 ) 188 ±2 Totl extrct ble cr bon (g OC kg 1 ) 128 ±2 Humic cids (g OC kg 1 ) 71.3 ±1.4 Ful vic cids (g OC kg 1 ) 7.0 ±0.3 Non-humic frc tion (g OC kg 1 ) 49.3 ±2.0 Dis solved orgnic cr bon (g OC kg 1 ) 15.0 ±0.1 Hydro pho bic DOM (g OC kg 1 ) 8.8 ±0.1 Hydro philic DOM (g OC kg 1 ) 6.3 ±0.1 SOUR mx (mg O2 g 1 VS h 1 ) b 1.9 ±0.3 Aver ge of trip li cte nl y sis ± stn drd error; ll dt except mois ture con tent nd SOUR mx re expressed on com post dry weight bsis. b Mx i mum spe cific oxy gen uptke rte nor ml ized to the con tent of vol tile solids (VS) nd me sured s described by Ad ni et l. (2003).

3 1294 E. Pugl is i et l. / Chemosphere 73 (2008) di tions, the fer men t tion ves sel ws equipped with ir dif fus ers nd n oxy gen probe to con stntly mon i tor dis solved oxy gen. A soft wre driven con trol sys tem helped to min tin dis solved oxygen con cen tr tions bove 2.0 mg L 1, by mens of inter mit tent ir bub bling through the sus pen sion. After 10 dys the com post suspen sion ws fil tered through cel lu lose fil ter pper, nd stored t 4 C. Orgnic C con cen tr tion of er ted com post te ws 1.19 g L 1. A frc tion of the com post te ws freeze-dried to obtin solid sm ple for chem i cl chr c ter iz tions CPMAS-NMR spec tros copy The molec u lr com po si tions of orgnic mte ri ls were evl u- ted by Cross-Polr i z tion Mgic Angle Spin ning cr bon-13 Nucler Mg netic Res o nnce spec tros copy (CPMAS- 13 C NMR). Exper i ments were con ducted on Bru ker AV300 instru ment oper t ing t crbon-13 res o nt ing fre quency of MHz min tin ing the rotor spin rte t Hz. All exper i ments were con ducted with pulse sequence CP with RAMP, recycle time of 1.5 s, nd n cqui si tion time of 20 ls. Fur ther detils on sm ple tret ment nd other oper tionl con di tions re reported in Šme jk lová et l., The differ ent chem i cl-shift regions of the spec tr were uto mt i clly inte grted to obtin the hydro philic C/hydro pho bic C (HI/HB) rtio for ech mte ril Lip ids frc tion tion Ali quots of ech orgnic mte ril (350 mg of com post, TEA nd HoDOM, 150 mg of Hi DOM) were oven dried t 40 C for 1 h. Lipids were frc tion ted ccord ing to their extrct bil ity from the rel tive originl mtrix. Struc tur lly unbound (SU) com pounds were extrcted using dichlo ro meth ne nd meth nol mix ture (2:1 v/v) for 2 h t room tem per ture. After extrc tion of struc turlly unbound lip ids, the wekly-bound com pounds (WB) were extrcted by tret ing the result ing ir-dried res i due with 15 ml of 12% boron tri fluor ide meth nol (BF 3 CH 3 OH) solu tion t 90 C for 12 h in poly eth yl ene bot tle. The detils of extrc tion pro ce dures nd GC MS deter mi n tion method re described in Fi o ren ti no et l., Com pounds still strongly-bound (SB) to the ir-dried resi due result ing from the BF 3 CH 3 OH trnse ste ri fi c tion were further extrcted in 100 ml of 1 M KOH in meth nol for 1 h t 70 C s reported by Nie rop et l., The sequen tilly extrcted frctions were cls si fied s unbound (SU, freely dis solved in orgnic sol vent), wekly bound (WB, sol u ble only fter mild trnse ste rifi c tion), nd strongly bound (SB, sol u ble only fter meth no lic lk line hydro ly sis) mte ril. Both WB nd SB extrcts were mde up of n orgnic nd n que ous phse. The qun ti fi c tion of the extrcted com pounds ws bsed on the sum of sig nl res observed in the GC MS chro mto grms. The qun ti t tive nl y sis ws con ducted by using cl i br tion curves of dif fer ent exter nl stn drds from Aldrich: tri dec noic cid, oct de c nol, 16-hydroxyhex dec noic cid, doc osndioic cid, b-sitos terol, nd cin nmic cid. Tri dec noic cid ws lso used s n inter nl stn drd to evlu te deriv ti z tion yields nd ste di ness of the chro mto grphic response. All GC MS nl y ses were con ducted in dupli ctes over three indi vid ul extrcted sm ples Soil An orchrd soil ws col lected t field site ner M ter, South- Est Itly; soil sm ples (7 cm dim e ter cores) were rn domly collected from the sur fce lyer (0 20 cm) t lest 2 m fr from trees, sieved through 2 mm sieve, pooled on site nd stored t 4 C before using for exper i ments. Soil hd lom texture (24.7% cly (<0.002 mm), 33.8% silt ( mm), 41.5% snd (>0.05 mm)), ph (H 2 O) of 6.7, nd totl orgnic C of 8.3 mg C g 1 d.w. soil Plnt cul ti v tion nd micro tome sm pling All exper i ments were con ducted in the rhi zo box sys tem described by Wen zel et l. (2001) (Fig. 1), which com prises n upper soil plnt com prt ment nd phys i clly sep rted lower rhi zo sphere com prt ment. Roots only ver ti clly pen e trte through nr row slit from the upper to the lower com prt ment, where they re sep rted from the rhi zo sphere soil by nylon mem brne, thus form ing thin root plne. We employed Nylon mem brnes of 7 lm mesh width, which exclude the pen e tr tion of root hirs in the rhi zo sphere soil (Wen zel et l., 2001). Amend ments were pplied only to the upper rhi zo box com prt ments by homo ge neously mix ing orgnic mte ri ls with soil. The lower rhi zo sphere soil com prt ments were not mended. Five tret ments were pplied, with 3 rep li ctes (3 rhi zo boxes) per tret ment (Tble 2): (i) con trol with out ny ddi tion; (ii) 11.5 g kg 1 of com post (COM); (iii) 67 mg kg 1 of the hydro pho bic frc tion of the DOM extrcted from com post (HoDOM); (iv) 67 mg kg 1 of the hydro philic frc tion of the DOM extrcted from com post (Hi DOM); (v) 57.7 ml kg 1 of te extrcted from com post (TEA). All mte ri ls were dis solved in deion ised wter before being dded to the upper rhi zo box com prt ments. All solu tions hd ph round 6. Although the totl pplied dose of com post ws three orders of mg ni tude lrger thn tht of extrcts, tret ments re firly com p r ble con sid er ing tht dose of 11.5 g kg 1 of com post brought to soil mg kg 1 of orgnic C s DOM. Fur ther more, sim i lr doses re Fig. 1. Cross sec tion of the rhi zo box sys tem, dpted from Wen zel et l. (2001). (1) soil plnt com prt ment; (2) trns pr ent crylic win dow; (3) nylon mem brne; (4) rhi zo sphere soil com prt ment nd (5) irri g tion wicks. Tble 2 Tret ments pplied to ech rhi zo box nd rel tive doses of ech mte ril pplied to soil in the top com prt ment Orgnic mte ril Applied dose Applied dose expressed in com post equiv lents (t h 1 ) Orgnic C dded Com post 11.5 g kg 1 Soil g kg 1 Soil (172.5 mg kg 1 Soil s DOM) HoDOM 67 mg kg 1 Soil mg kg 1 Soil Hi DOM 67 mg kg 1 Soil mg kg 1 Soil TEA 57.7 ml kg 1 Soil mg L 1 Soil

4 E. Pugl is i et l. / Chemosphere 73 (2008) obtined when doses of extrcts re expressed s com post equiv lents, i.e., the mount of pplied extrct rel tive to pplied compost (Tble 2). Mize seeds (Ze mys L.) were ger mi nted in Petri dishes nd pre-grown in the upper com prt ment of ech rhi zo box (6 seeds per ech box). Ten dys fter ger mi n tion, the upper nd the lower com prt ment of ech rhi zo box were con nected nd the roots llowed penetrting through the slit. At 25 dys fter ger min tion, the root plne ws fully devel oped in ech rhi zo box, nd the lower com prt ment ws sm pled using micro tome (Wen zel et l., 2001) in order to obtin soil sm ples t spe cific dis tnces from the root plne (0 2, 2 4 nd 6 4 mm). Soil sm ples from the upper com prt ment of the con trol tret ment, i.e., bulk soil without ny mend ment, were lso n lysed. Root bio mss yields for ech tret ment were, respec tively, (g dry weight): 0.29 ± 0.03 (control), 0.36 ± 0.06 (COM), 0.18 ± 0.04 (HoDOM), 0.26 ± 0.08 (Hi DOM), 0.35 ± 0.04 (TEA). All soil sm ples were fro zen imme di tely fter sm pling nd kept t 20 C before being n lysed. All exper i ments were con ducted in Binder KBWF 240 (Tuttlin gen Ger mny) phy to tron under the fol low ing con trolled exper i- men tl con di tions: 16 h light (120 le s 1 m 2 ) t 30 C, 8 h night t 20 C, 60% humid ity Soil chem i cl nl y ses Totl orgnic cr bon (TOC) con cen tr tions were deter mined by oxi d tion to CO 2 with pots sium dichro mte ccord ing to the Wlkley Blck method (Meth ods of Soil Anl y sis, 1996) nd expressed s g kg 1 of soil on dry weight bsis Bc te ril strin, growth con di tions nd bio s sy pro to col Cells cul tures nd bio s sys were cr ried out s described by Yeo mns et l. (1999). Cul tures of P. flu o res cens pucd607 (lux CDA BE from Vib rio fisc her ii, knr, mpr; Hm in-hn jn i et l., 1993) were grown in 250 ml Erlen meyer flsks con tin ing 100 ml of LB broth t 25 C, nd rotted t 200 rpm. Lte expo nen til cells were hr vested by cen tri fu g tion (5 min t 4000 g) t OD 550 of 2.0, which cor re sponds to con cen tr tion of P. flu o res cens pucd607 cells of round 3.00 E + 07 cells ml 1 (Yeo mns et l., 1999). The super n tnt ws decnted nd the cells resus pended in the sme vol ume of C-free M9 min i ml medium. The pro cess ws repeted nd the finl cell sus pen sion shken t 200 rpm in C-free M9 for 2 h t 25 C (str v tion step). Kn my cin ws dded t 50 lg ml 1 to ll cul tures. One mil li li tre li quot of strved cells were dded to 100 mg sm ples of soil col lected from rhi zo boxes in order to me sure in situ cr bon depo si tions. After 20 min of con tct, 800 ll of soil nd bc te ri slurry ws col lected nd me sured for light emis sion. Bios sys were cl i brted on dilu tion series of glu cose stn drd solutions in the rnge of con cen tr tions between 1 nd 10 mm of C con tent. Response of the bio sen sor ws lso tested on soil sm ples enriched with incres ing con cen tr tions of glu cose St tis ti cl nl y ses All st tis ti cl nl y ses were per formed using SPSS soft wre. TOC nd bio sen sor results from rhi zo boxes soil sm ples were studied by mens of fixed model nl y sis of vr i nce (ANOVA). The follow ing effects were stud ied: (1) Sm pling effect (cls si fi c tion vr i ble SAMP). The sm pling types were: bulk 0 dys (for con trol only), rhi zo sphere 0 2 mm, rhi zo sphere 2 4 mm, rhi zo sphere 4 6 mm. (2) Tret ment effect (cls si fi c tion vr i ble TREAT). The five tret ments were: con trol, com post, Hi DOM, HoDOM, nd TEA. (3) The inter c tion between the two pre vi ous effects (cls si fi ction vr i ble TREAT * SAMP). All sig nifi cnt effects were con firmed by Tu key s test (t P < 0.05) for com pr i son of mens. Cor re l tions between TOC nd bio sen sor dt were lso n lysed by mens of lin er regres sion. Sum of dif fer ent orgnic com pounds obtined in sequen til extrcts of dif fer ent fer til iz ers were lso n lysed by mens of fixed model nl y sis of vr i nce (ANOVA). Two dif fer ent effects were stud ied: the effect of the sequen til extrc tion nd the effect of the type of fer til izer. 3. Results 3.1. Chr c ter is tics of orgnic mte ri ls The spec trl infor m tion obtined from CPMAS-NMR spectr (Fig. 2) of the dif fer ent com post mte ri ls re sum m rized in Tble 3. The cr bon dis tri bu tion in the dif fer ent inter vls ws used to define the HI/HB rtio, n index of hydro phi lic ity of com plex orgnic mte ri ls (Spc cin i et l., 2000). This rtio indi cted, s expected, tht the Hi DOM frc tion ws the most hydro philic of ll frc tions, wheres the HoDOM ws the most hydro pho bic. The com post itself nd the TEA hd sim i lr HI/HB rtio. The qun ti t tive sum of com pounds (s percent of ini til orgnic cr bon in the n lysed sm ple) extrcted from dif fer ent 2.8. Lu mi nom e try nd bio sen sor response dt Light output ws me sured using Sys tem Sure lumi no meter Model (Nov Bio med i cl Wl thm MA, USA). In order to compre results from dif fer ent tril lumi nes cence dt were con verted from rel tive light units (RLU; 1 RLU equiv lent to 1 mv per 10 s) to rel tive glu cose cr bon units (RGU), fol low ing inter po l tion with cl i br tion curves of glu cose stn drd solu tions nd expressed in terms of lmol of glu cose C equiv lents per grm of soil. Bio sensor response dt were lso nor ml ized to the mount of Cr bon dded in ech tret ment, nd thus expressed s mg of relesed C per grm of pplied C. Fig. 2. CPMAS 13 C NMR spec tr of (A) com post; (B) TEA; (C) HoDOM nd (D) Hi- DOM.

5 1296 E. Pugl is i et l. / Chemosphere 73 (2008) Tble 3 Per cent ge dis tri bu tion of 13 C in res o nnce inter vls (ppm) nd hydro philic-c/hydro pho bic-c (HI/HB) rtios of orgnic mte ri ls ccord ing to CPMAS-NMR spec tros copy Orgnic mte ril Alkyl-C C N C O Aro mtic-c Cr boxyl-c Ketone-C HI/HB Com post TEA HoDOM Hi DOM HI/HB = [(50 71) + (71 100) + ( )/(0 50) + ( )]. com post nd com post-derived sm ples re reported in Tble 4. The SU com pounds, minly com posed of lip ids sol u ble in orgnic sol vents, were more bun dnt in TEA thn in the bulk com post, wheres HoDOM nd Hi DOM showed the low est bun dnce of SU com pounds. The WB com pounds, extrcted fter trnse ste ri fi ction, were pre dom i nnt in the orgnic frc tion from com post, nd, to lower extent, from TEA. The WB que ous frc tion ws lrger in HoDOM. On the other hnd, sig nifi cntly lower mount of WB orgnic mte ri ls ws detected in both the que ous nd orgnic frc tions of Hi DOM. A lrge mount of SB com pounds ws obtined fter meth no lic lk line hydro ly sis of com post, the mjor ity of which were in the orgnic frc tion. A lower mount of WB compounds ws found in TEA, though they were eqully dis trib uted in the que ous nd orgnic frc tions. The HoDOM sm ple pro duced little WB com pounds in the orgnic frc tions, wheres no strongly bound com pounds (SB) were pres ent in the Hi DOM extrct. The result obtined for frc tion tion of com post-derived mteri ls into pro gres sively less vil ble orgnic com pounds, gen er lly grees well with the sm ple chr c ter is tics shown by CPMAS-NMR spec tros copy. The Hi DOM, with the gret est hydro philic chr c ter, lso showed the low est mount of mte ril detected by GC MS, thus imply ing lrger con tent of low molec u lr-weight hydro philic compounds, such s sim ple cr bo hy drtes nd orgnic cids. Con versely, the hydro pho bic chr c ter of HoDOM is relted to the con tent of both SU nd WB com pounds in the sm ple mtrix (Tble 4) Totl orgnic cr bon con tent TOC orgnic vl ues in ll n lysed sm ples were very sim i lr, rng ing from min i mum of 8 to mx i mum of 10.2 g kg 1 soil. The two-wy fixed ANOVA model showed tht only the TREAT effect ws sig nifi cnt for TOC vl ues (P < 0.05). The con trol tret ment hd n ver ge TOC con tent of 8.52 g kg 1 of soil, wheres ll other orgnic mt ter tret ments hd sig nifi cntly lrger vl ues (8.81, 9.13, 9.06 nd 9.12 g kg 1 of soil, for COM, Hi DOM, HoDOM, nd TEA, respec tively). How ever, no sig nifi cnt dif fer ences were found for SAMP nd TREAT * SAMP effects. More over, TOC vl ues for the dif fer ent rhi zo sphere ly ers of ll tret ments were not st tis ti clly dif fer ent, ccord ing to Tu key s test for com pr i son of mens (Fig. 3) Pseu do mo ns flu o res cens pucd607 ssy Pre vi ous exper i ments (dt not shown) dem on strted tht the bio sen sor cn be pplied in situ on soil sm ples with dif fer ent textures. The emis sion of light in response to the glu cose stn drd solu tions in the pres ence nd bsence of soil ws lin er in the rnge between 1 nd 10 mm of glu cose Cr bon (R 2 > 0.97, P < 0.05), nd the two cl i br tion curves were not st tis ti clly dif fer ent (P = 0.097). The lin er regres sion obtined with glu cose stn drds in the bsence of soil ws used to con vert RLU vl ues obtined into rel tive glu cose cr bon units (RGU), tht re defined s lmol of glu cose C per grm of soil. Results of the fixed two wy ANOVA model for bio sen sors RGU dt showed sig nifi cnt dif fer ences (P < 0.001) for ll stud ied effects (i.e., TREAT, SAMP, nd TREAT * SAMP). Bio sen sor response t ech sm pling position for ech tret ment is plot ted in Fig. 3b, together with the results of Tu key s of the TREAT * SAMP inter c tion. Cr bon depo si tions regrd less of the sm pling position (TREAT effect), were sig nifi cntly dif fer ent in ll tret ments. Lrger vl ues were found for HoDOM (0.85 lmol glu cose C g 1 soil g 1 ), fol lowed by Hi DOM (0.75 lmol glu cose C g 1 soil g 1 ), TEA (0.74 lmol glucose C g 1 soil), con trol (0.53 lmol glu cose C g 1 soil), nd COM (0.40 lmol glu cose C g 1 soil). SAMP effect showed sig nifi cnt dif fer ences between the first lyer (0 2 mm) of soil rhi zo sphere (0.92 lmol glu cose C g 1 soil), nd the two fol low ing ly ers (2 4 nd 4 6 mm) hv ing RGU vl ues of 0.48 nd 0.57 lmol glu cose C g 1 soil, respec tively. Tble 4 Percent (nor ml ized to the orgnic cr bon con tent in ini til orgnic mte ri ls) of sum of orgnic com pounds observed in GC MS chro mto grms of sequen til extrcts, s struc tur lly unbound (SU), wekly-bound (WB), nd strongly-bound mol e cules (SB), nd of solid res i due remin ing fter extrc tions Orgnic mte ri ls Com post Te HoDOM Hi DOM F vlue SU Orgnic frc tion (±1.02) b B (±0.88) A 6.64 (±0.94) b C 7.69 (±0.78) C 583*** WB Aque ous frc tion 4.51 (±0.66) c B 1.39 (±0.38) c C (±0.75) A 0.57 (±0.12) b C 1280*** WB Orgnic Frc tion (±1.15) A 6.28 (±0.42) b B 0.87 (±0.11) c C 0.44 (±0.08) b C 3335*** SB Aque ous frc tion 0.00 d B 0.43±0.10 c A 0.00 c B 0.00 b B 52*** SB Orgnic Frc tion 6.72 (±0.96) c A 0.37 (±0.18) c B 0.18 (±0.09) c B 0.00 b B 133*** Solid res i due (±0.79) b A 0.99 (±0.23) c B 0.14 (±0.03) c B 0.00 b B 1325*** F vlue 1002*** 2672*** 1125*** 267*** ANOVA sig nifi cnt dif fer ences re indi cted by F vl ues (*P < 0.05, **P < 0.01, ***P < 0.005) for com pr i sons between rows nd col umns. Dt fol lowed by the sme minor let ter on ech col umn or by the sme cp i tl let ter on ech row re not st tis ti clly dif fer ent from ech other (Tu key s test, P < 0.05).

6 E. Pugl is i et l. / Chemosphere 73 (2008) b g OC / kg RCU (umol glucose-c/g ws scer tined tht this vlue ws not due to n out lier since the vr i nce for this sm ple (0.129, 2 degrees of free dom) ws lower thn the pooled vr i nce (0.598, 46 degrees of free dom). In order to hve bet ter com pr i son mong dif fer ent tretments, dt were lso nor ml ized to the mount of pplied dissolved cr bon, nd thus expressed in terms of mg of relesed C per grm of pplied C (Fig. 3c). By this pproch, it ws pos si ble to overcome the slight dif fer ences of pplied C mong tret ments (Tble 2) nd to esti mte the rtio between pplied nd depos ited C. As for RGU dt, lso for rtio dt the min effects TREAT, SAMP nd the inter c tion TREAT * SAMP were sig nifi cnt to ANOVA (P < 0.001). For the TREAT effect, HoDOM nd Hi DOM (respec tively, 0.61 nd 0.51 mg relesed C per grm pplied C) hd sig nifi cntly lrger rtios thn TEA nd COM tret ments (0.15 nd 0.06 mg relesed C per grm pplied C). SAMP effect showed the sme sig nifi cnce s tht found for RGU vl ues, with the 0 2 mm lyer (0.53 mg relesed C per grm pplied C) sig nifi cntly lrger thn the 2 4 mm nd 4 6 mm ly ers (respec tively, 0.25 nd 0.22 mg relesed C per grm pplied C). Tu key s test for com pr i son of mens (Fig. 3c) mong rtio dt showed higher level of strt i fi- upper mm 2-4 mm 4-6 mm control COM HiDOM HoDOM TEA b b bcd bc cde bcde cde cde de cde bcde de e de de c mg relesed C / g pplied C control COM HiDOM HoDOM TEA 0-2 mm 2-4 mm 4-6 mm WB queous frction 40 b bc bcd cde ef f ef cdef COM HiDOM HoDOM TEA def ef 10 ef 0 % of WB cqueous frction Fig. 3. Totl nd vil ble mounts of orgnic cr bon (mens ± stn drd devi tions, n = 3) in the upper com prt ment of the con trol rhi zo box nd in the first three rhi zo sphere ly ers (0 2, 2 4, nd 4 6 mm) of ech stud ied tret ment. Results of Tu key s test for com pr i son of mens for the TREAT * SAMP effect re reported s minor let ters bove ech br. () Totl orgnic cr bon con tent (TOC) s me sured with the pots sium dichro mte oxi d tion method; (b) vil ble cr bon esti mted by the response of P. flu o rescens pucd 607 nd expressed in terms of rel tive glu cose units (RGU), which re lmol of glu cose equiv lents per grm of soil; (c) mg of relesed C per grm of pplied C, cl cu lted s the rtio between the mount of dis solved C pplied in the upper com prt ments of the rhi zo boxes nd the RCU me sured in the lower rhi zo sphere ly ers. The con tent of WB que ous frc tion is reported s well for ech fer til izer. In HoDOM, Hi DOM nd TEA tret ments, sig nifi cnt dif fer ence between the first nd the fol low ing rhi zo sphere ly ers ws found (TREAT * SAMP effect), but not in con trol nd COM tret ments. This dif fer ence mong ly ers reflected lrger depo si tion of bio vilble com pounds in the lyer dj cent to roots. In con trol, where no orgnic mte ril ws dded to the upper rhi zo box com prt ment, no sig nifi cnt effects on cr bon depo sition were me sured. In fct, bio sen sor response, in terms of RGU, ws sim i lr in the bulk nd in the rhi zo sphere soil sm ples. Sim i- lrly, no sig nifi cnt dif fer ences were found for the COM tret ment between the three rhi zo sphere ly ers. Agin, the RGU vl ues were not sig nifi cntly lrger thn those obtined for the bulk soil of control (TREAT * SAMP effect). The Hi DOM, HoDOM nd TEA tret ments showed sim i lr biosen sor response. The first rhi zo sphere lyer showed sig nifi cntly lrger response thn the fol low ing two ly ers, which were not sttis ti clly dif fer ent between ech other. The C depo si tion ws lso lrger in the first lyer of the bove tret ments, thn in the bulk con trol soil. The lrg est RGU vlue (9.73 lmol glu cose C g 1 soil) ws obtined for the first rhi zo box lyer mended with HoDOM. It

7 1298 E. Pugl is i et l. / Chemosphere 73 (2008) c tion thn RGU (Fig. 3b). The 0 2 mm lyer of HoDOM tret ment hd still the lrg est vlue, fol lowed by the Hi DOm tret ment tht hd lyer vl ues lrger thn the TEA nd COM tret ments The lyers of the lt ters showed no sig nifi cnt dif fer ences mong them. The mount of orgnic com pounds in the WB que ous frc tion for ech dded mte ril were lso reported on sec ond ry xis of Fig. 3b in order to llow com pr i son between the rtio between relesed nd dded C, nd the most vil ble frc tion of C in the orgnic mte ri ls. The HoDOM hd the lrg est WB que ous frction nd lso stim u lted the lrg est relese of C in the first rhi zosphere lyer Cor re l tions between bio sen sor nd TOC vl ues Cor re l tions between bio sen sor nd TOC vl ues were ssessed either sep rtely for ech tret ment or by pool ing ll dt together, regrd less of tret ment type. Cor re l tions using the pooled dt ws neg li gi ble (R 2 = 0.09, 46 degrees of free dom), wheres, when the cor re l tions were ssessed within ech tret ment, lrger coe f i cients were found for the TEA tret ment only (R 2 = 0.78, 7 degrees of free dom, P < 0.001). This con firms tht the ssess ment of rhi zosphere C depo si tion by the P. flu o res cens pucd607 bio sen sor is sensi tive to chnges in the mount of bio vil ble-c nd, thus, more de qute thn TOC me sure ments. 4. Dis cus sion We evl u ted the rhi zo sphere C depo si tion in mize plnts fter soil tret ment with dif fer ent orgnic mte ri ls, by pplying the rhi zo box sys tem devel oped by Wen zel et l. (2001). This sys tem llowed the mend ments of orgnic mt ter in the upper plnt roots soil com prt ment nd the evl u tion of their effects on root exu d tion in the untreted lower com prtment. Since the lower com prt ment is dj cent to the com prtment con tin ing only roots, the me sured effects re restricted to plnt roots ctiv ity. The 7 lm nylon mem brne used in rhizo boxes pre vented the pen e tr tion of roots hir into the lower com prt ment, nd, thus, only the effect of root exu dtes com posi tion ws evl u ted. These exu dtes, being lbile sub strtes for fst grow ing het ero tro phic soil micro or gn isms, induce generl flux of vil ble C com pounds which chr c ter ize the soil rhi zo sphere ctiv ity. In our exper i ments, we fol lowed the cr bon chnges of rhizo sphere soil in such con trolled sys tem by bio sen sor ssy method. The bio sen sor response ws lrger in the untreted rhi zosphere com prt ments thn in the con trol bulk soil. Com pr ing the sen si tive bio sen sor results with those obtined by TOC nl y ses, it becme cler tht TOC deter mi n tion fils to detect the sig nifi cnt chnges occur ring in rhi zo sphere bio vil ble com pounds. This find ing seems to be in con trst with pre vi ous results on the com pr i son between P. flu o res cens pucd 607 response nd TOC vlues for exu dtes of Pln tgo lnceo l t plnts exposed to dif fer ent con cen tr tions of Pr qut (Por teous et l.,2000). How ever, such pre vi ous find ings were bsed on exu dtes col lected from plnts grow ing in cr bon-free rti fi cil micro cosm con tin ing syn thetic beds insted of soil. The only cr bon source in tht sys tem ws rep re sented by plnt root exu dtes, thereby jus ti fy ing the high corre l tions between TOC nd bio sen sor results. Con versely, in rel non-ster ile soil sys tems, the ddi tionl pres ence of soil orgnic C increses the di f iculty of ccu rtely evl u t ing the chnges in plnt C depo si tions by the less sen si tive TOC method, espe cilly dur ing the erly period of plnt devel op ment. In such rel con ditions, we showed tht the bio sen sor method, com bined with the rhi zo box sys tem, suc cess fully detected sig nifi cnt root-derived depo si tion of red ily bio vil ble-c com pounds in the soil rhi zosphere fol low ing tret ment with orgnic mend ments. Through the expres sion of dt in terms of RGU (rel tive glucose C units) nd cl cu l tion of rtios between the mount of relesed nd pplied C, it ws pos si ble to esti mte the pr ti tion of orgnic C pplied in cul tured soils. Accord ing to our esti mtes, mize plnts grown in rhi zo box sys tems relesed round 1 lmol glu cose C g 1 soil g 1, in their first weeks of life. When this mount ws relted to the qun tity of dis solved C in the orgnic mte ri ls, we showed tht pprox i mtely one prt per thou snd of C pplied in dis solved forms is relesed in the rhi zo sphere s vil ble C. After mend ment of solid bulk com post on the upper rhi zobox com prt ment, the mount of bio vil ble-c in the rhi zosphere ws not st tis ti clly dif fer ent from the con trol tret ment. Con versely, the incresed mount of bio vil ble cr bon substrtes found in rhi zo box sys tems treted with sol u ble com post frc tions is in line with their chem i cl chr c ter is tics. In fct, the pre dom i nnce of SU nd WB frc tions (Tble 4) indi cte tht these mte ri ls re rich in lbile com pounds, which my serve s red ily-vil ble C nd energy source to the micro bil biomss. More over, the most ctive sol u ble extrct from com post (HoDOM) (Fig. 2) hd lso the lrg est con tent of poten tilly biovil ble orgnic com pounds rep re sented by the WB (weklybound to the struc turl mtrix) que ous frc tion (Tble 4). Sim i lr obser v tions were mde by Swin nen et l. (1995) nd Cr vc et l. (2002), who reported n incresed rhi zo de po sition in plnts treted with org no-fer til iz ers or com posted mteri ls. The lrger increse in rhi zo de po si tion in the pres ence of solu ble com post extrcts, s com pred to the bulk com post, implies tht such frc tions re fr more ctive thn the bulk com post, most prob bly becuse of the higher vil bil ity of the bio c tive orgnic com pounds con tined in the sol u ble frc tions. At sim i lr lev els of dis solved orgnic cr bon, sol u ble extrcts hd n effect on plnt phys i ol ogy (t lest in terms of rhi zo de po si tion) tht ws more prom i nent thn tht of bulk com post from which they were extrcted. This con clu sion of our work my hve n importnt con se quence on the mn ge ment of soil fer til ity nd in the gri cul turl use of com posted mte ri ls. The suc cess ful com bined ppli c tion of rhi zo box nd gene reporter shown here might be extended to ppli c tions of other bio sen sors (e.g., gene report ers for nitro gen or phos phte) (Krgel und et l., 1995). Fur ther reserch should lso be con ducted in order to inves ti gte the bio sen sor response to spe cific chemi cl com pounds (e.g., sug rs, orgnic cids, phe nols nd mino cids) pres ent in root exu dtes, nd to strengthen the cor re l tions between exu d tion pro cesses nd spe cific prop er ties nd chem i cl nture of orgnic mend ments. Acknowl edge ments This reserch ws con ducted within the MIUR (Itl in Min is ter for Uni ver sity Reserch) pro ject Role of orgnic fer til iz tion in the mx i mi z tion of plnt nutri tion nd tten u tion of gri cul turl pol lu tion. Ken Kill hm nd Dom i nic Stnd ing re kindly cknowledged for sci en tific sup port nd for pro vid ing the bio sen sor strin. The dvice of Et to re Cpri on the rhi zo box exper i ments is lso grte fully cknowl edged. Ref er ences Ad ni, F., Gig liotti, G., Vl en tin i, F., L ri, R., Res pi r tion index deter mi ntion: com pr tive study of dif fer ent meth ods. Com post. Sci. 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