Plant Rhizobium symbiosis, seed nutraceuticals, and waste quality for energy production of Vicia faba L. as affected by crop management

Transcription

1 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 RESERCH Open ess Plnt Rhizoium symiosis, seed nutreutils, nd wste qulity for energy prodution of Vii f L. s ffeted y rop mngement Crmine mlfitno 1*, Leonrdo D. Gomez 2*, Pierre Frendo 3, Stefni De Psle 1, Olimpi Pepe 1, Rhel Simister 2, Vleri Ventorino 1, Din grelli 1, Crlo orrelli 1, Simon J. MQueen Mson 2 nd Ginlu Cruso 1 strt kground: rod en fits sustinle griulture model due to symiosis with Rhizoium, the seeds eing good soure of energy, proteins, polyphenols, nd fier. The lrge mount of rod en iomss residues n e employed for iofuel prodution, thus vlorizing the overll prodution proess. This reserh ws imed to investigte on the effets of frming mngement, suh s greenhouse ultivtion nd pproprite plnting time on the qulities of rod en seeds nd residul iomss for onversion into iofuel. The relted lnes of energy gin ssoited to oth ethnol yield nd nitrogen fertilizer sving due to Rhizoium nitrogen fixtion were ssessed. Methods: Reserh ws rried out on rod en in Portii, provine of Nples, southern Itly, sed on the ftoril omintion of two frming systems (open field, greenhouse) nd five plnting times: 27 Septemer nd 11 Otoer, to otin erly prodution; 25 Otoer, whih fell in the usul period for rod en plnting in the provine re; nd 8 Novemer nd 22 Novemer, for lte prodution. For eh of these ultivtion onditions, the qulity of seeds, in terms of protein, fier nd ntioxidnt onentrtions, nd of rop residul iomss were determined. In ddition, the energy yield s ethnol prodution from residul iomss nd nitrogen fertilizer sving due to Rhizoium tmospheri fixtion were ssessed. Results nd disussion: The highest plnt nitrogen uptke ws reorded under the fourth plnting time in open field nd the third in greenhouse, the verge umultion ttining 87% in residul iomss, 7.4% in pods, nd 5.6% in seeds. Seed protein ontent ws 12.6% higher in greenhouse thn in open field nd 16.2% higher under the ltest plnting time ompred to the erliest one. Seed polyphenol onentrtion ws higher in open field thn in greenhouse nd with the two erliest plnting times. Greenhouse grown iomss showed higher vlues of lignin, hemiellulose nd petin, ompred to open field, wheres the opposite trend ws for ellulose. Lignin showed derese from the first to the lst rop yle, opposite to ellulose, nd gluose ws the most represented monoshride. oth the highest theoretil ethnol nd overll energy prodution were highest with the fourth plnting time. Conlusions: Greenhouse mngement enled rod en plnts to umulte higher proteins in seeds, ut open field onditions resulted in etter residul iomss qulity for ethnol nd Rhizoium-depending energy prodution. Keywords: rod en, Greenhouse, Proteins, Polyphenols, Cellulose, iofuel *Correspondene: mlfit@unin.it; ldg3@york..uk 1 Deprtment of griulturl Sienes, University of Nples Federio II, vi Università 100, Portii, Itly 2 Deprtment of iology, Center for Novel griulturl Produts, University of York, Heslington, York YO10 5DD, UK Full list of uthor informtion is ville t the end of the rtile The uthor(s) This rtile is distriuted under the terms of the Cretive Commons ttriution 4.0 Interntionl Liense ( iveo mmons.org/lien ses/y/4.0/), whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( iveo mmons.org/ puli dom in/zero/1.0/) pplies to the dt mde ville in this rtile, unless otherwise stted.

2 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 2 of 13 kground rod en (Vii f L.) is vegetle rop grown worldwide, minly in Mediterrnen sin, United Kingdom, estern Europe, Chin, Indi, South meri, ustrli, with totl surfe re of 240,000 h [1]; Itly is mjor Europen produer, with 49,000 hetres devoted to this rop [2]. rod en shows fetures fitting sustinle griulture model [3], s it enefits from symiosis with Rhizoium terium to fix nitrogen from the tmosphere. Root Rhizoiumontining nodules uild up upon phenol root seretion ttrting miroorgnisms [4], depending on plnt environmentl nd growth onditions [5]. Notly, plnt phenolis t s signl-moleules, induing the nodultion ftor iosynthesis nd Rhizoium infetion [6]. However, under unfvorle onditions for symiosis triggering llelopthi sustnes in root exudtes exert ontrol tivity of Rhizoium infetions, reduing the terium reprodution rte in host plnt rhizosphere [4]. rod en seeds represent remrkle energy soure, providing 44 kl 100 g 1 of fresh seeds, nd they re rih in fier, lysine rih proteins, minerl nutrients, vitmins, nd ntioxidnts [7]. Notly, the high fier onentrtion is essentil for intestinl funtions regultion s well s for lood gluose nd holesterol ontrol [8]. Polyphenols t s ntioxidnts s well s protetive sreens ginst ultrviolet rditions [9] nd they re highly present in the seed tegument [10]. However, polyphenols exessive umultion relevnt to ell requirements n potentilly dmge lipids, proteins, nd nulei ids promoting hroni diseses suh s ner, rdiovsulr or neurodegenertive prolems [11]. rod en is mostly grown under field onditions, ut greenhouse environmentl onditions my llow for n effetive prevention of environmentl unlnes [12]. Plnting time is lso ruil in Vii f mngement in order to mximize seed qulity, s it is stritly mthed to the growing environment nd even ffets the rop yle in terms of possile ourrene of ioti nd/or ioti stresses [13]. Lrge mounts of rod en iomss residues re left on the field fter pod hrvest, whih n e employed for different uses, suh s iofuels nd iogs [14] or high vlue hemils, thus vlorizing the overll prodution proess. These residues ount for % of the totl rop iomss nd re hrterized y high polyshride ontent nd low lignin ( % iomss frtion nd % iomss frtion, respetively) whih hve effets on lignoellulosi iomss proessility [15, 16]. Indeed, the prodution of iofuel from iomss residues represents vlule opportunity to void onerns out ompromising food supply y using strh- or surose-sed feedstoks nd it is dvntgeous from eonomi, environmentl, nd strtegi points of view [17]. The gol of this reserh ws to ssess the effet of frming systems nd plnting time on: (i) the qulity of seeds, in terms of proteins, fiers nd ntioxidnts, nd rop residul iomss, (ii) the nitrogen uptke nd prtition in plnt prts, (iii) the energy yield s ethnol prodution from residul iomss, nd (iv) nitrogen fertilizer sving due to Rhizoium tmospheri fixtion. Methods Plnt mteril nd growth onditions rod en (Vii f L. mjor Hrtz)) ultivr gudule supersimoni ws grown in Nples, southern Itly (40 50 N, E, 17 m.s.l.), in Mediterrnen limte, in on sndy-lom soil field (76.2% snd, 15.8% silt, 8% ly, 1.9% orgni mtter, 0.11% totl N, ph 7.2, 340 µs m 1 EC); the trend of temperture nd PR re shown in Fig. 1. Comprisons were mde etween ten experimentl tretments, otined y the ftoril omintion of two frming systems (open field, greenhouse) nd five plnting times (27 Septemer, 11 Otoer, 25 Otoer, 8 Novemer, 22 Novemer). split plot design with three replites ws rrnged, where eh elementry plot ws 6.00 m 2 ; plnts were trnsplnted in single rows sped y 85 m from eh other nd the sping ws of 13 m long the rows, with n rel density of 9 pt m 2. Plnting times were hosen in order to ssess the effets of erlier nd lter trnsplnt dtes, referred to the ommon plnting dte of 25 Otoer in Nples provine, on the qulity of seeds nd of residul iomss s well s on Rhizoium nitrogen fixtion. Greenhouse onsisted of three-spn polytunnel, eh spn eing 30 m long, 5 m wide, 2 nd 3.5 m high t wll nd t roof, respetively. Eh yer the plnts were supplied with 90 kg h 1 of N, 75 kg h 1 of P 2 O 5 nd 200 kg h 1 of K 2 O. Nitrogen nd phosphorus were just given efore trnsplnting y using the Tim fertilizer integrted with mmonium sulfte, in order to ounterlne the slow initil growth of Rhizoium nd improving nutrient sorption [18]. Potssium supply ws ompleted on dressing y potssium sulfte pplition. Drip irrigtion ws tivted when the soil ville wter pity (WC) deresed to 80%. Pod hrvest ws performed every time the seeds hd ompleted their growth, strting with the first plnting time nd ending under the fifth trnsplnt, from 6 Ferury to 10 June in greenhouse nd from 18 Ferury to 16 June in open field, s n verge of the 2 reserh yers.

3 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 3 of 13 Fig. 1 Ten-dy mens of temperture nd P..R. in Nples (Itly): mens of nd Soil nd Rhizoium determintions The soil nitrogen ontent ws ssessed in ll tretments, t the eginning nd end of eh rop yle, in order to determine the ontriution of this soure to the totl plnt uptke of this element. Determintions of nodule numer were performed on rod en roots tken t 0 30 m depth, nd primry roots were used for hrterizing Rhizoium nd ssessing its effiieny. Rhizoium were isolted from fresh nodules ording to Vinent method [19] nd the teril strin ws verified s reommended y Grhm et l. [20], fter whih they were inuted t 28 C nd kept t 20 C in YM rodo on 25% (v/v) glyerol. Rhizoium genomi DN ws extrted using InstGene Mtrix (io-rd Lortories, Herules, C) nd the gene NODC ws mplified y nodcf nd nodcf primers [21]. PCR mixture s well s Rhizoium leguminosrum v. viie LPR1105 nd Rhizoium leguminosrum v. viie VF39 were used s positive ontrols [22]. Plnt nitrogen determintion Rndom plnt smples were tken in eh plot t the developmentl phse, vegettive growth (V), erly flowering (Fl), erly fruiting (Efr), mid fruiting (Mfr), lte fruiting (Lfr), oth in open field (OF), nd greenhouse (G). Roots of the olleted plnts were dethed nd the eril frtion ws seprted in its prts: vegettive iomss, pods, nd seeds. Eh prt ws dried t 60 C up to onstnt weight nd fter pulverized, Nitrogen of the smple powders of vegettive iomss nd pods ws determined y HCNS elementry nlyzer (E 1108, Fisons) in triplite. Nitrogen in seeds ws determined s desried in the next setion. Seed qulity nlysis rod en pods were rndomly smpled in eh plot, nd immeditely trnsferred to the lortory, where the seeds were extrted from the pods. From eh seed smple, 50 g of seeds ws rndomly drwn out nd homogenized with lender in liquid nitrogen for the determintions of sori id in triplite nd of solule solid ontent in duplite. Other 50 g of seeds ws frozen, lyophilized, nd then ground for the determintions of fier, protein nd totl polyphenol ontents in triplites. Solule solids, expressed s rix t 20 C, were ssessed using ellinghm nd Stnley digitl refrtometer, model RFM 81. sori id ws ssessed ording to Kmpfenkel et l. [23], totl polyphenols s previously desried [24]. Fier nd proteins were determined using hemometri method, with prinipl omponent regression method (PCR) pplying ner infrred spetrosopy (NIR). For PCR lirtion prmeters, the fier nd nitrogen ontents were determined for 20 smples.

4 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 4 of 13 Fier ws determined on the wshed nd dried residue from 1 g of lyophilized seeds extrted with petroleum ether nd then hydrolyzed for 30 min in 100 ml H 2 SO M t oiling point; nitrogen ws determined y HCNS nlyzer (Fisons E 1108) in triplite. Spetr etween 4000 nd 10,000 m 1 of powdered seeds were otined in refletne with NIR essory on Perkin Elmer Frontier infrred spetrophotometer equipped with Spetrum softwre nd hemometrilly proessed y Qunt+ softwre, oth from Perkin Elmer. The PCR prmeters for fier nd proteins were, respetively, s follows: numer of prinipl omponents, 5 nd 4; R 2, 91.0 nd 87.2; SEE, nd 1.395; SEP, nd 1.983; nd mens, 26.6 nd The protein ontent ws otined s nitrogen multiplied y Residul iomss determintions fter hrvest, plnt residul iomss ws ut off t ground level, inluding leves, shoots, stems, nd immture or dmged pods. The wste showed no fungl symptoms, nd therefore smples were rndomly olleted in eh plot nd immeditely trnsferred to the lortory, where they were dried in n oven t 70 C under vuum, t 15 kp pressure, until they rehed onstnt weight. fter ssessing the dry residue, the whole smples were refully milled using lortory mill, voiding segregtion of mterils elonging to different plnt prts. The finl mteril, omposed of prtiles 1 mm dimeter, ws stored in ir-tight gs t 20 C nd further dried just efore eing proessed. Determintion of lignin ws performed y etyl romide method. riefly, 4 mg of pulverized iomss ws treted with 0.25 ml of etyl romide in glil eti id (1:3 v/v) t 50 C frequently vortexing for 3 h. fter ooling, the mixture ws reovered with 1 ml of NOH 2 mol L 1, then dded with 0.17 ml of hydroxylmine HCl 0.5 mol L 1 nd diluted with glil eti id efore sorne mesurement t 280 nm for lignin quntifition tht ws performed using eer s Lw with L g 1 m 1 s extintion oeffiient. Contents of ellulose, hemiellulose, petin, rystlline ellulose, nd non-ellulosi monoshride were performed in ompline with previous study [25], s reported in the following. Holoellulose ws determined y delignifition of iomss dry powder (7.5 g) for 3 h t 75 C in mixture (240 ml wter, 0.75 ml glil eti id nd 2.25 g sodium hlorite) to whih further id nd hlorite mounts, equl to previous, were hourly dded. The resulting holoellulose residue ws dried t 105 C nd weighed fter wshing with old wter, wrm wter nd etone. The petin ontent ws determined y holoellulose weight loss fter petin extrtion. For this, holoellulose (1.3 g) ws treted t 75 C with potssium ette 0.6 mol L 1 (26 ml), nd dding mmonium oxlte 0.04 mol L 1 (26 ml) fter 3 h. The suspension ws filtered, then the residue ws wshed, dried t 105 C nd weighed. Cellulose ws isolted from holoellulose (3.8 g) y tretment with sodium hydroxide 4.4 mol L 1 (100 ml) for 30 min. The filtered residue of ellulose ws wshed with wrm wter, eti id 2 mol L 1, nd wter, then dried t 105 C nd weighed. The hemiellulose ws lulted s holoellulose minus ellulose nd petin. Cellulosi monoshride ws determined y hydrolysis of iomss dry powder (4 mg) treted with trifluoroeti id, TF, 2 mol L 1 (0.5 ml) in rgon fluxed vils t 100 C for 4 h. The pellets, resulting y drying vils in entrifugl evportor, were wshed with 2-propnol, vuum dried nd wter resuspended, then filtered with 0.45 mm PTFE filters nd nlyzed y HPEC. Crystlline ellulose ws determined y hydrolysis with 72% sulphuri id (90 ml) for 4 h of the TF hydrolysed pellets otined s ove nd wshed with wter nd etone. The hydrolizte ws dded of 1.89 ml wter nd heted for 4 h t 120 C, then gluose ws ssessed in superntnt y nthrone method. Nitrogen nd theoretil ethnol yield lultion Plnt nitrogen uptke ws ssessed s the nitrogen of the whole eril iomss (sum of the nitrogen in vegettive prts, pods nd seeds) nd it ws determined t eh phenologil phse y HCNS elementry nlyzer (E 1108, Fisons) in triplite, s desried in previous setions. The estimtion of nitrogen frtion rising from Rhizoium fixtion ws otined from the rtio etween the plnt nitrogen umulted t eh phse nd the nodule numers s men of nodules determined t ontiguous phses. The theoretil ethnol yield respet the iomss ws lulted onsidering the totl ellulose onversion in the smple, ording to the Ntionl Renewle Energy Lortory stndrds [26, 27] nd the following formul ws used: ellulose onversion = ([EtOH] f [EtOH] 0 ) (0.51 f [iomss] 1.111) 1, where [EtOH] f is the ethnol onentrtion t the end of the fermenttion minus ny ethnol produed from the enzyme nd medium; [EtOH] 0 is the ethnol onentrtion t the eginning of the fermenttion whih should e zero; [iomss] is the dry iomss onentrtion t the eginning of the fermenttion (ll onentrtions re in g L 1 ); f is the ellulose frtion y weight of dry iomss; 0.51 is the onversion ftor for gluose to ethnol sed on stoihiometri iohemistry of yest; onverts ellulose to equivlent gluose. The theoretil ethnol yield ws reported tking into ount the iomss yield per surfe re unit.

5 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 5 of 13 Clultion of energy deriving from ethnol nd nitrogen fertilizer sving The energy mount ssoited to ethnol ws lulted y multiplying its yield expressed in L h 1 y 21.4 MJ L 1 ethnol [28]. The energy gin relevnt to Rhizoium tmospheri fixtion ws otined from the fixed nitrogen mount multiplied y 78.2 MJ kg 1 needed for produing minerl nitrogen fertilizer [29]. Sttistil nlysis Dt were proessed y two-wy nlysis of vrine nd signifint min effets s well s intertions were reported. Men seprtions were performed through the Dunn multiple rnge test, with referene to 0.05 proility level, using SPSS softwre version 21. Dt expressed s perentge were sujeted to ngulr trnsformtion efore proessing. Correltions were performed with ll pirs of hemil prmeters using the softwre mentioned ove. Results nd disussion Plnt nitrogen uptke nd seed qulity s shown in Tle 1, the highest plnt nitrogen uptke ourred under the fourth plnting time in open field nd the third in greenhouse, wheres the lowest sorption orresponded to the ltest trnsplnt in oth frming systems. With regrd to nitrogen distriution mong the different plnt prts, the highest umultion in the residul iomss ws reorded in the erliest rop yle; differently, the pods nd the seeds ttined the highest nitrogen onentrtion in the fourth plnting time under open field onditions nd with the fifth trnsplnt in greenhouse. In the seeds, the protein ontent (Tle 2) showed 12.6% higher onentrtion in greenhouse thn in open field nd 16.2% inrese from the first to the lst plnting time. The protein onentrtions reorded in our reserh fell within the rnge reported y other uthors [7]. Notly, this qulity inditor ontent showed inresing trends with the temperture rise; indeed, protein iosynthesis in f en seeds is ffeted y the intertion mong geneti, ulturl nd limte ftors [30] nd it is quite intensive during the storge phse of seed development. The ltter is hrterized y swith in otyledon metolism from ell division to ell expnsion s well s y endopolyploidiztion [31] nd the eginning of storge protein umultion is linked with surose signl originting from the onset of surose/h+ symporter tivity [32]. Within the two mjor storge protein lsses, the synthesis of viilin strts out dys fter flower nthesis nd eses out 7 10 dys lter wheres the synthesis of legumin egins out 4 dys fter viilin synthesis [33]. In legume seeds, there re struturl linkges etween proteins nd fiers, ffeting seeds nutritive vlue whih is onneted to the vilility of proteins digested nd sored in the digestive trt. In ft, signifint protein perentge (23 43% of totl proteins) remins insolule nd less nutritionlly ville, s it is onneted to the insolule fier frtion [34]. Indeed, the proteins linked to insolule fier omponents (ellulose, hemiellulose, nd lignin) re not hydrolysed into lower moleulr weight ompounds suh s smll peptides nd mino ids [35]. Fier ws lso signifintly ffeted y frming system, s its ontent ws higher in greenhouse grown seeds thn in the open field ones, nd it grdully inresed from the erliest to the ltest plnting time (Tle 2). In previous reserh, lower fier levels were reported in f en seeds ompred to those deteted in our reserh [36]. Dry residue nd solule solids of f en seeds were not ffeted y frming system (Tle 2), ut they inresed with the trnsplnt dely. Totl polyphenols ontent (Tle 2) ws higher in open field grown seeds thn in the greenhouse ones (301 vs. 254 mg g 1 d.w., respetively). Moreover, polyphenols were more onentrted in the seeds otined with the first nd the seond trnsplnt (317 mg g 1 d.w. s n verge), wheres no signifint differenes were oserved in the lter yles (250 mg g 1 d.w. s n verge). Notly, in our reserh, the seed polyphenols Tle 1 Plnt nitrogen uptke nd nitrogen ontents in rod en plnt prts t the different plnting times in open field (OF) nd greenhouse (G) Plnting time Nitrogen uptke (kg h 1 ) Residul iomss N (%) Pod N (%) Seed N (%) OF G OF G OF G OF G 27 Septemer 322.4de 276.4f g 3.5f 3.0g 4.2f 11 Otoer de f e 8.9d 25 Otoer d d Novemer ef d Novemer 184.0g 198.1g e d 17.6 Within eh vrile, vlues followed y different letters re signifintly different ording to the Dunn test t p 0.05

6 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 6 of 13 Tle 2 Effets of frming system nd plnting time on the qulity of rod en seeds Tretment DR SS Fier (mg g 1 ) TP (mg g 1 ) PP (mg g 1 ) (mg g 1 ) mg g 1 rix Frming system Open field Greenhouse 193n.s. 8.9n.s. 240* 303* 25.4* 0.489* Plnting time 27 Septemer 171d Otoer Otoer Novemer Novemer DR dry residue, SS solule solids, fier, TP totl proteins, PP polyphenols, sori id, n.s. not sttistilly signifint differene * Signifint differene t p Within eh olumn, vlues followed y different letters re signifintly different ording to the Dunn test t p 0.05 On dry weight sis ontents displyed opposite trends to the ones reorded for proteins nd they fell in the rnge reported y [37]. These ompounds reportedly show positive orreltion with the ntioxidnt tivity [10, 38], wheres the orreltion with temperture is ontroversil, s it resulted positive in nn nd tomto [39, 40] ut dverse in lettue nd wtermelon [39, 41]. Phenyllnine mmonium-lyse (PL) is onsidered the min enzyme in the phenyl-propnoid uilding pthwy, tlyzing the l-phenyllnine turning into trns-innmi id, whih is the intermedite ompound in phenoli iosynthesis [42]. This enzyme tivity inreses in response to therml stress nd it is onsidered one of the min ell limtion symptoms ginst stress [43]. On the other hnd, phenol oxidtion is performed either y peroxidses (POD) or priority y polyphenols oxidses (PPO); the ltter enzyme tlyzes the oxidtion of o-diphenols to o-diquinons, s well s monophenols hydroxyltion [44]. oth enzymes hve een ssoited with physiologil dmge used y therml stress nd, in ft, under high or low temperture stress these enzymes re tivted, wheres the enzymes oxidizing the sme ompounds re inhiited [43, 44]. ordingly, solule phenoli ompounds n umulte s mehnism of limtion to overome high or low temperture stress [39]. sori id onentrtion (Tle 2) ws 57% higher in f en seeds grown in open field thn in the greenhouse, inresing from the first to the third plnting time (542 nd 656 mg g 1 d.w. respetively), fter whih it remined stedy up to the lst rop yle. In our reserh, onversely to polyphenols whose iosynthesis ws tivted y therml stress, sori id ws ffeted y light intensity, s its ontent ws higher in open field nd inresed with the trnsplnt dely. However, with regrd to the onnetions etween these two ntioxidnt ompounds, [45] reported the importnt role of polyphenols in preventing enzymti degrdtion of sori id. Yield nd ell wll hemil omposition of residul iomss The intertion etween frming system nd plnting time ws signifint on rod en residul iomss yield (Tle 3), whih ttined higher vlues in open field thn in greenhouse in ll the rop yles, exept for the ltest plnting time, in whih it did not show differenes etween the two frming systems. In open field, iomss showed n inresing trend from the first to the fourth plnting time nd then the iomss yield dropped. In greenhouse, the highest iomss ws reorded with the third nd the fourth trnsplnt. In previous reserh [46] rod en iomss grown in ustrli showed deresing trend with the plnting dely, whih ws similr to tht reorded in our reserh. s for ell wll hemil omposition of rod en residul iomss (Tle 3), greenhouse rops showed signifintly higher vlues thn the open field ones with regrd to lignin ontent (16.4 vs. 15.3%), hemielluloses (15.1 vs. 13.9%), nd petin (13.4 vs. 12.5%). Open field grown iomss showed higher ellulose ontent (44.5 vs. 46.0%), wheres no differene in rystlline ellulose ws deteted etween the two frming systems. In previous investigtions, [14, 28] lower lignin nd ellulose ut higher hemiellulose perentge in rod en iomss grown in open field were reported. s regrds the plnting times, the lignin ontent showed grdul derese from the first (17.7%) to the lst rop yle (13.7%); similr trend ws reorded for petin (from 14.1 to 11.5%). Conversely, ellulose nd rystlline ellulose perentge inresed with the

7 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 7 of 13 plnting dely (from 41.1 to 48.7% nd from 10.9 to 13.0% for ellulose nd rystlline ellulose respetively), wheres hemielluloses were not ffeted y the plnting time. Consistently with our findings, [47] reported derese in iomss lignin nd hemiellulose ontent with the plnting dely in spring grown sorghum. In the present work, lignin showed positive orreltion with petin (r = 0.61 t p < 0.01), ut negtive orreltion with totl ellulose (r = 0.73 t p < 0.01) nd with rystlline ellulose (r = 0.68 t p < 0.01). Totl nd rystlline ellulose, in turn, were positively orrelted to eh other (r = 0.72 t p < 0.01), ut negtively orrelted with petin (r = 0.59 t p < 0.01 nd r = 0.63 t p < 0.01, respetively). Finlly, no exmined omponent showed signifint orreltion with hemiellulose. The monoshride omposition in the hemiellulosi frtion is showed in Fig. 2. Gluose is the most represented monoshride (46.7%), followed y xylose (27.4%) nd gltose (9.9%), wheres gluuroni id nd fuose were deteted in tres. Similrly to our results, in previous reserh [28] the prevlene of gluns nd rinoxylns ws deteted in rod en hemiellulose. Some sugrs deresed from the first to the lst plnting time, suh s rinose (from 6.8 to 6.2%), gluose (from 45.9 to 43.2%), nd rhmnose (from 2.8 to 2.3%); glturoni id nd mnnose inresed with the plnting dely (from 4.5 to 6.4% nd from 3.9 to 5.5%, respetively); gltose nd xylose were not signifintly ffeted y plnting time. Theoretil ethnol yield The rod en theoretil ethnol yield ws signifintly ffeted y the intertion etween frming system nd plnting time (Fig. 3). In open field, the ethnol prodution inresed from the first to the fourth trnsplnt (from 4265 to 6552 L h 1, respetively) nd then deresed to the lowest level in the lst rop yle; under greenhouse onditions the highest vlues were shown y the third nd the fourth trnsplnt (5107 L h 1 s n verge) nd the lowest in the first nd in the fifth rop yles (3414 L h 1 s n verge). Moreover, the open field rops lwys produed higher theoretil ethnol yield thn the greenhouse ones, exept for the lst plnting time whih did not result in signifint differenes etween the two frming systems. The iomss qulity for ethnol prodution ws minly ffeted y the totl ellulose, whih inresed with the plnting time dely, rther thn y the hemiellulose whih did not signifintly hnge (Tle 3). The theoretil ethnol yield per ton of iomss dry weight lso displyed n inresing trend from the first (441 L Mg 1 d.w.) to the fifth plnting time (495 L Mg 1 d.w.). However, the theoretil ethnol yield ws stritly dependent on the rod en residul iomss mount (Tle 3), the two vriles showing similr trends. Consistently with our results, other uthors [48] reported the signifint effet of plnting time on ethnol prodution from sweet sorghum, whih inresed from mid Mrh to lte Mrh or erly pril nd then deresed. Moreover, the theoretil ethnol yields shown in our reserh re omprle with those otined in previous reserh onduted on veth, rye nd mize [49] nd muh higher thn tht reorded in tomto [50]. Energy yield from Rhizoium nitrogen fixtion nd ethnol The signifint intertion etween plnting time nd rop developmentl phse showed tht the numer of nodules on rod en roots, ontining Rhizoium (identified s Rhizoium leguminosrum iovr viie) generlly inresed from the vegettive phse until erly Tle 3 Effets of frming system nd plnting time on yield nd hemil omposition of rod en residul iomss on dry weight sis Tretment Yield (Mg h 1 ) Lignin (%) Totl ellulose (%) Crystlline ellulose (%) Hemiellulose (%) Petin (%) Frming system Open field Greenhouse 9.0* 16.4* 44.5* 12.0n.s. 15.1* 13.4* Plnting time 27 Septemer d 10.9d Otoer d Otoer Novemer Novemer 7.0d n.s. 11.5d Within eh olumn, vlues followed y different letters re signifintly different ording to the Dunn test t p 0.05 * Signifint differene t p 0.05.

8 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 8 of d Hemiellulose omposition (%) n.s. d d d d n.s. 0 r Gl Gl Gl Mn Rh Xyl 27 Sept 11 Ot 25 Ot 8 Nov 22 Nov Fig. 2 Hemiellulose monoshride omposition of rod en residul iomss t different plnting times. n.s. mens no signifint differenes, different letters men signifint differene within eh monoshride ording to Dunn multiple rnge test t p r, rinose; Gl, glturose; Gl, glturoni id; Gl, gluose; Mn, mnnose; Rh, rhmnose; Xyl, xylose L h d Open field Greenhouse 4000 e Sept 11 Ot 25 Ot 8 Nov 22 Nov Plnting time Fig. 3 Theoretil ethnol yield from rod en residul iomss t the different plnting times in open field nd greenhouse frming systems. Different letters men signifint differene ording to Dunn multiple rnge test t p 0.05: lowerse refers to omprisons etween plnting times within eh frming system, pitl to omprison etween frming systems within eh plnting time

9 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 9 of 13 Numer of nodules per m C V Fl Efr MFr Lfr 27 Septemer 11 Otoer 25 Otoer 8 Novemer 22 Novemer C C C Nitrogen uptke (g m -2 ) d d V Fl Efr MFr Lfr d d 27 Septemer 11 Otoer 25 Otoer 8 Novemer 22 Novemer Fig. 4 Numer of root nodules () nd nitrogen uptke () t the different phenologil phses nd plnting times; V, vegettive phse; Fl, erly flowering; Efr, erly fruiting; Mfr, mid-fruiting; Lfr, lte fruiting. Different letters men signifint differene ording to Dunn multiple rnge test t p 0.05: lowerse refers to omprison etween phenologil phses within eh plnting time, pitl letters to omprison etween plnting times within eh phse fruiting phse nd then it remined stedy until the rop end (Fig. 4). Moreover, the nodule numer ws highest from erly to lte fruiting with the seond to fourth trnsplnt, wheres it ws not ffeted y plnting time t vegettion phse. Plnt nitrogen uptke (Fig. 4) did not hnge etween the vegettive nd erly flowering phses, fter whih it inresed until erly fruiting phse under the first, seond nd ltest plnting times, nd until mid fruiting phse with the third nd fourth trnsplnting. Plnt nitrogen per root nodule (Fig. 5) showed the highest vlues during the vegettive phse, fter whih it remined onstnt until the mid fruiting phse nd delined t lst phse only for the third to fifth plnting times. Considering nitrogen fixtion ould entirely support the plnt growth from erly flowering to mid fruiting, nd tht Rhizoium effiieny ppered onstnt long ll rop yle, oth the highest vlue of plnt nitrogen per root nodule nd the lowest nodultion during vegettive phse suggest tht the plnt nitrogen is mostly uptken y soil fertilizer. This is supported y the ft tht plnt nitrogen per nodule did not signifintly hnge in different environmentl onditions relted to plnt developmentl phses, plnting times nd rop

10 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 10 of 13 nitrogen uptke (mg per nodule) C C C V Fl Efr MFr Lfr 27 Septemer 11 Otoer 25 Otoer 8 Novemer 22 Novemer Fig. 5 Nitrogen uptke per root nodule t the different phenologil phses nd plnting times; V, vegettive phse; Fl, erly flowering; Efr, erly fruiting; Mfr, mid-fruiting; Lfr, lte fruiting. Different letters men signifint differene ording to Dunn multiple rnge test t p 0.05: lowerse refers to omprison etween phenologil phses within eh plnting time, pitl to omprison etween plnting times within eh phse systems from erly flowering to mid fruiting phse. Further, Jensen et l. [51] reported tht rod en postplnting growth needs soil nitrogen vilility sine rod en root nodultion tkes time to estlish. On this sis, during the vegettive phse, the minerl frtion of nitrogen uptke in ll tretments ws estimted y sutrting from the totl plnt nitrogen umultion the liquot of the fixed nitrogen, the ltter expressed s the men Rhizoium fixtion tivity from erly flowering to mid fruiting phse (from 7.2 to 10.6 mg per nodule, under the seond to the lst plnting time, respetively). s reported in Tle 4, the mjority of nitrogen sored y plnts derived from the symioti fixtion, ttining the highest vlues with the fourth plnting time in open field nd with the third trnsplnt in greenhouse, nd it ws highly orrelted with the numer of root nodules (r = 0.97 t p < 0.01). The nitrogen gined from teril symiosis rnged from 82.8 to 92.5% under open field mngement nd from 84.0 to 87.6% in greenhouse out of totl plnt nitrogen, onsistently with previous reserh [52, 53] where this nitrogen soure ounted for 60 96%. The nitrogen gined from teril symiosis turned into redued need of minerl fertilizer supply up to 3.3 nd 2.6 Mg h 1 in open field nd greenhouse, respetively. This enles Vii f to produe food Tle 4 Nitrogen soures of rod en plnts t the different plnting times in open field (OF) nd greenhouse (G) Plnting time Root tive nodules Plnt N N fertilizer sving Fixed y Rhizoium Derived from fertilizers OF (no. m 2 ) G (no. m 2 ) OF (kg h 1 ) G (kg h 1 ) OF (kg h 1 ) G (kg h 1 ) OF (Mg h 1 ) G (Mg h 1 ) 27 Septemer d 267.0d 235.8e d 2.06e 11 Otoer d d 25 Otoer d Novemer d de 30.8d de 22 Novemer 555e 609e 152.5f 166.4f 31.5d 31.7d 1.33f 1.46f Within eh vrile, vlues followed y different letters re signifintly different ording to the Dunn test t p 0.05 n.s. not sttistilly signifint differene Root tive nodules refer to the highest nodule numer, reorded during the fruiting phse

11 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 11 of d GJ h e d N fixtion greenhouse Ethnol greenhouse N fixtion open field Ethnol open field Fig. 6 Totl rod en energy yield (ethnol plus tmospheri nitrogen fixtion) t the different plnting times in open field nd greenhouse frming systems. Different letters men signifint differene ording to Dunn multiple rnge test t p 0.05: lowerse refers to omprison etween plnting times within eh frming system, pitl to omprison etween frming systems within eh plnting time protein t low ost, whih represents mnufturing energy sving. The overll energy prodution onneted to the rod en rop systems onsisted of: (i) the energy ssoited to ethnol yield from rop residul iomss, rehing GJ h 1 under the fourth plnting time in open field nd GJ h 1 with the third trnsplnt in greenhouse, nd (ii) the energy derived from symiosis etween plnts nd Rhizoium, ttining the top level of 29.7 GJ h 1 under the fourth plnting time in open field nd of 23.1 GJ h 1 with the third trnsplnt in greenhouse (Fig. 6). With ll trnsplnting times, the open field ultivtion resulted in higher energy prodution thn the greenhouse mngement, exept for the lst trnsplnt for whih no differene were reorded. Conlusions The investigtion rried out on rod en, imed to evlute different frming mngements, showed the improving effets of greenhouse onditions on seed protein ontent ompred to open field. However, the ltter rop system enhned polyphenols nd sori id umultion in the seeds. Open field lso inresed ellulose in the iomss residues fter pod hrvest, whih resulted in the highest ethnol yield under the 8 Novemer trnsplnt. The most effiient symiosis etween Rhizoium nd rod en plnts ws reorded t the mid-utumn plnting times, 25 Otoer nd 8 Novemer, llowing to gin n importnt mount of energy onneted to nitrogen fertilizer sving. From our reserh, it n e inferred tht the pproprite omintion of frming system nd plnting time enles to vlorizing the overll rod en prodution proess. uthors ontriutions C ws involved in seed hemil nlysis, dt interprettion, nd mnusript writing; LDG interpreted hemil nlysis of residul iomss nd took prt to mnusript drfting; PF ddressed plnt Rhizoium reltionships nd ontriuted to mnusript drfting; SDP ws involved in the experimentl design setup nd mnusript ritil revision; OP supervised Rhizoium nlysis nd relevnt interprettion; RS performed hemil omposition ssys of residul iomss; VV performed Rhizoium determintions nd relevnt

12 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 12 of 13 proessing; D ws involved in seed hemil determintions nd relevnt proessing; C ontriuted to seed hemil nlysis nd residul iomss smpling; SJMM ritilly revised the mnusript; GC oneived the study nd ws minly involved in interprettion of dt nd in mnusript writing. ll uthors red nd pproved the finl mnusript. uthor detils 1 Deprtment of griulturl Sienes, University of Nples Federio II, vi Università 100, Portii, Itly. 2 Deprtment of iology, Center for Novel griulturl Produts, University of York, Heslington, York YO10 5DD, UK. 3 INR, CNRS, IS, Université Côte d zur, Nie, Frne. knowledgements The uthors wish to thnk Dr. Stefno Conti, Mr. Roerto Miello, Dr. Giuseppe Melhionn, Dr. Giuseppe Morno, Mr. Rosrio Noerino, nd Dr. Silvno Somm for their ssistne with the field nd lortory equipments. Competing interests The uthors delre tht they hve no ompeting interests. vilility of dt nd mterils dditionl dt my e ville on request to the uthors, plese ontt orresponding uthor. Consent for pulition ll uthors gree for sumitting this mnusript to Chemil nd iologil Tehnologies in griulture. Ethis pprovl nd onsent to prtiipte The uthors delre tht this study does not involve humn sujets, humn mteril nd humn dt. Funding This reserh ws prt of the IOMSSVL projet funded y the Ministry of griulture nd short-moility grnt ws provided y the University of Nples Federio II for rrying out nlyses t the Deprtment of iology of York University (United Kingdom). Pulisher s Note Springer Nture remins neutrl with regrd to jurisditionl lims in pulished mps nd institutionl ffilitions. Reeived: 29 Deemer 2017 epted: 6 June 2018 Referenes 1. FOSTT. t3.fo.org/rows e/q/qc/efos tt, ISTT. nnul rop dt. Istituto Nzionle di Sttisti, Rome, etcod e¼dcsp_colti VZ&Lng#. 3. Ndl S, Suso MJ, Moreno MT. Mngement of Vii f geneti resoures: hnges ssoited to the selfing proess in the mjor, equin nd minor groups. Genet Resour Crop Evol. 2003;50: Mkrov LE, Ltyshev SE, Putilin TE. The effet of the phenoli ompounds exuded y pe roots in drkness on the reprodution of Rhizoium. ppl iohem Miroiol. 2007;43: Lwson CGR, Rolfe G, Djordjevi M. Rhizoium inoultion indues ondition-dependent hnges in the flvonoid omposition of root exudtes from Trifolium suterrneum. ustr J Plnt Physiol. 1996;23: Long SR. Genes nd signls in the Rhizoium legume symiosis. Plnt Physiol. 2001;125: Ofuy ZM, khidue V. The role of pulses in humn nutrition: review. J ppl Si Environ Mng. 2005;9: Mrull MT, Medin C, De Diego M, Chvrri M, Zulet M, Mrtinez J, Noel-Suerville C, Higueret P, Portillo MP. Effets of the whole seed nd protein isolte of f en (Vii f) on the holesterol metolism of hyperholesterolemi rts. r J Nutr. 2001;85: Jnsen M, vn den Noort RE, Tn M, Prinsen E, Lgrimini LM, Thorneley RNF. Phenol-oxidizing peroxidses ontriute to the protetion of plnts from ultrviolet rdition stress. Plnt Physiol. 2001;126: Chie N, Gonzlez JL, Lopez-Mess M, ouslm M, Vliente M. Polyphenols ontent nd ntioxidnt pity of thirteen f en (Vii f L.) genotypes ultivted in Tunisi. Food Res Intern. 2011;44: Cho HY, Kleeerger SR. Nrf2 protets ginst irwy disorders. Toxiol ppl Phrmol. 2010;244: MDonld GK, disrwnto T, Knight R. Effet of time of sowing on flowering in f en (Vii f). ust J Exp gri. 1994;34: Shile S, hmed S, Finins C, ng MM, Skhuj PK. Survey of hoolte spot (otrytis fe) disese of f en (Vii f L.) nd ssessment of ftors influening disese epidemis in northern Ethiopi. Crop Prot. 2008;27: Petersson, Thomsen MH, Huggrd-Nielsen H, Thomsen -. Potentil ioethnol nd iogs prodution using lignoellulosi iomss from winter rye, oilseed rpe nd f en. iomss ioenerg. 2007;31: Shievno, D Imporzno G, dni F. Sustituting energy rops with orgni wstes nd gro-industril residues for iogs prodution. J Environ Mng. 2009;90: Liu H, Zhu JY, Fu S. Effets of lignin-metl omplextion on enzymti hydrolysis of ellulose. J gri Food Chem. 2010;58: Crdon C, Snhez OJ. Fuel ethnol prodution: proess design trends nd integrtion opportunities. ioresour Tehnol. 2007;98: Sánhez-Chávez E, Muñoz-Mrquez E, Gri-ñuelos ML, nhondo- Njer, Guerrero-Prieto VM, Nuñez-rrios, Ruiz-Sez JM, Romero- Monrel LM. Influene of nitrogen fertiliztion on K(+) nd C(2+) onentrtions nd on its ioinditors in roots nd leves of green en plnts. Spnish J gri Res. 2010;8: Vinent JM. mnul for the prtil study of root. nodule teri. IP Hndook 15. Oxford: lkwell; Grhm PH, Sdowsky MJ, Keyser HH, rnet YM, rdley RS, Cooper JE, De Ley DJ, Jrvis DW, Roslyky E, Strijdom W, Young PW. Proposed miniml stndrds for the desription of new gener nd speies of rootnd stem-nodulting teri. Intern J Syst teriol. 1991;4: Moshetti G, Peluso L, Protopp, nstsio M, Pepe O, Defez R. Use of nodultion pttern, stress tolerne, nodc gene mplifition, RPD-PCR nd RFLP-16S rdn nlysis to disriminte genotypes of Rhizoium leguminosrum iovr viie. Syst ppl Miroiol. 2005;28: Ventorino V, Chiurzzi M, ponte M, Pepe O, Moshetti G. Geneti diversity of nturl popultion of Rhizoium leguminosrum v. viie nodulting plnts of Vii f in the vesuvin re. Curr Miroiol. 2007;55: Kmpfenkel K, Vn Montgu M, Inzé D. Extrtion nd determintion of sorte nd dehydrosorte from plnt tissue. nl iohem. 1995;225: Cruso G, Conti S, Villri G, orrelli C, Melhionn G, Minutolo M, Russo G, mlfitno C. Effets of trnsplnting time nd plnt density on yield, qulity nd ntioxidnt ontent of onion (llium ep L.) in southern Itly. Si Horti. 2014;116: Gomez LD, mlfitno C, ndolfi, Simister R, Somm S, Erolno MR, orrelli C, MQueen-Mson SJ, Frusinte L, Cuiniello, Cruso G. Vlorising f en residul iomss: effet of frming system nd plnting time on the potentil for iofuel prodution. iomss ioenerg. 2017;107: Dowe N, Mmilln J. SSF experimentl protools lignoellulosi iomss hydrolysis nd fermenttion. Ntionl Renewle Energy Lortory (NREL), Tehnil Report TP , 2008;1: Cpred S. Introdution to iomss energy onversions. In: Gomez L, editor. Siene 2013, Chpter 4. o Rton, FL: CRC Press; Pkrinen, Mijl P, Stoddrd FL, Sntnen, Tuominen P, Kymäläinen M, Viikri L. Evlution of nnul ioenergy rops in the orel zone for iogs nd ethnol prodution. iomss ioenerg. 2011;35: Helsel ZR. Energy nd lterntives for fertilizer nd pestiide use. In: Fluk RC, editor. Energy in Frm Prodution, vol. 6. New York: Elsevier; p Hood-Niefer SD, Wrkentin TD, Chir RN, Vndenerg, Tyler RT. Effet of genotype nd environment on the onentrtions of strh nd protein in, nd the physiohemil properties of strh from, field pe nd fen. J Si Food gri. 2012;92:

13 mlfitno et l. Chem. iol. Tehnol. gri. (2018) 5:15 Pge 13 of orisjuk L, Heim U, Weer H, Wous U. Emryogenesis of Vii f L. histo differentition in reltion to strh nd storge protein synthesis. J Plnt Physiol. 1995;147: Roshe EG, lkmore D, Offler CE, Ptrik JW. Inresed pity for surose uptke leds to erlier onset of protein umultion in developing pe seeds. Funt Plnt iol. 2005;32: de Pe C, Delre V, Srsi Mugnozz GT, Mggini F, Cremonini R, Fredini M, Cionini PG. Legumin of Vii f mjor: umultion in developing otyledons, purifition, mrn hrteriztion nd hromosoml lotion of oding genes. Theor ppl Genet. 1991;83: Mrtin-Crejs M, Felii DM, guiler Y, enìtez V, Mollà E, Esten RM. Influene of germintion on the solule rohydrtes nd dietry fire frtions in non-onventionl legumes. Food Chem. 2008;107: rvo L. Polyphenols: hemistry, dietry soures, metolism, nd nutritionl signifine. Nutr Rev. 1998;56: Hedley CL. Crohydrtes in grin legume seeds: improving nutritionl qulity nd gronomi hrteristis. New York: CI Pulishing; Oomh D, Genevieve L, Clire L, Drover J, Hrrison JE, Olson M. Phenolis, phyti id, nd phytse in Cndin-grown low-tnnin f en (Vii f L.) genotypes. J gri Food Chem. 2011;59: Pstor-Cvd E, Jun R, Pstor JE, liz M, Girón-Clle J, Vioque J. ntioxidtive tivity in the seeds of 28 Vii speies from southern Spin. J Food iohem. 2011;35: Rivero RM, Ruiz JM, Gri PC, López-Lefere LR, Sánhez E, Romero L. Resistne to old nd het stress: umultion of phenoli ompounds in tomto nd wtermelon plnts. Plnt Si. 2001;160: Cml-Velázquez JH, Chi-Mnznero H, Cnhe-Ym JJ, Cstño E, Rodríguez-Zpt LC. Low temperture indue differentil expression genes in nn fruits. Si Horti. 2007;114: oo HO, Heo G, Gorinstein S, Chon SU. Positive effets of temperture nd growth onditions on enzymti nd ntioxidnt sttus in lettue plnts. Plnt Si. 2011;181: Dixon R, Piv NL. Stress-indued phenylpropnoid metolism. Plnt Cell. 1995;7: Leyv, Jrillo J, Slins J, Mrtinez-Zpter JM. Low temperture indues the umultion of phenyllnine mmoni-lyse nd hlone synthse messenger-rns of ridopsis thlin in light-dependent mnner. Plnt Physiol. 1995;108: MrtinezTellez M, Lfuente MT. Effet of high temperture onditioning on ethylene, phenyllnine mmoni-lyse, peroxidse nd polyphenol oxidse tivities in flvedo of hilled Fortune mndrin fruit. J Plnt Physiol. 1997;150: ltunky, Gӧkmen V. Effet of vrious nti-rowning gents on phenoli ompounds profile of fresh lettue (L. stiv). Food Chem. 2009;117: Conflone, Lizso JI, Ruiz-Nogueir, López-Cedrón F-X, Su F. Growth, PR use effiieny, nd yield omponents of field-grown Vii f L. under different temperture nd photoperiod regimes. Field Crops Res. 2010;115: Mhmood, Shhzd N, Honermeier. iomss nd iogs yielding potentil of sorghum s ffeted y plnting density, sowing time nd ultivr. Pk J ot. 2015;47: Xun TD, Phuong NT, Khng DT. Influene of sowing times, densities, nd soils to iomss nd ethnol yield of sweet sorghum. Sustinility. 2015;7: Crter MS, Huggrd-Nielsen H, Heiske S, Jensen M, Thomsen ST, Shmidt JE, Johnsen, mus P. Consequenes of field N 2 O emissions for the environmentl sustinility of plnt-sed iofuels produed within n orgni frming system. GC ioenerg. 2012;4: Erolno ME, Gomez LD, ndolfi, Simister R, Troise C, ngelino G, orrelli C, MQueen-Mson SJ, Evidente, Frusinte L, Cruso G. Residul iomss shrifition in proessing tomto is ffeted y ultivr nd nitrogen fertiliztion. iomss ioenerg. 2015;72: Jensen ES, Peoples M, Huggrd-Nielsen H. F en in ropping systems. Field Crops Res. 2010;115: Crrn C, de Vrennes, Rolston D. iologil nitrogen fixtion y f en, pe nd hikpe, under field onditions, estimted y the 15 N isotope dilution tehnique. Eur J gron. 1999;10: López-ellido L, López-ellido RJ, Redondo R, enítez J. F en nitrogen fixtion in whet-sed rottion under rinfed Mediterrnen onditions: effet of tillge system. Field Crops Res. 2006;98: