Effect of municipal biowaste derived biostimulant on nitrogen fate in the plant-soil system during lettuce cultivation

A main concern of agriculture is to improve plant nutrient efficiency to enhance crop yield and quality, and at the same time to decrease the environmental impact caused by the lixiviation of excess N fertilizer application. The aim of this study was to evaluate the potential use of biopolymers (BPs), obtained by alkaline hydrolysis of the solid anaerobic digestate of municipal biowastes, in order to face up these main concerns of agriculture. The experimental trials involved the application of BPs (at 50 and 150 kg/ha) alone or mixed with different amounts (100%, 60% and 0%) of mineral fertilizer (MF). Three different controls were routinely included in the experimental trials (MF 100%, 60% and 0%). The effect of BPs on lettuce was evaluated by monitoring growth parameters (fresh and dry weights of shoot and root, nitrogen use efficiency), and the N-flux in plant-soil system, taking into account the nitrate leached due to over irrigation events. The activities of enzymes involved in the nitrogen uptake (nitrate reductase, glutamate synthase and glutamine synthase), and the nitrogen form accumulated in the plant tissues (total N, protein and NO3−) were evaluated. The results show that the application to the soil of 150 kg/ha BPs allows to increase lettuce growth and nitrogen use efficiency, trough stimulation of N-metabolism and accumulation of proteins, and hence to reduce the use of MF by 40%, thus decreasing the nitrate leaching. These findings suggest that the use of BPs as biostimulant greatly contributes to reduce the consumption of mineral fertilizers, and to mitigate the environmental impact caused by nutrients leaching, according to European common agricultural policy, that encourages R&D of new bioproducts for sustainable eco-friendly agriculture.

Nowadays, the bioeconomy concept requires to exploit sustainable renewable biomasses to produce of fuels, chemicals, and agrochemicals which human population needs. Researchers are trying to valorise biomasses from different sources as alternative feedstocks, focusing these objectives 1 . These latter objectives are quite difficult to reach as they are dependent on the availability of biomasses, and the economic aspects related to their collection. So far, most of the R&D work on the valorisation of biomass as renewable feedstock focused on processing plants and crops to be used for fuel production, raising social concerns due to the exploitation of agricultural land for the production of non-food energy crops. On the contrary, the use of biowastes as feedstock could mitigate the popular discomfort for the environmental impact of the increasing wastes production and current disposal practices.
Municipal biowaste (MBW) is the most available and sustainable potentially renewable feedstock. As twothirds of world population is expected living in urban areas by 2050, and produce more wastes, the cities are crucial to the circular waste-based economy 2 . At present time, MBW is a social economic and environmental burden. Its valorization as feedstock producing valued added products would solve both problems. Currently, MBW is processed by anaerobic and aerobic fermentation, yielding biogas, anaerobic digestate and compost. The value of these products does not cover the processing costs. As collection and treatment costs are paid off by citizens' taxes, MBW and its digestate and compost represent negative cost feedstocks 3 . Converting MBW,   www.nature.com/scientificreports/ Determination of the nitrogen forms in lettuce tissues. The nitrogen chlorophyll content of lettuce leaves, related to the nitrogen status of the plant, was measured, before the second over-irrigation event, using in field condition a portable N-Tester (Konica, Minolta, Japan), as average of three different points of the last expanded leaf of each lettuce plant, for all treatments and replicates 27 . The tool provides a numeric three-digit dimensionless value that is commonly expressed as N-Tester value, and is used for leaf chlorophyll estimation in lettuce 28 . Total nitrogen was determined in leaves and roots by the Kjeldahl method, by digesting 2 g DW of tissues with concentrated sulphuric acid and selenium catalysis 29 .
Total protein extraction from lettuce tissues (root and leaf) was performed according to La Bella et al. 30 . Briefly, aliquots of lettuce leaves and roots were homogenized using an extraction buffer (1:1.25 w/v ratio) containing: 220 mM mannitol, 70 mM sucrose, 1 mM EGTA, 10 mM cysteine, and 5 mM HEPES-KOH pH 7.5. Samples were then filtered and centrifuged at 13,000 rpm for 30 min at 4 °C. The supernatant was recovered, and the total protein content was determined by the Bradford 31 method, using Bovine Serum Albumine (BSA) as a standard curve, and expressed as mg protein g -1 DW. All measurements were performed on 3 plants for treatment and replicates.
Nitrate (N-NO 3 ) concentration in leaves and roots, at the end of the trial, has been analysed on the fresh material. For each plant, 100 mg of fresh tissue was ground in liquid nitrogen and suspended in 10 mL of deionized water. Suspensions were incubated for 1 h at 45 °C and then centrifuged at 5,000 rpm for 15 min and filtered. The extract was used for nitrate spectrophotometric (U-2000, Hitachi, Tokyo, Japan) determination using the Griess reaction 32 .
Enzymatic activities related to nitrogen metabolism in lettuce tissues. Each enzymatic activity was assayed using an aliquot of the total protein extract, obtained as previously described, containing crude enzyme extract.
Nitrate reductase (NR) activity was measured according to Kaiser et al. 33 method. Briefly, a solution containing 100 mM KH 2 PO 4 and 100 mM KNO 3 was incubated at 28 °C for 15 min with the suitable amount of enzyme extract. The mixture was then centrifuged at 500 rpm, the supernatant was recovered, and the activity spectrophotometrically measured at 540 nm (Jasco V-530 UV-vis spectrophotometer, Tokyo, Japan), using a calibration curve, with known concentrations of NaNO 2 . Activity was expressed as Unit mg -1 protein.
Determination of the nitrogen forms in soil. The determination of nitrate nitrogen (NO 3 − N) was performed following the procedure described by Mulvaney 36 and Miranda et al. 32 . Soil samples were air dried and sieved at 2 mm. Nitrogen forms were extracted from soil (10 g) with 1 M KCl, under mechanical agitation for 60 min and further centrifugation at 3000 rpm for 10 min. Nitrites were detected in the supernatants, by using Griess solution, which was prepared by mixing 0.1% naphthalene ethylenediamine hydrochloride (NED) and 1% sulfonamide in phosphoric acid. The reaction was developed at room temperature for 20 min, then was spectrophotometrically analysed at 540 nm, using a NO 2 standard curve. Nitrate was measured by its reduction to nitrite by vanadium(III), and calculating its concentration in the supernatants by subtracting the amount of nitrite previously determined. N-NO 3 was expressed as mg N-form/g dry weight of soil (mg g -1 DW soil).
Total nitrogen was determined by the Kjeldahl method, by digesting 5 g of soil samples with concentrated sulphuric acid and selenium catalysis 37 .
Determination of the N-NO 3 in leached water. The nitrate content was determined in leached water after an extraction with 1 M KCl for 1 h, and then determined spectrophotometrically as above described for the soil, using Griess solution 32 .
Nitrogen use efficiency parameters. Nitrogen uptake efficiency (NUpE), nitrogen utilization efficiency (NUtE), and nitrogen use efficiency (NUE) were calculated according to Xu et al. 38 .

Results
Morphobiometric parameters of lettuce. The morphological traits of lettuce seedlings subjected to the BP treatments were measured, and the results are shown in Table 4. As expected, among controls, MF100% showed, for all the evaluated parameters, values greater than ones for MF60% and MF0% soil. The only exceptions were observed for root FW and DW, for which MF100% and MF60% registered similar values. At the shoot level, the best performances were obtained in the treatment BPs150 + MF100% and BPs150 + MF60%, recording for the FW of the edible portion, an increase of around 24% and 22% respect to the control MF100%, respectively. Moreover, also BPs50 + MF100% and BPs50 + MF60% showed significantly higher values than the control MF100% (e.g., shoot FW showed increases of 13% and 10%, respectively). Interestingly, the treatment BPs150 + MF0%, without added MF, recorded values always similar to MF100% in spite of the fact the applied nutrients were 1-2 order of magnitude lower. Similarly, at the root level, the highest values were obtained with the treatments BPs150 + MF100% and BPs150 + MF60%, recording a root FW 27% and 21% higher than the control MF100%, respectively, and a root length 17% and 19% higher than the control MF100%, respectively ( Table 4). All other treatments showed parameters not significantly different from the control MF100%, except for root length, in which the treatment BPs50 + MF0% showed a value similar to the control MF60%, and lower than MF100%.
Nitrogen forms in lettuce tissues. The nitrogen status of the plant was monitored in field using a N-Tester, prior to the second over-irrigation event, as described in the Material and Methods section. The values ( Fig. 1) showed that no significant differences were recorded among treatments. The total N content in leaves ( Fig. 2) significantly increased in the treatments with BPs150 + MF100% and BPs150 + MF60%, respect to the control with MF 100% (29% and 26%, respectively). The treatments BPs50 + MF100% and BPs50 + MF60% showed total N values similar to the control MF100%, thus indicating a Table 4. Morphological traits of lettuce seedlings subjected to BP treatments. Data are means ± SD. Values in the same column followed by different letters are significantly different (p < 0.05).

Treatment Shoot FW (g) Shoot DW (g) Root FW (g) Root DW (g) Root length (cm)
BPs150  Figure 3 reports the content of the total proteins extracted from lettuce tissues. The total protein content in leaves was strongly influenced by the treatments, recording a significantly increase in BPs150 + MF100% and BPs150 + MF60%, respect to the control with MF100% (32% and 28%, respectively). The treatments BPs50 + MF100% and BPs50 + MF60% also raised the protein content of the lettuce epigeal part, as compared to the control MF 100% (around 16%). Finally, in both the treatments with the two BPs dosage without mineral fertilizations (BPs150 + MF0% and BPs50 + MF0%), values always similar to MF100% and MF60% occurred. As previously reported for N total, a fertilization reduced of 40% leads to similar protein content as with the regular fertilization. At the root level, all the treatments showed not significant differences respect to the control MF100%, although they showed values higher than MF60%.
The N-NO 3 − content extracted from lettuce tissues is reported in Fig. 4. In leaves, due to the great variability of N-NO 3 − values in the replicates, no significant differences were observed among treatments. In roots a great variability of N-NO 3 − also occurred. However, in both cases, the highest value was recorded for BPs50 + MF100%. This value, although not significantly higher than those for most of the other treatments, was significantly higher than the lowest value recorded for the treatments BPs150 + MF100%, and BPs150 + MF0% and MF0%.  Table 2. NR activity, measured in lettuce leaves (Fig. 5A), increased respect to the control MF100% by about 68% under the treatment BPs50 + MF100%, and around 35% under the treatment BPs50 + MF60%. All other treatments showed NR activity values in the shoot similar to the control MF100%. In roots, the treatments BPs50 + MF100%, BPs150 + MF100%, and BPs50 + MF60% rapidly induced the activation of GS, reaching values of activity 43%, 30%, and 44%, respectively, higher than that measured in the control MF100%.
GOGAT activity in leaves showed a trend very similar to GS activity, recording the highest values under the treatments with BPs150 + MF100% (57%), BPs150 + MF60% (47%), and BPs50 + MF100% (42%), respect to the control MF 100%. The treatment BPs50 + MF60% showed an activity 25% higher than MF100%, whereas the treatments without MF (BPs150 + MF0% and BPs50 + MF0%) showed values of activity not significantly different from the control. As regard roots, all the treatments showed values of activity similar to the control MF100%, except the treatments BPs150 + MF100% and BPs150 + MF60%, which showed an increase respect to the control of 32% and 28%, respectively (Fig. 5C). Figures 6 and 7 report the N-NO 3 − and total N measured in soil at the end of the experimental trials. The N-NO 3 − data showed no significant differences between soils treated with the mineral fertilisers only (MF100% and MF60%) or with the MF-BS mixes. All the treatments with MF gave higher N-NO 3 − values than the values measured for the control MF0%. The treatments with BS only (BS150-MF0% and BS-MF0%) resulted not significantly different from MF0%. On the contrary, the soils treated with BPs exhibited the highest total N values, although these resulted not significantly different from values measured for all other treatments.

N-NO 3 − in soil.
The total N content in soils, at the end of the experimental trials, showed that not significant differences among treatments and controls MF60% and MF0% occurred (Fig. 7).

N-NO 3
− content in leached water. Figure 8 reports the N-NO 3 − contents in waters leached during the experimental trials. The data evidenced three groups of values significantly different one from the other. The MF100% and MF60% group showed the highest total average value (838 mg L -1 ). The second group, including the treatments with the BS-MF mixes, showed the highest total average value (471 mg L -1 ). The third group, including the BS150% + MF0%, BS50% + MF0% and MF0% treatments, showed the lowest total average value of 50 mg L -1 . In terms of reduction of N-NO 3 − leaching relatively to the first group, the second and third group exhibit reduction of 44% and 94%, respectively. Table 5

Discussion
Several studies evaluated the biostimulant effect of BPs on a wide range of crops 5 , but BPs for lettuce cultivation had never been tested. In lettuce at the shoot level, the treatments BPs150 + MF100% (+ 24% respect to MF100%) and BPs150 + MF60% (+ 22% respect to MF100%) determined a relevant increase of the FW of the edible portion, in accordance with the biostimulant effects observed for other species [10][11][12][13][14][15] . Moreover, the treatment with the highest amount of BPs without fertilization (BPs150 + MF0%) showed values of FW of the edible part comparable to MF100%, suggesting that BPs may be useful to ameliorate the use of the residual nutrients into the soil. The highest amount of BPs (150 kg/ha), both with MF100% or MF60%, determined also a positive effect at the root level, recording higher FW values in the treatments BPs150 + MF100% and BPs150 + MF60%, respect to the control (Table 4). Starting from the positive effect on the morphobiometric traits of the lettuce seedlings, the fate of the nitrogen (N) was investigated, as N represents the most important macronutrient in lettuce production for proper foliage growth and good green colour 39 . During lettuce cultivation, nitrogen status of the plant was monitored in field, using a non-invasive technique, and the results showed a great variability in the measurements with values not significantly different among the treatments (Fig. 1). N-test readings have been proven to be well correlated with the leaf chlorophyll content and/or leaf N concentration in several cereals such as Hordeum vulgare L. 40 , Zea mays L 41 , Oryza sativa L. 42 , and wheat 43 . These evidences suggest that, during the experimental trials, chlorophyll content keeps rather constant values. Moreover, according to Pennisi et al. 28 , who reported values of N-tester for lettuce ranging between 300 and 400, lettuce treated with 150 kg/ha BPs reached values ranging between 500-520, thus suggesting the presence of a great amount of chlorophyll in their leaves. On the contrary, significant differences were observed as regard the different forms of nitrogen accumulated in lettuce tissues at the end of the experimental period. The treatments BPs150 + MF100% and BPs150 + MF60% greatly affected the accumulation of total nitrogen (N) and proteins at the shoot level of the lettuce (Fig. 2  and 3). This increased protein content is compatible in order to support the enhanced growth of the epigeous part of lettuce 44 . However, the increased N absorption efficiency of the plant, on the other hand, may lead to nitrate accumulation 45 . Lettuce leaves can accumulate a wide range of nitrate, varying from 190 to 6600 mg kg −1 , depending on different factor such as species, individual plant, cultivation season, age, morphotype, climate, and fertilisation 46 . Risks related to high levels of nitrate are mainly related to methemoglobinemia, a disease affecting infants leading to anoxia or death, toxicity due to carcinogenic and mutagenic nitrosamine compounds, and associated to gastric cancer, due to the ingestion of N-nitroso compounds 47,48 . Moreover, a high nitrate levels in the edible part of baby leaf lettuce may determine a decrease of vitamins and hence of the nutritional profile 49 . Therefore, research is focusing on the use of techniques or treatments increasing N absorptions, but reducing its accumulation under form of nitrate. In Italy the presence of nitrate in lettuce is regulated by EU regulation N. 1258/2011, taking into account EFSA opinions 50,51 , indicating for lettuce cultivated in greenhouse a limit of nitrate corresponding to 4000 mg kg -1 , between 1 April-30 September, and 5000 mg kg -1 , between 1 October-30 March. Successfully, our results suggest that both the treatments 150BPs + MF100% and 150BPs + MF60% raised the total N accumulation in lettuce leaves (Fig. 2), by increasing the total protein content (Fig. 3), and nevertheless maintaining the levels of nitrate (Fig. 4) similar to those of control plants (MF100%, MF60% and MF0%). Interestingly, the highest value of nitrate, observed in BPs50 + MF100%, showed anyway a value (320 mg kg -1 FW) greatly lower than legal limits (4000-5000 mg kg -1 ).
In plants, nitrate may be metabolized both in shoots and roots, and the rate of its conversion is dependent on different environmental factors, type and amount of N supply, plant species, and age 52 . Nitrate reductase (NR) is a cytosolic enzyme that may be considered as the rate-limiting stage of the nitrate assimilation pathway, and it is considered to be a limiting factor for the growth and development of plants. NR, in the cytosol of plant cells, catalyses the reduction of NO 3 − into NO 2 − , and acts as a crucial point in the plant N metabolism 53 . Our results showed that, in the soil with MF100%, the treatments with both concentration of BPs, significantly increased NR activities in roots, whereas in leaves NR activities were higher in the treatments with the lower amount of BPs (BPs50 + MF100% and BPs50 + MF60%) (Fig. 5A). These results may be explained by the evidence that higher Table 5. Nitrogen efficiency parameters in lettuce seedlings subjected to BP treatments. Values in the same column followed by different letters are significantly different (p < 0.05). www.nature.com/scientificreports/ N accumulation in lettuce correspond to a higher NR activity during the initial stage of plant growth, whereas a decrease of NR activity during the final stage of plant growth may occur 54 .
In the primary metabolism involved in N assimilation, the glutamine synthetase (GS) and glutamate synthase (GOGAT) have also been proposed to play a key role through ammonium incorporation into carbon skeletons, by assimilating the cation into an organic form as glutamine and glutamate 55,56 . Both GS and GOGAT, significantly increased in treatments BPs150 + MF100% and BPs150 + MF60% (Fig. 5B,C), in accordance with an increased growth of lettuce, and a higher amount of total N and proteins. Supporting these results, the involvement of N metabolism in the enhanced growth of lettuce was also observed using other biostimulant types, such as microalgae-based extracts 27,30,57 , plant-based preparations containing triacontanol 58 , l-amino acid-based biostimulants 59 .
It is well known that nitrogen is distributed into the plant, in the fixed fraction into the soil, and in the leached water 39 . Our results showed that, although the N total of all the soils was quite similar (Fig. 7), significant differences were observed as regard nitrate concentrations (Fig. 6). Interestingly, all the soils subjected to fertilization, both MF100% and MF60%, showed amount of NO 3 − rather similar among them, and always greater than soils not fertilized (BPs150 + MF0%, BPs50 + MF0%, and MF0%). Meanwhile, NO 3 − amounts in leachates significantly decreased in all waters collected from the fertilized soils (both 100% and 60%) subjected to BP treatments (both concentrations) (Fig. 8). All these results taken together, suggest that nitrate keeps constant in the fertilized soils for two different reasons: i) in the control fertilized soils (MF100% and MF 60%), the residual amount of nitrate (Fig. 6), after the plant uptake, may be strictly linked to the loss of NO 3 − by lixiviation (Fig. 8); ii) on the contrary, the plants grown in fertilized soils and treated with BPs (BPs150 + MF100%, BPs50 + MF100%, BPs150 + MF60%, and BPs50 + MF60%) seem to uptake an higher amount of NO 3 − from the soil, in order to support a greater growth of lettuce (Table 4), by increasing total protein content in the edible portion, and hence greatly reducing the amount of leached nitrate in the waters (Fig. 8). This hypothesis is supported by the evidence that, among the mechanisms of action of biostimulants based on humic-like substances, the increased uptake of nutrients such as nitrogen from the soil is one of the main studied processes [60][61][62] .
In this context, the nitrogen use efficiency (NUE) is considered a further important parameter, being related to the produced biomass per unit of available N. This parameter takes into account two factors: N uptake efficiency (NUpE), representing the ability of the plant to absorb N from the soil, and N utilization efficiency (NUtE), representing the potentiality of the plant to transfer and utilize N in the biomass production of the different plant tissues 38 . Our results showed that BPs, in particular BPs150 + M100%, increased the NUE respect to the lettuce grown in MF100% (Table 4). According to Lemaire et al. 63 , higher NUE improves the yield and quality of the plant, and decreases the environmental impact caused by the lixiviation of excess N fertilizer application. Moreover, in lettuce cultivation, Navarro-Leòn et al. 59 , have recently shown that the use of L-amino acid-based biostimulants improves nitrogen use efficiency (NUE), associated to NO 3 − and total N accumulation in the plants. Our results hence suggest that 150 kg/ha BPs may be possible candidates to increase the lettuce growth, trough stimulation of N-metabolism, to reduce mineral fertilization, as the treatment BPs150 + MF60% showed results very similar to the treatment BPs150 + MF100%, and finally to decrease the nitrate concentration into groundwater. Table 6 shows summarises the effects of the different treatments on the measured parameters. It may be readily observed that, for all measured parameters, the treatments with the BPs-MF mixes rank first and exhibit the highest effects, compared to the treatments with MF only or BPs only, and with the control MF0%. Particularly significant is the N-NO 3 − in leached water 1575% increase measured for the treatment with MF100% and MF60%, relatively to the BPs150 + MF0% and BPs50 + MF0%, which together with the control MF0% trial exhibited the lowest N-NO 3 − value in leached water. This prospects that formulates containing both MF and BPs in the proper relative amounts can achieve the highest plant productivity, together with the lowest environmental impact from fertiliser leaching in waters through the soil and the best safest crop quality.
With reference to the goal of lowering the consumption of mineral fertilizers, and the consequent depletion of mineral fossil sources, production on energy intensive N compounds and related GHG production, and finally the European import of mineral fertilisers, by implementation of BPs as alternative/supplementation to commercial MF, Table 3 shows that, compared to the MF100% and MF60% treatments, the use of BPs150 + MF0% and BPs50 + MF0 implies a strong reduction of mineral fertilisers supplied. Generally, according to Table 3 data, the use of all BPs-MF mixes, except for BPs150 + MF100%, would result in a reduction of N, P, K amounts.

Conclusions
Considering all the concerns associated with nitrogen fertilization, nowadays it is essential to use new agronomic techniques able to increase NUE by plants and reduce the environmental impact linked to the lixiviation of nitrogen. In this context, the use of biostimulants has the potentiality to address some of the problems related to N fertilization. The present work has shown new evidences about BPs biostimulant properties on lettuce, a new species never tested before with BPs. Our results showed that 150 kg/ha BPs are able to increase lettuce growth, enhance NUE, and in the meantime reduce the loss of N thought lixiviation. In particular, the use of BPs in lettuce cultivation has shown to increase its growth, improve the nitrogen adsorption, thought the stimulation of N metabolism and the protein accumulation, allowing to reduce of 40% the consumption of mineral fertilizers. Moreover, BPs by increasing the N uptake are also effective to reduce the nitrate lixiviation trough the soil, thus contributing to mitigate the environmental impact caused by leaching.