www.nature.com/scientificreports OPEN Study on analytical characteristics of Nicotiana tabacum L., cv. Solaris biomass for potential uses in nutrition and biomethane production Antonella Fatica 1*, Francesco Di Lucia2, Stefano Marino1, Arturo Alvino1, Massimo Zuin3, Hayo De Feijter2, Boudewijn Brandt2, Sergio Tommasini2, Francesco Fantuz4 & Elisabetta Salimei 1 In order to limit the smoking tobacco sector crisis, a new non-GMO Nicotiana tabacum L. cv. Solaris was proposed as oil seed crop. Residues of oil extraction were successfully used in swine nutrition. The aim of this study was to explore the full potential of this innovative tobacco cultivar as multitasking feedstock non interfering with the food chain. In the triennium 2016–2018, samples from whole plant, inforescence and stem-leaf biomass were collected in three experimental sites and analysed for chemical constituents, including fbre fractions, sugars and starch, macro-minerals and total alkaloids. The KOH soluble protein content and the amino-acid profle were also investigated as well as the biochemical methane potential. All the analyses were performed according to ofcial methods and results were compared with values reported in literature for conventional lignocellulosic crops and agro-industry residues. The average protein content, ranging from 16.01 to 18.98 g 100 g−1 dry matter respectively for stem-leaf and whole plant samples, and their amino-acid profle are consistent with values reported for standard grass plant. These fndings suggest the potential use of cv. Solaris in industrial food formulations. Moreover, considering the average content of both fbre available for fermentations (72.6% of Neutral Detergent Fibre) and oils and fats (7.92 g 100 g−1 dry matter), the whole plant biomass of cv. Solaris showed good attitude to anaerobic fermentation, confrmed by the biochemical methane potential of whole plant (168 Nm3 t−1 organic matter). Similarly, results allow to defne the cv. Solaris biomass as a good quality forage apt to ensiling for its chemical composition. The low total alkaloids content of cv. Solaris, in average 0.3 g 100 g−1 dry matter, was previously reported not to afect growth performances and welfare traits of dairy heifers. These are the frst results showing the multitasking potential use of cv. Solaris biomass, that could allow the recovery of tobacco cultivation know-how especially in marginal areas. Te European Nicotiana tabacum L. cultivation has decreased considerably in recent years because restriction of subsidies1 and smoking tobacco production is reported to account for about 219,243 tons2. Italy is the largest tobacco producer in the European Union, with an amount of 21% of the total European tobacco production2. However, in Italy the crisis of the sector is particularly serious, with a reduction of both the production (from 110,000 tons in the 2007 to 46,060 in the 2017) and the cultivated areas, reduced from 32,000 hectares in 2007 to 14,548 hectares in 20172. Tobacco cultivation in Italy is concentrated in Campania, Umbria, Veneto and Toscana regions, where a strong cultivation know-how is available in terms of both technique and agricultural machinery. 1Dipartimento Agricoltura, Ambiente, Alimenti, Università degli Studi del Molise, via Francesco De Sanctis, 1, Campobasso, 86100, Italy. 2Sunchem BV, Amsterdam, The Netherlands. 3Agronomist Consultant, Pordenone, Italy. 4Scuola di Bioscienze e Medicina Veterinaria, Università degli Studi di Camerino, via Gentile III Da Varano, 62032, Camerino, Italy. *email: [email protected] SCIENTIFIC REPORTS | (2019) 9:16828 | https://doi.org/10.1038/s41598-019-53237-8 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Nicotiana tabacum L. cv. Solaris (lef) with reduced size and elevated number of fowers compared to traditional cultivar of smoking tobacco (right). In order to limit the crisis afecting the tobacco sector in Italy, thanks to 15 years of research, a new cultivar of Nicotiana tabacum L. was developed for maximizing the production of seeds with high content of oil used as bio- fuel or biomass source1. Tis cultivar, named Solaris, is a non-GMO developed and patented as “energy tobacco” (PCT/IB/2007/053412)3,4, registered in 119 countries and granted in over 75 countries, including USA, all Africa, EuroAsia, Italy, Russia and Australia. Te genetic improvement was addressed to a sustainable industrial “no food” culture, having no competition with plants produced as food for humans or animals. Nicotiana tabacum L. cv. Solaris compared with smoking tobacco varieties contains low levels of nicotine and maximizes the production of fowers/seeds, reducing leaf growth (Fig. 1). Te industrial uses of Nicotiana tabacum L. cv. Solaris as energy biomass were frstly reported by Grisan and colleagues1 that tested the tobacco Solaris cultivation as sustainable bioenergy chain in North and Central Italy. Te cold-press extraction from seeds showed that the content of oil, used as jet biofuel, in tobacco seeds is around 38–40% and the oil cake coproduct may be useful as a high protein and energy feed for livestock4. So, given the chemical and nutritional characteristics, the cake obtained from the extraction of oil from tobacco seeds, was successfully introduced in piglets’ diet5. Te strategy proposed by Grisan and colleagues1 included two seed harvests: the frst plant apexes collections carried out from the end of July to the frst decade of August (80–90 days afer transplanting), and the second seed harvest carried out in the second half of October (140–150 days afer transplanting) when, on the other hand, the environmental conditions can be critical. Consistently with its strong vegetative capacity and its short cultivation cycle (80–90 days the frst harvest)1, the Solaris tobacco biomass has been here investigated following an alternative strategy, i.e. 50–60 days afer the frst collection of the plant apexes (seeds), the total biomass harvesting may occur within the same vegetative sea- son. Te large amounts of biomass harvested afer the frst seed collection could be used in other sectors, such as for biomethane production6, animal nutrition or food industry, consistently with a circular supply chain (circular economy) approach. Te aspects related to alternative uses of biomass from Nicotiana tabacum cv. Solaris are at the moment unknown, therefore, aiming to contribute to the advances of knowledge on this innovative culture and its poten- tialities, the goal of the present trial was to study the chemical characteristics, including amino acid profle and mineral content, and the potential biomethane production of cv. Solaris biomass. Results were then compared with literature data on conventional plant materials used as source of biomass for nutrition, i.e. animal feeding and food supplement, and biomethane production. Materials and Methods Te study on Nicotiana tabacum L. cv. Solaris biomass has been carried out during the three-year period 2016– 2018 in three sites located in Vicenza (45°17′25.4″N 11°30′09″E), Chieti (42°10′0″N 14°20′0″E) and Perugia (42°59′0″N 12°25′0″E) provinces, adopting conventional farming techniques of the areas1. Te scalar collections of ffeen cv. Solaris tobacco samples were made in years 2016, 2017, 2018 for Vicenza province, in 2017 for Chieti province and in 2018 for Perugia province. Data on thermal-pluvio-metric conditions were collected by weather stations located in Vicenza, Chieti and Perugia provinces, near the study areas. Data were provided by ARPA Veneto7, HORT@ Agrometeorological Network8 and Regional Hydrographic Service9, respectively for Vicenza, Chieti and Perugia province. As depicted in Fig. 2(a–c), in the three years study the average temperature ranged monthly from 13.6 °C to 26.1 °C during the cultivation period (May–October), when total rainfalls ranged from 2.8 to 140.2 mm. Te feld experiment was carried out in each year and site in randomized blocks with three replications. Each plot was 50 m2 for a total surface of 600 m2. Tobacco plants were transplanted afer tillage carried out by plough- ing (moldboard plow - depth of 50 cm) and followed by secondary harrowing in order to reduce the clod size. Nitrogen, phosphorus and potassium were distributed based on the soil analysis, in total were distributed 120 kg SCIENTIFIC REPORTS | (2019) 9:16828 | https://doi.org/10.1038/s41598-019-53237-8 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 2. Termal-pluvio-metric diagrams for 2016, 2017 and 2018 about Vicenza7 (a), Chieti8 (b) and Perugia9 (c) province. of N, 60 kg of P2O5, 70 kg of K2O by fertigation system. Insecticide products (z-cypermethrin) were buried on the ground to prevent attacks of terricolous cutworms in the transplantation stage. During the season, fungicide treatments (cooper and metalaxil) and insecticide (indoxacarb) treatments were carried out to protect the matur- ing capsules from fungal disease and bollworm. In order to compare the efect of plant density, the transplant was performed mechanically on May, with two transplanting densities, i.e. 3.6 and 4.8 plants per m2. Te plants were arranged in rows 90 cm apart and the dis- tance between the plants on the row was 31 cm and 23 cm, respectively for the 3.6 and 4.8 plants per m2 density. Te herbicide treatment was performed the day afer the transplant using a clomazone based product. Drip irrigation (about 180 m3 ha−1 per watering) was adopted and the total amount ranged from about 1000 m3 ha−1 (6 waterings) to about 1450 m3 ha−1 (8 waterings), according to weather conditions and practices in use by local farmers. Te seed harvest was carried out manually by cutting the plant apexes (mature capsules) from the end of July to the frst decade of August (80–90 days afer transplanting). Te seeds collected were weighted and seed yield recorded.