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Chemical composition and yield of rhizome biomass of Arundo donax L. grown for biorefinery in the Mediterranean environment
Biomass and Bioenergy 107 (2017) 191–197
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Biomass and Bioenergy journal homepage: www.elsevier.com/locate/biombioe
Research paper
Chemical composition and yield of rhizome biomass of Arundo donax L. grown for biorefinery in the Mediterranean environment
MARK
Simona Proiettia, Stefano Moscatelloa, Massimo Fagnanob,∗∗, Nunzio Fiorentinob, Adriana Impagliazzob, Alberto Battistellia,∗ a b
Institute of Agro-Environmental and Forest Biology (IBAF), National Research Council (CNR), via Marconi 2, 05010, Porano, TR, Italy Department of Agricultural Sciences, University of Naples Federico II, Via Università 100, 80055, Portici, Naples, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords: Giant reed Biofuel Non-structural carbohydrate Cellulose Hemicellulose Lignin
The contribution of the rhizome to productivity of fermentable sugars and the detailed composition of rhizomes were analyzed in three mature stands of Arundo donax L. cultivated in three locations of variable fertility in the South of Italy. Although the average yearly aboveground dry biomass and rhizome amount showed large and significant differences among sites, (15.3 and 2.6 Mg ha−1 year−1 of rhizomes in the most and less productive sites respectively), rhizomes of all sites had more than 30% of the dry matter (DM) as non-structural carbohydrates (NSC). Sucrose and starch were the most abundant NSC but measurable amounts of glucose, fructose, galactose and of the valuable trisaccharide raffinose were also present. The amount of NSC in rhizomes affected their content of dry mater, and water extractives. The ash content also varied significantly among cultivation sites; the highest amount was recorded in rhizomes of the most productive site (Acerra). The abundance in cell wall components of rhizomes was similar to that of published values for the above ground biomass. The present results demonstrate that NSC content in rhizomes of mature stands is a conserved trait. Hence, rhizome biomass, thanks to its quantity and high fermentable sugars content, should be considered as a relevant fraction of the A. donax crop product whose utilization can increase the productivity and the environmental fingerprint of this crop, in view of its biomass utilization in biorefinery.
1. Introduction The implementation of biorefinery plants is only possible with an adequate supply of feedstock that do not compete with food production systems. This has fostered research on species able to grow in area were food production is not a benchmark. Arundo donax L., is one of the species that has recently received attention as a potential biomass producer in low input non-food systems for the temperate and hot zones. A. donax is a perennial rhizomatous wetland grass, widespread all over the world; it shows a very high adaptability to abiotic and biotic stresses as drought, flood [1], salinity [2] and pests, maintaining an elevated potential for biomass yield [3]. A. donax growth is very fast during spring and summer in Mediterranean environments, resulting in the accumulation of large amounts of biomass even with minimal agricultural input such as irrigation, fertilization and phytosanitary treatments [4,5]. This plant could be suitable also for the cultivation of marginal areas, where the use of other species is disadvantageous. Trials carried out in Italy have shown that A. donax could be a very
good feedstock for bioenergy production due to high yield potential [6,7]. Aboveground average dry biomass yield of 38 Mg ha−1 year−1 was recorded during growth from year 2–12 after planting [6], while Mantineo et al. [5] reported about the same production from the first three years of A. donax growth. A. donax was also able to grow in degraded or contaminated soils [8–10] or soils subjected to accelerated erosion [11]. This species was shown to have a favorable environmental impacts in marginal lands, due to carbon storage within the soil [12, 13; it was also suggested as being suitable in projects of phytoremediation of polluted soils [14]. For all of these advantages, giant reed is considered a very interesting non-food crop as source of biomass for the production of secondgeneration biofuel, bioenergy and other high value products for the chemical industry [6,15–18] with no competition for land with food crops. This has fostered research and improved the knowledge regarding yield and chemical characteristics of aboveground part of plant. However, most studies examining the potential of A. donax as a biomass producer for biorefinery have taken into account only the aboveground
Abbreviations: DM, Dry matter; NSC, Non-structural carbohydrates ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (M. Fagnano), [email protected] (A. Battistelli). http://dx.doi.org/10.1016/j.biombioe.2017.10.003 Received 10 March 2017; Received in revised form 29 September 2017; Accepted 6 October 2017 0961-9534/ © 2017 Elsevier Ltd. All rights reserved.
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Aerial biomass was harvested every year at the end of winter and dried at 70 °C to constant weight. Underground biomass samples were collected during winter, from sample areas of 1 m2, washed, weighted and dried at 70 °C until to constant weight. Two subsamples of 1 kg were collected from each plot (3 replicates x 3 sites), immediately frozen with dry ice, and sent to the laboratory for the chemical characterization.
biomass production [16]; with the exception of Nassi o Di Nasso et al. [19] who quantified also rhizome biomass accumulation. Rhizomes are modified stems that grow perpendicularly to the gravitational force, normally underground, and can produce stems and root from nodes, function as storage sites of reduced carbon and mineral nutrients, and affect plant survival under stressful conditions [19–21]. A. donax can produce large quantities of rhizomes that represent a relevant portion of its total crop biomass. Nassi o Di Nasso et al. [19] showed a rhizome dry biomass yield of 16 Mg ha−1year−1, for a three years old crop. Giant reed translocates nutrients to the rhizome at the end of the growing season [22], and remobilize them for regrowth of the new shoot at the beginning of the vegetation season, reducing fertilizers requirements. A number of studies have been carried out to estimate the seasonal dynamics of above and belowground resources allocation in rhizomatous perennial grasses such as Miscanthus, Panicum and Arundo [23,24], but the focus of existing studies was mineral and nitrogen partitioning. The present study is of particular importance as the carbohydrate content and partitioning in addition to the cell wall chemical characteristics of A. donax rhizome, similar to other rhizomatous grasses, are lacking. A. donax plantations can produce biomass for as long as 10–15 years, thereafter the yield declines dramatically and the crop stand must be removed. Upon removal of a giant reed plantation, or eventually with partial harvests during the plantation period, the rhizome could be used as a feedstock for bioenergy production or as a raw material for green chemistry applications, e.g. for the production of molecules of interest such as xylobiose and tetra- and penta-saccharides [25]. The aims of this work were: i) to evaluate the contribution of rhizome to total biomass yield by stands cultivated in sites of the Mediterranean area of different fertility; ii) to evaluate the quality of such rhizome biomass in terms of NSC (fermentable carbohydrates) and structural components. Our work highlights the potential role of rhizome biomass in increasing productivity of fermentable sugars; hence affecting the profitability and environmental fingerprint of A. donax cultivation for green chemistry and helping to improve the sustainability of biomass based agro-industrial systems in the Mediterranean area.
2.2. Analysis of NSC Soluble carbohydrates (glucose, fructose, sucrose, raffinose) and starch in the biomass were extracted for analysis as in Moscatello et al. [27]. In brief, 10 mg of dry material was extracted in 1 ml of 50% ethanol/water at 80 °C for 45 min. Soluble sugars were recovered in the supernatant after centrifugation at 16000g for 5 min. Starch in the pellet was hydrolyzed enzymatically to glucose after washing and autoclaving. The supernatant containing soluble carbohydrates, and the glucose solution obtained after starch hydrolysis were filtered on 0.2 μm nylon filters (GE-Whatman, Maidstone, UK), then analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Thermo Scientific™ Dionex™ ICS5000, Sunnyvale, CA U.S.A.) consisting of a dual pump system (quaternary analytical pump plus isocratic pump), a pulsed amperometric detector, and an analytical CarboPac PA20 column (3 mm × 150 mm) with guard column. The detection cell contained a gold working electrode (1.0 mm in diameter) and an Ag/AgCl reference electrode. Pulsed amperometric detection was carried out with the following waveform: E1 = +0.10 V (t1 = 0.4 s), E2 = −2.00 V (t1 = 0.01 s), E3 = +0.60 V (t = 0.01 s), E4 = −0.10 V (t = 0.06 s). The integration range began at 0.2 s and ended at 0.4 s. The electrical signal was integrated in nanocoulomb (nC). Runs were carried out at 30 °C using an injection valve with a 5 μl injection loop. Mobile phase was NaOH at a flow rate 0.5 ml min−1. Sugars were eluted under the following conditions: 0–12 min, 10 mM; 12–25 min, 80 mM; 25–39 min, 240 mM; 39–55 min, 10 mM. Regeneration of amperometric electrode was done with a post-column addition of concentrated sodium hydroxide (300 mM) using a secondary pump at a flow rate of 0.25 ml min−1. The carbohydrate standard solutions were prepared using HPLC grade reagents (Sigma, Steinheim, Germany).
2. Materials and methods
2.3. Analytical methods for the determination of chemical composition of rhizome
2.1. Biomass sampling Giant reed rhizomes were collected from 3 experimental fields located in marginal land areas of the Campania Region, Southern Italy: Acerra, (40° 59′N, 14° 20′E, 26 m a.s.l.), from a 6 year stand (2009–2014) in polluted soils of a Vesuvius plain; Sant’Angelo dei Lombardi (40° 55′ N, 15° 07′ E, 700 m a.s.l.) from an 11 year stand (2004–2014) in a hilly area of Southern Apennine subjected to accelerate erosion; and Bellizzi (Torre Lama) (40° 37′N, 14° 58′E, 30 m a.s.l), from a 7 year stand (2008–2014) in a coastal plain subjected to soil salinization. Details of cropping systems are reported in Fiorentino et al. [9], Fagnano et al. [11] and Impagliazzo et al. [26], respectively. In all sites the same low input cropping system was adopted with low doses on N fertilizers and no irrigation. Physico-chemical characteristics of the soils of the three locations are reported in Table 1.
Chemical analyses were performed in accordance to the standard biomass analytical methods provided by the National Renewable Energy Laboratory (NREL) as detailed in Santi et al. [28]. The collected samples were milled with a MF10 IKA mill (Werke GmbH & Co. KG) and sieved to screen particles until 500 μm in size. After milling, the samples were oven dried for 24 h at 45 °C and then used for the determination of the biomass chemical composition. In brief, the relative moisture was determined by placing the samples at 105 °C for 24 h in a ventilated oven. The ash content was quantified after ignition of dried samples at 575 ± 25 °C for 24 h using a muffle furnace equipped with a ramping program (Nabertherm P300 Germany). A two-step extraction was used to quantify extractives in the rhizome biomass samples. Approximately 1.5 g of dried sample was placed in cellulose extraction thimbles, 100 mm length by 25 mm diameter (GE-Whatman, Maidstone, UK). The first step was performed with water, by extraction in 25 ml of deionized water at 50 °C for 30 min. This step was repeated 4 times to a final extraction volume of 100 ml for each sample. The second step was performed, using 100% ethanol on the water insoluble material, following the same procedure described for the first step. Water and ethanol extracts were quantified gravimetrically; the water soluble components were freeze-dried, while the ethanol extractable material was dried using a rotavapor (VV 2000 Heidolph Instruments, Schwabach, Germany).
Table 1 Soil characteristics in the tree cultivation sites. N-tot, total nitrogen; SOM, soil organic matter; N-NO3, nitric nitrogen; N-NH4, ammonia nitrogen.
Acerra Bellizzi S. Angelo dei Lombardi
Sand %
Silt %
Clay %
N-tot %
SOM %
N−NO3 ratio N−NH4
pH
55.7 34.2 36.9
27.5 26.1 24.6
16.7 39.6 38.0
0.16 0.09 0.08
2.64 1.57 1.03
1.25 0.55 0.40
7.7 7.8 8.2
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Yearly aboveground biomass productivity reported in recent studies spans from less than 10 Mg ha−1 year−1 to more than 40 Mg ha−1 year−1 [1,3]. Our stands grew on site with medium to high productivity. The Acerra site, located in the Vesuvius plain, has a high content of minerals and organic matter [8]. Its above ground productivity was high and similar to the best recorded values at national and international level [3]. The aboveground productivity at Bellizzi, in the coastal plain at risk of salinization, was relatively low which is not surprising because of the low soil content of mineral and organic matter at this site [26]. The relatively low aboveground biomass productivity recorded at Sant’Angelo dei Lombardi can be attributed to the severe climatic conditions of this internal hilly area of the Southern Apennine, [11]. These values were lower than the ones measured in other field experiments in Italy [6] but similar to results from experiments in marginal lands [30]. Literature data on belowground biomass accumulation in A. donax is particularly scarce. It is known that roots of A.donax can reach very deep soil layers and be abundant even at 1 m in depth [31]. Sánchez et al. [32,33] found that shoot/root ratio can be affected by environmental stresses but did not quantify the absolute effect on rhizome biomass. Nassi o Di Nasso et al. [19] found that in marginal lands of Central Italy, young stands increases the partitioning of biomass in rhizomes over the years reaching about 16 Mg ha−1of DM allocated in rhizome at the end of the third year. On an yearly basis this value would be larger than the one we recorded in the less productive site of Sant’Angelo dei Lombardi (average of 11 years of accumulation) but similar to that recorded at Bellizzi (average of 7 years of accumulation) and one third of what we recorded in the more fertile site of Acerra (average of 6 years of accumulation. In the same study [19] it was shown that the ratio of below and aboveground biomass can change with stand age with values close to 2, at the end on the first growing year, and values of 0.7 and 0.8 at the end of the second and third year. The rhizome/aerial biomass ratio measured in the sites of Acerra and Bellizzi is consistent, although slightly lower, with that shown for the second and third year of growth, by Nasso o Di Nasso et al. [19] while that of the less productive site of Sant’Angelo dei Lombardi is largely lower. It is known that rhizomes in perennial plants contribute to nutrient and carbohydrate balance at the plant level [34] by accumulating resources during the mid and late vegetative season, and re-supplying them to the aerial part of the plant to support spring sprouting [22]. This function is controlled at the genetic and environmental level, can be regulated by multiple factors [35] and is of particular importance for re-growth and productivity of rhizomatous crops undergoing aboveground sequential harvesting [21]. Nassi o di Nasso et al. [19], showed that there is a linear relationship, in a young A. donax stand, between the dry weight of rhizomes and the number of buds, hence the low rhizome biomass in the Sant’ Angelo dei Lombardi stand can be linked to its low productivity. Our data confirms the abundant accumulation of DM in rhizome of mature stands, and shows that this phenomenon is affected by general conditions, such as the age of the stand and the fertility of the site. We also show that not only biomass accumulation is (in absolute terms) variable, but also allocation (in relative terms among different plant parts) can be strongly affected by stand age and
Lignin, hemicellulose and cellulose content was determined in extractive free material following NREL protocol (TP-510-42618 [29]) with 72% H2SO4 treatment at 30 °C for 1 h and subsequently using 4% H2SO4 treatment at 121 °C for 1 h in an autoclave. After the two-step acid hydrolysis, the autoclaved solution was vacuum filtered using a calibrated glass filter (GF/A 55 mm diameter GE-Whatman, Maidstone, UK). In the hydrolyzed liquor the lignin is fractionated into acid soluble lignin and acid insoluble material (Klason lignin). The Klason lignin was determined gravimetrically from the solid residue on the glass filter after drying at 105 °C for 24 h. In addition, the acid soluble lignin was measured using UV–Vis spectrophotometer (Jenway 6715 Bibby Scientific) at 205 nm, with the extinction coefficient of 110 l g−1cm−1. The filtrate was subsequently analyzed for monosaccharides resulting from the acid hydrolysis (glucose, arabinose, galactose, and xylose). The monomeric sugars were analyzed by high-performance anion exchange chromatography, with pulsed amperometric detection as described for the analysis of non-structural carbohydrate, with an analytical CarboPac SA10 column (4 mm × 250 mm) with the guard column. Runs were carried out at 45 °C. NaOH (1 mmol l−1) was used as mobile phase at a flow rate 1.0 ml min−1 with a post-column addition of concentrated NaOH (300 mmol l−1). The post-column addition was made using a secondary pump, at a flow rate 0.5 ml min−1. The eluents and the carbohydrate standard solutions were prepared using HPLC grade reagents (Sigma, Steinheim, Germany). The IC method required minimum sample preparation and the analytes were separated without interferences. Hemicellulose content was determined as the sum of arabinose, galactose, and xylose amount, while cellulose was quantified on the value of glucose corrected for the starch content in the samples. 2.4. Statistical analysis The parameters of the chemical composition of biomass from Arundo rhizome were analyzed by One-Way analysis of variance (ANOVA) with the crop site as the random factor. Differences between averages were tested by an LSD test for a significance level of P = 0.05. Regression analyses between the percentage of DM and NSC, and between the percentage of sucrose and starch were carried out estimating the correlation by the Pearson coefficient (r) for a significance level of P = 0.05. All statistical analyses were done by ANOVA using the STATISTICA 8 software package (StatSoft for Windows 1998). 3. Results and discussion 3.1. Contribution of rhizome quantity to total biomass yield Below and above ground biomass production and partitioning was significantly different in the three experimental fields (Table 2). Acerra was by far the most productive site in terms of both below and aboveground cumulated and yearly biomass production. Bellizzi overcame Sant’ Angelo dei Lombardi in terms of belowground accumulation capacity but not in terms of yearly aboveground biomass accumulation. Belowground biomass was more than one third of the total biomass accumulated in the Acerra and Bellizzi sites. Sant’Angelo dei Lombardi was the site with the lowest partitioning of biomass in rhizomes.
Table 2 Production parameters. Relevant production parameters of Arundo donax stands cultivated in the Campania Region. Values in bracket close to the name of the site indicate the age of the stand at the time of harvest. Values in bracket below data are standard errors. On the same columns, values with different letters were statistically different for P = 0.05. Columns with no letters indicate that the F test for the ANOVA was not significant for P = 0.05. Experimental sites (age of crop)
Rhizome yield (DM Mg ha−1)
Rhizome yield per year (DM Mg ha−1 year−1)
Cumulated aerial biomass yield (DM Mg ha−1)
Aerial yield per year (DM Mg ha−1 year−1)
Rhizome/aerial biomass ratio
Acerra (6 years) Bellizzi (7 years) S. Angelo dei Lombardi (11 years)
91.9 ± 6.9 a 64.2 ± 1.3 b 29.0 ± 4.2
15.32 9.17 2.64
168.8 ± 7.3 a 101.1 ± 6.5 b 170.2 ± 20.8 a
28.10 14.44 15.47
0.55 ± 0.05 a 0.64 ± 0.05 a 0.17 ± 0.00 b
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Fig. 2. Dry Matter (%) measured from each site with the correlation between NSC (%) and DM (%) inlaid. On the same row, values with different letters were statistically different for P = 0.05. Values (bars) with different letters were statistically different, for P = 0.05. Regression was statistically significant for P = 0.05.
Fig. 1. (A) NSC and Total Carbohydrates as % DM at each production site, (B) Sucrose, Starch, Glucose, Fructose, Raffinose as % DM from each site, with an inverse correlation between Sucrose and Starch as % DM inlaid. For each component, values (bars) with different letters were statistically different, among sites, for P = 0.05. Regression was statistically significant for P = 0.05.
site fertility. The fact that rhizome biomass can strongly increase the total biomass production of the crop suggests to include this information in models of A. donax economic and environmental performances. Rhizome yield data is, at the moment, not considered in the economic profitability evaluation [36] nor in the Life Cycle Assessment of this crop [12,13], but its inclusion in future analyses would guarantee a more complete evaluation of the economic and environmental sustainability of the giant reed crop. Finally, more research is needed to optimize crop lifetime and management in order to maximize the total biomass yield considering both reeds and rhizomes.
3.2. NSC in rhizomes NSC were the highest component fraction in the rhizomes. The total amount of NSC was on average 33% of the rhizome DM. Statistically significant differences were recorded among sites; however, their content was higher than 30% of the DM even in Acerra, the site with the lowest amount of NSC. (Fig. 1A). The high NSC content affected directly and positively the DM content of rhizomes (Fig. 2, insert). Sucrose was the main NSC in rhizome (Fig. 1B), representing on average 87% of all soluble carbohydrates and 67% of total NSC. Large and statistically significant differences were recorded between Sant’ Angelo dei Lombardi and the other two sites. Other soluble sugars found in measurable quantities in all samples were glucose, fructose and raffinose, but these were in low amounts (close to 1% of the DM as a total) (Fig. 1B). Galactose was found in measurable quantities, but lower than 0.1%, only
Fig. 3. (A) Xylose, Arabinose and Galactose as main hemicellulose monomers in rhizomes as % DM across each production site, (B) Relative abundances of Xylose, Arabinose and Galactose in hemicellulose a % across each production site. For each component, values (bars) with different letters were statistically different, among sites, for P = 0.05. Values (bars) with ns indicate that the F test for the ANOVA was not significant for P = 0.05.
in samples from Sant’ Angelo dei Lombardi (data not shown). Starch content was relevant and variable among sites, it was lower than 4% of the DM in Acerra, almost twice and three times that amount in Bellizzi and in Sant’ Angelo dei Lombardi respectively (Fig. 3 B) and hence it accounted for the 12.7% of total NSC in Acerra, 23.2% in Bellizzi and
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Table 3 Composition of Arundo donax rhizomes (% of DM). Content of ash, extractives, and structural cell wall component in rhizomes. Data are calculated as the % of the total dry matter of rhizomes. Values represent the mean value of six replicates ± standard deviation. On the same row, values with different letters were statistically different for P = 0.05. Rows with no letters indicate that the F test for the ANOVA was not significant for P = 0.05. Component
Experimental Farms Acerra (6 years)
Bellizzi (7 years)
S.Angelo dei Lombardi (11 years)
Ash
7.38 ± 0.11a
5.89 ± 0.21b
4.97 ± 0.16c
Water Extractives Ethanol Extractives Total Extractives
29.02 ± 1.81 3.59 ± 0.20 32.61 ± 1.95
33.47 ± 1.84 3.16 ± 0.33 36.64 ± 1.65
31.59 ± 0.57 2.98 ± 0.21 34.57 ± 0.41
Lignin
Soluble Klason Total
1.24 ± 0.03b 19.05 ± 0.28a 20.29 ± 0.28a
1.38 ± 0.07b 17.78 ± 0.33 ab 19.16 ± 0.32ab
1.62 ± 0.03a 17.10 ± 0.67b 18.71 ± 0.67b
Structural Carbohydrate
Cellulose Hemicellulose Total
28.02 ± 0.67a 13.89 ± 0.16a 41.92 ± 0.70a
23.47 ± 0.53b 11.94 ± 0.28b 35.41 ± 0.66b
22.42 ± 0.33b 11.10 ± 0.43b 33.52 ± 1.68b
these aspects deserve more studies. It is remarkable that the abundance of NSC was a very conservative trait of the rhizome biomass, irrespective of the quantity of specific carbohydrates and of the productivity of the site, its age and rhizome biomass accumulation. This aspect is relevant in terms of reliability of the quality characteristics of the rhizome biomass. For the biorefinery industry, stable quality characteristics of the feedstock is a relevant end very valuable trait.
39.8% in Sant’Angelo dei Lombardi. A significant inverse relationship was evident between sucrose and starch contents in rhizomes (Fig. 1B insert). In the site of Sant’ Angelo dei Lombardi sucrose and starch amounts were almost similar (starch/sucrose = 0.87) while the ratio among them decreased to 0.44 and to 0.15 in Bellizzi and Acerra, respectively. Similarly, other soluble carbohydrates (glucose, fructose and raffinose) were low, in relative terms, where sucrose was high. Because of the reciprocal changes of sucrose VS starch and of sucrose VS other soluble sugars, differences in the concentration of total soluble carbohydrates and of total NSC among different sites where much smaller, albeit still statistically significant, than the differences among single carbohydrates. Particularly relevant is the occurrence of raffinose, a trisaccharide with physiological role in plant response to abiotic stresses like cold, desiccation and water stress [37], and with a potential market value several times higher than that of sucrose glucose and fructose. Decruyenaere and Holt [38] reported the presence of raffinose in rhizomes of A. donax on samples collected from natural stands. This is the first time that appreciable amounts of raffinose (0.46%, 0.21% and 0.14% of the DM content in Sant’Angelo dei Lombardi, Bellizzi and Acerra, respectively) are quantified in A. donax rhizome biomass produced with dedicated crops. Taking into account the high rhizome biomass produced, the total raffinose present in rhizome was more than 100 kg ha−1 for all sites and considering its high commercial value raffinose can be a target for biorefinery. The concentration of raffinose can change depending on the season and physiological status of the plant. Hence, further studies are needed to elucidate the role of raffinose for A. donax stress tolerance and to investigate potential ways to both increase its content and set up sustainable and profitable ways to extract and purify it from the biomass of A. donax crops. Rhizomes are storage organs that function as both a sink and source of photo-assimilates to buffer production and use of carbohydrates at the plant level. In storage organs of plants, sugar composition changes during the year in relation to the photo-assimilate supply or demand, growth phase, stress conditions, and/or environmental parameters [22,39]. For instance, chilling acclimation and the establishment of freezing resistance promote inter-conversion of different carbohydrates [40,41]. Furthermore, carbohydrate reserves remobilization resupplies for spring sprouting. In this study, rhizomes were harvested during the winter resting period, so we can infer that reserves were at their maximum level. We demonstrated that rhizomes of A. donax, from cultivated stands, even when productivity of the site spans among large boundaries, accumulate a very large amount of easily fermentable sugars that include sucrose, starch and minor quantities of glucose, fructose, raffinose and, occasionally, galactose. Changes in relative amounts of the different NSC depend on seasonality and physiology and
3.3. Other components of the rhizomes The composition of rhizome (structural components) was similar to the composition of the aerial plant biomass, considering the variability of analytical methods and of biomass production systems reported in the literature [18,42,43] with the exception of ashes and extractives. Ash content in rhizomes was statistically different among the three plantation sites. It was higher in Acerra than in Bellizzi where it was in turn higher than in Sant’Angelo dei Lombardi. Interestingly the highest ash content was in rhizomes of the most productive site of Acerra. The content of ashes found in rhizomes was higher, on average, than that normally found on A. donax aboveground biomass [3,44], particularly when leaves are excluded [45,46]. Nassi o Di Nasso [19] found that rhizomes recover mineral nutrients from the aerial plant parts in the late phases of the growing season, store them over the winter and remobilize them as required in the following growing season. The mineral nutrient storage function of A. donax rhizomes justifies the relatively high content of ashes and the positive link with productivity shown in the site of Acerra in comparison with the other two. A high ash content can be a negative trait for biorefinery, but this depends largely on the scheme adopted. Extractives were very high in rhizomes from all three sites, with the water fraction accounting for more than 90% of the total extractives. This is in line with the very high content of soluble carbohydrates of rhizomes and shows that rhizomes do not accumulate large amount of fatty storage compounds. The amount of extractive found in rhizome was higher than that reported for the aboveground biomass of A. donax biomass [16,45,47], and of other species commonly used as feedstock for bioenergy production [48,49]. Only in the case of sugarcane, extractives are higher than in A. donax rhizomes (about 47%) [49]. Statistically significant differences were recorded among production sites for all the structural (cell wall) components in rhizomes (Table 3). However, it should be noted that data in Table 3 are calculated as percentage of the total dry biomass of the rhizomes. NSC are a labile component, scarce in the aboveground biomass, that can change with seasonality and physiological status. Hence, we have also calculated the percentage of the structural components in rhizome with reference to the total amount of DM after subtracting the amount of NSC (Table 4). 195
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Table 4 Relative composition of Arundo donax rhizomes (% of DM). Relative content of ash, lignin, hemicellulose and cellulose. Data are calculated as the % of the total dry matter of rhizomes after the subtraction of the non-structural carbohydrates present on the materials characterized. On the same row, values with different letters were statistically different for P = 0.05. Rows with no letters indicate that the F test for the ANOVA was not significant for P = 0.05. Component
Experimental Farms
Ash
Acerra (6 years)
Bellizzi (7 years)
S. Angelo dei Lombardi (11 years)
10.66 ± 0.05 a
9.05 ± 0.41 b
7.49 ± 0.35 c
Lignin
Soluble Insoluble Total
1.80 ± 0.05 c 27.58 ± 0.44 29.39 ± 0.47
2.08 ± 0.09 b 26.88 ± 0.85 28.96 ± 0.85
2.43 ± 0.07 a 25.64 ± 0.60 28.08 ± 0.57
Structural Carbohydrate
Cellulose Hemicellulose Total
42.23 ± 1.65 20.12 ± 0.33 a 62.36 ± 1.88 a
39.23 ± 1.40 18.10 ± 0.77 b 57.33 ± 2.08 ab
39.84 ± 0.66 16.66 ± 0.46 b 56.50 ± 0.85 b
shows that the potential productivity of A. donax should be profoundly revised considering rhizome as a biomass product. Rhizomes would add more than 30% product to the cumulative crop biomass productivity, and even more in terms of fermentable sugars. Hexoses and pentoses are valuable compounds in biomass used for second-generation bioethanol, for biogas production and, more generally, for any biorefinery scheme including bioreactor fermentation. Rhizomes add approximately an amount of 67, 45 and 19 Mg ha−1year−1 of fermentable sugars, (82% as hexoses, on average among the three sites) to the productivity of Acerra, Bellizzi and Sant’Angelo dei Lombardi. This would be a very relevant proportion of the total fermentable sugars output of the A. donax crop, it would increase its economic relevance and ameliorate its energy and CO2 balance. The possibility to recover raffinose from rhizomes should be carefully considered, since it has prebiotic characteristics [52] and hence possibly high market value. In conclusion we showed here that, looking at the belowground biomass could merit positive insight on how to increase the potential productivity of poly-annual rhizomatous grasses and possibly their environmental and economical performances.
This is a necessary exercise in this case, to compare the composition of rhizomes with that of published aboveground components. After “depuring” values from the influence of the NSC, statistically significant differences are recorded only for the amounts of soluble lignin, and hemicelluloses (compare Tables 3 and 4). Soluble lignin is higher in the rhizome of Sant’ Angelo dei Lombardi and lower in those of Acerra. However, the insoluble lignin content does not vary among sites and the same occurs for the total lignin content. Opposite differences are recorded with respect to hemicelluloses whose content is higher in Acerra than in the other two sites. Since the content of cellulose is not affected by the site of cultivation, the total content of cellulose plus hemicellulose does not show statistically significant differences among sites. Xylose is the most abundant monomer in the rhizome hemicellulose, accounting for more than 70% of the total, followed by arabinose and galactose. Xylose and arabinose absolute values where significantly higher in the rhizomes of Acerra than in those of the other two sites. However, this was not caused by a change in the hemicellulose composition as shown by similar relative abundance, among monomers, in the different sites (Fig. 3B). To our knowledge, this is the first accurate analysis of the composition of A. donax rhizomes obtained from mature cultivated stands. For this reason, we can only compare our cell wall component analysis data with those available for the aboveground biomass, which are surprisingly variable in the literature [3,50]. This can be due to factors such as the time of harvest (vegetative VS resting period) [51], the presence or absence of leaves [46], and, amongst others, different measuring procedures. Hence, taking into due consideration the variability of published data, we can observe that the structural components abundance in A. donax rhizome is quite similar to the most frequently reported values in aboveground biomass. In particular, considering the data referred to DM “depured” of NSC, hemicellulose content and xylose abundance were in the lower range, while cellulose and lignin contents were in the higher range of published data referred to aboveground A. donax biomass [15]. The structural components of rhizome are similar to that of several lignocellulosic biomasses indicated as suitable for biorefinery.
Acknowledgments This work was supported by grant from the Ministero dell’Università e della Ricerca Scientifica—Industrial research project “Development of green technologies for production of BIOchemicals and their use in preparation and industrial application of POLImeric materials from agricultural biomasses cultivated in a sustainable way in Campania region (BioPoliS)” grant number: PON03PE_00107_1, funded in the frame of Operative National Programme Research and Competitiveness 2007–2013 D. D. Prot. N. 713/Ric. 29/10/2010, and by the Ministero Italiano delle Politiche Agricole Alimentarie e Forestali, Project: “EFFBIOETA2 – Bioetanolo di II generazione da biomasse italiane: qualità del feedstock, efficienza di conversione ottimizzazione d'uso dei residui”. We also express our thanks to Peter Downey for his revision of English language and to Roberto Maiello, Enzo Leone and Eugenio Cozzolino for their assistance in the hard work to collect rhizomes.
4. Conclusions
Appendix A. Supplementary data
In this study, for the first time, the detailed composition of rhizomes harvested from mature cultivated stands of A. donax was analyzed together with their quantity. We found that A. donax rhizomes accumulate a very high amount of easily fermentable NSC, mainly in the forms of sucrose and starch. We also showed that accumulation of large amounts of NSC in rhizome during winter resting is a highly conservative trait at least in poly-annual stands cultivated for the production of biomass in the Mediterranean area. The combination of the high amount of biomass in rhizome and of their high quality composition in terms of fermentable carbohydrates
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Research paper
Chemical composition and yield of rhizome biomass of Arundo donax L. grown for biorefinery in the Mediterranean environment
MARK
Simona Proiettia, Stefano Moscatelloa, Massimo Fagnanob,∗∗, Nunzio Fiorentinob, Adriana Impagliazzob, Alberto Battistellia,∗ a b
Institute of Agro-Environmental and Forest Biology (IBAF), National Research Council (CNR), via Marconi 2, 05010, Porano, TR, Italy Department of Agricultural Sciences, University of Naples Federico II, Via Università 100, 80055, Portici, Naples, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords: Giant reed Biofuel Non-structural carbohydrate Cellulose Hemicellulose Lignin
The contribution of the rhizome to productivity of fermentable sugars and the detailed composition of rhizomes were analyzed in three mature stands of Arundo donax L. cultivated in three locations of variable fertility in the South of Italy. Although the average yearly aboveground dry biomass and rhizome amount showed large and significant differences among sites, (15.3 and 2.6 Mg ha−1 year−1 of rhizomes in the most and less productive sites respectively), rhizomes of all sites had more than 30% of the dry matter (DM) as non-structural carbohydrates (NSC). Sucrose and starch were the most abundant NSC but measurable amounts of glucose, fructose, galactose and of the valuable trisaccharide raffinose were also present. The amount of NSC in rhizomes affected their content of dry mater, and water extractives. The ash content also varied significantly among cultivation sites; the highest amount was recorded in rhizomes of the most productive site (Acerra). The abundance in cell wall components of rhizomes was similar to that of published values for the above ground biomass. The present results demonstrate that NSC content in rhizomes of mature stands is a conserved trait. Hence, rhizome biomass, thanks to its quantity and high fermentable sugars content, should be considered as a relevant fraction of the A. donax crop product whose utilization can increase the productivity and the environmental fingerprint of this crop, in view of its biomass utilization in biorefinery.
1. Introduction The implementation of biorefinery plants is only possible with an adequate supply of feedstock that do not compete with food production systems. This has fostered research on species able to grow in area were food production is not a benchmark. Arundo donax L., is one of the species that has recently received attention as a potential biomass producer in low input non-food systems for the temperate and hot zones. A. donax is a perennial rhizomatous wetland grass, widespread all over the world; it shows a very high adaptability to abiotic and biotic stresses as drought, flood [1], salinity [2] and pests, maintaining an elevated potential for biomass yield [3]. A. donax growth is very fast during spring and summer in Mediterranean environments, resulting in the accumulation of large amounts of biomass even with minimal agricultural input such as irrigation, fertilization and phytosanitary treatments [4,5]. This plant could be suitable also for the cultivation of marginal areas, where the use of other species is disadvantageous. Trials carried out in Italy have shown that A. donax could be a very
good feedstock for bioenergy production due to high yield potential [6,7]. Aboveground average dry biomass yield of 38 Mg ha−1 year−1 was recorded during growth from year 2–12 after planting [6], while Mantineo et al. [5] reported about the same production from the first three years of A. donax growth. A. donax was also able to grow in degraded or contaminated soils [8–10] or soils subjected to accelerated erosion [11]. This species was shown to have a favorable environmental impacts in marginal lands, due to carbon storage within the soil [12, 13; it was also suggested as being suitable in projects of phytoremediation of polluted soils [14]. For all of these advantages, giant reed is considered a very interesting non-food crop as source of biomass for the production of secondgeneration biofuel, bioenergy and other high value products for the chemical industry [6,15–18] with no competition for land with food crops. This has fostered research and improved the knowledge regarding yield and chemical characteristics of aboveground part of plant. However, most studies examining the potential of A. donax as a biomass producer for biorefinery have taken into account only the aboveground
Abbreviations: DM, Dry matter; NSC, Non-structural carbohydrates ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (M. Fagnano), [email protected] (A. Battistelli). http://dx.doi.org/10.1016/j.biombioe.2017.10.003 Received 10 March 2017; Received in revised form 29 September 2017; Accepted 6 October 2017 0961-9534/ © 2017 Elsevier Ltd. All rights reserved.
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Aerial biomass was harvested every year at the end of winter and dried at 70 °C to constant weight. Underground biomass samples were collected during winter, from sample areas of 1 m2, washed, weighted and dried at 70 °C until to constant weight. Two subsamples of 1 kg were collected from each plot (3 replicates x 3 sites), immediately frozen with dry ice, and sent to the laboratory for the chemical characterization.
biomass production [16]; with the exception of Nassi o Di Nasso et al. [19] who quantified also rhizome biomass accumulation. Rhizomes are modified stems that grow perpendicularly to the gravitational force, normally underground, and can produce stems and root from nodes, function as storage sites of reduced carbon and mineral nutrients, and affect plant survival under stressful conditions [19–21]. A. donax can produce large quantities of rhizomes that represent a relevant portion of its total crop biomass. Nassi o Di Nasso et al. [19] showed a rhizome dry biomass yield of 16 Mg ha−1year−1, for a three years old crop. Giant reed translocates nutrients to the rhizome at the end of the growing season [22], and remobilize them for regrowth of the new shoot at the beginning of the vegetation season, reducing fertilizers requirements. A number of studies have been carried out to estimate the seasonal dynamics of above and belowground resources allocation in rhizomatous perennial grasses such as Miscanthus, Panicum and Arundo [23,24], but the focus of existing studies was mineral and nitrogen partitioning. The present study is of particular importance as the carbohydrate content and partitioning in addition to the cell wall chemical characteristics of A. donax rhizome, similar to other rhizomatous grasses, are lacking. A. donax plantations can produce biomass for as long as 10–15 years, thereafter the yield declines dramatically and the crop stand must be removed. Upon removal of a giant reed plantation, or eventually with partial harvests during the plantation period, the rhizome could be used as a feedstock for bioenergy production or as a raw material for green chemistry applications, e.g. for the production of molecules of interest such as xylobiose and tetra- and penta-saccharides [25]. The aims of this work were: i) to evaluate the contribution of rhizome to total biomass yield by stands cultivated in sites of the Mediterranean area of different fertility; ii) to evaluate the quality of such rhizome biomass in terms of NSC (fermentable carbohydrates) and structural components. Our work highlights the potential role of rhizome biomass in increasing productivity of fermentable sugars; hence affecting the profitability and environmental fingerprint of A. donax cultivation for green chemistry and helping to improve the sustainability of biomass based agro-industrial systems in the Mediterranean area.
2.2. Analysis of NSC Soluble carbohydrates (glucose, fructose, sucrose, raffinose) and starch in the biomass were extracted for analysis as in Moscatello et al. [27]. In brief, 10 mg of dry material was extracted in 1 ml of 50% ethanol/water at 80 °C for 45 min. Soluble sugars were recovered in the supernatant after centrifugation at 16000g for 5 min. Starch in the pellet was hydrolyzed enzymatically to glucose after washing and autoclaving. The supernatant containing soluble carbohydrates, and the glucose solution obtained after starch hydrolysis were filtered on 0.2 μm nylon filters (GE-Whatman, Maidstone, UK), then analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Thermo Scientific™ Dionex™ ICS5000, Sunnyvale, CA U.S.A.) consisting of a dual pump system (quaternary analytical pump plus isocratic pump), a pulsed amperometric detector, and an analytical CarboPac PA20 column (3 mm × 150 mm) with guard column. The detection cell contained a gold working electrode (1.0 mm in diameter) and an Ag/AgCl reference electrode. Pulsed amperometric detection was carried out with the following waveform: E1 = +0.10 V (t1 = 0.4 s), E2 = −2.00 V (t1 = 0.01 s), E3 = +0.60 V (t = 0.01 s), E4 = −0.10 V (t = 0.06 s). The integration range began at 0.2 s and ended at 0.4 s. The electrical signal was integrated in nanocoulomb (nC). Runs were carried out at 30 °C using an injection valve with a 5 μl injection loop. Mobile phase was NaOH at a flow rate 0.5 ml min−1. Sugars were eluted under the following conditions: 0–12 min, 10 mM; 12–25 min, 80 mM; 25–39 min, 240 mM; 39–55 min, 10 mM. Regeneration of amperometric electrode was done with a post-column addition of concentrated sodium hydroxide (300 mM) using a secondary pump at a flow rate of 0.25 ml min−1. The carbohydrate standard solutions were prepared using HPLC grade reagents (Sigma, Steinheim, Germany).
2. Materials and methods
2.3. Analytical methods for the determination of chemical composition of rhizome
2.1. Biomass sampling Giant reed rhizomes were collected from 3 experimental fields located in marginal land areas of the Campania Region, Southern Italy: Acerra, (40° 59′N, 14° 20′E, 26 m a.s.l.), from a 6 year stand (2009–2014) in polluted soils of a Vesuvius plain; Sant’Angelo dei Lombardi (40° 55′ N, 15° 07′ E, 700 m a.s.l.) from an 11 year stand (2004–2014) in a hilly area of Southern Apennine subjected to accelerate erosion; and Bellizzi (Torre Lama) (40° 37′N, 14° 58′E, 30 m a.s.l), from a 7 year stand (2008–2014) in a coastal plain subjected to soil salinization. Details of cropping systems are reported in Fiorentino et al. [9], Fagnano et al. [11] and Impagliazzo et al. [26], respectively. In all sites the same low input cropping system was adopted with low doses on N fertilizers and no irrigation. Physico-chemical characteristics of the soils of the three locations are reported in Table 1.
Chemical analyses were performed in accordance to the standard biomass analytical methods provided by the National Renewable Energy Laboratory (NREL) as detailed in Santi et al. [28]. The collected samples were milled with a MF10 IKA mill (Werke GmbH & Co. KG) and sieved to screen particles until 500 μm in size. After milling, the samples were oven dried for 24 h at 45 °C and then used for the determination of the biomass chemical composition. In brief, the relative moisture was determined by placing the samples at 105 °C for 24 h in a ventilated oven. The ash content was quantified after ignition of dried samples at 575 ± 25 °C for 24 h using a muffle furnace equipped with a ramping program (Nabertherm P300 Germany). A two-step extraction was used to quantify extractives in the rhizome biomass samples. Approximately 1.5 g of dried sample was placed in cellulose extraction thimbles, 100 mm length by 25 mm diameter (GE-Whatman, Maidstone, UK). The first step was performed with water, by extraction in 25 ml of deionized water at 50 °C for 30 min. This step was repeated 4 times to a final extraction volume of 100 ml for each sample. The second step was performed, using 100% ethanol on the water insoluble material, following the same procedure described for the first step. Water and ethanol extracts were quantified gravimetrically; the water soluble components were freeze-dried, while the ethanol extractable material was dried using a rotavapor (VV 2000 Heidolph Instruments, Schwabach, Germany).
Table 1 Soil characteristics in the tree cultivation sites. N-tot, total nitrogen; SOM, soil organic matter; N-NO3, nitric nitrogen; N-NH4, ammonia nitrogen.
Acerra Bellizzi S. Angelo dei Lombardi
Sand %
Silt %
Clay %
N-tot %
SOM %
N−NO3 ratio N−NH4
pH
55.7 34.2 36.9
27.5 26.1 24.6
16.7 39.6 38.0
0.16 0.09 0.08
2.64 1.57 1.03
1.25 0.55 0.40
7.7 7.8 8.2
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Yearly aboveground biomass productivity reported in recent studies spans from less than 10 Mg ha−1 year−1 to more than 40 Mg ha−1 year−1 [1,3]. Our stands grew on site with medium to high productivity. The Acerra site, located in the Vesuvius plain, has a high content of minerals and organic matter [8]. Its above ground productivity was high and similar to the best recorded values at national and international level [3]. The aboveground productivity at Bellizzi, in the coastal plain at risk of salinization, was relatively low which is not surprising because of the low soil content of mineral and organic matter at this site [26]. The relatively low aboveground biomass productivity recorded at Sant’Angelo dei Lombardi can be attributed to the severe climatic conditions of this internal hilly area of the Southern Apennine, [11]. These values were lower than the ones measured in other field experiments in Italy [6] but similar to results from experiments in marginal lands [30]. Literature data on belowground biomass accumulation in A. donax is particularly scarce. It is known that roots of A.donax can reach very deep soil layers and be abundant even at 1 m in depth [31]. Sánchez et al. [32,33] found that shoot/root ratio can be affected by environmental stresses but did not quantify the absolute effect on rhizome biomass. Nassi o Di Nasso et al. [19] found that in marginal lands of Central Italy, young stands increases the partitioning of biomass in rhizomes over the years reaching about 16 Mg ha−1of DM allocated in rhizome at the end of the third year. On an yearly basis this value would be larger than the one we recorded in the less productive site of Sant’Angelo dei Lombardi (average of 11 years of accumulation) but similar to that recorded at Bellizzi (average of 7 years of accumulation) and one third of what we recorded in the more fertile site of Acerra (average of 6 years of accumulation. In the same study [19] it was shown that the ratio of below and aboveground biomass can change with stand age with values close to 2, at the end on the first growing year, and values of 0.7 and 0.8 at the end of the second and third year. The rhizome/aerial biomass ratio measured in the sites of Acerra and Bellizzi is consistent, although slightly lower, with that shown for the second and third year of growth, by Nasso o Di Nasso et al. [19] while that of the less productive site of Sant’Angelo dei Lombardi is largely lower. It is known that rhizomes in perennial plants contribute to nutrient and carbohydrate balance at the plant level [34] by accumulating resources during the mid and late vegetative season, and re-supplying them to the aerial part of the plant to support spring sprouting [22]. This function is controlled at the genetic and environmental level, can be regulated by multiple factors [35] and is of particular importance for re-growth and productivity of rhizomatous crops undergoing aboveground sequential harvesting [21]. Nassi o di Nasso et al. [19], showed that there is a linear relationship, in a young A. donax stand, between the dry weight of rhizomes and the number of buds, hence the low rhizome biomass in the Sant’ Angelo dei Lombardi stand can be linked to its low productivity. Our data confirms the abundant accumulation of DM in rhizome of mature stands, and shows that this phenomenon is affected by general conditions, such as the age of the stand and the fertility of the site. We also show that not only biomass accumulation is (in absolute terms) variable, but also allocation (in relative terms among different plant parts) can be strongly affected by stand age and
Lignin, hemicellulose and cellulose content was determined in extractive free material following NREL protocol (TP-510-42618 [29]) with 72% H2SO4 treatment at 30 °C for 1 h and subsequently using 4% H2SO4 treatment at 121 °C for 1 h in an autoclave. After the two-step acid hydrolysis, the autoclaved solution was vacuum filtered using a calibrated glass filter (GF/A 55 mm diameter GE-Whatman, Maidstone, UK). In the hydrolyzed liquor the lignin is fractionated into acid soluble lignin and acid insoluble material (Klason lignin). The Klason lignin was determined gravimetrically from the solid residue on the glass filter after drying at 105 °C for 24 h. In addition, the acid soluble lignin was measured using UV–Vis spectrophotometer (Jenway 6715 Bibby Scientific) at 205 nm, with the extinction coefficient of 110 l g−1cm−1. The filtrate was subsequently analyzed for monosaccharides resulting from the acid hydrolysis (glucose, arabinose, galactose, and xylose). The monomeric sugars were analyzed by high-performance anion exchange chromatography, with pulsed amperometric detection as described for the analysis of non-structural carbohydrate, with an analytical CarboPac SA10 column (4 mm × 250 mm) with the guard column. Runs were carried out at 45 °C. NaOH (1 mmol l−1) was used as mobile phase at a flow rate 1.0 ml min−1 with a post-column addition of concentrated NaOH (300 mmol l−1). The post-column addition was made using a secondary pump, at a flow rate 0.5 ml min−1. The eluents and the carbohydrate standard solutions were prepared using HPLC grade reagents (Sigma, Steinheim, Germany). The IC method required minimum sample preparation and the analytes were separated without interferences. Hemicellulose content was determined as the sum of arabinose, galactose, and xylose amount, while cellulose was quantified on the value of glucose corrected for the starch content in the samples. 2.4. Statistical analysis The parameters of the chemical composition of biomass from Arundo rhizome were analyzed by One-Way analysis of variance (ANOVA) with the crop site as the random factor. Differences between averages were tested by an LSD test for a significance level of P = 0.05. Regression analyses between the percentage of DM and NSC, and between the percentage of sucrose and starch were carried out estimating the correlation by the Pearson coefficient (r) for a significance level of P = 0.05. All statistical analyses were done by ANOVA using the STATISTICA 8 software package (StatSoft for Windows 1998). 3. Results and discussion 3.1. Contribution of rhizome quantity to total biomass yield Below and above ground biomass production and partitioning was significantly different in the three experimental fields (Table 2). Acerra was by far the most productive site in terms of both below and aboveground cumulated and yearly biomass production. Bellizzi overcame Sant’ Angelo dei Lombardi in terms of belowground accumulation capacity but not in terms of yearly aboveground biomass accumulation. Belowground biomass was more than one third of the total biomass accumulated in the Acerra and Bellizzi sites. Sant’Angelo dei Lombardi was the site with the lowest partitioning of biomass in rhizomes.
Table 2 Production parameters. Relevant production parameters of Arundo donax stands cultivated in the Campania Region. Values in bracket close to the name of the site indicate the age of the stand at the time of harvest. Values in bracket below data are standard errors. On the same columns, values with different letters were statistically different for P = 0.05. Columns with no letters indicate that the F test for the ANOVA was not significant for P = 0.05. Experimental sites (age of crop)
Rhizome yield (DM Mg ha−1)
Rhizome yield per year (DM Mg ha−1 year−1)
Cumulated aerial biomass yield (DM Mg ha−1)
Aerial yield per year (DM Mg ha−1 year−1)
Rhizome/aerial biomass ratio
Acerra (6 years) Bellizzi (7 years) S. Angelo dei Lombardi (11 years)
91.9 ± 6.9 a 64.2 ± 1.3 b 29.0 ± 4.2
15.32 9.17 2.64
168.8 ± 7.3 a 101.1 ± 6.5 b 170.2 ± 20.8 a
28.10 14.44 15.47
0.55 ± 0.05 a 0.64 ± 0.05 a 0.17 ± 0.00 b
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Fig. 2. Dry Matter (%) measured from each site with the correlation between NSC (%) and DM (%) inlaid. On the same row, values with different letters were statistically different for P = 0.05. Values (bars) with different letters were statistically different, for P = 0.05. Regression was statistically significant for P = 0.05.
Fig. 1. (A) NSC and Total Carbohydrates as % DM at each production site, (B) Sucrose, Starch, Glucose, Fructose, Raffinose as % DM from each site, with an inverse correlation between Sucrose and Starch as % DM inlaid. For each component, values (bars) with different letters were statistically different, among sites, for P = 0.05. Regression was statistically significant for P = 0.05.
site fertility. The fact that rhizome biomass can strongly increase the total biomass production of the crop suggests to include this information in models of A. donax economic and environmental performances. Rhizome yield data is, at the moment, not considered in the economic profitability evaluation [36] nor in the Life Cycle Assessment of this crop [12,13], but its inclusion in future analyses would guarantee a more complete evaluation of the economic and environmental sustainability of the giant reed crop. Finally, more research is needed to optimize crop lifetime and management in order to maximize the total biomass yield considering both reeds and rhizomes.
3.2. NSC in rhizomes NSC were the highest component fraction in the rhizomes. The total amount of NSC was on average 33% of the rhizome DM. Statistically significant differences were recorded among sites; however, their content was higher than 30% of the DM even in Acerra, the site with the lowest amount of NSC. (Fig. 1A). The high NSC content affected directly and positively the DM content of rhizomes (Fig. 2, insert). Sucrose was the main NSC in rhizome (Fig. 1B), representing on average 87% of all soluble carbohydrates and 67% of total NSC. Large and statistically significant differences were recorded between Sant’ Angelo dei Lombardi and the other two sites. Other soluble sugars found in measurable quantities in all samples were glucose, fructose and raffinose, but these were in low amounts (close to 1% of the DM as a total) (Fig. 1B). Galactose was found in measurable quantities, but lower than 0.1%, only
Fig. 3. (A) Xylose, Arabinose and Galactose as main hemicellulose monomers in rhizomes as % DM across each production site, (B) Relative abundances of Xylose, Arabinose and Galactose in hemicellulose a % across each production site. For each component, values (bars) with different letters were statistically different, among sites, for P = 0.05. Values (bars) with ns indicate that the F test for the ANOVA was not significant for P = 0.05.
in samples from Sant’ Angelo dei Lombardi (data not shown). Starch content was relevant and variable among sites, it was lower than 4% of the DM in Acerra, almost twice and three times that amount in Bellizzi and in Sant’ Angelo dei Lombardi respectively (Fig. 3 B) and hence it accounted for the 12.7% of total NSC in Acerra, 23.2% in Bellizzi and
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Table 3 Composition of Arundo donax rhizomes (% of DM). Content of ash, extractives, and structural cell wall component in rhizomes. Data are calculated as the % of the total dry matter of rhizomes. Values represent the mean value of six replicates ± standard deviation. On the same row, values with different letters were statistically different for P = 0.05. Rows with no letters indicate that the F test for the ANOVA was not significant for P = 0.05. Component
Experimental Farms Acerra (6 years)
Bellizzi (7 years)
S.Angelo dei Lombardi (11 years)
Ash
7.38 ± 0.11a
5.89 ± 0.21b
4.97 ± 0.16c
Water Extractives Ethanol Extractives Total Extractives
29.02 ± 1.81 3.59 ± 0.20 32.61 ± 1.95
33.47 ± 1.84 3.16 ± 0.33 36.64 ± 1.65
31.59 ± 0.57 2.98 ± 0.21 34.57 ± 0.41
Lignin
Soluble Klason Total
1.24 ± 0.03b 19.05 ± 0.28a 20.29 ± 0.28a
1.38 ± 0.07b 17.78 ± 0.33 ab 19.16 ± 0.32ab
1.62 ± 0.03a 17.10 ± 0.67b 18.71 ± 0.67b
Structural Carbohydrate
Cellulose Hemicellulose Total
28.02 ± 0.67a 13.89 ± 0.16a 41.92 ± 0.70a
23.47 ± 0.53b 11.94 ± 0.28b 35.41 ± 0.66b
22.42 ± 0.33b 11.10 ± 0.43b 33.52 ± 1.68b
these aspects deserve more studies. It is remarkable that the abundance of NSC was a very conservative trait of the rhizome biomass, irrespective of the quantity of specific carbohydrates and of the productivity of the site, its age and rhizome biomass accumulation. This aspect is relevant in terms of reliability of the quality characteristics of the rhizome biomass. For the biorefinery industry, stable quality characteristics of the feedstock is a relevant end very valuable trait.
39.8% in Sant’Angelo dei Lombardi. A significant inverse relationship was evident between sucrose and starch contents in rhizomes (Fig. 1B insert). In the site of Sant’ Angelo dei Lombardi sucrose and starch amounts were almost similar (starch/sucrose = 0.87) while the ratio among them decreased to 0.44 and to 0.15 in Bellizzi and Acerra, respectively. Similarly, other soluble carbohydrates (glucose, fructose and raffinose) were low, in relative terms, where sucrose was high. Because of the reciprocal changes of sucrose VS starch and of sucrose VS other soluble sugars, differences in the concentration of total soluble carbohydrates and of total NSC among different sites where much smaller, albeit still statistically significant, than the differences among single carbohydrates. Particularly relevant is the occurrence of raffinose, a trisaccharide with physiological role in plant response to abiotic stresses like cold, desiccation and water stress [37], and with a potential market value several times higher than that of sucrose glucose and fructose. Decruyenaere and Holt [38] reported the presence of raffinose in rhizomes of A. donax on samples collected from natural stands. This is the first time that appreciable amounts of raffinose (0.46%, 0.21% and 0.14% of the DM content in Sant’Angelo dei Lombardi, Bellizzi and Acerra, respectively) are quantified in A. donax rhizome biomass produced with dedicated crops. Taking into account the high rhizome biomass produced, the total raffinose present in rhizome was more than 100 kg ha−1 for all sites and considering its high commercial value raffinose can be a target for biorefinery. The concentration of raffinose can change depending on the season and physiological status of the plant. Hence, further studies are needed to elucidate the role of raffinose for A. donax stress tolerance and to investigate potential ways to both increase its content and set up sustainable and profitable ways to extract and purify it from the biomass of A. donax crops. Rhizomes are storage organs that function as both a sink and source of photo-assimilates to buffer production and use of carbohydrates at the plant level. In storage organs of plants, sugar composition changes during the year in relation to the photo-assimilate supply or demand, growth phase, stress conditions, and/or environmental parameters [22,39]. For instance, chilling acclimation and the establishment of freezing resistance promote inter-conversion of different carbohydrates [40,41]. Furthermore, carbohydrate reserves remobilization resupplies for spring sprouting. In this study, rhizomes were harvested during the winter resting period, so we can infer that reserves were at their maximum level. We demonstrated that rhizomes of A. donax, from cultivated stands, even when productivity of the site spans among large boundaries, accumulate a very large amount of easily fermentable sugars that include sucrose, starch and minor quantities of glucose, fructose, raffinose and, occasionally, galactose. Changes in relative amounts of the different NSC depend on seasonality and physiology and
3.3. Other components of the rhizomes The composition of rhizome (structural components) was similar to the composition of the aerial plant biomass, considering the variability of analytical methods and of biomass production systems reported in the literature [18,42,43] with the exception of ashes and extractives. Ash content in rhizomes was statistically different among the three plantation sites. It was higher in Acerra than in Bellizzi where it was in turn higher than in Sant’Angelo dei Lombardi. Interestingly the highest ash content was in rhizomes of the most productive site of Acerra. The content of ashes found in rhizomes was higher, on average, than that normally found on A. donax aboveground biomass [3,44], particularly when leaves are excluded [45,46]. Nassi o Di Nasso [19] found that rhizomes recover mineral nutrients from the aerial plant parts in the late phases of the growing season, store them over the winter and remobilize them as required in the following growing season. The mineral nutrient storage function of A. donax rhizomes justifies the relatively high content of ashes and the positive link with productivity shown in the site of Acerra in comparison with the other two. A high ash content can be a negative trait for biorefinery, but this depends largely on the scheme adopted. Extractives were very high in rhizomes from all three sites, with the water fraction accounting for more than 90% of the total extractives. This is in line with the very high content of soluble carbohydrates of rhizomes and shows that rhizomes do not accumulate large amount of fatty storage compounds. The amount of extractive found in rhizome was higher than that reported for the aboveground biomass of A. donax biomass [16,45,47], and of other species commonly used as feedstock for bioenergy production [48,49]. Only in the case of sugarcane, extractives are higher than in A. donax rhizomes (about 47%) [49]. Statistically significant differences were recorded among production sites for all the structural (cell wall) components in rhizomes (Table 3). However, it should be noted that data in Table 3 are calculated as percentage of the total dry biomass of the rhizomes. NSC are a labile component, scarce in the aboveground biomass, that can change with seasonality and physiological status. Hence, we have also calculated the percentage of the structural components in rhizome with reference to the total amount of DM after subtracting the amount of NSC (Table 4). 195
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Table 4 Relative composition of Arundo donax rhizomes (% of DM). Relative content of ash, lignin, hemicellulose and cellulose. Data are calculated as the % of the total dry matter of rhizomes after the subtraction of the non-structural carbohydrates present on the materials characterized. On the same row, values with different letters were statistically different for P = 0.05. Rows with no letters indicate that the F test for the ANOVA was not significant for P = 0.05. Component
Experimental Farms
Ash
Acerra (6 years)
Bellizzi (7 years)
S. Angelo dei Lombardi (11 years)
10.66 ± 0.05 a
9.05 ± 0.41 b
7.49 ± 0.35 c
Lignin
Soluble Insoluble Total
1.80 ± 0.05 c 27.58 ± 0.44 29.39 ± 0.47
2.08 ± 0.09 b 26.88 ± 0.85 28.96 ± 0.85
2.43 ± 0.07 a 25.64 ± 0.60 28.08 ± 0.57
Structural Carbohydrate
Cellulose Hemicellulose Total
42.23 ± 1.65 20.12 ± 0.33 a 62.36 ± 1.88 a
39.23 ± 1.40 18.10 ± 0.77 b 57.33 ± 2.08 ab
39.84 ± 0.66 16.66 ± 0.46 b 56.50 ± 0.85 b
shows that the potential productivity of A. donax should be profoundly revised considering rhizome as a biomass product. Rhizomes would add more than 30% product to the cumulative crop biomass productivity, and even more in terms of fermentable sugars. Hexoses and pentoses are valuable compounds in biomass used for second-generation bioethanol, for biogas production and, more generally, for any biorefinery scheme including bioreactor fermentation. Rhizomes add approximately an amount of 67, 45 and 19 Mg ha−1year−1 of fermentable sugars, (82% as hexoses, on average among the three sites) to the productivity of Acerra, Bellizzi and Sant’Angelo dei Lombardi. This would be a very relevant proportion of the total fermentable sugars output of the A. donax crop, it would increase its economic relevance and ameliorate its energy and CO2 balance. The possibility to recover raffinose from rhizomes should be carefully considered, since it has prebiotic characteristics [52] and hence possibly high market value. In conclusion we showed here that, looking at the belowground biomass could merit positive insight on how to increase the potential productivity of poly-annual rhizomatous grasses and possibly their environmental and economical performances.
This is a necessary exercise in this case, to compare the composition of rhizomes with that of published aboveground components. After “depuring” values from the influence of the NSC, statistically significant differences are recorded only for the amounts of soluble lignin, and hemicelluloses (compare Tables 3 and 4). Soluble lignin is higher in the rhizome of Sant’ Angelo dei Lombardi and lower in those of Acerra. However, the insoluble lignin content does not vary among sites and the same occurs for the total lignin content. Opposite differences are recorded with respect to hemicelluloses whose content is higher in Acerra than in the other two sites. Since the content of cellulose is not affected by the site of cultivation, the total content of cellulose plus hemicellulose does not show statistically significant differences among sites. Xylose is the most abundant monomer in the rhizome hemicellulose, accounting for more than 70% of the total, followed by arabinose and galactose. Xylose and arabinose absolute values where significantly higher in the rhizomes of Acerra than in those of the other two sites. However, this was not caused by a change in the hemicellulose composition as shown by similar relative abundance, among monomers, in the different sites (Fig. 3B). To our knowledge, this is the first accurate analysis of the composition of A. donax rhizomes obtained from mature cultivated stands. For this reason, we can only compare our cell wall component analysis data with those available for the aboveground biomass, which are surprisingly variable in the literature [3,50]. This can be due to factors such as the time of harvest (vegetative VS resting period) [51], the presence or absence of leaves [46], and, amongst others, different measuring procedures. Hence, taking into due consideration the variability of published data, we can observe that the structural components abundance in A. donax rhizome is quite similar to the most frequently reported values in aboveground biomass. In particular, considering the data referred to DM “depured” of NSC, hemicellulose content and xylose abundance were in the lower range, while cellulose and lignin contents were in the higher range of published data referred to aboveground A. donax biomass [15]. The structural components of rhizome are similar to that of several lignocellulosic biomasses indicated as suitable for biorefinery.
Acknowledgments This work was supported by grant from the Ministero dell’Università e della Ricerca Scientifica—Industrial research project “Development of green technologies for production of BIOchemicals and their use in preparation and industrial application of POLImeric materials from agricultural biomasses cultivated in a sustainable way in Campania region (BioPoliS)” grant number: PON03PE_00107_1, funded in the frame of Operative National Programme Research and Competitiveness 2007–2013 D. D. Prot. N. 713/Ric. 29/10/2010, and by the Ministero Italiano delle Politiche Agricole Alimentarie e Forestali, Project: “EFFBIOETA2 – Bioetanolo di II generazione da biomasse italiane: qualità del feedstock, efficienza di conversione ottimizzazione d'uso dei residui”. We also express our thanks to Peter Downey for his revision of English language and to Roberto Maiello, Enzo Leone and Eugenio Cozzolino for their assistance in the hard work to collect rhizomes.
4. Conclusions
Appendix A. Supplementary data
In this study, for the first time, the detailed composition of rhizomes harvested from mature cultivated stands of A. donax was analyzed together with their quantity. We found that A. donax rhizomes accumulate a very high amount of easily fermentable NSC, mainly in the forms of sucrose and starch. We also showed that accumulation of large amounts of NSC in rhizome during winter resting is a highly conservative trait at least in poly-annual stands cultivated for the production of biomass in the Mediterranean area. The combination of the high amount of biomass in rhizome and of their high quality composition in terms of fermentable carbohydrates
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