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Genetic improvement through breeding is one of the key approaches to increasing biomass supply. This paper documents the breeding progress to date for four perennial biomass crops (PBCs) that have high output-input energy ratios: namely Panicum virgatum (switchgrass), species of the genera Miscanthus (miscanthus), Salix (willow) and Populus (poplar). For each crop, we report on the size of germplasm collections, the efforts to date to phenotype and genotype, the diversity available for breeding and on the scale of breeding work as indicated by number of attempted crosses. We also report on the development of faster and more precise breeding using molecular breeding techniques. Poplar is the model tree for genetic studies and is furthest ahead in terms of biological knowledge and genetic resources. Linkage maps, transgenesis and genome editing methods are now being used in commercially focused poplar breeding. These are in development in switchgrass, miscanthus and willow generating large genetic and phenotypic data sets requiring concomitant efforts in informatics to create summaries that can be accessed and used by practical breeders. Cultivars of switchgrass and miscanthus can be seed-based synthetic populations, semihybrids or clones. Willow and poplar cultivars are commercially deployed as clones. At local and regional level, the most advanced cultivars in each crop are at technology readiness levels which could be scaled to planting rates of thousands of hectares per year in about 5 years with existing commercial developers. Investment in further development of better cultivars is subject to current market failure and the long breeding cycles. We conclude that sustained public investment in breeding plays a key role in delivering future mass-scale deployment of PBCs.

Genetic improvement through breeding is one of the key approaches to increasing biomass supply. This paper documents the breeding progress to date for four perennial biomass crops (PBCs) that have high output-input energy ratios: namely Panicum virgatum (switchgrass), species of the genera Miscanthus (miscanthus), Salix (willow) and Populus (poplar). For each crop, we report on the size of germplasm collections, the efforts to date to phenotype and genotype, the diversity available for breeding and on the scale of breeding work as indicated by number of attempted crosses. We also report on the development of faster and more precise breeding using molecular breeding techniques. Poplar is the model tree for genetic studies and is furthest ahead in terms of biological knowledge and genetic resources. Linkage maps, transgenesis and genome editing methods are now being used in commercially focused poplar breeding. These are in development in switchgrass, miscanthus and willow generating large genetic and phenotypic data sets requiring concomitant efforts in informatics to create summaries that can be accessed and used by practical breeders. Cultivars of switchgrass and miscanthus can be seed-based synthetic populations, semihybrids or clones. Willow and poplar cultivars are commercially deployed as clones. At local and regional level, the most advanced cultivars in each crop are at technology readiness levels which could be scaled to planting rates of thousands of hectares per year in about 5 years with existing commercial developers. Investment in further development of better cultivars is subject to current market failure and the long breeding cycles. We conclude that sustained public investment in breeding plays a key role in delivering future mass-scale deployment of PBCs.

The dwindling availability of agricultural land, caused by factors such as rapid population growth, urban expansion, and soil contamination, has significantly increased the pressure on food production. To address this challenge, cultivating non-food crops on contaminated land has emerged as a promising solution. This approach not only frees up fertile soil for food production but also mitigates human exposure to contaminants. This work aimed to examine the impact of soil contamination with Cd, Pb, Ni, and Zn on the growth, productivity, metal accumulation, and the tolerance of five lignocellulosic non-food crops: switchgrass (Panicum virgatum L.), biomass sorghum (Sorghum bicolor L. Moench), giant reed (Arundo donax L.), African fodder cane (Saccharum spontaneum L. spp. aegyptiacum Willd. Hackel), and miscanthus (Miscanthus × giganteus Greef et Deu.). A two-year pot experiment was conducted in Greece, Italy, and Portugal, following the same protocols and applying various levels of metals: Cd (0, 4, 8 mg kg−1), Pb and Zn (0, 450, 900 mg kg−1), and Ni (0, 110, 220 mg kg−1). The experimental design was completely randomized, with three replicates for each treatment. The results showed that switchgrass and sorghum generally maintained their height and productivity under Cd and Pb stress but were adversely affected by high Zn and Ni concentrations. Giant reed and African fodder cane showed reduced height and productivity at higher Ni and Zn levels. Miscanthus exhibited resilience in height but experienced productivity reductions only at the highest Zn concentration. Heavy metal uptake varied among crops, with switchgrass and sorghum showing high Cd and Pb uptake, while giant reed accumulated the most Cd and Zn. Miscanthus had the highest Ni accumulation. The tolerance indices indicated that switchgrass and sorghum were more tolerant to Cd and Zn at lower concentrations, whereas miscanthus had lower tolerance to Cd but a higher tolerance to Zn at higher concentrations. Giant reed and African fodder cane demonstrated stable tolerance across most heavy metals. Accumulation indices highlighted the effectiveness of switchgrass and sorghum in Cd and Pb uptake, while miscanthus excelled in Ni and Zn accumulation. The cluster analysis revealed similar responses to heavy metal stress between African fodder cane and giant reed, as well as between sorghum and miscanthus, with switchgrass displaying distinct behavior. Overall, the study highlights the differential tolerance and accumulation capacities of these crops, indicating the potential for phytoremediation applications and biomass production in heavy metal-contaminated soils. [ABSTRACT FROM AUTHOR]

To date, economically and energy-costly vegetative propagation using rhizomes and tissue culture are the only options for the cultivation of Miscanthus spp. Some genotypes of miscanthus produce fertile seeds, offering a valid alternative to vegetative propagation. A preliminary study has been conducted on the seeds of two hybrids of miscanthus obtained via interspecific cross-pollination between M. sacchariflorus and M. sinensis: 'GRC14' (maternal: M. sacchariflorus) and 'GRC10B' (maternal: M. sinensis). Seeds were assessed for germination traits in a laboratory (at 25 °C in the dark) just after panicle harvest, and during 1-year storage at room temperature or at 8 °C. In a second experiment, the effects of gibberellic acid (GA3) solution at different concentrations (0, 50, 100, 300, 500 ppm) on the germination of freshly matured seeds were assessed. Poor germination just after harvest (<30%) indicates the occurrence of a physiological dormancy. Indeed, two months later, germination rose up to 76.7% in 'GRC14' and 50.8%, in 'GRC10B', and peaked at 95.6% in 'GRC14' and at 78% in 'GRC10B', 6 months after harvest. After a total of 12 months, germination was significantly reduced in both hybrids (≈60%). Seeds stored at room temperature lost dormancy earlier than those stored at 8 °C. Overall, germination was significantly improved by GA3, but the extent of the GA effect was genotype-dependent. In conclusion, a low establishment rate may result from direct seeding when fresh seed is used in the field. In this case, the use of GA3 is a possible strategy to ameliorate the impact of dormancy on seed germination. In the case of delayed sowings in late winter–early spring, seeds stored at room temperature after harvest may better perform than those stored at 8 °C. [ABSTRACT FROM AUTHOR]

Growth in planted areas of Miscanthus for biomass in Europe has stagnated since 2010 due to technical challenges, economic barriers and environmental concerns. These limitations need to be overcome before biomass production from Miscanthus can expand to several million hectares. In this paper, we consider the economic and environmental effects of introducing seed based hybrids as an alternative to clonal M. x giganteus (Mxg). The impact of seed based propagation and novel agronomy was compared with current Mxg cultivation and used in 10 commercially relevant, field scale experiments planted between 2012 and 2014 in the United Kingdom, Germany, and Ukraine. Economic and greenhouse gas (GHG) emissions costs were quantified for the following production chain: propagation, establishment, harvest, transportation, storage, and fuel preparation (excluding soil carbon changes). The production and utilization efficiency of seed and rhizome propagation were compared. Results show that new hybrid seed propagation significantly reduces establishment cost to below £900 ha-1. Calculated GHG emission costs for the seeds established via plugs, though relatively small, was higher than rhizomes because fossil fuels were assumed to heat glasshouses for raising seedling plugs (5.3 and 1.5 kg CO2 eq. C Mg [dry matter (DM)]-1), respectively. Plastic mulch film reduced establishment time, improving crop economics. The breakeven yield was calculated to be 6 Mg DM ha-1 y-1, which is about half average United Kingdom yield for Mxg; with newer seeded hybrids reaching 16 Mg DM ha-1 in second year United Kingdom trials. These combined improvements will significantly increase crop profitability. The trade-offs between costs of production for the preparation of different feedstock formats show that bales are the best option for direct firing with the lowest transport costs (£0.04 Mg-1 km-1) and easy on-farm storage. However, if pelleted fuel is required then chip harvesting is more economic. We show how current seed based propagation methods can increase the rate at which Miscanthus can be scaled up; ∼×100 those of current rhizome propagation. These rapid ramp rates for biomass production are required to deliver a scalable and economic Miscanthus biomass fuel whose GHG emissions are ∼1/20th those of natural gas per unit of heat. [ABSTRACT FROM AUTHOR]

Field trials in Europe with Miscanthus over the past 25 years have demonstrated that interspecies hybrids such as M. × giganteus ( M × g) combine both high yield potentials and low inputs in a wide range of soils and climates. Miscanthus hybrids are expected to play a major role in the provision of perennial lignocellulosic biomass across much of Europe as part of a lower carbon economy. However, even with favourable policies in some European countries, uptake has been slow. M × g, as a sterile clone, can only be propagated vegetatively, which leads to high establishment costs and low multiplication rates. Consequently, a decade ago, a strategic decision to develop rapidly multiplied seeded hybrids was taken. To make progress on this goal, we have (1) harnessed the genetic diversity in Miscanthus by crossing and progeny testing thousands of parental combinations to select several candidate seed-based hybrids adapted to European environments, (2) established field scale seed production methods with annual multiplication factors >1500×, (3) developed the agronomy for establishing large stands from seed sown plug plants to reduce establishment times by a year compared to M × g, (4) trialled a range of harvest techniques to improve compositional quality and logistics on a large scale, (5) performed spatial analyses of yield potential and land availability to identify regional opportunities across Europe and doubled the area within the bio-climatic envelope, (6) considered on-farm economic, practical and environmental benefits that can be attractive to growers. The technical barriers to adoption have now been overcome sufficiently such that Miscanthus is ready to use as a low-carbon feedstock in the European bio-economy. [ABSTRACT FROM AUTHOR]

A general obstacle to the development of perennial grasses is the relatively high cost of propagation and planting. The objective of the present study was to investigate new propagation and planting methods of giant reed ( Arundo donax L.), miscanthus ( Miscanthus x giganteus Greef et Deuter) and switchgrass ( Panicum virgatum L.). Field and open-air pot trials were carried out in four different locations across Europe: hydro-seeding of switchgrass was tested in field trials at the experimental farm of the University of Bologna, Italy; stem propagation and bud activation methods of miscanthus were evaluated in field experiments in Péteri, Hungary; giant reed rhizome and stem propagations were compared in a field trial in Aliartos, Greece; finally, an open-air pot trial was carried out in Catania, Italy, using single-node stem cuttings of giant reed. Hydro-seeding emerged as a feasible and promising technique for switchgrass to ensure prompt seed emergence and weed control during plant establishment. Direct stem plantings of miscanthus were successful, and activated stem-buds were able to sprout in field conditions; however, timely stem transplant was determinant for shoot density and biomass yield. In giant reed, rhizome propagation showed a higher stem density and biomass yield than stem propagation; however, the yield gap was not significant from the second year onwards. Single-node rooting was mainly driven by air temperature. Nodes from basal stems showed higher rooting rates than median and apical ones. Growth regulator pretreatments enhanced rooting rate only at transplanting times under suboptimal air temperatures. In general, these experiments provided insights into propagation strategies aimed at enhancing the establishment phase of perennial grasses. [ABSTRACT FROM AUTHOR]

Giant reed ( Arundo donax) and Miscanthus spp. were tested to evaluate their tolerance and phytoremediation capacity in soils contaminated with heavy metals. Giant reed was tested under 450 and 900 mg Zn kg, 300 and 600 mg Cr kg, and 450 and 900 mg Pb kg contaminated soils, while the Miscanthus genotypes M. × giganteus, M. sinensis, and M. floridulus were tested on 450 and 900 mg Zn kg contaminated soils, along 2 years. Giant reed biomass production was negatively affected by the contamination; however, yield reduction was only significant under 600 mg Cr kg soil. Zn contamination reduced significantly M. × giganteus production but not M. sinensis or M. floridulus yields. Yet, M. × giganteus was also the most productive. Both grasses can be considered as indicators, once metal concentration in the biomass reflected soil metal concentration. Regarding giant reed experiments, higher modified bioconcentration factors (mBCFs, 0.3-0.6) and translocation factors (TFs, 1.0-1.1) were obtained for Zn, in the contaminated soils, followed by Cr (mBCFs, 0.2-0.4, belowground organs; TFs, 0.2-0.4) and Pb (mBCFs, 0.06-0.07, belowground organs; TFs, 0.2-0.4). Metal accumulation also followed the same pattern Zn > Cr > Pb. Miscanthus genotypes showed different phytoremediation potential facing similar soil conditions. mBCFs (0.3-0.9) and TFs (0.7-1.5) were similar among species, but highest zinc accumulation was observed with M. × giganteus due to the higher biomass production. Giant reed and M. × giganteus can be considered as interesting candidates for Zn phytoextraction, favored by the metal accumulation observed and the high biomass produced. A. donax and Miscanthus genotypes showed to be well suited for phytostabilization of heavy metal contamination as these grasses prevented the leaching of heavy metal and groundwater contamination. [ABSTRACT FROM AUTHOR]

Abstract: In the present study the effect of temperature, reaction time and dilute oxalic acid (OA) concentration during steam-pretreatment of Miscanthus × gigantueus has been evaluated using the combined severity factor (CS). At the highest CS glucan and lignin content in the water insoluble fraction (WIF) increased, while xylan content decreased. While glucose recovery in the water soluble fraction (WSF) was found at low concentration when mild CS were used (≤5.0 g L−1 at CS ≤ 2.17), xylose and arabinose concentrations were higher at low-mild CS (1.58–2.17) with a concentration peak at CS 2.03 (39.9 and 3.2 g L−1 for xylose and arabinose, respectively). The decrease in pentoses coincided with inhibitory formation in the WSF, namely acetic acid, furfural, HMF and phenolic compounds. Glucan conversion rose from 46.1% at CS 1.54 to 91.2% at CS 2.76. Likewise, maximum ethanol concentration was achieved at CS 2.76, corresponding to 20.2 g L−1 and a volumetric ethanol productivity of 0.28 g L−1 h−1. Negative correlations have been found between xylan vs. glucan conversion and xylan vs. ethanol production, suggesting that decreasing the xylan content in WIF increases both saccharification rate and ethanol concentration (R 2 0.91 and R 2 0.93, respectively). On the other hand, a positive correlation was found between ethanol production and glucan conversion (R 2 0.93). Fermentation of WSF by Scheffersomyces (Pichia) stipitis CBS 6054 at CS 1.54 produced 12.1 g L−1 of ethanol after 96 h incubation with a volumetric ethanol productivity of 0.13 g L−1 h−1. [Copyright &y& Elsevier]

Abstract: Miscanthus × giganteus is one of the most promising biomass crops for non-food utilisation. Taking into account its area of origin (Far East), its temperature and rainfall requirements are not well satisfied in Mediterranean climate. For this purpose, a research was carried out with the aim of studying the adaptation of the species to the Mediterranean environment, and at analysing its ecophysiological and productive response to different soil water and nitrogen conditions. A split plot experimental design with three levels of irrigation (I1, I2 and I3 at 25%, 50% and 100% of maximum evapotranspiration (ETm), respectively) and three levels of nitrogen fertilisation (0kgha−1: N0, 60kgha−1: N1 and 120kgha−1: N2 of nitrogen) were studied. The crop showed a high yield potential under well-watered conditions (up to 27tha−1 of dry matter). M. × giganteus, in Mediterranean environment showed a high yield potential even in very limited water availability conditions (more than 14tha−1 with a 25% ETm restoration). A responsiveness to nitrogen supply, with great yield increases when water was not limiting, was exhibited. Water use efficiency (WUE) achieved the highest values in limited soil water availability (between 4.51 and 4.83gl−1), whilst in non-limiting water conditions it decreased down to 2.56 and 3.49gl−1 (in the second and third year of experiment, respectively). Nitrogen use efficiency (NUE) decreased with the increase of water distributed (from 190.5gg−1 of I0 to 173.2gg−1 of I2); in relation to N fertilisation it did not change between the N fertilised treatments (N1 and N2), being much higher in the unfertilised control (177.1gg−1). Radiation use efficiency (NUE) progressively declined with the reduction of the N fertiliser level (1.05, 0.96 and 0.86gd.m.MJ−1, in 1994, and 0.92, 0.91 and 0.69gd.m.MJ−1, in 1995, for N2, N1 and N0, respectively). [Copyright &y& Elsevier]

Low iLUC risk feedstocks, such as lignocellulosic no-food crops, have been indicated as sustainable crops for the transition to a bio-based economy. Given the high output to input ratio and the environmental benefits that can be obtained from renewable heat production replacing fossil fuels, the present study addressed the biomass yield, CO2-sequestration, and life cycle assessment of giant reed (Arundo donax L.) and miscanthus (Miscanthus × giganteus Greef et Deuter) growing under different soil water availability and nitrogen fertilization for three consecutive growing seasons in a semiarid Mediterranean environment. Giant reed outperformed miscanthus, showed a higher CO2-sequestration and a lower overall environmental impact. In case of both crops, the irrigation effect was significant, while the one of nitrogen fertilization was not apparent. While giant reed responded positively to reduced irrigation, compared to its highest level, as the plantation became older, miscanthus needed high water volume to get most out its potential yield. Nonetheless, the growing season had also a significant effect on both crops, mainly when low yields were achieved following the establishment year. Unlike the environmental benefits in the impact categories "non-renewable energy use" and "global warming potential", environmental burdens concerning ozone depletion, acidification, and eutrophication were observed, indicating that further improvements of the evaluation of impact assessment associated with bioenergy production might be necessary. [ABSTRACT FROM AUTHOR]

Predictions of future climate scenarios indicate that yields from perennial biomass crops (PBCs) growing in the Mediterranean region are likely to decline due to prolonged drought. Among PBCs, Miscanthus grasses with C4 photosynthesis combine high yield potentials and water use efficiencies. However, the standard commercial clone M. x giganteus (Mxg), with minimal stomatal regulation, is too sensitive to drought for reliable yields in the Mediterranean regions. This paper screened a diverse panel of thirteen Miscanthus genotypes (M. sinensis, M. floridulus, M. sacchariflorus and Mxg) to identify which types could maximize yield under summer drought conditions typical in the South Mediterranean climate. In the second growing season, significant differences were observed for plant height (from 63 to 185 cm), stem number (from 12 to 208 stems plant−1), biomass yield (from 0.17 to 6.4 kg DM plant−1) and whole crop water use efficiency (from 0.11 to 7.0 g L−1). Temporal variation in net photosynthesis, stomatal conductance, transpiration rate and instantaneous water use efficiency identified different strategies adopted by genotypes, and that genotypes selected from M. floridulus and M. sinensis were better adapted to rainfed conditions and could produce six times more biomass than the Mxg. These accessions are being used as parents in experimental breeding aimed at producing future seed-based drought resilient hybrids. [ABSTRACT FROM AUTHOR]

Miscanthus, a C4 perennial grass native to Eastern Asia, is being bred to provide biomass for bioenergy and biorenewable products. Commercial expansion with the clonal hybrid M. × giganteus is limited by low multiplication rates, high establishment costs and drought sensitivity. These limitations can be overcome by breeding more resilient Miscanthus hybrids propagated by seed. Naturally occurring fast growing indigenous Miscanthus species are found in diverse environments across Eastern Asia. The natural diversity provides for plant breeders, the genetic resources to improve yield, quality, and resilience for a wide range of climates and adverse abiotic stresses. The challenge for Miscanthus breeding is to harness the diversity through selections of outstanding wild types, parents, and progenies over a short time frame to deploy hybrids that make a significant contribution to a world less dependent on fossil resources. Here are described the strategies taken by the Miscanthus breeding programme at Aberystwyth, UK and its partners. The programme built up one of the largest Miscanthus germplasm collections outside Asia. We describe the initial strategies to exploit the available genetic diversity to develop varieties. We illustrate the success of combining diverse Miscanthus germplasm and the selection criteria applied across different environments to identify promising hybrids and to develop these into commercial varieties. We discuss the potential for molecular selections to streamline the breeding process. [ABSTRACT FROM AUTHOR]

Miscanthus is a high-yielding lignocellulosic perennial biomass crop. The low multiplication rate of clonal rhizome propagation is a bottleneck to upscaling plantation areas of feedstock needed to supply and expand the bioeconomy. Novel seeded Miscanthus hybrids are currently being developed to overcome this bottleneck by increasing annual multiplication rates from approximately 10 to over 1000 times. We describe a series of field experiments in southern Italy using agronomic methods to optimise multiplication rates through (i) planting configurations and densities, (ii) ratio of seed parents to pollen parents (iii) supplemental pre-dawn misting to increase humidity during pollination. In these trials the seed-bearing M. sinensis started flowering 2–3 weeks earlier than the M. sacchariflorus pollen parent. Earlier experiments indicated that flowering in M. sacchariflorus was mostly determined by photoperiod while in M. sinensis it was modulated by endogenous signals. Consequently, a second set of experiments were conducted to delay flowering time in M. sinensis : (iv) mid-season stem cut back, (v) oversupply of nitrogen, and (vi) undersupply of water. Across all treatments and years, the multiplication rates varied from 140 to 1300 seeds m². Reducing the proportion of the pollen parent plants (M. sacchariflorus) from 50 % to 25 % did not reduce seed yield per plant. This therefore increases the seed yield per m² and reduces seed production upscaling costs. Flowering time and duration in M. sinensis was significantly impacted by mid-season cutting and water stress, but not by nitrogen supply rates. Mid-season shoot cutting reduced number of flowers per plant (77 %), seed quantity (47 %), seed size (46 %), and resulted in seeds with a low germination rate of 39 %. High M. sinensis planting densities produced higher seed yields in the first year. However, in subsequent years higher density plots were more susceptible to autumn lodging lowering seed production by loss of panicles. Pre-dawn misting to prolong pollen life and stigma receptivity had no significant effects on seed production. This study demonstrates the importance of flowering time synchronization in the open field for commercial seed production. The limited effect of agronomic efforts to reduce the interspecies flowering time gap emphasises the importance of genetic factors in controlling flowering time. The most impactful intervention to change flowering time and improve parental synchronisation was mid-season cutting, while this method reduced seed production when applied to the seed parent it could be ideal for pollen parents. • Commercially relevant in field optimisation of Miscanthus hybrid seed production. • Field flowering synchronisation manipulation for production of interspecies seed. • Parental ratios ≥ 3:1 can increase seed per m² without impacting pollination rate. • Under field conditions reduced watering delayed M. sinensis flowering. • Mid-season cutting was most effective for flowering delay but reduced seed set. [ABSTRACT FROM AUTHOR]

Sustainable production of high value chemicals, biofuels and bioenergy relies on the replacement of fossil oil with low production cost feedstock and low environmental impact technologies. The present study evaluated the dry matter yield and feedstock properties of three different Arundo donax L. clones (Fontane Bianche, Piazza Armerina and Capo d’Orlando), two Miscanthus species ( M . × giganteus and M . sinensis “Goliath”) and Saccharum spontaneum L. spp. aegyptiacum (Willd.) Hack., all grown side-by-side in rainfed conditions for three consecutive years in semi-arid Mediterranean environment. Significant differences were observed among species, as well as among giant reed clones. F. Bianche and Saccharum were the highest yielding species in all growing seasons, followed by P. Armerina and C. Orlando, which did not differ. Out of Miscanthus, Giganteus overyielded Goliath. All species exhibited the highest dry matter yields in the wettest growing season. Saccharum and giant reed clones showed higher biomass water content than both Miscanthus. Ash content was the lowest in Giganteus and the highest in F. Bianche. Low heating value was the highest in Saccharum, both Miscanthus and giant reed P. Armerina. Generally, the bulk density was greater in giant reed and Saccharum than Miscanthus. Cellulose was the highest in both Miscanthus and hemicellulose in Saccharum. Lignin content was the lowest in giant reed clones. Several significant correlations occurred between biomass yield and raw material characteristics, as well as within feedstock properties. To support the development of a suitable bioenergy chain based on perennial grasses, besides biomass yield, feedstock properties should be rigorously considered. Further research is still required in crop management practices, logistic and technology front in order to tackle bottlenecks for improved quality of herbaceous biomass. [ABSTRACT FROM AUTHOR]

• Long-term perennial grasses were compared in two locations and four growing seasons. • The main driving factor of bioethanol production was the biomass yield. • Optimization of harvest time allowed to gain more ethanol per unit mass. • Giant reed was the most chemically consistent feedstock among study factors. • Bioethanol biorefineries may gain fourfold the energy invested. Advanced biofuel production requires feedstock with specific composition in terms of quantity and quality. Decisions about the harvest time of perennial energy grasses have important implications for bioconversion process as well as for the life-span of the plant stand. Four-year data from long term-field trials were used to identify the optimal crop-specific harvest date (autumn vs. winter), in relation to advanced biofuel production. Three different perennial energy grasses (i.e., switchgrass, giant reed and miscanthus) were grown at Bologna and Catania (Italy), under northern and southern Mediterranean climate, respectively, adopting a low input management. In addition to the biomass yield and composition, the net energy value (NEV) from agricultural activities, and the energy return on investment (EROI) at the biorefinery gate was also evaluated. At Bologna, switchgrass biomass yield and quality (hemicellulose, cellulose and acid detergent lignin – ADL) were significantly enhanced when harvested in winter. At Catania, giant reed productivity, cellulose and ADL content were unaffected by harvest date, while hemicellulose and ash content decreased in winter. Biomass composition of miscanthus was more stable in Catania than Bologna, however, yield and components resulted significantly higher in Bologna than Catania, due mainly to the more even precipitation distribution throughout the vegetative crop development. The NEV was positive for all energy crops, treatments and locations, with the highest value for miscanthus grown at Bologna. The EROI of lignocellulosic bioethanol ranged from 4.16 for switchgrass in the autumn harvest at Bologna to 4.37 for miscanthus grown at Catania, which showed also the highest theoretical bioethanol production (169.2 kg h−1) at a feeding rate of 300 kg h−1 of raw material. Nonetheless, miscanthus grown at Bologna attained the highest bioethanol yield (12,254 L ha−1). This study proved that investigated perennial grasses are worth to be grown as lignocellulosic feedstock under low input for at least one decade, upon which Mediterranean region biorefineries may gain fourfold the energy invested. [ABSTRACT FROM AUTHOR]