Short-rotation Intensive Culture Research Articles

Modern and future forestry based on biotechnology

AbstractForest is the most important terrestrial component of the Earth's biosphere, containing nearly 80% of terrestrial biodiversity. However, climate change and population growth are exacerbating deforestation and forest degradation. Reforestation efforts aim to restore ecosystems, but the effectiveness of traditional tree plantations in repairing the ecological function of native forests is controversial. The ideal scenario for forestry in the future is to meet the growing demand for forest products while keeping forest sustainable development. One possible way forward is to greatly improve the productivity and adaptability of forests through molecular breeding to use fewer Short Rotation Intensive Culture (SRIC) plantations to produce sufficient forest products. In the last 2 decades, advances in biotechnologies such as high‐throughput sequencing and genome editing are transforming tree breeding and forestry. Here, we propose possible future directions for modern forestry, including molecular design tree breeding, agroforestry and mixed farming, sustainable and biosphere friendly forestry.

Ectomycorrhizal Fungi Dominated the Root and Rhizosphere Microbial Communities of Two Willow Cultivars Grown for Six-Years in a Mixed-Contaminated Environment.

There is a growing interest in plant microbiome’s engineering to optimize desired functions such as improved phytoremediation. This study is aimed at examining the microbial communities inhabiting the roots and rhizospheres of two Salix miyabeana cultivars that had been grown in a short-rotation intensive culture (SRIC) system for six years in a soil contaminated with the discharge from a petrochemical factory. DNA was extracted from roots and rhizospheric soils, and fungal ITS and bacterial and archaeal 16S rDNA regions were amplified and sequenced using Illumina MiSeq technology. Cultivars ‘SX61’ and ‘SX64’ were found to harbor a similar diversity of fungal, bacterial, and archaeal amplicon sequence variants (ASVs). As expected, a greater microbial diversity was found in the rhizosphere biotope than in the roots of both cultivars, except for cultivar ‘SX64’, where a similar fungal diversity was observed in both biotopes. However, we found that microbial community structures were cultivar- and biotope-specific. Although the implication of some identified taxa for plant adaptability and biomass production capacity remains to be explored, this study provides valuable and useful information regarding microbes that could potentially favor the implantation and phytoremediation efficiency of Salix miyabeana in mixed contamination sites in similar climatic environments.

Willows Used for Phytoremediation Increased Organic Contaminant Concentrations in Soil Surface

The Salix genus includes shrub species that are widely used in phytoremediation and various other phytotechnologies due to their advantageous characteristics, such as a high evapotranspiration (ET) rate, in particular when cultivated in short rotation intensive culture (SRIC). Observations made in past field studies suggest that ET and its impact on soil hydrology can also lead to increases in soil pollutant concentrations near shrubs. To investigate this, sections of a mature willow plantation (seven years old) were cut to eliminate transpiration (Cut treatment). Soil concentrations of polychlorinated biphenyls (PCBs), aliphatic compounds C10–C50, polycyclic aromatic hydrocarbons (PAHs) and five trace elements (Cd, Cr, Cu, Ni and Zn) were compared between the Cut and the uncut plots (Salix miyabeana ‘SX61’). Over 24 months, the results clearly show that removal of the willow shrubs limited the contaminants’ increase in the soil surface, as observed for C10–C50 and of 10 PAHs under the Salix treatment. This finding strongly reinforces a hypothesis that SRIC of willows may facilitate the migration of contaminants towards their roots, thus increasing their concentration in the surrounding soil. Such a “pumping effect” in a high-density willow crop is a prominent characteristic specific to field studies that can lead to counterintuitive results. Although apparent increases of contaminant concentrations contradict the purification benefits usually pursued in phytoremediation, the possibility of active phytoextraction and rhizodegradation is not excluded. Moreover, increases of pollutant concentrations under shrubs following migration suggest that decreases would consequently occur at the source points. Some reflections on interpreting field work results are provided.

Short Rotation Intensive Culture of Willow, Spent Mushroom Substrate and Ramial Chipped Wood for Bioremediation of a Contaminated Site Used for Land Farming Activities of a Former Petrochemical Plant.

The aim of this study was to investigate the bioremediation impacts of willows grown in short rotation intensive culture (SRIC) and supplemented or not with spent mushroom substrate (SMS) and ramial chipped wood (RCW). Results did not show that SMS significantly improved either biomass production or phytoremediation efficiency. After the three growing seasons, RCW-amended S. miyabeana accumulated significantly more Zn in the shoots, and greater increases of some PAHs were found in the soil of RCW-amended plots than in the soil of the two other ground cover treatments’ plots. Significantly higher Cd concentrations were found in the shoots of cultivar ‘SX61’. The results suggest that ‘SX61’ have reduced the natural attenuation of C10-C50 that occurred in the unvegetated control plots. The presence of willows also tended to increase the total soil concentrations of PCBs. Furthermore, we found that many contaminant concentrations were subject to seasonal oscillations, showing average increases throughout the whole experimental site after a growing period, while showing significantly different variations, such as lesser increases or even decreases, after a dormant period. These observations suggest that contaminants may have leached or degraded faster in untreated conditions, and conversely to have mobilized towards trees through water flow driven by plant transpiration during growing seasons.

Reflections on the contributions of Populus research at Rhinelander, Wisconsin, USA

The United States Department of Agriculture Forest Service established three regional Institutes of Forest Genetics in the United States in the 1950s to improve trees for reforestation and improve the management of forests. The institute in Rhinelander, Wisconsin, started in 1956 as part of the Lake States Forest Experiment Station. Since that time, the “Rhinelander Lab” has undergone changes in research priorities, organizational changes, and name changes while becoming an international center of forest scientific excellence. Many of the researchers’ key findings over the years were published in the Canadian Journal of Forest Research. In this paper, for the 50th anniversary edition of the Journal, we reflect upon one part of those accomplishments: the history of the contributions of Populus L. research at Rhinelander. We discuss major research programs and the scientists conducting this work, including (i) physiology of wood formation, (ii) short rotation intensive culture and short rotation woody crops, (iii) intensively cultured plantations, (iv) physiology and utilization of short rotation poplar yields, (v) breeding and selection, (vi) biotechnology and molecular genetics, (vii) atmospheric pollution and climate change, (viii) phytotechnologies, and (ix) ecosystem services. Also, we describe four major international conferences held in Rhinelander and (or) hosted by Rhinelander researchers.

Preventing unwanted spread of invasive fungal species in willow (Salix spp.) plantations

In North America, biomass production of fast-growing trees represents an important forestry and agroforestry practice for bioenergy, bioproducts and phytoremediation. In Canada, imported cuttings are used as the propagation material to establish Short Rotation Intensive Culture (SRIC) of willow. Profiling the mycodiversity of willow cuttings is vital for early diagnosis, prevention or control of exotic diseases. It also helps to identify disease tolerant willow genotypes to increase the survival rate of cuttings and the establishment of high-density tree plantations, free of introduction of alien pathogenic species to the Canadian ecosystems. In this study, a total of 82 fungal taxa were isolated and identified from asymptomatic willow cuttings imported in Canada. The most abundant phylum was Ascomycota, although some Basidiomycota (Agaricales and Tremellales) were also detected. The most abundant fungal taxa belonged to Hypocreales, while the most abundant species belonged to Kabatiella in Dothideales. In vitro and in planta bioassays results, combined with polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analyses, revealed that potentially pathogenic species belonging to Glomeraceae, Dermateaceae, Diaporthaceae and Venturiaceae may originate from the willow cuttings, which could be transmitted to tree plantations and spread via standard management and coppice practices. A newly discovered Lecythophora–Coneochaeta complex was the most frequently observed. Pathogenic Cadophora spp. were also detected; they are a potential threat to the health of willow during long cold-storage of cuttings. Among cultivars, SV1 (Salix dasyclados) showed low infection rates and demonstrates the possibility of risk stratification according to willow genotypes.

Evaluating sampling designs and deriving biomass equations for young plantations of poplar and willow clones

Short-rotation intensive culture (SRIC) for bioenergy production is at its pre-commercial stage in Canada. To be economically viable, these types of plantations need an accurate examination of actual yields, which requires precise and efficient estimation methods (i.e., specific allometric equations and sampling methods). At six SRIC plantations from three Canadian provinces (Quebec, Ontario and Alberta), 6 willow and 10 poplar clones were sampled and clone allometric equations were developed to estimate plant biomass. A stem selection approach was successfully used to develop plant allometric equations, reducing the number of stems to be measured by up to 81% in coppiced plantations relative to traditional stem equations. Clone-specific equations were more accurate than equations for groups of clones, but the difference in terms of RMSE% was generally small (less than 5%). Using extensive measurements of all the plants inside a plantation and a simulation approach, we also compared five sampling methods (simple random sampling, stratified sampling, systematic sampling, random and systematic cluster sampling) to estimate total biomass inside the plantation. Simple random sampling and stratified random sampling were the most efficient methods (i.e., increased precision for equal sample size) for the estimation of average plant biomass, survival and total plantation biomass. Stratified random sampling (based on the position inside the plantation) made it possible to reduce the sample size as compared to simple random sampling, but only at higher levels of precision (e.g., 25 less plants at 5% precision). Applications of sampling using remote sensing techniques and GIS are briefly discussed.

Forecasting the deployment of short-rotation intensive culture of willow or hybrid poplar: Insights from a Delphi study

Short-rotation intensive culture (SRIC) of willow (Salix spp.) or hybrid poplar (Populus spp.) is currently at a precommercial stage with a potential to be applied economically across important areas to produce lignocellulosic biomass and environmental services in Canada. A two-round Delphi survey was conducted among 50 experts to assess the future deployment of SRIC in this country. The total area in 10 years (2011 base year) was forecasted as 1330, 4100, and 11 400 ha under pessimistic, realistic, and optimistic scenarios, respectively. The deployment of SRIC in the next decade depends mainly on the development of the demand for SRIC biomass and services, that of production technologies, and the establishment of policies and programs promoting its application. In the short term, research and development (R&D) and policy initiatives should be funded or implemented by various stakeholders to facilitate the deployment of the system. On average, respondents deemed that the potential for long-term (20 years) deployment of SRIC in Canada was good. Some of the conclusions and the methodological approach of this study could apply to short-rotation woody crop systems and to agroforestry systems in Canada and elsewhere.

Evaluating the biomass production of coppiced willow and poplar clones in Michigan, USA, over multiple rotations and different growing conditions

Willow and poplar are both important crops for Short Rotation Intensive Culture (SRIC). Here, we report the results of biomass yield trials at three locations in Michigan, where the average annual yield of twelve willow and two poplar clones where compared over three- or four-year growth periods after coppicing. On average, fast-growing hybrid poplars (NM5 and NM6) were more productive than any of the willow clones tested by about 30%. Poplar and willow revealed different growth patterns after successive harvests; poplars grew quickly and consistently while willow growth was initially slower but increased over time. Willows at the intensively managed site in southern Michigan were usually 2–5 times more productive than at the two less intensively managed sites in southern Michigan and northern Michigan, but poplars at both southern sites were similarly more productive than at the northern site. The annual yield of clones was further standardized to account for differences in growing degree days (GDD) between sites in southern and lower Michigan, which caused differences in the relative ranking of some willow clones to change. The results suggest that GDD-standardized yield rates may be useful for comparisons of clonal performance over larger regions. Decomposition of clonal yield rates into individual stool yield and survival rates revealed a strong positive correlation (r2 = 0.94) between them, indicating that faster-growing clones also had higher survival rates. The best performing clones for biomass production were also identified.

Changes in root-associated fungal assemblages within newly established clonal biomass plantations of Salix spp.

Fungal assemblages associated with the roots of willow clones (Salix spp.) grown under short rotation intensive culture (SRIC) were isolated in order to evaluate their relationship with plant physiological (health) status, clone, geography and plant age. The assemblages were highly dominated by Fusarium (F. culmorum, F. redolens and F. equiseti), Alternaria and Phomopsis (Ascomycota); while fungi belonging to Basidiomycota and Zygomycota were only marginally present. The results suggested a shift from assemblages dominated by potentially pathogenic fungi during the first year of plant establishment to assemblages with an increased presence of potentially beneficial fungi on two-year old plants. Among the potentially pathogenic fungi, Gibberella/Fusarium and Neonectria taxa generalists appeared to be the most important threats. On the other hand, Alternaria, Penicillium and Phomopsis taxa were identified as potentially beneficial fungi given their dominance in roots of healthy plants. Differences between the assemblages of different willow clones were also evident but their implications in relation with genotype adaptability and biomass production capacity remain to be explored. Root-associated fungal assemblages appear to be useful to monitor the impacts of SRICs on the rhizosphere ecology and their study may enlighten effective ways to a proper management of these crop systems.

Performance of 14 hybrid poplar clones grown in Beijing, China

Evaluating the performance of poplar clones to be used in short-rotation intensive culture (SRIC) and selecting superior clones are critical to increase wood production and ecological effects. This study described the growth and aboveground biomass production of 14 hybrid poplar clones after three growing seasons, evaluated the clones with cluster analysis on multiple basis. At the end of each growing season, total tree height, basal diameter and biomass were measured. Highest production was found for clone NE-353 and DN-70 with mean annual biomass production 22 Mg ha 1 year 1 . Lowest performance was observed for clone 311-93 and 309-74 with biomass production 0.29 and 0.75 Mg ha 1 year 1 . Cluster analysis was conducted with survival and tree volume index, all measured at the end of the third growing season. The clones were grouped into four classes. Clone R-247, DN-70 and Simplot had higher survival and highest tree volume index, appeared to well adapted to the region; clone 52-225, 58-280, NE-353, DN-34, PC-01 and OP-367 exhibited moderate survival and Growth; clone DN-14273, R-419 showed moderate survival and low growth; clone 195529, 311-93 and 309-74 performed poorest with lowest survival and tree volume index and these clones may not suitable for biomass producers in this region. a 2010 Published by Elsevier Ltd.

Performance of Salix viminalis and Populus nigra×Populus maximowiczii in short rotation intensive culture under high irrigation

Abstract On a plantation established in 2004 from stem cuttings at a density of 20,000 trees per hectare, we investigated growth and nutritional plant response to a high hydraulic regime for two species ( Salix viminalis and Populus nigra × Populus maximowiczii ), using a comparative approach with measurements from irrigated and control plots. The plantation was irrigated from June to September 2005 with about 140 mm per day. The equivalent of 120 Kg NO 3 –N, 40 Kg P 2 O 5 –P and 85 Kg K 2 O–K per hectare per year was applied by means of irrigation with wastewater. No mortality occurred and stem biomass production of both poplar and willow species were not statistically different on irrigated and control areas. However, S. viminalis revealed to be more tolerant to flooded conditions since these corresponded more closely to its nutritional requirements (foliar concentration of 20 mgN g −1 ). The capacity of S. viminalis to withstand waterlogged conditions could play an important role in the sustainability of a plantation for the filtration of effluent at low pollutant concentration.

Rapid biomass estimation using optical stem density of willow (Salix spp.) grown in short rotation

Abstract Quick and accurate biomass estimation of willow (Salix spp.) grown under short rotation intensive culture (SRIC) is essential for carbon accounting and management decisions. Currently, most estimates of tree biomass, including willow, rely on measurement of stem diameter. This is a suitable approach for single-stem species but for measurements of multi-stem species such as willow, there is an increase in the time and effort required as well as the need to include site, clone and age specific information. Therefore, we developed a new method which calculates optical stem density from digital photographs taken at predetermined locations and angles within a plantation during the fall or winter when the willow is without leaves. We then calibrated a mathematical model using destructive sampling to convert the measurements of optical stem density into estimates of biomass. The method produced very strong relationships (adjusted r2 = 0.97) between the predicted and actual harvested biomass for the plots studied. Being new, the method still requires further testing and possibly adjustments for different planting designs and clones.

05/02161 The effect of moisture content on retention of fluorocarbon tracers on sand

Various cultural factors influence the biological and economic potentials of Eucalyptus grandis and slash pine as biomass energy crops for southern and northern Florida, respectively. On muck soil, superior E. grandis progenies may produce up to 70 dry Mg ha−1 in 24 months. However, coppicing success on muck soil has been inconsistent. Clones superior in frost-resilience and vigor and propagated as rooted cuttings have increased productivity potential on sandy soils. Break-even costs GJ−1 range from just under US$2 for E. grandis short-rotation intensive culture (SRIC) on muck soils (10 000 trees ha−1 with a 6-year rotation, i.e. seedling/coppice/coppice cycles of 2 years each) to US$4.00 GJ−1 on sandy soils. Planting density, but not coppice rotation, is sensitive to the discount factor. Through 8 years, slash pine yields responded well to soil amendments on typical sites and to high planting densities. Fertilization and vegetation control increased productivity some seven times over conventional culture through 5 years. For slash pine SRIC, higher discount rates increased initial stocking levels and shortened rotations. Break-even costs for slash pine are within the range of values estimated for E. grandis.

Field performance and biomass production of 12 willow and poplar clones in short-rotation coppice in southern Quebec (Canada)

Twelve clones of fast growing trees (willow and poplar) were planted in 1999 under short-rotation intensive culture (SRIC) on an abandoned farmland in southern Quebec. The plantation was established at a density of 18,000 trees per hectare from stem cuttings and no fertilizer and irrigation were applied. Trees performances were measured at regular interval during four growing seasons. The aims of the experiment were to compare the growth, insect and disease resistance of these clones in order to select those that have good potential for use as commercial biomass energy crops in northern regions of North America. The follow up of the growing performance has shown statistically significant differences between the clones. Poplar clones registered the highest aboveground biomass yield after 4 growing seasons (from 66.48 to 72.20 tDM ha-1). The best willow biomass productivity was obtained from clones SX64 (67.58 tDM ha-1) and clone SX61 (62.34 tDM ha-1). Only one willow clone S301 (Salix interior × S. eriocephala) was sensitive to leaf rust (Melampsora spp.) and clones SVQ (S. viminalis) and SV1 (S. dasyclados) were more prone to insect attacks. The results proved that some clones of S. miyabeana and S. sachalinensis were more productive and more resistant to insect and disease damage than S. viminalis which has been widely planted in SRIC in southern Quebec since many years.

Growth and nutrition response of young sweetgum plantations to repeated nitrogen fertilization on two site types

Short-rotation intensive tree culture is being investigated in the southern United States as a method of producing hardwood fiber, but little is known about the early productivity and nutritional needs of these systems, especially on different site types. We studied the growth and foliar nutrition response of two sweetgum ( Liquidambar styraciflua L.) plantations on a converted agricultural field and a pine cutover site to biannual applications of three nitrogen (N) fertilizer rates: 0, 56, and 112 kg N ha −1 . The trees did not respond to treatment at any age on the agricultural field site, but the fertilized trees on the cutover site had about 60% greater biomass at ages 5 and 6. Fertilization doubled foliar biomass on the cutover site in the years fertilizer was applied. Stem biomass was directly related to foliar biomass, but the relationship was age-specific at both sites. Stem biomass was also related to the foliar N concentration. Foliar critical values of N were about 18 g N kg −1 . Foliage phosphorus (P) and potassium (K) contents were diluted by the N fertilization-induced growth responses at both sites. Fertilization of young intensive-culture sweetgum plantations is necessary for optimum foliar N concentrations and foliar and stem biomass production, but is site-specific.

04/01374 Experimental investigation of the decomposition and calcinations of calcium-enriched bio-oil: Sotirchos, S. and Smith, A. R. Industrial & Engineering Chemistry Research, 2003, 42, (10), 2245–2255

The gradual and then rapid reduction of crude oil prices, the impact of U.S. Gramm-Rudman-Hollings legislation (to reduce budget deficits) on R&D (research and development) budgets, the partial elimination of renewable-energy tax credits, and the possible elimination of all energy tax credits and forgiveness via tax reform, are all taking their toll on energy research and commercialization ventures. However, it appears that “biofuels” will survive and continue to exhibit modest increases in contributions to primary energy demand. A comprehensive assessment of renewable-technology options has shown that biofuels is the only option capable of making significant contributions to all energy sectors. The total contribution of renewable energy to primary energy demand in the U.S.A. is about 7.2EJ/y (6.8 quad/y); biofuels provide about 40% of the total. Research on short-rotation intensive-culture (SRIC) trees and herbaceous and aquatic biomass has identified specific species for development. Some of these species appear to be near economic feasibility as energy crops now. Commercialization of biomass and waste combustion systems has shown a spurt in growth because of the business opportunities offered by laws encouraging cogeneration. Thermochemical gasification research on advanced technology systems has reached the point where scale-up is being considered. Commercialization of state-ofthe-art technologies has slowed, but applications for small-scale producer gasifiers, particularly in developing countries, continue to expand. Thermochemical liquefaction research has concentrated mainly on high-temperature pyrolytic conversion methods that can increase yields and selectivities. Significant research achievements have been made in anaerobic digestion, but whatever its future as a large-scale source of methane, it is clear that digestion capacity will have to increase many-fold before biological methane can attain its potential. The mandated reduction in lead content of gasolines presents substantial marketing opportunities to alcohol-fuel producers. If projections for ethanol-fuel markets materialize, conversion of low-cost lignocellulosic materials will be necessary. Research to develop this technology is now in progress.

Genetic variation of the bud and leaf phenology of seventeen poplar clones in a short rotation coppice culture.

Leaf phenology of 17 poplar ( Populus spp.) clones, encompassing spring phenology, length of growth period and end-of-year phenology, was examined over several years of different rotations. The 17 poplar clones differed in their latitude of origin (45 degrees 30'N to 51 degrees N) and were studied on a short rotation experimental field plantation, situated in Boom (province of Antwerpen, Belgium; 51 degrees 05'N, 04 degrees 22'E). A similar, clear pattern of bud burst was observed during the different years of study for all clones. Clones Columbia River, Fritzi Pauley, Trichobel (Populus trichocarpa) and Balsam Spire (Populus trichocarpa x Populus balsamifera) from 45 degrees 30'N to 49 degrees N reached bud burst (expressed as day of the year or degree day sums) almost every year earlier than clones Wolterson (Populus nigra), Gaver, Gibecq and Primo (Populus deltoides x Populus nigra) (50 degrees N to 51 degrees N). This observation could not be generalised to end-of-season phenology, for which a yearly returning pattern for all clones was lacking. Late bud burst and early leaf fall of some clones (Beaupré, Boelare, IBW1, IBW2, IBW3) was brought about by increasing rust incidence during the years of observation. For these clones, the variability in leaf phenology was reflected in high coefficients of variation among years. The patterns of genetic variation in leaf phenology have implications for short rotation intensive culture forestry and management of natural populations. Moreover, the variation in phenology reported here is relevant with regard to the genetic mapping of poplar.

Energy feedstock characteristics of willow and hybrid poplar clones at harvest age

Woody biomass feedstock produced from willow and hybrid poplar can be converted into bioenergy via thermochemical and biochemical processes. Variation in key properties that relate to the quality of biomass feedstock and determine its value for energy conversion were determined at rotation age (3 years), in 30 willow and seven hybrid poplar clones, grown in a short-rotation intensive culture (SRIC) system in central NY. Substantial clonal variation in the concentrations of nitrogen (2.9– 5.0 g kg −1 ), phosphorus (0.4– 0.8 g kg −1 ) potassium (1.2– 2.4 g kg −1 ), sodium (0.09– 0.20 g kg −1 ), calcium (3.9– 8.9 g kg −1 ), magnesium (0.2– 0.6 g kg −1 ), ash (13.2– 27.2 g kg −1 ) and bark percentage (3.6–8.1%) was found in stem (bark+wood) samples. A lower amount of variation was documented for specific gravity (0.33– 0.48 g cm −3 ) and percent moisture (49–56%). Bark had a higher concentration of inorganic elements and ash, relative to wood. Willow clones as a group had a higher specific gravity, bark percentage and calcium concentration relative to hybrid poplar clones, which had a higher potassium concentration. The two groups were similar in terms of the concentrations of other elements and ash. Clonal variation in these characteristics present opportunities for manipulating feedstock quality through selection, breeding and plantation management.

High biomass yield achieved by Salix clones in SRIC following two 3-year coppice rotations on abandoned farmland in southern Quebec, Canada

Two species of willow, Salix discolor and S. viminalis, were planted in 1995 under short-rotation intensive culture on two abandoned farmland sites: sandy site (S1) and clay site (S2). After three seasons of growth the two species were coppiced. In the spring of the first season following coppicing, one dose of composted sludge equivalent to 100kg of “available” Nha−1 was applied to some plots (T1) while others were left unfertilized (T0). The aims of the experiment were to compare the growth performance and nutrients exported by willow species planted on marginal sites with different soil characteristics and to assess the impact of fertilization with wastewater sludge on yields during a second rotation cycle.Over three seasons, willow height, diameter and aboveground biomass were greater for S. viminalis than for S. discolor on all fertilized plots. The best growth performance of two willows were obtained on the clay site. S. viminalis, planted on the fertilized plots of the clay site, had the highest biomass yield (70.36tDMha−1). The application of a dose of wastewater sludge (100kg of “available” Nha−1) was not enough to satisfy all nutritional requirements of willows for the period of growth. Over the second rotation the nutrients removed from the soil by willows (in kg per ton of dry mass harvested) were: from 5.3 to 7.5 for N; from 0.6 to 0.9 for P; from 1.8 to 3 for K; from 4.2 to 7.2 for Ca and from 0.4 to 0.7 for Mg.