- Open Access
Efficient hydrogen production from the lignocellulosic energy crop Miscanthus by the extreme thermophilic bacteria Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana
© de Vrije et al.; licensee BioMed Central Ltd. 2009
- Received: 19 March 2009
- Accepted: 17 June 2009
- Published: 17 June 2009
The production of hydrogen from biomass by fermentation is one of the routes that can contribute to a future sustainable hydrogen economy. Lignocellulosic biomass is an attractive feedstock because of its abundance, low production costs and high polysaccharide content.
Batch cultures of Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana produced hydrogen, carbon dioxide and acetic acid as the main products from soluble saccharides in Miscanthus hydrolysate. The presence of fermentation inhibitors, such as furfural and 5-hydroxylmethyl furfural, in this lignocellulosic hydrolysate was avoided by the mild alkaline-pretreatment conditions at a low temperature of 75°C. Both microorganisms simultaneously and completely utilized all pentoses, hexoses and oligomeric saccharides up to a total concentration of 17 g l-1 in pH-controlled batch cultures. T. neapolitana showed a preference for glucose over xylose, which are the main sugars in the hydrolysate. Hydrogen yields of 2.9 to 3.4 mol H2 per mol of hexose, corresponding to 74 to 85% of the theoretical yield, were obtained in these batch fermentations. The yields were higher with cultures of C. saccharolyticus compared to T. neapolitana. In contrast, the rate of substrate consumption and hydrogen production was higher with T. neapolitana. At substrate concentrations exceeding 30 g l-1, sugar consumption was incomplete, and lower hydrogen yields of 2.0 to 2.4 mol per mol of consumed hexose were obtained.
Efficient hydrogen production in combination with simultaneous and complete utilization of all saccharides has been obtained during the growth of thermophilic bacteria on hydrolysate of the lignocellulosic feedstock Miscanthus. The use of thermophilic bacteria will therefore significantly contribute to the energy efficiency of a bioprocess for hydrogen production from biomass.
- Chemical Oxygen Demand
- Lignocellulosic Biomass
In view of the transition to hydrogen as a major energy carrier in the future, new routes for hydrogen production need to be explored. The production of hydrogen from biomass is one of the options for contributing to the supply of exploitable renewable resources. Hydrogen can be produced from a vast range of biomass, using thermochemical, as well as fermentative, processes. Carbohydrates, such as sugars, starch or (hemi)cellulose, are the prime substrates for fermentative processes. For future sustainability of the energy supply, the utilization of (hemi)cellulose is of prime interest, as this component is most abundant in crops that can be grown for the purpose of energy supply.
To date, many studies have been done on fermentative hydrogen production from pure sugars and from feedstocks, such as by-products from the agricultural and food industry, municipal waste, or wastewaters . However, only a few studies describe the production of hydrogen from lignocellulosic biomass (reviewed in ). Some of these feedstocks were offered as solid materials, such as office paper waste , wheat straw waste , delignified wood fibres , and a variety of cellulosic waste materials . Other types of biomass, such as Miscanthus , paper sludge , corn stover , and corn stalks  were first pretreated and/or hydrolyzed to obtain a soluble substrate of mixed sugars and oligosaccharides. Bacterial consortia from anaerobic digester sludge or cow dung compost, as well as single cultures of mesophilic (Clostridium acetobutylicum) and thermophilic bacteria (Clostridium thermocellum, Caldicellulosiruptor saccharolyticus and Thermotoga elfii), have been used as hydrogen producers. The reported hydrogen yields on these lignocellulosic substrates varied greatly from approximately 10% to more than 80% of the theoretical value, which is 4 mol of hydrogen per mol of hexose. The diversity of the applied feedstocks and pretreatment methods hardly allow a comparison of hydrogen production efficiency.
It is proposed that research should focus on the conversion of biomass to fermentable substrates to obtain a successful introduction of cellulosic biomass for the production of biofuels . The fermentability of lignocellulose is improved by pretreatment of the biomass. This is required to overcome the recalcitrance of the lignocellulosic complex by altering its structure, which makes the cellulose and hemicelluloses accessible to the enzymes . One of the methods of interest is pretreatment with an alkaline agent at relatively low temperatures (<100°C). One of the main effects of the alkali is the disruption of the intermolecular bonding between xylan and other biomass components, such as lignin or other hemicellulosic components, resulting in increased porosity of the lignocellulosic biomass. In addition, applying alkali in a water solution generally leads to swelling of cell wall material, thereby increasing the internal surface of the lignocellulosic matrix. These effects (that is, increase of porosity; swelling of fibrous material) lead to an increase in enzymatic degradability, as the lignocellulosic matrix is more accessible for enzymes. Another effect, less well understood, is the modification of lignin during alkaline pretreatment. Depending on the alkali used and the biomass type, alkaline pretreatment may lead to lignin depolymerisation, and (partial) dissolution of lignin components.
This study describes the efficiency of hydrogen production from Miscanthus, a lignocellulosic energy crop. Miscanthus is a rapidly growing perennial C4 grass with relatively high yields of 8 to 15 ton dry weight per ha in Western European regions, which requires only low inputs of nutrients for cultivation. It has been studied for years because of its potential for future energy supply. Previously, we have compared mechanical methods of pretreatment, milling and extrusion, in combination with chemical treatment. The applied methods were aimed at the high conversion of polysaccharides and high yields of monomeric sugars . In the present study, the effect of various chemicals used for alkali pretreatment on the fermentability of the feedstock is investigated. For this, two extreme thermophilic bacteria were used, the cellulolytic bacterium Caldicellulosiruptor saccharolyticus of the order Clostridiales , and the moderately halophilic bacterium Thermotoga neapolitana of the order Thermotogales , which grow at optimum temperatures of 70 and 80°C, respectively. Thermophilic bacteria are superior with respect to hydrogen yield [15–17], due to favourable thermodynamical conditions at high temperatures, and reduced variety in by-product formation. Furthermore, many thermophilic bacteria, including T. neapolitana and C. saccharolyticus, are able to utilize a wide range of substrates for growth from simple sugars to complex carbohydrates [18, 19]. Unlike T. neapolitana, C. saccharolyticus is capable of growth on crystalline cellulose , and on lignocellulosic feedstocks, although degradation of these substrates was limited . Therefore, the pretreatment was followed by enzymatic hydrolysis of Miscanthus to prepare fermentable substrates. The soluble fraction, that is, the hydrolysate, will be a complex mixture of monomeric C6 and C5 sugars, and di- and oligosaccharides. The production of hydrogen from all of these sugars is required to develop an efficient process. The results showed that both thermophiles were able to consume most, if not all, soluble carbohydrates present in the Miscanthus hydrolysate. Hydrogen was produced at high yields of more than 75% of the theoretical value.
Comparison of alkaline pretreatment methods
Effect of alkaline pretreatment on Miscanthus biomass.
Lignin, % of insoluble fraction
% of initial dry matter
Effect of different methods for alkaline pretreatment and enzymatic hydrolysis on the Miscanthus hydrolysate composition.
Sugars (g l-1)
The selection of an optimal pretreatment method will not only be determined by the efficiencies of the biomass pretreatment, and the enzymatic hydrolysis of the polysaccharides, but also by the fermentability of the hydrolysates. Hydrolysates were prepared from batches of approximately 200 g of milled Miscanthus, which were pretreated using NaOH or Ca(OH)2, titrated with different acids prior to enzymatic hydrolysis, and hydrolyzed with commercial enzyme preparations, after which the sugar composition of the hydrolysates was determined (Table 2). In addition, the acid usage in the preparation of hydrolysate IV was reduced by circa 50% through washing with water prior to pH adjustment. Glucose and xylose were the main monosaccharides in the hydrolysates. The highest amount of monomeric sugars was found in hydrolysate III, which has been prepared by pretreatment with the highest amount of alkali. This resulted in a polysaccharide conversion efficiency of circa 55%.
Fermentability of hydrolysates on small scale and inhibitory compounds
Fermentability of Miscanthus hydrolysates by C. saccharolyticus and T. neapolitana.
IC20 (g sugars l-1)a
5 to 7.5
7.5 to 10
7.5 to 10
Effect of chemicals on the growth of C. saccharolyticus and T. neapolitana and the production of hydrogen and organic acids (acetic and lactic acid).
10 to 30
35 to 50
1 to 2
2 to 4
1 to 2
2 to 4
Salts, such as calcium chloride, potassium phosphate, and sodium sulfate, also inhibit C. saccharolyticus (Table 4). The presence of calcium and sulfate ions in hydrolysate I (Table 2) probably contributed to the strong inhibition with this hydrolysate. The inhibition of C. saccharolyticus was not observed with hydrolysate II, because the concentration of calcium ions was reduced through formation of an insoluble salt when phosphoric acid, instead of sulfuric acid, was used for pH adjustment. The growth of T. neapolitana appeared to be stimulated by the presence of calcium ions up to 50 mmol l-1, and the organism is less sensitive for phosphate and sulfate (Table 4). The inhibition of fermentation by hydrolysate I and II is, therefore, likely to be caused by other components in these hydrolysates.
Inhibitory compounds may be generated during the pretreatment and hydrolysis of lignocellulosic biomass. These include degradation products of sugars, such as the aldehydes furfural and 5-hydroxymethyl furfural (HMF). The effects of 0 to 4 g l-1 of furfural and HMF on the fermentability of glucose were tested in small flasks. Both C. saccharolyticus and T. neapolitana were inhibited by these compounds. C. saccharolyticus appeared to be more sensitive (Table 4). Actively growing cells of C. saccharolyticus fully metabolized furfural to mainly furfuryl alcohol (>80%). However, at 4 g l-1, hardly any growth was observed and the furfural was no longer converted. Similar results were obtained with HMF, although the conversion products were not identified. T. neapolitana also metabolized furfural and HMF. Furfural was partly converted to furfuryl alcohol, while other conversion products were not identified. Previous reports also showed inhibition of growth of other bacteria by similar concentrations of furfural and HMF (IC50 at 1 to 4 g l-1; [23, 24]), but a stimulatory effect (up to 3 g l-1) on the growth of Clostridium beijerinckii has also been reported . The reduction of the furaldehydes to alcohols has been mentioned earlier as a detoxification step under both aerobic and anaerobic conditions . In yeast, alcohol dehydrogenases and xylose reductase were responsible for the reduction of furfural and HMF [26, 27]. Bacterial enzymes involved in furaldehyde reduction have not been identified yet, but the reduction appeared to be constitutive with reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) acting as the electron donor . The concentration of furaldehydes in hydrolysate I and II was less than 0.1 g l-1 and, therefore, the inhibition of T. neapolitana was not caused by these compounds.
Hydrolysate preparation on bench scale and fermentability
Composition of Miscanthus hydrolysate prepared at the bench scale.
COD, g l-1
Di- and/or oligosaccharides
During enzymatic hydrolysis, the pH of the slurry decreased from the initial pH of 5.2 to 4.3. Besides the release of organic acids from the lignocellulosic material during hydrolysis, this decline can be ascribed to lactic acid formation by contaminating bacteria. The relative amount of other, unknown, organic compounds amounted to 21% of the total COD of the hydrolysate. Because of the mild process conditions (moderate temperature, ambient pressure, and no extreme acidic pH), sugar degradation products are not expected to be formed. Indeed, furfural and HMF were not detected in the hydrolysate (detection limit 10 mg l-1).
Anaerobic batch fermentations under controlled conditions in a bioreactor
Fermentation parameters of 1 l batch cultures of C. saccharolyticus at 72°C and T. neapolitana at 80°C grown on a glucose/xylose (7:3, w w-1) mixture and on Miscanthus hydrolysate.
On pure sugars at 10 g l-1, carbon balances were more than 90%. At increased substrate concentrations the carbon balances often became less, indicating that more non-identified products were formed. Part of these products were volatile components, since COD balances, including the gaseous hydrogen, were still incomplete and volatile components in the gas stream were not collected. The large amount of non-defined products in the T. neapolitana fermentation at high hydrolysate concentration was due to the Maillard reactions.
Hydrogen yield and productivity
The molar yields of products on the consumed substrates are shown in Table 7. The hydrogen yields (YH2) at low-sugar concentrations were generally more than 3 mol H2 per mol hexose, with a maximum of 3.4 mol per mol hexose obtained in fermentations with C. saccharolyticus. For C. saccharolyticus, the hydrogen yield after growth on hydrolysate was equally high as that from pure sugars. The yields with cultures of T. neapolitana on hydrolysate varied between 89% to almost 100% of the yield on pure sugars. The yield dropped substantially at the highest substrate concentrations. The biomass yield (Yxs) of both thermophiles also decreased with increasing substrate concentrations.
Molar yields and maximal volumetric hydrogen productivity (QH2, max) of C. saccharolyticus and T. neapolitana batch cultures grown on a glucose/xylose (7:3, w w-1) mixture and on Miscanthus hydrolysate.
mol (mol C6)-1
g (mol C6)-1
mmol l-1 h-1
Most of the hydrogen was produced during the exponential growth phase, but even when the cell density decreased, hydrogen was still produced, although often at a lower rate (Figure 5). The hydrogen production rate by T. neapolitana was very similar on pure sugars and on Miscanthus hydrolysate. The productivity by C. saccharolyticus on hydrolysate was lower than the productivity on pure sugars (Figure 5), which is in line with the lower sugar consumption rate. Apparently, C. saccharolyticus is inhibited by the hydrolysate.
The maximum volumetric hydrogen productivity (QH2, max) occurred at the late exponential growth phase of the thermophiles. The highest QH2, max of 14.5 mmol l-1 h-1 was observed with T. neapolitana that was fermenting 10 g l-1 of pure sugars (Table 7). The maximum p H2 measured in the off gas was 4.6 kPa. In the C. saccharolyticus fermentations, hydrogen pressures of circa 4 kPa were found, which is significantly lower than the critical value of 10 to 20 kPa. For T. neapolitana the critical value has not been established yet.
Cultures of C. saccharolyticus and T. neapolitana produced hydrogen and acetic acid as the main organic acid during the fermentation of sugars in hydrolysates prepared from the lignocellulosic energy crop Miscanthus. The mild conditions of alkaline treatment enabled the optimized pretreatment protocol to be compatible with the thermophilic fermentations. The formation of potential inhibitors, for example, sugar degradation products, was negligible. For future applicability on an industrial scale this one-stage process needs to be further optimized with respect to alkali usage, for instance by regeneration and recycling of the alkali agent.
Both thermophiles, C. saccharolyticus and T. neapolitana, appeared to be able to simultaneously and completely utilize all soluble monomeric C5 and C6 sugars, di- and oligosaccharides up to a total sugar concentration of 17 g l-1. The capacity of co-fermenting glucose and xylose by C. saccharolyticus was recently confirmed by whole-genome transcriptome analysis . Simultaneous and complete substrate utilization from complex feedstocks, such as the hydrolysates of lignocellulosic biomass, will add to an energy-efficient process and is a major advantage in industrial scale production facilities.
The observed hydrogen yields resulting from the thermophilic fermentations were 74 to 85% of the theoretical value of 4 mol per mol hexose in fermentations with circa 17 g l-1 total sugars. These are amongst the highest hydrogen yields obtained in the fermentation of sugars in lignocellulosic hydrolysates reported to date. C. saccharolyticus offers the advantage of a nearly 10% higher hydrogen yield during growth on Miscanthus hydrolysates as compared to T. neapolitana. However, the rate of substrate consumption and hydrogen production by T. neapolitana was higher. C. saccharolyticus seemed to be hampered by the increase in ionic strength of the culture medium during fermentation and showed lower hydrogen productivity in the stationary phase. Because T. neapolitana is a moderately halophilic organism, it tolerates conditions with higher ionic strength, and possibly with higher osmolalities.
Cultures growing on Miscanthus hydrolysates had low volumetric hydrogen productivity with a maximum of 13 mmol l-1 h-1 and a mean productivity of circa 7 mmol l-1 h-1. Higher volumetric hydrogen productivities have been reported for systems with higher cell densities, such as the carrier-induced granular sludge bed (CIGSB) bioreactor. Productivities of more than 300 mmol H2 l-1 h-1 have been observed, but the hydrogen yield of circa 50% on pure sucrose was low .
The COD reduction of the culture medium containing Miscanthus hydrolysate was limited to 30% because the carbohydrates were only partially oxidized to organic acids. More hydrogen can potentially be produced from the organic acids in the effluent by photobacteria, which use light as an extra energy source. Theoretically, another 4 moles of H2 and 2 moles of CO2 can be produced from acetic acid. The first experiment was done with supernatant that was obtained from a culture of T. neapolitana after growth on Miscanthus hydrolysate. Rhodobacter capsulatus, a purple non-sulfur bacterium, was able to grow and produce hydrogen on the supernatant, which was diluted twice, and supplemented with Fe(III)-citrate . The yield of the two fermentations was circa 4.5 moles of hydrogen per mole hexose, that is, 37% of the theoretical value of 12 moles of hydrogen per mole of glucose. Further research is aimed at improving the hydrogen production efficiency of this combined thermophilic and photoheterotrophic fermentation .
Biomass pretreatment and enzymatic hydrolysis
Miscanthus giganteus was collected in the spring of 2004 from a location in Groningen, The Netherlands and consisted primarily of stems. The dry matter content of the harvested stems was circa 80 to 85% on a wet weight basis. The total carbohydrate and lignin content on a dry weight basis was 63% (including 42% glucose, 19% xylose, and 1% arabinose) and 23%, respectively.
Alkaline pretreatment experiments at the lab scale (2 l pulp mixer, Quantum Mark V reactor) were done with milled Miscanthus (Retsch SM 200 mill equipped with a 2 mm screen). An amount of 225 g of biomass (circa 200 g dry matter) was pretreated with 9 to 12% Ca(OH)2 or NaOH (w w-1 dry matter, for details see Tables 1 and 2) at a solid:liquid ratio of 0.125 (w w-1). Pretreatment was done at 85°C for 16 h. One batch of pretreated Miscanthus was washed three times with demineralised water. Prior to enzymatic hydrolysis, the pH of the pretreated material was adjusted to 4.8 to 5.1 with 17% phosphoric, 20% sulfuric or 25% acetic acid (v v-1). Enzymatic hydrolysis of the pretreated biomass was done using commercial enzyme preparations (Cellubrix and Novozymes 188 from Novozymes, Bagsvaerd, Denmark and GC 220 from Genencor, Rochester, NY, USA). The amount of enzyme added per 100 g dry matter was 28, 9, and 13 ml of Cellubrix, Novozymes 188 and GC 220, respectively (for details see Table 2). The enzyme concentration was selected to warrant a similar cellulase activity of 15 IFPU per g dry matter, on the basis of cellulase activity measurements . Incubation was done at 50°C for 24 h.
A pretreatment and hydrolysis experiment was also done at the bench scale in a 10 l stirred vessel. An amount of 1.35 kg (circa 1 kg dry matter) of milled Miscanthus (Pallmann type PS 3–5 knife mill equipped with a screen of 10 mm × 10 mm square opening) was added to the vessel, together with NaOH, under continuous mixing (90 rpm). The amount of NaOH was 9% (w w-1) at a solid:liquid ratio of 0.125 (w w-1). The pretreatment was done for 6 h at 75°C. The material was then dewatered using a manual piston press to make a slurry of 275 g dry matter l-1. The pH of the remaining viscous pulp was adjusted to 5 using a 20% (v v-1) acetic acid solution. Enzymatic hydrolysis was done by a fed-batch procedure of GC 220 addition. The incubation was at 50°C for 24 h.
Hydrolysates were collected after neutralization of the pH and removal of the solids of the enzymatically hydrolyzed material by centrifugation. The hydrolysates were stored at -15°C until use.
Microorganisms and medium
C. saccharolyticus DSM 8903 and T. neapolitana DSM 4359 were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). The culture medium consisted of (per l) KH2PO4 0.3 g, K2HPO4 0.3 g, MgCl2.6H2O 0.4 g, NH4Cl 0.9 g, yeast extract 1.0 g, cysteine-HCl 0.75 g, FeCl3.6H2O 2.5 mg, SL-10 trace elements 1 ml, and resazurin 0.5 mg. The pH was adjusted to 7.0 at room temperature. NaCl (5 g per l) was added to the culture medium of T. neapolitana. The culture medium for flask experiments was supplemented with 50 mM 4-morpholine propanesulfonic acid (MOPS) to increase the buffering capacity of the medium. A mixture of glucose and xylose (7:3, w w-1) or Miscanthus hydrolysate sugars were used as the carbon source. The medium was made anoxic by flushing with N2. The experiments were carried out under non-sterile conditions. C. saccharolyticus and T. neapolitana were grown at 72 and 80°C, respectively.
The fermentability of hydrolysates was tested using flasks of 118 ml with 20 ml culture medium under a nitrogen atmosphere. The total monosaccharide concentration was 10 g l-1 coming from the pure sugar mixture, the hydrolysate, or a combination of both. The flasks were inoculated with 5% (v v-1) of a preculture that was grown overnight on the same pure sugar mixture. After 16 and 40 h, samples were withdrawn from the headspace (duplicate gas sample of 0.2 ml) and the culture medium (single sample of 1 ml) for analyses of the hydrogen production, cell density, pH, and organic acid production. The experiments were carried out in duplicate (two flasks per condition). The inhibition of compounds was tested using the same method, except that 10 g l-1 of glucose was used as the carbon source.
Batch fermentations under controlled conditions were carried out in a jacketed 2 l bioreactor (Applikon, Delft, The Netherlands) with a working volume of 1 l. The pH was controlled at circa 6.8 (measured at room temperature) by automatic addition of 2 N NaOH. The cultures were continuously stirred at 350 rpm and sparged with N2 at 7 l h-1. Inoculation was done by adding 10% (v v-1) of a preculture that was grown overnight on the glucose/xylose mixture. A fermentation was considered to have ended when the hydrogen concentration was less than 0.2% in the off gas. Samples of 7 ml were regularly taken from the culture medium for measurement of the cell density and substrate and product analyses. Hydrogen and CO2 were measured in the off gas each hour. Data are from one representative fermentation per condition.
Determination of the acid-soluble and acid-insoluble lignins was performed according to the Tappi method . Organic acids were analyzed by high performance liquid chromatography (HPLC) using a Shodex ionpak KC811 column (Waters, The Netherlands), as described earlier . Monosaccharides, di- and oligosaccharides, furfural, HMF and furfuryl alcohol were analyzed by HPLC using an Altech IOA-1000 column at 90°C, with 3 mM sulfuric acid as the mobile phase (0.4 ml per min), followed by detection by differential refractometry. Fructose was determined enzymatically (Megazyme International Ireland Ltd, Bray, Ireland). Hydrogen in the headspace of the serum bottles and hydrogen and CO2 production in the bioreactors were measured as previously described . COD measurements of the culture medium and hydrolysates were done using the LCK test kit 014 of Hach Lange (Düsseldorf, Germany). The hydrolysis of di- and oligosaccharides in hydrolysates to monomeric sugars was done by the addition of concentrated sulfuric acid (95 to 97%) to a final concentration of 1 M and incubation at 95°C for 1 h. The optical density of the cultures was measured against a water blank at 580 nm after dilution of the culture broth with deionised water. The cell dry weight was determined from the highest value of the optical densities using the relation CDW (g l-1) = (0.377 × OD580) + 0.011 for C. saccharolyticus  and CDW (g l-1) = (0.528 × OD580) for T. neapolitana. The molecular weight of T. neapolitana was assumed to be the same as the measured value for C. saccharolyticus, that is, 24.6 g (mol C)-1 .
Yield and productivity
The amount of consumed substrate, including non-defined organic compounds, was used for calculating product yields. Yields were expressed as mol product per mol C6 sugar. Because the theoretical hydrogen and acetate yields per C-mol are equal for glucose and xylose , the molar amount of xylose was converted to a molar amount of hexose. The consumed unknown organic compounds were determined from COD measurements. They were considered to be carbohydrates with the same product yield as for glucose and xylose. COD in mmol O2 l-1 was converted to mmol hexose l-1, according to the equation C6H12O6 + 6O2 → 6CO2 + 6H2O. The maximum volumetric hydrogen productivity was calculated from the time interval with the highest percentage of hydrogen in the off gas.
This study was financially supported by the Commission of the European Communities, Sixth Framework Programme, Priority 6, Sustainable Energy Systems (019825 HYVOLUTION), the Dutch Programme EET (Economy, Ecology, Technology), a joint initiative of the Ministries of Economic Affairs, of Education, Culture and Sciences and of Housing, Spatial Planning and the Environment (EETK03028 BWPII), and the Dutch Ministry of Agriculture, Nature and Food Quality. Mr. A. Drenth (Agromiscanthus B.V.) is acknowledged for the supply of Miscanthus.
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