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Tim Sieker1 Andreas Neuner2 Darina Dimitrova3 Nils Tippkotter1 Kai Mufer1 Hans-Jorg Bart3 Elmar Heinzle2 Roland Ulber1
1

Research Article

Ethanol production from grass silage by simultaneous pretreatment, saccharication and fermentation: First steps in the process development
Grass silage provides a great potential as renewable feedstock. Two fractions of the grass silage, a press juice and the ber fraction, were evaluated for their possible use for bioethanol production. Direct production of ethanol from press juice is not possible due to high concentrations of organic acids. For the ber fraction, alkaline peroxide or enzymatic pretreatment was used, which removes the phenolic acids in the cell wall. In this study, we demonstrate the possibility to integrate the enzymatic pretreatment with a simultaneous saccharication and fermentation to achieve ethanol production from grass silage in a one-process step. Achieved yields were about 53 g ethanol per kg silage with the alkaline peroxide pretreatment and 91 g/kg with the enzymatic pretreatment at concentrations of 8.5 and 14.6 g/L, respectively. Furthermore, it was shown that additional supplementation of the fermentation medium with vitamins, trace elements and nutrient salts is not necessary when the press juice is directly used in the fermentation step.
Keywords: Bioethanol / Ethanol / Grass / Lignocellulose / Process integration Received: September 14, 2010; Received: June 6, 2011; accepted: June 9, 2011 DOI: 10.1002/elsc.201000160

Institute of Bioprocess Engineering, University of Kaiserslautern, Kaiserslautern, Germany Biochemical Engineering, Saarland University, Saarbrucken, Germany . Chair of Separation Science and Technology, University of Kaiserslautern, Kaiserslautern, Germany

Introduction

Grasses belong to the most important crops world wide. They are used for food and grazing and also provided the energy for mobility and industry over many centuries [1]. Nowadays, they are receiving increasing interest for the same purpose, since an intense search for renewable resources is going on. Thirteen percent of the EU 27s area are covered by permanent grassland [2]. Due to breeding results and changing agricultural technologies, the area needed for feeding cattle is decreasing, so that surplus grassland can be used for growing renewable resources. Data on the surplus grassland are available only from regional studies. For the German region of BadenWurttemberg, currently 132 000 ha or 21% of the grassland are . not needed anymore for the production of feed. By 2015, this area will increase to 26% of the total grassland in BadenWurttemberg [3]. At the average German grassland yield of . 8 t/ha [4] about 1.3 106 t haymass could be harvested from

Correspondence: Professor Roland Ulber (ulber@mv.uni-kl.de), Institute of Bioprocess Engineering, University of Kaiserslautern, Gottlieb-Daimler-StraXe 44, 67663 Kaiserslautern, Germany Abbreviations: FAE, ferulic acid esterase; SSF, simultaneous saccharication and fermentation

this area. In addition to the declining grassland area required for feeding cattle, German laws ascertain the conservation of grassland according to cross compliance rules. For this purpose, a decrease of grassland area of more than 5% compared to the 2003 status is prohibited. If it should decrease by more than 8% compared to 2003, the growing of new grassland could be instructed by the federal state governments [5, 6]. Although grasses have many advantages when used as renewable resources compared to one-season energy crops [7, 8], the storage is a major problem, since grasses decay very fast. This would limit their applicability as feedstock in Europe to the growth period from May to October. For this reason, haymaking and ensiling have been developed and optimized for the long-term storage of grasses in agriculture. In this study, ensiling, a wet storage mode, is used. During the ensiling process, lactic acid is produced by Lactobacilli, which causes conservation of the grass clippings for up to 1 year, provided that anaerobic conditions are maintained [9]. Besides the conservatory effect, lactic acid is among the 30 most promising chemicals as dened by the US Department of Energy (http://www1. eere.energy.gov/biomass/pdfs/35523.pdf [31.08.2010][10]). So

Additional corresponding author: Tim Sieker

E-mail: tim.sieker@mv.uni-kl.de

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a valuable component can be isolated from the silage as byproduct. Similar to other lignocellulosic feedstocks, grass lignocelluloses require a pretreatment prior to saccharication for the production of fuel ethanol or bulk chemicals. But compared to wood-derived lignocelluloses, the grass cell wall shows major differences. The most important is the low lignin content of below 10%, which contributes to the fast decay. Some tissues are even completely free of lignin [11, 12]. Nonetheless, a simple saccharication of the cellulose and hemicellulose is not possible, since phenolic acids, mainly ferulic and p-coumaric acid, hinder the hydrolysis [13, 14]. These are ester-bound to arabinose side-chains of the hemicelluloses and mask the hemicelluloses physically. They can also dimerize and by that crosslink the hemicelluloses. Furthermore, they anchor the lignin to the hemicelluloses [15]. The ester-bonds between the phenolic acids and the hemicelluloses can be released by alkaline conditions [16]. Therefore, an alkaline peroxide pretreatment [17] has been established, which releases the phenolic acids from the cell wall and also oxidizes part of the lignin. As the fast decomposition of the grass during storage indicates, microorganisms also have ways to overcome the recalcitrance of grass lignocelluloses against hydrolysis. For this purpose, ferulic acid esterases (FAE) are used. The effect of FAEs on saccharication of grass lignocelluloses has been shown before [13, 18]. Since the FAEs act synergistically with cellulases and hemicellulases in natural systems, the integration of a pretreatment by FAEs into a simultaneous saccharication and fermentation (SSF) approach for the production of ethanol from grass-silage is investigated. Furthermore, the utilization of all components of the silage is aspired. This can be achieved by a biorenery concept as shown in Fig. 1. In this concept, the silage is at rst separated into a liquid and a solid fraction by pressing. From the liquid fraction (the press juice) the organic acids, mainly the lactic acid, are removed. The crude extract can either be used for the production of lysine,

for which genetically optimized Corynebacterium glutamicum strains are under construction [19], or the lactic acid can be further puried. The lignocellulose contained in the bers of the solid phase (the press cake) is enzymatically saccharied and used for fermentations, for example, for the production of fuel ethanol. The rafnate from the lactic acid extraction is used for the supplementation of the hydrolysates. Residues from the fermentation processes are to be used for biogas production. The parts of the concept are currently under investigation. First results for the ethanol fermentation stage are presented here and have also been partially published in [19].

Materials and methods

The silage was taken from a local farm (Munchweiler an der . Alsenz, Hofgut Neumuhle, Dienstleistungszentrum l. ndlicher a . Raum (DLR) Westpfalz). The silage is made from a grass mixture with perennial ryegrass as main component and is from the rst cut in May. It has been ensiled for 1 year and was stored at 201C afterwards. As determined by a simplied nutritional value analysis carried out by the Landwirtschaftliche Untersuchungs und Forschungsanstalt Speyer (Speyer, Germany) via near infrared spectroscopy, the used silage contains approximately 236 g cellulose, 176 g hemicellulose, 249 g non-ber carbohydrates and 22 g Lignin per kg dry mass. Dry matter content is 420 g/kg at a pH of 5.01. The silage quality is rated very good by the analyzing institute. The press juice was produced with a manually driven highpressure tincture press (type HP2, Fischer GmbH, Neuss, Germany). Per approach 500 g silage moist mass were pressed at 100 bar for 40 min. This resulted in 0.748 L press juice (dry mass content 23.3%) and 0.754 kg press cake (dry mass) per kg silage dry mass (equivalent to 2.4 kg moist mass). The press juice contains about 20 g/L glucose, 17 g/L xylose/galactose, 44 g/L fructose/arabinose and 44 g/L lactic acid. The rafnate

Figure 1. Flow scheme of the biorenery project under investigation [19].

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was prepared by extracting silage press juice with the same volume of 0.724 M Cyphos IL 104 (Cytec Industries, NJ, USA) in decanol at 251C for 2 h. Phase separation was carried out by centrifugation. Cellulase (Celluclast 1.5 L, 700 endoglucanase units/g), b-glucosidase (Novozym 188, 250 cellobiase units/g) and hemicellulase (Shearzyme 500 L, 500 fungal xylanase units/g) were taken from Novozymes Biomass kit (Novozymes A/S, Bagsvaerd, Denmark). FAE preparations were provided by Biocatalysts (Biocatalysts, Cardiff, Wales, UK). Depol 740 L (36 units/g) was taken for the pretreatment and Depol 670 L, a mixture of FAE with several other activities including cellulase (1200 cellulase units/g, 800 pectinase units/g), was taken for the integrated approaches. All enzymes were technical liquid preparations. Activities are as described by the manufacturer. If not stated otherwise, the press cake used for hydrolysis or fermentation was dried at 501C and chipped to bers of 12 mm length prior to the experiments. For the alkaline peroxide pretreatment, press cake was added to a 0.6% H2O2 solution to a concentration of 40120 g/L and the solution was adjusted to pH 11 with 10 M NaOH. The approaches were incubated in 25 mL total volume in an overhead-mixer (IntelliMixer, Neolab Migge Laborbedarf-Vertriebs GmbH, Heidelberg, Germany) at 25 rpm and 501C for 24 h. The experiments for the enzymatic hydrolysis and the enzymatic pretreatment were carried out likewise at 40120 g press cake per liter in a McIlvaine buffer consisting of 490 mL/L of a 21 g/L citric acid solution and 510 mL/L of a 35.6 g/L Na2HPO4 solution at pH 5. Enzyme loadings per gram substrate were 4 mg Celluclast 1.5 L (160 mL), 0.5 mg Novozym 188 (16 mL), 0.7 mg Shearzyme 500 L (53.3 mL) and, in case of the enzymatic pretreatment, 8.6 mg Depol 740 L (750 mL). Saccharomyces cerevisiae (DSM 70449) and Pachysolen tannophilus (DSM 70352) were used for the fermentation. The latter is able to utilize Xylose for the production of ethanol [2022]. The microorganisms were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Precultures of both organisms were grown overnight at 301C and 140 rpm on a complex medium containing 10 g/L sugar, 3 g/L yeast extract, 3 g/L malt extract and 5 g/L soy bean peptone to a cell concentration of 6 g/L. For S. cerevisiae 10 g/L glucose and for P. tannophilus 10 g/L xylose were used as carbon source. The screening experiments for the ethanol production were carried out in 25 mL head-space GC vials with a culture volume of 10 mL. The vials were sealed with Butyl/PFTE-caps and ushed with nitrogen after inoculation and sampling to ensure anaerobic conditions. Fermentations were carried out in a rotary shaker (Ecotron, Infors HT, Bottmingen, Switzerland) at 301C and 200 rpm. Fermentations for the investigation of the inuence of lactic acid were carried out as described above. Lactic acid was added from an autoclaved stock solution to the required concentrations and the pH was adjusted to 5 using sterile 10 M NaOH. The approaches were inoculated with 1 mL of the S. cerevisiae preculture. For the fermentation experiments, 120 g/L substrate in McIlvaine buffer, pH 5, were used. The enzyme solutions employed for the SSF were as stated above. For the simultaneous

pretreatment, saccharication and fermentation 16 mg Depol 670 L (0.5 mL) were used per gram substrate. The required nitrogen and phosphorus sources, vitamins and trace elements were supplied by adding 3.3% of silage press juice or stock solutions of the substances as described by Schlegel (3 g/L (NH4)2SO4, 2.5 g/L Na2HPO4, 2 g/L MgSO4, 1 mL/L trace element stock solution (per liter: 100 mg MnCl 4H2O, 200 mg CoCl2 6H2O, 25 mg NiCl2 6H2O, 2 mg CuCl2 2H2O, 70 mg ZnCl2, 50 mg NaMoO4 2H2O, 3 mg Na2SeO3 5H2O), 3 mL/L vitamin stock solution (per liter: 2 mg Biotin, 20 mg Niacin, 10 mg Thiamin, 10 mg 4-Aminobenzoate, 5 mg Pantothenate, 50 mg Pyridoxamine, 20 mg Cyanocobalamin) [23]). The fermentations were inoculated with 5% of each preculture. Sugar concentrations were determined by HPLC (300 8 mm Reprogel-Ca11-column, Dr. Maisch GmbH (Ammerbruch-Entringen, Germany), eluent water, 801C, 0.5 mL/min, detection: RI). Xylose and galactose as well as fructose and arabinose could not be separated by this method, so they are shown together. For the ethanol analysis, a headspace GC was used (Clarus 500, Perkin Elmer (Waltham, MA, USA), with 30 m 250 mm Elite-Wax-capillary (Perkin Elmer), head-space sampler at 801C, GC at 601C, carrier helium at 120 kPa, detection: FID). Organic acids were measured by HPLC (300 4.6 mm Reprogel-H1-column, Dr. Maisch GmbH, eluent 9 mM H2SO4, 551C, 0.5 mL/min, detection: UV).

Results and discussion

The easiest way to gain a fermentation medium from grass silage would be the utilization of a press juice. By the utilization of only this fraction it would be possible to press the silage on the farm and leave the solid fraction, the press cake, behind, which could be used as fodder. This approach would reduce the logistic costs [24]. Only little literature is available on the use of silage press juice as cultivation medium [2527], none of these dealing with the production of ethanol. A major hindrance for the fermentation of the press juice is the high concentration of organic acids. Besides several other organic acids, it contains 4050 g/L lactic acid and 10 g/L acetic acid. As shown in Fig. 2, lactic acid has a strong inhibitory effect on the growth of S. cerevisiae under anaerobic conditions. Narendranath et al. reported that the inhibitory effect of mixtures of organic acids is even higher than the effect of one acid alone [28]. However, this effect was expected because organic acids are the key factor for the conservative effect of ensiling. The direct fermentation of the sugars contained in the press juice to ethanol should therefore not be possible. Consequently, neither growth nor ethanol production could be observed during fermentation of the press juice. A possibility to get lactic acid concentrations that are suitable for ethanol production is to dilute the press juice. The result of this fermentation of press juice that was diluted 1:5 with water is shown in Fig. 3. Only low ethanol concentrations of 4 g/L were formed from the diluted press juice. To get higher ethanol concentrations, a fed-batch approach was tested. The batch phase was carried out like the approach described above. After 6 h, when the ethanol production of the batch approach

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Figure 2. Inuence of lactic acid concentration on the growth of S. cerevisiae under anaerobic conditions in a semi-dened medium. R2 of the linear t is 0.92.

Figure 4. Inuence of different press juice concentrations in the hydrolysate. In case of the 0 mL/L approach salts, vitamins and trace elements as described in [22] were used. Shown data are mean values of duplicates.

Figure 3. Batch and Fed-Batch Fermentation using silage press juice as medium. In both cases, the press juice for the batchphase was diluted 1:5 with water in a culture volume of 25 mL. During the fed-batch approach, the feed started after 6 h with 1 mL/h for 18 h. Shown data are mean values of duplicates.

declined, 1 mL/h undiluted press juice was added, which was kept going for another 18 h. Due to the feed, the lactic acid concentration increased from 10 to 30 g/L. By the fed-batch approach, the fourfold ethanol concentration compared to the batch approach could be reached before the ethanol production declined owing to the increasing organic acid concentration. Nonetheless, the yields stayed unsatisfactory with 11 g/kg for the batch and 14 g/kg for the fed-batch approach, calculated per kg silage dry mass (see also Fig. 6). Due to the unsatisfactory yields achieved with the press juice, the hydrolysis of the bers was tested. The pretreatment was carried out by alkaline peroxide. A problem of chemical pretreatment methods is that the salts that are present in the silage and are required for the fermentation are washed out from the silage. For the hydrolysates, supplementation with nitrogen und phosphorus sources as well as vitamins and trace elements became necessary. To avoid these additional costs, the

supplementation of the hydrolysate with the silage press juice was tested. As shown in Fig. 4, the addition of 2% v/v press juice to the hydrolysate is sufcient to achieve productivities slightly higher than with nutritional salts, vitamins and trace elements. The more the press juice added, the higher is the productivity up to a press juice level of 10%. Addition of 20% press juice resulted in a productivity increase that is much smaller than the increase from 5 to 10% press juice, probably due to the inhibition by lactic acid. Furthermore, the addition of 10% press juice in a 100 g/L approach during hydrolysis is equivalent to the complete utilization of the press juice. According to the scheme presented in Fig. 1, the press juice is used for the production of lactic acid and is not available for the supplementation of fermentations. Therefore, it was tested whether it is possible to use the rafnate from the lactic acid extraction instead of the original press juice for the supplementation of the hydrolysates. The productivity achieved with the rafnate (0.75 g L1 h1) is 86% of the productivity with the press juice (0.87 g L1 h1), indicating that also the waste stream rafnate can be used as a cheap source for nutritional salts. The extraction is carried out with a liquid ion exchange system, so that some of the nutritional salts are expected to be co-extracted with the organic acids, which explains the decrease in productivity with the rafnate. Due to the fact that so far only small samples from the process for lactic acid isolation are available, a low efciency was assumed for the screening experiments. For this reason, only 3.3% v/v press juice were added to the following fermentations instead of the 10% that would be possible. Therefore, the yields of the following experiments somewhat underestimate the possible results. Alkaline peroxide pretreated silage was either used for saccharication and subsequent fermentation or as substrate in an SSF approach. The process with two subsequent steps yielded 52 and 41 g/kg for approaches with 40 and 120 g/L, respectively. This is about 3.7-fold higher than when fermenting the press juice (see Fig. 5), but still far below the theoretical yield of 300 g/kg. Since the phenolic acids can also be removed enzymatically, a pretreatment by FAE was established. This is already

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Figure 5. Comparison of ethanol yields in different approaches for ethanol production from silage. Approach 1: batch fermentation of silage press juice, diluted 1:5. 2: Fed-batch fermentation of silage press juice. 3: Batch fermentation of hydrolysate after alkaline peroxide pretreatment with 40 g/L silage press cake. 4: Likewise, with 120 g/L silage press cake. 5: SSF with 120 g/L alkaline peroxide pretreated silage press cake. 6: SPSF with 120 g/L press cake after mechanical pretreatment. 7: SPSF with 120 g/L press cake without mechanical pretreatment. All approaches were carried out as duplicates.

Figure 6. Inuence of the integration of pretreatment and saccharication. In approach 1, pretreatment and hydrolysis are carried out in two subsequent steps of 24 h each with removal of the buffer between the steps. The displayed sugar concentrations are the sums of pretreatment and saccharication. In approach 2, the buffer was not replaced between the subsequent steps. Approach 3 is a fully integrated approach. All enzymes for pretreatment were added at once and pretreatment and saccharication were carried out simultaneously within 24 h. Control: Saccharication without FAE. All approaches were incubated at 501C and 25 rpm in a rotary shaker. Shown data are mean values of duplicates.

described in literature [13, 18], but the described processes always are two-step approaches with separate pretreatment and saccharication. All enzymes used in pretreatment and saccharication have approximately the same optimum conditions (pH 5, 501C). Due to this fact, it was investigated whether pretreatment and saccharication can be integrated to a single step. The results are shown in Fig. 6. The combination of enzymatic pretreatment and enzymatic hydrolysis is possible without losses in the sugar concentration and yield. No inhibition of the saccharolytic enzymes by the released phenolic acids is obvious at the used silage concentrations. By this combination, only half the process time is required for pretreatment and saccharication. The next step was to integrate the process described above within the fermentation step. The integration of saccharication and fermentation is a well-known strategy for process intensication in biorenery concepts [2931], resulting in shortened process times and higher yields [32, 33]. The possibility to conduct an SSF on silage was demonstrated by SSF with alkaline peroxide pretreated silage, giving at 120 g/L presscake with 53 g/kg a 22% higher yield than the subsequent hydrolysis and fermentation of this substrate. By addition of an FAE to the enzyme cocktail used for saccharication, the unpretreated silage could be used in the SSF. By this, the process time and investment costs for the pretreatment plant as well as for necessary downstream steps could be reduced. The results are presented in Fig. 5 in comparison to the other approaches. By simultaneous pretreatment, saccharication and fermentation the yields could be further increased to 91 g/kg compared to 53 g/kg in the SSF with alkaline peroxide pretreated silage, due to the fact that easily available sugars are extracted from the silage by the alkaline peroxide pretreatment.

This is about one-third of the theoretical yield. A major disadvantage of the alkaline peroxide pretreatment was the requirement for drying and chipping of the press cake, which requires a high energy input and thereby increases the process costs. In the simultaneous pretreatment, saccharication and fermentation approach, the results for dried and mechanically pretreated and undried not mechanically pretreated silage are the same (Fig. 5), so that by the enzymatic pretreatment drying and grinding steps could be removed from the concept. This is due to the disintegration of the grass bers during simultaneous pretreatment and saccharication, by which the enzymes gain access to deeper regions of the ber with the ongoing process, which was the aim of the mechanical pretreatment.

Concluding remarks

Different fractions of grass silage were tested for their possible use for the production of ethanol. The silage press juice cannot be used as a fermentation medium due to the high concentrations of organic acids. Nonetheless, this press juice can serve as a source for nitrogen, vitamins and trace elements in fermentations of hydrolysates, eliminating the requirement for additional supplementation and thus reducing the costs of the chosen approach. It was proven that it is also possible to use the rafnate from lactic acid isolation for this purpose. The saccharication of grass lignocelluloses requires a pretreatment, as it is true for all lignocellulosic materials. Due

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to the special structure of grass lignocelluloses, this can be carried out enzymatically. It was shown that it is possible to integrate the enzymatic pretreatment and the saccharication into one step, and furthermore to integrate the enzymatic pretreatment into an SSF, resulting in a simultaneous pretreatment, saccharication and fermentation. By this, the yields could be increased compared to an SSF after alkaline peroxide pretreatment. The mechanical pretreatment including the drying step required for most chemical pretreatments could be eliminated from the concept. A process for the production of ethanol from grass silage could be established, which only requires three process steps (pressing, simultaneous pretreatment, saccharication and fermentation, and distillation). In the fermentation, waste streams from other processes of the green biorenery can be utilized. Nonetheless, ethanol yields and concentration are still about factor ve too low for an industrial process. Further work will therefore focus on increasing the ethanol concentrations by fed-batch approaches and on decreasing enzyme loadings, which are the main cost factor in the described process, as well as fermentations without the used buffer system. Also, the acceptability of silages from later cuts for the described process, which typically have a higher lignin content, the isolation of the phenolic acids and the suitability of the fermentation residues for biogas production will be investigated.

The presented work was funded by the Fachagentur Nachwachsende rderkennzeichen 22025407). The authors thank Dr. P. Rohstoffe (Fo Huck from the Dienstleistungszentrum L. andlicher Raum Westpfalz for providing the silage. The authors have declared no conict of interest

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