Extraction of high molecular mass hemicelluloses prior to ethanol production Alkali steam pretreatment of wheat and barley straw Elisabeth Joelsson Department of Chemical Engineering Lund University P.O. Box 124, SE-22100 Lund, Sweden 2008 Abstract The aim of this study was to investigate the possibility to extract large hemicellulose chains from wheat and barley straw by alkali steam pretreatment followed by enzymatic hydrolysis to extract glucose. The hemicelluloses, that mainly consist of xylan, could then be used to manufacture barrier films and the glucose to produce bioethanol. To receive as high as possible yield of large xylan chains the influence of three operating conditions were studied: reactor temperature, residence time and alkali concentration. The conditions were choosen in such a way that they were possible to implement in industrial scale. The enzymatic hydrolysis was preformed during 72 hours at a fibre content of 5% and with three different enzymes; Novozyme 188, Celluclast 1.5 L and Multifect Xylanase. The results showed that it was possible to receive a better yield of large xylan chains, during moderate process conditions, with barley straw than with wheat straw. The best results were obtained with a NaOH concentration of 1.5%, a temperature of 190 C and a residence time of about 20 minutes giving a yield of large xylan chains of approximately 25-3 of the xylan retrieved in the straw. The results from the enzymatic hydrolysis also show that it was possible to extract more than 7 of the glucose in the residual material from the steam pretreatment when executing the hydrolysis at 5% fiber content. Introduction Increasing oil prices and the great impact of fossil fuels on the environment has arisen the interest to reveal new alternative sources, which could be used as energy supplier and replace fossil based materials such as plastics. In the search of new renewable raw materials it has been natural to study the agricultural and forest industry. At present the main cereal crops produced in Europe are wheat and barley with an amount of approximately 120 million tons wheat and 60 million tons barley grain per year. This could be a considerable energy supply; however it could also be questioned if it is ethically right to use human food for energy production. Therefore it would be interesting to see if it could be possible to use the waste material resulting from cereal crops to replace oil-based products. This would convert the waste material into a high-value added product that can help reduce the green-house effect without competing with the food production. For every kilo of wheat and barley grain that is produced 1.3 kg straw is received. This would mean that only in Europe the yield of wheat and barley straw approximately would reach 160 and 80 million tons, respectively. Straw consists to a great extent of cellulose and hemicelluloses which could be extracted and used to make a variety of products. Two products that can be derived from cellulose and hemicelluloses are bio-ethanol and bio-plastics. Bio-ethanol can be used as energy supply especially in the transportation sector which would reduce the emission of fossil carbon dioxid to the environment. The bio-plastic could be used to replace oil based films that today are used as 1
oxygen barrier films in the packing industry. That could make cardboard and plastic packing s biodegradable and easier to dispose which would be a benefit to the environment [1,2,3]. The aim of this work was to investigate the possibility to utilize wheat and barley straw for more high-added value purposes than is the case today. The study has been concentrated on two major steps. The first step was to extract hemicelluloses larger than 4 kda from the straw by alkali impregnation followed by steam pretreatment to reach a yield as high as possible with optimized conditions. The hemicelluloses are then going to be used for production of barrier films. The second step was to investigate the possibility to extract sugar from the residual material, from the first step, with enzymatic hydrolysis for ethanol production. This will make it possible to achieve two valuable products from a low cost material. Alkali impregnation followed by steam explosion was used in this study because it is a relatively simple process that can be made in industrial scale. Alkali treatment is as a contrary to acid impregnation more kind to the material, protecting it from breaking down entirely into monomeric sugar. A total degradation of the polymer chains would prevent the possibility to obtain the two earlier mentioned products in the process [3]. Materials and methods Raw material The raw material consisted of wheat and barley straw and was provided by two local farmers. The material which had a dry matter of approximately 9 was cut into smaller pieces and stored at room temperature. Pretreatment The raw material was divided into batches containing 300 gram dry matter each. The batches were pretreated separately through alkali impregnation followed by steam explosion to find the optimum condition to extract high-molecular-mass hemicelluloses. The alkali solutions were made of NaOH and altered between the three different concentrations 1, 1.5 and 2%. The NaOH solution was sprayed onto the straw during agitation for 30 minutes giving the material a dry mater of 3. The batches were then separately steam pretreated in a pretreatment unit comprising a 10 L reactor, described elsewhere [4], during different residence times and temperatures. The residence times were 10, 20, 30, 40, 50 minutes and the temperatures were 180 and 190 C. The pretreatments were made in two separate screenings, where the first one contained batches from both wheat and barley and the second only contained barley. The results from the different settings were compared by severity factor. The severity factor would give a measurement of the harshness of the treatment due to the residence time and temperature used. The calculation of the severity factor was made according to equation 1. ( T Tref ) Log ( R 0 ) = Log t exp (1) 14.75 Where t is the residence time in the reactor, T is the temperature used and T ref is a reference temperature set to 100ºC [ 5]. Filtration The steam pretreated material consisted of a slurry of solid material and hydrolyzed liquid, which were separated through filtration. The filtration was made in a manual 3-L press resulting in a liquid phase containing hemicelluloses and a solid residual phase that were subjected to enzymatic hydrolysis. The ph of the liquids from the different batches were measured and afterwards the solutions were filtrated through a 1µm filter and stored in separate flasks before analysis. The analyses were later made to determine the sugar concentration and the size distribution of the sugar molecules in the liquid. 2
Enzymatic hydrolysis Enzymatic hydrolysis was made on six of the pretreated batches to investigate the amount of glucose that was possible to extract from the residula material. The batches, of which three came from the wheat straw and three from the barley straw, were selected due to its high yields of xylan and moderate severity factors. The solid residual material from the choosen batches were washed to eliminate any dissolved suger left that came from the liquid phase and then the experiment were set up to run as a duplicate. The amount of washed material needed were calculated according the total solid (TS) in the washed material and the desire to enzymaticaly hydrolysis 40 gram substrate in a total volume of 800 gram giving a fiber content of 5%. The washed material corresponding to 40 gram substrate was placed in a glas flask and the enzymes were prepered in an beaker with about 300 ml Millpore water and the ph was regulated to approximately five with sodium hydroxide at the same time. The utilized enzymes and amount were the following; 2.08 gram Novozyme 188 (500 β- glucosidase IU/g) (Novozyme A/S, Bagsvaerd, Denmark), 9.28 gram Celluclast 1.5 L (60 FPU/g, 30 β-glucosidase IU/g) (Novozyme A/S, Bagsvaerd, Denmark ) and 2 gram Multifect Xylanase (43gprotein/ml) (Genecorn international, inc, Rochester NY, USA). This corresponds to a FPU of approximatley 14 FPU/g substrate and 33 IU/ g substrate. The mixture of enzyms was poured into the glas flask and Millipore water was added up to 800 gram. The flasks were afterward placed with a lid and stirring equipment in a water bath at 40-50 C and the time recorded. Samples were withdrawn after approximately 4,6,21, 31, 47, 51 and 72 hours and filtrated through a 0.2 µm filter before analysed to investigate the glucoses concentration. Analytical methods Raw material analysis To examine the content of the two different types of straw and the residual material two analysis were made according to the standardized method of the NREL (National Renewable Energy Laboratory). In the first one, concentrating on the straw, the structural carbohydrates, lignin and ash were determined and in the second one, made for the residual material, only the carbohydrates were determined [6]. High Performance Anion Exchange Chromatography To determine the concentration of polysaccharides in the liquid phases highperformance anion-exchange chromatography (HPAEC) which is described elsewhere [7], was used. Because the detector could only analyse monomeric sugars the polysaccharides in addition had to be degrade with acid hydrolysis according to a standardized NREL method (Ruiz and Ehrman, 1996) before run in the HPLC [8]. Size exlusive chromatography To decide the size distribution and approximated molecular mass of the arabinoxylan size exclusion chromatography (SEC) was used which is described elsewhere [9]. The system controller was programmed to separate the outgoing fluid into nine fractions, where the first four fractions contained molecules larger than 4 kda. The sugar concentration in the fractions were later analysied with HPLC as described earlier. Results Raw material analysis The results of the raw material analysis made on wheat and barley straw are presented in table 1 and the composition of the residual material used in the enzymatic hydrolysis is presented in table 2. 3
Table 1. The composition of wheat and barley straw. Components Wheat straw Barley straw Glucan 41,8% 36,3% Xylan 25,4% 21,7% Galactan 0,7% 1, Arabinan 2,5% 2,6% Mannan 0, 0, Lignin 28, 30,4% Ash 2,1% 0,3% Table 2. The composition of the solid residual. Batch Arabinan Galactan Glucose Xylan Wheat1 3% 2% 48% Wheat3 3% 2% 49% 23% Wheat4 3% 2% 52% 18% Barley7 3% 2% 5 19% Barley9 3% 2% 49% 19% Barley10 3% 2% 5 18% Screening The results from the first screening gave that the total yield of xylan was somewhat better for the barley batches than the wheat batches. This can be seen in Figure 1 showing the increasing yield with the severity factor. Furthermore it can be seen in Figure 2 that the ph are declining with harsher severity factor. Comparing the different NaOH concentrations in Figure 2 it also shows that the declinings are greater fore the ones impregnated with 1 % NaOH than the ones impregnated with stronger solutions. % xylan yield 4 35% 3 25% 15% 5% Total xylan yield with 1 % NaOH pretreatment 3,6 3,7 3,8 3,9 4 4,1 4,2 4,3 factor Figure 1. The total yield of xylan extracted at 1% NaOH impregnation for wheat and barley, based on the amount of xylan in the material before pretreatment. Yield Barley Yield w heat ph 12 10 8 6 4 2 0 ph-change at 1, 1,5 and 2 % NaOH concentration 3,6 3,7 3,8 3,9 4 4,1 4,2 4,3 Barley NaOH 1% Barley NaOH 1.5 % Barley NaOH 2% Wheat NaOH 1% Figure 2. The declining of the ph in the wheat and barely batches after pretreatment with different NaOH concentrations. The size distribution was measured in order to study the yield of xylan chains over 4 kda. The yield was plotted against the severity factor and the results are presented in Figure 3. The figure shows that the xylan yields were much higher for the barley batches than for the wheat batches. This was probably due to the great declining in the ph for the wheat batches, presented earlier, which most likely degraded the xylan polymer chains into too small chains. Therefore the second screening were targeting on barley straw and an optimsation was performed at 190 C and a residence time range of 10-30 minutes, due to the high xylan yield and moderat severity factor shown in Figure 3. Xylan yield % 25% 15% 5% Yield of xylan chains bigger than 4kDa 3,6 3,8 4 4,2 4,4 Barley 190 Wheat 190 Barley 180 Wheat 180 Figure 3. The xylan yield of molecules with molecule mass > 4kDa that is possible to extract from barley and wheat straw at certain pretreatment conditions In the second screening the temperature was kept constant at 190 C and the NaOH concentration and the residence time were altered. The results fore the overall xylan yield 4
are plotted against the severity fatcors and the results are presented in Figure 4. In the diagram it is shown that the highest overall yield is obtained at 1.5% NaOH concentration. % xylan yield 4 3 Total xylan yield for 1.5 and 2% NaOH 3,6 3,7 3,8 3,9 4 4,1 4,2 Barley NaOH 1.5% Barley NaOH 2% Figure 4. The yield of xylan extracted at 1.5 and 2% NaOH impregnation based on the amount of xylan in the material before pretreatment The size distribution investigation of the second screening are shown in Figure 5 with the results from the barley batches run at 1% NaOH as a reference. Comparing the results it shows that the best results for extracting large xylan molecules were given at 1.5 % NaOH concentration, 190 C and approximately 20 minutes recidence time. Comparing the declining of the ph and xylan yield for large molecules at 1.5 and 2% with 1 % it can furthermore be seen that a higher ph seems to give a better yield. Xylan yield % 3 25% 15% 5% Yield of xylan bigger than 4kDa 3,6 3,8 4 4,2 4,4 Barley 190ºC 1.5% Barley 190ºC 2% Barley 190ºC 1% Figure 5. The xylan yield of large molecule chains possible to extract from barley straw at certain pretreatment conditions Enzymatic hydrolysis The extracted glucose from the enzymatic hydrolysis was compared to the total amount of glucose in the residual material giving the glucoses yield. The average glucoses yield from the two duplicates were then plotted against the recidence time as can be seen in figure 6. Yield glucose % Extracted yield of glucose from the solid material 8 7 6 5 4 3 0 500 1000 1500 2000 2500 3000 3500 Time min Barley 190 C 10 min Barley 190 C 20 min Barley 180 C 30 min Wheat 190 C 10 min Wheat 190 C 20 min Wheat 180 C 30 min Figure 6. The extracted yield of glucose from the residual material for the chosen wheat and barley batches. The results shows that it was possible to obtain a yield of over 7 glucoses from some of the single samples at a fibre content of 5%. Discussion and conclusions The first screening made with barley and wheat straw with 1% NaOH impregnation shows that the total yield of xylan is increasing with increasing severity factor. It also shows that the ph is decreasing substantially with a higher severity factor. Comparing the two materials it could be seen that it was possible to reach a better yield of xylan from the barley straw than from the wheat straw. This was also true when investigating the size distribution where the barley batches contained a larger yield of big molecule chains. This was due to a lower reduction of ph in the barley batches which prevents the chains to degrade to a great extend in the barley batches. Overall this gave a better result for the batches containing barley straw since the investigation was targeting on receiving a high yield of xylan chains larger than 4 kda that could be used for production of barrier films. When studying the outcome of the time and temperature influence on the barley batches it showed that the batches run at 5
180 C and 30 to 50 minutes could compete with the batches run at 190 C in giving a good yield of large molecule chains. However the residence time used in the trials at 180 C are probably too long relative to the extra yield gained. The conditions used in the batch run at 190 C and 20 minutes therefore appeared to be the most suitable to investigate further in a second screening due to the high yield of large xylan chains and reasonable residence time in the batch. The second screening was done to investigate the influence of the NaOH impregnation on the xylan yield and at the same time trying to optimise the chosen conditions from screening one. In this screening it was shown that the impregnation at 1.5% gave an overall higher yield of xylan compared to the 1% impregnation. For the batches impregnated with 2% NaOH however the results were reversed. It was also shown that the ph was remarkable higher in the batches from the second run compared to the batches from screening one due to the elevated NaOH concentrations. In the size distribution the effect of the elevated ph could be noticed since both the 1.5 and 2% NaOH impregnations gave higher yields of xylan chains over 4 kda compared with the batches impregnated by 1% NaOH. The higher ph hindered the chains from being cut into shorter chains to a greater extent than in the first barley batches. When combining the results from the total yield of xylan and the yield from the size distribution it was shown that the impregnation made at both 1.5 and 2% gave a substantially higher yield of large xylan chains than the impregnation made at 1%. This even though the total yield from the 2% NaOH impregnation was lower. When comparing the yield of long molecule chains after treatment with 1.5 and 2 % NaOH and a residence time of 20 minutes it was shown that the yield for 1.5% was in the range of 25-3 and the yield for 2% was located in the range of 20-25%. Subsequently the optimal conditions to extract large xylan molecules from barley straw at low alkali concentration that could be used to make barrier films from would be at 1.5% NaOH concentration, a residence time of about 20 minutes and a temperature of 190 C. This setting would make it possible to implement the process industrial due to the fact that low alkali concentrations and moderate conditions are used. During the first screening an enzymatic hydrolysis was also preformed to investigate the possibility to extract glucose from the residual material that could be used to produce ethanol. The investigation was made on residual material received from both wheat and barley batches. The result showed that it was possible to extract as much as 7 of the glucose found in the waste material when running the experiments with 5% fibre content. According to earlier studies this yield could probably be increased by using lower fibre content. Consequently the study has shown that it could be possible to receive a reasonable high yield of both large xylan chains and glucose from the wheat and barley straw at moderate process conditions and low costs when summarizing the results. References 1 Eurostat. ttp://epp.eurostat.ec.europa.eu/cache/ity_off PUB/KS-ED-08-001/EN/KS-ED-08-001- EN.PDF. date 081108. 2 D.Montane, X. Farriol, J.Salvadó, P. Jollezand, E. Chornet, Application of steam explosion to the fractionation and rapid vaporphase alkaline pulping of wheat straw, Biomass and Bioenergy. 14:3 (1998) 261-276. 3 http://chalmersnyheter.chalmers.se/article.jsp?articl e=5676 6
4 E. Palmqvist, B. Hahn-Hägerdal, M. Galbe, S. Larsson, K. Stenberg, Z. Szengyel, et al Design and operation of bench-scale process development unit for production of ethanol from lignocellulosics. Bioresource Technology 58:2 (1996) 171-179 5 R.P. Overend and E.Chornet, Fraction of lignocellulosics by steam-aqueous pretreatments, Philosophical Transactions of the Royal Society of London. Series A, Mathematical and Physical Sciences, 321:1561 (1987) 523-536. 6 A. Sluiter, B. Hames, R.Ruiz, C. Scarlata, J. Sluiter, D.Tempelton, D. Crocker, Laboratory Analytcial Procedure, Determination of structural carbonhydrates and lignin in biomass, Version 2007, National Renewable Energy Laboratory Midwest Research Istitute for the Department of Energy, USA. 7 A. Andersson. Roos, T. Persson, H. Krawczyk, G. Zacchi and H. Stålbrand, Extraction of water-soluble hemicelluloses from barley husks, Bioresource Technology. 100:2 (2009) 763-769. 8 R. Ruiz and T.Erhman, Laboratory Analytcial Procedure-014 (1996), Dilute Acid Hydrolysis Procedure for Determination of Total Sugars in the Liquid Fraction of Process Sample, National Renewable Energy Laboratory Midwest Research Istitute for the Department of Energy, USA. 9 T.Persson, E Dinh and A.-S. Jönsson. Improvement of arabinoxylan isolation from barley husks. Food and Bioproducts Processing, doi:10.1016/j.fbp.2008.10.001 7