Steam Pretreatment Optimisation for Sugarcane Bagasse in Bioethanol Production

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1 Steam Pretreatment Optimisation for Sugarcane Bagasse in Bioethanol Production Johan Sendelius Master of Science Thesis 2005 Department of Chemical Engineering, Lund University, Sweden Abstract: Steam pretreated sugarcane bagasse was enzymatically hydrolysed by a blend of Celluclast 1.5L and Novozym 188 cellulase mixtures. This was done to evaluate different pretreatment parameters (time, temperature and impregnating agent) with the intention of finding conditions which generate high sugar yields. The pretreatment conditions assessed were: temperature: 180, 190 and 205 ºC; time: 5 and 10 minutes; and impregnating agents: water, 2% SO 2 by weight of water in the bagasse and finally 0.25 g H 2 SO 4 per 100 g dry matter. To avoid end-product inhibition the enzymatic hydrolyses were performed with 2% DM loads under continuous stirring. Hydrolysis was carried out for 72 h and temperature was kept at 40 ºC, ph was initially 4.8. After evaluation, four pretreatment conditions were examined in an SSF process with 5% DM by weight in order to see fermentability behaviour and performance of the materials under presence of inhibitors generated under the pretreatment process. In the hydrolysis experiments SO 2 - pretreatment at temperatures 180 and 190 ºC for 5 minutes (samples 7 and 13) gave rise to overall yields of % based on glucose content. When performing SSF experiments ethanol yields of 80% of theoretical were obtained for three of the conditions tested. The high total xylose yields for pretreatment sample 7 made it the most suitable condition, since pentose sugar fermentation will be employed in the future. Overall, hemicellulose removal rather than delignification were evident as the most important action during the pretreatment process. Moreover, this study indicates that smaller particle sizes in the raw bagasse may improve the performance of the material. INTRODUCTION Since the 20 th century, our major energy demand has been supplied by fossil fuels such as: oil, coal, and natural gas. Fossil fuels originate from deceased organisms that lived several million years ago and by time have been embedded in the earth s crust. Incineration of this fossil remains results in a net-increase of today s carbon dioxide levels. Environmental issues such as the threatening increase in temperature caused by the greenhouse effect and the fact that fossil fuels are non-renewable resources, has increased the interest in producing fuels from renewable resources, e.g. biomass. Ethanol as well as other biofuels produced from plant biomass, is an alternative to fossil fuels. Ethanol does not add to a net-co 2 atmospheric increase, thus there is in theory no contribution to global warming. Combustion of ethanol results in relatively low emissions of volatile organic compounds, carbon monoxide and nitrogen oxides [1]. In order to introduce ethanol as a largescale transportation fuel, the production cost must be lowered to about the same level as petrol and diesel. Today the production cost of ethanol from lignocellulose is still too high, which is the major reason why ethanol has not made its breakthrough yet. When producing

2 ethanol from maize or sugarcane the raw material constitutes about 40-70% of the production cost [2]. By using cheaper waste products from forestry, agriculture and industry, the costs may be lowered. However, it is of economical concern obvious to make use of the feedstock as efficient as possible, e.g. to improve the production process and as a result achieve higher ethanol yields [3], [4]. Production of ethanol is historically a wellknown process and in principal it is carried out by fermentation of plant sugars into ethanol using strains of yeast. However, plant biomass is made of polymers of sugars, ordered in a matrix called lignocellulose, which is not as easily fermented. In order to produce ethanol, this material must undergo degradation to for the yeast more accessible components, e.g. mono- and dimers of sugars. This degradation can be made by hydrolysis of biomass using added enzymes, called enzymatic hydrolysis (EH). To help the enzymes to perform well and degrade the lignocellulose efficiently, the fibres in the raw material need to be accessible to the enzymes. A pretreatment in some way is needed to expose the fibres. If the pretreatment is too harsh, liberated sugars can be degraded to enzyme- and yeast-inhibiting compounds lowering the overall yields. On the other hand, if too weak pretreatment conditions are used this will result in low enzyme accessibility and the same drawbacks. Industrially, the pretreated material is mainly thought to be hydrolysed and fermented in two different steps: separate hydrolysis and fermentation (SHF); or in one single step: simultaneous saccharification and fermentation (SSF). The aim of this study was to compare and optimise various steam pretreatment conditions with respect to final ethanol yield, using sugarcane bagasse as feedstock. Enzymatic hydrolyses were performed in lab scale to evaluate pretreatments that were made. To include the level of fermentability, material from four of the most promising pretreatments was fermented in an SSFprocess. Total ethanol- and sugar yields for the tested conditions were evaluated to eventually find the most satisfying pretreatment condition. MATERIALS AND METHODS Raw Material Sugarcane bagasse from the sugar plant Usina Bom Jesus (Cabo/PE, Brazil) was kindly supplied to the laboratory in Lund. The material was stored in plastic buckets at approximately 5ºC in a refrigerated room. Before the analyses and operations the bagasse was rinsed in water to remove soil- and gravel particles and then pressed to remove a considerable part of the water. The dry matter content (DM) of the bagasse was initially 48%. Steam Pretreatment Previously the equipment has been described in detail [5].The equipment consists of a 10L pressure reactor followed by a flash cyclone tank to accumulate the pretreated bagasse. Bagasse equivalent to 300 grams of dry matter was loaded into the pre-heated reactor. The bagasse was treated by saturated steam produced by an electric boiler, and this was done at temperatures of 180, 190 or 205ºC during 5 or 10 minutes. See Table 1. Since too high temperatures were suspected by Martín [6] as the reason for obtaining low yields, the pretreatments in this study were carried out using comparably low temperatures (<210ºC). When impregnating the bagasse with sulphur dioxide, the bagasse was put in a plastic bag and then SO 2 -gas was added from a gas container corresponding to 2.0% by weight based on the water content of the bagasse. The bag was then sealed and the gas was let to distribute and impregnate the bagasse by first distributing the material and then let it settle for about two hours at low temperature, 5ºC. Sulphuric acid was used as impregnation by mixing with bagasse in a plastic bucket with dilute acid to get a concentration of 0.25g H 2 SO 4 /100g dry bagasse. The bucket was covered and kept at room temperature overnight.

3 Table 1. The table shows the tested pretreatment conditions and the corresponding sample numbers used. The (*) means that H 2 SO 4 instead of SO 2 was used, and the values are in grams per 100g dry bagasse, compared to the SO 2 -experiments where the amount is % of total water weight in the bagasse. Sample 18 had the same condition as number 14 but instead, sieved bagasse was used. Sample Raw Material Proc variables # DW T SO 2 t Log (g) ( o C) (%) (min) R , ,0 5 3, , , , ,0 5 3, ,0 10 3, ,0 10 4, ,0 10 3, ,25* 10 3, ,25* 10 4, ,25* 10 3,36 Enzymatic Hydrolysis The pretreated material was separated from the soluble sugars. This was done by stirring the bagasse in hot water for one hour in a plastic jar using an electric stirrer. After the stirring the bagasse was vacuum filtrated and rinsed thouroghly by hot distilled water. 10 g DM of washed bagasse was then placed in a glass vessel and was then diluted to a final concentration of 2% by weight with addition of acetate buffer solution and a mixture of cellulolytic enzymes. The buffer solution used was 0.1 M NaAc and had recently been produced prior to the experiments. The cellulases added were Celluclast 1.5L supplemented by β- glucosidase preparation Novozym 188 kindly given by Novo Industri A/S, Bagsvaerd Denmark. The activity of Celluclast 1.5L is 65 FPU/g and 17 β-glucosidase IU/g, while the enzymatic activity of Novozyme 188 is 376 β-glucosidase IU/g [7]. A fixed amount of enzymes; 2.32 g Celluclast and 0.52 g Novozym188 was added in each hydrolysis flask. The vessel holding 500 g of material at ph 4.8 was then kept in a water bath (40ºC) and the content was continuously stirred for 72 hours. Enzymatic hydrolysis of the pretreated washed bagasse where in all cases done in duplicates. Samples were taken for carbohydrate analysis after 0, 2, 4, 6, 24, 48 and 72 hours. For mass balance purposes and quantification of liberated sugars the liquid and solid fraction was eventually separated by vacuum filtration. Enzymatic hydrolysis was also performed on ethanol and hydrogen peroxide washed pretreatment condition #13. This was to see if hydrolysis was improved if a great part of the redistributed lignin in the pretreated material was removed before hydrolysis. The pretreated bagasse was rinsed thoroughly in 96% ethanol (denoted 13E) or hydrogen peroxide (denoted 13P or 13PW), respectively, before the hydrolysis experiments were made. SSF Experiments The SSF trials were made at 5% DM pretreated non-washed bagasse concentration in 2L fermentors (Labfors, Infors AG, Switzerland) where the content had a total weight of 1400 g as follows: 70 g DM of pretreated bagasse was put in a fermentor flask, distilled water was added and then the fermentor was sealed and put in autoclave (121 ºC, 10 min). Along with the fermenters, a flask of nutrients (0.5 g/l (NH4) 2 HPO 4, g/l MgSO 4 *7H 2 O and 1.0g/L of dry yeast extract) were put in the autoclave. Proportional to the DM in the EHtrials ((70/10)*2.32))=16.24 g of non-sterile Celluclast and 3.64 g of Novozym 188 was

4 then added to the room temperature fermentors. Finally, 7 g DM of baker s yeast, Saccharomyces cerevisiae (Jästbolaget, Rotebro, Sweden), corresponding to 5 g/l together with the rest of the water was added. The ph was automatically adjusted to ph 5.0 by addition of small amounts of 10% NaOH water solution. SSF was performed in duplicates for 72 hours at ph and temperature was maintained at 37 ºC. Samples where taken at 0, 2, 4, 6, 8, 12, 24, 36, 48, 72 hours and analysed for ethanol, sugars and by-products. In some cases additional samples were taken after 72 hours. In the initial phase the yeast rapidly ferments soluble sugars from the pretreated bagasse. This can create extensive foaming and when it became a problem anti-foam agent (Dow Corning TM Antifoam RD Emulsion) was added in small amounts until the foam settled. Composition Analysis To calculate sugar yields both the solid bagasse and process liquids must be analysed by composition. The produced solids were treated according to the National Renewable Energy Laboratory (NREL) standard procedure Determination of Structural Carbohydrates and Lignin in Biomass [8]. The time-consuming method is roughly carried out by sieving dried sample and then extracting the sugars by first adding 74%- followed by 4% sulphuric acid. The solids left are assumed to contain lignin and some ash, while the sugars and the acid soluble lignin are distributed in the liquid fraction. Samples are taken from the liquid fraction and processed as described below. However, some of the sugars are degraded by the strong acid and this is compensated by a reference degradation of a duplicate of sugar standards along with the other samples. All sugar composition analyses were done in duplicates. A more detailed description can be found in the literature mentioned. The liquids produced after the solid material analysis and the liquid phase after each pretreatment step, as well as before, after and during EH and SSF were analysed as described in NREL s procedure Determination of Sugars, By-products and Degradation Products in Liquid Samples [9]. Sugar standards were also used in this HPLC analysis, and these where made in excess previous to the analyses. The samples were stored in a fridge at -18ºC when not analysed in the HPLC to prevent spoilage by microbes. RESULTS & DISCUSSION Results from the Pretreatment The pretreatment generated material with variation in visual appearance. Since steam condensed, the pretreated material was turned into a slurry containing solid particles. In general, higher severity gave a darker and more fragmented slurry. Sample number 14 and 18, showed obvious difference in hemi-cellulose sugar extraction. Sample 18 was sieved and it resulted in 69.3% higher xylose extraction and 89.8% higher arabinose extraction during pretreatment compared to 14. Additionally, glucose extraction was substantially higher in the sieved bagasse. Enzymatic Hydrolysis Results The sugar yields give an estimation of how much of the bound sugars can be liberated during hydrolysis. These glucose yields based on pretreated material can be seen in Figure1

5 1 0.9 Glucose Yields in Enzymatic Hydrolysis a-sample b-sample Yield E PW Sample Number Figure 1. The figure shows the glucose yields achieved during the enzymatic hydrolysis. Yields are ranging from 0 to 1, where 1 means 100% hydrolysation. The tests were carried out in duplicates, thus both a- and b-sample are shown. By comparing yields for number 14 and 18, the sieved sample showed higher sugar yields. In average the sieved samples obtained 27.7% higher glucose yields compared with the non-sieved bagasse. In general, the SO 2 -impregnated bagasse turned out to generate a pretreated material which gave the highest sugar yields. The sample which showed highest glucose yields was sample number 13; in average 86.3%, while xylose yields were in average 72.0%. Sample number 7 gave highest xylose yields, in average 84.3%, but the glucose yields were in average 73.2%, which is lower than number13. By comparing yields for number 13E and 13PW with number 13 one can see that removing soluble lignin by washing with ethanol or hydrogen peroxide did not have apparent positive effects. However, the removing of lignin must be made on more conditions to see the effects, since this number 13 had good performance (86% glucose yields) in the EH, the possibility to improve it is low. Total Yields When incorporating the pretreatment step in calculating the yields, thus obtaining the overall yields of glucose and xylose, one gets a total picture of the combined effect of both pretreatment and enzymatic hydrolysis. This total sugar yield is actually the most important yield since it deals with all process steps from the raw bagasse to fermentable sugars. If sugars are degraded during the pretreatment step one can get high yields during enzymatic hydrolysis anyway. However, this loss of sugars will have negative impact on total yields. In Figures 2 and 3 on next page the total yields for glucose and xylose are shown respectively.

6 1 Total Yields of Glucose Yield E P Pretreatment Number Figure 2. Duplicate values of total yields of glucose for the different pretreatment conditions. 1 Total Yields of Xylose Yield E P Pretreatment Number Figure 3. Duplicate values of total yields of xylose is shown for each pretreatment condition. For total glucose yields, SO 2 -impregnation generally gave the best results, but this was more uncertain when looking at total xylose yields. Of the non-sieved bagasse samples, number 13 (190ºC, 2% SO 2, 5 min) gave the highest glucose yields, in average 96.3% and number 7 (180ºC, 2% SO 2, 5 min) gave the highest xylose yields, 66.6% in average. Number 18 with sieved bagasse reached xylose yields of 72.6% which compared to the same non-sieved bagasse pretreatment number 14 was an improvement by 80.6%. The same comparison as above but with total glucose yield gives an improvement of using small particle sizes by 40.5%.

7 Effects of Hemicellulose Removal during Pretreatment According to the data, the removal of xylose indicates positive impact on the cellulose hydrolysis and consequently the glucose yields, which is shown in Figure 4. Glucose Yield Effect of Xylose Extraction during Pretreatment on Total Glucose Yield R 2 = Figure 4. The experimental relation between xylose extraction during pretreatment and total glucose yield. Higher xylose extraction show higher glucose yields Xylose Extraction (%) 80 SSF Results Fermentations could not be done on all the pretreatment conditions; only four was chosen. When choosing which pretreatment condition to do fermentations on, total yield of glucose and xylose was used as evaluation parameters. Number 13 was chosen because it had the highest glucose yield; number 7 was used for its high xylose yield and good glucose yield. Number 15 was chosen as a reference, to see the effect of fermenting a moderately performing material. Number 16 must be seen as a test of extending the experimental track sheet having high temperature (205ºC) and short time (5 minutes) as pretreatment parameters. The higher temperature is likely to generate larger amounts of inhibitors which decrease the ethanol yields. The results of the SSFexperiments can be seen in Table 2. Table 2. This table shows ethanol produced in the SSF experiments and the total ethanol yield. Total ethanol yield was compared with the theoretical yield of fermentation. SSF Lactic Acid Ethanol Produced Total Ethanol Yield SAMPLE (g/l) (g) (% of Theoretical) 7 a b a b a b a b Three of the pretreatment samples showed similar results; approximately 80% of theoretical ethanol yield. The three samples reached ethanol maxima in about the same time. Number 16 reached the lowest yields, but this was partly probably due to the contamination of lactic acid bacteria.

8 CONCLUSION The SO 2 -impregnation showed best sugar yields of the pretreatment impregnation methods tested. The most prominent tested pretreatment condition was: SO 2 -impregnation with a temperature of 180ºC during 5minutes, which gave an overall ethanol yield of 80%, based on theoretical value. Overall, glucose yields indicated to be dependant on hemicellulose removal in the pretreatment step. Smaller particle size gave higher yields, by comparing sample 14 & 18. This may indicate that grinding of the raw bagasse can improve total yields. Hydrolysis rates generally decreased over time, which is the same behaviour as found in literature. REFERENCES [1] Bailey, BK. Performance of ethanol as a transportation fuel. In: Wyman CE (editor). Handbook on bioethanol: production and utilization. Taylor and Francis, Bristol, Pa. pp [2] Claassen, P. A. M., van Lier, J.B., Lopez Contreras, A.M., van Niel, E. W., Sijtsma, L., Stams, A. J. M., de Vries, S.S., Weusthuis, R.A Utilisation of biomass for the supply of energy carriers. Appl. Microbiol. Biotechnol. 52: [3] Hinman, N. D., J. Schell, C. J. Riley, P. W. Bergeron, and P. J. Walter Preliminary estimate of the cost of ethanol production for SSF technology. Appl. Biochem. Biotechnol : [4] Lynd, L. R Ethanol production from lignocellulosic substrates using thermophilic bacteria: critical evaluation of potential and review, Adv. Biochem. Eng. Biotechnol. 38: [5] Palmqvist, E., Hahn-Hägerdahl, B., Galbe, M., Larsson, M., Stenberg, K., Szengyel, Z., Tengborg, C., Zacchi, G Design and operation of a bench-scale process development unit for the production of ethanol from lignocellulosics. Bioresource Technol. 58: [6] Martín, C., Galbe, M., Nilvebrant, N. O., Jönsson, L. J Comparison of the fermentability of enzymatic hydrolyzates of sugarcane bagasse pretreated by steam explosion using different impregnating agents. Appl. Biochem. Biotechnol : [7] Söderström, J., Pilcher, L., Galbe, M., Zacchi, G Two-step steam pretreatment of softwood with SO 2 impregnation for ethanol production. Appl. Biochem. Biotechnol. vol [8] Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D NREL Biomass Program: Determination of structural carbohydrates and lignin in biomass. Biomass Analysis Technology Team, Laboratory Analytical Procedure Department of Energy, United States of America. [9] Sluiter, A., Hames, B., Ruiz, R., Sluiter, J., NREL Biomass Program: Determination of Sugars, By-products and Degradation Products in Liquid Samples. Biomass Analysis Technology Team, Laboratory Analytical Procedure Department of Energy, United States of America.

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