Optimization of the pretreatment of wheat straw for production of bioethanol

Eva-Lena Jakobsson Department of Chemical Engineering, Lund University

Abstract
Bioethanol has some advantages over petrol as fuel. Bioethanol is made from biomass and it is renewable. As the biomass grows it consumes as much carbon dioxide as it forms during the combustion of bioethanol, which makes the net contribution to the green house effect zero. Wheat straw is a good raw material for ethanol production, since wheat straw has a rather high content of cellulose. To make the cellulose more accessible to enzymes the straw was pre-treated with steam at different temperatures (190, 200, 210 C) and residence times (2, 5, 10 min). Sulphuric acid was used as a catalyst; the straw was impregnated with sulphuric acid before pre-treatment. The evaluation of the pre-treatment was made by enzymatic hydrolysis and fermentation tests. The pre-treatment at 190 C and 10 min resulted in the best overall yield for glucose as well as for xylose. 100 g wheat straw yielded 42.6 g glucose and 22.0 g xylose.

Introduction
Gases that retain parts of the radiation from the sun surround the earth. But lately the concentration of these gases has increased; the result is an increased green house effect and therefore global warming [1]. By using bioethanol instead of petrol the emissions of green house gases decreases. The cellulose in biomass can be hydrolysed to sugars that can be fermented into ethanol. Wheat straw has a rather large content of cellulose, roughly 34 % [2]. The aims of this study were to do a literature study of previous work and then an experimental study of steam pretreatment of wheat straw. The results of the pre-treatment were evaluated with enzymatic hydrolysis and fermentation.

Wheat straw
The cell walls of wheat straw consist mostly of cellulose fibres. Cellulose is a linear crystalline polymer of (1-4)--Dglucose [3]. Hemicellulose is an amorphous and partly crystalline polymer, which mostly consist of (1-4)--xylose. Hemicellulose is also present in the cell walls. Lignin, a phenolic polymer, binds the fibres together. Hemicellulose and lignin protects the cellulose, which is why the wheat straw has to be pre-treated before the enzymatic hydrolysis.

Pre-treatment
The goal for the pre-treatment is to make it easier for the enzymatic hydrolysis. There are many ways of pretreating the material;

it can be done chemically, physically, biologically or as a combination of these. The pre-treatment with steam can be done with and without catalysts; a higher yield is obtained with a catalyst [4]. In this study the pre-treatment was preformed with steam pre-treatment with sulphuric acid as a catalyst. In the steam pre-treatment the straw is exposed to steam with high pressure. The water inside the cells evaporates and as the pressure around the straw drops the straw explodes. [5] The explosion increases the specific area. Parts of the hemicellulose decomposes to acids which catalyses the decomposition of hemicellulose and lignin, and releases the cellulose. The steam pre-treatment is affected by the temperature of the steam, the residence time in the reactor, the size of the particles, moisture content and the concentration of the catalyst. Log R is a function of residence time and temperature and can be used as a measurement to compare pre-treatment conditions.
(T - Tref ) Log ( R0 ) = Log t exp 14.75 t = residence time T = temperature Tref = reference temperature (100C)

Enzymatic Hydrolysis
Hydrolysis of the cellulose can be done with acids or with enzymes. The disadvantages when using acids are [5]: No selectivity A relatively low yield By-products formation Need of high temperature (140160 C) Demand of neutralisation after the hydrolysis Corrosion problems The enzymes on the other hand are selective, results in a relatively high yield and the hydrolysis is done at lower temperature. There are three different enzymes needed for the enzymatic hydrolysis of cellulose. Endo-glucanase splits the long chains of cellulose to shorter. Cellobiohydrolase cleaves units of two linked glucose molecules (cellobiose) from the ends of the cellulose chain. Finally -glucosidase cleaves cellobiose into glucose molecules. Most cellulases and -glucosidase have an optimum at 505 C and pH 4.0-5.0 [6]. Cellobiose inhibits cellulases, but the concentration of cellobiose is rather low since -glucosidase cleaves cellobiose. Glucose on the other hand inhibits glucosidase [6]. During the steam pretreatment some aromatic compounds are formed, from the lignin, which can inhibit both the hydrolysis and the fermentation [7]. The enzymes used in this study were from commercial enzyme mixtures from Novozymes A/S (Bagsvrd, Denmark), Celluclast 1.5 L and Novozyme 188. Material from the pre-treatment was dissolved in acetate buffer, pH 4.8. A total of 500 ml liquid with 2% dry material, 2.32 g Celluclast and 0.52 g Novozyme was used. The hydrolysis was performed at 40 C. During the hydrolysis samples were

(I)

In this study the temperature was varied between 190 C and 210 C and the residence time between 2 and 10 min, but the concentration of the sulphuric acid was set to 0.2 % by weight. The slurry from the pre-treatment was filtrered before enzymatic hydrolysis.

withdrawn from the liquid and these samples were analysed with HPLC.

Results
The results are divided into results of pretreatment, yield in the enzymatic hydrolysis, the total yield of sugars and fermentation. Pre-treatment Stream pre-treatment with sulphuric acid as catalyst decomposes lignin. Mainly the acid soluble lignin, but to some part even the acid insoluble lignin, is affected. The more severe the pre-treatment were the less of the lignin was left. Parts of the carbohydrates can be hydrolysed during the pre-treatment. Analysis of the pre-treated material shows that it is primarily xylan that decomposes, which means the hemicellulose. This can be seen in Figure 1. At higher temperatures and longer residence times parts of the glucan can decompose as well.
Content of glucan and xylan in raw material and washed material 80 70 60 50 40 30 20 10 0 Raw 10 10 10 2 5 2 5 2 5

Fermentation
The sugar formed in the hydrolysis is fermented into ethanol. The most common microorganism for this purpose is Saccharomyces cerevisiae, which is the same that is used in ordinary baking yeast [8]. S. cerevisiae ferment glucose and mannose. In this study fermentation was done on the filtrate from the pre-treatment. Fermentation was done in order to control if the possible by-products from the pretreatment affected the fermentation. The filtrates were analysed with HPLC for determination of the concentration of fermentable sugars. Glucose was added so that all the filtrates and the reference had the same concentration of fermentable sugars, 50 g/l. The fermentation conditions were 30 C and pH 5.5. Samples were withdrawn during the fermentation and analysed with HPLC.

Content (%)

Glucan Xylan

SSF
Hydrolysis and fermentation can be done simultaneously and this is called simultaneous saccharification and fermentation, SSF. The glucose produced in SSF ferments directly into ethanol. The result of this is that the concentration of glucose is low, and therefore results in a lower inhibition of -glucosidase. The conditions must be a compromise since hydrolysis of cellulose and fermentation of glucose is performed in the same reactor at the same time. SSF was not investigated experimentially in this study.

190 C

200 C

210 C

Figure 1: The content of glucan and xylan in raw material and in pretreated washed material.

Since the pre-treatment decomposes both lignin and hemicellulose it is a good way of making the cellulose more available for the enzymes used in the hydrolysis. Enzymatic hydrolysis The yield over the enzymatic hydrolysis can be used as a measurement of how good

the pre-treatment was. The pre-treatment at 200 C and 10 min resulted in the highest yield of glucose, 96 %. A weak trend that more severe pre-treatment conditions resulted in a higher yield of glucose over the enzymatic hydrolysis can be seen in Figure 1.
Yield over the EH
1 0,8 0,6 0,4 0,2 0 2,95 3,25 3,35 3,54 3,64 3,65 3,94 3,94 4,24 Log R

glucose and xylose for the different pretreatment conditions as well as how much glucose resp. xylose that was produced in the hydrolysis and in the filtrate respectively.
Total yield
1,2 1 0,8 Yield 0,6 0,4 0,2 From enzymatic hydrolysis From filtrate

Yield

Glucose Xylose

0 2 5 10 2 5 10 2 5 10 2 5 10 2 5 10 2 5 10 190 C 200 C Glucose 210 C 190 C 200 C Xylose 210 C

Figure 3: The overall yield of glucose and xylose as function of pre-treatment conditions.

Figure 2: Yield of glucose and xylose over the enzymatic hydrolysis for different severity in the pre-treatment.

The highest yield of xylose was achieved for pretreatment at 190 C and 10 min. Xylose showed a higher yield at lower temperature than glucose. A reason for this is that xylose is decomposed at more severe pretreatment conditions. Overall sugar yield The overall sugar yield is based on the amount of sugar formed during the enzymatic hydrolysis and the amount of sugar in the filtrate compared with the theoretical amount of sugar in the raw material. Some of the experiment resulted in a higher yield than 100 %, which is impossible. However the results are comparable since all the experiments were done in the same way, by the same person. The highest yield of glucose was achieved for pre-treatment at 190 C and 10 min. Xylose resulted in somewhat higher yield for the pre-treatment at 190 C and 5 min compared to 10 min. The difference was small and is most likely in the margin of error. Figure 3 shows the overall yield of

Fermentation There were by-products present in the filtrate such as HMF and furfural. The amount of by-products formed per amount of raw material increased with increased severity in the pre-treatment. The fermentation tests showed that all filtrates were able to ferment. Most of the filtrates fermented at least as well as the reference did. However the filtrates from pretreatments at the highest temperature, i.e. 210 C, resulted in a lower ethanol yield than the reference. The yield of ethanol varied from 74 % and 100 % for the different filtrates.

Conclusions
The high content of cellulose in wheat straw, 34 %, makes it suitable as raw material for production of bioethanol. Steam pre-treatment with sulphuric acid as catalyst works well and results in a high sugar yield. Pre-treatment at 200 C for 10 min resulted in the highest yield of glucose

in the enzymatic hydrolysis. Pre-treatment at 190 C and 10 min resulted in the highest overall yield of sugars, i.e. 110% of the glucose and 79 % of the xylose. It was possible to ferment all of the filtrates. At pre-treatment at higher severity (210 C) more by-products were formed, which made the filtrates somewhat harder to ferment.

Chemical Engineering I, Lund Sweden, (1996) 6 Tengberg, C. Bioethanol Production: Pretreatment and Enzymatic Hydrolysis of Softwood Doktorsavhandling, Department of Chemical Engineering I, Lund, Sweden, (2000) Galbe, M. Ethanol from wood, An experimental study of pretreatment and hydrolysis process simulation Doktorsavhanling, Department of Chemical Engineering I, Lund, Sweden, (1994) Galbe, M.; Zacchi, G. A review of the production of ethanol from softwood Applied Biochemistry and Biotechnology, 59, 618-628, (2002)

References
1 Warfinge, P. Miljkemi: miljvetenskap i biogeokemiskt perspektiv KFS i Lund AB, 1997 Alfani, F.; Gallifuoco, A.; Saporosi, A.; Spera, A.; Cantarella, M. Comparison of SHF and SSF processes for the bioconversion of steam-exploded wheat straw Journal of Industrial Microbiology & Biotechnology, 25 (4), 194-192, (2000) Danielsson, N-. Processteknologi Cellulosateknik Massatillverkning Avdelningen fr Kemisk Teknologi, Lund, Sweden (1996) Schell, D.J.; Torget, R.; Power, A.; Walter, P.J.; Grohmann, K.; Hinman, N.D. A Technical and Economic Analysis of Acid-Catalyzed Steam Explosion and Dilute Sulfuric Acid Pretreatments Using Wheat Straw or Aspen Wood Chips Applied Biochemistry and Biotechnology, 28/29, 87-97, (1991) Hellqvist, L. Conversion of straw to ethanol Optimisation of the pretreatment step Examensarbete, Department of