production of ethanol from biomass research in...

Journal of Scientific & Industrial Research Vol. 64, November 2005, pp. 905-919

Production of ethanol from biomass Research in Sweden

Mats Galbe, Gunnar Lidén and Guido Zacchi*

Chemical Engineering, Lund University, P O Box 124, S-221 00 Lund, Sweden

Ethanol produced from various lignocellulosic materials such as wood, agricultural and forest residues has the potential to be a valuable substitute for, or complement to, gasoline. This paper reviews the research activities in Sweden on development of the technology for ethanol production from lignocellulosics. The paper focuses on hemicellulose and cellulose hydrolysis and fermentation as well as on process integration and techno-economic evaluation of the overall process.

Keywords: Biomass, Ethanol, Fermentation, Hydrolysis, Lignocellulosic materials IPC Code: C12Q 1/34

Introduction

Replacement of gasoline by liquid fuels produced from renewable sources is a high-priority goal in many countries worldwide. It is driven by the aims of a secure and sustainable energy supply and a desire to diminish the green house effect. The transportation sector (25% of the total energy consumption in Sweden) is totally dependent on imported fossil fuels and thus extremely vulnerable to any market disturbance. This is also the case for the rest of European Union (EU). The transportation sector is the main reason for the EU failure to meet the Kyoto targets as it is expected that 90 percent of the increase of CO2 emissions between 1990 and 2010 will be coming from this sector. In order to encourage greater use of biofuels, EU has set a target of 2 percent substitution of gasoline and diesel with biofuels in 2005 on an energy basis. In 2010, this percentage would increase (5.75%).

One such fuel is ethanol produced from biomass such as agricultural waste and forest residues. Although CO2 is emitted during the combustion of bioethanol, the same amount will be assimilated when new biomass is produced. Thus, total net emissions of CO2 are essentially zero. Bioethanol has also several other advantages: i) It is non-toxic; ii) It is a liquid at room temperature; iii) It can be blended (up to 20% without modifications of the engine) with gasoline

and used in most modern spark-ignited combustion engines; iv) Flexible fuel vehicles can run on pure ethanol, pure gasoline or any blend in between; v) It can be produced from sugar, starch and cellulose containing crops and residues; and vi) It can also replace diesel fuel in compression-ignition engines by using an emulsifier.

Despite advantages of bioethanol, Brazil and the US are still the only countries that produce large quantities of fuel ethanol from sugar cane and corn, respectively1. In 2004, Brazil produced 15 million m3 ethanol per year and the US about 14 million m3. The production capacity of ethanol is growing very rapidly in the US. Since 2000, there has been an increase in capacity of 109 percent and since 2003 the increase is 30 percent2. Fuel ethanol production is considerably more modest in the EU with about 0.5 million m3, where Spain is the largest producer. However, The Netherlands, Italy and Portugal have also shown interest in ethanol. In Sweden, an ethanol plant based on wheat and barley (capacity, 50 000 m3/y) came into operation in 20013 and another (10 000 m3/y) was obtained from fermentation of spent sulphite liquor from a pulp mill in Örnsköldsvik. The total consumption is about 260 000 m3, of which the main part is imported, mainly from Brazil.

In Sweden, fuel ethanol is used in a variety of ways; it is blended (up to 5%) in almost all gasoline, used as E85 for flexible fuel vehicles (FFV) and also used as neat alcohol in buses in Stockholm. Today more than 12000 Ford Focus FFV cars have been sold and from 2005 also Volvo and SAAB introduce new

____________________ *Author for correspondence Tel: +46 46 2228297, Fax: +46 46 2224526 E-mail: [email protected]

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FFV models into the Swedish market, which is expected to result in a rapid increase of FFV cars in the near future. This will also lead to an increased number of filling stations with E85, which at present are around 250. A recent government report suggests a law that all large filling stations must have a biofuel pump.

However, to compete with gasoline, production cost must be substantially lowered. The cost of the raw material constitutes one of the major contributions to the total production cost, which makes it necessary to use the cheapest raw materials possible. When using sugar- or a starch- containing feedstock, such as sugarcane or maize, the raw material accounts for 40-70 percent of the total ethanol production cost and the production cost is also dependent on animal feed as a by-product4. Lignocellulosic biomass, such as fast-growing trees, grass, aquatic plants, waste products (including agricultural and forestry residues) and municipal and industrial waste, is an abundant and relatively cheap raw material and is the only real option for replacement of a large fraction of the gasoline used today. The main by-product is lignin, which can be used as a solid fuel for production of heat and/or electricity for which there are no foreseeable market limits. The potential of using lignocellulosic biomass for energy production is even more apparent when one realises that it is the most abundant renewable organic component in the biosphere. It accounts for approx 50 percent of the biomass in the world, with an estimated annual production of 10-50 x 1012 kg4.

Ethanol production from lignocellulosic materials comprises following main steps: hydrolysis of hemicellulose, hydrolysis of cellulose, fermentation, separation of lignin, recovery and concentration of ethanol and wastewater handling (Fig. 1). Some of the most important factors to reduce the cost are: efficient utilisation of raw material by high ethanol yields, high productivity, high ethanol concentration in the feed to distillation and process integration in order to reduce capital cost and energy demand. A part of the lignin, remaining part of the biomass, can be burnt to provide heat and electricity for the process and the surplus is sold as a co-product for heat and power application. It is also necessary to minimize internal energy demand and maximize production of solid fuel.

Ecologically sustainable potential of woody biomass for fuel production is estimated to be 130 TWh/y in Sweden around 2020 (Parrika 1997). Total consumption of woody biomass for fuel production in Sweden in 1995 was 42 TWh/y. This is assumed to increase to 90 TWh/y by 2020 due to the need for increased heat and power generation from biomass as a consequence of the shutdown of some of Sweden’s nuclear power plants. Even so, there would be sufficient amounts of woody biomass for the production of bioethanol to be able to replace gasoline (> 20%) and diesel used today. In Sweden, several comprehensive studies have been performed on ethanol production from lignocellulosic materials. Most of the studies were performed on spruce as this is the most abundant biomass in Sweden but also other materials, such as salix, wheat straw, corn stover, sugarcane bagasse, Paja brava and tobacco stalks.

Fig. 1 Simplified flowsheet for a base case for ethanol production

GALBE et al: PRODUCTION OF ETHANOL FROM BIOMASS - RESEARCH IN SWEDEN

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This review presents the recent status of the research in Sweden on production of fuel-ethanol from lignocellulosics with focus on the activities at Lund University. Research Activities in Sweden Major Research Initiatives and Main Actors

There has been a continuous support for research on alternative fuels in Sweden for the past 30 years (Fig. 2). This research has been financially supported by the government via the Swedish Energy Agency, or its predecessor NUTEK (the Swedish National Board for Technical and Industrial Development), in several initiatives focusing on ethanol production from biomass. Research aiming at ethanol production from cellulose-rich materials was initiated at Lund University in the 1980's. In 1993, the Swedish ethanol development program was established with an increased level of governmental funding. A program was initiated, rather than merely a support of individual projects. The total funding of the programme amounted to 45 million SEK (5 M Euro) for a three-year period, which was later extended by one year. The first ethanol program was followed by a larger dedicated 7-year program on ethanol production from lignocellulosic material with a funding of 210 MSEK (23 million Euros). This

program comprised more than 10 different projects and was completed in 2004. The research concerned microbiology, enzymology and engineering.

At Chalmers University, Göteborg, research concerning dewatering of hydrolysates as well as fermentation of hexoses was carried out. At the Department of Forest Products, various methods to increase the filterability of the lignin were studied. Related issues concern solubilization of lignin. Fundamental work regarding hexose fermentation in yeast was carried out at the Department of Molecular biotechnology. Prime issues were starvation response, hexose transport and glycerol metabolism5-9. The Department of Chemical Engineering studies, in collaboration with University College of Boras, use of alternative fermentation organisms of the genera Rhizopus and Mucor for fermentation of multiple sugars in dilute-acid hydrolyzates. Furthermore, fermentation technology studies primarily focused on immobilized cell systems10-12.

Detoxification and characterization of inhibitors was studied in collaboration between Deptt. of Biochemistry, Karlstad University and STFI-Packforsk (the Swedish Forest Research Laboratory). The efficiency of various methods for detoxification, including overliming, enzymatic treatment by laccase, ion exchange, has been assessed. Furthermore, the

Fig. 2 R & D and production of ethanol from biomass in Sweden


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mechanism behind the sugar degradation in overliming detoxification was elucidated13-15.

The Bio-Alcohol Fuel Foundation (BAFF)16, former known as the Swedish Ethanol Development Foundation (SSEU), is supported by a group of industrial companies. The aim of the foundation is to promote implementation of bio-alcohol in the transport sector, including aspects such as production of fuel, fuel distribution and acquisition of vehicles to the Swedish market. BAFF is also engaged in international networking activities. SSEU was established in 1983 and it has played an active role in promoting the introduction of ethanol on the Swedish fuel market. Up to the middle of the 1990´s, the organization also supported ethanol research projects in industry and at universities. The Swedish Pilot Plant

Parallel to the research projects in the program, there was development of a Swedish pilot plant unit (Fig. 3), with the aim to prove process concepts and to provide reliable design data for a full-scale process. The pilot plant (16 million Euro), situated in Örnsköldsvik, was built during the final phase of the 7-y programme and was officially opened in May 2004. The plant (capacity, 2,000 kg dry material/day) was designed to be operational for both a two-stage dilute acid hydrolysis process and an enzymatic process, with a dilute acid pretreatment step. The two-

stage hydrolysis reactor was designed to be operated in either co-current or counter-current mode. In the enzymatic process, fermentation will take place simultaneously with the hydrolysis, an SSF process. Swedish company, ETEK, is responsible for operation of the plant17. The location is adjacent to the company, SEKAB, which produces ethanol from spent sulphite liquor. A core competence of SEKAB is the purification of ethanol to meet desired specifications, and the company purifies and distributes biomass-based ethanol for many different applications, including bio-ethanol as a fuel for motor vehicles. International Collaboration

Swedish researchers have actively participated in international collaborations in several of the research projects supported by the EU, such as contract Nos. ENK6-2002-00604 (TIME), QLK3-1999-00080 (Bio-Hug) and NNE5-2001-00685 (Babilafuente). Sweden has been also participating in the task group on Biotechnology for the conversion of lignocellulosics within the IEA (International Energy Agency) since the middle of 1980s. This group is now referred to as IEA task group 39 - Liquid biofuels from biomass - and currently holds 10 countries as members. Workshops are regularly arranged and short updates are published on the current status of implementation of ethanol as a fuel in the

Fig. 3 Schematic process layout of the Swedish Pilot Plant, Örnsköldsvik, Sweden


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membership countries18. Abilateral collaboration supported by STINT (the Swedish Foundation for International Cooperation in Research and Higher Education) was initiated with Brazil involving researchers from Lund University and the Federal University of Rio de Janeiro, the University of Pernambuco in Recife and Universidade de Brasilia. Research in Lund

Research on ethanol production has a long tradition at Lund University. The departments of Applied Microbiology, Biochemistry, Analytical Chemistry and Chemical Engineering started a co-operation project covering the whole chain from pretreatment of the raw material to the final recovery of ethanol. In Lund, the focus has been on the enzymatic route (Fig. 3), which probably will have a greater potential in terms of overall yield and raw material utilisation than will the acid-hydrolysis procedure. The department of Applied Microbiology focuses on development of new genetically modified organisms, mainly originating from baker’s yeast. The aim is to develop an industrial microorganism able to ferment all sugars available in biomass and to make them tolerant to the inhibitory compounds that are present in wood hydrolysates. Detoxification of fermentation feedstocks is also studied. The department of Biochemistry studies fundamental aspects of enzymatic hydrolysis of cellulose and hemicellulose and the interaction of the enzymes with lignocellulosic material. The aim is to optimize the enzymatic hydrolysis step to give a cost-efficient hydrolysis of the cellulose after pretreatment. Specifically, the aims are to minimize the addition of enzymes and to understand the interaction between

enzymes and cellulose as well as between enzymes and lignin.

The department of Chemical Engineering performs research on pretreatment, enzymatic hydrolysis, simultaneous saccharification and fermentation (SSF), process integration and techno-economic process evaluation. Developments of fermentation technologies, mainly fed-batch techniques, to overcome inhibition and to increase productivity are also important research areas. A process development unit (PDU) has been constructed in which 5-10 kg of raw material can be processed through all stages to the final product (ethanol) and co-products19. The unit is very flexible and capable of handling various raw materials (agricultural residues and forest residues); it can be used to study various process steps, individually or integrated, and various process configurations including recirculation of process streams. This is a national facility, which can be used both for applied research within the universities and research institutes, and for commissioned research for industries. It can also be used for commissioned work to institutes and industries outside Sweden.

Raw Materials

The main lignocellulosic source in Sweden available for ethanol production is residues from softwood, mainly spruce. It is widely accessible at a reasonable cost. Other raw materials are several species of hardwood, straws and agricultural residues from various countries in Europe and Americas20-25. Bagasse, the residue from sugarcane; Paja Brava, a type of sturdy grass from Bolivia’s high planes; tobacco stalks from Cuba; and corn stover from Hungary and Italy; all of these starting materials have been used in the experimental studies performed in Lund. In general, hardwood species contain more pentose sugars than do softwood species (Table 1).

Conversion Processes Pretreatment

The enzymatic process is considered to be the most attractive way to degrade cellulose to glucose. However, enzyme-catalysed conversion of cellulose to glucose is very slow unless the biomass has been subjected to pretreatment, as native cellulose is well protected by a matrix of hemicellulose and lignin. Steam pretreatment, often called steam explosion, is one of the most widely used methods for pretreatment of lignocellulosics. The raw material is first chipped and then treated with high-pressure saturated steam.

Table 1 Composition of various lignocellulosic materials (% of dry material)

Material Glu Man Gal Xyl Ara Lig Ref

Sugar cane bagasse

40.2 0.5 1.4 22.5 2.0 25.2 26

Corn stover 36.1 1.8 2.5 21.4 3.5 17.2 27 Salix 41.5 3.0 2.1 15.0 1.8 25.2 28 Paja Brava 32.2 0.3 1.4 22.7 3.7 23.1 29 Radiata pine 42.8 11.3 2.5 5.9 1.6 27.2 30 Spruce wood 41.9 14.3 n.d. 6.1 1.2 27.1 31 Spruce bark 27.3 3.2 n.d. 3.2 4.1 37.1 31 Douglas fir wood

46.1 14.0 2.7 3.9 1.1 27.3 32

Douglas fir bark

14.7 2.5 1.6 1.8 2.8 38.8 32

Glu=glucan, Man=mannan, Gal=galactan, Xyl=xylan, Ara=arabinan, Lig=lignin, n.d.=not detected


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Typical temperatures used are 160-240°C, (corresponding to a pressure of 6-34 bar), which are maintained for several sec to a few min, after which the pressure is released. During pre-treatment, some of the raw material, predominantly hemicellulose, is solubilized and found in the liquid phase as oligomeric and monomeric sugars. The cellulose in the solid phase is then more accessible to the enzymes. Steam pretreatment can be improved by using an acid catalyst, such as H2SO4 or SO2. The acid increases the recovery of hemicellulosic sugars, and also improves the enzymatic hydrolysis of the solid residue. The use of an acid catalyst in steam pretreatment results in an action similar to cooking with acid, but with less liquid. It is especially important to use an acid catalyst for softwood, since the hemicellulose in softwood contains less acetylated groups and autohydrolysis cannot occur to the same extent as in hardwood.

Steam pretreatment with addition of a catalyst is the method for hydrolysis and enzymatic digestibility improvement that is closest to commercialization. It has been widely tested in the Iotech pilot plant (Canada), the Souston pilot plant (France) and in the pilot plant in Örnsköldsvik (Sweden) and is used in a demonstration scale ethanol plant at Iogen (Canada). Several studies of one-step and two-step steam pretreatment of spruce (Table 2), including enzymatic hydrolysis, have been performed25,33-38. The highest sugar yields were achieved for two-step pretreatment with either SO2 impregnation or H2SO4 impregnation in both steps, while material treated with H2SO4 in the first step and SO2 in the second did not result in a high overall yield25,33.

For pretreatment with SO2 impregnation in both steps, maximum overall sugar yield (82%) was obtained for pretreatment conditions of 190°C for 2 min and 220°C for 5 min25. Pretreatment conditions of 180°C for 10 min with 0.5% H2SO4 and 200°C for 2 min with 2% H2SO4 resulted in an overall sugar yield of 77 percent25. The sugar yields obtained with two-step steam pretreatment was higher than one-step steam pretreatment, using similar conditions in the assay with enzymatic hydrolysis. During pre-treatment, sugars released from the hemicellulose can be subjected to thermal degradation to furans while lignin can be partially hydrolysed. Besides causing a loss of potential ethanol, some of the degradation products have been identified as powerful inhibitors. It is therefore important to choose conditions that do not generate toxic hydrolysates. Some of the substances present in the slurry are furfural and 5-hydroxymethylfurfural (HMF), which are the results of degradation of pentose and hexoses, respectively. Furfural may react further to yield formic acid, or it may polymerise. HMF can be converted to formic acid and levulinic acid. These compounds are known to cause a lag phase in fermentation of sugars. Depending on the raw material, acetyl groups in various amounts are also liberated during steam pretreatment.

Impregnation with dilute H2SO4 followed by pretreatment at a high combined severity (high temperature and/or long residence time) resulted in materials that were not fermentable25. Impregnation with SO2, however, was successful in creating fermentable materials for all investigated pretreatment severities.

Two other materials for which the pretreatment has been optimised recently are salix28 and corn stover39. For salix, highest glucose yield (92%) was obtained for pretreatment at 200ºC for 8 min after impregnation with 0.5% H2SO4. The maximum glucose yield was similar to that obtained when steam pretreatment was performed with non- and SO2-impregnated Salix chips. However, maximum xylose yield (86%) obtained for pretreatment at 190ºC for 4 min after impregnation with 0.5% H2SO4, was higher than that obtained using SO2 or non-impregnated material. The overall yield of sugars was 55.6 g glucose + xylose per 100g dry raw material, corresponding to about 88 percent of the theoretical based on the content in the raw material. Pretreatment of SO2 impregnated corn stover (dry matter, 40%) at 200ºC for 5 min resulted

Table 2 Optimized pretreatment conditions for spruce in one- or two-step procedures

Impregnating agent

H2SO4 SO2

Step 1

Catalyst, % 0.5 3 Temperature, °C 180 190 Time, min 10 2 Step 2

Catalyst, % 1 3 Temperature, °C 210 210 Time, min 2 5 One-step pretreatment Catalyst, % 2.5 Temperature, °C 215 Time, min 5


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in a glucose yield of 92 percent of the theoretical and a xylose yield of 66 percent. The maximum xylose yield (84%) obtained with pretreatment at 190ºC for 5 min. At these conditions, the glucose yield was 90 percent.

Dried Paja brava material, which was pre-steamed, impregnated with dilute H2SO4 (0.5 or 1.0% by wt), was steam pretreated at 170-230°C for a reaction time of 3-10 min29. The highest yield of xylose (indicating efficient hydrolysis of hemicellulose) was found at a temperature of 190°C, and a reaction time of 5 to 10 min, while considerably higher temperatures (230°C) were required for hydrolysis of cellulose. The Paja brava straw was found to be surprisingly resistant with respect to hydrolysis of cellulose. Furthermore, the maximum glucose yield obtained was a mere 30 percent of the theoretical. With respect to the hydrolysis of hemicellulose, however, reasonable sugar yields were obtained using dilute acid hydrolysis.

In a study using bagasse, conditions evaluated were: non-impregnated, SO2-impregnated (1.1% w/w DM) or H2SO4-impregnated (1g acid/100 g DM)22. The steam pretreatment was performed at 205°C and a residence time of 10 min. The highest yield of xylose (16.2%) and total sugars (52.9%) were obtained using SO2-impregnation. When H2SO4 or SO2 was used, glucose yield was about the same (35-36%), but the total sugar yield was less for H2SO4 (42.3 %). Enzymatic Hydrolysis (EH) and Fermentation

Following pretreatment, EH and fermentation can be run separately as separate hydrolysis and fermentation (SHF), or simultaneously as SSF. The advantage of performing enzymatic hydrolysis and

fermentation separately is that both steps can be performed at optimal pH and temperature, e.g. enzymatic hydrolysis at 40-50°C at pH 4.8 and fermentation at 30-37°C at pH 5.5. However, one great disadvantage of SHF is the end-product inhibition of the enzymes40. The released glucose and cellobiose inhibit further hydrolysis of the cellulose. This makes it difficult to reach high ethanol concentrations in the fermenter tank, which results in high-energy demand in the distillation step. In SSF, the glucose is immediately consumed by the fermenting organism, removing the end-product inhibition, which makes it possible to reach high ethanol concentrations. The ethanol produced can also act as an inhibitor in enzymatic hydrolysis, but not to the same extent as cellobiose or glucose. The main disadvantage of SSF is the difficulty to recycle yeast. After SSF, the yeast will be mixed with the solid residue after fermentation, mainly consisting of lignin. Recycling of yeast is much easier in the SHF case, where fermentation is performed without the lignin residue.

The following results are based on enzymatic hydrolysis, performed with a cellulase activity of 15 FPU/g water-insoluble solids (WIS) using a mixture of Celluclast 1.5 l and Novozyme 188, both kindly donated by Novozymes A/S, Bagsværd, Denmark and fermentation with 5 g/l of Saccharomyces cerevisiae (ordinary baker’s yeast), which ferments glucose and mannose to ethanol, in SSF at 37°C and pH 5.0. The yield of sugar and ethanol was determined in a process development unit, where pretreatment was performed in a 10-l reactor and SSF or EH were performed in 30-l reactors. EH and SSF of the whole slurry after the second pretreatment step were performed, at a concentration of 5% WIS, to determine the yield of sugars and ethanol. The liquid after the first pretreatment step was also fermented41.

When SSF and EH were performed at the same WIS content and enzymatic activity, ethanol yield in SSF exceeded the sugar yield in EH in both pretreatment cases (Fig. 4). Thus, SSF is a better process alternative if the yield is main priority. Comparison of acid catalyst used showed higher yields for the SO2-case in both SSF and EH. The overall ethanol yield in the SSF alternative for the SO2-case reached 81 percent of the theoretical, corresponding to 357 litres per metric ton dry raw material.41

Fig. 4 Overall yields obtained with two-step pretreatment of spruce


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An investigation on enzymatic digestibility of cellulosic substrates by Eriksson et al

42 showed a significant impact of using detergents in EH. Different types of detergents such as Tween and SDS increased conversion of cellulose. Tween-20 was best among the different types of ionic and non-ionic surfactants investigated. Using Celluclast cellulose hydrolysis was improved in presence of non-ionic surfactants, while charged surfactants had an adverse effect on hydrolysis. Among the non-ionic surfactants, Tween and Triton had the greatest positive effect of the investigated surfactants. It was found that the reaction time might be reduced (50%) while still maintaining the same yield. The most likely conclusion from studies using steam-pretreated spruce (SPS) and delignified SPS is that there is a reduction in unproductive binding if surfactants are added42

Influence of Tween-20 on the performance of SSF, in terms of final ethanol yield and productivity, was also investigated43. SSF was initially run with different concentrations of Tween-20 in order to determine the optimal concentration. Addition of 2.5 g/l of Tween-20 increased ethanol yield (8%) compared with the reference case without any detergent. The productivity was increased and the same ethanol yield as that reached after 72 h (without detergent) was reached after 24 h when 2.5 g/l of Tween was added. The increase in ethanol yield may be attributed to a combination of increased hydrolysis rate and improved yeast fermentation rather than only one factor. The exact mechanisms of Tween-20 as an enhancer for both enzyme hydrolysis and sugar fermentation are not yet known. The potential of reducing the enzyme loading in SSF by surfactant

addition was also investigated. SSF was performed with different enzyme loads [24, 12 and 6% Celluclast (w/w WIS)] with and without the addition of Tween-20. The enzyme loading could be reduced (with addition of 2.5 g/l Tween-20) by as much as 50 percent without a reduction in ethanol yield and productivity. It was also possible to further decrease the enzyme load (75%) without affecting the ethanol yield, but the residence time required to reach the final ethanol yield increased to 120 h (Table 3).

These results have potential economical implications since the price of Tween-20 is lower than price of enzymes. Addition of Tween-20 also decreased enzyme adsorption onto the residual solids after SSF. This could make it possible to recover enzyme from liquid fraction after SSF. Also SSF of both salix (Sassner P, Lund University, unpublished data) and corn stover pretreated44 at optimal conditions using bakers yeast resulted in high ethanol yields. With salix an overall ethanol yield (84%), based on the total glucan and mannan content in the raw material, was obtained using 7% WIS. SSF on pretreated SO2 impregnated corn stover, with higher dry matter content, showed that concentration of water insoluble substances in SSF could be increased (from 5% to 10%) without the ethanol yield (70%) being affected. It was also shown that the amount of yeast used in the SSF could be decreased from 5 g/l to 2g/l without any effect at all on the ethanol yield and only minor decrease of the productivity. The use of yeast cultivated on the pretreatment hydrolysate at these conditions did not improve the ethanol yield and only slightly improved the productivity at the beginning of the SSF. Fermentation of Hemicellulose Hydrolysates In situ

Detoxification by Fed-batch Techniques

Chemical hydrolysis of lignocellulose gives rise to inhibitors of different kinds such as furans, phenols and carboxylic acids45-52. One of the principal advantages of enzymatic processes is that the severe conditions required to degrade the cellulose are avoided and thereby the inhibition is largely avoided. However, substrates requiring relatively harsh pretreatment will still contain inhibitors. Adding a separate detoxification step adds to the cost and may give sugar losses14. Alternatively, in situ

detoxification, i.e. use of the intrinsic conversion capacity of the microbes of several inhibitors can be applied. Methods of fed-batch processes have been developed for this purpose53-56. These processes give a

Table 3 The influence of enzyme loading with and without the addition of 2.5 g/l Tween-20 on the ethanol yield (% of theoretical) in SSF (of steam pretreated spruce), and on the residence time required to reach the maximum ethanol concentration

SSF Run Cellulose

FPU/g Time to reach max. yield (h)

Yield % of theoretical

Without Tween

Case 1 44 72 85.2 Case 2 22 96 74.5 Case 3 11 140 71.3

With Tween

Case 1 44 48 92.0

Case 2 22 72 87.4 Case 3 11 120 87.0

FPU; Filter paper activity unit


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microbial conversion of furans and possibly other aldehyde inhibitors, and allow a high productivity while maintaining the viability of the yeasts.

Importantly, strains that show very similar and strong inhibition in batch processes may perform very differently in fed-batch processes (Fig. 5)56. Fed-batch technique is also important in SSF processes. However, the main reason for using fed-batch in SSF is to be able to handle the high viscosity of broth with a high WIS. By adding the substrate gradually, the viscosity of the broth can be kept at a manageable level due to the solubilization of the cellulose by the EH. The effects on cell viability are less pronounced than in dilute acid hydrolysates57. Adaptation of Yeast

A drawback in the SSF process is that the yeast (and the enzymes) cannot be reused. For this reason, concentration of yeast should be kept at a minimum. An added problem is that the yeast may be gradually inhibited and loose viability. The choice of strain and cultivation procedure has shown very important58. Pre-cultivation of the yeast on hydrolysate was found to result in yeast with a higher fermentation capacity in hydrolysate (Fig. 6). This is beneficial for both fermentation of hemicellulose hydrolysates and for the combined EH and fermentation step in SSF57. Significant improvement of yeast performance for yeast grown on hydrolysate underlines the importance of an efficient propagation of yeast on hydrolysate. In an on-going study, yeast could be grown on dilute acid hydrolysates at yields of more than 0.42 g/g (Nilsson et al, Lund University, unpublished data). However, there was a trade-off between yield and specific growth rate. In a fully implemented process this must of course be determined from an overall process economy point-of-view. Pentose Fermentation

Lignocellulosic materials have a wide variation in composition. In general, softwood contains more hexoses (glucose and mannose), while hardwood and agricultural residues are richer in xylose and arabinose (Table 1). This poses a problem since most naturally occurring organisms produce xylitol and not ethanol from pentoses. Some organisms (Pichia

stipitis), can produce ethanol from xylose under certain conditions, but production rate is low. It also requires micro-aerobic conditions, which may be possible to maintain in lab-scale, but is more difficult in full-scale production.

By introducing xylose-fermenting capabilities in glucose fermenting microorganisms, overall ethanol yield can be increased, which is important, as raw

Fig. 5 Differences in strain performance between two different yeast strains during fed-batch fermentation of hydrolyzate. Carbon dioxide Evolution Rate, CER (–) and glucose concentration (-●-) are shown for fed-batch fermentation of dilute-acid hydrolysate using the strain TMB 3000 (Fig. A) and CBS 8066 (Fig. B). Batch fermentations (0.5 l) on glucose were made during the first 15-17 h to produce initial cell mass49

Fig. 6 Comparison of ethanol production from hemicellulose hydrolysate with Baker's yeast pre-cultivated at different conditions51


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material cost is a major cost in ethanol production59-62. S. cerevisiae has been used for a long time for industrial ethanol production from sugar and starch and is a very robust microorganism with high resistance towards ethanol and inhibiting substances present in lignocellulose hydrolysates63. A stable xylose-utilising recombinant strain, TMB3001, was constructed by Eliasson et al

64 with over-expression of the XYL1 and XYL2 genes from Pichia stipitis encoding for xylose reductase and xylitol dehydrogenase, and of the endogenous XKS1 encoding for xylulokinase. With this strain, anaerobic ethanol formation from xylose was obtained for the first time with a recombinant S. cerevisiae. However, xylose fermentation rates and ethanol yields (0.21 g/g xylose) were low in comparison to those achieved on glucose. The strain was also used in fermentation of hydrolysates from sugarcane bagasse65.

To make the yeast more tolerant and to improve the ethanol formation, genes for the enzymes mentioned above were introduced into an industrial strain of S. cerevisiae, USM21 resulting in the transformed yeast TMB 3399. This was then further mutated and the best mutant, selected for improved growth on xylose, was named TMB 340066. These two strains were then tested for fermentation performance and robustness and compared with some other pentose-fermenting recombinant S. cerevisiae strains in defined medium containing a mixture of glucose and xylose and on lignocellulosic hydrolysates67. TMB3400 appeared to be the most promising strain as it showed the highest fermentation rate and was somewhat resistant to the inhibitors in the lignocellulose hydrolysate. However, fermentation in a minimal medium containing 50 g/l each of glucose and xylose resulted in a substantial production of xylitol (0.41 g/g xylose) and the ethanol yield from xylose was 0.24 g/g. This is mainly due to the redox problem, which seems to be an inherent property of strains engineered with xylose reductase and xylitol dehydrogenase.

TMB3400 has been used in SSF of slurry of corn stover that had been steam pretreated at 200°C for 5 min after SO2-impregnation. This resulted in an estimated yield of 0.46 g ethanol/g xylose and very low xylitol formation. This is probably due to the effect of degradation products emanating from lignin and carbohydrates that could affect the redox imbalance, and also the effect of slow release of glucose during the SSF.

Recirculation of Process Streams

It is of great importance to minimize liquid streams to and from a full-scale plant. Not only does fresh water add to the overall production cost, but also wastewater treatment constitutes a cost, which may be considerable if large volumes have to be processed. Like in all industrial plants, recirculation streams are part of the ethanol-production process. However, substances present in some of the process streams may act inhibiting to the enzymes and the fermenting organism. The lignin is mostly in the solid phase after steam pretreatment, but some degradation occurs and a wide range of phenolic compounds is released. Typically, vanillin, coniferyl aldehyde, ketones, hydroquinone and a multitude of aromatic substances are present in the slurry. The total concentration is, however, low.

There are a number of potential process streams to utilize for recirculation68,69. Depending on the choice of location, different scenarios can be identified (Fig. 7). If condensate from the evaporation step is recirculated (RC), concentrations of highly volatile inhibitors are reduced in the preceding stripping unit, but it still contains some medium-volatile compounds. This may have some effect on the fermenting organism, negative or positive. The flows to the distillation and evaporation steps are unaffected, while flow to wastewater treatment will be smaller. If recirculation before distillation (RBD) is applied, the flows to both the distillation and the evaporation steps are reduced. To avoid accumulation of solids, this option requires a filtration step prior to distillation. A third option is to use the stream after the stripper, which will reduce the flow to the evaporator only.

All the alternatives will probably have some effect on the performance of SSF. When fresh water is replaced using RBD-alternative, concentrations of all substances will increase–also that of ethanol–in the SSF step. Recirculation of stillage, as in the RAD case, will cause an accumulation of non-volatile substances in the SSF step. Given the large number of compounds involved and the synergism of the compounds, it is impossible to theoretically predict the impact of recirculation.

Alkasrawi et al68 investigated the effect of

recirculating the streams before and after distillation in an SSF-based process with spruce wood as raw material. In the study SSF was carried out batch wise with an initial concentration of 5% WIS. It was concluded that a large portion of the fresh water added to the SSF step could be replaced with


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recirculated process streams without affecting the final ethanol yield or the productivity. In the RBD case it was possible to reach a concentration factor (CF) of 1.5 and in the RAD case a CF of 2.5. The CF represents the ratio between non-volatile compounds in SSF with recirculation and non-volatile compounds in SSF without recirculation. Thus, higher the CF, the more non-volatile compounds including potential inhibitors are present in the SSF. Stenberg et al

70 performed similar studies on an SHF-based process with 5% WIS in the hydrolysis step. It was possible to replace fresh water (50%) with recirculated stillage without any negative effect on either hydrolysis or fermentation. Techno-Economic Evaluations

All experimental data obtained in the process development unit has been used to perform technical and economic evaluation of various process alternatives. Commercial process simulation programs, Aspen Plus and Icarus Process Evaluator, from Aspen Tech Inc (Cambridge, MA, USA), which were adapted for the purpose, were used. The whole process, from wood to ethanol as well as the production of solid fuel, has been evaluated. The approach is to establish a base case, which has been used as a reference when evaluating alternative process configurations and to study the influence of various process parameters. The base case configuration is for a production unit (capacity, 200, 000 tons of dry spruce). The simulated process consists of the following process steps: pretreatment, SSF, distillation, dewatering of solids, evaporation,

drying, and pellet- and steam production. In the economic evaluation, a feedstock handling area and an off-site area are included as well.

The overall ethanol yield (70%) for the SSF process was theoretical based on the glucan, mannan and galactan in the raw material, based on a one-step pretreatment. In the SHF process, yield was estimated to be 62 percent. In the base case, both SSF and SHF were performed at 5% WIS62. Use of SSF process results in a lower ethanol production cost (Table 4). The production costs were estimated to 5.12 and 5.66 SEK/l for the SSF and SHF base cases, respectively. The main reasons for higher cost for SHF process are the capital cost being higher and the overall ethanol yield being lower. If SHF can be improved (without additional costs) to reach the same ethanol yield as the SSF process, its production cost would be lower than SSF. A significant cost reduction could also be achieved if the residence time in the enzymatic hydrolysis step could be shortened without affecting the ethanol yield. At a residence time of around 30 h, compared to the base case value of 96 h, production cost is same as the SSF base case. However, both a higher yield and a shorter residence time would require higher enzyme loadings or a more efficient enzyme cocktail.

The income from the solid fuel is crucial to the economics of both SSF and SHF process. Since the base cases investigated require large amount of energy, production of solid fuel is low. It is possible to reduce the addition of fresh water either by running the process at a higher concentration of WIS or by recirculation of the process streams both alternatives

Fig. 7 Recirculation of process streams RC: Recirculation of condensate; RAD: Recirculation after distillation; RBD: Recirculation before distillation


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with a considerable reduction in the cost of ethanol (Fig. 8). Recent SSF-runs have actually been performed with up to 12% WIS. This results in ethanol concentrations around 4.5% (w/w), which reduces energy demand in the distillation and evaporation steps considerably leading to a larger amount of lignin co-product for sale.

A major drawback of SSF process is the addition of yeast required. The residue slurry leaving SSF step contains lignin and cellulose, along with the yeast. Recirculation is thus very difficult to perform, although if it could be performed it would reduce the production cost significantly. Another option is, of course, to lower the concentration of yeast. This can

be made by using adapted yeast to overcome possible inhibition from the sugar and lignin degradation products present in the hydrolysate. This is especially important at high WIS concentration as the concentration of inhibitors is increased proportionally to the increase in WIS. The maximum ethanol concentration is reached already after 24 h and is higher than that obtained with normal bakers yeast after 72 h (Fig. 9).

The cumulative effect of the most promising alternatives was evaluated for the SSF process. The effect of a combination of higher concentration of non-soluble solid material, the recycling of process streams and lower concentration of yeast was investigated. A realistic case would be a process involving non-soluble solid material (8%) and recycling of the stillage stream (26%), which together with a 50 percent reduction in the yeast requirement would result in an ethanol production cost of 3.81 SEK/l (Table 4). This is a decrease (26%) compared with the SSF base case.

Conclusions

Steam pretreatment, with small additions of SO2 or H2SO4, has been shown to be a very effective pretreatment method prior to enzymatic hydrolysis or SSF. It has resulted in overall sugar yields around 90 percent of the theoretical value for several raw materials (spruce, salix and corn stover). Overall ethanol yields (75-80%) of the theoretical value have been achieved using SSF for several raw materials. The steam pretreatment procedure for spruce was optimized using one- or two-step pretreatment with addition of either SO2 or H2SO4. The total sugar yield using two steps was higher.

Regarding high ethanol yield, SSF is superior to SHF. In experiments using only 2 g/l cultivated yeast

Fig. 8 Influence of WIS in SHF and SSF on the ethanol production cost

Fig. 9 Ethanol yield in SSF with 8% WIS (pretreated softwood) using various yeasts P-BY = normal package yeast (bakers yeast); C-BY bakers yeast cultivated on hydrolysate from pretreatment; C-TMB TMB3000 cultivated on hydrolysis

Table 4 Effect of improvements in SSF on the production cost

Cost (SEK/L) SSF-5%

SHF-5%

SSF*-8%

Wood 1.45 1.67 1.45 Enzymes 0.74 0.51 0.74 Yeast 0.53 0.00 0.16 By-products -0.09 -0.09 -0.67 Other chemicals and utilities 0.37 0.46 0.29 Labour, maintenance, insurance 0.63 0.89 0.56 Capital 1.48 2.21 1.26 Total 5.12 5.66 3.81

* Improvements including 8% non-soluble solid material, recirculation of process stream after distillation (26%) and reduction of yeast load to 2 g/l


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at 10% WIS, about 76 percent ethanol was reached. This is equivalent to about 325 l of ethanol per 1000 kg spruce. Ethanol concentrations (> 4%, w/w) have also been reached, which are high enough to allow for distillation at moderate costs. The residue after SSF is a lignin-rich material, which is well suited for pellet production or for generation of internal process steam.

It is important to reduce fresh water requirements and wastewater streams. By recirculation of various process streams, it is possible to more or less eliminate fresh water addition, which also has a positive effect on the energy demand. The impact of different flowsheet alternatives has been successfully modelled using Aspen Plus and Icarus Process Evaluator.

Although much progress has been made during the last two decades, major several challenges remain. It is imperative that organisms, which ferment all the sugars in the raw material is made available, since raw material cost constitutes a major part of production cost. The enzyme efficiency must be improved and enzyme production cost must be less than it is today. Since energy requirement is very much dependent on the downstream processing equipment, it is also important to run SSF at high dry matter contents to reach reasonable ethanol concentrations. High solids contents also affect the evaporation step positively. Improved process integration will also be beneficial with respect to the ethanol cost. The results are from experiments in non-continuously operating equipment. The whole process must be verified in the fully integrated pilot plant (capacity, 2000 kg raw material/day), which has been taken into operation in the middle of Sweden by Etek Etanolteknik AB (Ethanol Technology Corp.). This will provide detailed data that are necessary for scale-up to a full-scale production plant.

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