BUDAPEST UNIVERSITY OF TECHNOLOGY AND ECONOMICS Department of Agricultural Chemical Technology
P H .D. THESIS
B IOETHANOL PRODUCTION :
P RE - TREATMENT AND ENZYMATIC HYDROLYSIS OF CORN STOVER
A
UTHOR: V
ARGAE
NIKŐS
UPERVISOR: D
R. R
ÉCZEYK
ATALIN2003
Varga Enikő
B IOETHANOL PRODUCTION :
P RE - TREATMENT AND ENZYMATIC HYDROLYSIS OF CORN STOVER
(Ph.D. Thesis)
A CKNOWLEDGEMENTS
Elöljáróban köszönettel tartozom Dr. Réczey Katalin témavezetőmnek. Munkámat, szakmai fejlődésemet minden elképzelhető szempontból támogatta, és tanulmányaim során bármikor bizalommal fordulhattam hozzá tanácsért vagy segítségért.
Dr Anne Belinda Thomsen, for her invaluable advice and constructive commands through the half year spent in her lab. Thank you for teaching me, how to write a scientific paper and how two see the substance behind the details.
Professor Guido Zacchi, for his never-ending constructive criticism.
Köszönet illeti Babits Miklósnét, felbecsülhetetlen technikusunkat, aki gyakorlati tapasztalataival megkönnyítette laboratóriumi munkámat.
Szeretnék köszönetet mondani a csoportban dolgozó valamennyi munkatársamnak, hogy egy kitűnő közösség megteremtésével segítették munkámat.
Végezetül és leginkább szeretnék köszönetet mondani Szüleimnek, akik végtelen türelemmel és szeretettel álltak mellettem ez alatt a 4 év alatt is és mindenben támogattak.
L IST OF A CRONYMS
CBU Cellobiose unit
DM dry matter
DMC Direct microbial conversion (enzyme production, hydrolysis and fermentation in the same stage with special microorganisms).
E “X” fuel There are two main types of ethanol-blended gasoline in the World: low-level and high-level ethanol blends. High-level ethanol blends are often
E 85 blended in a proportion of 85% ethanol with 15% gasoline, and are called E85.
Low-level ethanol blends are widely available, in proportions of 5-10%
E 10 ethanol blended with gasoline (E05 and E10 respectively). Ethanol is a non- corrosive and relatively non-toxic alcohol and it can be used directly as fuel (most commonly in Brazil), or as an octane - enhancing gasoline additive (throughout the United States, Canada and Europe). Blends of 5-15% ethanol with gasoline can be used in all gasoline-powered automobiles, without engine or carburettor modification.
EC European Commission
ECC Enzymatic cellulose conversion, 100%
11 .
1 ⋅
⋅
= ⋅ m
V ECC c
where c is the concentration of D-glucose after enzymatic hydrolysis (g/L), V is the total volume (L), and m the weight of cellulose before enzymatic hydrolysis (g). The 1.11 factor converts the cellulose concentration to the equivalent glucose concentration.
ECOFIN The Council of Finance Ministers in the EU ETBE fuel additive (ethyl-tertier-butyl-ether)
EU European Union
FPA Filter Paper Activity FPU Filter Paper Unit
GC gas chromatography
HPLC high-performance liquid chromatography OPEC Organisation of Petroleum Exporting Countries
Oxygenates These are compounds, such as alcohols and ethers (ETBE), which contain oxygen in their molecular structure. Oxygenates improve combustion efficiency, thereby reducing polluting emissions. Many oxygenates, such as
RI refractive index
SC-CO2 Supercritical carbon-dioxide
SHF Separate hydrolysis and fermentation
SSF Simultaneous saccharification and fermentation TS two-step pre-treatment
TS-1 1% NaOH & 1% HCl TS-2 5% NaOH & 1% HCl TS-3 1% Ca(OH)2 & 1% HCl TS-4 1% NaOH & 1% H2SO4
Tween 80 surfactant (polyoxyethylene sorbitan monooleate)
IN CONNECTION WITH THE BIOFUELS
CO2 Carbon dioxide, a normal product of burning fuel, is non-toxic, but contributes to the greenhouse effect (global warming). All petroleum (hydrocarbon) fuels cause increased atmospheric carbon dioxide levels because they represent the combustion of fossilized carbon. By contrast, using renewable fuels, such as ethanol, does not increase atmospheric carbon dioxide levels. The carbon dioxide formed during combustion is balanced by that utilized during the annual growth of plants used to produce ethanol.
CO Carbon monoxide a poisonous gas produced by incomplete combustion. Vehicles operating at colder temperatures (in winter months, during engine warm-up or in stop- and-go traffic) produce significant quantities of this deadly gas, which is of particular concern in urban areas. Research shows that transportation sources account for over two-thirds of this pollutant. In the U.S. and in Europe (in Sweden, France, Spain, Germany) many cities have mandated the use of "oxygenated" gasolines, such as ethanol blends, to reduce carbon monoxide emissions.
L IST OF PUBLICATIONS
T
HIS THESIS IS BASED ON THE FOLLOWING PAPERS:
VARGA E., SZENGYEL ZS., RÉCZEY K. 2002. Chemical pre-treatment of corn stover. Appl.
Biochem. Biotech. 98-100:73-87.
VARGA E., SCHMIDT A.S., RÉCZEY K., THOMSEN A.B. 2003. Pretreatment of corn stover using wet oxidation to enhance enzymatic digestibility. Appl. Biochem. Biotech.
104:37-49.
VARGA E., ZACCHI G., RÉCZEY K. 2003. Optimization of steam pretreatment for corn stover to enhance enzymatic digestibility, Appl. Biochem. Biotech. In press
VARGA E., KLINKE H.B., RÉCZEY K., THOMSEN A.B. 2003. High solid simultaneous saccharification and fermentation of wet oxidised corn stover to ethanol, Biotechnol.
Bioeng.. (Manuscript (number: 03-570) submitted: on 10th October 2003.) OTHER RELATED PUBLICATIONS BY THE SAME AUTHOR:
VARGA E., KÁDÁR ZS., SCHUSTER K.C., GAPES J. R., SZENGYEL ZS., RÉCZEY K. 2002.
Possible substrates for Acetone–Butanol and Ethanol fermentation based on organic by-products. Hungarian Journal of Industrial Chemistry 30, 19-25.
KÁLMÁN G., VARGA E., RÉCZEY K. 2002. Dilute Sulphuric Acid Pretreatment of Corn Stover at Long Residence Times. Chemical and Biochemical Engineering Quarterly 16(4):151-157.
KÁDÁR ZS., VARGA E., RÉCZEY K. 2001. New Substrates of Biofuel, Magyar Mezőgazdaság, V 56. N. 19, pp. 32-33.
POSTER AND ORAL PRESENTATIONS:
VARGA E., KÁDÁR ZS, RÉCZEY K. 1999. Possible substrates for ABE fermentation International Conference on the Applied Acetone Butanol Fermentation. Krems, Austria, 16-18. September 1999.
KÁDÁR ZS., VARGA E., RÉCZEY K. 1999. Possible substrates for ethanol fermentation.
3rd European Motor Biofuels Forum, Brussels, Belgium, 10-13. October 1999.
VARGA E., RÉCZEY K. 1999. Enzymatic hydrolysis of the rest of fruit juice processing.
Chemist-conference ‘99. 26-28. November, Budapest
VARGA E., RÉCZEY K. 2000. Pretreatment and enzymatic hydrolysis of corn stover.
„Lippay János - Vas Károly” Scientific Conference, Budapest, 6-7. November 2000.
VARGA E., RÉCZEY K. 2001. Pretreatment of corn stover to enhance the enzymatic digestibility. 23. February, Budapest, Scientific Conference (KÉKI),
VARGA E. 2001. New substrates of the alcohol fermentation - pretreatment of the lignocellulosic materials. 28. February, Budapest, BUTE „Industrial Open Day”, Bioenergy, Biofuels symposium
VARGA E., ÁDÁM J., SZENGYEL ZS., RÉCZEY K. 2002. Chemical Pretreatment of corn stover. 4-9. May, Colorado, USA. 23th Symposium on Biotechnology for Fuels and Chemicals.
VARGA E., KLINKE H., RÉCZEY K., THOMSEN A.B. 2002. Enzymatic hydrolysis and fermentation of wet oxidised corn stover. 26-27. April, Floriade, Netherlands, International Congress & Trade Show Green-Tech® 2002 with European Symposium Industrial Crops and Products.
VARGA E., KLINKE H., RÉCZEY K., THOMSEN A.B. 2002. Enzymatic hydrolysis and simultaneous saccharification and fermentation of wet oxidised corn stover.
28. April – 1 May, Gatlinburg, USA 24th Symposium on Biotechnology for Fuels and Chemicals.
VARGA E., RÉCZEY K. 2002. Pre-treatment of corn stover at high temperature. 8 – 10 April, Veszprém, Hungary, Conference entitled: Technical Chemical Days’03,
VARGA E., ZACCHI G., RÉCZEY K. 2003. Optimisation of steam pre-treatment for corn stover to enhance enzymatic digestibility. 4 – 7 May, Breckenridge USA 25th Symposium on Biotechnology for Fuels and Chemicals.
C
ONTENTS1. INTRODUCTION...10
2. BACKGROUND:...16
ETHANOL PRODUCTION FROM LIGNOCELLULOSIC SUBSTRATE...16
2.1. LIGNOCELLULOSIC BIOMASS... 16
2.2. PRETREATMENT PROCESSES... 19
2.2.1. Biological pretreatment ... 20
2.2.2. Physical pretreatment... 21
2.2.3. Physico-chemical pretreatment... 21
2.2.3.1. Steaming/Steam explosion... 21
2.2.3.2. Wet oxidation ... 22
2.2.3.3. AFEX process... 25
2.2.3.4. CO2 explosion... 25
2.2.4. Chemical pretreatment ... 26
2.2.4.1. Acidic pretreatment ... 26
2.2.4.2. Alkaline pretreatment... 27
2.2.4.3. Organosolv process... 27
2.2.4.4. Ozonolysis ... 28
2.3. HYDROLYSIS PROCESSES... 28
2.3.1. Acid hydrolysis ... 29
2.3.2. Enzymatic hydrolysis... 30
2.3.2.1. Surfactant effect in enzymatic hydrolysis ... 31
2.4. FERMENTATION FOR BIOETHANOL PRODUCTION... 33
2.4.1. Separate hydrolysis and fermentation (SHF) ... 33
2.4.2. Simultaneous saccharification and fermentation (SHF)... 33
2.4.3. Simultaneous saccharification and co-fermentation (SSCF)... 34
2.4.4. Direct microbial conversion (DMC)... 34
3. MATERIALS AND METHODS...36
3.1. RAW MATERIAL... 36
Analysis of raw material ... 36
3.2. PRETREATMENTS... 39
3.2.1. Chemical pretreatments ... 39
Alkaline and acidic pretreatments... 39
Combined alkaline and acidic pretreatments (two-step pretreatments) ... 39
Pretreatments with dilute base and acid ... 39
3.2.4. Supercritical CO
2pretreatment ... 43
3.3. ENZYMATIC HYDROLYSIS... 44
3.3.1. Effect of ultrasonic waves during enzymatic hydrolysis... 45
3.3.2. Enzyme activity measurement ... 46
3.4. ANALYSIS OF THE LIQUID FRACTION AFTER PRETREATMENTS... 46
3.4.1. Poly- and monosaccharide analysis of liquid fraction... 46
3.4.2. Carboxylic acids and phenols analysis... 47
3.5. SIMULTANEOUS SACCHARIFICATION AND FERMENTATION (SSF) AFTER WET- OXIDATION... 47
3.6. FERMENTATION FOLLOWING STEAM PRETREATMENT... 48
4. RESULTS AND DISCUSSION...50
4.1. CHEMICAL PRETREATMENTS ON YIELDS AND COMPOSITION... 50
4.1.1. Effect of chemical pretreatments on yield and composition ... 50
Effect of two-step pretreatments on corn stover... 52
4.1.2. Enzymatic hydrolysis... 53
4.1.3. Effect of supercritical pretreatment on yield and composition ... 57
4.1.4. Conclusions on chemical pretreatments ... 58
4.2. STEAM PRETREATMENT... 58
4.2.1. Effect of steam pretreatment ... 58
4.2.2. Enzymatic hydrolysis... 63
4.2.3. Fermentability test... 64
4.2.4. Conclusions on steam pretreatments ... 65
4.3. WET OXIDATION PRETREATMENT... 66
4.3.1. Effect of wet oxidation ... 66
4.3.2. Enzymatic hydrolysis... 71
4.3.3. SSF ... 74
4.3.3.1. Effect of substrate concentration on ethanol yield ... 74
4.3.3.2. Effect of enzyme loading ... 76
4.3.3.3. Conversion of phenols and carboxylic acids by SSF ... 78
4.3.4. Conclusions on wet-oxidation pretreatments ... 79
4.4. COMPARISON OF THE DIFFERENT PRETREATMENT PROCESSES... 80
4.5. ESTIMATE OF PRODUCTION COST OF FUEL ETHANOL... 83
4.5.1. Production cost of starch based bioethanol... 83
4.5.2. Production cost of lignocellulose based bioethanol... 83
5. CONCLUSIONS AND FUTURE POSSIBILITIES...86
6. REFERENCES...88
1. I NTRODUCTION
In October of 1973, the Organisation of Petroleum Exporting Countries (OPEC) decreased the output of oil, which quadrupled the oil prices and tipped the world's leading industrial powers into crisis. This dramatic increasing in oil prices turned the world’s interest to the alternative fuels (Mably, 2003). Then the main motivation was to become independent of petroleum market and to reduce the cost of expensive oil imports. However, the emphasis today is on reducing pollution and helping to satisfy the Kyoto protocol, established in 1997, by limiting the global net emission of carbon dioxide (CO2).
Energy from the sun heats the earth's surface; in turn, the earth radiates energy back into space. Atmospheric greenhouse gases (water vapour, carbon dioxide, methane, nitrous oxide, and ozone) trap some of the outgoing energy, retaining heat somewhat like the glass panels of a greenhouse. Without this natural phenomenon, which is refereed to as the
“greenhouse effect”, temperatures would be much lower than they are now, and life as known today would not be possible. However, problems may arise when the atmospheric concentration of greenhouse gases increases.
Since the beginning of the industrial revolution, atmospheric concentrations of carbon dioxide have increased nearly 30% from 280 ppm to 365 ppm, methane concentrations have more than doubled, and nitrous oxide concentrations have risen by about 15% (WMO, 2002). Very powerful greenhouse gases have appeared in the atmosphere, that are not naturally occurring, include hydrofluorocarbons (HFCs), perfluorocarbons (PFCs), and sulphur hexafluoride (SF6), which are generated in a variety of industrial processes.
Parallel with the increasing green house gas concentration, the Earth's global mean surface temperature has risen by about 0.6-0.9°C since the late 19th century, but the warming of the atmosphere is continuing (Schneider 1989). The Word’s nine warmest years all occurred in the last ten years, among of these 2001 was the warmest year of the world on record (WMO, 2002), but the year of 2003 could take the lead.
There is new and stronger evidence that most of the warming over the last 50 years is attributable to human activities, in consequence of the additional release of carbon dioxide, which is the primary global warming gas.
The OECD countries consume the 57% of the world’s energy (Table 1.1.) and contribute more than half of the world’s total emission of CO2. The USA is one of the countries that has the biggest energy demand, and corresponding to this, discharges the most, emitting about one-fifth of total global greenhouse gases (U.S. Ministry of Environmental, 2003).
Energy consumption and also the CO2 concentration has increased steadily over the last century and it has increased significantly even in the last 10 years, which is shown in
Opposite to this trend, energy demand of Hungary has decreased in the last 10 years, but unfortunately it could be explained with the industrial decline after the political changes in the latest 1980’s and not with conscious, energy saving behaviour or with new, energy friendly technologies.
Estimating future emissions is difficult, because it depends on demographic, economic, technological, policy, and institutional developments, but it is fact, that the world population has been growing and even more countries have been becoming industrialised.
Figure 1.1. shows the share of the oil consumption in various industrial fields.
Table 1.1. Primary energy consumption by energy source (Mtoe, million tone oil equivalent)
2001 1991
Oil Gas Carbon Nuclear Water Summa Summa World 3510.6 2164.3 2255.1 601.2 594.5 9125.7 8147.2
OECD 2189.6 1167.2 1108.2 518.8 291.0 5274.8 4606.7
USA 1066.3 650.4 590.9 202.6 129.7 2639.9 2300.7
Asia 972.7 274.7 1020.7 115.0 128.8 2511.9 1886.2
Europe 760.2 423.0 344.1 225.0 142.4 1894.7 1770.7 EU (15) 637.1 343.3 212.5 201.6 84.4 1478.9 1345.6
FSU 169.6 493.6 180.4 51.2 54.9 949.7 1375.3
Middle East 206.4 181.3 8.0 –– 1.5 397.2 264.3
Hungary 6.8 10.7 3.1 3.2 –– 23.8 25.6
One way of reducing both environmental effects and dependence on fossil fuels is to use alternative, renewable energy sources. In accordance with this trend the European Commission (EC) has decided to increase the market share of renewable energy to 12%
until 2010.
The rate of renewable energy in the total energy consumption in Hungary was only 3.2% in 2001, mainly low-efficiency direct burning, but the using of alternative fuels in the transport sector was nearly negligible. In half a year, Hungary will be a member of the EU, which underlines these problems and pushes to decide to overcome these deficiencies in the near future.
As the transportation sector is practically 100% dependent on oil and it is responsible for the greatest and even more increasingly proportion of CO2 emission, the biofuel, including fuel ethanol, is a topical issue both in the European Union (EU) and in the USA. (Mielenz, 2001). In 2000 the EC has developed a strategy to obtain 20% share in the biofuel market by the year 2020 (Vermeersch 2002). To reach these aims the EC published an action plan and two directive proposals in November 2001 to encourage increased use of biofuels in the transport sector (http://www.platts.com/features/biofuels/europe.shtml).
The objectives of the action plan and the directives are to:
• Help reduce the European Union's dependence on external oil supply.
• Contribute to EU greenhouse gas emission reduction targets as agreed in the Kyoto Protocol.
• Meet the objective of substituting 20% of diesel and gasoline fuels by alternative fuels in the road transport sector by 2020.
The first directive calls for the establishment of a minimum level of biofuels as a proportion of fuels sold from 2005, starting at 2% and reaching 5.75% of fuels sold in 2010. Whether the targets of the first directive will be indicative or mandatory is still to be decided.
The second directive deals with tax issues, seeking to give EU member states the option of offering tax breaks on pure or blended biofuels used in either heating or motor fuel. The Council of Finance Ministers (Ecofin) has allowed 100% relief as long as the targets are indicative, while the EC's draft proposed a 50% duty cut on biofuels.
In Europe, contrary to the US, biodiesel is the dominating biofuel. France, Germany, Italy, Austria and Belgium together produced 701.6 kton of biodiesel in 2000, according to EurObserver, the EU research institute on renewable energy.
The European ethanol production is much smaller than biodiesel production. In 2000, the three main ethanol producting country, France, Spain and Sweden together produced a total of 216 kton ethanol (Table 1.2.). France is the biggest European producer but does not use ethanol in its pure form. It transforms the alcohol into fuel oxygenate ETBE (ethyl- tertier-butyl-ether).
While Spain produced only 80 kton of ethanol in 2000, one Spanish company, Abengoa, is investing heavily in ethanol production. In the last two years, the company has spent 382 million Euro on its bioethanol production and is now the second biggest producer in the world with a 15% market share. (http://www.platts.com/features/biofuels/europe.shtml)
Table 1.2. Biodiesel, ethanol and ETBE production in the EU in the year of 2000.
BIODIESEL ETHANOL ETBE (KTON)
Austria 27.6 - -
Belgium 20 - -
Germany 246 - -
Italy 78 - -
France 328.6 91 193
Spain - 80 170
Sweden 1 45 0
In the USA there is also a considerable growth in oxygenated fuel additive improved gasoline (e.g E10 and E 85 fuels) (Knapp et al., 1998). However, as a result, the US transportation sector now consumes only about 4540 million litre of ethanol annually, about 1% of the total consumption of gasoline (Sun and Cheng 2002).
Figure 1.1. The distribution of the world’s oil consumption
The use of fuel ethanol will significantly reduce net carbon dioxide emissions, when it replace fossil fuels, because fermentation-derived ethanol is already part of the global carbon cycle.
Apart from a very low net emission of CO2 to the atmosphere, the combustion of bioethanol in generally results in the emission of low levels of uncombusted hydrocarbons, carbon monoxide (CO), nitrogen oxides (NOx) and exhaust volatile organic compounds (VOCs). However, of major environmental concern regarding the increased use of ethanol fuels is the increased exhaust emission of reactive aldehydes, such as acetaldehyde and formaldehyde. Thus, a key factor with respect to the possible of ethanol on urban air quality will be the durability and effectiveness of catalyst system for aldehyde control.
The T model invented by Henry Ford in the early 1900’s was originally developed to run on ethanol. Today, all cars with a catalyst can be run on a mixture of 90% petrol and 10%
ethanol without engine modification. New cars can even use mixtures containing up to 20% ethanol. There are also new engines available, which can run on pure ethanol, and so- called flexible fuel vehicles (FFV) which are able to use mixtures of 0-85% ethanol in petrol. Ethanol can also replace diesel fuel in compression-ignition engines, however to be able to mix diesel with ethanol an emulsifier is needed (Wheals et al., 1999).
Fuel ethanol is used in a variety of ways (Table 1.3.). However, today the major use of ethanol is as an oxygenated fuel additive. Mixing ethanol and petrol has several
8%
25%
8% 5%
54%
power plant oil for heating not for energetic use petrochemistry transport
advantages. The higher octane number of ethanol (96-113) increases the octane number of the mixture, reducing the need for toxic, octane-enhancing additives. Ethanol also provides oxygen for the fuel, which will lead to the reduced emission of CO and uncombusted hydrocarbons (Mielenz, 2002).
Table 1.3. Common ethanolic motor-fuel formulations
FUEL ETHANOL CONTENT (% V/V)
Hydrous ethanol (Álcool, in Brazil) 95.5%
E 85 (North America, Sweden) 85
Gasoline (Brazil) 24
E 10 (gasohol, North America, Canada, Sweden) 10
ETBE (France, Spain) 7.6
Oxygenated fuel (USA, Canada) 5.7
Biodiesel (EU) 15
In additional to these points, several positive consequences of an introduction of biofuel are to be considered as additional assets, e.g. improvement of the trade balance, employment safeguarding, creation of jobs, social and tax revenues.
Despite the advantages of bioethanol, Brazil and North America are still the only two countries that produce large quantities of fuel ethanol, from sugar cane and maize, respectively. Brazil produces 12 million m3 ethanol per year and the USA about half of this amount. Fuel ethanol production is considerably more modest in the European Union, where the three dominant producer France, Spain and Sweden together produced a total of 475 000 m3 per year. However, other European countries such as Austria, Italy, Poland and Portugal have shown interest in the oxygenate ETBE (ethyl tertiary butyl ether) produced from ethanol.
The efficiency of ethanol production has steadily increased, but tax relief will be required to make fuel ethanol commercially viable compared with oil. Thus current bioethanol research is driven by the need to reduce the production cost. Historically, the projected cost of lignocellulosic biomass-based-ethanol has dropped from about US$ 1.22 / L to about US$ 0.35 / L because of the continuous improvement in pretreatment, enzymatic application and fermentation techniques. When using feedstocks such as sugar cane or maize, the raw material accounts for 40 to 70% of the total production cost (calculation is showed in the “list of Acronyms”) (Worley et al., 1992, Claassen et al., 1999). However using high cellulose containing agricultural residues, for example, corn stover, the production cost could be reduced significantly in the next 15 years (Lynd 1996). The potential of using lignocellulosic biomass for energy production is even more apparent when one realises that it is the most abundant renewable organic components in the
resource converted into useful energy at a 65% efficiency could supply all of the world’s present energy needs (Clausen and Gaddy 1983).
Corn is a major crop in Hungary, produced at nearly 8 million tons in every year.
Assuming the 1:1.3 ratio (depending on the harvesting time) of dry matter of corn grain to dry matter of corn stover, the equivalent mass dry mass residue was approximately 11 million tons. Corresponding its amount, corn stover is the most abundant agricultural residue in Hungary. (Statistical Annual Reviews, 2002). Less, then 10% of this amount has been used as an animal feet, the major portion of corn stover has traditionally been removed from the field by the practice of open-field burning. This practice plays an important role in protecting and improving soil quality, however it has a negative influence on the air quality and it wastes a huge amount of potential energy source. In defend of the air quality a new law has been brought recently, which prohibit the open air burning in the fields in Hungary.
The US, which is the main corn producer country in the world, produced approximately 216 million tons of corn grain in 2001. Associated with corn production is a corresponding annual production of approximately 254 million tons of corn stover (Sokhansanj et al, 2002). In spite of its large quantities, currently only 6% is collected and used for animal feeding and bedding.
However corn stover consisting of 70% carbohydrates (cellulose and hemicellulose) is a promising raw material for fuel ethanol (Hahn-Hägerdal 1996). Due to structural features such as lignin, acetyl groups and cellulose crystalinity, lignocellulosic biomass must be pre-treated to enhance its digestibility before microbial conversion into liquid fuels (Kaar and Holtzapple 1998).
The most of the commonly used, effective fractionation methods break down the lignin structure and solubilise the hemicellulose fraction (Schmidt and Thomsen 1998). However during these pretreatments, degradation products such as acetic acid, formic acid and phenol monomers (Klinke et al, 2002) could be formed, which are well known inhibitors for the fermenting microorganisms, e.g. Saccharomyces cerevisiae.
The absence of these by-products, heterogeneity in feedstock and the influence of different process conditions on microorganisms and enzymes make the biomass-to-ethanol process complex.
2. B ACKGROUND :
E THANOL PRODUCTION FROM LIGNOCELLULOSIC SUBSTRATE
2.1. L
IGNOCELLULOSIC BIOMASSLignocellulosic biomass from different sources may appear outwardly quite different, but the chemical composition is in fact very similar. It primarily comprises three major fractions: cellulose, hemicellulose and lignin, besides the rest is typically much smaller amounts of minerals (ash), soluble phenolics and fatty acids and other compounds often termed extractives. Figure 2.1. A-C shows the three main components of lignocellulose.
The composition of different herbaceous residues are given in Table 2.1. Although the compositions are quite similar, corn stover generally contains more lignin than other lignocellulosic materials.
Table 2.1. Percent Composition of lignocellulosic material on a Dry-Wt basis CORN
STOVERA
WHEAT STRAWB
SWITCH-
GRASSA
BARLEY STRAWC
RICE STRAWD
SORGHUM STRAWC
Glucan 36.0 38.0 32.2 37.5 37.2 32.5
Xylan 19.8 22.4 20.3 15.0 24.4 16.9
Galactan 1.3 2.1 - 1.7 1.3 0.2
Arabinan 2.8 3.6 3.7 3.9 2.8 2.1
Mannan - 0.7 0.4 1.3 0.4 0.8
Total glycan 59.9 66.8 56.6 59.4 66.1 52.5
Klason lignin 26.9 14.5 19.5 13.8 15.0 14.5
Acid-soluble lignin 1.9 nd 3.7 nd nd nd
Ash 7.2 7.2 7.1 10.8 8.9 10.1
Acetyl groups 1.4 1.4 2.35 2.0 4.4 6.2
Other 2.7 10.4 10.8 14 5.6 16.7
a Fenske et al. 1998
b Bjerre et al. 1996
c Wilke et al. 1981
d Vlasenko et al. 1997
Cellulose comprises between 35 and 50% of the total dry mass and consists of long chains of ß-anhydroglycose unit linked by ß1,4-glucoside bonds. The degree of polymerisation i.e. the number of glucose molecules included in a cellulose chain is normally in the range of 7500 to 15000 for plant cellulose. The cellulose molecules are organised in elementary
Between the elementary fibrils, there are microfibrils contain regions, which are less ordered, and often called “amorphous regions”. These regions are particularly susceptible to enzymatic hydrolysis, but about 50-90% of the cellulose in lignocellulosic material forms crystalline structure. The cellulose is provided by hemicellulose and lignin, which formed a physical protection of the cellulose hydrolysis.
Figure 2.1. A. Structure of cellulose
Hemicellulose represents to about 25% of total lignocellulosic mass, and, like cellulose, its monomer units can also be fermented to ethanol. In contrast to cellulose is a branched polymer of sugars whose units include mostly aldopentoses, such as xylose and arabinose and some aldohexoses, such as glucose, mannose and galactose. Various substitutes, e.g.
acetyl groups or uronicacid groups are attached to the main chain or the branches and the DP ranges from 20 to 300. The variety of linkages, branching, and different monomer units contribute to the complex structure of hemicellulose and thereby its variety of conformations and function. Within biomass, hemicellulose links covalently to lignin and through hydrogen bonds to cellulose. However the hemicellulose is much more easily broken down than crystalline cellulose. (Bringham et al., 1996)
Figure 2.1.B. Structure of hemicellulose
Lignin, the third significant fraction in biomass, is one of the most abundant and important polymeric organic substance in the plant world. Lignin increases the mechanical strength to such an extent that even the 100 m height trees remain upright. Although there are a
great number of microorganisms, which are able to utilise hemicellulose or cellulose, relatively few strains have the ability to decompose the lignin effectively. Lignin is a highly complex, three-dimensional polymer of different phenylpropane units, which are bound together by ether and carbon-carbon bonds. A few lignin structures have been elucidated but in general their structures remain unknown. The lignin fraction of biomass remains as a solid after most hydrolysis methods and can impact fermentation. It is often burned as boiler fuel because of its high energy-content.
Figure 2.1.C. Structure of lignin
E
THANOL PRODUCTION FROM LIGNOCELLULOSIC SUBSTRATEEthanol could be produced in various ways according to the various feedstocks. Although the production of ethanol from cane sugar is a relatively simple process and known since several hundred years, complexity increases when ethanol is produced from corn or wheat starch, as these processes require enzymes to hydrolyse starch to glucose prior fermentation. Production of ethanol from lignocellulosic biomass (Figure 2.2.), which was investigated in this thesis, requires even more extensive processing to release the polymeric sugars in cellulose and hemicellulose.
Figure 2.2. Schematic flowchart of the “ethanol from lignocellulosic biomass” process
2.2. P
RETREATMENT PROCESSESAlthough the most lignocellulosic biomass is rich in carbohydrates, it is an insoluble substrate with a complex structure. Thus the polysaccharides are not directly available for bio-conversion as the lignin component and the regular and cross-linked polymers in lignocellulosics form a very efficient physical barrier. Figure 2.3. shows schematically the complex structure of lignocellulose. Cellulose fibers are embedded in a sheat of hemicellulose and lignin and held together by hydrogen and van der Waals bonds, as it was mentioned above. There are several ways to increase the digestibility of cellulose before it is exposed to enzymes: physical, chemical and biological processes and combination of these have been used for pretreatment of lignocellulosic material.
The purpose of all pretreatments is to remove lignin and hemicellulose, reduce cellulose crystalinity and increase the porosity of the materials. All kind of pretreatment must meet the following requirements:
• Fractionation of the raw material into high quality feedstock for further processing/conversion,
• Avoid the degradation or loss of carbohydrate,
• Avoid the formation of by-products inhibitory to the subsequent hydrolysis and fermentation processes,
• Have a minimum consumption of energy and chemicals,
• Be cost-effective with low operating and capital cost.
Figure 2.3. Schematic figure of the complex structure of lignocellulose
2.2.1. Biological pretreatment
The category of biological pretreatments comprises the techniques of applying lignin-, and hemicellulose-solubilising microorganisms, such as brown-, white- and soft-rot fungi, to render lignocellulosic materials suitable for enzymatic digestion (Schurz 1978,
and Cheng 2002). However the rate of hydrolysis in most biological pretreatment processes is very low and the most lignin-solubilising microorganisms also solubilise or consume cellulose (Ghosh and Shingh 1993, Han 1978). In order to prevent the loss of cellulose, development for a cellulase less mutant is necessary.
2.2.2. Physical pretreatment
Pretreatment techniques, which do not involve chemical application called physical treatments. Most physical pretreatments, as high energy irradiation, dry, wet, and vibratory ball milling are mainly reported to be time-consuming, ineffective and energy- intensive, therefore expensive (Fan et al., 1982, Chang et al., 1981, Lin et al., 1981 ).
However most pretreatment techniques studied appear to employ particles passing a 3 mm or smaller rejection screen (Alvo and Belkacemi 1997). This primary size reduction is an essential step in the conversion of lignocellulosic material to fuel ethanol, thus it is not considered as a mechanical pretreatment in this work. On the other hand some reviews indicated that milling alone can result yields near to the theoretical maximum from some sources of lignocellulose (e.g. straws, woods) (Koullas et al., 1992, Millet et al., 1976).
However it has to be mentioned, that the required particle size was <10-3 mm, which needs an extremely great portion of energy.
2.2.3. Physico-chemical pretreatment
2.2.3.1. Steaming/Steam explosion
Steaming or steam explosion is an extensively investigated pretreatment method. A vast amount of literature can be found, treating several types of raw materials (Clark and Mackie 1987, Eklund et al., 1995). In this method, chipped biomass is treated with high- pressure saturated steam and then the pressure is swiftly reduced, which makes the materials undergo an explosive decompression. Steam explosion is initiated at a temperature of 160-260°C (corresponding pressure 0.69-4.83 MPa) for several seconds to a few minutes before the material is exposed to atmospheric pressure. During this treatment the presence of moisture initiates an auto- hydrolysis reaction catalysed by organic acids, which are initially formed from acetyl groups, liberated from the biomass. The action mode of steaming/ steam explosion is therefore similar to that chemical pretreatment with acid, as described bellow.
Ninety percent efficiency of enzymatic hydrolysis has been achieved in 24 h for poplar chips treated by steam explosion compared to only 15% hydrolysis of untreated chips (Grous et al., 1986). Similar result was achieved for steam pre-treated corn stover, (Varga et al., 2003) however it is reported less effective for softwoods (Stenberg et al., 1998). The factors that affect steam explosion pretreatment are residence time, temperature, chip size
and moisture content (Duff and Murray 1996). Optimal hemicellulose solubilisation and hydrolysis can be achieved by either high temperature and short residence time (270°C, 1 min) or lower temperature and longer residence time (190°C, 10 min). Recent studies indicate that lower temperature and longer residence time are more favourable (Wright 1998). Impregnation with sulphuric acid, sulphur-dioxide or carbon-dioxide prior to steam pretreatment can effectively improve enzymatic hydrolysis, decrease the production of inhibitory compounds and lead to more complete removal of hemicellulose (Tenborg et al., 1998) even when softwood are considered.
Limitations of steam explosion include destruction of portion of the xylan fraction, incomplete disruption of the lignin-carbohydrate matrix, and generation of compounds that may be inhibitory to microorganisms used in downstream processes.
An advantage of steam pretreatment is that it is one of a very few fractionation methods that have been tested in pilot scale, and commercial equipment is available. The process can be performed either in batch reactors, as in the “Masonite” process, or in continuos reactors, as in the “Stake” process (Heitz et al., 1991, Glasser and Wright 1998).
2.2.3.2. Wet oxidation
Wet oxidation (WO), a reaction involving oxygen and water at elevated temperature and pressure, was presented in the early 1980’s to pre-treat lignocellulose as an alternative to the well studied steam explosion (McGinnis et al., 1983a,b). The most important process parameters of wet oxidation are the reaction temperature and residence time like for steaming.
In the early studies of wet oxidation, usually the heating and the cooling times of the reaction have been very long (up to 30 min), and a low reaction temperature was needed to obtain fractionation. In the temperature range between 120-170°C it is mainly the hemicellulose fraction that is dissolved, but also the lignin and the cellulose fractions are affected to a smaller extent. At temperatures higher than 170°C, the reaction becomes increasingly more oxidative and causes a considerable amount of fragmentation and oxidation of the biomass, in particular the lignin, leading to the formation of organic acids and small amounts of neutral organic compounds. Studies using monosaccharides as model compounds indicate that most of the monosaccharides (88-100%) can be recovered after wet oxidation at 154°C The oligosaccharides are probably even more stable to the partial hydrolysis of the biomass, since the glycosidic linkages in the oligosaccharides are more stable to oxidation than the aldehyde group of the monosaccharide (McGinnis et al., 1983).
Recently, studies of wet oxidation of various herbaceous materials, e.g. corn stover and wheat straw employ a reactor with very short heating and cooling times, about 1-2 minutes.
Hence, a higher temperature can be used without extensive degradation of the polysaccharides. At 185°C and at 190°C very high recoveries for cellulose (above 95%) and reasonable recoveries for hemicellulose (about 60%) were found (Schmidt et al., 1998,
degradation. The extraction of the lignin fraction depends on the type of the raw material and the applied chemical during the pretreatment.
The principals both of the wet oxidation and the steam explosion processes are shown in Figure 2.4. using the plant residue, corn stover as an example. The steam pretreatment is carried out in generally in the presence of inorganic acid, while wet oxidation uses mainly alkaline addition. Following steam pretreatment the pressure is swiftly reduced (1-2 sec), which makes the materials undergo an explosive decompression, but after wet oxidation the pressure decreases slightly (15-20 min). The major difference between wet oxidation and steam explosion pretreatment is that the wet oxidation reaction is more complete due to the presence of oxygen.
Compared to other pretreatment processes, wet oxidation has the advantages that it is suitable for a wide range of biomasses, including both hardwoods, softwoods and agricultural residues, mainly for the generation of an easily accessible cellulose fraction due to the crystalline structure of cellulose is opened during the process. Organic molecules, including lignin, decompose to CO2, H2O and simpler and more oxidised organic compounds, mainly to low-molecular-weight carboxylic acids. This method appears to produce fewer by-products, like furfural and hydroxymethyl-furfural (Bjerre et al., 1996, Ahring et al., 1999).
Figure 2.4. A typical wet oxidation and the steam explosion concept
Under the conditions of wet oxidation, aliphatic aldehydes and saturated carbon bonds are very reactive, hence the sugar degradation products, which are known inhibitors of
microbial growth, are not expected to be produced at high concentration. The principle of the treatment is illustrated in Figure 2.5.
Apart from polysaccharides available for fermentation some degradation products were formed during the treatment (Klinke et al., 1999, 2002). these were identified as phenolic compounds and low molecular weight carboxylic acids that might be potential inhibitors during fermentation of the sugar fractions. The inhibitors were produced by decomposition of the plant constituents (lignin, hemicellulose, cellulose, pectin, wax) due to thermal degradation and oxidation. By alkaline wet oxidation, the production of furans seemed to be avoided (Bjerre et al., 1996, Schmidt et al., 1998) due to further oxidation of these compounds to carboxylic acids. The furans, 2-furfural and 5-hydroxymethil-2-furfural are common degradation products from steam explosion (Von Sivers and Zacchi 1996) due to thermal decomposition of xylose and glucose, respectively (also elucidated in Figure 2.5.).
Figure 2.5. A simplified flow diagram of the fractionation and formation of degradation products by WO of wheat straw
2.2.3.3. AFEX process
The ammonia fiber/freezer explosion (AFEX) process, which concept is similar to steam explosion is another type of physico-chemical pretreatment in which lignocellulosic materials are exposed to liquid ammonia at moderate temperatures from 25°C to 90°C, and elevated pressures for a period of time, from 10 to 60 min. (Holtzapple et al., 1991, 1992).
When the reaction is complete, the pressure is explosively released which disrupts the fibrous structure. After the explosion, to reduce the cost and protect environment, ammonia must be recycled (Holtzapple et al., 1994). In an ammonia recovery process, a superheated ammonia vapour with a temperature up to 200°C was used to vapourise and strip the residual ammonia in the pre-treated biomass and the evaporated ammonia was then withdrawn from the system by a pressure controller for recovery.
AFEX pretreatment appears to be more effective on agricultural residues and of various herbaceous crops and grasses, including alfalfa, wheat straw, corn stover and rice straw, etc. than on substrates derived from wood (Holtzapple et al., 1991.). Although testing on woody substrates has not been extensively reported. Over 90% hydrolysis of cellulose and hemicellulose has been obtained after AFEX pretreatment of Bermuda grass, but hydrolysis for the biomass with high lignin-content (20-25%) such as aspen wood chips was reported as only 50%. AFEX pretreatment can significantly improve the saccharification rates
The ammonia pretreatment does not produce inhibitors for the downstream biological processes, so water wash is not necessary (Dale and Moreira 1982, Mes-Hartree et al., 1987). The AFEX pretreatment does not significantly solubilise hemicellulose compared to acid pretreatment or acid catalysed steam explosion. (Mes-Hartree et al., 1988)
Mes-Hartree et al. compared the steam and ammonia pretreatment for enzymatic hydrolysis of aspen wood, wheat straw and alfalfa steams and found that steam explosion solubilise the hemicellulose, while AFEX did not.
2.2.3.4. CO2 explosion
Similar to steam and ammonia explosion pretreatment, CO2 explosion is also used for pretreatment of lignocellulosic materials. It was hypothesized that CO2 would form carbonic acid and increase the hydrolysis rate. Dale and Moreira (Dale 1982) used this method to pretreat alfalfa (4 kg CO2/kg fiber at the pressure of 5.62 MPa) and obtained 75% of the theoretical glucose released during 24 h of the enzymatic hydrolysis. The yields were relatively low compared to steam or ammonia explosion pretreatment, but high compared to the enzymatic hydrolysis without pretreatment. It was found, that CO2
explosion was more cost-effective than ammonia explosion and did not cause the formation of inhibitory compounds that could occur in steam explosion.
2.2.4. Chemical pretreatment
Chemical pretreatments can be simple; such as soaking the biomass in sodium hydroxide at room temperature, or more complicated as, for example, treating the material with acidic or basic catalysis at high-temperature (Stenberg et al., 2000a,b, Kaar et al., 1998, Holtzapple et al., 1991). Especially these latest methods are very effective. During chemical pretreatment processes, hemicellulose and/or lignin may be hydrolysed to their monomeric constituents and lignin-cellulose-hemicellulose interactions are partially disrupted, thus increasing the enzymatic digestibility of cellulose.
2.2.4.1. Acidic pretreatment
Concentrated acids such as H2SO4 and HCl have been used to pretreat lignocellulosic materials. Although they are powerful agents for cellulose hydrolysis, concentrated acids are toxic, corrosive and hazardous and require reactors that are resistant to corrosion. In addition, the concentrated acid must be recovered after hydrolysis to make the process economically feasible (Von Sivers and Zacchi 1996).
Dilute acid hydrolysis has been successfully developed for pretreatment of lignocellulosic materials. The dilute sulphuric acid pretreatment can achieve high reaction rates and significantly improve cellulose hydrolysis at high temperature (Esteghlalian et al., 1997), but at moderate temperature the saccharification yields of cellulose is quite poor.
(McMillan 1994).
However, if the hydrolysis of the hemicellulose fraction is the aim of the experiments, low temperature is more favourable. Recently developed dilute acid (mainly sulphuric acid) hydrolysis processes use less severe conditions and achieve high xylan to xylose conversion yields. Achieving high xylan to xylose conversion yields is necessary to achieve high overall process economics because xylan accounts for up to a third of the total carbohydrate in many lignocellulosic materials (Hinman et al., 1992).
Wilke et al. (1981) used dilute sulphuric acid pretreatment efficiently for saccharification of hemicellulose in various herbaceous materials, e.g. wheat straw and rice straw. However the obtained conversion of cellulose to glucose was only 40% and the overall yield of all polysaccharides by enzymatic hydrolysis was also quite low, around 60%.
Although dilute acid pretreatment can significantly improve the cellulose hydrolysis, its cost is usually higher than some physico-chemical pretreatment processes such as steam explosion or AFEX. A neutralization of pH is necessary before enzymatic hydrolysis or fermentation processes.
2.2.4.2. Alkaline pretreatment
Some bases, e.g. sodium-, potassium-, calcium-, and ammonium-hydroxide are appropriate chemicals for pretreatment of lignocellulose, but the effect of alkaline pretreatment depends on the lignin content of the materials. Alkaline pretreatment techniques are basically delignification processes, however, generally a significant amount of hemicellulose solubilise as well. During these mechanisms the porosity of the lignocellulosic materials increases with the removal of the crosslinks of intermolecular ester bonds between xylan hemicelluloses and other components, for example, lignin and other hemicellulose (McMillan 1994). Dilute NaOH treatment of lignocellulosic materials caused swelling, leading to an increase in internal surface area, a decrease in the degree of polymerization, a decrease in crystallinity, separation of structural linkages between lignin and carbohydrates, and disruption of the lignin structure. Compared with acid processes, alkaline pretreatment results in lower degradation of sugars.
The digestibility of NaOH-treated hardwood increased from 14% to 55% with the decrease of lignin content from 24-55% to 20%. However, no effect of dilute NaOH pretreatment was observed for softwoods with lignin content greater than 26% (Millet et al., 1976).
Dilute NaOH pretreatment was also effective for the hydrolysis of straws with relatively low lignin content of 10-18% (Bjerre et al., 1996).
MacDonald et al. (1983) obtained 77.5% overall conversion applying a dilute sodium hydroxide pretreatment at high temperature for corn stover. Elshafei et al. (1991) achieved nearly the theoretical conversion maximum for cellulose, by soaking the corn stover in 1.0 M sodium hydroxide for 24 h at room temperature. Kaar et al. (2000) reported 88.0%
conversion of cellulose to glucose using slake lime as a pretreatment for 4 h at 120°C.
Moreover, lime gives the possibility to recover calcium quite easily, as an insoluble calcium carbonate.
Ammonia was also used for the pretreatment to remove lignin. Iyer et al. (1996) described an ammonia recycled percolation process (temperature, 170°C; ammonia concentration, 2.5-20%; reaction time 1 h) for the pretreatment of corn cobs/stover mixture and switchgrass. The efficiency of delignification was 60-80% for corn cobs and 65-85% for switchgrass.
2.2.4.3. Organosolv process
In the organosolv process, an organic or aqueous organic solvent mixture alone, or with addition of an acid (HCl or H2SO4), or rarely alkaline catalyst is used to break the internal lignin and hemicellulose bonds. Organic solvents, such as methanol, ethanol, acetone, ethylene glycol, etc. are used in this process (Chum et al., 1988). If the process is conducted at high temperature (T>185°C), there is no need for acid addition, as it is
believed that organic acids released from the wood or from other lignocellulosic material act as catalysts for the rupture of the lignin – carbohydrate complex. However usually, a high yield of xylose can be obtained with the addition of acid (Wright 1988). Both the hemicellulose and the lignin fraction are solubilized, while the cellulose remains as a solid.
The cellulose fraction from organosolv pretreatment is susceptible to enzymatic hydrolysis.
Chum et al. (1988) reported more, than 85% conversion of cellulose to glucose, following pretreatment at 195°C.
However, because organic solvents are costly, potentially dangerous, and inflammable and their use requires high-pressure equipment, the process is perceived as complex and expensive. (Schell et al., 1991.) Solvents used in the process need to be drained from the reactor, evaporated, condensed and recycled to reduce the cost. Removal of solvents from the system is also necessary because the solvents may be inhibitory to the growth of organisms, enzymatic hydrolysis, and fermentation.
2.2.4.4. Ozonolysis
Ozone can be used to degrade lignin and hemicellulose in many herbaceous materials.
Ben-Ghedalia used this technique successfully for wheat straw (Ben-Ghedalia and Miron 1981). The degradation was essentially limited to lignin and hemicellulose was slightly attacked, but cellulose was hardly affected. The yield of enzymatic hydrolysis increased from 20% to 57%, following 60% removal of the lignin from wheat straw in ozone pretreatment. Ozonolysis pretreatment has the following advantages:
(1) it effectively removes lignin;
(2) it does not produce toxic residues for the downstream processes; and (3) the reactions are carried out at room temperature and pressure.
However, a large amount of ozone is required, making the process expensive.
2.3. H
YDROLYSIS PROCESSESMost processes for the production of ethanol from lignocellulosic materials have similar designs based on feedstock handling, hydrolysis, fermentation and distillation. Big difference usually lies in how the hydrolysis step is performed. Therefore, the processes have been divided into two big groups, according to the design of the hydrolysis steps, enzymatic and acid hydrolysis processes (Figure 2.6.).
Figure 2.6. Schematic flowchart of the “ethanol from lignocellulosic biomass” process
2.3.1. Acid hydrolysis
Acid hydrolysis of plant lignocellulosic biomass has been known since 1819. (Von Sivers 1989). Acid hydrolysis can be performed with several types of acids, including sulphuric, hydrochloric, hydrofluoric, phosphoric, nitric and formic acid. These acids may be either concentrated or diluted. Processes involving concentrated acids are operated at low temperature and give high yields (e.g. 90% of theoretical glucose yield), but the large amount of acids causes problems both in economical and environmental aspect.
Furthermore, when sulphuric acid is used the neutralisation process produces large amounts of gypsum. However, the process has attached some new interest due to novel economic methods for acid recovery proposed.
Dilute acid hydrolysis is fast and easy to perform and it has the advantage of the relatively low acid consumption, but is hampered by non-selectivity and by-product formation.
Namely, high temperatures are required to achieve acceptable rates of conversion of cellulose to glucose, but high temperatures increase also the rates of hemicellulose sugar decomposition and equipment corrosion. Under these conditions, xylose degrades to furfural and glucose degrades to 5-hydroxymethyl furfural (HMF), both of which are toxic
to microorgaisms and can also cause inhibition in the subsequent fermentation stage. The maximum yield of glucose is obtained at high temperature and short residence time, but even under these conditions the glucose yield is only between 50% and 60% of the theoretical.
A two stage acid hydrolysis process enables the hemicellulose and cellulose to be degraded separately under conditions appropriate for each reaction. In a pre-hydrolysis or pretreatment stage under rather mild conditions the relatively easily hydrolysed hemicellulose is removed. These enables the second acid hydrolysis step to proceed under harsher conditions without degrading the hemicellulose sugars to furfural and other substances. Using a two-stage dilute acid hydrolysis process, recovery yields of as much as 70-98% of xylose, arabinose and galactose were obtained from corn stover. However the yield of glucose 60% was still quite low (Wilke et al 1981).
On the top of the Figure 2.6. is the concentrated acid process that uses sulphuric acid to hydrolysis lignocellulosic biomass prior to fermentation to ethanol. Very efficient recycling is required for the process to be cost effective. The second part of Figure shows the two stage acid hydrolysis process prior fermentation. The processes depicted along the bottom of the figure is the separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes.
2.3.2. Enzymatic hydrolysis
The biodegradation of lignocellulosics has been investigated since the 1960’s (Philipidis et al., 1993, 1996). Using cellulase enzymes as catalyst, a very specific conversion of cellulose is performed. There is wide range of microorganisms, both bacteria and fungi, which produce enzymes capable of hydrolysing cellulose. Of all these microorganisms, Trichoderma species have been most extensively studied for cellulase production.
(Coughlan et al., 1992)
During the experimental work of this thesis, a commercial available cellulase (Celluclast 1.5 L from Novozymes) was used in the enzymatic hydrolysis, which is also a cellulase enzyme complex by Trichoderma reesei.
Cellulases are usually a mixture of several cellulolytic enzymes. At least three major groups of cellulases are involved in the hydrolysis process (Parisi 1989):
(1) endoglucanases (EG), which are highly active on amorphous cellulose, creating free chain ends;
(2) exoglucanases or cellobiohydrolases (CBH), which degrade the molecule further by removing cellobiose units from the free chain-ends, and
(3) ß-glucosidase, which hydrolyses cellobiose to produce glucose (Coughlan et al., 1988).
Strictly speaking, “cellulases” is defined as the endo- and exocellulases and does not include ß-glucosidase. However it has a very important role in hydrolysis, since cellobiose is highly inhibitory to many cellulases and is unusable by most microorganisms.
ß-glucosidase hydrolyses cellobiose to glucose which is much less inhibitory and highly fermentable. (Holtapple et al., 1991) On the other hand ß-glucosidase is inhibited by glucose. Cellulases are inhibited by end-products, thus the build-up of any of these products decrease the efficiency of hydrolysis.
Because of the specification of cellulases it is considered to have the potential of higher yields than acid hydrolysis. Other advantages of enzymatic hydrolysis are the mild operating conditions and the high quality sugar products. The maximum cellulase activity for most cellulases and ß-glucosidase occurs at 50±5°C and a pH 4.0-5.0.(Saddler and Gregg 1998) In addition to these, enzymes are naturally occurring compounds which are biodegradable and therefore environmental friendly. However nowadays the enzymatic reactions are quite slow and the biomass must be pre-treated to improve the yields and kinetics.
2.3.2.1. Surfactant effect in enzymatic hydrolysis
Cellulase enzyme loading of 10 – 20 FPU/g cellulose is often used in laboratory studies because it provides a hydrolysis profile with high level of glucose yield in a reasonable time (24-72 h) at a reasonable enzyme cost (Gregg and Saddler, 1996).
High cellulose conversion requires high enzyme loading, which has strong negative effects on the process economy. Thus, methods to increase enzyme effectiveness are important for reduction of enzyme consumption. Addition of surfactants to enzymatic hydrolysis of lignocellulose gives a possibility to increase the conversion of cellulose into soluble sugars, without increasing the enzyme loading.
It is believed, that enzymatic hydrolysis of cellulose consists of three steps: adsorption of cellulase enzymes onto the surface of the cellulose, the biodegradation of cellulose to fermentable sugars, and desorption of cellulase. Cellulase activity decreases during the hydrolysis. The irreversible adsorption of cellulase on cellulose is partially responsible for this deactivation (Converse et al., 1988).
Adsorption of enzymes to cellulose during hydrolysis has been shown to decrease in the presence of surfactant (Castanon et al., 1981, Helle et al., 1993). Different mechanisms have been proposed for the positive effect of surfactant addition in the enzymatic hydrolysis of cellulose. The surfactant could change the nature of the substrate, e.g. by increasing the available cellulose surface or by removing inhibitory lignin (Helle et al., 1993). Based on kinetic analysis, Kaar and Holtzapple (1998) have found indications that surfactants could promote the availability of reaction sites, which would increase the
hydrolysis rate. The surfactant could also increase the stability of the enzymes and thus, reduce enzyme denaturation during the hydrolysis. Surfactant effects on enzyme- substrate interaction have been proposed, e.g. adsorbed enzymes are prevented from inactivation by addition of surfactant which facilitates desorption of enzymes from substrate (Park et al., 1992).
Enhancement of cellulose hydrolysis by adding surfactants to the hydrolysis mixture has been reported by several authors (Helle et al., 1993, Ooshima et al., 1986, Castanon and Wilke 1981, Kaar et al., 1998) and different cellulose substrates have been studied.
Castanon and Wilke (1981) showed that conversion of newspaper was increased by 14%
after 48 h hydrolysis by the addition of Tween 80. They also investigated the effect of Tween additives for enhancing enzymatic saccharification on non-native cellulose and cellulose analogs such as newsprint, microcrystalline cellulose and carboxymethylcellulose (CMC). These researchers postulated that Tween 80 assists the desorption of enzyme from substrate. Park et al. (1991), also working with newspaper examined several surfactants and found Tween to be among the best performers, resulted 10% increase in the cellulose conversion. Increased hydrolysis by addition of surfactants has also been reported for steam-exploded wood (Helle et al., 1993, Erikkson et al., 2002, Alkasrawi et al., 2003), bagasse (Kurakae et al, 1994), and lime-pre-treated corn stover (Kaar et al., 1998). Eriksson et al. (2002) found, that addition of non-ionic surfactant increased significantly the conversion of steam-pretreated spruce. With addition of Tween 20 at 2.5 g L-1 it was possible to lower the enzyme loading by 50% and at the same time retain cellulose conversion.
Ooshima et al. (1986) compared amorphous cellulose with different types of crystalline celluloses (Avicel, tissue paper and reclaimed paper). They showed that the higher the crystallinity of the substrate, the more positive was the effect of the added surfactant. It was also observed, that the surfactant effect is higher at low cellulase concentration. In addition to these Eriksson et al. (2002) published, that the effect of the surfactants was significantly lower, when delignified cellulose substrate was used. They proposed, that the dominating mechanism for surfactant effects in substrates containing lignin, be found in the role of surfactant on enzyme interaction with lignin surfaces. Surfactant adsorption to lignin prevents unproductive binding of enzymes to lignin.
The surfactants used in the enzymatic hydrolysis include non-ionic Tween 20, (polyoxyethylene sorbitan monolaurate) and Tween 80 (polyoxyethylene sorbitan monooleate) (Kaar et al., 1998), polyoxyethylene glycol (Park et al., 1992), sophorolipid, rhamnolipid, and bacitracin (Helle et al., 1993). Inhibitory effects have been observed with cationic Q-86W at high concentration and anionic surfactant Neopelex F-25, thus non - ionic surfactants are therefore believed to be more suitable for enhancing the cellulose hydrolysis.
After the published positive results of the surfactant on enzymatic hydrolysis, it was suspected that surfactant could increase the enzymatic saccharification of a native lignocellulose like corn stover. Preliminary experiments were carried out on ground corn stover improving the enzyme efficiency using Tween 80 supernatant. It was added to the mixture of corn stover and sodium citrate buffer (pH 4.8), before the loading of cellulase. The dry matter (DM) content in the mixture was 5% and the enzyme loading was 25 FPU/ g DM. The recommended Tween loading, according to the results of Kaar et al (1998) was 0.15 g Tween/g dry biomass.
The achieved conversion of ground corn stover with Tween 80 was 21% compared to 19% without addition of Tween. Thus, contrary to my all expectations the conversion of cellulose to glucose increased by only 8% after 24 h hydrolysis by the addition of Tween 80.
2.4. F
ERMENTATION FOR BIOETHANOL PRODUCTIONSeveral enzyme-catalysed processes have been emphasised for the conversion of lignocellulosic biomass into ethanol: separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF) and direct microbial conversion (DMC).
2.4.1. Separate hydrolysis and fermentation (SHF)
The main advantage of separate hydrolysis and fermentation (SHF) process is the ability to carry out both the hydrolysis and the fermentation under optimal conditions, e.g., enzymatic hydrolysis at 40-50°C, which is optimal temperature of the cellulases as it was mentioned, and fermentation at about 30°C, which is required temperature for most ethanol fermenting microorganisms, e.g. Saccharomyces cerevisae. The major drawback of SHF is that the released sugars inhibit the cellulases during hydrolysis, therefore the hydrolysis rate in SHF is strongly affected by end-product inhibition (Alfani et al., 2000)
2.4.2. Simultaneous saccharification and fermentation (SHF)
The simultaneous saccharification and fermentation (SSF) process was published in 1977 by Takagi et al (Takagi et al., 1977, Gauss et al., 1976) and has proven to be a promising alternative to SHF. The key to the SSF process is its ability to rapidly convert sugars into ethanol avoiding their build-up in their fermentation broth. In SSF the produced glucose is immediately consumed by the fermenting microorganism. The ethanol produced can also act as an inhibitor in the conversion process, but sugars in the enzymatic hydrolysis of cellulose, are much more inhibitory than ethanol is (Takagi 1984). Thus SSF process can achieve greater rates, yields, and concentrations than competing process now known. (Wyman et al., 1992) The SSF process also eliminates expensive equipment and
