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Production of bioethanol from lignocellulosic materials via the biochemical

pathway: A review

Mustafa Balat

Sila Science & Energy Company, University Mah, Trabzon, Turkey

a r t i c l e i n f o

Article history:Received 1 January 2010

Accepted 15 August 2010

Available online 6 September 2010

Keywords:

Bioethanol

Lignocellulosic material

Pretreatment

Enzymatic hydrolysis

Fermentation

a b s t r a c t

Bioethanol is by far the most widely used biofuel for transportation worldwide. Production of bioethanolfrom biomass is one way to reduce both consumption of crude oil and environmental pollution. Bioeth-anol can be produced from different kinds of raw materials. These raw materials are classified into three

categories of agricultural raw materials: simple sugars, starch and lignocellulose. The price of the rawmaterials is highly volatile, which can highly affect the production costs of the bioethanol. One major

problem with bioethanol production is the availability of raw materials for the production. Lignocellu-losic biomass is the most promising feedstock considering its great availability and low cost, but the

large-scale commercial production of fuel bioethanol from lignocellulosic materials has still not beenimplemented.

2010 Elsevier Ltd. All rights reserved.

1. Introduction

Today, the transportation sector worldwide is almost entirelydependent on petroleum-based fuels. It is responsible for 60% ofthe world oil consumption [1]. In addition, transportation sectoraccounts for more than 70% of global carbon monoxide (CO) emis-sions and 19% of global carbon dioxide (CO2) emissions [2]. CO2emissions from a gallon of gasoline are about 8 kg [3]. Aroundthe world, there were about 806 million cars and light trucks onthe road in 2007[4]. These numbers are projected to increase to1.3 billion by 2030 and to over 2 billion vehicles by 2050[5]. Thisgrowth will affect the stability of ecosystems and global climate aswell as global oil reserves. The dramatic increase in the price ofpetroleum, the finite nature of fossil fuels, increasing concernsregarding environmental impact, especially related to greenhousegas (GHG) emissions, and health and safety considerations are forc-ing the search for new energy sources and alternative ways to

power the worlds motor vehicles. An alternative fuel must betechnically feasible, economically competitive, environmentallyacceptable, and readily available[6]. Numerous potential alterna-tive fuels have been proposed, including bioethanol, biodiesel,methanol, hydrogen, boron, natural gas, liquefied petroleum gas(LPG), FischerTropsch fuel, p-series, electricity, and solar fuels.

Biomass-based fuels, also known as biofuels offer manyadvantages over petroleum-based fuels[7]: (1) biofuels are easilyavailable from common biomass sources, (2) they are represent aCO2-cycle in combustion, (3) biofuels have a considerable environ-

mentally friendly potential, (4) there are many benefits the envi-ronment, economy and consumers in using biofuels, and (5) theyare biodegradable and contribute to sustainability. The major ben-efits of biofuels are given inTable 1. The use of biofuels can con-tribute to the mitigation of GHG emissions, provide a clean andtherefore sustainable energy source, and increase the agriculturalincome for rural poor in developing countries. Developing coun-tries have a comparative advantage for biofuel production becauseof greater availability of land, favorable climatic conditions foragriculture and lower labour costs. However, there may be othersocio-economic and environmental implications affecting the po-tential for developing countries to benefit from the increased glo-bal demand for biofuel [8]. Large-scale production of biofuelsoffers an opportunity for certain developing countries to reducetheir dependence on oil imports. In developed countries there isa growing trend towards employing modern technologies and effi-cient bioenergy conversion using a range of biofuels, which are

becoming cost-wise competitive with fossil fuels[9].Biofuels are made from bio-based materials through thermo-

chemical processes such as pyrolysis [10,11], gasification[12,13],liquefaction [14], supercritical fluid extraction [15], supercriticalwater liquefaction [16] and biochemical [17]. Thermo-chemicalreforming of biomass concerns the processes of catalytic andnon-catalytic pyrolysis as well as the gasification, which aims atthe maximization of the production of energetically exploitableliquid and gaseous products.

Biofuels include bioethanol, biomethanol, vegetable oils,biodiesel, biogas, biosynthetic gas (bio-syngas), bio-oil, bio-char,FischerTropsch liquids, and biohydrogen. The term biofuels canrefer to fuels for direct combustion for electricity production, but

0196-8904/$ - see front matter 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.enconman.2010.08.013

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Energy Conversion and Management 52 (2011) 858875

Contents lists available at ScienceDirect

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is generally used for liquid fuels for transportation sector [18].Renewable liquid biofuels for transportation have recently at-tracted huge attention in different countries all over the world be-cause of its renewability, sustainability, common availability,regional development, rural manufacturing jobs, reduction ofGHG emissions, and its biodegradability[19].

Bioethanol is by far the most widely used biofuel for transpor-tation worldwide. Bioethanol and bioethanol/gasoline blends havea long history as alternative transportation fuels. It has been usedin Germany and France as early as 1894 by the then incipientindustry of internal combustion engines (ICEs)[20]. Brazil has uti-lized bioethanol as a fuel since 1925. By that time, the productionof bioethanol was 70 times bigger than the production and con-sumption of petrol[21]. The use of bioethanol for fuel was wide-

spread in Europe and the United States until the early 1900s.Because it became more expensive to produce than petroleum-based fuel, especially after World War II, bioethanols potentialwas largely ignored until the oil crisis of the 1970s [22]. Sincethe 1980s, there has been an increased interest in the use of bio-ethanol as an alternative transportation fuel.

To ensure that good bioethanol is produced, with reference toGHG benefits, the following demands must be met[23]: (1) bioeth-anol plants should use biomass and not fossil fuels, (2) cultivationof annual feedstock crops should be avoided on land rich in carbon(above and below ground), such as peat soils used as permanentgrassland, (3) by-products should be utilized efficiently in orderto maximize their energy and GHG benefits, and (4) nitrous oxideemissions should be kept to a minimum by means of efficient fer-

tilization strategies, and the commercial nitrogen fertilizer utilizedshould be produced in plants which have nitrous oxide gas clean-ing. Bioethanol is a fuel derived from renewable sources of feed-stock; typically plants such as wheat, sugar beet, corn, straw, andwood. Bioethanol is an alternative fuel that is produced almost en-tirely from food crops. It represents an important, renewable liquidfuel for motor vehicles. Producing bioethanol as a transportationfuel can help reduce CO2 buildup in two important ways: by dis-placing the use of fossil fuels, and by recycling the CO 2 that is re-leased when it is combusted as fuel. An important advantage ofcrop-based bioethanol is its GHG benefits[24].

2. Bioethanol as a transportation fuel

The alcohols are oxygenates fuels that the alcohol molecule hasone or more oxygen, which decreases to the combustion heat.

Practically, any of the organic molecules of the alcohol family canbe used as a fuel. The alcohols can be used for motor fuels aremethanol (CH3OH), bioethanol (C2H5OH), propanol (C3H7OH),butanol (C4H9OH). However, only methanol and bioethanol fuelsare technically and economically suitable for internal combustionengines (ICEs)[24].

Bioethanol is ethyl alcohol, grain alcohol, or chemically C 2H5OH

or EtOH. It has high octane number (108)[25], both permit the ris-ing of the compression ratio and gives lower emission[26]. Octanenumber is a measure of the gasoline quality for prevention of earlyignition, which leads to cylinder knocking. The fuels with higheroctane numbers are preferred in spark-ignition ICEs. An oxygenatefuel such as bioethanol is provides a reasonable antiknock value[3]. Disadvantages of bioethanol include its lower energy densitythan gasoline (but about 35% higher than that of methanol), its cor-rosiveness, low flame luminosity, lower vapor pressure (makingcold starts difficult), miscibility with water, and toxicity to ecosys-tems[27], increase in exhaust emissions of acetaldehyde, and in-crease in vapor pressure (and evaporative emissions) whenblending with gasoline. Physical and chemical properties of bioeth-anol, methanol and gasoline are given inTable 2[28].

Bioethanol has been used as a modern biofuel, applied directlyas a gasoline improver or gasoline subsistent, or in the form ofETBE (ethyl tertiary butyl ether) for currently added syntheti-cally-produced octane enhancers and in bioethanoldiesel blendswith particular purpose to reduce the emissions of exhaust gasses[29].Bioethanol is most commonly blended with gasoline in con-centrations of 10% bioethanol to 90% gasoline, known as E10 andnicknamed gasohol. Bioethanol can be used as a 5% blend withpetrol under the European Union (EU) quality standard EN 228.This blend requires no engine modification and is covered by vehi-cle warranties. With engine modification, bioethanol can be usedat higher levels, for example, E85[30]. Some countries has exer-cised biofuel program both form bioethanolgasoline blendprogram such as the United States (E10 and for flexible-fuelvehicle-FFV E85), Canada (E10 and for FFV E85), Sweden (E5 and

for FFV E85), India (E5), Australia (E10), Thailand (E10), China

Table 1

Major benefits of biofuels.

Economic impacts Sustainability

Fuel diversity

Increased number of rural manufacturing jobsIncreased income taxes

Increased investments in plant and equipment

Agricultural development

International competitiveness

Reducing the dependency on imported petroleum

Environmental impacts Greenhouse gas reductions

Reducing of air pollution

Biodegradability

Higher combustion efficiency

Improved land and water use

Carbon sequestration

Energy security Domest ic targets

Supply reliability

Reducing use of fossil fuels

Ready availability

Domestic distribution

Renewability

Table 2

Physical and chemical properties of ethanol, methanol and gasoline.

Property Methanol

CH3OH

Ethanol

C2H5OH

Gasoline

C4-C12

Molecular weight (g/mol) 32 46 114

Specific gravity 0.789 (298 K) 0.788 (298 K) 0.739

(288.5 K)

Vapor density rel. to air 1.10 1.59 3.04.0Liquid density (g cm3

at 298 K)

0.79 0.79 0.74

Boiling point (K) 338 351 300518

Melting point (K) 175 129 Heat of evaporation (Btu/lb) 472 410 135

Heating value (kBTU gal1)

Lower 58 74 111

Upper 65 85 122

Tank design pressure (psig) 15 15 15

Viscosity (cp) 0.54 1.20 0.56

Flash point (K) 284 287 228

Flammability/explosion

limits

(%) Lower (LFL) 6.7 3.3 1.3

(%) Upper (UFL) 36 19 7.6

Auto ignition

temperature (K)

733 636 523733

Solubility in H2O (%) Miscib.

(100%)

Miscib.

(100%)

Negl. (0.01)

Azeotrope with H2O None 95% EtOH Immiscible

Peak flame temperature (K) 2143 2193 2303Minimum ignition energy

in air (mJ)

0.14 0.23

M. Balat / Energy Conversion and Management 52 (2011) 858875 859

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(E10), Columbia (E10), Peru (E10), Paraguay (E7), Brazil (E20, E25and FFV any blend).

The reduced CO2emissions mean that bioethanol is good for theenvironment. Using bioethanol-blended fuel for automobiles cansignificantly reduce petroleum use and exhaust GHG emission[24]. On a life-cycle basis, not all biofuels are equal in terms of

environmental benefits.Fig. 1demonstrates the lower GHG emis-sions resulting from the use of biofuels compared to gasoline ona life-cycle basis. AsFig. 1illustrates, corn-based bioethanol offersrather limited benefits, as it reduces GHG emissions by only 18%compared to gasoline. In contrast, sugarcane and cellulosic bioeth-anol result in almost 90% lower emissions[31].

The net energy balance of biomass to bioethanol conversion isthe key parameter that explains the interest in using bioethanolfuel instead of fossil gasoline. From a life-cycle assessment (LCA)viewpoint, the ratio of the energy content of bioethanol to thenet non-renewable primary energy (allocated to bioethanol) con-sumed in the whole production process from biomass productionto its conversion into bioethanol. As the approach is LCA oriented,the energy input must be estimated in terms of primary energy

[32]. Studies have shown that corn-based bioethanol yields 2030% more energy, typically fossil fuel energy, than is consumedin making it. On the other hand, sugarcane and cellulosic bioetha-nol yield renewable energy nine times worth the fossil energy usedto produce them[31].

3. An overview of bioethanol feedstocks

Bioethanol can be produced from different kinds of raw materi-als. The raw materials are classified into three categories of agricul-tural raw materials: sucrose-containing feedstocks (e.g. sugar cane,sugar beet, sweet sorghum and fruits), starch materials (e.g. corn,milo, wheat, rice, potatoes, cassava, sweet potatoes and barley)and lignocellulosic materials (e.g. wood, straw and grasses). Cur-

rently, a focus is on bioethanol production from crops, such as corn,wheat, sugar cane, as well as on highly abundant agriculturalwastes.

One major problem with bioethanol production is the availabil-ity of raw materials for the production. The availability of feedstockfor bioethanol can vary considerably from season to season and de-pends on geographic locations. Locally available agricultural bio-mass will be used for the bioethanol production[33]. For a givenproduction line, the comparison of the feedstocks includes severalissues[34]: (1) chemical composition of the biomass, (2) cultiva-tion practices, (3) availability of land and land use practices, (4)use of resources, (5) energy balance, (6) emission of greenhousegases, acidifying gases and ozone depletion gases, (7) absorptionof minerals to water and soil, (8) injection of pesticides, (9) soil

erosion, (10) contribution to biodiversity and landscape valuelosses, (11) farm-gate price of the biomass, (12) logistic cost (trans-

port and storage of the biomass), (13) direct economic value of thefeedstocks taking into account the co-products, (14) creation ormaintain of employment, and (15) water requirements and wateravailability.

Brazil utilizes sugarcane for bioethanol production while theUnited States and Europe mainly use starch from corn, and fromwheat and barley, respectively. Sugarcane as a biofuel crop hasmuch expanded in the last decade, yielding anhydrous bioethanol

(gasoline additive) and hydrated bioethanol by fermentation anddistillation of sugarcane juice and molasses[35]. Brazils sugarcaneyield averages about 82.4 tons/ha[36]. The yield of bioethanol perhectare, currently at around 6650 l/ha (Table 3)[37]. Brazil is thelargest single producer of sugarcane with about 31% of global pro-duction[35]. It has nearly 9 million hectares of sugarcane undercultivation. Sugar beet crops are grown in most of the EU-25 coun-tries, and yield substantially more bioethanol per hectare thanwheat.

The United States is predominantly a producer of bioethanol de-rived from corn, and production is concentrated in Midwesternstates with abundant corn supplies[38]. Feedstock availability isnot expected to be a constraint for bioethanol production overthe next decade. Corn is expected to remain the predominant feed-

stock in the United States, although its share likely will declinemodestly by 2015. Corn-based bioethanol production in most ofthe countries assessed is limited, especially compared to the Uni-ted States. Only Canada reported explicit plans for significant fu-ture development of corn-based bioethanol, although China hasused corn as a feedstock in the past and Argentina is looking atthe possibility of corn as biofuel feedstock in the future[39].

4. Lignocellulosic-biomass materials

4.1. Availability of lignocellulosic material

The price of the raw materials is also highly volatile, which canhighly affect the production costs of the bioethanol[40]. Lignocel-

lulosic materials serve as a cheap and abundant feedstock, which isrequired to produce fuel bioethanol from renewable resources atreasonable costs. In 2007 the US Department of Energy providedmore than US$1 billion toward lingocellulosic bioethanol projects,with the goal of making the fuel cost competitive at US$1.33 pergallon by 2012[41]. The level of support provided by the EU isfar less, but is still significant (approximately US$68 million in2006)[41].

Lignocellulosic materials can be classified in four groups basedon type of resource: (1) forest residues, (2) municipal solid waste,(3) waste paper, and (4) crop residue resources. Literature reportsseveral papers on utilization of various lignocellulosic waste mate-rials such as rice straw [42], corn stover [43], switchgrass [44],palm bagasse[45], etc.

Lignocellulosic materials could produce up to 442 billion litersper year of bioethanol [46]. Rice straw is one of the abundant

Fig. 1. Reduction in GHG emissions, compared to gasoline, by bioethanol produced

from a variety of feedstocks (on a life-cycle basis) [31].

Table 3

Bioethanol yields from different energy crops.

Country Energy crop Bioethanol yield (l/ha)

Brazil Sugarcane, 100% 6641USA Corn, 98% 3770

Sweet sorghum, 2% 1365

China Corn, 70% 2011Wheat, 30% 1730

EU-27 Wheat, 48% 1702

Sugar beet, 29% 5145

Canada Corn, 70% 3460

Wheat, 30% 1075

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lignocellulosic waste materials in theworld. It is annually producedabout 731 million tons which is distributed in Africa (20.9 milliontons), Asia (667.6 million tons), Europe (3.9 million tons), America(37.2 million tons) and Oceania (1.7 million tons). This amount ofrice straw can potentially produce 205 billion liters bioethanol peryear, which is the largest amount from a single biomass feedstock[47].

4.2. Chemical structure and basic components of lignocellulosic

materials

Chemical composition of lignocellulosic materials is a key factoraffecting efficiency of biofuel production during conversion pro-cesses. The structural and chemical composition of lignocellulosicmaterials is highly variable because of genetic and environmentalinfluences and their interactions[48]. A typical chemical composi-tion of lignocellulosic materials is 48 wt.% C, 6 wt.% H, and 45 wt.%O, the inorganic matter being a minor component[49]. The proxi-mate analysis of rice straw and wheat straw shows components asfollow: volatile matter (65.47%, 75.27%), fixed carbon (15.86%,17.71%) and ash (18.67%, 7.02%), respectively[50].

Lignocelluloses consist mainly of cellulose, hemicellulose andlignin; these components build up about 90% of dry matter inlignocelluloses, with the rest consisting of e.g. extractive and ash[51]. The basic structure of all woody biomass consists of three ba-sic polymers: cellulose (C6H10O5)x, hemicelluloses such as xylan(C5H8O4)m, and lignin [C9H10O3(OCH3)0.91.7]n in trunk, foliage,and bark. The proportion of these wood constituents varies be-tween species, and there are distinct differences between hard-woods and softwoods. Cellulose + hemicellulose contents aremore in hardwoods (78.8%) than softwoods (70.3%), but lignin ismore in softwoods (29.2%) than hardwoods (21.7%)[52]. The struc-tural composition of various types of lignocellulosic-biomassmaterials are given inTable 4[53].

Cellulose and hemicellulose, which typically make up two-

thirds of cell wall dry matter, are polysaccharides that can behydrolyzed to sugars and then fermented to bioethanol. Processperformance, i.e. Bioethanol yield from biomass, is directly relatedto cellulose, hemicellulose, and individual sugar concentration inthe feedstock [54]. The lignin cannot be used for bioethanolproduction.

Cellulose, the major componentof plant biomass (3060% of totalfeedstockdry matter), is a linear polymer of glucose; theorientationof the linkages and additional hydrogen bonding make the polymerrigid and difficult to break. In hydrolysis the polysaccharide isbroken down to free sugar molecules by the addition of water [55].

This process is also known as saccharification. Theproduct, glucose,is a six-carbon sugar.

Hemicellulose (2040% of total feedstock dry matter) is a short,highly branched polymer of five-carbon (pentoses) and six-carbon(hexoses) sugars. Specifically, hemicellulose contains xylose andarabinose (five-carbon sugars) and galactose, glucose, and man-nose (six-carbon sugars). Hemicellulose is more readily hydrolyzed

compared to cellulose because of its branched, amorphous nature[48].The dominant sugars in hemicelluloses are mannose in soft-woods and xylose in hardwoods and agriculture residues [56].

Lignin (1525% of total feedstock dry matter) is an aromaticpolymer synthesised from phenylpropanoid precursors. The basicchemical phenylpropane units of lignin (primarily syringyl, guaia-cyl and p-hydroxy phenol) are bonded together by a set of linkagesto form a very complex matrix[57]. This matrix comprises a vari-ety of functional groups, such as hydroxyl, methoxyl and carbonyl,which impart a high polarity to the lignin macromolecule [58].Softwood and hardwood lignins belong to the first and second cat-egory, respectively. Softwoods generally contain more lignin thanhardwoods[59]. Lignin contents on a dry basis in both softwoodsand hardwoods generally range from 20% to 40% by weight andfrom 10% to 40% by weight in various herbaceous species, suchas bagasse, corncobs, peanut shells, rice hulls and straws [60]. Lig-nin is one of the drawbacks of using lignocellulosic-biomass mate-rials in fermentation, as it makes lignocellulose resistant tochemical and biological degradation[56].

5. Bioethanol from lignocellulosic materials via the biochemical

pathway

Biochemical conversion of lignocellulosic materials throughsaccharification and fermentation is a major pathway for bioetha-nol production from biomass. Bioconversion of lignocellosics tobioethanol is difficult due to: (1) the resistant nature of biomassto breakdown; (2) the variety of sugars which are released when

the hemicellulose and cellulose polymers are broken and the needto find or genetically engineer organisms to efficiently fermentthese sugars; (3) costs for collection and storage of low densitylignocellosic materials. Generic block diagram of bioethanol pro-duction from lignocellulose materials is given inFig. 2[61]. The ba-sic process steps in producing bioethanol from lignocellulosicmaterials are: pretreatment, hydrolysis, fermentation and productseparation/distillation.

5.1. Pretreatment of lignocellulosic materials

The recalcitrance of lignocellulose is one of the major barriers tothe economical production of bioethanol. The technical approachto overcome recalcitrance has been pretreatment of biomass feed-

stock to remove the barriers and make cellulose more accessible tohydrolytic enzymes for conversion to glucose [62]. The goals ofpretreatment on lignocellulosic material are depicted in Fig. 3[63].If the pretreatment is not efficient enough the resultant resi-due is not easily hydrolyzable by cellulase enzyme and if it is moresevere, result is the production of toxic compounds which inhibitthe microbial metabolism[64].

Pretreatment has been viewed as one of the most expensiveprocessing steps within the conversion of biomass to fermentablesugar[65]. There is huge scope in lowering the cost of pretreat-ment process through extensive R&D approaches. Pretreatmentof cellulosic biomass in cost effective manner is a major challengeof cellulose to bioethanol technology research and development[66]. Taherzadeh and Karimi [56]has summarized the prerequi-

sites for an ideal lignocellulose pretreatment; it should: (1) pro-duction of reactive cellulosic fiber for enzymatic attack, (2)

Table 4

Composition of various types of lignocellulosic-biomass materials (% dry weight).

Mat erial Cellulose Hemicelluloses Lignin Ash Extractives

Algae (green) 2040 2050

Cotton, flax,

etc.

8095 520

Grasses 2540 2550 1030

Hardwoods 45 2 30 5 20 4 0.6 0.2 5 3Hardwood

barks

2240 2038 3055 0.8 0.2 6 2

Softwoods 42 2 27 2 28 3 0.5 0.1 3 2

Softwood

barks

1838 1533 3060 0.8 0.2 4 2

Cornstalks 3947 2631 35 1216 13

Wheat straw 3741 2732 1315 1114 7 2

Newspapers 4055 2540 1830

Chemical

pulps

6080 2030 210


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avoiding destruction of hemicelluloses and cellulose, (3) avoidingformation of possible inhibitors for hydrolytic enzymes and fer-menting microorganisms, (4) minimizing the energy demand, (e)reducing the cost of size reduction for feedstocks, (5) reducing

the cost of material for construction of pretreatment reactors, (6)producing less residues, and (7) consumption of little or no chem-ical and using a cheap chemical. Pretreatment is crucial for ensur-ing good ultimate yields of sugars from both polysaccharides.Hydrolysis without preceding pretreatment yields typically

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0.694.83 MPa for several seconds to a several minutes before thematerial is exposed to atmospheric pressure for cooling [85].

Uncatalyzed steam explosion refers to a pretreatment tech-nique in which lignocellulosic biomass is rapidly heated by high-pressure steam without addition of any chemicals. The biomass/steam mixture is held for a period of time to promote hemicellu-lose hydrolysis, and terminated by an explosive decompression[86]. Negro et al.[87]studied steam explosion and liquid hot water

methods for pretreatment of poplar (Populus nigra) biomass. Thebest results were obtained in steam explosion pretreatment at483 K and 4 min, taking into account cellulose recovery above95%, enzymatic hydrolysis yield of about 60%, and 41% xyloserecovery in the liquid fraction. Addition of H2SO4 (or SO2) or CO2[typically 0.33% (w/w)] in steam explosion can decrease timeand temperature, effectively improve hydrolysis, decrease the pro-duction of inhibitory compounds, and lead to complete removal ofhemicellulose[68]. H2SO4is a strong catalyst that highly improvesthe hemicellulose removal but also easily yields inhibitory sub-

stances[88]. Ballesteros et al. [89] applied acid-catalyzed steamexplosion pretreatment of wheat straw for bioethanol productionby varying the temperature (433473 K), the residence time (5,10 or 20 min) and the acid concentration [H2SO4 0.9% (w/w)].According to results of this study, the best pretreatment conditionsto obtain high conversion yield to bioethanol (approx 80% of theo-retical) of cellulose-rich residue after steam explosion are 463 Kand 10 min or 473 K and 5 min, in acid-impregnated straw. Using

a H2SO4-catalyzed steam explosion process for pretreatment ofSalix chips, at 473 K for either 4 or 8 min using 0.5% sulfuric acid,resulted in glucose recovery about 92% and 86% xylose recoveryafter enzymatic hydrolysis[90]. SO2 appears more appealing thanH2SO4 in steam explosion since the former requires milder andmuch less expensive reactor material, generates less gypsum,yields more xylose, and produces more digestible substrate withhigh fermentability[76]. The treatment can be carried out by 14% SO2 (w/w substrate) at elevated temperatures, e.g. 433503 K,for a period of e.g. 10 min [56]. The main drawback of SO2 is itshigh toxicity, which may pose safety and health risks. However,SO2 is used in various industrial processes using established tech-niques[91].

Two-step pretreatment has been suggested in several studies as

a means of increasing the sugar recovery [92,93]. In the first step,steam is performed using low temperature to solubilize hemicellu-

Table 5

Advantages and disadvantages of various pretreatment processes for lignocellulosic materials [68].

Pretreatment process Advantages Limitations and disadvantages

Mechanical comminut ion Reduces cellulose crystall inity Power consump tion usually higher t han inherent biomass energySteam exp losion Causes hemicellulose degradation

and lignin transformation; cost-effective

Destruction of a portion of the xylan fraction;

incomplete disruption of the lignin-carbohydrate matrix;

generation of compounds inhibitory to microorganisms

AFEX Increases accessible surface area,

removes lignin and hemicellulose to an extent;does not produce inhibitors for down-stream processes

Not efficient for biomass with high lignin content

CO2explosion Increases accessible surface area; cost-effective;

does not cause formation of inhibitory compounds

Does not modify lignin or hemicelluloses

Ozonolysis Reduces lignin content; does not produce toxic residues Large amount of ozone required; expensive

Acid hyd rolysis Hyd rolyzes hemicellulose t o xy loseand other sugars; alters lignin structure

High cost; equipment corrosion; formation of toxic substances

Alkaline hydrolysis Removes hemicelluloses and lignin;

increases accessible surface area

Long residence times required; irrecoverable

salts formed and incorporated into biomass

Organosolv Hydrolyzes lignin and hemicelluloses Solvents need to be drained from the reactor,

evaporated, condensed, and recycled; high cost

Pyrolysis Produces gas and liquid products High temperature; ash production

Pulsed electrical field Ambient conditions; disrupt s p lant cells; Process needs more research

Biological Simple equipment degrades lignin

and hemicelluloses; low energy requirements

Rate of hydrolysis is very low

Fig. 4. Energy requirements for ball milling municipal solid waste [78].

Table 6

Pyrolysed cellulosic materials[75].

Of fice paper Newspaper Cardboard

Reducing sugar (mg/ml) 0.14 0.11 0.11


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losic fraction, and cellulose fraction is subjected to a second steamexplosion pretreatment step at a temperature higher than 483 K. Itoffers some additional advantages (higher bioethanol yields, betteruse of raw material and lower enzyme dosages during steamexplosion)[81].

5.1.2.2. Ammonia fiber explosion. Ammonia fiber explosion (AFEX) is

one of the alkaline physico-chemical pretreatment processes. Inthis process, the material is subjected to liquid ammonia at hightemperature and pressure, and a subsequent fast decompression,similar to the steam explosion, which causes a fast saccharificationof the lignocellulosic material [94]. In a typical AFEX process, thedosage of liquid ammonia is 12 kg ammonia/kg dry biomass,the temperature is 363 K, and the residence time is 30 min[68,74]. The effective parameters in the AFEX process are ammonialoading, temperature, water loading, blowdown pressure, time,and number of treatments[56]. This system does not directly lib-erate any sugars, but allows the polymers (hemicellulose and cel-lulose) to be attacked enzymatically and reduced to sugars[95].AFEX pretreatment yields optimal hydrolysis rates for pretreatedlignocellulosics with close to theoretical yields at low enzymeloadings (65% lig-nin reduction in cotton stalk treated with 2% NaOH for 90 min at394 K/15 psi. Cellulases produced by Bacillus subtilis for the sac-charification of wheat straw, rice straw and bagasse were usedby Akhtar et al. [111]. Pretreatment of these substrates with 2%NaOH was found to be more effective for increasing the saccharifi-cation. The saccharification rates of 33.0%, 25.5% and 35.5% were

obtained with 2% NaOH pretreated wheat straw, rice straw and ba-gasse, respectively. In a study[112], a combination of NaOH treat-ment and homogenization was used as a pretreatment to enhancethe enzymatic hydrolysis of corn stover. The highest glucose yield(6.25 g/l) was obtained when the corn stover was pretreated by acombination of 1.0 N NaOH treatment and homogenization (Fig. 5).

Lime (Ca(OH)2) as compared to NaOH and KOH has lower costand less significant safety requirements. It can be recovered fromhydrolyzate by reaction with CO2, so that formed carbonate canthen be reconverted to lime [113]. To make lime as efficient asother alkalis in enhancing the digestibility of lignocellulose, appro-priate pretreatment conditions need to be employed [114]. Lime,water, and an oxidizing agent (air or O2) are mixed with the bio-mass at temperatures ranging from 313 to 423 K for a period rang-

ing from hours to weeks[115]. Two types of lime treatment havebeen explored: (1) short-term and (2) long-term. Short-term lime


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pretreatment involves boiling the biomass with a lime loading of0.1 g Ca(OH)2/g dry biomass at temperatures of 358408 for 13 h [116]. Long-term pretreatment involves using the same limeloading at lower temperatures (313328 K) for 46 weeks in thepresence of air[116]. Lime has been used to pretreat switchgrass(373 K for 2 h)[117], wheat straw (394 K for 1 h)[118], corn stover(373 K for 13 h)[119], and poplar wood (423 K for 6 h with 14-atmoxygen)[120]. Saha and Cotta [118] obtained maximum total sugaryield (451 3mg g1 straw; glucose, 252 6 mg; xylose, 173 3 mg; arabinose, 27 2 mg; 65% conversion) by lime pretreatment(100mg g1 straw, 394 K, 1 h). The authors also investigated the

effects of pH (3.56.5) and temperature (298343 K) on the enzy-matic hydrolysis of lime pretreated wheat straw (8.6%, w/v) using acombination of three enzymes (cellulose,b-glucosidase, and hemi-cellulase), each enzyme at a dose level of 0.05 ml g1 substrate.Fig. 6shows the effect of pH and temperature on the enzymatichydrolysis of lime pretreated wheat straw.

Alkaline peroxide is one of the effective pretreatment methodsthat can improve the enzymatic hydrolysis by delignification of lig-nocellulosic materials. In thismethod, the lignocellulosesare soakedin pH-adjusted water (e.g. to pH 1112 using NaOH) containingH2O2at room temperatures for a period of time (e.g. 624 h)[56].

5.1.3.3. Acid pretreatment. Acid pretreatments normally aim forhigh yields of sugars from lignocellulosic materials. Acid pretreat-

ment involves the use of sulfuric, nitric, or hydrochloric acids to re-move hemicellulose components and expose cellulose forenzymatic digestion [107]. The acid pretreatment can operateeither under a high temperature and low acid concentration (diluteacid pretreatment) or under a low temperature and high acid con-centration (concentrated acid pretreatment) [56]. Dilute acidhydrolysis has been successfully developed for pretreatment of lig-nocellulosic materials. The dilute acid pretreatment works fairlywell on agricultural feedstocks, such as corn stover and rice/wheatstraw[62]. While dilute acid pretreatments are known to improveenzymatic hydrolysis, their cost is relatively high compared tophysico-chemical pretreatments [44]. This pretreatment methodgives high reaction rates and significantly improves cellulosehydrolysis[47]. There are primarily two types of dilute acid pre-

treatment processes: low solids loading (510% [w/w]), high tem-perature (T> 433 K), continuous-flow processes and high solids

loading (1040% [w/w]), lower temperature (T< 433 K), batch pro-cesses [121]. In general, higher pretreatment temperatures and

shorter reactor residence times result in higher soluble xyloserecovery yields and enzymatic cellulose digestibility. Higher-tem-perature dilute acid pretreatment has been shown to increase cel-lulose digestibility of pretreated residues[122]. Depending on thesubstrate and the conditions used, between 80% and 95% of thehemicellulosic sugars can be recovered by dilute acid pretreatmentfrom the lignocellulosic material [47,123,124]. Silverstein et al.[107]reported 95% xylan reduction in cotton stalk treated with2% H2SO4 for 90 min at 394 K/15 psi.

In recent years, treatment of lignocellulosic biomass with dilutesulfuric acid has been primarily used as a means of hemicellulosehydrolysis and pretreatment for enzymatic hydrolysis of cellulose[125]. Sulfuric acid at concentrations usually below 4 wt.%, hasbeen of the most interest in such studies as it is inexpensive and

effective[68]. Dilute sulfuric acid pretreatment (0.22.0% sulfuricacid, 394493 K) of lignocellulose serves three important functionsin the conversion process[121]: (1) hydrolysis of the hemicellulosecomponents to produce a syrup of monomeric sugars, (2) exposureof cellulose for enzymatic digestion by removal of hemicelluloseand part of the lignin, and (3) solubilization of heavy metals whichmay be contaminating the feedstock. In spite of these benefits, di-lute sulfuric acid has some important disadvantages[76]: (1) cor-rosion that mandates expensive materials of construction, (2)acidic prehydrolyzates must be neutralized before the sugars pro-ceed to fermentation, (3) gypsum has problematic reverse solubil-ity characteristics when neutralized with inexpensive calciumhydroxide, (4) formation of degradation products and release ofnatural biomass fermentation inhibitors are other characteristics

of acid pretreatment, (5) disposal of neutralization salts is needed,and (6) biomass particle size reduction is necessary.

Fig. 5. Effect of NaOH concentration on enzymatic hydrolysis of corn stover

pretreated by combined NaOH treatment and homogenization (raw mate-

rial = 2 mm corn stover, pretreatment conditions = NaOH treatment + homogeniza-

tion, hydrolysis conditions = 20 GCU cellulose/g substrate at 323 K, PH 4.8) [112].

Fig. 6. (A) Effect of pH on the enzymatic hydrolysis of lime pretreated wheat straw

at 318 K and (B) effect of temperature on the enzymatic hydrolysis of limepretreated wheat straw at pH 5.0. Yield of total sugars at: (a) 6, (b) 24, and (c) 72 h

[118].


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5.2.2. Enzymatic hydrolysis

Acid hydrolysis has a major disadvantage where the sugars areconverted to degradation products like tars. This degradation canbe prevented by using enzymes favoring 100% selective conversionof cellulose to glucose. When hydrolysis is catalyzed by such en-zymes, the process is known as enzymatic hydrolysis [137]. Enzy-matic hydrolysis of natural lignocellulosic materials is a very slowprocess because cellulose hydrolysis is hindered by structuralparameters of the substrate, such as lignin and hemicellulose con-tent, surface area, and cellulose crystallinity [138]. Utility cost ofenzymatic hydrolysis is low compared to acid or alkaline hydroly-sis because enzyme hydrolysis is usually conducted at mild condi-tions (pH 4.8) and temperature (318323 K) and does not have acorrosion problem [74]. The enzymatic hydrolysis has currentlyhigh yields (7585%) and improvements are still projected (85

95%), as the research field is only a decade young[55]. Comparisonof process conditions and performance of three cellulose hydrolysisprocesses is given inTable 8. Enzymatic hydrolysis is an environ-mentally friendly alternative that involves using carbohydratedegrading enzymes (cellulases and hemicellulases) to hydrolyzelignocelluloses into fermentable sugars[44].

5.2.2.1. Enzymatic hydrolysis of cellulose. Cellulose is typicallyhydrolyzed by an enzyme called cellulase. These enzymes are pro-duced by several microorganisms, commonly by bacteria and fun-gi. These microorganisms can be aerobic or anaerobic, mesophilicor thermophilic. Bacteria belonging to Clostridium, Cellulomonas,

Bacillus, Thermomonospora, Ruminococcus, Bacteriodes, Erwinia,

Acetovibrio, Microbispora, and Streptomycescan produce cellulaseseffectively [74]. Fungi such as Sclerotium rolfsii, P. chrysosporiumandspecies ofTrichoderma, Aspergillus, Schizophyllum and Penicili-umare used to produce cellulases[139]. Mutant strains ofTricho-derma sp. (T. viride, T. reesei, T. longibrachiatum) have long beenconsidered to be the most productive and powerful destroyers ofcrystalline cellulose[140]. Commercial products of variousT. reeseiisolates have been available for a long time in cereal foods applica-tions, the brewing industry, fruit and vegetable processing andhave also been widely evaluated and applied in relation to bioeth-anol production processes. T. reesei secretes high amounts of en-zymes, up to 100 g l1 [141].

Cellulase is a group of enzymes that synergistically hydrolyzescellulose (Fig. 9)[142]. The widely accepted mechanism for enzy-

matic cellulose hydrolysis involves synergistic actions by endoglu-canses (EG, endo-1,4-b-D-glucanases, or EC 3.2.1.3.), exoglucanasesor cellobiohydrolases (CBH, 1,4-b-D-glucan cellobiohydrolases, orEC 3.2.1.91.), andb-glucosidases (BGL, cellobiases or EC 3.2.1.21).EG hydrolyze accessible intramolecular b-1,4-glucosidic bonds ofcellulose chains randomly to produce new chain ends; CBH proc-essively cleave cellulose chains at the ends to release soluble cello-biose or glucose; and BGL hydrolyze cellobiose to glucose in orderto eliminate cellobiose inhibition[143]. BGL complete the hydroly-sis process by catalyzing the hydrolysis of cellobiose to glucose.The supplementation of b-glucosidase in hydrolysis is requireddue to its insufficient amount from T. reesei, to prevent cellulasesinhibition resulted from cellobiose accumulation[114]. During cel-lulose hydrolysis, the solid substrate characteristics vary, includ-

ing: (1) changes in the cellulose chain end number resultingfrom generation by EG and consumption by CBH and (2) changesin cellulose accessibility resulting from substrate consumptionand cellulose fragmentation[143].

There are different factors that affect the enzymatic hydrolysisof cellulose, namely, substrates, cellulase activity, reaction condi-tions (temperature, pH as well as other parameters), and a strongproduct inhibition. To improve the yield and rate of enzymatichydrolysis, research has been focused on optimizing the hydrolysisprocess and enhancing the cellulase activity[74]. The rate of enzy-matic hydrolysis of cellulose is dependent upon several structuralfeatures of the cellulose. The cellulose features known to affectthe rate of hydrolysis include: (1) molecular structure of cellulose,(2) crystallinity of cellulose, (3) surface area of cellulose fiber, (4)

degree of swelling of cellulose fiber, (5) degree of polymerization,and (6) associated lignin or other materials[144]. A low substrate

Fig. 8. Dilute acid hydrolysis (first-stage and two-stages) and separate fermentation of pentose and hexose sugars [66].

Table 7Yields of bioethanol by concentrated sulfuric acid hydrolysis from cornstalks [53].

Amount of cornstalk (kg) 1000Cellulose content (kg) 430

Cellulose conversion and recovery

efficiency (% dry weight)

76

Bioethanol stoichiometric yield (% dry weight) 51

Glucose fermentation efficiency (% dry weight) 75

Bioethanol yield from glucose (kg) 130

Amount of cornstalk (kg) 1000

Hemicelluloses content (kg) 290

Hemicelluloses conversion and recovery

efficiency (% dry weight)

90

Bioethanol stoichiometric yield (% dry weight) 51

Xylose fermentation efficiency (% dry weight) 50

Bioethanol yield from xylose (kg) 66

Total bioethanol yield from 1000 kg of cornstalks 196 kg

(225.7 L = 59 gallons)


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concentration gives low yield and rate, and a high cellulase dosagemay increase the costs disproportional [55]. A cellulase dosageof 10FPU (filter paper units) per gram of biomass is often used in labora-torystudies because it provides a hydrolysis profilewith high levelsof glucose yield in a reasonable time (4872 h) at a reasonable en-zyme cost[145]. Chen et al.[146]investigated effects of cellulasedosage on the enzymatic hydrolysis of dilute acid-treated corncob.Hydrolysis experiments were performed with 100 g l1 substrateand different dosages ofT. reesei ZU-02 cellulase (FPU g1 substrate)at pH 4.8 and 323 K. Results of this study are shown in Fig. 10. As

shown inFig. 10, reducing sugar concentration and hydrolysis yieldhada similar variation trend,thatis, both increasedsharplywithcel-lulasedosage varying from 10 to 20 FPU g1 substrate, and basicallyleveled off from 20 to 30 FPU g1 substrate.

One limitation with using cellulase is that there is a reduction inrates due to end product (cellobiose and glucose) inhibition. Simul-taneous saccharification and fermentation (SSF) overcomes thisproblem by hydrolyzing cellulose and fermenting the hydrolysisproduct at the same time[147].

5.2.2.2. Enzymatic hydrolysis of hemicelluloses. There is a great inter-est in the enzymatic hydrolysis of xylan because of possible appli-cations in ruminal digestion, waste treatment, fuel and chemicalproduction, and paper manufacture[148]. Unlike cellulose, xylans

are chemically quite complex, and their degradation requires mul-tiple enzymes. Xylan-degrading enzymes are produced by a widevariety of fungi and bacteria such as Trichodrema spp.[149,150],Penicillium spp. [151,152], Talaromyces spp. [151,153]Aspergillusspp.[154], and Bacillusspp.[155].

Enzymatic hydrolysis of xylan involves a multi-enzyme system,including endoxylanase, exoxylanase, -xylosidase,a-arabinofura-nosidase,a-glucoronisidase, acetyl xylan esterase, and ferulic acidesterase[156].Table 9presents most important enzyme activitiesrequired for hydrolysis of xylooligosaccharides obtained fromhardwoods and herbaceous type materials[113]. The endoxylan-ase attacks the main chains of xylans and b-xylosidase hydrolyzesxylooligosaccharides to xylose. The a-arabinofuranosidase anda-glucuronidase remove the arabinose and 4-0-methyl glucuronic

acid substituents, respectively, from the xylan backbone [157].Hemicellulolytic esterases include acetyl esterases which hydro-

lyze the acetyl substitutions on xylose moieties, and feruloyl ester-ases which hydrolyze the ester bond between the arabinosesubstitutions and ferulic acid. Feruloyl esterases aid the release

of hemicellulose from lignin and renders the free polysaccharideproduct more amenable to degradation by the other hemicellulases[158].

As in cellulase systems, xylan-degrading systems also exhibit.While the number of enzymes required for xylan hydrolysis ismuch greater than for cellulose hydrolysis, accessibility to the sub-strate is easier since xylan does not form tight crystalline struc-tures[44].

5.3. Fermentation

The supernatant from enzymatic hydrolysis of lignocellulosescan contain both six-carbon (hexoses) and five-carbon (pentoses)sugars (if both cellulose and hemicellulose are hydrolyzed).

Depending on the lignocellulose source, the hydrolysate typicallyconsists of glucose, xylose, arabinose, galactose, mannose, fucose,

Table 8

Comparison of process conditions and performance of three hydrolysis processes [55].

Consumables Temperature (K) Time Glucose yield (%) Available

Dilute acid

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and rhamnose[44]. One ton of glucan, galactan, or mannan yields1.11 tons of six-carbon sugars and could be fermented theoreti-cally into 172.0 gallons of bioethanol[159]. One ton of arabinanor xylan yields 1.14 tons of five-carbon sugars and could be fer-mented theoretically into 176.0 gallons of bioethanol[159]. Micro-organisms can be used to ferment all lignocellulose-derived sugarsto bioethanol.

5.3.1. Microorganisms related to bioethanol fermentation

Microorganisms for bioethanol fermentation can best be de-scribed in termsof their performance parameters and other require-

ments such as compatibility with existing products, processes andequipment. The performance parameters of fermentation are: tem-perature range, pH range, alcohol tolerance, growth rate, productiv-ity, osmotic tolerance, specificity, yield, genetic stability, andinhibitor tolerance. The characteristics required for an industriallysuitable microorganism are summarized inTable 10[160].

Traditionally,Saccharomyces cerevisiae and Zymomonas mobilishave been used for bioethanol fermentation. They are capable ofefficiently fermenting glucose into bioethanol, but are unable toferment xylose [44]. Natural xylose-fermenting yeasts, such asPichia stipitis, Candida shehatae, and Candida parapsilosis, canmetabolize xylose via the action of xylose reductase (XR) to con-vert xylose to xylitol, and of xylitol dehydrogenase (XDH) to con-vert xylitol to xylulose. Therefore, bioethanol fermentation from

xylose can be successfully performed by recombinantS. cerevisiaecarrying heterologous XR and XDH from P. stipitis, and xylulokinase(XK) from S. cerevisiae [161]. In bacteria, a xylose isomerase (XI)converts xylose to xylulose, which after phosphorylation, is metab-olized through the pentose phosphate pathway (PPP) [162].

The most frequently used microorganism for fermenting bio-ethanol in industrial processes is S. cerevisiae, which has provedto be very robust and well suited to the fermentation of lignocellu-losic hydrolysates[91]. S. cerevisiaecan easily ferment hexoses, buthardly xylose in lignocellulose hydrolysates, because S. cerevisiaelacks enzymes that convert xylose to xylulose[163]. However, thisyeast can ferment xylulose [164]. For xylose-using S. cerevisiae,high bioethanol yields from xylose also require metabolic engi-neering strategies to enhance the xylose flux[165].

Bacteria, such asZ. mobilis,Escherichia coli and Klebsiella oxytoca,have attracted particular interest, given their rapid fermentation,

which can be minutes compared to hours for yeasts[102].Z. mobi-lis, a Gram-negative bacterium, is well recognized for its ability toefficiently produce bioethanol at high rates from glucose, fructose,and sucrose. When Z. mobilis and S. cerevisiae were compared fortheir efficiency to produce bioethanol from glucose and starchhydrolysate, higher yield was observed for Z. Mobilis [166]. Com-parative performance trials on glucose have shown that Z. mobiliscan achieve 5% higher bioethanol yields and up to 5-fold higherbioethanol volumetric productivity compared to traditionalS. cere-visiaeyeast[167]. It has a theoretical yield of 97%[94]. Z. mobilisefficiently produces bioethanol from the hexose sugars glucoseand fructose but not from pentose sugars, although a xylose fer-mentingZ. mobiliswas generated by introducing a xylose-metabo-lizing pathway from E. coli [165]. Modified Z. mobilis has the

advantages of requiring a minimum of nutrients, growing at lowpH and high temperatures, and it is considered generally recog-nized as safe (GRAS). A comparison of modified Z. mobilis andE. coli showing their respective advantages is shown in Table 11[94].E. coliandK. oxytocanaturally metabolize arabinose, such thatthe ethanologenic strains ferment all lignocellulose-derived sugars[165]. The construction ofE. colistrains to selectively produce bio-ethanol was one of the first successful applications of metabolicengineering [168]. E. coli, as a biocatalyst for bioethanol produc-tion, has ability to ferment a wide spectrum of sugars, no require-ments for complex growth factors, and prior industrial use (e.g. forproduction of recombinant protein). The major disadvantagesassociated with using E. coli cultures are a narrow and neutralpH growth range (6.08.0), less hardy cultures compared to yeast,

and public perceptions regarding the danger ofE. colistrains. Thelack of data on the use of residual E. colicell mass as an ingredientin animal feed is also an obstacle to its application [160,169]. K.oxytocais an enteric bacterium found growing in paper and pulpstreams as well as around other sources of wood. The microorgan-ism is capable of growing at a pH at least as low as 5.0 and temper-atures as warm as 308 K. It can grow on a wide variety of sugarsincluding hexoses and pentoses, as well as on cellobiose and cello-triose[160].

Thermophilic anaerobic bacteria have also been extensivelyexamined for their potential as bioethanol producers. These bacte-ria includeThermoanaerobacter ethanolicus [170], Clostridium ther-mohydrosulfuricum [171], Thermoanaerobacter mathranii [172],Thermoanaerobium brockii[173],Clostridium thermosaccharolyticum

[174], etc. Thermophilic anaerobic bacteria have a distinct advan-tage over conventional yeasts for bioethanol production in their

Table 9

Relevant enzymatic activities for enzymatic posthydrolysis of xylooligosaccharides [113].

Enzyme EC Hydrolyzed linkage Substrate Main product

Endoxylanase 3.2.1.8 Internal b-1,4 Main chain OligomersExoxylanase n.c. Terminal b-1,4 (reducing end) Main chain Xylose, xylobiose

b-Xylosidase 3.2.1.37 Terminal b-1,4 (non-reducing end) Oligomers Xylose

Arabinosidase 3.2.1.55 Side groups Arabinose

Glucoronisidase 3.2.1.139 Side groups Methylglucuronic acids

Acetyl xylan esterases 3.1.1.72 Ester bond Side groups Acetic acidFeruloyl esterases 3.1.1.73 Ester bond Side groups Ferulic acid

n.c.: not yet classified.

Table 10

Important traits for bioethanol fermentation process[160].

Trait Requirement

Bioethanol yield >90% of theoretical

Bioethanol tolerance >40 g l1

Bioethanol productivity >1 g l1 h1

Robust grower and simple growth

requirements

Inexpensive medium

formulation

Able to grow in undiluted hydrolysates Resistance to inhibitors

Culture growth conditions retard

contaminants

Acidic pH or higher

temperatures

Table 11

Comparison between modified Z. mobilis and E. coli [94].

Z. mobilis E. coli

Bioethanol (g l1) 62 27

Bioethanol yielda (%) 97 90

Bioethanol productivity (g l1 h1) 1.29 0.92

a Estimation from the theoretical yields.


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ability to use a variety of inexpensive biomass feedstocks and theirability to withstand temperature extremes[175]. The low bioetha-nol tolerance of thermophilic anaerobic bacteria (313 K, may be favorable for enzy-matic hydrolysis, whereas the low pH can surely inhabit the lacticacid production and the high temperature may affect adversely thefungal cell growth[187]. Trichoderma reeseicellulases, which con-stitute the most active preparations, have optimal activity at pH4.5 and 328 K. ForSaccharomyces cultures SSF are typically con-trolled at pH 4.5 and 310 K[160]. A typical fermentation will take57 days, depending on the accessibility of the cellulose and initialsolids loading of the fermentation. The long residence time maymake contamination control difficult in a continuous process, butmay be manageable in a batch process[188].

Major advantages of SSF as described by Sun and Cheng[74], in-clude: (1) increase of hydrolysis rate by conversion of sugars thatinhibit the cellulase activity; (2) lower enzyme requirement; (3)higher product yields; (4) lower requirements for sterile conditionssince glucose is removed immediately and bioethanol is produced;(5) shorter process time; and (6) less reactor volume. The majoradvantage of SSF is that the immediate consumption of sugars bythe microorganism produces low sugar concentrations in the fer-mentor, which significantly reduces enzyme inhibition comparedto SHF [188]. The main disadvantage of SSF lies in different temper-ature optima for saccharification (323 K) and fermentation (308 K)[189].

The most important process improvement made for the enzy-matic hydrolysis of biomass is the introduction of SSF, which hasbeen improved to include combines the cellulase enzymes and fer-menting microbes in one vessel to improve the bioethanol produc-

tion economics. The technology has been improved to include theco-fermentation of multiple sugar substrates, i.e., simultaneoussaccharification of both cellulose (to glucose) and hemicellulose(to xylose), and co-fermentation of both glucose and xylose bygenetically engineered microbes in the same broth[136].

5.3.3.3. Direct microbial conversion (DMC). Direct microbial conver-sion (DCM) combines cellulase production, cellulose hydrolysisand glucose fermentation into a single step. This process is attrac-tive in that it reduces the number of reactors, simplifies operation,and reduces the cost of chemicals [121]. DCM seems the logicalendpoint in the evolution of bioethanol production from lignocel-lulosic materials. Application of DCM entails no operating cost orcapital investment for dedicated enzyme production (or purchase),reduced diversion of substrate for enzyme production, and com-patible enzyme and fermentation systems[55]. The disadvantagesare low bioethanol yields, caused by byproduct formation (acetate,lactate), low tolerance of the microorganism to bioethanol (3.5% w/v), and limited growth in hydrolysate syrups[162].

5.4. Product and solids recovery

As biomass hydrolysis, and fermentation technologies approachcommercial viability, advancements in product recovery technolo-gies will be required. For cases in which fermentation products aremore volatile than water, recovery by distillation is often the tech-nology of choice. Distillation technologies that will allow the eco-

nomic recovery of dilute volatile products from streamscontaining a variety of impurities have been developed and


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commercially demonstrated[190]. A distillation system separatesthe bioethanol from water in the liquid mixture.

The first step is to recover the bioethanol in a distillation or beercolumn, where most of the water remains with the solids part. Theproduct (37% bioethanol) is then concentrated in a rectifying col-umn to a concentration just below the azeotrope (95%)[55]. Theremaining bottoms product is fed to the stripping column to re-

move additional water, with the bioethanol distillate from strip-ping being recombined with the feed to the rectifier [191]. Therecovery of bioethanol in the distillation columns in the plant isfixed to be 99.6% to reduce bioethanol losses[192].

After the first effect, solids are separated using a centrifuge anddried in a rotary dryer. A portion (25%) of the centrifuge effluent isrecycled to fermentation and the rest is sent to the second andthird evaporator effects. Most of the evaporator condensate is re-turned to the process as fairly clean condensate (a small portion,10%, is split off to waste water treatment to prevent build up oflow-boiling compounds) and the concentrated syrup contains1520% by weight total solids[193].

6. Bioethanol economy

Considering that up to now the cost of bioethanol was consider-ably higher than the cost of fossil gasoline supply, national govern-ments had to enact special policies in order to encourageproduction and use of bioethanol in the transportation sector. Ingeneral, the following three main approaches can be distinguishedin the implementation of biofuels supporting policies and regula-tion: (1) taxation-based policies, (2) agriculture-based policies/subsidies, and (3) fuel mandates[34]. At present, the developmentand promotion of biofuels are mainly driven by the agriculturalsector and green lobbies rather than the energy sector. In fact, mostbiofuel programs depend on subsidies and government programs,which can lead to market distortion and is costly for governments.Nevertheless, at sustained high oil prices and with a steady pro-

gression of more efficient and cheaper technology, biofuels couldbe a cost-effective alternative in the near future in many countries[194].

The cost for bioethanol production can vary substantiallydepending on several factors, e.g. feedstock costs and by-productsrevenues, cost of process energy, investment costs (related to thetype of feedstock), plant location and transportation cost andfinancing costs[195]. Brazilian bioethanol is far more competitivethan that produced in the United States from corn or in Europefrom sugar beet, because of shorter processing times, lower labourcosts, lower transport costs and input costs [196].Bioethanol pro-duction from sugarcane is very economical in Brazil because of twoprimary reasons. Brazil dropped support of sugar prices to supportthe bioethanol industry with government established mandates for

the blending of bioethanol with gasoline. This drastically loweredthe cost of the feedstock, sugarcane, and created a demand forand supported the price of bioethanol. In addition, Brazils vastland area of cultivatable acreage means that land devoted to sugar-cane production for bioethanol is not in competition with land de-voted for food production [197]. Bioethanol from sugarcane inBrazil costs US$0.230.29/l[198], while in the EU and the UnitedStates sugar beet and corn-derived bioethanol cost US$0.29/l[199]and US$0.53/l[200], respectively. Other efficient sugar pro-ducing countries such as Pakistan, Swaziland and Zimbabwe haveproduction costs similar to Brazils[194].

The cost of raw material, which varies considerably betweendifferent studies (US$22US$61 per metric ton dry matter), andthe capital costs, which makes the total cost dependent on plant

capacity, contribute most to the total production cost [165]. Thecost and availability of feedstock was crucial because in most bio-

fuels, feedstock represents 6075% of the total bioethanol produc-tion cost[3]. Estimates of the costs of bioethanol production fromdifferent feedstock are shown inTable 12. The cost figures can becompared with the cost of producing gasoline of around US$0.70/l at oil prices of US$100 per barrel [201].

Bioethanol production generally utilizes derivatives from foodcrops such as corn grain and sugarcane, but the limited supply ofthese crops can lead to competition between their use in bioetha-nol production and food provision[202]. Using food crops to pro-duce bioethanol raises major nutritional and ethical concerns.

Nearly 60% of humans in the world are currently malnourished,so the need for grains and other basic foods is critical. Growingcrops for fuel squanders land, water, and energy resources vitalfor the production of food for people[203]. In 2007, when US retailfood prices rose 4% above 2006 levels and twice as fast as overallcore inflation (2.3%), consumers took notice [204]. The bioetha-nol-driven surge in corn demand has fueled a sharp rise in cornprices. For example, the futures contract for March 2007 corn onthe Chicago Board of Trade, rose from US$2.50 per bushel in Sep-tember 2006 to a contract high of over US$4.16 per bushel in Jan-uary 2007 (a rise of 66%). This sharp rise in corn prices owes itsorigins largely to increasing corn demand spurred by the rapidexpansion of corn-based bioethanol production capacity in theUnited States since mid-2006 [205]. Higher corn prices were, in

part, driven by demand to make bioethanol and these higher priceseffectively bid acres away from other crops that provided lower re-turns, such as soybeans, wheat, and hay[204]. Using corn for bio-ethanol increases the price of US beef, chicken, pork, eggs, breads,cereals, and milk from 10% to 30%[203].

Lignocellulosic biomass is the most promising feedstock consid-ering its great availability and low cost, but the large-scale com-mercial production of fuel bioethanol from lignocellulosicmaterials has still not been implemented. Today the productioncost of bioethanol from lignocellulose is still too high, which isthe major reason why bioethanol has not made its breakthroughyet. Pretreatment has been viewed as one of the most expensiveprocessing steps in cellulosic biomass to fermentable sugars con-version with costs as high as US$0.08/l bioethanol produced[86].

Enzyme pricing is assumed such that the total contribution of en-zymes to production costs is about US$0.04/l of bioethanol withsome variation depending upon actual bioethanol yields resultingfrom the particular pretreatment approach [206]. Significantgrowth of the bioethanol industry will depend on the developmentof new processes that convert cellulosic biomass from non-foodcrops and waste materials into bioethanol [207].

7. Conclusion

Recently, there has been growing interest in biofuels due to therising energy costs and environmental problems. Bioethanol is byfar the most widely used biofuel for transportation worldwide. Itwill continue to be developed as a transport fuel produced in trop-

ical latitudes and traded internationally, for use primarily as a gas-oline additive.

Table 12

Estimates of the costs of bioethanol production from different feedstock (US$/l)[201].

Feedstock Production cost

rangea in 2005

Projected production

cost rangea in 2030

Sugarcane 0.200.50 0.200.35

Corn 0.600.80 0.350.55

Sugar beet 0.620.82 0.400.60

Wheat 0.700.95 0.450.65

Lignocellulose 0.801.10 0.250.65

a Excluding any subsidies to bioethanol production.


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Bioethanol production generally utilizes derivatives from foodcrops such as corn grain and sugarcane, but the limited supply ofthese crops can lead to competition between their use in bioetha-nol production and food provision. The price of the raw materials isalso highly volatile, which can highly affect the production costs ofthe bioethanol. Lignocellulosic materials serve as a cheap andabundant feedstock, which is required to produce fuel bioethanol

from renewable resources at reasonable costs.Lignocellulosic biomass can be converted to bioethanol byhydrolysis and subsequent fermentation. Lignocellulose is oftenhydrolyzed by acid treatment; the hydrolysate obtained is thenused for bioethanol fermentation by microorganisms such as yeast.Because such lignocellulose hydrolysate contains not only glucose,but also various monosaccharides (e.g. xylose, mannose, fructose,galactose, and arabinose) and oligosaccharides, microorganismsshould be required to efficiently ferment these sugars for the suc-cessful industrial production of bioethanol.

Acknowledgement

The author would like to thank Professor Ayhan Demirbasfor

his very considerable help and encouragement throughout thecourse of this work.

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