Handbook of plant based biofuels - Chapter 11 potx

Current processes for the conver-sion of biomass to ethanol involve chemical and/or enzymatic hydrolysis of cellulose and hemicellulose to the respective sugars and subsequent fermentati

Lignocellulosic Biomass

Part III Hydrolysis

and Fermentation

Ramakrishnan Anish and Mala Rao

AbstrAct

Lignocellulose is the most abundant natural renewable resource and is one of the preferred choices for the production of bioethanol As a substrate for bioethanol production it has a barrier in its complex structure, which resists hydrolysis For lignocellulose to be amenable to fermentation, treatments are necessary that release

contents

Abstract 159

11.1 Introduction 160

11.2 Hydrolysis of Lignocellulosic Biomass 160

11.2.1 Acid Hydrolysis 160

11.2.1.1 Dilute Acid Hydrolysis 160

11.2.1.2 Concentrated Acid Hydrolysis 162

11.3 Enzymatic Hydrolysis of Lignocellulosic Biomass 163

11.3.1 Factors Governing Enzymatic Hydrolysis 164

11.3.2 Detoxification 166

11.3.2.1 Biological Detoxification Methods 166

11.3.2.2 Physical Detoxification Methods 167

11.3.2.3 Chemical Detoxification Methods 167

11.4 Fermentation of Lignocellulosic Biomass to Ethanol 167

11.4.1 Separate Hydrolysis and Fermentation (SHF) 167

11.4.2 Direct Microbial Conversion (DMC) 167

11.4.3 Simultaneous Saccharification and Fermentation (SSF) 168

11.5 Recombinant DNA Approaches 168

11.6 Conclusions and Future Prospects 169

References 170

monomeric sugars, which can be converted to ethanol by microbial fermentation The current state of the art on acid and enzymatic hydrolysis of lignocellulose and subse-quent microbial fermentation to ethanol are described in this chapter Approaches for detoxification of the lignocellulose hydrolysate for effective fermentation to ethanol are also discussed

11.1 IntroductIon

The rapid depletion of fossil fuels coupled with the increasing demands for transpor-tation fuels has necessitated research focus on alternative renewable energy sources Lignocellulose is the most abundant renewable resource, abundantly available for conversion to fuels On a worldwide basis, terrestrial plants produce 1.3 × 1010 metric tons of wood per year (equivalent to 7 × 109 metric tons of coal) or about two-thirds

of the world’s energy requirement (Demain, Newcomb, and Wu 2005) Agriculture and other sources provide about 180 million tons of cellulosic feedstock per year Furthermore, tremendous amounts of cellulose are available as municipal and indus-trial wastes causing pollution problems Lignocellulosic biomass includes materi-als such as agricultural and forestry residues, municipal solid waste, and industrial wastes Herbaceous and woody crops can also be used as a source of biomass Ligno-cellulosic biomass can be used as an inexpensive feedstock for production of renew-able fuels and chemicals

Lignocellulosic biomass is made up of cellulose, hemicellulose, and a cementing material, lignin Cellulose is a linear polymer of glucose, whereas hemicellulose is a branched heteropolymer of d-xylose, L-arabinose, D-mannose, D-glucose, D-galac-tose and D-glucuronic acid Lignin is a complex, hydrophobic, cross-linked aromatic polymer that interferes with the hydrolysis process Current processes for the conver-sion of biomass to ethanol involve chemical and/or enzymatic hydrolysis of cellulose and hemicellulose to the respective sugars and subsequent fermentation to ethanol Enzymatic processes are highly specific and are carried out under mild conditions of temperature and pH and do not create a corrosion problem The process requires the use of expensive biocatalysts Dilute acid hydrolysis is fast and easy to perform but

is hampered by nonselectivity and by-product formation

11.2 hydrolysIs of lIgnocellulosIc bIomAss

The most commonly considered hydrolysis processes are the concentrated hydro-chloric acid process, the two-step dilute acid hydrolysis, and enzymatic hydroly-sis During the hydrolysis of lignocellulosic materials a wide range of compounds are released which are inhibitory to microbial fermentation The composition of the inhibitors differs depending on the type of lignocelluIosic hydrolysates

11.2.1 a cid H ydrolySiS

11.2.1.1 dilute Acid hydrolysis

Dilute acid hydrolysis of biomass is, by far, the oldest technology for converting biomass to ethanol The first attempt at commercializing a process for producing

ethanol from the wood was carried out in Germany in 1898 It involved the use of dilute acid to hydrolyze the cellulose to glucose, and was able to produce 7.6 liters of ethanol per 100 kg of wood waste (18 gal per ton)

The hydrolysis occurs in two stages to accommodate the differences between the hemicellulose and the cellulose (Harris et al 1985) and to maximize the sugar yields from the hemicellulose and cellulose fractions of the biomass The first stage

is operated under milder conditions to hydrolyze the hemicellulose, while the sec-ond stage is optimized to hydrolyze the more resistant cellulose fraction The liquid hydrolysates are recovered from each stage, neutralized, and fermented to ethanol The National Renewable Energy Laboratory (NREL), a facility of the U.S Department of Energy (DOE) operated by Midwest Reseach Institute, Bettelle, out-lined a process whereby the hydrolysis is carried out in two stages to accommodate the differences between hemicellulose and cellulose The first stage can be operated under milder conditions, which maximize yield from the more readily hydrolyzed hemicellulose The second stage is optimized for hydrolysis of the more resistant cellulose fraction NREL has reported the results for a dilute acid hydrolysis of soft-woods in which the conditions of the reactors were as follows: Stage 1, 0.7% sulfuric acid, 190°C, and a 3-minute residence time; Stage 2, 0.4% sulfuric acid, 215°C, and

a 3-minute residence time The liquid hydrolysates are recovered from each stage and fermented to alcohol Residual cellulose and lignin left over in the solids from the hydrolysis reactors serve as boiler fuel for electricity or steam production These bench-scale tests confirmed the potential to achieve yields of 89% for mannose, 82%

for galactose, and 50% for glucose Fermentation with Saccharomyces cerevisiae

achieved ethanol conversion of 90% of the theoretical yield (Nguyen 1998)

The degradation of the lignocellulosic structure often requires two steps, first, the prehydrolysis in which the hemicellulose structure is broken down, and second, the hydrolysis of the cellulose fraction in which lignin will remain as a solid by-product The two hydrolyzed streams are fermented to ethanol either together or separately, after which they are mixed together and distilled (Figure 11.1) During the degrada-tion of the lignocellulosic structure, not only fermentable sugars are released, but a

Lignocellulosic Material Prehydrolysis

Fermentation Pentose Hydrolysis

Fermentation Hexose Lignin

Distillation Ethanol

Hemicellulose fraction

Cellulose fraction

fIgure 11.1 Flow chart for ethanol production from lignocellulosic biomass.

broad range of compounds, some of which might inhibit the fermenting microorgan-ism The prehydrolysis process can be performed by physical, chemical, or biologi-cal methods such as steam pretreatment, milling, freeze explosion, acid treatment (hydrochloric acid, phosphoric acid, sulfuric acid, sulfur dioxide), alkaline treatment (sodium hydroxide, ammonia), or treatment with organic solvents (ethanol, ethylene glycol) or white rot fungi (Vallander and Eriksson 1990; Saddler, Ramos, and Breuil 1993) In the prehydrolysis step, the hemicellulose is liquefied, resulting in a mixture

of mono- and oligosaccharides The hydrolysis of the cellulose is usually performed

by weak acids or by enzymes (Olsson and Hahn-Hägerdal 1996)

11.2.1.2 concentrated Acid hydrolysis

This process is based on concentrated acid decrystallization of the cellulose followed

by dilute acid hydrolysis to sugars at near theoretical yields The separation of acid from the sugars, acid recovery, and acid reconcentration are critical operations The fermentation converts sugars to ethanol A process was developed in Japan in which the concentrated sulfuric acid was used for the hydrolysis The process was commer-cialized in 1948 The remarkable feature of their process was the use of membranes

to separate the sugar and acid in the product stream The membrane separation, a technology that was way ahead of its time, achieved 80% recovery of acid (Wenzl 1970)

The concentrated sulfuric acid process was also commercialized in the former Soviet Union However, these processes were only successful during times of national crisis, when economic competitiveness of ethanol production could be ignored Con-centrated hydrochloric acid has also been utilized and in this case, the prehydrolysis and hydrolysis are carried out in one step Generally, acid hydrolysis procedures give rise to a broad range of compounds in the resulting hydrolysate, some of which might negatively influence the subsequent steps in the process A weak acid hydrolysis pro-cess is often combined with a weak acid prehydrolysis

In 1937, the Germans built and operated commercial concentrated acid hydroly-sis plants based on the use and recovery of hydrochloric acid Several such facilities were successfully operated During World War II, researchers at the U.S Department

of Agriculture’s Northern Regional Research Laboratory in Peoria, Illinois, further refined the concentrated sulfuric acid process for corn cobs They conducted pro-cess development studies on a continuous propro-cess that produced a 15 to 20% xylose sugar stream and a 10 to 12% glucose sugar stream, with the lignin residue remain-ing as a by-product The glucose was readily fermented to ethanol at 85 to 90% of theoretical yield Research and development based on the concentrated sulfuric acid process studied by the USDA (and which came to be known as the “Peoria Process”) picked up again in the United States in the 1980s, particularly at Purdue University and at the Tennessee Valley Authority (TVA) (Broder, Barrier, and Lightsey 1992) Among the improvements added by these researchers were recycling of dilute acid from the hydrolysis step for pretreatment, and improved recycling of sulfuric acid Minimizing the use of sulfuric acid and recycling the acid cost effectively are criti-cal factors in the economic feasibility of the process (see http://www1.eere.energy gov/biomass/printable_versions/concentrated_acid.html) The conventional wisdom

in the literature suggests that the Peoria and TVA processes cannot be economical because of the high volumes of acid required (Wright and d’Agincourt 1984) The improvements in the acid sugar separation and recovery have opened the door for commercial application Two companies, Arkenol and Masada, in the United States are currently working with DOE and NREL to commercialize this technology by taking advantage of niche opportunities involving the use of biomass as a means of mitigating waste disposal or other environmental problems (http://www1.eere.energy gov/biomass/concentrated_acid.html) Minimizing the use of the sulfuric acid and recycling the acid cost effectively are the critical factors in the economic feasibility

of the process U.S Patent 5,366,558 (Brink 1994) describes the use of two “stages”

to hydrolyze the hemicellulose sugars and the cellulosic sugars in a countercurrent process using a batch reactor, which results in poor yields of glucose and xylose using a mineral acid Further, the process scheme is complicated and the economic potential on a large scale to produce inexpensive sugars for fermentation is low U.S Patent 5,188,673 employs concentrated acid hydrolysis which has the benefit of high conversion of biomass, but suffers from low product yields due to degradation and the requirement of acid recovery and recycling Sulfuric acid concentrations used are 30 to 70 weight percent at temperatures less than 100°C Although 90% hydro-lysis of the cellulose and hemicellulose is achieved by this process, the concentrated acids are toxic, corrosive, and hazardous and require reactors that are resistant to corrosion In addition, the concentrated acid must be recovered after the hydrolysis

to make the process economically feasible (Von Sivers and Zacchi 1995) A multi-function process for hydrolysis and fractionation of lignocellulosic biomass to sepa-rate hemicellulosic sugars using mineral acids like sulfuric acid, phosphoric acid,

or nitric acid has been described (Torget et al., U.S Patent 6,022,419) A process for treatment of hemicellulose and cellulose in two different configurations has also been described (Scott and Piskorz, U.S Patent 4,880,473) Hemicellulose is treated with dilute acid in a conventional process The cellulose is separated out from the prehydrolysate and then subjected to pyrolysis at high temperatures Further, the process step between the hemicellulose and cellulose reactions requires a drying step with a subsequent high-temperature pyrolysis step at 400 to 600°C for conversion of the cellulose to fermentable products A 70% yield of glucose was obtained from the hydrolysis of lignocellulose under extremely low acid and high temperature condi-tions by autohydrolysis (Ojumu and Ogunkunle 2005)

11.3 enzymAtIc hydrolysIs of lIgnocellulosIc bIomAss

The enzymatic hydrolysis or saccharification of lignocellulosic biomass is preceded

by a pretreatment process in which the lignin component is separated from the cel-lulose and hemicelcel-lulose to make it amenable to the enzymatic hydrolysis The lignin interferes with hydrolysis by blocking the access of the cellulases to the cellulose and by irreversibly binding the hydrolytic enzymes Therefore, the removal of the lignin can dramatically increase the hydrolysis rate (McMillan 1994) For the effi-cient enzymatic hydrolysis of lignocellulosic biomass a pretreatment step is neces-sary Various pretreatment processes and the enzymes involved in hydrolysis have been described in different chapters

11.3.1 f actorS G overninG e nzymatic H ydrolySiS

There are different factors that affect the enzymatic hydrolysis of cellulose, namely, substrates, cellulase activity, and reaction conditions (temperature, pH, as well as other parameters) To improve the yield and rate of enzymatic hydrolysis, research has been focused on optimizing the hydrolysis process and enhancing the cellulase activity The yield and initial rate of enzymatic hydrolysis of cellulose is affected mainly by the substrate concentration At low substrate levels, an increase of sub-strate concentrations yields an increase in the reaction rate of the hydrolysis and the products (Cheung and Anderson 1997) However, substrate inhibition is caused at high substrate concentration, which considerably lowers the rate of hydrolysis The ratio of the enzyme to substrate in the hydrolysis reaction is crucial to establish the level of substrate inhibition (Huang and Penner 1991) The hydrolysis of cellulosic substrates by the enzymes depend to a large extent on the structural features of the substrate, such as cellulose crystallinity, degree of cellulose polymerization, surface area, and content of lignin (Table 11.1)

The yield and rate of hydrolysis of the cellulosic substrate can be increased to a certain extent by increasing the dosage of the cellulases in the process, but that would significantly increase the cost of the process Cellulase dosage of 10 FPU/g cellulose

is often used in laboratory studies because it provides a hydrolysis profile with high levels of glucose yield in a reasonable time (48–72 h) at a reasonable enzyme cost (Gregg and Saddler 1996) Depending on the type and concentration of the sub-strates, cellulase enzyme could be used in the hydrolysis (7–33 FPU/g substrate) The adsorption of the cellulase enzymes onto the surface of the cellulose, the biodegrada-tion of cellulose to fermentable sugars, and desorpbiodegrada-tion of the cellulase are three steps involved in enzymatic hydrolysis of the cellulose The cellulase activity decreases during hydrolysis because of the irreversible adsorption of the cellulase on the cel-lulose (Converse et al 1988) The celcel-lulose surface property can be modified and the irreversible binding of the cellulase can be minimized by the addition of surfactants during the hydrolysis The ionic surfactants Q-86W (cationic) at high concentration and Neopelex F-25 (anionic) have been shown to have an inhibitory effect (Ooshima,

tAble 11.1

structural properties potentially limiting enzymatic hydrolysis of

cellulosic fibers at different structural levels

Microfibril Degree of polymerization

Crystallinity Cellulose lattice structure Fibril Structural composition (lignin content and distribution)

Particle size (fibril dimension) Fiber Available surface area

Degree of fiber swelling Pore structure and distribution

From Mansfield et al 1999 Biotechnol Progr 15: 804–816 With permission.

Sakata, and Harano 1986), hence, the nonionic surfactants such as Tween 20, 80 (Wu and Ju 1998), polyoxyethylene glycol (Park et al., 1992), Tween 81, Emulgen 147, amphoteric Anhitole 20BS, cationic Q-86W (Ooshima, Sakata, and Harano 1986), sophorolipid, rhamnolipid, and bacitracin (Helle, Duff, and Cooper 1993) have been used to enhance the cellulose hydrolysis Cellulose conversion with 2% (w/v) F68 and 2 g/l cellulase reached 52%, compared to 48% conversion with 10 g/l cellulase

in a surfactant-free system (Wu and Ju 1998) However, Tween 20 was highly

inhibi-tory to D clausenii even at a low concentration of 0.1% Use of a cellulase mixture

from different microorganisms, or a mixture of cellulases and other enzymes, in the hydrolysis of cellulosic materials was studied by Excoffier, Toussaint, and Vignon (1991) The addition of β-glucosidases into the Trichoderma reesei cellulases

sys-tem achieved better saccharification than the syssys-tem without β-glucosidases The β-glucosidase hydrolyzes the cellobiose, which is an inhibitor of the cellulase activ-ity The saccharification of the cellulose is reported to be faster when supplemented with additional β-glucosidase There are few organisms that secrete complete

cel-lulase, for example, Penicillium funiculosum with high β-glucosidases activity (Rao, Seeta, and Deshpande 1983) A mixture of hemicellulases or pectinases with cellu-lases exhibited a significant increase in cellulose conversion (Beldman et al 1984)

A 90% enzymatic saccharification of 8% alkali-treated sugarcane bagasse has been

reported when a mixture of the cellulases (dose, 1.0 FPU/g substrate) from

Aspergil-lus ustus and Trichoderma viride was used (Mononmani and Sreekantiah 1987) The

use of the cellulase mixture of the commercial Cellucast and Novozyme prepara-tions has achieved a nearly complete saccharification of steam-explosion pretreated

Eucalyptus viminalis chips (substrate concentration of 6% and enzyme loading of

10 FPU/g cellulose) (Ramos, Brueil, and Saddler 1993) Baker, Adney, and Nieves

(1994) reported a new thermostable endoglucanase from Acidothermus

cellulolyti-cus E1 and an endoglucanase from T fusca E5 that exhibited striking synergism with

T reesei CBH1 in the saccharification of the microcrystalline cellulose The cellu-lases can be recovered from the liquid supernatant or the solid residues and recycled Enzyme recycling can effectively increase the rate and yield of the hydrolysis and lower the enzyme cost (Mes-Hartree, Hogan, and Saddler 1987) The efficiency of cellulose hydrolysis gradually decreases with each recycling step (Ramos, Brueil, and Saddler 1993)

Recently, the enzymatic hydrolysis of lignocellulosic biomass has been opti-mized using enzymes from different sources and mixing in an appropriate propor-tion using a statistical approach of factorial design A twofold reducpropor-tion in the total protein required to reach glucan to glucose and xylan to xylose hydrolysis targets (99% and 88% conversion, respectively), thereby validating this approach toward enzyme improvement and process cost reduction for lignocellulose hydrolysis (Kim, Kang, and Lee 1997, Berlin et al 2005)

Many studies have been presented over the years aiming to understand the inhib-iting factors in enzymatic hydrolysis of lignocellulose substrates Reasons for low yield of fermentable sugars in enzymatic conversion include reduced accessible sur-face area of cellulose in the lignocellulose complex, leading to restricted access for enzymes; restricted pore volume of the substrate (Eklund et al 1990; Mooney et al 1998); slow enzyme kinetics for crystalline cellulose (Fan et al 1980); and obstacles

in the structure of cellulose leading to unproductive enzyme binding (Eriksson, Karlsson, and Tjerneld 2002; Väljamäe et al., 1998) Lignin has also been identified

to have a high binding affinity for cellulase proteins (Lu et al 2002; Berlin et al 2005) Both addition of lignin (Sewalt et al 1997) and the composition of lignin have been shown to be responsible for inhibitory factors for the degradation of cellulose

It was recently found that cellulases lacking cellulose binding module (CBM) also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic module (Berlin et al 2005)

An enhancement in enzymatic hydrolysis of softwood lignocellulosic by non-ionic surfactants and polymers was observed It was suggested that ethylene oxide containing surfactants and polymers such as polyethylene glycol bind to lignin by hydrophobic interaction and hydrogen bonding and helps to reduce the unproductive binding of enzymes, thus yielding more fermentable sugars (Börjesson, Peterson, and Tjerneld 2007)

11.3.2 d etoxification

Biological, physical, and chemical methods have been employed for detoxification (the specific removal of inhibitors prior to fermentation) of lignocellulosic hydro-lysates (Olsson and Hahn-Hägerdal, 1996) The methods of detoxification change depending on the source of the lignocellulosic hydrolysate and the microorganism being used The lignocellulosic hydrolysates vary in their degree of inhibition and different microorganisms have different inhibitor tolerances Several reports on adaptation of yeasts to inhibiting compounds in lignocellulosic hydrolysates are found in the literature (e.g., Amartey and Jeffries 1996; Buchert, Puls, and Poutanen 1988; Nishikawa, Sutcliffe, and Saddler 1988)

11.3.2.1 biological detoxification methods

Biological methods of treatment make use of specific enzymes or microorgan-isms that act on the toxic compounds present in hydrolysates and change their composition Treatment with the enzymes peroxidase and laccase, obtained from

the ligninolytic fungus Trametes versicolor, has been shown to increase

maxi-mum ethanol productivity in a hemicellulose hydrolysate of willow two to three times due to their action on acid and phenolic compounds (Jönsson et al 1998)

The filamentous soft-rot fungus Trichoderma reesei has been reported to degrade

inhibitors in a hemicellulose hydrolysate obtained after steam pretreatment of wil-low, resulting in around three times increased maximum ethanol productivity and four times increased ethanol yield (Palmqvist et al 1997) Acetic acid, furfural, and benzoic acid derivatives were removed from the hydrolysate by treatment with

T reesei The use of microorganism has also been proposed to selectively remove inhibitors from lignocellulose hydrolysates Adaptation of a microorganism to the hydrolysate is another interesting biological method for improving the fermenta-tion of hemicellulosic hydrolysate media

11.3.2.2 physical detoxification methods

Hydrolysate concentration by vacuum evaporation is a physical detoxification method for reducing the concentration of volatile compounds such as acetic acid, furfural, and vanillin present in the hydrolysate However, physical detoxification increases moderately the concentration of nonvolatile toxic compounds and consequently the degree of fermentation inhibition

11.3.2.3 chemical detoxification methods

Chemical detoxification includes precipitation of toxic compounds and ionization of some inhibitors under certain pH values, the latter being able to change the degree of toxicity of the compounds (Mussatto 2002) Toxic compounds may also be adsorbed

on activated charcoal (Dominguez, Gong, and Tsao 1996; Mussatto and Roberto 2001), on diatomaceous earth (Ribeiro et al 2001) and on ion exchange resins (Lars-son et al 1999; Nilvebrant et al 2001)

11.4 fermentAtIon of lIgnocellulosIc

bIomAss to ethAnol

The hydrolysis of lignocellulosic biomass yields reducing sugars Once the sugars are available, its fermentation to ethanol is not a difficult task as many technologies have been developed Essentially, there are three different types of processes by which this can be achieved, namely,

1 Separate hydrolysis and fermentation (SHF)

2 Direct microbial conversion (DMC)

3 Simultaneous saccharification and fermentation (SSF)

SSF has been shown to be the most promising approach to biochemically convert cellulose to ethanol in an effective way (Wright, Wyman, and Grohmann 1988)

11.4.1 S eParate H ydrolySiS and f ermentation (SHf)

This is a conventional two-step process where the lignocellulose is hydrolyzed using enzymes to form reducing sugars in the first step and the sugars thus formed are

fer-mented to ethanol in the second step using Saccharomyces or Zymomonas (Bisaria

and Ghose 1981; Philippidis 1996) The advantage of this process is that each step can be carried out at its optimum conditions

11.4.2 d irect m icroBial c onverSion (dmc)

This process involves three major steps, namely, enzyme production, hydrolysis

of the lignocellulosic biomass, and the fermentation of the sugars, all occurring in one step (Hogsett et al 1992) The relatively lower tolerance of the ethanol is the main disadvantage of this process A lower tolerance limit of about 3.5% has been reported as compared to 10% of ethanologenic yeasts Acetic acid and lactic acid are

also formed as by-products in this process in which a significant amount of carbon

is utilized (Klapatch et al 1994) Neurospora crassa is known to produce ethanol

directly from cellulose/hemicellulose, because it produces both cellulase and xyla-nase and also has the capacity to ferment the sugars to ethanol anaerobically (Desh-pande et al 1986)

11.4.3 S imultaneouS S accHarification and f ermentation (SSf)

The saccharification of lignocellulosic biomass by enzymes and the subsequent

fer-mentation of the sugars to ethanol by yeast such as Saccharomyces or Zymomonas

take place in the same vessel in this process (Glazer and Nikaido 1995) The com-patibility of both saccharification and fermentation processes with respect to various conditions, such as pH, temperature, substrate concentration, etc., is one of the most important factors governing the success of the SSF process The main advantages of using SSF for ethanol bioconversion are:

Enhanced rate of lignocellulosic biomass (cellulose and hemicellulose) due

•

to removal of the sugars that inhibit cellulase activity

Lower enzyme loading

•

Higher product yield

•

Reduced inhibition of the yeast fermentation in case of continuous recovery

•

of the ethanol

Reduced requirement for aseptic conditions, resulting in increasing

eco-•

nomics of the process (Deshpande, Siva Raman, and Rao 1984; Schell et al 1988; Wright, Wyman, and Grohmann 1988; Philippidis and Smith 1995) Because several inhibitory compounds are formed during hydrolysis of the raw material, the hydrolytic process has to be optimized so that inhibitor formation can

be minimized When low concentrations of inhibitory compounds are present in the hydrolysate, detoxification is easier and fermentation is cheaper The choice of detoxification method has to be based on the degree of microbial inhibition caused

by the compounds As each detoxification method is specific to certain types of compounds, better results can be obtained by combining two or more different methods Another factor of great importance in the fermentative processes is the cultivation conditions, which, if inadequate, can stimulate the inhibitory action of the toxic compounds

SSF seems to offer a better option for commercial production of ethanol from

lignocellulosic biomass Penicillium funiculosum cellulase and Saccharomyces

uvarum cells have been reported to be used for SSF (Deshpande et al 1981)

11.5 recombInAnt dnA ApproAches

Recombinant DNA methods are being used currently for lignocellulosic hydrolysis

and fermentation to ethanol Genetic manipulations of Saccharomyces cerevisiae and Z mobilis have been explored for improving their ability to utilize lignocel-lulosic biomass S cerevisiae has been engineered with arabinose metabolizing