In the fermentation process, sugars are transformed into ethanol by addition of microoganism. Ethanol production from sugars has been commercially dominated by the yeast *S. cereviseae* (Tanaka, 2006). Practically, yeast cells are often exposed in multiple stress environments. Therefore, it is helpful to fermentation efficiency and economic benefits to breed the yeast strains with tolerance against the multiple-stress such as temperature, ethanol, osmotic pressure, and so on (Cakar et al., 2005). Yeast strain improvement strategies are numerous and often complementary to each other, a summary of the main technologies is shown in Table 2. The choice among them is based on three factors: (1) the genetic nature of traits (monogenic or polygenic), (2) the knowledge of the genes involved (rational or blind approaches) (3) the aim of the genetic manipulation (Giudici et al ., 2005; Gasch et al.,

environmental parameters.

**ethanol**

2000 ).

**yeast to produce bioethanol**

Table 2. Summary of the main genetic improvement strategies. Dpt Desired phenotype

It is difficult to improve the multi-tolerance of the yeast by rational genetic engineering technology before its mechanism completely clarified. Nevertheless, for quantitative traits, the number of responsible genes QTLs is so great that a "gene-by-gene" engineering strategy is impossible to perform. In these cases, blind strategies, such as genome shuffling (Zhang et al., 2002), could be applied in order to obtain quickly strains with recombinant traits. Genome shuffling is an accelerated evolutionary approach that, on the base of the recursive multiparental protoplast fusion, permits obtaining the desired complex phenotype more rapidly than the normal breeding methods (Figure 6). Genome shuffling technology can bring a rapidly improvement of breeding a hybrid with whole-genome random reorganization. After the initial strains in various long term evolution experiments (Figure 7), we successfully applied the genome shuffling technology that combines the advantage of multi- parental recursive fusion with the recombination of entire genomes normally associated with conventional mutant breeding to selecting the multiple-stress resistant yeast (Figure 8).

#### **5.3.2 Continuous fermentation**

Traditionally, ethanol has been produced batch wise. However, high labor costs and the low productivity offered by the batch process have led many commercial operators to consider the continuous fermentation. Continuous fermentation can be performed in different kind of bioreactors – stirred tank reactors or plug flow reactors. Continuous fermentation often gives a higher productivity, offers ease of control and is less labor intensive than batch fermentation (Cheng et al., 2007). However contamination is more serious in this operation (Skinner & Leathers, 2004). In the fuel ethanol industry, control of bacterial contamination is achieved by acidification and using antibiotics such as penicillin G, streptomycin, tetracycline (Aquarone E,1960; Day et al., 1954), virginiamycin(Hamdy et al., 1996; Hynes et

Simultaneous Production of Sugar and Ethanol

Fig. 7. Approach for evolutionary engineering

Fig. 8. Multiple-stress Resistant Yeast

from Sugarcane in China, the Development, Research and Prospect Aspects 83

Fig. 6. Protoplast fusion of the genome shuffling process

al., 1997; Islam et al., 1999), monensin(Stroppa et al., 2000), or mixtures thereof. Fig 9 shows the process of continuous fermentation of molasses and sugarcane juice to produce ethanol. A high cell density of microbes in the continuous fermenter is locked in the exponential phase, which allows high productivity and overall short processing of 6 - 12 h as compared to the conventional batch fermentation (30 - 60 h). This results in substantial savings in labor and minimizes investment costs by achieving a given production level with a much smaller plant.

Fig. 6. Protoplast fusion of the genome shuffling process

plant.

al., 1997; Islam et al., 1999), monensin(Stroppa et al., 2000), or mixtures thereof. Fig 9 shows the process of continuous fermentation of molasses and sugarcane juice to produce ethanol. A high cell density of microbes in the continuous fermenter is locked in the exponential phase, which allows high productivity and overall short processing of 6 - 12 h as compared to the conventional batch fermentation (30 - 60 h). This results in substantial savings in labor and minimizes investment costs by achieving a given production level with a much smaller

Fig. 7. Approach for evolutionary engineering

Fig. 8. Multiple-stress Resistant Yeast

Simultaneous Production of Sugar and Ethanol

Fig. 10. Yeast immobilized in Polyvinyl Alcohol

immobilization.

production) animal feeds.

from Sugarcane in China, the Development, Research and Prospect Aspects 85

Fig. 11. Scanning electron micrographs of the middle part of the support after yeast

sugarcane supported biocatalyst was equally efficient to that described in the literature for ethanol fermentation. Sugarcane pieces were found suitable as support for yeast cell immobilization in fuel ethanol industry. The sugarcane immobilized biocatalysts showed high fermentation activity. The immobilized yeast would dominate in the fermentation broth due to its high populations and lower fermentation time, that in relation with low price of the support and its abundance in nature, reuse availability make this biocatalyst attractive in the ethanol production as well as in wine making and beer production. After a long period of using, spent immobilized supports can be used as protein-enriched( SCP

Fig. 9. Continuous fermentation of molasses and sugarcane juice to produce ethanol

#### **5.3.3 Sugarcane pieces as yeast supports for alcohol production from sugarcane juice and molasses**

A limitation to continuous fermentation is the difficulty of maintaining high cell concentration in the fermenter. The use of immobilized cells circumvents this difficulty. Immobilization by adhesion to a surface (electrostatic or covalent), entrapment in polymeric matrices or retention by membranes has been successful for ethanol production (Godia et al., 1987). The applications of immobilized cells have made a significant advance in fuel ethanol production technology. Immobilized cells offer rapid fermentation rates with high productivity – that is, large fermenter volumes of mash put through per day, without risk of cell washout. In continuous fermentation, the direct immobilization of intact cells helps to retain cells during transfer of broth into collecting vessel. Moreover, the loss of intracellular enzyme activity can be kept to a minimum level by avoiding the removal of cells from downstream products (Najafpour, 1990). Immobilization of microbial cells for fermentation has been developed to eliminate inhibition caused by high concentration of substrate and product and also to enhance ethanol productivity and yield. Neelakantam (2004) demonstrated that a high yeast inoculation at the start of the sugarcane juice fermentation allows the yeast outgrow the contaminant bacteria and inhibit its growth and metabolism.

Varies immobilization supports for variety of products have been reported such as polyvinyl alcohol (PVA, see Fig10), alginates (Kiran Sree, 2000; Corton et al., 2000), Apple pieces (Kourkoutas et al., 2006), orange peel (S.plessas, 2007), and delignified cellulosic residues (Kopsahelis, 2006; Bardi & Koutinas, 1994). We applied sugarcane pieces as yeast supports for alcohol production from sugarcane juice and molasses(Fig 11).The results(Liang et al.,2008) showed ethanol concentrations (about 77g/l or 89.76g/l in average value) , and ethanol productivities (about 62.76 g/l.d or 59.55g/l .d in average value)were high and stable, and residual sugar concentrations were low in all fermentations(0.3- 3.6g/l)with conversions ranging from 97.7-99.8%, showing efficiency(90.2-94.2%) and operational stability of the biocatalyst for ethanol fermentation. the results presented in this paper (see table 3), according to initial concentration of sugars in the must, showed that the

Fig. 10. Yeast immobilized in Polyvinyl Alcohol

Fig. 9. Continuous fermentation of molasses and sugarcane juice to produce ethanol

**juice and molasses**

**5.3.3 Sugarcane pieces as yeast supports for alcohol production from sugarcane**

A limitation to continuous fermentation is the difficulty of maintaining high cell concentration in the fermenter. The use of immobilized cells circumvents this difficulty. Immobilization by adhesion to a surface (electrostatic or covalent), entrapment in polymeric matrices or retention by membranes has been successful for ethanol production (Godia et al., 1987). The applications of immobilized cells have made a significant advance in fuel ethanol production technology. Immobilized cells offer rapid fermentation rates with high productivity – that is, large fermenter volumes of mash put through per day, without risk of cell washout. In continuous fermentation, the direct immobilization of intact cells helps to retain cells during transfer of broth into collecting vessel. Moreover, the loss of intracellular enzyme activity can be kept to a minimum level by avoiding the removal of cells from downstream products (Najafpour, 1990). Immobilization of microbial cells for fermentation has been developed to eliminate inhibition caused by high concentration of substrate and product and also to enhance ethanol productivity and yield. Neelakantam (2004) demonstrated that a high yeast inoculation at the start of the sugarcane juice fermentation allows the yeast outgrow the contaminant bacteria and inhibit its growth and metabolism. Varies immobilization supports for variety of products have been reported such as polyvinyl alcohol (PVA, see Fig10), alginates (Kiran Sree, 2000; Corton et al., 2000), Apple pieces (Kourkoutas et al., 2006), orange peel (S.plessas, 2007), and delignified cellulosic residues (Kopsahelis, 2006; Bardi & Koutinas, 1994). We applied sugarcane pieces as yeast supports for alcohol production from sugarcane juice and molasses(Fig 11).The results(Liang et al.,2008) showed ethanol concentrations (about 77g/l or 89.76g/l in average value) , and ethanol productivities (about 62.76 g/l.d or 59.55g/l .d in average value)were high and stable, and residual sugar concentrations were low in all fermentations(0.3- 3.6g/l)with conversions ranging from 97.7-99.8%, showing efficiency(90.2-94.2%) and operational stability of the biocatalyst for ethanol fermentation. the results presented in this paper (see table 3), according to initial concentration of sugars in the must, showed that the

Fig. 11. Scanning electron micrographs of the middle part of the support after yeast immobilization.

sugarcane supported biocatalyst was equally efficient to that described in the literature for ethanol fermentation. Sugarcane pieces were found suitable as support for yeast cell immobilization in fuel ethanol industry. The sugarcane immobilized biocatalysts showed high fermentation activity. The immobilized yeast would dominate in the fermentation broth due to its high populations and lower fermentation time, that in relation with low price of the support and its abundance in nature, reuse availability make this biocatalyst attractive in the ethanol production as well as in wine making and beer production. After a long period of using, spent immobilized supports can be used as protein-enriched( SCP production) animal feeds.

Simultaneous Production of Sugar and Ethanol

Fig. 12. Ethanol separation and dehydration.

from Sugarcane in China, the Development, Research and Prospect Aspects 87

Table 3. Fermentation parameters(average value)obtained in batch fermentation with *Saccharomyces cerevisiae,* immobilized on various carriers, at 30℃

#### **5.3.4 Ethanol purification and water recovery**

Distillation and molecular - sieve absorption are used to recover ethanol from the raw fermentation beer. The flow sheet of this section is presented in Figure 12 and figure 13. Distillation itself is a two-way progress include heating and cooling. That could be possible to save much steam and cooling water if we take good advantage of the heat exchange in the system. Due to its energy-saving, so far negative pressure distillation system has been popular in China. Take molasses alcohol as an example, compare to air distillation system, negative pressure distillation system could save approximately 2t steam per ton 95% (v/v) alcohol. The system showed in figure contains 3 columns, which is .fractioning column 1, fractioning column 2, and separating methanol column respectively. Making use of the different boiling points the alcohol in the fermented wine is separated from the main resting solid components. The remaining product is hydrated ethanol with a concentration of 95% (v/v). Further dehydration is normally done by molecular-sieve absorption, up to the specified 99.7°GL in order to produce anhydrous ethanol which is used for blending with pure gasoline to obtain the country's E10 mandatory blend. The fermented mash which contains 10~13 %(v/v) alcohol is preheated by the alcohol gas from the top of the first column and gas is cooled simultaneously. Then the gas stream is cooled by 3 heat exchangers, the cooler is water. Subsequently the liquid distillate which contains 30% (v/v) alcohol is feeding on the middle tray of column 2. Wastewater of column 1 is heated by the alcohol gas from the top of column 2 in the reboiler, meanwhile the steam flash evaporated in the vacuum bottom. The waste goes to anaerobic jar and then aeration tank. Cooled alcohol is pumped back to the top trays of column 2. Fusel oil is extracted from the middle trays of the column 2. Liquid distillate contains 95 %( v/v) alcohol and exceeded methanol amount. In order to decrease the concentration of aldehyde and methanol, one more column is needed. The 96%v/v alcohol with 4% water is feeding on the molecular-sieve absorption system.Finally 99.5%v/v ethanol which could be added to the gas to make gasohol is achieved.

Residual sugar (g/l)

Grape must 206 80 30.8 85 26 85

Glucose 120 45 1.4 45.0 24.0 98

molasses 187 30 8.8 51.4 42.7 95.3

Glucose 125 9 4 51.4 128.3 96.8 molasses 128 14 2 58.9 100.1 98.4

extract 124 12 2.3 55.3 110.4 98.1

Molasses 154 27 2.3 77.12 62.76 98.5

juice 176 32 0.85 89.76 59.55 99.5

Ethanol (g/l)

Ethanol productivity (g/l.d)

Conversion (%)

Ferm.time (h)

Table 3. Fermentation parameters(average value)obtained in batch fermentation with

Distillation and molecular - sieve absorption are used to recover ethanol from the raw fermentation beer. The flow sheet of this section is presented in Figure 12 and figure 13. Distillation itself is a two-way progress include heating and cooling. That could be possible to save much steam and cooling water if we take good advantage of the heat exchange in the system. Due to its energy-saving, so far negative pressure distillation system has been popular in China. Take molasses alcohol as an example, compare to air distillation system, negative pressure distillation system could save approximately 2t steam per ton 95% (v/v) alcohol. The system showed in figure contains 3 columns, which is .fractioning column 1, fractioning column 2, and separating methanol column respectively. Making use of the different boiling points the alcohol in the fermented wine is separated from the main resting solid components. The remaining product is hydrated ethanol with a concentration of 95% (v/v). Further dehydration is normally done by molecular-sieve absorption, up to the specified 99.7°GL in order to produce anhydrous ethanol which is used for blending with pure gasoline to obtain the country's E10 mandatory blend. The fermented mash which contains 10~13 %(v/v) alcohol is preheated by the alcohol gas from the top of the first column and gas is cooled simultaneously. Then the gas stream is cooled by 3 heat exchangers, the cooler is water. Subsequently the liquid distillate which contains 30% (v/v) alcohol is feeding on the middle tray of column 2. Wastewater of column 1 is heated by the alcohol gas from the top of column 2 in the reboiler, meanwhile the steam flash evaporated in the vacuum bottom. The waste goes to anaerobic jar and then aeration tank. Cooled alcohol is pumped back to the top trays of column 2. Fusel oil is extracted from the middle trays of the column 2. Liquid distillate contains 95 %( v/v) alcohol and exceeded methanol amount. In order to decrease the concentration of aldehyde and methanol, one more column is needed. The 96%v/v alcohol with 4% water is feeding on the molecular-sieve absorption system.Finally 99.5%v/v ethanol

Initial sugar (g/l)

Carrier Medium

Raisin

Sugarcane

**5.3.4 Ethanol purification and water recovery**

*Saccharomyces cerevisiae,* immobilized on various carriers, at 30℃

which could be added to the gas to make gasohol is achieved.

Apple pieces (Y. Kourkoutas et al.,2001)

figs(Bekatorou et

Dried

al., 2002)

Spent grains (Kopsahelis et al.,2006)

Orange peel (S.plessas et al.,2007)

Sugarcane pieces present study

Fig. 12. Ethanol separation and dehydration.

Simultaneous Production of Sugar and Ethanol

Table 4. The profits of the three different modes

**6. Conclusions**

from Sugarcane in China, the Development, Research and Prospect Aspects 89

Various technologies have been identified for immediate increases in the efficiency and sustainability of current and future sugarcane ethanol. In conclusion, recycle utilization design are seems to be suitable for sugarcane bioethanol development, for example, recycling of byproducts of sugarcane in the fields reduces chemical fertilizers application rates, reducing water consumption with closure of water-processing circuits and the use of bagasse to generate electricity or to manufacture bagasse polymer composites (Xu et al., 2010), improving the energy balance of ethanol production; as well as in production and

Fig. 14. Recycle utilization design for sugarcane bioethanol development

product only

sugar combined fuel ethanol

Ethanol product only

Project Sugar

Sugarcane milled(t/d) 100 100 100 Fuel ethanol (t) - 1 7 Sugar (t) 12 11.5 - Molasses (t) 3 - - Sugar product costs(RMB/t sugar) 4000 3970 - Fuel ethanol product costs (RMB) - 1000 5500 Total costs (RMB) 48000 46655 38500 Fuel ethanol product incomes (RMB) - 8000 56000 Sugar product incomes (RMB) 72000 69000 - Molasses incomes (RMB) 2700 - - Total incomes (RMB) 74700 77000 56000 profits (RMB) 26700 30345 17500

Fig. 13. Ethanol dehydration with molecular sieve bed

#### **5.4 Economic analysis for simultaneous production of sugar and ethanol from sugarcane**

Based on the economic analysis, the profits of the three different modes in 5,000 tons sugarcane pressed plants are showed in table 4. There are high costs of fermentation and distillation for sugarcane directly for ethanol fermentation due to low concentration of sugarcane juice, and about 15 tons waste water need treatment. It is also uneconomic to produce fuel ethanol using concentrated juice because of high energy costs. Therefore, that sugarcane is used directly for fuel ethanol production does not reflect its best economic benefits and flexible market response capacity.

In traditional opinion, people prefer to produce sugar as possible as they can rather than use more molasses to produce ethanol. They think that it is uneconomic to produce 1 ton ethanol with nearly 2 tons sugar consumption. In fact, we can achieve the maximized economic benefits applying "the simultaneous production of sugar and ethanol " mode, in which we boil the A-syrup that have the good characteristic of low energy consumption, to produce the top-grade white sugar production. B-green syrup and second pressed juice are mixed to produce the fuel ethanol. Costs of the ethanol production can be greatly reduced.

According to the calculations, it will bring more economic benefits while employ "the simultaneous production of sugar and ethanol" mode.

Table 4. The profits of the three different modes

#### **6. Conclusions**

88 Bioethanol

Fig. 13. Ethanol dehydration with molecular sieve bed

benefits and flexible market response capacity.

simultaneous production of sugar and ethanol" mode.

production can be greatly reduced.

**sugarcane**

**5.4 Economic analysis for simultaneous production of sugar and ethanol from**

Based on the economic analysis, the profits of the three different modes in 5,000 tons sugarcane pressed plants are showed in table 4. There are high costs of fermentation and distillation for sugarcane directly for ethanol fermentation due to low concentration of sugarcane juice, and about 15 tons waste water need treatment. It is also uneconomic to produce fuel ethanol using concentrated juice because of high energy costs. Therefore, that sugarcane is used directly for fuel ethanol production does not reflect its best economic

In traditional opinion, people prefer to produce sugar as possible as they can rather than use more molasses to produce ethanol. They think that it is uneconomic to produce 1 ton ethanol with nearly 2 tons sugar consumption. In fact, we can achieve the maximized economic benefits applying "the simultaneous production of sugar and ethanol " mode, in which we boil the A-syrup that have the good characteristic of low energy consumption, to produce the top-grade white sugar production. B-green syrup and second pressed juice are mixed to produce the fuel ethanol. Costs of the ethanol

According to the calculations, it will bring more economic benefits while employ "the

Various technologies have been identified for immediate increases in the efficiency and sustainability of current and future sugarcane ethanol. In conclusion, recycle utilization design are seems to be suitable for sugarcane bioethanol development, for example, recycling of byproducts of sugarcane in the fields reduces chemical fertilizers application rates, reducing water consumption with closure of water-processing circuits and the use of bagasse to generate electricity or to manufacture bagasse polymer composites (Xu et al., 2010), improving the energy balance of ethanol production; as well as in production and

Fig. 14. Recycle utilization design for sugarcane bioethanol development

Simultaneous Production of Sugar and Ethanol

671-675

61-68

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Developing the sugarcane ethanol provides a novel option for utilization of the sugar industry, and it will be also helpful to the fuel ethanol development in China.

#### **7. Acknowledgements**

This research work was supported by Ministry of Science and Technology of China, (NCSTE-2006-JKZX-023), Guangdong Science and Technology Department (2010A010500005) and the Natural Science Foundation of Guangdong Province<PHONE_NUMBER>1006220). We specially thank Guangdong Key Laboratory of Sugarcane Improvement and Biorefinery, Guangzhou Sugarcane Industry Research Institute for supporting this research.

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**5**

,

*Italy*

**Hydrolysis of Lignocellulosic Biomass:**

**Technologies and Future Perspectives**

Bioethanol can be produced from several different biomass feedstocks: sucrose rich feedstocks (e.g. sugar-cane), starchy materials (e.g. corn grain), and **lignocellulosic biomass.**  This last category, including biomass such as corn stover and wheat straw, woody residues from forest thinning and paper, is promising especially in those countries with limited lands availability. In fact, residues are often widely available and do not compete with food production in terms of land destination. The process converting the biomass biopolymers to fermentable sugars is called **hydrolysis**. There are two major categories of methods employed. The first and older method uses acids as catalysts, while the second uses enzymes called **cellulases**. Feedstock **pretreatment** has been recognized as a necessary upstream process to remove lignin and enhance the porosity of the lignocellulosic materials

Cellulases are proteins that have been conventionally divided into three major groups: **endoglucanase**, which attacks low cristallinity regions in the cellulose fibers by endoaction, creating free chain-ends; **exoglucanases** or cellobiohydrolases which hydrolyze the 1, 4 glycocidyl linkages to form cellobiose; and **β-glucosidase** which converts cellooligosaccharides and disaccharide cellobiose into glucose residues. In addition to the three major groups of cellulose enzymes, there are also a number of other enzymes that attack hemicelluloses, such as **glucoronide**, **acetylesterase**, **xylanase**, **β-xylosidase**, **galactomannase** and **glucomannase**. These enzymes work together synergistically to attack cellulose and hemicellulose. Cellulases are produced by various **bacteria and fungi** that can

The use of enzymes in the hydrolysis of cellulose is more effective than the use of inorganic catalysts, because enzymes are highly specific and can work at mild process conditions. In spite of these advantages, the use of **enzymes in industrial processes** is still limited by

prior to the enzymatic process (Zhu & Pan, 2010; Kumar et al., 2009).

have cellulolytic mechanisms significantly different.

**1. Introduction**

Corresponding Author

**Current Status of Processes and**

*2ENEA Italian National Agency for New Technologies, Energy and the Sustainable Economical Development, Rotondella (MT)*

Alessandra Verardi1, Isabella De Bari2

Emanuele Ricca1 and Vincenza Calabrò1

*1Department of Engineering Modeling, University of Calabria, Rende (CS)*

## **Hydrolysis of Lignocellulosic Biomass: Current Status of Processes and Technologies and Future Perspectives**

Alessandra Verardi1, Isabella De Bari2 , Emanuele Ricca1 and Vincenza Calabrò1 *1Department of Engineering Modeling, University of Calabria, Rende (CS) 2ENEA Italian National Agency for New Technologies, Energy and the Sustainable Economical Development, Rotondella (MT) Italy*

#### **1. Introduction**

Bioethanol can be produced from several different biomass feedstocks: sucrose rich feedstocks (e.g. sugar-cane), starchy materials (e.g. corn grain), and **lignocellulosic biomass.**  This last category, including biomass such as corn stover and wheat straw, woody residues from forest thinning and paper, is promising especially in those countries with limited lands availability. In fact, residues are often widely available and do not compete with food production in terms of land destination. The process converting the biomass biopolymers to fermentable sugars is called **hydrolysis**. There are two major categories of methods employed. The first and older method uses acids as catalysts, while the second uses enzymes called **cellulases**. Feedstock **pretreatment** has been recognized as a necessary upstream process to remove lignin and enhance the porosity of the lignocellulosic materials prior to the enzymatic process (Zhu & Pan, 2010; Kumar et al., 2009).

Cellulases are proteins that have been conventionally divided into three major groups: **endoglucanase**, which attacks low cristallinity regions in the cellulose fibers by endoaction, creating free chain-ends; **exoglucanases** or cellobiohydrolases which hydrolyze the 1, 4 glycocidyl linkages to form cellobiose; and **β-glucosidase** which converts cellooligosaccharides and disaccharide cellobiose into glucose residues. In addition to the three major groups of cellulose enzymes, there are also a number of other enzymes that attack hemicelluloses, such as **glucoronide**, **acetylesterase**, **xylanase**, **β-xylosidase**, **galactomannase** and **glucomannase**. These enzymes work together synergistically to attack cellulose and hemicellulose. Cellulases are produced by various **bacteria and fungi** that can have cellulolytic mechanisms significantly different.

The use of enzymes in the hydrolysis of cellulose is more effective than the use of inorganic catalysts, because enzymes are highly specific and can work at mild process conditions. In spite of these advantages, the use of **enzymes in industrial processes** is still limited by

Corresponding Author

Hydrolysis of Lignocellulosic Biomass:

Current Status of Processes and Technologies and Future Perspectives 97

Fig. 1. Lignocellulosic materials: composition of major compounds (Kumar, 2009)

**(%)**

Coastal bermudagrass 25 35.7 6.4 Corn Cobs 45 35 15 Cotton seed hairs 80-95 5-20 0 Grasses 25-40 35-50 10-30 Hardwoods steam 40-55 24-40 18-25 Leaves 15-20 80-85 0 Newspaper 40-55 25-40 18-30 Nut shells 25-30 25-30 30-40 Paper 85-99 0 0-15

Primary wastewater solids 8-15 NA 24-29 Softwoods stems 45-50 25-35 25-35 Solid cattle manure 1.6-4.7 1.4-3.3 2.7-5.7 Sorted refuse 60 20 20 Swine waste 6.0 28 NA Switchgrass 45 31.4 12.0

Waste papers from chemical pulps 60-70 10-20 5-10

Table 1. Composition of some common sources of biomass (Sun and Cheng, 2002)

Wheat straw 30 50 15

**Hemicellulose (%)**

**Lignin (%)**

**Lignocellulosic materials Cellulose**

several factors: most enzymes are relatively unstable at high temperatures, the costs of enzyme isolation and purification are high and it is quite difficult to recover them from the reaction mixtures. Currently, extensive research is being carried out on cellulases with **improved thermostability**. These enzymes have high specific activity and increased flexibility. For these reasons they could work at low dosages and the higher working temperatures could speed up the hydrolysis reaction time. As consequence, the overall process costs could be reduced. Thermostable enzymes could play an important role in assisting the liquefaction of concentrated biomass suspensions necessary to achieve ethanol concentrations in the range 4-5 wt%.

**The immobilization of enzymes** has also been proposed to remove some limitations in the enzymatic process (Hong et al., 2008). The main advantage is an easier recovery and reuse of the catalysts for more reaction loops. Also, enzyme immobilization frequently results in improved thermostability or resistance to shear inactivation and so, in general, it can help to extend the enzymes lifetime.

This chapter contains an overview of the lignocellulosic hydrolysis process. Several process issues will be deepened: cellulase enzyme systems and hydrolysis mechanisms of cellulose; commercial mixtures; currents limits in the cellulose hydrolysis; innovative bioprocesses and improved biocatalysts.

#### **2. Structure of lignocellulose biomass**

Lignocellulosic biomass is typically nonedible plant material, including dedicated crops of wood and grass, and agro-forest residues. Lignocellulosics are mainly composed of **cellulose, hemicellulose, and lignin**.

Cellulose is a homopolysaccharide composed of β-D-pyranose units, linked by β-1, 4 glycosidic bonds. Cellobiose is the smallest repetitive unit and it is formed by two glucose monomers. The long-chain cellulose polymers are packed together **into microfibrils** by hydrogen and van der Waals bonds. Hemicellulose and lignin cover the microfibils (Fig.1). Hemicellulose is a mixture of polysaccharides, including pentoses, hexoses and uronic acids. Lignin is the most complex natural polymer consisting of a predominant building block of phenylpropane units.  Lignocellulosic materials also contain small amounts of pectin, proteins, extractives (i.e. no- structural sugars, nitrogenous material, chlorophyll and waxes) and ash (Kumar et al., 2009).

The composition of the biomass constituents can vary greatly among various sources (Table 1). Accurate measurements of the biomass constituents, mainly lignin and carbohydrates, are of prime importance because they assist tailored process designs for the maximum recovery of energy and products from the raw materials.

Since 1900, researchers have developed several methods to measure the lignin and carbohydrates content of lignocellulosic biomass. Globally recognized Organizations, such as American Society for Testing and Materials (ASTM), Technical Association of the Pulp and Paper Industry (TAPPI) and National Renewable energy and Laboratory (NREL) have developed methods to determine the chemical composition of biomass, based on modifications of the two main procedures developed by Ritter (Ritter et al., 1932) and by Seaman (Saeman et al., 1954), (Table 2).

several factors: most enzymes are relatively unstable at high temperatures, the costs of enzyme isolation and purification are high and it is quite difficult to recover them from the reaction mixtures. Currently, extensive research is being carried out on cellulases with **improved thermostability**. These enzymes have high specific activity and increased flexibility. For these reasons they could work at low dosages and the higher working temperatures could speed up the hydrolysis reaction time. As consequence, the overall process costs could be reduced. Thermostable enzymes could play an important role in assisting the liquefaction of concentrated biomass suspensions necessary to achieve ethanol

**The immobilization of enzymes** has also been proposed to remove some limitations in the enzymatic process (Hong et al., 2008). The main advantage is an easier recovery and reuse of the catalysts for more reaction loops. Also, enzyme immobilization frequently results in improved thermostability or resistance to shear inactivation and so, in general, it can help to

This chapter contains an overview of the lignocellulosic hydrolysis process. Several process issues will be deepened: cellulase enzyme systems and hydrolysis mechanisms of cellulose; commercial mixtures; currents limits in the cellulose hydrolysis; innovative bioprocesses

Lignocellulosic biomass is typically nonedible plant material, including dedicated crops of wood and grass, and agro-forest residues. Lignocellulosics are mainly composed of

Cellulose is a homopolysaccharide composed of β-D-pyranose units, linked by β-1, 4 glycosidic bonds. Cellobiose is the smallest repetitive unit and it is formed by two glucose monomers. The long-chain cellulose polymers are packed together **into microfibrils** by hydrogen and van der Waals bonds. Hemicellulose and lignin cover the microfibils (Fig.1). Hemicellulose is a mixture of polysaccharides, including pentoses, hexoses and uronic acids. Lignin is the most complex natural polymer consisting of a predominant building block of phenylpropane units.  Lignocellulosic materials also contain small amounts of pectin, proteins, extractives (i.e. no- structural sugars, nitrogenous material, chlorophyll and waxes) and

The composition of the biomass constituents can vary greatly among various sources (Table 1). Accurate measurements of the biomass constituents, mainly lignin and carbohydrates, are of prime importance because they assist tailored process designs for the maximum

Since 1900, researchers have developed several methods to measure the lignin and carbohydrates content of lignocellulosic biomass. Globally recognized Organizations, such as American Society for Testing and Materials (ASTM), Technical Association of the Pulp and Paper Industry (TAPPI) and National Renewable energy and Laboratory (NREL) have developed methods to determine the chemical composition of biomass, based on modifications of the two main procedures developed by Ritter (Ritter et al., 1932) and by

concentrations in the range 4-5 wt%.

extend the enzymes lifetime.

and improved biocatalysts.

ash (Kumar et al., 2009).

**2. Structure of lignocellulose biomass**

recovery of energy and products from the raw materials.

Seaman (Saeman et al., 1954), (Table 2).

**cellulose, hemicellulose, and lignin**.

Fig. 1. Lignocellulosic materials: composition of major compounds (Kumar, 2009)

Table 1. Composition of some common sources of biomass (Sun and Cheng, 2002)

Hydrolysis of Lignocellulosic Biomass:

Current Status of Processes and Technologies and Future Perspectives 99

DOE describing the value added chemicals that can be produced from biomass (Werpy, 2004). Figure 2 displays a general biorefinery scheme for the production of specialty

Besides ethanol, several other products can be obtained following the hydrolysis of the carbohydrates in the lignocellulosic materials. For instance, xylan/xylose contained in hemicelluloses can be thermally transformed into furans (2-furfuraldeyde, hydroxymethil furfural), short chain organic acids (formic, acetic, and propionic acids), and cheto compounds

Fig. 2. Scheme of a lignocellulosic biorefinery. The shape of each step describes the type of

Furfural can be further processed to form some building blocks of innovative polymeric materials (i.e. 2, 5-furandicarboxylic acid). In addition, levulinic acid could be formed by the degradation of hydroxymethil furfural (Demirabas, 2008). Another product prepared either by fermentation or by catalytic hydrogenation of xylose is xylitol (Bozell & Petersen, 2010). Furthermore, through the chemical reduction of glucose it is possible to obtain several products, such as sorbitol (Bozell & Petersen, 2010). The residual lignin can be an intermediate product to be used for the synthesis of phenol, benzene, toluene, xylene, and other aromatics. Similarly to furfural, lignin could react to form some polymeric materials

Ethanol is the most common renewable fuel recognized as a potential alternative to petroleum-derived transportation fuels. It can be produced from lignocellulosic materials in

process used, chemical, biological, and physical (legend) (FitzPatrick et al., 2010)

**4. Production for ethanol from lignocellulosic biomass**

(i.e. polyurethanes) (Demirabas, 2008).

(hydroxy-1-propanone, hydroxy-1-butanone) (Güllü, 2010; Bozell & Petersen, 2010).

polymers, fuel, or composite materials (FitzPatrick et al., 2010).

Table 2. Methods provided by globally recognized organizations for the chemical composition of biomass (Sluiter et al., 2010)

#### **3. Products from lignocellulosic biomass**

Lignocellulosic biomass is a potential source of several bio-based products according to the **biorefinery** approach. Currently, the products made from bioresources represent only a minor fraction of the chemical industry production. However, the interest in the bio-based products has increased because of the rapidly rising barrel costs and an increasing concern about the depletion of the fossil resources in the near future (Hatti-Kaul et al., 2007). The goal of the biorefinery approach is the generation of energy and chemicals from different biomass feedstocks, through the combination of different technologies (FitzPatrick et al., 2010).

The biorefinery scheme involves a multi-step biomass processing. The first step concerns the feedstock pretreatment through physical, biological, and chemical methods. The outputs from this step are **platform (macro) molecules or streams** that can be used for further processing (Cherubini & Ulgiati, 2010). Recently, a detailed report has been published by

**Method Title Method Title Title**

D 1106-96 (2007)

ASTM D1915- 63 (1989) withdrawn, replaced by D5896

AST D5896-96 Standard Test

Table 2. Methods provided by globally recognized organizations for the chemical composition

Lignocellulosic biomass is a potential source of several bio-based products according to the **biorefinery** approach. Currently, the products made from bioresources represent only a minor fraction of the chemical industry production. However, the interest in the bio-based products has increased because of the rapidly rising barrel costs and an increasing concern about the depletion of the fossil resources in the near future (Hatti-Kaul et al., 2007). The goal of the biorefinery approach is the generation of energy and chemicals from different biomass feedstocks, through the combination of different technologies (FitzPatrick et al.,

The biorefinery scheme involves a multi-step biomass processing. The first step concerns the feedstock pretreatment through physical, biological, and chemical methods. The outputs from this step are **platform (macro) molecules or streams** that can be used for further processing (Cherubini & Ulgiati, 2010). Recently, a detailed report has been published by

T 13 os 54; Later T222 om-06

Lignin in Wood (original) Acid- Insoluble Lignin in Wood and Pulp (later)

Composition of Extractive –Free Wood and Wood Pulp by Gas-Liquid Chromatography

T249 cm-00 Carboydrate

of biomass (Sluiter et al., 2010)

2010).

**3. Products from lignocellulosic biomass**

**TAPPI ASTM NREL**

Standard Test Method for Cromatographic Analysis of Chemically Refined Cellulose (1996)

Standard Test Method for Chromatographic Analysis of Chemically Refined Cellulose (1996)

Method for Carbohydrate Distribution of Cellulosic Material

Method for Determination of Acid-Insoluble Residue in Biomass

Carbohydrates in Biomass by High Performance Liquid Chromatography

E1721 Standard Test

E1758 Determination of

Determination of Structural Carbohydrates and Lignin in Biomass

DOE describing the value added chemicals that can be produced from biomass (Werpy, 2004). Figure 2 displays a general biorefinery scheme for the production of specialty polymers, fuel, or composite materials (FitzPatrick et al., 2010).

Besides ethanol, several other products can be obtained following the hydrolysis of the carbohydrates in the lignocellulosic materials. For instance, xylan/xylose contained in hemicelluloses can be thermally transformed into furans (2-furfuraldeyde, hydroxymethil furfural), short chain organic acids (formic, acetic, and propionic acids), and cheto compounds (hydroxy-1-propanone, hydroxy-1-butanone) (Güllü, 2010; Bozell & Petersen, 2010).

Fig. 2. Scheme of a lignocellulosic biorefinery. The shape of each step describes the type of process used, chemical, biological, and physical (legend) (FitzPatrick et al., 2010)

Furfural can be further processed to form some building blocks of innovative polymeric materials (i.e. 2, 5-furandicarboxylic acid). In addition, levulinic acid could be formed by the degradation of hydroxymethil furfural (Demirabas, 2008). Another product prepared either by fermentation or by catalytic hydrogenation of xylose is xylitol (Bozell & Petersen, 2010). Furthermore, through the chemical reduction of glucose it is possible to obtain several products, such as sorbitol (Bozell & Petersen, 2010). The residual lignin can be an intermediate product to be used for the synthesis of phenol, benzene, toluene, xylene, and other aromatics. Similarly to furfural, lignin could react to form some polymeric materials (i.e. polyurethanes) (Demirabas, 2008).

#### **4. Production for ethanol from lignocellulosic biomass**

Ethanol is the most common renewable fuel recognized as a potential alternative to petroleum-derived transportation fuels. It can be produced from lignocellulosic materials in

Hydrolysis of Lignocellulosic Biomass:

(Jones & Semrau, 1984).

200-230°C (Wyman, 1999).

(Galbe & Zacchi, 2002).

hydrolytic enzymes (Alvira et al., 2010).

time to contain the process costs.

1 News on: *Sci Focus Direct on Catalysts*, 2005

**4.2 Pretreatment**

Current Status of Processes and Technologies and Future Perspectives 101

hemicellulose sugars decomposition thus causing the formation of toxic compounds such as furfural and 5-hydroxymethyl-furfural (HMF). These compounds inhibit yeast cells and the subsequent fermentation stage, causing a lower ethanol production rate (Larsson et al., 1999; kootstra et al., 2009). In addition, these compounds lead to reduction of fermentable sugars (Kootstra et al., 2009). In addition, high temperatures increase the equipment corrosion

In 1999, the BC International (BCI) of United States has marketed a technology based on two-step dilute acid hydrolysis: the first hydrolysis stage at mild conditions (170-190°C) to hydrolyze hemicellulose; the second step at more severe conditions to hydrolyze cellulose

In 1991, the Swedish Ethanol Development Foundation developed the CASH process. This is a two-stage dilute acid process that provides the impregnation of biomass with sulphur dioxide followed by a second step in which diluted hydrochloric acid is used. In 1995, this foundation has focused researches on the conversion of softwoods using sulphuric acid

A pretreatment step is necessary for the enzymatic hydrolysis process. It is able to remove the lignin layer and to decristallize cellullose so that the hydrolytic enzymes can easily access the biopolymers.The pretreatment is a critical step in the cellulosic bioethanol technology because it affects the quality and the cost of the carbohydrates containing streams (Balat et al., 2008). Pretreatments methods can be classified into different categories: physical, physiochemical,

On the whole, the final yield of the enzymatic process depends on the combination of several factors: biomass composition, type of pretreatment, dosage and efficiency of the

The use of enzymes in the hydrolysis of cellulose is more advantageous than use of chemicals, because enzymes are highly specific and can work at mild process conditions. Despite these advantages, the use of enzymes in industrial applications is still limited by several factors: the costs of enzymes isolation and purification are high; the specific activity of enzyme is low compared to the corresponding starch degrading enzymes. As consequence, the process yields increase at raising the enzymatic proteins dosage and the hydrolysis time ( up to 4 days) while, on the contrary, decrease at raising the solids loadings. One typical index used to evaluate the performances of the cellulase preparations during the enzymatic hydrolysis is the **conversion rate** to say the obtained glucose concentration per time required to achieve it (g glucose/L/h/). Some authors reported conversion rates of softwoods substrates (5%w/v solids loading) in the range 0.3-1.2 g/L/h (Berlin et al., 2007). In general, compromise conditions are necessary between enzymes dosages and process

In 2001, the cost to produce cellulase enzymes was 3-5\$ per gallon of ethanol (0.8-1.32\$/liter ethanol), (Novozymes and NREL)1. In order to reduce the cost of cellulases for bioethanol production, in 2000 the National Renewable Laboratory (NREL) of USA has started collaborations with Genencor Corporation and Novozymes. In particular, in 2004, Genencor has achieved an estimated cellulase cost in the range \$0.10-0.20 per gallon of ethanol (0.03-

chemical, biological, electrical, or a combination of these (kumar et al., 2009), (Table 3).

various ways characterized by common steps: hydrolysis of cellulose and hemicellulose to monomeric sugars, fermentation and product recovery (fig 3). The main differences lie in the hydrolysis phase, which can be performed by dilute acid, concentrated acid or enzymatically (Galbe & Zacchi, 2002).

#### **4.1 Acid hydrolysis**

The main advantage of the acid hydrolysis is that acids can penetrate lignin without any preliminary pretreatment of biomass, thus breaking down the cellulose and hemicellulose polymers to form individual sugar molecules. Several types of acids, concentrated or diluted, can be used, such as sulphurous, sulphuric, hydrocloric, hydrofluoric, phosphoric, nitric and formic acid (Galbe & Zacchi, 2002). Sulphuric and hydrochloric acids are the most commonly used catalysts for hydrolysis of lignocellulosic biomass (Lenihan et al., 2010).

The acid concentration used in the **concentrated acid hydrolysis** process is in the range of 10-30%. The process occurs at low temperatures, producing high hydrolysis yields of cellulose (i.e. 90% of theoretical glucose yield) (Iranmahboob et al., 2002).

Fig. 3. Process for production ethanol from lignocellulosic biomass. The circle in the scheme indicates two alternative process routes: simultaneous hydrolysis and fermentation (SSF); separate hydrolysis and fermentation (SHF).

However, this process requires large amounts of acids causing corrosion problems to the equipments. The main advantage of the **dilute hydrolysis process** is the low amount of acid required (2-5%). However this process is carried out at high temperatures to achieve acceptable rates of cellulose conversion. The high temperature increases the rates of hemicellulose sugars decomposition thus causing the formation of toxic compounds such as furfural and 5-hydroxymethyl-furfural (HMF). These compounds inhibit yeast cells and the subsequent fermentation stage, causing a lower ethanol production rate (Larsson et al., 1999; kootstra et al., 2009). In addition, these compounds lead to reduction of fermentable sugars (Kootstra et al., 2009). In addition, high temperatures increase the equipment corrosion (Jones & Semrau, 1984).

In 1999, the BC International (BCI) of United States has marketed a technology based on two-step dilute acid hydrolysis: the first hydrolysis stage at mild conditions (170-190°C) to hydrolyze hemicellulose; the second step at more severe conditions to hydrolyze cellulose 200-230°C (Wyman, 1999).

In 1991, the Swedish Ethanol Development Foundation developed the CASH process. This is a two-stage dilute acid process that provides the impregnation of biomass with sulphur dioxide followed by a second step in which diluted hydrochloric acid is used. In 1995, this foundation has focused researches on the conversion of softwoods using sulphuric acid (Galbe & Zacchi, 2002).

#### **4.2 Pretreatment**

100 Bioethanol

various ways characterized by common steps: hydrolysis of cellulose and hemicellulose to monomeric sugars, fermentation and product recovery (fig 3). The main differences lie in the hydrolysis phase, which can be performed by dilute acid, concentrated acid or

The main advantage of the acid hydrolysis is that acids can penetrate lignin without any preliminary pretreatment of biomass, thus breaking down the cellulose and hemicellulose polymers to form individual sugar molecules. Several types of acids, concentrated or diluted, can be used, such as sulphurous, sulphuric, hydrocloric, hydrofluoric, phosphoric, nitric and formic acid (Galbe & Zacchi, 2002). Sulphuric and hydrochloric acids are the most commonly used catalysts for hydrolysis of lignocellulosic biomass (Lenihan et al., 2010). The acid concentration used in the **concentrated acid hydrolysis** process is in the range of 10-30%. The process occurs at low temperatures, producing high hydrolysis yields of

Fig. 3. Process for production ethanol from lignocellulosic biomass. The circle in the scheme indicates two alternative process routes: simultaneous hydrolysis and fermentation (SSF);

However, this process requires large amounts of acids causing corrosion problems to the equipments. The main advantage of the **dilute hydrolysis process** is the low amount of acid required (2-5%). However this process is carried out at high temperatures to achieve acceptable rates of cellulose conversion. The high temperature increases the rates of

separate hydrolysis and fermentation (SHF).

cellulose (i.e. 90% of theoretical glucose yield) (Iranmahboob et al., 2002).

enzymatically (Galbe & Zacchi, 2002).

**4.1 Acid hydrolysis**

A pretreatment step is necessary for the enzymatic hydrolysis process. It is able to remove the lignin layer and to decristallize cellullose so that the hydrolytic enzymes can easily access the biopolymers.The pretreatment is a critical step in the cellulosic bioethanol technology because it affects the quality and the cost of the carbohydrates containing streams (Balat et al., 2008). Pretreatments methods can be classified into different categories: physical, physiochemical, chemical, biological, electrical, or a combination of these (kumar et al., 2009), (Table 3).

On the whole, the final yield of the enzymatic process depends on the combination of several factors: biomass composition, type of pretreatment, dosage and efficiency of the hydrolytic enzymes (Alvira et al., 2010).

The use of enzymes in the hydrolysis of cellulose is more advantageous than use of chemicals, because enzymes are highly specific and can work at mild process conditions. Despite these advantages, the use of enzymes in industrial applications is still limited by several factors: the costs of enzymes isolation and purification are high; the specific activity of enzyme is low compared to the corresponding starch degrading enzymes. As consequence, the process yields increase at raising the enzymatic proteins dosage and the hydrolysis time ( up to 4 days) while, on the contrary, decrease at raising the solids loadings. One typical index used to evaluate the performances of the cellulase preparations during the enzymatic hydrolysis is the **conversion rate** to say the obtained glucose concentration per time required to achieve it (g glucose/L/h/). Some authors reported conversion rates of softwoods substrates (5%w/v solids loading) in the range 0.3-1.2 g/L/h (Berlin et al., 2007). In general, compromise conditions are necessary between enzymes dosages and process time to contain the process costs.

In 2001, the cost to produce cellulase enzymes was 3-5\$ per gallon of ethanol (0.8-1.32\$/liter ethanol), (Novozymes and NREL)1. In order to reduce the cost of cellulases for bioethanol production, in 2000 the National Renewable Laboratory (NREL) of USA has started collaborations with Genencor Corporation and Novozymes. In particular, in 2004, Genencor has achieved an estimated cellulase cost in the range \$0.10-0.20 per gallon of ethanol (0.03-

<sup>1</sup> News on: *Sci Focus Direct on Catalysts*, 2005

Hydrolysis of Lignocellulosic Biomass:

Alkaline hydrolysis

**Biological** Several fungi

electrical field in the range of 5-20 kV/cm,

**5. Enzymatic hydrolysis: Cellulases**

**Electrical** Pulsed

2008).

& Ghisolfi Group.

Current Status of Processes and Technologies and Future Perspectives 103

**Advantages Disadvantages**

Residual salts in biomass

High costs due to the solvents recovery

Slow hydrolysis rates

Process needs more research

Removes hemicelluloses and lignin; increases accessible surface area

Hydrolyzes lignin and hemicelluloses

Degrades lignin and hemicelluloses; low energy requirements

Ambient conditions; disrupts plant cells; simple equipment

**conditions**

Low temperature; Long time high. Concentration of the base; For soybean straw: ammonia liquor (10%) for 24 h at room temperature

without addition of catalysts (oxalic, salicylic, acetylsalicylic acid)

(brown-, white- and soft-rot fungi

~2000 pulses of 8 kV/cm

0.05\$/liter ethanol) in NREL´s cost model (Genencor, 2004)2. Similarly, collaboration between Novozymes and NREL has yielded a cost reduction in the range \$0.10-0.18 per gallon of ethanol (0.03-0.047\$/liter ethanol), a 30-fold reduction since 2001 (Mathew et al.,

Unlike the acid hydrolysis, the enzymatic hydrolysis, still has not reached the industrial scale. Only few plants are available worldwide to investigate the process (pretreatment and bioconversion) at demo scale. More recently, the steam explosion pretreatment, investigated for several years in Italy at the ENEA research Center of Trisaia (De Bari et al., 2002, 2007), is now going to be developed at industrial scale thanks to investments from the Italian Mossi

**5.1 Cellulolytic capability of organisms: Difference in the cellulose-degrading strategy**  Different strategies for the cellulose degradation are used by the cellulase-producing microorganisms: aerobic bacteria and fungi secrete soluble extracellular enzymes known as *non complexed cellulase system*; anaerobic cellulolytic microorganisms produce *complexed cellulase systems*, called *cellulosomes* (Sun et al., 2002). A third strategy was proposed to explain the cellulose-degrading action of two recently discovered bacteria: the aerobic

*Cytophaga hutchinsonii* and the anaerobic *Fibrobacter succinogenes* (Ilmén et al., 1997).

2 Genencor, relations, 21 October 2004, avaible from: http:/genencor.com/cms/connect/

genencor/media\_relations/news/archive/2004/gen\_211004\_en.htm

Table 3. Methods for biomass lignocellulosic pretreatment (Kumar et al., 2009)

 **Operating**

Organosolv 150-200 °C with or

**Advantages Disadvantages**

Power consumption higher than inherent biomass energy

Destruction of a portion of the xylan fraction; incomplete distruption of the lignin-carboydrate matrix; generation of inhibitory compounds; less effective for softwoods

Do not modify lignin neither hydrolyzes hemicellulose;

Do not modify lignin neither hydrolyzes hemicellulose;

It is not suitable for biomass with high lignin content (such as woods and nut shells) Does not modify lignin neither hydrolyze hemicelluloses

Expensive for the ozone required;

High cost of oxygen and alkaline catalyst

Equipment corrosion; formation of toxic substances

Reduces cellulose critallinity

Causes hemicellulose auto hydrolysis and lignin transformation; cost-effective for hardwoods and agricultural residues

Increases accessible surface area, removes lignin and hemicellulose;

Increases accessible surface area, removes lignin and hemicellulose;

Do not produce inhibitors for downstream processes. Increases accessible surface area, does not cause formation of inhibitory compounds

content; does not produce toxic residues

Efficient removal of lignin; low formation of inhibitors; low energy demand

Hydrolyzes hemicellulose to xylose and other sugar; alters lignin structure

**conditions**

Room temperature Energy input < 30Kw per ton biomass

160-260°C (0. 69- 4.83MPa) for 5-15 min

90°C for 30 min.1- 2kg ammonia /kg dry biomass

150-170°C for 14 min Fluid velocity 1cm/min

5.62 Mpa 160 bar for 90 min at 50 °C under supercritical carbon dioxide

Ozonolysis Room temperature Reduce lignin

min

Type I: T>160°, continuous-flow process for low solid loading 5-10%,)- Type II: T<160°C, batch process for high solid loadings (10-40%)

CO2 explosion 4kg CO2/kg fiber at

Wet oxidation 148-200°C for 30

 **Operating**

**Physical** Chipping

**Chemical** Acid

hydrolysis: dilute-acid pretreatment

**Physiochemical**  Grinding Milling

Steam pretreatment

AFEX (Ammonia fiber explosion method)

ARP (Ammonia recycle percolation method)

Table 3. Methods for biomass lignocellulosic pretreatment (Kumar et al., 2009)

0.05\$/liter ethanol) in NREL´s cost model (Genencor, 2004)2. Similarly, collaboration between Novozymes and NREL has yielded a cost reduction in the range \$0.10-0.18 per gallon of ethanol (0.03-0.047\$/liter ethanol), a 30-fold reduction since 2001 (Mathew et al., 2008).

Unlike the acid hydrolysis, the enzymatic hydrolysis, still has not reached the industrial scale. Only few plants are available worldwide to investigate the process (pretreatment and bioconversion) at demo scale. More recently, the steam explosion pretreatment, investigated for several years in Italy at the ENEA research Center of Trisaia (De Bari et al., 2002, 2007), is now going to be developed at industrial scale thanks to investments from the Italian Mossi & Ghisolfi Group.

### **5. Enzymatic hydrolysis: Cellulases**

#### **5.1 Cellulolytic capability of organisms: Difference in the cellulose-degrading strategy**

Different strategies for the cellulose degradation are used by the cellulase-producing microorganisms: aerobic bacteria and fungi secrete soluble extracellular enzymes known as *non complexed cellulase system*; anaerobic cellulolytic microorganisms produce *complexed cellulase systems*, called *cellulosomes* (Sun et al., 2002). A third strategy was proposed to explain the cellulose-degrading action of two recently discovered bacteria: the aerobic *Cytophaga hutchinsonii* and the anaerobic *Fibrobacter succinogenes* (Ilmén et al., 1997).

 2 Genencor, relations, 21 October 2004, avaible from: http:/genencor.com/cms/connect/ genencor/media\_relations/news/archive/2004/gen\_211004\_en.htm

Hydrolysis of Lignocellulosic Biomass:

Current Status of Processes and Technologies and Future Perspectives 105

 *Third cellulose-degrading strategy*. The third strategy was recently proposed to explain the cellulose-degrading behavior of two recently sequenced bacteria: *Cytophaga hutchinsonii*  and *Fibrobacter succinogenes* (Ilmén, 1997). *C. hutchinsonii* is an abundant aerobic cellulolytic soil bacterium (Fägerstam & Petterson, 1984), while *F. succinogenes* is an anaerobic rumen bacterium which was isolated by the Rockville, (Maryland), and San

Diego (California) Institute of Genomic Research (TIGR) (Mansfield et al., 1998). In the aerobic *C. hutchinsonii* no genes were found to code for CBM and in the anaerobic *F*. *succinogenes* no genes were identified to encode dockerin and scaffoldin. Thus, a third cellulose degrading mechanism was proposed. It includes the binding of individual cellulose molecules by outer membrane proteins of the microrganisms followed by the transport into the periplasmic space where they are degraded by endoglucanases

Most cellulase enzymes are relatively unstable at high temperatures. The maximum activity for most fungal cellulases and β-glucosidase occurs at 50±5°C and a pH 4.5- 5 (Taherzadeh & Karimi, 2007; Galbe & Zacchi, 2002). Usually, they lose about 60% of their activity in the temperature range 50–60 °C and almost completely lose activity at 80°C (Gautam et al., 2010). However, the enzymes activity depends on the hydrolysis duration and on the source of the enzymes (Tengborg et al., 2001). In general, cellulases are quite difficult to use for

As mentioned before, the enzyme production costs mainly depend on the productivity of the enzymes-producing microbial strain. Filamentous fungi are the major source of cellulases and mutant strains of *Trichoderma* (*T. viride, T. reesei, T. longibrachiatum*) have long

that can be included in the cellulosome are lichenases.

Fig. 4. Schematic representation of a cellulosoma

**5.2 Characteristics of the commercial hydrolytic enzymes**

(Ilmén, 1997).

prolonged operations.

The concept of cellulosome was firstly discovered in the thermophilic cellulolytic and anaerobic bacterium, *Clostridium thermocellum* (Wyman, 1996). It consists of a large number of proteins, including several cellulases and hemicellulases. Other enzymes

 *Non-complexed cellulase system*. One of the most fully investigated non*-complexed cellulase system* is the *Trichoderma reesei* model. *T. reesei* (teleomorph *Hypocrea jecorina*) is a saprobic fungus, known as an efficient producer of extracellular enzymes (Bayer et al., 1998). Its non-complexed cellulase system includes two cellobiohydrolases, at least seven endoglucanases, and several β-glucosidases. However, in *T. reesei* cellulases, the amount of ß-glucosidase is lower than that needed for the efficient hydrolysis of cellulose into glucose. As a result, the major product of hydrolysis is cellobiose. This is a dimer of glucose with strong inhibition toward endo- and exoglucanases so that the accumulation of cellobiose significantly slows down the hydrolysis process (Gilkes et al., 1991). By adding ß-glucosidase to cellulases from either external sources, or by using co-culture systems, the inhibitory effect of cellobiose can be significantly reduced (Ting et al., 2009).

It has been observed that the mechanism of cellulose enzymatic hydrolysis by *T.reesei* involves three simultaneous processes (Ting et al., 2009):

Cellulosomes are protuberances produced on the cell wall of the cellulolytic bacteria grown on cellulosic materials. These protuberances are stable enzyme complexes tightly bound to the bacteria cell wall but flexible enough to bind strongly to cellulose (Lentig & Warmoeskerken, 2001). A cellulosome contains two types of subunits: *non-catalytic subunits*, called *scaffoldins*, and *enzymatic subunits*. The scaffoldin is a functional unit of cellusome, which contain multiple copies of *cohesins* that interact selectively with domains of the *enzymatic subunits*, CBD (cellulose binding domains) and CBM (carbohydrates binding modules). These have complementary cohesins, called dockerins, which are specific for each bacterial species (Fig. 4) (Gilligan & Reese, 1954; Lynd et al., 2002; Arai et al., 2006;).

For the bacterial cell, the biosynthesis of a cellulosome enables a specific adhesion to the substrate of interest without competition with other microorganisms. The cellulosome allows several advantages: (1) synergism of the cellulases; (2) absence of unspecific adsorption (McCarter & Whiters, 1994; Zhang & Lynd, 2004). Thanks to its intrinsic Lego-like architecture, cellulosomes may provide great potential in the biofuel industry.

 *Non-complexed cellulase system*. One of the most fully investigated non*-complexed cellulase system* is the *Trichoderma reesei* model. *T. reesei* (teleomorph *Hypocrea jecorina*) is a saprobic fungus, known as an efficient producer of extracellular enzymes (Bayer et al., 1998). Its non-complexed cellulase system includes two cellobiohydrolases, at least seven endoglucanases, and several β-glucosidases. However, in *T. reesei* cellulases, the amount of ß-glucosidase is lower than that needed for the efficient hydrolysis of cellulose into glucose. As a result, the major product of hydrolysis is cellobiose. This is a dimer of glucose with strong inhibition toward endo- and exoglucanases so that the accumulation of cellobiose significantly slows down the hydrolysis process (Gilkes et al., 1991). By adding ß-glucosidase to cellulases from either external sources, or by using co-culture systems, the inhibitory effect of cellobiose can be significantly reduced (Ting

It has been observed that the mechanism of cellulose enzymatic hydrolysis by *T.reesei*

1. Chemical and physical changes in the cellulose solid phase. The chemical stage includes changes in the degree of polymerization, while the physical changes regard all the modifications in the accessible surface area. The enzymes specific

2. Primary hydrolysis. This process is slow and involves the release of soluble intermediates from the cellulose surface. The activity involved in this step is the

3. Secondary hydrolysis. This process involves the further hydrolysis of the soluble fractions to lower molecular weight intermediates, and ultimately to glucose. This step is much faster than the primary hydrolysis and *β-glucosidases* play a role for the

 *Complexed cellulase system.* Cellulosomes are produced mainly by anaerobic bacteria, but their presence have also been described in a few anaerobic fungi from species such as *Neocallimastix*, *Piromyces*, and *Orpinomyces* (Tatsumi et al., 2006; Watanabe & Tokuda, 2010). In the domain Bacteria, organisms possessing cellulosomes are only found in the phylum *Firmicutes*, class *Clostridia*, order *Clostridiales* and in the *Lachnospiraceae* and *Clostridiaceae* families. In this latter family, bacteria with cellulosomes are found in various clusters of the genus Clostridium (McCarter & Whiters, 1994; Wilson, 2008). Cellulosomes are protuberances produced on the cell wall of the cellulolytic bacteria grown on cellulosic materials. These protuberances are stable enzyme complexes tightly bound to the bacteria cell wall but flexible enough to bind strongly to cellulose (Lentig & Warmoeskerken, 2001). A cellulosome contains two types of subunits: *non-catalytic subunits*, called *scaffoldins*, and *enzymatic subunits*. The scaffoldin is a functional unit of cellusome, which contain multiple copies of *cohesins* that interact selectively with domains of the *enzymatic subunits*, CBD (cellulose binding domains) and CBM (carbohydrates binding modules). These have complementary cohesins, called dockerins, which are specific for each bacterial species (Fig. 4) (Gilligan & Reese, 1954;

For the bacterial cell, the biosynthesis of a cellulosome enables a specific adhesion to the substrate of interest without competition with other microorganisms. The cellulosome allows several advantages: (1) synergism of the cellulases; (2) absence of unspecific adsorption (McCarter & Whiters, 1994; Zhang & Lynd, 2004). Thanks to its intrinsic Lego-like architecture, cellulosomes may provide great potential in the biofuel industry.

involves three simultaneous processes (Ting et al., 2009):

function involved in this step is the *endoglucanase*.

et al., 2009).

*cellobiohydrolase.*

secondary hydrolysis.

Lynd et al., 2002; Arai et al., 2006;).

The concept of cellulosome was firstly discovered in the thermophilic cellulolytic and anaerobic bacterium, *Clostridium thermocellum* (Wyman, 1996). It consists of a large number of proteins, including several cellulases and hemicellulases. Other enzymes that can be included in the cellulosome are lichenases.

 *Third cellulose-degrading strategy*. The third strategy was recently proposed to explain the cellulose-degrading behavior of two recently sequenced bacteria: *Cytophaga hutchinsonii*  and *Fibrobacter succinogenes* (Ilmén, 1997). *C. hutchinsonii* is an abundant aerobic cellulolytic soil bacterium (Fägerstam & Petterson, 1984), while *F. succinogenes* is an anaerobic rumen bacterium which was isolated by the Rockville, (Maryland), and San

Fig. 4. Schematic representation of a cellulosoma

Diego (California) Institute of Genomic Research (TIGR) (Mansfield et al., 1998). In the aerobic *C. hutchinsonii* no genes were found to code for CBM and in the anaerobic *F*. *succinogenes* no genes were identified to encode dockerin and scaffoldin. Thus, a third cellulose degrading mechanism was proposed. It includes the binding of individual cellulose molecules by outer membrane proteins of the microrganisms followed by the transport into the periplasmic space where they are degraded by endoglucanases (Ilmén, 1997).

#### **5.2 Characteristics of the commercial hydrolytic enzymes**

Most cellulase enzymes are relatively unstable at high temperatures. The maximum activity for most fungal cellulases and β-glucosidase occurs at 50±5°C and a pH 4.5- 5 (Taherzadeh & Karimi, 2007; Galbe & Zacchi, 2002). Usually, they lose about 60% of their activity in the temperature range 50–60 °C and almost completely lose activity at 80°C (Gautam et al., 2010). However, the enzymes activity depends on the hydrolysis duration and on the source of the enzymes (Tengborg et al., 2001). In general, cellulases are quite difficult to use for prolonged operations.

As mentioned before, the enzyme production costs mainly depend on the productivity of the enzymes-producing microbial strain. Filamentous fungi are the major source of cellulases and mutant strains of *Trichoderma* (*T. viride, T. reesei, T. longibrachiatum*) have long

Hydrolysis of Lignocellulosic Biomass:

**FPU (U/ml)a** **Cellobiase (U/ml)b**

Bio-feed beta L <5 12 8 *T. longibrachiatum*

Cellulase 2000L 10 nd 7 *T. longibrachiatum*

Cellulyve 50L 24 nd 34 *T. longibrachiatum*

Energex L <5 19 28 *T. longibrachiatum*

GC220 116 215 64 *T. longibrachiatum*

GC440 <5 70 29 *T. longibrachiatum*

GC880 <5 86 43 *T. longibrachiatum*

Rohament CL 51 28 44 *T. longibrachiatum T.*

Spezyme CP 49 nd 41 *T. longibrachiatum*

Ultraflo L <5 20 18 *T. longibrachiatum*

Viscozyme L <5 23 27 *T. longibrachiatum*

Viscostar 150L 33 111 40 *T. longibrachiatum T.*

during the hydrolysis reaction of filter paper Whatman No.1

**activity(U/ml)a**

Novozymes 188 <5 1,116 57 *A.niger* Novozymes

A) One FPU (filter paper unit) is the amount of enzyme that forms 1 mol of reducing sugars/min

B) One CBU (cellobiase unit) corresponds to the amount of enzyme which forms 2 mol of glucose/min

Biocellulase A 32 5 55 *A. niger* Quest Intl.

Cellulase AP 30 K 60 4.5 60 *A. niger* Amano

Table 5. Commercial cellulases able to work at temperature ranging from 50 to 60°C.

**Commercial mixture**

Cellubrix (Celluclast)

from cellobiose

Table 4. Commercial cellulases

**Commercial mixture Β-glucosidase**

Current Status of Processes and Technologies and Future Perspectives 107

**Proteins**

56 136 43 *T. longibrachiatum*

**(U/ml)c Source Supplier**

Novozymes (Bagsvaerd, Denmark)

Novozymes

Rodhia –Danisco (Vinay, France)

Lyven (Colombelles France)

Novozymes

Genencor-Danisco (Rochester, USA)

Genencor

Genencor

Rhom-AB Enzymes (Rajamäki, Finland)

Genencor

Novozymes

Novozymes

Dyadic (Jupiter, Usa)

(Sarasota, Fl)

Enzyme Inc.

*T. reesei*

*A. niger*

*T. reesei*

*T. reesei*

*T. reesei*

*T. reesei*

*T. reesei*

*T. reesei*

*reesei*

*T. reesei*

*T. reesei*

*T. reesei*

*reesei*

**pH Temperature (°C) Source Supplier**

been considered to be the most productive (Gusakov et al., 2007; Galbe & Zacchi, 2002). Preparations of cellulases from a single organism may not be highly efficient for the hydrolysis of different feedstocks. For example, *Thrichoderma reesei* produces endoglucanases and exoglucanases in large quantities, but its β-glucosidase activity is low, resulting in an inefficient biomass hydrolysis. For this reason, the goal of the enzymes producing companies has been to form cellulases cocktails by enzymes assembly (multienzyme mixtures) or to construct engineered microrganisms to express the desired mixtures (Mathew et al., 2008). Enzyme mixtures often derive from the co-fermentation of several micro-organisms (Ahamed & Vermette, 2008; Kabel et al., 2005; Berlin et al., 2007), (Table 4). All the commercial cellulases listed in table 4 have an optimal condition at 50°C and pH of 4.0-5.0. More recently, some enzymes producers have marked new mixtures able to work in a higher temperature ranging from 50 to 60°C (Table5).

In 2010, new enzymes were produced by two leading companies, Novozymes and Genencor, supported by the USA Department of Energy (DOE). Genencor has launched four new blends: **Accelerase®1500, Accelerase®XP, Accelerase®XC and Accelerase®BG**. Accelerase®1500 is a cellulases complex (exoglucanase, endoglucanase, hemi-cellulase and β-glucosidase) produced from a genetically modified strain of *T. reesei.* All the other Accelerase are accessory enzymes complexes: Accelerase®XP enhances both xylan and glucan conversion; Accelerase®XC contains hemicellulose and cellulase activities; Accelerase® BG is a β-glucosidase enzyme. In February 2010, Genencor has developed an enzyme complex known as **Accellerase®Duet** which is produced with a genetically modified strain of *T. reesei* and that contains not only exoglucanase, endoglucanase, βglucosidase, but includes also xylanase. This product is capable of hydrolyzing lignocellulosic biomass into fermentable monosaccharides such as glucose and xylose (Genencos, 2010)3. Similarly, Novozymes has produced and commercialized two new enzymatic mixtures: **cellic Ctec**, and **cellic Htec**. **Cellic CTec** is used in combination with Cellic HTec and this mixture is capable to work with a wide variety of pretreated feedstocks, such as sugarcane bagasse, corn cob, corn fiber, and wood pulp, for the conversion of the carbohydrates in these materials into simple sugars (Novozyme, 2010)4.

In order to meet the future challenges, innovative bioprocesses for the production of new generation of enzymes are needed. As already described, conventional cellulases work within a range of temperature around 50°C and they are typically inactivated at temperatures above 60-70 °C due to disorganization of their three dimensional structures followed by an irreversible denaturation (Viikari et al., 2007). Some opportunities of process improvement derive from the use of thermostable enzymes.

#### **5.3 Enzymes for the cellulose liquefaction: Thermophilic enzymes**

The thermophilic microrganisms can be grouped in thermophiles (growth up to 60 °C), extreme thermophiles (65-80 °C) and hyperthermophiles (85-110 °C). The unique stability of the enzymes produced by these microrganisms at elevated temperatures, extreme pH and high pressure (up to 1000 bar) makes them a valuable resource for the industrial

<sup>3</sup> Genencor, products, 14 January 2010, avaible from: http:// www.genencor.com/ wps/ wcm/ connect/ genencor/ genencor/ products and services/ business development/ biorefineries/ products/ accellerase product line en.htm 4 Novozyme, brochure, 29 January 2010, Viable from: http:// www.bioenergy. novozymes.com/ files/

documents/ Final%20Cellic%20Product%20Brochure\_ 29Jan2010.pdf

been considered to be the most productive (Gusakov et al., 2007; Galbe & Zacchi, 2002). Preparations of cellulases from a single organism may not be highly efficient for the hydrolysis of different feedstocks. For example, *Thrichoderma reesei* produces endoglucanases and exoglucanases in large quantities, but its β-glucosidase activity is low, resulting in an inefficient biomass hydrolysis. For this reason, the goal of the enzymes producing companies has been to form cellulases cocktails by enzymes assembly (multienzyme mixtures) or to construct engineered microrganisms to express the desired mixtures (Mathew et al., 2008). Enzyme mixtures often derive from the co-fermentation of several micro-organisms (Ahamed & Vermette, 2008; Kabel et al., 2005; Berlin et al., 2007), (Table 4). All the commercial cellulases listed in table 4 have an optimal condition at 50°C and pH of 4.0-5.0. More recently, some enzymes producers have marked new mixtures able

In 2010, new enzymes were produced by two leading companies, Novozymes and Genencor, supported by the USA Department of Energy (DOE). Genencor has launched four new blends: **Accelerase®1500, Accelerase®XP, Accelerase®XC and Accelerase®BG**. Accelerase®1500 is a cellulases complex (exoglucanase, endoglucanase, hemi-cellulase and β-glucosidase) produced from a genetically modified strain of *T. reesei.* All the other Accelerase are accessory enzymes complexes: Accelerase®XP enhances both xylan and glucan conversion; Accelerase®XC contains hemicellulose and cellulase activities; Accelerase® BG is a β-glucosidase enzyme. In February 2010, Genencor has developed an enzyme complex known as **Accellerase®Duet** which is produced with a genetically modified strain of *T. reesei* and that contains not only exoglucanase, endoglucanase, βglucosidase, but includes also xylanase. This product is capable of hydrolyzing lignocellulosic biomass into fermentable monosaccharides such as glucose and xylose (Genencos, 2010)3. Similarly, Novozymes has produced and commercialized two new enzymatic mixtures: **cellic Ctec**, and **cellic Htec**. **Cellic CTec** is used in combination with Cellic HTec and this mixture is capable to work with a wide variety of pretreated feedstocks, such as sugarcane bagasse, corn cob, corn fiber, and wood pulp, for the conversion of the

In order to meet the future challenges, innovative bioprocesses for the production of new generation of enzymes are needed. As already described, conventional cellulases work within a range of temperature around 50°C and they are typically inactivated at temperatures above 60-70 °C due to disorganization of their three dimensional structures followed by an irreversible denaturation (Viikari et al., 2007). Some opportunities of process

The thermophilic microrganisms can be grouped in thermophiles (growth up to 60 °C), extreme thermophiles (65-80 °C) and hyperthermophiles (85-110 °C). The unique stability of the enzymes produced by these microrganisms at elevated temperatures, extreme pH and

to work in a higher temperature ranging from 50 to 60°C (Table5).

carbohydrates in these materials into simple sugars (Novozyme, 2010)4.

**5.3 Enzymes for the cellulose liquefaction: Thermophilic enzymes**

high pressure (up to 1000 bar) makes them a valuable resource for the industrial

3 Genencor, products, 14 January 2010, avaible from: http:// www.genencor.com/ wps/ wcm/ connect/ genencor/ genencor/ products and services/ business development/ biorefineries/

products/ accellerase product line en.htm 4 Novozyme, brochure, 29 January 2010, Viable from: http:// www.bioenergy. novozymes.com/ files/

improvement derive from the use of thermostable enzymes.

documents/ Final%20Cellic%20Product%20Brochure\_ 29Jan2010.pdf

A) One FPU (filter paper unit) is the amount of enzyme that forms 1 mol of reducing sugars/min during the hydrolysis reaction of filter paper Whatman No.1

B) One CBU (cellobiase unit) corresponds to the amount of enzyme which forms 2 mol of glucose/min from cellobiose

Table 4. Commercial cellulases

Table 5. Commercial cellulases able to work at temperature ranging from 50 to 60°C.

Hydrolysis of Lignocellulosic Biomass:

β-glucosidase *Pyrococcus*

Endoglucanase *Pyrococcus*

Endoglucanase *Acidothermus*

**Enzymes Organism pH**

*furiousus*

*Pyrococcus horikoshi*

*furiousus*

*Pyrococcus horikoshi*

*cellulolyticus*

*Anaerocellum thermophilum*

*Clostridium stercorarium*

*Clostridium thermocellum*

*Clostridium thermocellum*

*Rhodothermus marinus*

*Thermotoga marittima*

*Thermotoga neapolitana*

*stercorarium*

**Enzymes Organism pH**

Exoglucanase *Clostridium*

**Enzymes Organism pH**

Current Status of Processes and Technologies and Future Perspectives 109

**Archea**

**Bacteria**

**Fungal**

**T optimum (°C)**

**optimum**

**T optimum (°C)**

**optimum**

**T optimum (°C)**

**Stability (half life)** *Refs.*

**Stability (half life) Refs.**

*1994*

*1998*

*al., 2004*

*al., 2004*

*1996*

*1998*

*Bronnenmeier K et al., 1991*

*Bergquist et al., 2004*

*Romaniec et al. 1992*

*Bergquist et al., 2004*

> *K, et al., 1995*

> > *1995*

*K et al., 1990*

5 102 13h at 110°C *Ma & Adams,*

6 100 15h at 90°C *Rahman et al.,*

6 100 40h at 95°C *Bergquist et*

6-6.5 100 19h at 100°C *Bergquist et*

5.0 83 Inactivated at 110°C *Sakon J. et al.*

5-6 95-100 40min at 100°C *Zverliv et al.,*

days

remained after 50h at 60°C

remained after 48h at 60°C

remained after 3.5h at 100°C, 80% after 16h at 90°C

6.0-7.5 95 2h at 95°C *Bronnenmeier*

6.0 95 ˃240min at 100°C *Bok JD et al.,*

5-6 75 3 days at 70°C *Bronnenmeier*

**Stability (half life) Refs.**

6-6.5 90 Stable for several

6.6 70 33% of activity

7.0 70 50% of activity

7.0 95 50% of activity

**optimum**

bioprocesses that run at harsh conditions (Demain et al., 2005). Of special interest is the thermoactivity and thermostability of these enzymes in the presence of high concentrations of organic solvents, detergents and alcohols. On the whole, thermophilic enzymes have an increased resistance to many denaturing conditions such as the use of detergents which can be often the unique efficient mean to obviate the irreversible adsorption of cellulases on the substrates. Furthermore, the utilization of high operation temperatures, which cause a decrease in viscosity and an increase in the diffusion coefficients of substrates, have a significant influence on the cellulose solubilization. It is worth noting that, differently from the mesophilic enzymes, most thermophilic cellulases did not show inhibition at high level of reaction products (e.g. cellobiose and glucose). As consequence, higher reaction rates and higher process yields are expected (Bergquist et al., 2004). The high process temperature also reduces any contamination of the fermentation medium.

Several cellulose degrading enzymes from various thermophilic organisms have been investigated. These include cellulases mainly isolated from anaerobic bacteria such as *Anaerocellum thermophilum* (Zverlov et al., 1998), *Clostridium thermocellum* (Romaniec et al., 1992), *Clostridium stercorarium* (Bronnenmeier et al., 1991; Bronnenmeier & Staudenbauer, 1990) and *Caldocellum saccharolyticum* (Te'o V et l., 1995), *Pyrococcus furiosus* (Ma & Adams, 1994), *Pyrococcus horikoshi* (Rahman et al.,1998), *Rhodothermus* strains (Hreggvidsson et al., 1996), *Thermotoga sp*., (Ruttersmith et al., 1991), *Thermotoga marittima* (Bronnenmeier et al., 1995), *Thermotoga neapolitana* (Bok et al., 1998).

Xylanase have been detected in *Acidothermus cellulolyticus* in different *Thermus*, *Bacillus*, *Geobacillus*, *Alicyclobacillus* and *Sulfolobales* species (Sakon et al., 1996).

Although many cellulolytic anaerobic bacteria such as *Clostridium thermocellum* produce cellulases with high specific activity, they do not produce high enzymes quantities. Since the anaerobes show limited growth, most researches on thermostable cellulases production have been addressed to aerobic species. Several mesophilic or moderately thermophilic fungal strains are also known to produce enzymes stable and active at high temperatures. These enzymes are produced from species such as *Chaetomium thermophila* (Venturi et al., 2002), *Talaromyces emersonii* (Grassick et al., 2004), *Thermoascus aurantiacus* (Parry et al., 2002). They may be stable at temperatures around 70 °C for prolonged periods. Table 6 summarizes some of thermostable enzymes isolated from Archea, Bacteria and Fungi.

During the last decade several efforts have been devoted to develop different mixtures of selected thermostable enzymes. In 2007, mixtures of thermostable enzymes, including cellulases from *Thermoascus auranticus*, *Thrichoderma reseei*, *Acremonium thermophilum* and *Thermoascus auranticus*, have been produced by ROAL, Finland (Viikari et al., 2007). Multienzyme mixtures were also reconstituted using purified *Chrysosporium lucknowense* enzymes (Gusakov et al., 2005).

Despite the noticeable advantages of thermostable enzymes, cultivation of thermophiles and hyperthermophyles requires special and expensive media, and it is hampered by the low specific growth rates and product inhibition (Krahe et al., 1996; Schiraldi et al., 2002;Turner et al., 2007). Large scale commercial production of thermostable enzymes still remains a challenge also dependent on the optimization of their production from mesophilic microorganisms.

#### **6. Immobilization of enzymes**

Thanks to the latest breakthroughs in the research for improving the enzymes, nowadays most enzymes are produced for a commercially acceptable price. Nonetheless, the industrial

bioprocesses that run at harsh conditions (Demain et al., 2005). Of special interest is the thermoactivity and thermostability of these enzymes in the presence of high concentrations of organic solvents, detergents and alcohols. On the whole, thermophilic enzymes have an increased resistance to many denaturing conditions such as the use of detergents which can be often the unique efficient mean to obviate the irreversible adsorption of cellulases on the substrates. Furthermore, the utilization of high operation temperatures, which cause a decrease in viscosity and an increase in the diffusion coefficients of substrates, have a significant influence on the cellulose solubilization. It is worth noting that, differently from the mesophilic enzymes, most thermophilic cellulases did not show inhibition at high level of reaction products (e.g. cellobiose and glucose). As consequence, higher reaction rates and higher process yields are expected (Bergquist et al., 2004). The high process temperature also

Several cellulose degrading enzymes from various thermophilic organisms have been investigated. These include cellulases mainly isolated from anaerobic bacteria such as *Anaerocellum thermophilum* (Zverlov et al., 1998), *Clostridium thermocellum* (Romaniec et al., 1992), *Clostridium stercorarium* (Bronnenmeier et al., 1991; Bronnenmeier & Staudenbauer, 1990) and *Caldocellum saccharolyticum* (Te'o V et l., 1995), *Pyrococcus furiosus* (Ma & Adams, 1994), *Pyrococcus horikoshi* (Rahman et al.,1998), *Rhodothermus* strains (Hreggvidsson et al., 1996), *Thermotoga sp*., (Ruttersmith et al., 1991), *Thermotoga marittima* (Bronnenmeier et al.,

Xylanase have been detected in *Acidothermus cellulolyticus* in different *Thermus*, *Bacillus*,

Although many cellulolytic anaerobic bacteria such as *Clostridium thermocellum* produce cellulases with high specific activity, they do not produce high enzymes quantities. Since the anaerobes show limited growth, most researches on thermostable cellulases production have been addressed to aerobic species. Several mesophilic or moderately thermophilic fungal strains are also known to produce enzymes stable and active at high temperatures. These enzymes are produced from species such as *Chaetomium thermophila* (Venturi et al., 2002), *Talaromyces emersonii* (Grassick et al., 2004), *Thermoascus aurantiacus* (Parry et al., 2002). They may be stable at temperatures around 70 °C for prolonged periods. Table 6 summarizes some of thermostable enzymes isolated from Archea, Bacteria and Fungi. During the last decade several efforts have been devoted to develop different mixtures of selected thermostable enzymes. In 2007, mixtures of thermostable enzymes, including cellulases from *Thermoascus auranticus*, *Thrichoderma reseei*, *Acremonium thermophilum* and *Thermoascus auranticus*, have been produced by ROAL, Finland (Viikari et al., 2007). Multienzyme mixtures were also reconstituted using purified *Chrysosporium lucknowense*

Despite the noticeable advantages of thermostable enzymes, cultivation of thermophiles and hyperthermophyles requires special and expensive media, and it is hampered by the low specific growth rates and product inhibition (Krahe et al., 1996; Schiraldi et al., 2002;Turner et al., 2007). Large scale commercial production of thermostable enzymes still remains a challenge also dependent on the optimization of their production from mesophilic microorganisms.

Thanks to the latest breakthroughs in the research for improving the enzymes, nowadays most enzymes are produced for a commercially acceptable price. Nonetheless, the industrial

*Geobacillus*, *Alicyclobacillus* and *Sulfolobales* species (Sakon et al., 1996).

reduces any contamination of the fermentation medium.

1995), *Thermotoga neapolitana* (Bok et al., 1998).

enzymes (Gusakov et al., 2005).

**6. Immobilization of enzymes**

Hydrolysis of Lignocellulosic Biomass:

enzymes (Boller et al., 2002).

Yuan et al., 1999).

**6.1 Immobilization of cellulases**

above 45 °C (Yuan et al., 1999).

temperature (Dourado et al., 2002).

particles of enzyme are generated (Brady & Joordan, 2009).

Current Status of Processes and Technologies and Future Perspectives 111

**Cross-linking** results in the formation of enzyme aggregates by using bifunctional reagent, like glutaraldehyde, able to bind enzymes to each other without resorting to any support. In 1996, cross-linked enzyme crystals (CLEC; St. Clair and Navia 1992) were commercialized by Altus Biologics (Margolin, 1996). However the CLEC formation requires laborious and expensive processes of protein purification and it is applicable only to crystallisable enzymes. In addition, only one kind of enzyme can be used in the CLEC formation (Brady & Joordan, 2009). In 2001 a less-expensive method, known as CLEA (cross linked enzyme aggregates) was developed in Sheldon´s Laboratory and commercialized by CLEA Technologies (Netherlands), (Sheldon et al., 2005). Recently a new method has been developed, especially suitable for lipase immobilization. It is defined Spherezymes and it is based on the formation of a water-in-oil emulsion, in which lipases and surfactant are dissolved. Following the addition of a bifunctional cross-linker, permanent spherical

The most interesting immobilization procedures are in the area of covalent binding. Supports containing epoxy groups are widely used in the immobilization by covalent binding because these generate intense multipoint covalent attachment with different nucleophiles present on the surface of the enzyme molecules (Mateo et al., 2007). One limitation of the epoxy supports is the slow reaction of immobilization. To overcome this problem, Mateo and coworkers have designed epoxy supports able to ensure a mild physical adsorption of the enzymes followed by a very fast intramolecular covalent binding with the material epoxy groups. These supports were used to immobilize and stabilize enzymes such as glutaryl acylase (Mateo et al., 2001), βgalactosidase from *Thermus sp*. (Pessela et al., 2003), and peroxidase (Abad et al., 2002). Epoxy supports, known as **Sepabeads®** are marketed by Resindion s.r.l. and quickly have begun to supersede another commercial support, known as Eupergit. This last is a microporous, epoxyactivated acrylic beads with a diameter of 100-250µ, used for a wide variety of different

In literature, only few papers are available on the cellulases immobilization. This is due to the fact that cellulose is not soluble and some immobilization techniques, such as enzymes entrapment, impede the interaction enzyme-substrate. Immobilization of cellulases via covalent bonds appears to be the most suitable technique. Besides the enzyme stabilization, the covalent-immobilization allows the use of supported enzymes for several cycles of reactions (Brady & Joordan, 2009; Li et al., 2007; Mateo et al., 2007; Dourado et al., 2002;

In 1999, Yuan and coworkers, immobilized cellulases onto acrylamide grafted acrylonitrile copolymer membranes (PAN) by means of glutaraldehyde. They showed that the enzyme stability was increased after the immobilization process. Also, the activity of the immobilized cellulases was higher than the free cellulases at pH 3 - 5 and at temperatures

In 2002, cellulases from *T. reesei* were immobilized on Eudragit L-100 by researchers of the University of Minho (Portugal). They used the commercial mixture Celluclast® 1.5L supplied by Novozymes (Denmark). This method allowed to improve the stability of the enzymes without significant loss of its specific activity. The adsorption of cellulases on Eudragit lowered the enthalpy of denaturation, but affected only slightly the denaturation

Table 6. Thermostable cellulases

utilization of cellulases could be even more convenient by improving their stability in longterm operations and by developing methods/processes for the downstream recovery and reuse. These objectives can be achieved by the **immobilization of the enzymes** (Cao, 2005).

The main advantages of the enzyme immobilization are:

The main methods of enzyme immobilization can be classified into four classes: support binding (carrier), entrapment, encapsulation and cross-linking.

**Support binding** is based on fixing the enzyme to the external or internal surface of a substrate, by physical (adsorption), ionic or covalent bonding. **Adsorption** is a simple and inexpensive method of immobilization, and does not modify the enzyme chemical structure. However, it does not produce strong bonds between enzyme and substrate and this could cause a progressive lost of the enzyme from the support. **Ionic-binding** determines a strong bond between enzyme and support. The supports may be functionalized with a variety of chemical groups to achieve the ionic interaction, including quaternary ammonium, diethylaminoethyl and carboxymethyl derivates (Brady & Joordan, 2009). **Covalent binding** is the most widely used method of immobilization. Here the amino group of lysine is typically used as point of covalent attachment (Brady & Joordan, 2009). Lysine is a very common amino-acid in proteins, often localized on the surface of proteins. It has a good reactivity and provides acceptable bonds stability (Krenkova & Forest, 2004). Supports containing *epoxy groups* are widely used in the immobilization by covalent binding. These can react with lysine and with many other nucleophilic groups on the protein surface (e.g. Cys, Hys, and Tyr). Epoxy groups also react, in a slower way, with carboxylic groups (Mateo et al., 2007). The support used in this immobilization method is typically a prefabricated carrier, such as synthetic resins, biopolymers, inorganic polymers such as silica or zeolites.

**Entrapment** is based on inclusion of the enzyme in a polymer network (i.e. organic polymer, silica sol-gel). Unlike the previous methods, entrapment requires the synthesis of the polymeric network in the presence of the enzyme (Sheldon, 2007). This method has the advantage of protecting the enzyme from direct contact with the environment, reducing the effects of mechanical sheer and hydrophobic solvents. However, low amount of enzymes can be immobilized (Lalonde & Margolin, 2002).

**Encapsulation** is a method similar to entrapment, but, in this case, the enzyme is enclosed in a membrane that acts as a physical barrier around it (Cao L., 2005). The disadvantage is that entrapping or encapsulating matrix offer a certain resistance to the substrates diffusion.

utilization of cellulases could be even more convenient by improving their stability in longterm operations and by developing methods/processes for the downstream recovery and reuse. These objectives can be achieved by the **immobilization of the enzymes** (Cao, 2005).

5. enhanced stability under storage and operational conditions (e.g. towards denaturation

The main methods of enzyme immobilization can be classified into four classes: support

**Support binding** is based on fixing the enzyme to the external or internal surface of a substrate, by physical (adsorption), ionic or covalent bonding. **Adsorption** is a simple and inexpensive method of immobilization, and does not modify the enzyme chemical structure. However, it does not produce strong bonds between enzyme and substrate and this could cause a progressive lost of the enzyme from the support. **Ionic-binding** determines a strong bond between enzyme and support. The supports may be functionalized with a variety of chemical groups to achieve the ionic interaction, including quaternary ammonium, diethylaminoethyl and carboxymethyl derivates (Brady & Joordan, 2009). **Covalent binding** is the most widely used method of immobilization. Here the amino group of lysine is typically used as point of covalent attachment (Brady & Joordan, 2009). Lysine is a very common amino-acid in proteins, often localized on the surface of proteins. It has a good reactivity and provides acceptable bonds stability (Krenkova & Forest, 2004). Supports containing *epoxy groups* are widely used in the immobilization by covalent binding. These can react with lysine and with many other nucleophilic groups on the protein surface (e.g. Cys, Hys, and Tyr). Epoxy groups also react, in a slower way, with carboxylic groups (Mateo et al., 2007). The support used in this immobilization method is typically a prefabricated carrier, such as synthetic resins, biopolymers, inorganic polymers such as

**Entrapment** is based on inclusion of the enzyme in a polymer network (i.e. organic polymer, silica sol-gel). Unlike the previous methods, entrapment requires the synthesis of the polymeric network in the presence of the enzyme (Sheldon, 2007). This method has the advantage of protecting the enzyme from direct contact with the environment, reducing the effects of mechanical sheer and hydrophobic solvents. However, low amount of enzymes

**Encapsulation** is a method similar to entrapment, but, in this case, the enzyme is enclosed in a membrane that acts as a physical barrier around it (Cao L., 2005). The disadvantage is that entrapping or encapsulating matrix offer a certain resistance to the substrates diffusion.

4.0 60 60min at 60°C *Venturi L. Et*

3.6 78 34 min at 80°C *Grassik A.,*

at 75°C

4.5 75 98h at 70°C and 41h

*al., 2002*

*Parry N., 2002*

*2004*

Endoglucanase *Chaetomium*

Table 6. Thermostable cellulases

Exoglucanase (CBH IA)

silica or zeolites.

*termphilum*

*Thermoascus aurantiacus*

*Talaromyces emersonii*

The main advantages of the enzyme immobilization are:

3. minimal or no protein contamination of the product

by heat or organic solvents or by autolysis) (Sheldon, 2007).

binding (carrier), entrapment, encapsulation and cross-linking.

1. more convenient handling of enzymes 2. easy separation from the product

4. possible recovery and reuse of enzymes

can be immobilized (Lalonde & Margolin, 2002).

**Cross-linking** results in the formation of enzyme aggregates by using bifunctional reagent, like glutaraldehyde, able to bind enzymes to each other without resorting to any support. In 1996, cross-linked enzyme crystals (CLEC; St. Clair and Navia 1992) were commercialized by Altus Biologics (Margolin, 1996). However the CLEC formation requires laborious and expensive processes of protein purification and it is applicable only to crystallisable enzymes. In addition, only one kind of enzyme can be used in the CLEC formation (Brady & Joordan, 2009). In 2001 a less-expensive method, known as CLEA (cross linked enzyme aggregates) was developed in Sheldon´s Laboratory and commercialized by CLEA Technologies (Netherlands), (Sheldon et al., 2005). Recently a new method has been developed, especially suitable for lipase immobilization. It is defined Spherezymes and it is based on the formation of a water-in-oil emulsion, in which lipases and surfactant are dissolved. Following the addition of a bifunctional cross-linker, permanent spherical particles of enzyme are generated (Brady & Joordan, 2009).

The most interesting immobilization procedures are in the area of covalent binding. Supports containing epoxy groups are widely used in the immobilization by covalent binding because these generate intense multipoint covalent attachment with different nucleophiles present on the surface of the enzyme molecules (Mateo et al., 2007). One limitation of the epoxy supports is the slow reaction of immobilization. To overcome this problem, Mateo and coworkers have designed epoxy supports able to ensure a mild physical adsorption of the enzymes followed by a very fast intramolecular covalent binding with the material epoxy groups. These supports were used to immobilize and stabilize enzymes such as glutaryl acylase (Mateo et al., 2001), βgalactosidase from *Thermus sp*. (Pessela et al., 2003), and peroxidase (Abad et al., 2002). Epoxy supports, known as **Sepabeads®** are marketed by Resindion s.r.l. and quickly have begun to supersede another commercial support, known as Eupergit. This last is a microporous, epoxyactivated acrylic beads with a diameter of 100-250µ, used for a wide variety of different enzymes (Boller et al., 2002).

#### **6.1 Immobilization of cellulases**

In literature, only few papers are available on the cellulases immobilization. This is due to the fact that cellulose is not soluble and some immobilization techniques, such as enzymes entrapment, impede the interaction enzyme-substrate. Immobilization of cellulases via covalent bonds appears to be the most suitable technique. Besides the enzyme stabilization, the covalent-immobilization allows the use of supported enzymes for several cycles of reactions (Brady & Joordan, 2009; Li et al., 2007; Mateo et al., 2007; Dourado et al., 2002; Yuan et al., 1999).

In 1999, Yuan and coworkers, immobilized cellulases onto acrylamide grafted acrylonitrile copolymer membranes (PAN) by means of glutaraldehyde. They showed that the enzyme stability was increased after the immobilization process. Also, the activity of the immobilized cellulases was higher than the free cellulases at pH 3 - 5 and at temperatures above 45 °C (Yuan et al., 1999).

In 2002, cellulases from *T. reesei* were immobilized on Eudragit L-100 by researchers of the University of Minho (Portugal). They used the commercial mixture Celluclast® 1.5L supplied by Novozymes (Denmark). This method allowed to improve the stability of the enzymes without significant loss of its specific activity. The adsorption of cellulases on Eudragit lowered the enthalpy of denaturation, but affected only slightly the denaturation temperature (Dourado et al., 2002).

Hydrolysis of Lignocellulosic Biomass:

Current Status of Processes and Technologies and Future Perspectives 113

A major requirement in cost-efficient lignocellulosics-to-ethanol process is to employ reactor systems yielding the maximal conversion of the cellulose with the minimal enzyme dosage. As consequence, one of the most important parameter for the design and operation of bioreactors for lignocellulosic conversion is the effective use of the biocatalysts to obtain high specific rates of cellulose conversion (namely the yield of glucose obtained per amount of enzymes). The maximization of the product concentration, i.e. the amount of glucose obtained per liquid volume, is also an important parameter as well as the optimization of the volumetric productivity, in this case the rate of glucose formation per reactor volume. When the hydrolysis is carried out with high dry matter contents, hence high cellulose levels, the product concentration will drive up. For this reason, some recent researches have been finalized into attempting the enzymatic biomass conversion at high-solids loads (Jørgensen et al., 2007; Tolan, 2002). The most important problem of high solid loadings is related to the fact that the viscosity of the reaction mixture is very high and the rheology of the mixture has to be well studied: normal stress might become very significant during bioconversion. In particular, mixing and mass transfer limitations, and, presumably increased inhibition by intermediates come into play. Various fed-batch strategies have been attempted with the scope of supplying the substrate without reaching excessive viscosities and unproductive enzyme binding to the

As said, the currently employed cellulolytic enzyme systems, that include the widely studied *T. reesei* enzymes, are significantly inhibited by the hydrolysis products cellobiose and glucose. This inhibition retards the overall conversion rate of lignocellulosics-to glucose (Gan et al., 2002; Katz and Reese, 1968). Product inhibition is particularly significant during processing at high substrate loadings mainly because the glucose concentration is higher than that obtained in diluted biomass suspensions. (Kristensen et al., 2009; Rosgaard et al., 2007a). As consequence, both the conversion rate and the glucose yields achievable in batch processing of lignocellulose are reduced (Rosgaard et al., 2007b; Tengborg et al., 2001). General criteria in the bioreactor design and in the selection of the operating conditions could be: use of reactors or reaction regimes that allow a rapid reduction of the glucose concentration; running of the reactions at low to medium substrate concentrations in order to maintain higher conversion rates and hence obtain higher volumetric productivity of the

The integration of the bioreactor with a separation unit (reaction–separation hybrids) has shown promising results with product inhibited or equilibrium limited enzyme-catalyzed conversions, because it is possible to remove the products as they are formed (Ahmed et al., 2001; Gan et al., 2002). In this regard, membrane (bio) reactors could be a viable process configuration. Unlike the SSF approach in which the glucose consumption is carried out by the microrganisms simultaneously available in the hydrolyzate, the use of membrane bioreactors would accomplish the same function without any compromise in the reaction temperature. A membrane (bio-) reactor is a multifunction reactor that combines the reaction with a separation, namely in this case product removal by membrane separation, in one integrated unit, i.e. in-situ removal, or alternatively in two or more separate units. The membrane bioreactors hitherto used for the separation in enzymatic processes have been mainly ultraand nanofiltration (Pinelo et al., 2009). However, the use of this technology is limited by the bank-up of unreacted lignocellulosics (lignin and particularly recalcitrant cellulose) in largescale and/or continuous processing (Andrić et al. 2010, b). Already in the past, some authors

**8. Innovative bioreactor geometries and process strategies**

substrate (Rosgaard et al., 2007a; Rudolf et al., 2005).

reactor (Andrić et al. 2010, a).

In 2006, Li and coworkers*,* immobilized cellulase enzymes by means of liposomes. These are phospholipid vesicle, ranging in size from 25 nm to 1µm. In this method, glutaraldehydeactivated liposome bound to the enzyme thus forming the liposome-cellulase complex. Following this step, the complex was immobilized on chitosan-gel. The immobilized enzyme by the liposome molecules showed efficiency higher by 10% compared to the enzyme immobilized in chitosan-gel without liposome. The immobilized cellulase-liposome complex showed a loss of activity of 20% with respect to the original value after six cycles of reaction. Therefore, liposome-binding cellulase appeared to prevent or limit the enzyme deactivation (Li et al., 2007).

In recent investigations, two commercial cellulase enzymes (Celluclast 1.5 and Novozym 188) were immobilized on epoxy Sepabeads® support (Resindion s.rl.). The preliminary data showed that 60% of loaded Celluclast proteins were adsorbed by the support and that more than 90% of these proteins remained stably linked even after repeated washings (Verardi et al., 2011).

#### **7. Process strategies for the hydrolysis and fermentation of lignocellulosics**

After the pretreatment step, the bioconversion of lignocellulosic materials includes the biopolymers hydrolysis and the sugar fermentation. These two steps can be performed separately (SHF, separate hydrolysis and fermentation) or simultaneously (SSF, simultaneous saccharification and fermentation). SSF technology is generally considered more advantageous than SHF technology, for several reasons:

However, the optimum temperature for the enzymatic hydrolysis is typically higher than that of fermentation. Therefore, in SHF process, the temperature for the enzymatic hydrolysis can be optimized independently from the fermentation temperature, whereas a compromise must be found in SSF process (Olofsson et al., 2008). Another obstacle of the SSF process is the difficulty to carry out continuous fermentation by recirculating and reusing the yeast due to the presence of the solid residues from the hydrolysis.

High solids loadings are usually required to obtain high ethanol levels in the fermentation broths (**high gravity fermentation**). In particular, solids loadings of pretreated biomass up to 30% (w/w) could be necessary to reach an ethanol concentration of 4-5 wt% that is considered a threshold level for a sustainable distillation process. However, increasing the amount of the solids content in a bioreactor, the hydrolytic performances of the enzymes mixture tends to worsen. In particular, the high initial substrate consistency causes a **viscosity increase** (Sassner et al., 2006) that is an obstacle toward the homogeneous and effective distribution of the enzymes in the bioreactor. This problem could be partly overcome by using thermostable enzymes. In particular, the hydrolysis could be carried out in two steps: a former step at elevated temperatures with thermostable hydrolytic enzymes producing the liquefaction of biomass (SHF); the latter step, aimed at completing the biomass saccharification, could be carried out at milder temperatures by using the SSF approach (Olofsson et al., 2008).

In 2006, Li and coworkers*,* immobilized cellulase enzymes by means of liposomes. These are phospholipid vesicle, ranging in size from 25 nm to 1µm. In this method, glutaraldehydeactivated liposome bound to the enzyme thus forming the liposome-cellulase complex. Following this step, the complex was immobilized on chitosan-gel. The immobilized enzyme by the liposome molecules showed efficiency higher by 10% compared to the enzyme immobilized in chitosan-gel without liposome. The immobilized cellulase-liposome complex showed a loss of activity of 20% with respect to the original value after six cycles of reaction. Therefore, liposome-binding cellulase appeared to prevent or limit the enzyme

In recent investigations, two commercial cellulase enzymes (Celluclast 1.5 and Novozym 188) were immobilized on epoxy Sepabeads® support (Resindion s.rl.). The preliminary data showed that 60% of loaded Celluclast proteins were adsorbed by the support and that more than 90% of these proteins remained stably linked even after repeated washings

**7. Process strategies for the hydrolysis and fermentation of lignocellulosics**  After the pretreatment step, the bioconversion of lignocellulosic materials includes the biopolymers hydrolysis and the sugar fermentation. These two steps can be performed separately (SHF, separate hydrolysis and fermentation) or simultaneously (SSF, simultaneous saccharification and fermentation). SSF technology is generally considered

reduced end product inhibition because of the rapid conversion of glucose into ethanol

reduced contamination by unwanted microorganisms thanks to the presence of ethanol

However, the optimum temperature for the enzymatic hydrolysis is typically higher than that of fermentation. Therefore, in SHF process, the temperature for the enzymatic hydrolysis can be optimized independently from the fermentation temperature, whereas a compromise must be found in SSF process (Olofsson et al., 2008). Another obstacle of the SSF process is the difficulty to carry out continuous fermentation by recirculating and

High solids loadings are usually required to obtain high ethanol levels in the fermentation broths (**high gravity fermentation**). In particular, solids loadings of pretreated biomass up to 30% (w/w) could be necessary to reach an ethanol concentration of 4-5 wt% that is considered a threshold level for a sustainable distillation process. However, increasing the amount of the solids content in a bioreactor, the hydrolytic performances of the enzymes mixture tends to worsen. In particular, the high initial substrate consistency causes a **viscosity increase** (Sassner et al., 2006) that is an obstacle toward the homogeneous and effective distribution of the enzymes in the bioreactor. This problem could be partly overcome by using thermostable enzymes. In particular, the hydrolysis could be carried out in two steps: a former step at elevated temperatures with thermostable hydrolytic enzymes producing the liquefaction of biomass (SHF); the latter step, aimed at completing the biomass saccharification, could be carried out at milder temperatures by using the SSF

reusing the yeast due to the presence of the solid residues from the hydrolysis.

more advantageous than SHF technology, for several reasons: reduced number of the process steps (Koon Ong, 2004)

deactivation (Li et al., 2007).

by yeast (Viikari et al., 2007)

approach (Olofsson et al., 2008).

(Elumalai & Thangavelu, 2010).

(Verardi et al., 2011).

#### **8. Innovative bioreactor geometries and process strategies**

A major requirement in cost-efficient lignocellulosics-to-ethanol process is to employ reactor systems yielding the maximal conversion of the cellulose with the minimal enzyme dosage. As consequence, one of the most important parameter for the design and operation of bioreactors for lignocellulosic conversion is the effective use of the biocatalysts to obtain high specific rates of cellulose conversion (namely the yield of glucose obtained per amount of enzymes). The maximization of the product concentration, i.e. the amount of glucose obtained per liquid volume, is also an important parameter as well as the optimization of the volumetric productivity, in this case the rate of glucose formation per reactor volume.

When the hydrolysis is carried out with high dry matter contents, hence high cellulose levels, the product concentration will drive up. For this reason, some recent researches have been finalized into attempting the enzymatic biomass conversion at high-solids loads (Jørgensen et al., 2007; Tolan, 2002). The most important problem of high solid loadings is related to the fact that the viscosity of the reaction mixture is very high and the rheology of the mixture has to be well studied: normal stress might become very significant during bioconversion. In particular, mixing and mass transfer limitations, and, presumably increased inhibition by intermediates come into play. Various fed-batch strategies have been attempted with the scope of supplying the substrate without reaching excessive viscosities and unproductive enzyme binding to the substrate (Rosgaard et al., 2007a; Rudolf et al., 2005).

As said, the currently employed cellulolytic enzyme systems, that include the widely studied *T. reesei* enzymes, are significantly inhibited by the hydrolysis products cellobiose and glucose. This inhibition retards the overall conversion rate of lignocellulosics-to glucose (Gan et al., 2002; Katz and Reese, 1968). Product inhibition is particularly significant during processing at high substrate loadings mainly because the glucose concentration is higher than that obtained in diluted biomass suspensions. (Kristensen et al., 2009; Rosgaard et al., 2007a). As consequence, both the conversion rate and the glucose yields achievable in batch processing of lignocellulose are reduced (Rosgaard et al., 2007b; Tengborg et al., 2001).

General criteria in the bioreactor design and in the selection of the operating conditions could be: use of reactors or reaction regimes that allow a rapid reduction of the glucose concentration; running of the reactions at low to medium substrate concentrations in order to maintain higher conversion rates and hence obtain higher volumetric productivity of the reactor (Andrić et al. 2010, a).