**Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass**

Amy Philbrook, Apostolos Alissandratos and Christopher J. Easton

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55309

### **1. Introduction**

[23] Mita N, Tawaki S. I, Hiroshi U, and Kobayashi S. 2003. Laccase-catalyzed oxidative

[24] Nazari K, Esmaeili N, Mahmoudi A, Rahimi H, and Moosavi-Movahedi A. 2007. Per‐ oxidative phenol removal from aqueous solutions using activated peroxidase bioca‐

[25] Nugroho Prasetyo E, Kudanga T, Ostergaard L, Rencoret J, Gutierrez A, del Rio J. C, Ignacio Santos J, Nieto L, Jimenez-Barbero J, Martinez A. T, Li J, Gellerstedt G, Lep‐ ifre S, Silva C, Kim S Y, Cavaco-Paulo A, Seljebakken Klausen B, Lutnaes B. F, Nyan‐ hongo G. S, and Guebitz G. M. 2010. Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility. Bioresource

[26] Ouyang X, Qiu X, Chen P. 2006. Physicochemical characterization of calcium ligno‐ sulfonate-A potentially useful water reducer. Colloids Surf A 282-283: 489-497.

[27] Reihmann M, and Ritter H. 2006. Synthesis of phenol polymers using peroxidases in

[28] Riva S. 2006. Laccases: blue enzymes for green chemistry. Trends in Biotechnology

[29] Rodrigues S, Pinto G. A. S, and Fernandes F. A. N. 2008. Optimization of ultrasound extraction of phenolic compounds from coconut (Cocos nucifera) shell powder by re‐

[30] Venkateswaran P, Palanivelu K. 2006. Recovery of phenol from aqueous solution by supported liquid membrane using vegetable oils as liquid membrane. J Hazardous

[31] Widsten P, Kandelbauer A. 2008. Laccase applications in the forest products indus‐

[32] Wu Y, Taylor K. E, Biswas N, Bewtra, J. K. 1999. Kinetic model-aided reactor design for peroxidase-catalyzed removal of phenol in the presence of polyethylene glycol. J

[33] Yang D, Qiu X, Pang Y, Zhou M. 2008. Physicochemical properties of calcium ligno‐ sulfonate with different molecular weights as dispersant in aqueous suspension J.

[34] Zhou M, Qiu X, Yang D, Lou H. 2006. Properties of different molecular weight so‐ dium lignosulfonate fractions as dispersant of coal-water slurry. J. Dispersion Sci.

sponse surface methodology. Ultrasonics Sonochem 15: 95-100.

polymerization of phenols. Macromolecular Bioscience 3: 253-257.

talyst. Enzyme Microb Technol 41: 221-233.

38 Environmental Biotechnology - New Approaches and Prospective Applications

Advances in Polymer Science Vol. 194, pp. 1-49.

try: A review. Enzyme Microb Technol 42: 293-307.

Chem Technol Biotech 74: 519-526.

Disper. Sci. Technol. 29: 1296-1303.

Technol 10: 5054-5062.

24: 219-226.

Mater 131: 146-152.

Technol 27: 851-856.

Fuels and chemicals derived from biomass are regarded as an environmentally friendly alternative to petroleum based products. The concept of using plant material as a source for fuels and commodity chemicals has been embraced by governments to alleviate dependence on the volatile petroleum market. This trend is driven not only by economics but also by social and political factors. Global warming has been associated with CO2 emissions largely originating from the combustion of fossil fuels.[1] This, together with depleting and finite carbon fossil fuel resources, and insecurity of petroleum supplies has prompted a shift towards biofuels and biomaterials.[1] The use of biomass as an economically competitive source of transport fuel was initiated by the fuel crisis in 1970 and its commercialization was led by the USA and Brazil.[2] In 2010, the USA and Brazil processing corn and sugarcane, respectively, produced 90% of the world's bioethanol. In 2008, the "food for fuel" debate emerged sparked by concerns that the use of arable land for bioethanol and biodiesel crops was placing pressure on food demand for a growing world population.[3] In June 2011, the World Bank and nine other international agencies produced a report advising governments to cease biofuel subsidies as the use of food stock for fuel production was linked to increasing food prices.[4] Subsidies were thus ended in the USA when their Senate voted overwhelmingly to end billions of dollars in bioethanol subsidies.[5] This reform resulted in USA bioethanol plants recording losses in the first quarter of 2012[6] and is foreseen as the end of bioethanol production from corn at least in the USA.

Emerging from the "food for fuel" debate, the concept of commercializing second generation biofuels was embraced by governments as a route to produce biofuels without diminishing global food supplies.[7]. Second generation biofuels address concerns over designating arable land to grow food crops for fuel production as lignocellulosic biomass may consist of waste

© 2013 Philbrook et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Philbrook et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

materials such as plant residues.[8] In many proposed biorefinery setups, the food portion of the crop is to be used for human consumption and the waste residues, for example, the leaves and stalks, are to be processed for biofuels and chemicals.[8] An illustration of the processes for 1st, 2nd and 3rd generation biofuel production is shown in Figure 1. Third generation biofuel production, the generation of biodiesel from algae, is included in the diagram for completeness.

All three of the processes outlined in Figure 1 rely on biotechnology for the conversion of biomass to fuels. First generation bioethanol production traditionally incorporates two biological transformations. The first stage uses commercialized saccharification biotechnology which depolymerises starch into fermentable glucose units. The second stage is the fermenta‐ tion of sugar units to ethanol and again uses commercialized biotechnology generally with yeast extracts.[1] Although the use of lignocellulosic biomass is socially widely supported, the processes for its conversion are more complex and therefore more costly. The major costadding component of 2nd generation bioethanol production compared to the 1st is the pretreat‐ ment step as the removal of the lignin is required for cellulose accessibility.[9] Whilst 1st generation bioethanol production converts substrates high in starch (mainly corn, sugarbeet and sugarcane), the effective utilization of lignocellulosic biomass requires at least separation, if not complete conversion, of all plant components. The composition of plant material includes lignin, cellulose and hemicelluloses and a diagram illustrating how these components relate is shown in Figure 2. The percentage of these three plant components varies with species (Figure 2) further complicating the processing of such biomass.

It has been reported that the separation and use of all plant components is required for environmentally and economically viable biorefineries.[1, 8, 9] The application of biotechnol‐ ogy for all aspects of biomass conversion avoids toxic by-products and high energy inputs encountered with chemical, thermal and mechanical processes often used. It is due to these energy and environmental concerns that biochemical methods are feverously being investi‐ gated, as enzymatic processes are largely environmentally benign and low in energy demand. Processes for the transformation of biomass need to be carbon efficient, otherwise the envi‐ ronmental objectives of biomass utilization are negated. It is with this in mind that the current chapter is focused on advances using environmentally benign biocatalysts.

theoretical sugar yield can be obtained from lignocellulose without pretreatment.[10, 11] Currently, the biomass pretreatment step for producing 2nd generation bioethanol is the most expensive component of the process after the raw material.[12] Thermal and mechanical methods are energy intensive and therefore carbon costly as they indirectly produce CO2. Chemical techniques result in contamination of the biomass producing biochemical inhibitors as by-products[13, 14] and require costly neutralization processes.[15] Removal of the lignin fraction using microorganisms has several advantages compared to other pretreatment methods. Firstly, microorganisms function under ambient conditions thus eliminating thermal and electrical energy inputs. When compared to chemical pretreatment methods, biochemical pretreatment does not result in chemical by-products that often inhibit cellulose hydrolysis.

**Figure 1.** Examples of processes to produce 1st generation bioethanol from corn, 2nd generation bioethanol from

Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass

http://dx.doi.org/10.5772/55309

41

corn waste residues and 3rd generation biodiesel from algae.

In nature, fungi are responsible for the biodegradation of lignin, thus the majority of research into biochemical pretreatments has focused on fungi for the delignification of biomass. Early research in the area was led by the pulp and paper industry and focussed on fungal treatment as a method for removing the lignin fraction from wood to facilitate cellulose accessibility and to lower pulping energy costs. In 1982, Eriksson and Vallander were able to achieve a 23% reduction in refining energy by incubating wood chips with the white-rot fungus

The use of biochemical techniques for processing of lignocellulosic biomass is covered herein. This includes the bioprocessing of the plant components, lignin, cellulose and hemicellulose and is focused on progress made in their biochemical conversion not only to ethanol but also to value-added chemicals according to biomass fraction. The review of the literature is concentrated on biocatalytic advances in the past decade and is delineated by the plant-derived substrate. Strategies for the commercialization of 2nd generation biofuels and commodity chemicals are discussed.

### **2. Biochemical pretreatment**

Pretreatment of lignocellulosic biomass is required to increase holocellulose (cellulose and hemicellulose) accessibility for its hydrolysis into fermentable sugars and only 20% of the Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass http://dx.doi.org/10.5772/55309 41

materials such as plant residues.[8] In many proposed biorefinery setups, the food portion of the crop is to be used for human consumption and the waste residues, for example, the leaves and stalks, are to be processed for biofuels and chemicals.[8] An illustration of the processes for 1st, 2nd and 3rd generation biofuel production is shown in Figure 1. Third generation biofuel production, the generation of biodiesel from algae, is included in the diagram for completeness. All three of the processes outlined in Figure 1 rely on biotechnology for the conversion of biomass to fuels. First generation bioethanol production traditionally incorporates two biological transformations. The first stage uses commercialized saccharification biotechnology which depolymerises starch into fermentable glucose units. The second stage is the fermenta‐ tion of sugar units to ethanol and again uses commercialized biotechnology generally with yeast extracts.[1] Although the use of lignocellulosic biomass is socially widely supported, the processes for its conversion are more complex and therefore more costly. The major costadding component of 2nd generation bioethanol production compared to the 1st is the pretreat‐ ment step as the removal of the lignin is required for cellulose accessibility.[9] Whilst 1st generation bioethanol production converts substrates high in starch (mainly corn, sugarbeet and sugarcane), the effective utilization of lignocellulosic biomass requires at least separation, if not complete conversion, of all plant components. The composition of plant material includes lignin, cellulose and hemicelluloses and a diagram illustrating how these components relate is shown in Figure 2. The percentage of these three plant components varies with species

It has been reported that the separation and use of all plant components is required for environmentally and economically viable biorefineries.[1, 8, 9] The application of biotechnol‐ ogy for all aspects of biomass conversion avoids toxic by-products and high energy inputs encountered with chemical, thermal and mechanical processes often used. It is due to these energy and environmental concerns that biochemical methods are feverously being investi‐ gated, as enzymatic processes are largely environmentally benign and low in energy demand. Processes for the transformation of biomass need to be carbon efficient, otherwise the envi‐ ronmental objectives of biomass utilization are negated. It is with this in mind that the current

The use of biochemical techniques for processing of lignocellulosic biomass is covered herein. This includes the bioprocessing of the plant components, lignin, cellulose and hemicellulose and is focused on progress made in their biochemical conversion not only to ethanol but also to value-added chemicals according to biomass fraction. The review of the literature is concentrated on biocatalytic advances in the past decade and is delineated by the plant-derived substrate. Strategies for the commercialization of 2nd generation biofuels and commodity

Pretreatment of lignocellulosic biomass is required to increase holocellulose (cellulose and hemicellulose) accessibility for its hydrolysis into fermentable sugars and only 20% of the

(Figure 2) further complicating the processing of such biomass.

40 Environmental Biotechnology - New Approaches and Prospective Applications

chapter is focused on advances using environmentally benign biocatalysts.

chemicals are discussed.

**2. Biochemical pretreatment**

**Figure 1.** Examples of processes to produce 1st generation bioethanol from corn, 2nd generation bioethanol from corn waste residues and 3rd generation biodiesel from algae.

theoretical sugar yield can be obtained from lignocellulose without pretreatment.[10, 11] Currently, the biomass pretreatment step for producing 2nd generation bioethanol is the most expensive component of the process after the raw material.[12] Thermal and mechanical methods are energy intensive and therefore carbon costly as they indirectly produce CO2. Chemical techniques result in contamination of the biomass producing biochemical inhibitors as by-products[13, 14] and require costly neutralization processes.[15] Removal of the lignin fraction using microorganisms has several advantages compared to other pretreatment methods. Firstly, microorganisms function under ambient conditions thus eliminating thermal and electrical energy inputs. When compared to chemical pretreatment methods, biochemical pretreatment does not result in chemical by-products that often inhibit cellulose hydrolysis.

In nature, fungi are responsible for the biodegradation of lignin, thus the majority of research into biochemical pretreatments has focused on fungi for the delignification of biomass. Early research in the area was led by the pulp and paper industry and focussed on fungal treatment as a method for removing the lignin fraction from wood to facilitate cellulose accessibility and to lower pulping energy costs. In 1982, Eriksson and Vallander were able to achieve a 23% reduction in refining energy by incubating wood chips with the white-rot fungus

Different species of white-rot fungi, *Echinodontium taxodii,*[22, 23]*, Coriolus versicolor*[24] and *Trametes versicolor*[23]*,* have been studied for their ability to degrade lignin to promote cellulose digestibility in bamboo residues. Zeng, Yang et al.[22] recently reported a 29% decrease in lignin content in bamboo treated with *Echinodontium taxodii* however the aim of the work was to improve the thermal decomposition of the bamboo and not to recovery and utilize the holocellulose. Zhang, Xu et al*.*[24] reported an increase in saccharification rate of 37% when bamboo residues were incubated with *Coriolus versicolor.* Zhang, Yu et al*.*[23] compared two species of white-rot fungus for their effectiveness in increasing sugar yields and found that incubation with both *Trametes versicolor* and *Echinodontium taxodii* improved sugar yields 5.15 and 8.75 times respectively. Cornstalk and corn stover have been pretreated with different fungal strains for delignification. Impressive results were reported by Wan and Li[25] who pretreated corn stover with *Ceriporiopsis subvermispora* and measured a 31.59% reduction in lignin with only a 6% loss in cellulose. In 2010, Dias et al*.*[26] reported a nearly 4-fold increase in saccharification of wheat straw treated with basidiomycetous fungi Euc-1 and *Irpex lacteus*. *Dichomitus squalens*,[27] *Pleurotus ostreatus*[28] and *Phaerochaete chrysosporium*[28] were applied to rice straw with varying effects (Table 1), with the most notable reported by Bak et al.,[27] being a 58.1% theoretical glucose yield of rice straw treated with *Dichomitus squalens*. The biochemical pretreatment of cotton stalks was studied by Shi et al. who reported 33.9% lignin reduction[29] using submerged fungus cultivation and 27.6% lignin reduction[30] using solid state cultivation of the same fungus, *Phanerochaete chrysosporium*. Hideno et al*.*[31] applied *Grifola frondosa* for the pretreatment of sawdust matrix and reported a 21% reduction in lignin

Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass

Substrate Species Findings Duration Ref Bamboo *Echinodotium taxodi* 29% reduction in lignin 30 days [22]

saccharification of 47.3%

Corn Stover *Ceriporiopsis subvermispora* 31.59% lignin degradation with less than 6% cellulose loss

Corn Stover *Ceriporiopsis subvermispora* Lignin degradation reached 45% 30 days [33] Corn Stover *Irpex lacteus CD2* 66.4% saccharification ratio 25 days [34]

Corn Stover *Cyanthus stercoreus* 3‐ to 5‐fold improvement in enzymatic digestibility 29 days [12]

Wheatstraw *Irpex lacteus* 3‐fold increase in saccharification 46 days [26] Rice straw *Dichomitus squalens* 58% theoretical glucose yield for remaining glucan 15 days [27] Rice straw *Pleurotus ostreatus* 39% degradation of lignin with 79% cellulose retention 48 days [35] Rice straw *Phaerochaete chrysosporium* 64.9% of maximum glucose yield from recovered glucan 15 days [28]

> *Phaerochaete chrysosporium* 33.9% lignin degradation with 18.4% carbohydrate availability

*Coriolus versicolor* Enhanced saccharification rate of 37% 2 days [24]

*Echinodontium taxodii* 5.15‐fold increase in sugar yields 120 days [23]

*Trametes versicolor* 8.75‐fold increase in sugar yields 120 days [23]

*Phaerochaete chrysosporium* 27.6% lignin degradation 14 days [30]

*Grifola frondosa* 21% reduction in lignin and 90% recovery of cellulose 2 days [31]

34.3% reduction in lignin with a maximum enzyme

4‐fold increase in saccharification 46 days [26]

15 days [32]

http://dx.doi.org/10.5772/55309

43

18 days [25]

14 days [29]

with 90% cellulose recovery.

Bamboo residues

Bamboo culms

Bamboo culms

Cotton stalks

Cotton stalks

Sawdust matrix

pCornstalk *Phanerochaete chrysosporium*

Wheat straw Basidiomycetous fungi Euc‐1

**Table 1.** Fungal strains studied for pretreatment of lignocellulosic biomass.

**Figure 2. Diagram of plant components cellulose, hemicelluloses and lignin and a graphical representation of their weight percentage according to biomass source[10]. Figure 2.** Diagram of plant components cellulose, hemicelluloses and lignin and a graphical representation of their weight percentage according to biomass source[10].

*Phanerochaete chrysosporium* for 2 weeks.[16] Messner and Srebotnik[17] studied the same species of fungus and reported similar results. Later studies by Akhtar et al*.*[18] also found substantial energy savings in pulping when the wood was treated with *Ceriporiopsis subvermispora*. Others[19] studied soft and hard wood processes and reported a reduction in refining energy from incubation with strains of white-rot fungi of 33% for soft-wood pulp and more than 50% for hardwood. More recently, Liew et al*.*[20] reported a lignin loss of 26.9% in biopulping studies with *Acacia mangium* wood chips when incubated with the whiterot fungi *Trametes versicolour*. It has been reported that the separation and use of all plant components is required for environmentally and economically viable biorefineries.(1, 8, 9) The application of biotechnology for all aspects of biomass conversion avoids toxic by-products and high energy inputs encountered with chemical, thermal and mechanical processes often used. It is due to these energy and environmental concerns that biochemical methods are feverously being investigated, as enzymatic processes are largely environmentally benign and low in energy demand. Processes for the transformation of biomass need to be carbon efficient, otherwise the environmental objectives of biomass utilization are negated. It is with this in mind that the current chapter is focused on advances using environmentally benign biocatalysts.

The amount of lignin present in the biomass directly affects enzymatic digestion of the holocel‐ lulose fraction. For example, a decrease from 22% to 17% lignin in biomass samples doubles the sugar yield and samples with 26% lignin result in virtually no sugar.[21] However, the effective‐ ness of pretreatment is not only measured by the decrease in lignin content but also by holocel‐ lulose recovery and ultimately the saccharification percentage. Table 1 summarizes recent studies conducted on the pretreatment of different biomass substrates. As stated earlier and depicted in Figure 2, the amount of lignin, cellulose and hemicellulose varies greatly with biomass source and it is therefore logical to assess the effectiveness of fungal pretreatment according to substrate. It is important to note that direct comparisons are not always possible as the experi‐ mental techniques and measurements vary within many of the cited studies. For example, the fungal incubation times listed in Table 1 vary from 2 to 120 days. The use of biochemical techniques for processing of lignocellulosic biomass is covered herein. This includes the bioprocessing of the plant components, lignin, cellulose and hemicellulose and is focused on progress made in their biochemical conversion not only to ethanol but also to value-added chemicals according to biomass fraction. The review of the literature is concentrated on biocatalytic advances in the past decade and is delineated by the plant-derived substrate. Strategies for the commercialization of 2nd generation biofuels and commodity chemicals are discussed.

Different species of white-rot fungi, *Echinodontium taxodii,*[22, 23]*, Coriolus versicolor*[24] and *Trametes versicolor*[23]*,* have been studied for their ability to degrade lignin to promote cellulose digestibility in bamboo residues. Zeng, Yang et al.[22] recently reported a 29% decrease in lignin content in bamboo treated with *Echinodontium taxodii* however the aim of the work was to improve the thermal decomposition of the bamboo and not to recovery and utilize the holocellulose. Zhang, Xu et al*.*[24] reported an increase in saccharification rate of 37% when bamboo residues were incubated with *Coriolus versicolor.* Zhang, Yu et al*.*[23] compared two species of white-rot fungus for their effectiveness in increasing sugar yields and found that incubation with both *Trametes versicolor* and *Echinodontium taxodii* improved sugar yields 5.15 and 8.75 times respectively. Cornstalk and corn stover have been pretreated with different fungal strains for delignification. Impressive results were reported by Wan and Li[25] who pretreated corn stover with *Ceriporiopsis subvermispora* and measured a 31.59% reduction in lignin with only a 6% loss in cellulose. In 2010, Dias et al*.*[26] reported a nearly 4-fold increase in saccharification of wheat straw treated with basidiomycetous fungi Euc-1 and *Irpex lacteus*. *Dichomitus squalens*,[27] *Pleurotus ostreatus*[28] and *Phaerochaete chrysosporium*[28] were applied to rice straw with varying effects (Table 1), with the most notable reported by Bak et al.,[27] being a 58.1% theoretical glucose yield of rice straw treated with *Dichomitus squalens*. The biochemical pretreatment of cotton stalks was studied by Shi et al. who reported 33.9% lignin reduction[29] using submerged fungus cultivation and 27.6% lignin reduction[30] using solid state cultivation of the same fungus, *Phanerochaete chrysosporium*. Hideno et al*.*[31] applied *Grifola frondosa* for the pretreatment of sawdust matrix and reported a 21% reduction in lignin with 90% cellulose recovery.


**Table 1.** Fungal strains studied for pretreatment of lignocellulosic biomass.

*Phanerochaete chrysosporium* for 2 weeks.[16] Messner and Srebotnik[17] studied the same species of fungus and reported similar results. Later studies by Akhtar et al*.*[18] also found substantial energy savings in pulping when the wood was treated with *Ceriporiopsis subvermispora*. Others[19] studied soft and hard wood processes and reported a reduction in refining energy from incubation with strains of white-rot fungi of 33% for soft-wood pulp and more than 50% for hardwood. More recently, Liew et al*.*[20] reported a lignin loss of 26.9% in biopulping studies with *Acacia mangium* wood chips when incubated with the white-

It has been reported that the separation and use of all plant components is required for environmentally and economically viable biorefineries.(1, 8, 9) The application of biotechnology for all aspects of biomass conversion avoids toxic by-products and high energy inputs encountered with chemical, thermal and mechanical processes often used. It is due to these energy and environmental concerns that biochemical methods are feverously being investigated, as enzymatic processes are largely environmentally benign and low in energy demand. Processes for the transformation of biomass need to be carbon efficient, otherwise the environmental objectives of biomass utilization are negated. It is with this in mind that the current chapter is focused on advances using environmentally benign

**Figure 2. Diagram of plant components cellulose, hemicelluloses and lignin and a graphical representation of their weight percentage according to biomass source[10].**

**Figure 2.** Diagram of plant components cellulose, hemicelluloses and lignin and a graphical representation of their

Weight (%)

Beechwood Rice Husk Bark Grass

The amount of lignin present in the biomass directly affects enzymatic digestion of the holocel‐ lulose fraction. For example, a decrease from 22% to 17% lignin in biomass samples doubles the sugar yield and samples with 26% lignin result in virtually no sugar.[21] However, the effective‐ ness of pretreatment is not only measured by the decrease in lignin content but also by holocel‐ lulose recovery and ultimately the saccharification percentage. Table 1 summarizes recent studies conducted on the pretreatment of different biomass substrates. As stated earlier and depicted in Figure 2, the amount of lignin, cellulose and hemicellulose varies greatly with biomass source and it is therefore logical to assess the effectiveness of fungal pretreatment according to substrate. It is important to note that direct comparisons are not always possible as the experi‐ mental techniques and measurements vary within many of the cited studies. For example, the

The use of biochemical techniques for processing of lignocellulosic biomass is covered herein. This includes the bioprocessing of the plant components, lignin, cellulose and hemicellulose and is focused on progress made in their biochemical conversion not only to ethanol but also to value-added chemicals according to biomass fraction. The review of the literature is concentrated on biocatalytic advances in the past decade and is delineated by the plant-derived substrate. Strategies for the commercialization of 2nd generation biofuels

fungal incubation times listed in Table 1 vary from 2 to 120 days.

and commodity chemicals are discussed.

rot fungi *Trametes versicolour*.

weight percentage according to biomass source[10].

42 Environmental Biotechnology - New Approaches and Prospective Applications

biocatalysts.

### **3. Bioconversion of lignin to chemicals and fuels**

During biochemical pretreatment, the lignin fraction is metabolized by the microorganism. In chemical and thermal pretreament processes the lignin fraction often remains intact and is thus able to be separated and utilized. After separation, microorganisms could in principle transform the lignin into materials, chemicals and fuels. Despite efforts over a long period of time, research into the bioconversion of lignin into economically viable products is still in its infancy, primarily because of the complex and irregular structure of lignin (Figure 3). However, advancements for the valorization of lignin are actively being pursued, as lignin is the second most abundant carbon source in nature and contains valuable phenolic building blocks within its structure.[36]

Although biochemical pretreatment methods generally use fungi as the lignin degrading microorganism, it is unlikely that usable lignin-derived materials and chemicals will result from fungal processes as white-rot fungi are known to mineralize lignin.[40] Thus bacterial conversion of lignin into chemicals and fuels constitutes an attractive method for the valori‐ zation of lignin. Classes of bacteria capable of degrading lignin have been identified as Actinomycetes, α-Proteobacteria and γ-Proteobacteria.[41-44] Recently, a range of metabolites (Figure 4) have been isolated from the bacterial degradation of lignocelluloses. [40] Metabolites A and B have been observed from lignocelluloses processed by the bacteria *Pseudomonas putida* mt-2[43], *Rhodococcus jostii* RHA1[43] and *Sphinogobium* sp. SYK-6.[42] Furthermore *Sphinogobium* sp. metabolizes β-aryl ether linked aromatics to vanillin.[42] Compounds C, D, E and G were identified using GC-MS as bacterial degradation products of Kraft lignin.[45] Ferulic acid as well as compounds F, H, I and J were identified by GC-MS as products of waste paper effluent treated with *Aeromonas formicans*.[46] There are established chemical and biochemical methods for converting lignin derived monomers, like those observed in the bacterial degradation of lignin (Figure 4), into simpler aromatics like phenol (Figure 3) as well

Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass

as hydrocarbons like cyclohexane (Figure 3).

O

OH

COOH

**(E) (F)**

> OH OR

RO

OCH3

R=H R=CH3

OH

COOH

OH

H3CO

OH

**Figure 4.** Compounds isolated after bacterial conversion of lignin.[40]

**4. Biochemical conversion of cellulose**

OCH3

The use of starchy feedstock, such as corn and sugar cane, is problematic in relation to food sustainability and biodiversity. Therefore, as mentioned, the focus in second generation biofuel production processes has been on biomass consisting mainly of cellulose. A high percentage

COOH

COOH

**(A) (B) (C) (D)**

OH

COOH

COOH

**(G) (H) (I)**

OH

CHO

http://dx.doi.org/10.5772/55309

45

OCH3 OCH3

H3CO

COOH

**(J)**

R=H R=CH3

OH OR

Although lignin has traditionally been burned as an inefficient energy source by-product from bioethanol or pulping production, lignin derived value-added products are necessary to improve biomass conversion economics.[37] Lignin has been used in the manufacture of wood adhesives as a component of phenol-formaldehyde resins (LPF resins).[38, 39] Lignin-derived commodity chemicals have been targeted through chemical and biological routes (Figure 3). Vanillin and cinnamic acid are subunits of the complex lignin structure and are used com‐ mercially as food sweeteners, as additives for fragrances and as precursors for pharmaceuti‐ cals. Phenol is the most widely used starting material in the plastic and resin industry and phenolic monomers have also been targeted from lignin. After the depolymerisation of lignin into monomeric units, the substituted monomers are precursors of a range of products including fuels such as cyclohexane.

**Figure 3.** Examples of chemicals targeted from lignin.

Although biochemical pretreatment methods generally use fungi as the lignin degrading microorganism, it is unlikely that usable lignin-derived materials and chemicals will result from fungal processes as white-rot fungi are known to mineralize lignin.[40] Thus bacterial conversion of lignin into chemicals and fuels constitutes an attractive method for the valori‐ zation of lignin. Classes of bacteria capable of degrading lignin have been identified as Actinomycetes, α-Proteobacteria and γ-Proteobacteria.[41-44] Recently, a range of metabolites (Figure 4) have been isolated from the bacterial degradation of lignocelluloses. [40] Metabolites A and B have been observed from lignocelluloses processed by the bacteria *Pseudomonas putida* mt-2[43], *Rhodococcus jostii* RHA1[43] and *Sphinogobium* sp. SYK-6.[42] Furthermore *Sphinogobium* sp. metabolizes β-aryl ether linked aromatics to vanillin.[42] Compounds C, D, E and G were identified using GC-MS as bacterial degradation products of Kraft lignin.[45] Ferulic acid as well as compounds F, H, I and J were identified by GC-MS as products of waste paper effluent treated with *Aeromonas formicans*.[46] There are established chemical and biochemical methods for converting lignin derived monomers, like those observed in the bacterial degradation of lignin (Figure 4), into simpler aromatics like phenol (Figure 3) as well as hydrocarbons like cyclohexane (Figure 3).

**Figure 4.** Compounds isolated after bacterial conversion of lignin.[40]

### **4. Biochemical conversion of cellulose**

**3. Bioconversion of lignin to chemicals and fuels**

44 Environmental Biotechnology - New Approaches and Prospective Applications

its structure.[36]

including fuels such as cyclohexane.

OCH3 OCH3

HO

O

**cinnamic acid**

**Figure 3.** Examples of chemicals targeted from lignin.

O OH

**-aryl ether linkage**

OCH3

OH

O

OH

CH3

O

O

O

OCH3 OH O HO

OH

**vanillin phenol benzene cyclohexane**

**di-aryl ether linkage**

O OCH3

OH

OH

OCH3

**biphenyl linkage**

HO

OCH3

OH OCH3

During biochemical pretreatment, the lignin fraction is metabolized by the microorganism. In chemical and thermal pretreament processes the lignin fraction often remains intact and is thus able to be separated and utilized. After separation, microorganisms could in principle transform the lignin into materials, chemicals and fuels. Despite efforts over a long period of time, research into the bioconversion of lignin into economically viable products is still in its infancy, primarily because of the complex and irregular structure of lignin (Figure 3). However, advancements for the valorization of lignin are actively being pursued, as lignin is the second most abundant carbon source in nature and contains valuable phenolic building blocks within

Although lignin has traditionally been burned as an inefficient energy source by-product from bioethanol or pulping production, lignin derived value-added products are necessary to improve biomass conversion economics.[37] Lignin has been used in the manufacture of wood adhesives as a component of phenol-formaldehyde resins (LPF resins).[38, 39] Lignin-derived commodity chemicals have been targeted through chemical and biological routes (Figure 3). Vanillin and cinnamic acid are subunits of the complex lignin structure and are used com‐ mercially as food sweeteners, as additives for fragrances and as precursors for pharmaceuti‐ cals. Phenol is the most widely used starting material in the plastic and resin industry and phenolic monomers have also been targeted from lignin. After the depolymerisation of lignin into monomeric units, the substituted monomers are precursors of a range of products

> The use of starchy feedstock, such as corn and sugar cane, is problematic in relation to food sustainability and biodiversity. Therefore, as mentioned, the focus in second generation biofuel production processes has been on biomass consisting mainly of cellulose. A high percentage

of cellulose (usually 35-50% dry weight) is consistently found in all plants despite the vast genetic diversity that is observed within the plant kingdom.[47] For the production of ethanol, cellulose is exposed during pretreatment, hydrolysed by either chemical or enzymatic hydrolysis and then fermented into ethanol. The material, once stripped from other biopoly‐ mers surrounding it within the plant structure, also appears to have characteristics independ‐ ent of plant taxa. Cellulose is a linear polymer, composed of glucose monomers held together by β-1,4-glucosidic bonds (Figure 5), in contrast to α-1,4-bonds found in other common glucans such as starch and glycogen. Through interchain and intrachain hydrogen bonding as well as Van der Waals forces, cellulose chains self-assemble on biosynthesis into protofibrils, then microfibrils, which are in turn packed into fibres with high crystallinity, imparting the material with high tensile strength and water insolubility.[48] These very properties that make it a suitable structural polysaccharide are the cause of the main difficulties associated with the use of biomass rich in cellulose, for the generation of products through fermentation. The addi‐ tional energy required to break down the rigid structure of cellulose is one of the main obstacles towards commercialization of lignocellulosic biomass processing.

Cellulases, the enzymes responsible for cellulose hydrolysis, differ from other glucoside hydrolases in that they are able to catalyse hydrolysis of β-1,4-glucosidic bonds. Cellulases vary significantly and belong to several glycoside hydrolase families.[49] The main differences between them relate to their mode of action. While endoglucanases are thought to randomly hydrolyse the amorphous fraction of cellulose, exoglucanases process the polysaccharide preferentially from a reducing or non-reducing end, releasing cellobiose (cellobiohydrolases) or glucose (glucanohydrolases).[47] An important feature of the exoglucanase structure is a distinct domain termed the carbohydrate binding module (CBM), which allows the enzyme to remain attached to the cellulose chain during catalytic action. This aids enzymatic action upon crystalline material by bringing the catalytic domain closer to the substrate and has been suggested to also help catalysis by peeling fragments of cellulose from the cellulosic surface. [50] β-Glucosidases are the third general category of cellulolytic enzymes; they act upon bonds in soluble cellobiose or cellodextrins formed by the action of the other two types of cellulases. The different types of cellulases act in coordination to efficiently hydrolyse cellulose, display‐ ing synergy and, depending on the host, may or may not form stable complexes of highmolecular weight.[51] These complexes, although beneficial to penetration of cellulosic material *in vivo*, when used in bioprocessing are generally considered problematic.[52]

The microorganism to receive by far the most attention in relation to sourcing of cellulolytic enzymes has been *Trichoderma reesei*.[53] This fungus was identified by E.T. Reese as the culprit for the rapid destruction of allied forces' cotton tents during WWII. Since its isolation, it has been extensively studied in relation to its cellulolytic capability and various cellulase hyper‐ producing strains have been developed, with RUT C30 currently the benchmark strain for production of cellulases in high yields.[54] One of the problems associated with this fungus has been the low expression of β-glucosidases, the enzymes responsible for liberation of glucose from short oligosaccharides. This, however, has been overcome with genetic engi‐ neering and supplementation of commercial preparations with foreign β-glucosidases.[55] Other promising fungal sources for cellulases exist, such as *Acremonium*, *Penicillium* and

**Figure 5.** Cellulose and hemicellulose, their sugar units and some potential chemical targets organized by carbon

Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass

http://dx.doi.org/10.5772/55309

47

chain number.

**Figure 5.** Cellulose and hemicellulose, their sugar units and some potential chemical targets organized by carbon chain number.

of cellulose (usually 35-50% dry weight) is consistently found in all plants despite the vast genetic diversity that is observed within the plant kingdom.[47] For the production of ethanol, cellulose is exposed during pretreatment, hydrolysed by either chemical or enzymatic hydrolysis and then fermented into ethanol. The material, once stripped from other biopoly‐ mers surrounding it within the plant structure, also appears to have characteristics independ‐ ent of plant taxa. Cellulose is a linear polymer, composed of glucose monomers held together by β-1,4-glucosidic bonds (Figure 5), in contrast to α-1,4-bonds found in other common glucans such as starch and glycogen. Through interchain and intrachain hydrogen bonding as well as Van der Waals forces, cellulose chains self-assemble on biosynthesis into protofibrils, then microfibrils, which are in turn packed into fibres with high crystallinity, imparting the material with high tensile strength and water insolubility.[48] These very properties that make it a suitable structural polysaccharide are the cause of the main difficulties associated with the use of biomass rich in cellulose, for the generation of products through fermentation. The addi‐ tional energy required to break down the rigid structure of cellulose is one of the main obstacles

Cellulases, the enzymes responsible for cellulose hydrolysis, differ from other glucoside hydrolases in that they are able to catalyse hydrolysis of β-1,4-glucosidic bonds. Cellulases vary significantly and belong to several glycoside hydrolase families.[49] The main differences between them relate to their mode of action. While endoglucanases are thought to randomly hydrolyse the amorphous fraction of cellulose, exoglucanases process the polysaccharide preferentially from a reducing or non-reducing end, releasing cellobiose (cellobiohydrolases) or glucose (glucanohydrolases).[47] An important feature of the exoglucanase structure is a distinct domain termed the carbohydrate binding module (CBM), which allows the enzyme to remain attached to the cellulose chain during catalytic action. This aids enzymatic action upon crystalline material by bringing the catalytic domain closer to the substrate and has been suggested to also help catalysis by peeling fragments of cellulose from the cellulosic surface. [50] β-Glucosidases are the third general category of cellulolytic enzymes; they act upon bonds in soluble cellobiose or cellodextrins formed by the action of the other two types of cellulases. The different types of cellulases act in coordination to efficiently hydrolyse cellulose, display‐ ing synergy and, depending on the host, may or may not form stable complexes of highmolecular weight.[51] These complexes, although beneficial to penetration of cellulosic material *in vivo*, when used in bioprocessing are generally considered problematic.[52]

The microorganism to receive by far the most attention in relation to sourcing of cellulolytic enzymes has been *Trichoderma reesei*.[53] This fungus was identified by E.T. Reese as the culprit for the rapid destruction of allied forces' cotton tents during WWII. Since its isolation, it has been extensively studied in relation to its cellulolytic capability and various cellulase hyper‐ producing strains have been developed, with RUT C30 currently the benchmark strain for production of cellulases in high yields.[54] One of the problems associated with this fungus has been the low expression of β-glucosidases, the enzymes responsible for liberation of glucose from short oligosaccharides. This, however, has been overcome with genetic engi‐ neering and supplementation of commercial preparations with foreign β-glucosidases.[55] Other promising fungal sources for cellulases exist, such as *Acremonium*, *Penicillium* and

towards commercialization of lignocellulosic biomass processing.

46 Environmental Biotechnology - New Approaches and Prospective Applications

*Chrysosporium* strains.[52] Their cellulase properties are comparable to those of *T. reesei*, however, they are unlikely to replace it as the standard enzyme source due to the amount of improvement already achieved with the latter. Bacterial cellulases have been the focus of some attention due to the higher robustness observed with some hyperthermophilic enzymes, making them more adaptable to the harsh conditions of industrial processes.[56] However, the production of cellulases as part of complexed systems (cellulosomes) in anaerobes, as well as the much lower protein yields in bacteria, means that interest in these enzymes is mainly restricted to their heterologous expression in fungi and use in consolidated bioprocessing (CBP, see *Consolidated fermentation*).[47, 56]

**6. Fermentation to fuels and chemicals**

reducing the overall cost of the production process.

advances in their genetic manipulation.[67]

uct toxicity issues.[123]

The vast amount of available know-how, due to the fact that this process is one of man's earliest biotechnological applications, continues to set the use of *Saccharomyces cerevisiae* for the production of ethanol as the benchmark fermentation system employed for second generation biofuel processes. This yeast's properties, particularly in relation to robustness, toxicity, ethanol productivities approaching the theoretical maximum and ease of genetic manipulation make it an extremely suitable microorganism for the fermentation step of lignocellulose conversion.[61, 62] As a result, much has already been accomplished in production of efficient yeast strains for the conversion of hexoses from starchy feedstock in first generation biofuel production. The issues that require addressing for carrying over these microorganisms to second generation biofuel production processes relate to tolerance to by-products of lignocel‐ lulose pretreatment and digestion, and the ability to ferment pentoses generated by the hemicellulosic fraction of the biomass. Furthermore, the possibility of combining efficient pentose and hexose utilisation as well as production of lignocellulose hydrolysing enzymes within a single host would allow the combination of hydrolysis and fermentation steps, greatly

Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass

http://dx.doi.org/10.5772/55309

49

Other types of microorganisms have also been investigated as alternatives, mainly for the coproduction of other compounds. A recent review by Jang et al*.*[63] lists organisms according to their corresponding C2-C6 platform chemical products (Figure 3 and Table 2). Anaerobic clostridial strains have been of particular interest due to their ability to efficiently generate butanol as well as their tolerance to other common metabolites (acetate, lactate) which, they are able to use as nutrients for the further production of alcohols.[64, 65] As a result the use of microorganisms such as *Clostridium acetobutylicum* has been proposed for acetone-butanolethanol (ABE) bioprocesses, since butanol is an attractive alternative to ethanol as a biofuel due to its lower vapour pressure and higher energy density.[66] *Clostridia* are also interesting because of the broad spectrum of chemicals that they are able to produce, as well as recent

Related types of yeast have also been investigated as alternatives in order to produce microorganisms with superior properties. Thermophilic yeasts show increased ability to work at elevated temperatures which may present great advantages, particularly in relation to *in situ* evaporative removal of the product in batch processes, a procedure that is generally considered essential for the reduction of down-stream costs as well as minimising prod‐

The pretreatment and hydrolysis of the complex mixture of lignocellulose, unlike the simple hydrolysis of starch, yields a number of additional by-products. These may pose problems to the growth of the microorganism fermenting the simple sugars as feedstock for fuel or chemical production. Toxic compounds encountered in lignocellulosic hydrolysates normally consist of phenolic compounds, weak organic acids and furan aldehydes.[61] Complex strategies have been employed to combat the effects of the presence of these compounds. Genetic engineering approaches have aimed at overexpression of pathways which, metabolise the inhibitors.[62]

### **5. Biochemical conversion of hemicellulose**

Hemicellulose (Figure 5) is a mixture of several different polysaccharides, the composition of which varies from plant to plant as well as within the same plant.[57] While cellulose is built from a single building block, a number of different monomers compose hemicellulosic heteropolymers including pentoses, hexoses and sugar acids. Commonly xylans, glucomann‐ ans, arabinogalactans and different types of glucans are found in hemicellulose. Xylans are comprised of β-1,4-linked xyloses interspersed with arabinose and glucuronic acid, while glucomannans are a mixture of β-1,4-linked glucose with α-1,6-substituted mannose side chains (Figure 5]. The presence of acetyl substitutions on hydroxyl groups of carbohydrates in hemicellulose is not completely understood, but may pose difficulties in hydrolysis due to the generation of acetate which acts as an enzyme and microorganism inhibitor.[58] The network of hemicellulosic chains is highly branched, cross-linking with cellulose microfibrils and lignin, creating a very compact material from which plant cell walls are composed. It is generally agreed that economically viable bioprocessing of lignocellulosic biomass requires efficient extraction and conversion of the hemicellulosic sugars.

As is the case with cellulases, hemicellulases constitute a useful tool for the generation of fermentable sugars from hemicellulose and are sometimes classed as cellulases themselves. Due to the diversity of components and complexity of structure found in this polymer it is only natural that a myriad of enzymes with different catalytic functions have been produced by microorganisms to effectively attack this matrix.[53, 59] Therefore, for example, endoxyla‐ nases, exoxylanases and β-xylosidases have been identified to break the linkages between xylose moieties, while esterases releasing acetyl and ferulic acid groups are also found amongst this category of enzymes. These enzymes sometimes display relative promiscuity towards the type of bond they hydrolyse, making it extremely difficult to measure a specific enzymatic activity. They also display significant synergy between themselves as well as with other lignocellulose hydrolysing enzymes.[52] As expected, microorganisms that express cellulose degrading enzymes also possess the ability to degrade hemicellulosic polymers. Accordingly it has been highlighted that *Trichoderma* and *Penicillium* fungi contain efficient hemicellulolytic catalysts.[59] Another group of fungi identified for their important xylan degrading capabili‐ ties have been *Aspergillus* spp.[60]

### **6. Fermentation to fuels and chemicals**

*Chrysosporium* strains.[52] Their cellulase properties are comparable to those of *T. reesei*, however, they are unlikely to replace it as the standard enzyme source due to the amount of improvement already achieved with the latter. Bacterial cellulases have been the focus of some attention due to the higher robustness observed with some hyperthermophilic enzymes, making them more adaptable to the harsh conditions of industrial processes.[56] However, the production of cellulases as part of complexed systems (cellulosomes) in anaerobes, as well as the much lower protein yields in bacteria, means that interest in these enzymes is mainly restricted to their heterologous expression in fungi and use in consolidated bioprocessing

Hemicellulose (Figure 5) is a mixture of several different polysaccharides, the composition of which varies from plant to plant as well as within the same plant.[57] While cellulose is built from a single building block, a number of different monomers compose hemicellulosic heteropolymers including pentoses, hexoses and sugar acids. Commonly xylans, glucomann‐ ans, arabinogalactans and different types of glucans are found in hemicellulose. Xylans are comprised of β-1,4-linked xyloses interspersed with arabinose and glucuronic acid, while glucomannans are a mixture of β-1,4-linked glucose with α-1,6-substituted mannose side chains (Figure 5]. The presence of acetyl substitutions on hydroxyl groups of carbohydrates in hemicellulose is not completely understood, but may pose difficulties in hydrolysis due to the generation of acetate which acts as an enzyme and microorganism inhibitor.[58] The network of hemicellulosic chains is highly branched, cross-linking with cellulose microfibrils and lignin, creating a very compact material from which plant cell walls are composed. It is generally agreed that economically viable bioprocessing of lignocellulosic biomass requires efficient

As is the case with cellulases, hemicellulases constitute a useful tool for the generation of fermentable sugars from hemicellulose and are sometimes classed as cellulases themselves. Due to the diversity of components and complexity of structure found in this polymer it is only natural that a myriad of enzymes with different catalytic functions have been produced by microorganisms to effectively attack this matrix.[53, 59] Therefore, for example, endoxyla‐ nases, exoxylanases and β-xylosidases have been identified to break the linkages between xylose moieties, while esterases releasing acetyl and ferulic acid groups are also found amongst this category of enzymes. These enzymes sometimes display relative promiscuity towards the type of bond they hydrolyse, making it extremely difficult to measure a specific enzymatic activity. They also display significant synergy between themselves as well as with other lignocellulose hydrolysing enzymes.[52] As expected, microorganisms that express cellulose degrading enzymes also possess the ability to degrade hemicellulosic polymers. Accordingly it has been highlighted that *Trichoderma* and *Penicillium* fungi contain efficient hemicellulolytic catalysts.[59] Another group of fungi identified for their important xylan degrading capabili‐

(CBP, see *Consolidated fermentation*).[47, 56]

**5. Biochemical conversion of hemicellulose**

48 Environmental Biotechnology - New Approaches and Prospective Applications

extraction and conversion of the hemicellulosic sugars.

ties have been *Aspergillus* spp.[60]

The vast amount of available know-how, due to the fact that this process is one of man's earliest biotechnological applications, continues to set the use of *Saccharomyces cerevisiae* for the production of ethanol as the benchmark fermentation system employed for second generation biofuel processes. This yeast's properties, particularly in relation to robustness, toxicity, ethanol productivities approaching the theoretical maximum and ease of genetic manipulation make it an extremely suitable microorganism for the fermentation step of lignocellulose conversion.[61, 62] As a result, much has already been accomplished in production of efficient yeast strains for the conversion of hexoses from starchy feedstock in first generation biofuel production. The issues that require addressing for carrying over these microorganisms to second generation biofuel production processes relate to tolerance to by-products of lignocel‐ lulose pretreatment and digestion, and the ability to ferment pentoses generated by the hemicellulosic fraction of the biomass. Furthermore, the possibility of combining efficient pentose and hexose utilisation as well as production of lignocellulose hydrolysing enzymes within a single host would allow the combination of hydrolysis and fermentation steps, greatly reducing the overall cost of the production process.

Other types of microorganisms have also been investigated as alternatives, mainly for the coproduction of other compounds. A recent review by Jang et al*.*[63] lists organisms according to their corresponding C2-C6 platform chemical products (Figure 3 and Table 2). Anaerobic clostridial strains have been of particular interest due to their ability to efficiently generate butanol as well as their tolerance to other common metabolites (acetate, lactate) which, they are able to use as nutrients for the further production of alcohols.[64, 65] As a result the use of microorganisms such as *Clostridium acetobutylicum* has been proposed for acetone-butanolethanol (ABE) bioprocesses, since butanol is an attractive alternative to ethanol as a biofuel due to its lower vapour pressure and higher energy density.[66] *Clostridia* are also interesting because of the broad spectrum of chemicals that they are able to produce, as well as recent advances in their genetic manipulation.[67]

Related types of yeast have also been investigated as alternatives in order to produce microorganisms with superior properties. Thermophilic yeasts show increased ability to work at elevated temperatures which may present great advantages, particularly in relation to *in situ* evaporative removal of the product in batch processes, a procedure that is generally considered essential for the reduction of down-stream costs as well as minimising prod‐ uct toxicity issues.[123]

The pretreatment and hydrolysis of the complex mixture of lignocellulose, unlike the simple hydrolysis of starch, yields a number of additional by-products. These may pose problems to the growth of the microorganism fermenting the simple sugars as feedstock for fuel or chemical production. Toxic compounds encountered in lignocellulosic hydrolysates normally consist of phenolic compounds, weak organic acids and furan aldehydes.[61] Complex strategies have been employed to combat the effects of the presence of these compounds. Genetic engineering approaches have aimed at overexpression of pathways which, metabolise the inhibitors.[62]


Another approach is adaptation of the microorganism to an inhibitor rich environment through evolutionary processes. It has been observed that the stress imposed stimulates changes in the resulting strains, usually in relation to glycolytic enzyme activity, levels of intracellular materials and expression of inhibitor metabolising enzymes, which impart increased tolerance. The new strains are generally able to grow at higher hydrolysate, and consequently inhibitor, concentrations thus reducing processing time and cost.[124] Cell viability is also threatened by the target products of fermentation, as these may cause damage to cell membranes and interfere with physiological processes. Tolerance to these compounds without decreasing the process yield may be achieved by regulation of membrane transporters such as efflux pumps, modification of the membrane composition or regulation of heat shock proteins that have been found to be linked to stress response in cells.[125] An added benefit to the increase of tolerance in some cases may be an increase in product yield.[126, 127]

Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass

**Strain Inhibitor Approach Reference**

*S. cerevisiae* PK113-7D formate, acetate Expression of formate dehydrogenase structural gene

*S. cerevisiae* vanillin Overexpression of laccase gene *lac*A from *Trametes sp.*

*S. cerevisiae* ethanol, glucose Global transcriptional machinery engineering, also

**Table 3.** Examples of engineering microorganisms for improved tolerance to inhibitors in lignocellulosic biomass

Microorganisms naturally capable of fermenting pentoses such as *Pichia stipitis*, *Kluyveromyces marxianus*, *Clostridium saccharolyticum* and *Thermoanaerobacter ethanolicus* exist and may well be employed in processes for the production of ethanol as well as other chemicals.[62] However, considerable effort has been put into engineering pentose fermentation capability into strains traditionally used for ethanol production, such as *S. cerevisiae,* with great success. This yeast is able to take up pentose with hexose transporters, however the ability to metabolise these sugars had to be introduced with expression of bacterial and fungal gene insertion. This has also led towards engineering hexose/pentose efficient cofermentation, something that has not been identified in native microorganisms. The ability to coferment xylose, arabinose and glucose has been successfully introduced to *S. cerevisiae*, however modern approaches to metabolic engineering need to be employed in order to improve on this, concentrating more on non-traditional aspects of cell engineering, such as catabolism repression mechanisms and

*C. acetobutylicum* butanol Overexpression of GroESL heat shock protein [127] *E. coli* isobutanol Simultaneous disruption of five unrelated genes [132]

Deletion of *HRK*1 gene regulating membrane transporter activity

FAHD2

AH28-2

improved ethanol yield by 15% [126]

biogasoline RND efflux pumps heterologously expressed [131]

[128]

http://dx.doi.org/10.5772/55309

51

[129]

[130]

*S. cerevisiae* acetate

*E. coli* biodiesel,

processing.

stress response.[133]

Related types of yeast have also been investigated as alternatives in order to produce microorganisms with superior properties. Thermophilic yeasts show increased ability to work at elevated temperatures which may present great advantages, particularly in **Table 2.** Current status of the production of platform chemicals using microorganisms. Duplicated with permission.[63]

relation to *in situ* evaporative removal of the product in batch processes, a procedure that is generally considered essential for the

The pretreatment and hydrolysis of the complex mixture of lignocellulose, unlike the simple hydrolysis of starch, yields a number of additional by-products. These may pose problems to the growth of the microorganism fermenting the simple sugars as feedstock for fuel or chemical production. Toxic compounds encountered in lignocellulosic hydrolysates normally consist of phenolic

Figure 6. Current status of the production of platform chemicals using microorganisms. Duplicated with permission.[63]

reduction of down-stream costs as well as minimising product toxicity issues.[123]

Another approach is adaptation of the microorganism to an inhibitor rich environment through evolutionary processes. It has been observed that the stress imposed stimulates changes in the resulting strains, usually in relation to glycolytic enzyme activity, levels of intracellular materials and expression of inhibitor metabolising enzymes, which impart increased tolerance. The new strains are generally able to grow at higher hydrolysate, and consequently inhibitor, concentrations thus reducing processing time and cost.[124] Cell viability is also threatened by the target products of fermentation, as these may cause damage to cell membranes and interfere with physiological processes. Tolerance to these compounds without decreasing the process yield may be achieved by regulation of membrane transporters such as efflux pumps, modification of the membrane composition or regulation of heat shock proteins that have been found to be linked to stress response in cells.[125] An added benefit to the increase of tolerance in some cases may be an increase in product yield.[126, 127]

Platform

3- Hydroxypropio nic acid

1,2 propanediol *C.* 

Succinic acid Engineered rumen

Fumaric acid *R. arrhizus* 

C5 Itaconic acid *Aspergillus terreus*  IFO-6365

3-

2-methyl-1-

3-methyl-1-

chemical Leading host Substrates and/or conditions Titer

*S. cerevisiae* Cellobiose, xylose, and glucose, batch

C2 Ethanol *S. cerevisiae* Ammonia fiber expansion (AFEX)-corn stover

50 Environmental Biotechnology - New Approaches and Prospective Applications

C3 Propionic acid P. acidipropionici Glycerol, fed-batch fermentation in fibrous bed

Lactic acid *Sporolactobacillus* Glucose, fed-batch supplemented with 40g/L

C4 Butyric acid *C. tyrobutyricum* Glucose fed-batch fermentation 32.5-

hydroxyvalerate *P. putida* Glucose and levulinic acid, flask batch

Xylitol *C. tropicalis* Xylose, oxygen-limited condition with cell

Figure 6. Current status of the production of platform chemicals using microorganisms. Duplicated with permission.[63]

reduction of down-stream costs as well as minimising product toxicity issues.[123]

*C. tyrobutyricum* Glucose, repeated fed-batch fermentation by

(g/L)

41.7

146

immobilized cells in a fibrous bed bioreactor 86.9 0.46 1.1 [87]

0.38-

0.92-

0.42 0.24-0.68 [85,

0.88 1.8-2.8 [88,

1.1 1.9-2.5 [93,

0.36 0.20-0.29 [102,

86]

89]

92]

94]

103]

(CS)-hydrolysates, batch fermentation 40 0.46 0.8 [68]

fermentation 48 0.37 0.8 [69]

bioreactor 106 0.56 0.035 [73]

peanut meal 207 0.93 3.8 [74]

*Z. mobilis* Glucose and xylose, batch fermention 62 0.46 1.29 [70] *E. coli* Xylose, batch fermentation 23.5 0.48 n/a [71]

*E.coli* Glucose, fed-batch fermentation 138 0.86 3.5 [75]

*K. pneumonia* Glycerol, fed-batch fermentation 16 n/a 0.01 [76] *E. coli* Glycerol, fed-batch fermentation 38.7 0.34 0.53 [77]

*E. coli* Glucose, batch-fed fermentation 13.6 0.15 0.28 [80]

*thermosaccharolyticum* Glucose, anaerobic batch fermentation 9.1 0.20 0.35 [81] *E. coli* Glycerol, batch fermentation 5.6 0.21 0.077 [82]

*E. coli* Glucose, fed-batch 10L fermentation 135 0.51 3.5 [84]

*E. coli* Glucose, fed-batch fermentation 73-87 0.8-1.0 0.7-0.9 [90-

*S. cerevisia* Glucose, fed-batch fermentation 59 0.31 0.19 [96] *E. coli* Glucose, two-stage fermentation 33.9 0.47 1.06 [97]

NRRL 2582 Glucose, batch fermentation 97.7 0.81 1.02 [98]

*C. glutamicum* Glucose, batch fermentation 2.2 n/a 0.01 [100]

*C. glutamicum* Glucose, fed-batch fermentation 13.0 0.20 0.33 [105]

*S. marcescens* Glucose, fed-batch fermentation 152 0.46 2.67 [108]

*E. coli* Glucose, flask batch culture 6 0.61 0.06 [111]

*E. coli* Glucose and threonine, flask batch cultivate 1.3 n/a n/a [112] *E. coli* Glucose, flask batch cultivation 0.81 n/a n/a [112]

*E. coli* Glucose and xylose, fed-batch fermentation 38 n/a n/a [117]

fermentation 82-85 0.54 0.57 [110]

cultivation 5.3 n/a n/a [112]

recycling 1.82 0.85 12.0 [116]

Acetic acid *A. aceti* Ethanol, batch fermentation 111.7 n/a 0.6 [72]

Propanol *E. coli* Glucose, flask culture 3.9 n/a 0.04 [78] Iso-propanol *C. acetobutylicum* Glucose, anaerobic flask culture 5.1 n/a n/a [79]

1,3 propanediol *C. acetobutylicum* Glycerol, anaerobic fed-batch fermentation 83.6 0.54 1.70 [83]

bacteria Glucose, anaerobic fed-batch fermentation 52-106 0.76-

Malic acid *Aspergillus flavus* Glucose, batch fermentation 113 0.95 0.59 [95]

GABA *L. brevis* NCL912 Glucose and glutamate, fed-batch fermentation 103.7 n/a n/a [99]

1-butanol *C. acetebutylicum* Glucose, anaerobic batch fermentation 16.7 n/a 0.31 [101] *E. coli* Glucose, batch cultivation 14-15 0.33-

Isobutanol *E. coli* Glucose, batch cultivation 20 n/a n/a [104]

1,4-butanediol *E. coli* Glucose, microaerobic fed-batch fermentation 18 n/a 0.15 [106] 2,3-butanediol *K. pneumonia* SDM Glucose, fed-batch fermentation 150 0.48 4.21 [107]

Putrescine *E. coli* Glucose, fed-batch culture 24.2 n/a 0.75 [109]

Glucose and corn steep, flask and 100 L batch

1-pentanol *E. coli* Glucose 0.5 n/a n/a [113]

butanol *E. coli* Glucose 1.25 n/a 0.17 [114]

butanol *E. coli* Glucose 1.28 n/a 0.11 [115]

Cadaverine *E. coli* Glucose, fed-batch fermentation 9.61 n/a 0.12 [118] C6 Glucaric acid *E. coli* Glucose, flask culture 2.5 n/a n/a [119] Anthranilic acid *E. coli* Glucose, fed-batch cultivation 14 0.20 0.41 [120] Phenol *P. putida* S12 Glucose, flask batch culture 0.14 3.5 0.006 [121] Catechol *P. putida* ML2 3-Dehydroshikimate 4.2 n/a 0.12 [122]

Related types of yeast have also been investigated as alternatives in order to produce microorganisms with superior properties. Thermophilic yeasts show increased ability to work at elevated temperatures which may present great advantages, particularly in relation to *in situ* evaporative removal of the product in batch processes, a procedure that is generally considered essential for the

**Table 2.** Current status of the production of platform chemicals using microorganisms. Duplicated with permission.[63]

The pretreatment and hydrolysis of the complex mixture of lignocellulose, unlike the simple hydrolysis of starch, yields a number of additional by-products. These may pose problems to the growth of the microorganism fermenting the simple sugars as feedstock for fuel or chemical production. Toxic compounds encountered in lignocellulosic hydrolysates normally consist of phenolic

*C. glutamicum* Glucose, fed-batch fermentation 140-

Yield (g/g)

Productivity (g/L/h) Ref


**Table 3.** Examples of engineering microorganisms for improved tolerance to inhibitors in lignocellulosic biomass processing.

Microorganisms naturally capable of fermenting pentoses such as *Pichia stipitis*, *Kluyveromyces marxianus*, *Clostridium saccharolyticum* and *Thermoanaerobacter ethanolicus* exist and may well be employed in processes for the production of ethanol as well as other chemicals.[62] However, considerable effort has been put into engineering pentose fermentation capability into strains traditionally used for ethanol production, such as *S. cerevisiae,* with great success. This yeast is able to take up pentose with hexose transporters, however the ability to metabolise these sugars had to be introduced with expression of bacterial and fungal gene insertion. This has also led towards engineering hexose/pentose efficient cofermentation, something that has not been identified in native microorganisms. The ability to coferment xylose, arabinose and glucose has been successfully introduced to *S. cerevisiae*, however modern approaches to metabolic engineering need to be employed in order to improve on this, concentrating more on non-traditional aspects of cell engineering, such as catabolism repression mechanisms and stress response.[133]

### **7. Consolidated fermentation**

One of the great advantages of biochemical methods of biomass conversion is that they all require mild conditions, which makes them relatively compatible, allowing for potential consolidation of processing steps. This has been identified as an area of great potential in relation to process optimization, cost reduction and ultimately biorefinery commercialization. Instead of applying four distinct biochemical processing steps (cellulose production, cellulose hydrolysis, hexose fermentation, pentose hydrolysate fermentation), a setup termed Separate Hydrolysis and Fermentation (SHF), two or more steps may be consolidated leading to alternate process configurations for biomass conversion.[47] This requires generation of biocatalysts with properties suited to the optimum processing conditions, or engineering of microorganisms with more than one processing capability. Simultaneous Saccharification and Fermentation (SSF) involves performing cellulase-catalysed cellulose hydrolysis in the cellulose hydrolysate fermenter, after the enzymes are produced in a separate fermentation. Further consolidation may include cofermentation of the hemicellulose hydrolysate, either by a separate pentose utilising microorganism or by an engineered strain capable of efficient cofermentation of hexoses and pentoses. This configuration is termed Simultaneous Saccha‐ rification and Cofermentation (SSCF). The most desirable setup that minimises utility costs is direct fermentation of biomass to the product of choice with the aid of a cellulase expressing, hexose/pentose cofermenting microorganism. This approach was first introduced in 1996 as consolidated bioprocessing (CBP).[53, 134]

**8. Future outlook for commercialization**

ute to the economic viability of these processes.

cialization of any process based on lignocellulosic feedstock.

commercialized.

Inedible crops are a renewable and sustainable source of fuels and chemicals and it has been estimated that replacing corn with cellulosic stock would result in an 82% increase in bioe‐ thanol production. Despite this, 2nd generation biofuel and chemical production is yet to be

Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass

http://dx.doi.org/10.5772/55309

53

Finding economical pretreatment methods has been recognized as one of the hurdles to commercializing 2nd generation biofuels and chemicals. The results listed in Table 1 show that fungal treatment can reduce lignin content of biomass and in most cases improve sugar yields. However, chemical methods are deemed more economical at present, mainly due to the long incubation times as well as the loss of holocellulose during biological pretreatment. With further screening studies, it is to be expected that fungal strains with selectivity for lignin and faster metabolic processing will be discovered, thus reducing the overall process cost. Bacteria present advantages for biotechnological applications in terms of growing times and being more prone to metabolic manipulation. As discussed above, bacterial strains have been identified that convert lignin into valuable phenolic monomers. The conversion of all plant components, and in particular the lignin fraction, is the basis for 2nd generation biorefineries, where a vast array of products may be prepared in conjunction with a central fermentation for biofuel production. Therefore integration of bacterial utilization of lignin will greatly contrib‐

The cost of hydrolytic enzyme production greatly influences the overall cost of cellulosic biomass conversion thus hindering commercialization.[135] There have been great strides forward in this respect with the estimated cost being driven down from US\$5.40 to US\$0.20 per gallon of ethanol produced, according to claims of major enzyme producers.[136] The use of such information however in techno-economical analysis of biomass conversion is prob‐ lematic. These values relate to the production of a specific target, usually ethanol, and depend on a range of variables other than enzyme production. Klein-Marcuschamer et al. prepared a model for the calculation of the cost for the production of cellulases from *T. reesei* that could then be applied to another model for the estimation of its contribution to the cost of ethanol production.[137] The results showed that there is systematic underestimation of the contribu‐ tion of enzyme costs to biofuels production in the literature, as conservative calculations pointed to around US\$1.00 per gallon ethanol. The authors highlighted that approaches aiming to decrease enzyme loading in the pretreatment steps should become a focus point. It seems that lowering the enzyme production cost will be a significant obstacle towards the commer‐

Despite the hurdles that need to be overcome for commercialization, there is much anticipation from federal governements that biofuels and chemicals derived from lignocellulosic biomass will play a central role in overcoming fossil fuel dependence. In October 2012 the EU com‐ mission issued a proposal stating that advanced biofuel development has to be encouraged due to their high greenhouse gas savings and lower risk of land use change, and this should be mirrored in post-2020 renewable energy policies.[138] In accordance with this a directive was proposed to limit the allowed contribution of food crop derived biofuels, towards the 10%

**Figure 6.** Consolidated fermentation processes.

### **8. Future outlook for commercialization**

**7. Consolidated fermentation**

52 Environmental Biotechnology - New Approaches and Prospective Applications

consolidated bioprocessing (CBP).[53, 134]

**Figure 6.** Consolidated fermentation processes.

**Production of**

**Hydrolases Saccharification Hexose**

<sup>O</sup> <sup>O</sup> O O O

O O <sup>O</sup> <sup>O</sup> O O O

O O O O O O O

> O O

**Fermentation**

**Pentose Fermentation**

**SHF**

**SSF**

**SSCF**

**CBP**

OH OH HO HO O

OH O OHH HO O HO

One of the great advantages of biochemical methods of biomass conversion is that they all require mild conditions, which makes them relatively compatible, allowing for potential consolidation of processing steps. This has been identified as an area of great potential in relation to process optimization, cost reduction and ultimately biorefinery commercialization. Instead of applying four distinct biochemical processing steps (cellulose production, cellulose hydrolysis, hexose fermentation, pentose hydrolysate fermentation), a setup termed Separate Hydrolysis and Fermentation (SHF), two or more steps may be consolidated leading to alternate process configurations for biomass conversion.[47] This requires generation of biocatalysts with properties suited to the optimum processing conditions, or engineering of microorganisms with more than one processing capability. Simultaneous Saccharification and Fermentation (SSF) involves performing cellulase-catalysed cellulose hydrolysis in the cellulose hydrolysate fermenter, after the enzymes are produced in a separate fermentation. Further consolidation may include cofermentation of the hemicellulose hydrolysate, either by a separate pentose utilising microorganism or by an engineered strain capable of efficient cofermentation of hexoses and pentoses. This configuration is termed Simultaneous Saccha‐ rification and Cofermentation (SSCF). The most desirable setup that minimises utility costs is direct fermentation of biomass to the product of choice with the aid of a cellulase expressing, hexose/pentose cofermenting microorganism. This approach was first introduced in 1996 as

Inedible crops are a renewable and sustainable source of fuels and chemicals and it has been estimated that replacing corn with cellulosic stock would result in an 82% increase in bioe‐ thanol production. Despite this, 2nd generation biofuel and chemical production is yet to be commercialized.

Finding economical pretreatment methods has been recognized as one of the hurdles to commercializing 2nd generation biofuels and chemicals. The results listed in Table 1 show that fungal treatment can reduce lignin content of biomass and in most cases improve sugar yields. However, chemical methods are deemed more economical at present, mainly due to the long incubation times as well as the loss of holocellulose during biological pretreatment. With further screening studies, it is to be expected that fungal strains with selectivity for lignin and faster metabolic processing will be discovered, thus reducing the overall process cost. Bacteria present advantages for biotechnological applications in terms of growing times and being more prone to metabolic manipulation. As discussed above, bacterial strains have been identified that convert lignin into valuable phenolic monomers. The conversion of all plant components, and in particular the lignin fraction, is the basis for 2nd generation biorefineries, where a vast array of products may be prepared in conjunction with a central fermentation for biofuel production. Therefore integration of bacterial utilization of lignin will greatly contrib‐ ute to the economic viability of these processes.

The cost of hydrolytic enzyme production greatly influences the overall cost of cellulosic biomass conversion thus hindering commercialization.[135] There have been great strides forward in this respect with the estimated cost being driven down from US\$5.40 to US\$0.20 per gallon of ethanol produced, according to claims of major enzyme producers.[136] The use of such information however in techno-economical analysis of biomass conversion is prob‐ lematic. These values relate to the production of a specific target, usually ethanol, and depend on a range of variables other than enzyme production. Klein-Marcuschamer et al. prepared a model for the calculation of the cost for the production of cellulases from *T. reesei* that could then be applied to another model for the estimation of its contribution to the cost of ethanol production.[137] The results showed that there is systematic underestimation of the contribu‐ tion of enzyme costs to biofuels production in the literature, as conservative calculations pointed to around US\$1.00 per gallon ethanol. The authors highlighted that approaches aiming to decrease enzyme loading in the pretreatment steps should become a focus point. It seems that lowering the enzyme production cost will be a significant obstacle towards the commer‐ cialization of any process based on lignocellulosic feedstock.

Despite the hurdles that need to be overcome for commercialization, there is much anticipation from federal governements that biofuels and chemicals derived from lignocellulosic biomass will play a central role in overcoming fossil fuel dependence. In October 2012 the EU com‐ mission issued a proposal stating that advanced biofuel development has to be encouraged due to their high greenhouse gas savings and lower risk of land use change, and this should be mirrored in post-2020 renewable energy policies.[138] In accordance with this a directive was proposed to limit the allowed contribution of food crop derived biofuels, towards the 10% 2020 renewable transportation fuel objective, to only 5%. This means that a greater contribution from lignocellulosic biomass and especially agricultural waste derived biofuels will be required. Experts including those from Shell Corporation recognized that substantial research and development from industry and academia is still required in order to achieve this target. However, it is generally agreed upon that the rapid advances in enzyme, microbial and plant engineering as well as biocatalyst optimization suggest that biochemical processes are much more likely to provide the necessary breakthroughs that will propel second generation biofuels and chemicals into the marketplace.

[7] Sierra R, Smith A, Granda C, Holtzapple MT. Producing fuels and chemicals from

Biochemical Processes for Generating Fuels and Commodity Chemicals from Lignocellulosic Biomass

http://dx.doi.org/10.5772/55309

55

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### **Author details**

Amy Philbrook, Apostolos Alissandratos and Christopher J. Easton\*

\*Address all correspondence to: easton@rsc.anu.edu.au

CSIRO Biofuels Research Cluster, Research School of Chemistry, Australian National Uni‐ versity, Canberra ACT, Australia

### **References**


[7] Sierra R, Smith A, Granda C, Holtzapple MT. Producing fuels and chemicals from lignocellulosic biomass. Chem Eng Prog. 2008 Aug;104(8):S10-S8.

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CSIRO Biofuels Research Cluster, Research School of Chemistry, Australian National Uni‐

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54 Environmental Biotechnology - New Approaches and Prospective Applications

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**Chapter 4**

**Synergistic Effects of Pretreatment**

**Process on Enzymatic Digestion of**

Additional information is available at the end of the chapter

Prihardi Kahar

**1. Introduction**

http://dx.doi.org/10.5772/54949

**Rice Straw for Efficient Ethanol Fermentation**

Lignocellulose, the most abundant renewable biomass produced by plants from photosyn‐ thesis, has a yearly supply of approximately 200 billion metric tons worldwide (Ragauskas et al., 2006; Zhang et al., 2006). Lignocellulosic biomass is widely expected to be a major resource for biorefineries, including bioethanol (Lin and Tanaka, 2006). The composition of lignocellu‐ lose varies depending on plant species, plant parts, growth conditions, etc. (Ding and Himmel, 2006; Zhang and Lynd, 2004), and their structures are rigid and low degradable against cellulase enzymes. In general, the lignocellulose structure is composed by three major components: crystalline cellulose, amorphous hemicellulose and non-sugar lignin. Cellulose microfibrils are coated with hemicellulose matrices building holocellulose structures and severely protected by lignin outside. The structures are rigidly packed to form a physical barrier for cellulase access to cellulose chains (Mansfield et al., 1999). To hydrolyze them efficiently into sugars, a high dosage of commercial available cellulase enzymes is required. At a current technical stage, 20 g-cellulase is needed to hydrolysis 1 kg cellulose at 70% for 5 days (Gusakov, 2011; Roche et al., 2009). However, the baseline production cost of cellulase is still expensive as reported to be \$10.14/kg (Klein-Marcuschamer et al., 2012). If the recalcitrance problem remains unresolved, it is not feasible for high-solids enzymatic saccharification.

The ability of cellulase access to cellulose chains within microfibrils will be limited even if lignin is completely removed from the cellulose structure, because its ability is generally limited to accessing the outer layer of the microfibrils (Mansfield et al., 1999). Although cellulose can be slowly eroded by surface shaving or planning, cellulose chains in highly ordered and tightly packed regions of microfibrils must be disintegrated by delamination,

> © 2013 Kahar; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Kahar; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

