**1. Introduction**

Much of Earth's recent geologic history is dominated by periods of extensive glaciation, with relatively low global mean temperatures and correspondingly low atmospheric CO2 concen‐ trations [1]. The current interglacial period stands out as an anomaly because the atmospheric CO2 concentration has risen sharply above the range of approximately 180-280 parts per million by volume that has defined the past 420,000 years to reach levels that are nearly 40% higher than the biosphere has experienced over this time frame [2]. This rapid increase in CO2 concentration is primarily due to the release of ancient fixed atmospheric CO2 into the modern atmosphere through the combustion of fossil fuel resources over the past 200 years. Since it is clear from ice core records that atmospheric CO2 concentration has a strong positive correlation to global temperature, it is expected that changes to global climate are forthcoming [3]. There are substantial uncertainties regarding the ability of terrestrial and oceanic carbon sinks to absorb this anthropogenic CO2 on time scales that are relevant to human society [2], so the continued release of ancient CO2 into the modern atmosphere at current rates carries with it an important risk of inducing climate changes of unknown amplitude along with a host of ancillary changes that are difficult to predict with certainty. This has led to the search for alternatives to fossil fuels to meet a rising global energy demand, and one such option is the use of extant organic matter to produce energy. This resource contains carbon that was fixed from the modern atmosphere, which means it does not result in a net increase in atmospheric CO2 upon combustion.

© 2013 Canam 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 Canam 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.

Meeting the world's energy demands requires resources that are abundant and inexpensive to produce. Biomass from forestry and agricultural activities is certainly a candidate, as hundreds of millions of tonnes of agricultural waste from rice, wheat, corn, and other crops are produced worldwide, which could generate billions of litres of ethanol [4]. For ethanol, butanol, methane, and other biofuels to be produced economically, however, requires an integrated approach, with a number of value-added co-products produced in addition to the energy – a "biorefinery" that stands in analogy to petroleum refineries that produce both energy and a wide range of petroleum-based chemicals and products [5-7]. The biorefinery concept is hardly new, as the industrial-scale bacterial fermentation of starch to acetone and butanol (A-B) was developed a century ago. These A-B fermentations were done on an industrial scale in the West during World War I and persisted into the 1950's. They continued in Russia until late in the Soviet era, ultimately using corn cobs and other agricultural residues as input [8]. However, releasing the energy and co-product potential of plant-based material requires energy inputs and processing steps, as discussed below; this hinders the ability of biofuels to compete economically with petroleum resources, which have been exposed to millions of years of geological energy input to reach their current biochemical state.

penetration, which greatly decreases the yields of fermentable sugars and negatively affects the overall process of energy production from these resources to the extent that it is uneco‐ nomical [5, 16]. To overcome this limitation, some form of pretreatment of the biomass is required for economical and efficient production of biofuels by any of the strategies described

Pretreatment of Lignocellulosic Biomass Using Microorganisms: Approaches, Advantages, and Limitations

http://dx.doi.org/10.5772/55088

183

The purpose of this chapter is to review the various pretreatment options available for lignocellulosic biomass, with particular emphasis on agricultural residues and on strategies that exploit the natural metabolic activity of microbes to increase the processability of the biomass. These microbial-based strategies can be effective pretreatments on their own or, more probably, can be used in combination with thermomechanical pretreatments in order to provide a cost-effective means to make lignocellulosic substrates available for conversion to biofuels by microorganisms. The key advantages and disadvantages of this strategy will be presented along with a vision for how microbial pretreatment can be integrated into an

economical biorefinery process for biofuels and co-product production.

positive benefits to the overall economics of biofuels processes.

**2. Mechanical, thermomechanical and thermochemical pretreatments**

An early, essential mechanical pretreatment step is comminution, or mechanical particle size reduction, to transform the biomass from its native state into a suitable substrate for further pretreatment and energy production [17]. This step is often not considered in the energy balance of biofuels processes, but it is important to keep in mind that particle size reduction involves energy input that can influence the effective energy yield of these processes [22]. While smaller particle sizes are often considered to be more desirable for yields of fermentable sugars, sizes smaller than about 0.4-0.5 mm provide no additional benefit [17, 23], and the process becomes economically unfeasible at even smaller particle sizes [22]. Methods for mechanical size reduction include wet milling, dry milling, ball milling or vibratory ball milling, and other forms of chipping and grinding of biomass [4, 17]. Regardless of the method employed, particle size reduction requires energy input; therefore, strategies that facilitate the production of biomass in the proper size range while minimizing energy input will provide

A wide range of options is available for preparing ground biomass for further processing. One of the most common and simple technologies for rendering the carbohydrate fraction available for biofuels production is the application of a dilute solution of sulfuric acid (0.5%-2%) at temperatures of 140°C – 180°C with residence times of 10-30 minutes [24]. This process leaves a residue that is depleted in hemicellulose but retains most of the cellulose intact, making it an ideal substrate for enzymatic hydrolysis to yield fermentable sugars for ethanol production. There is a range of conditions for acid hydrolysis that will result in more or less carbohydrate remaining in the solid fraction, with the most severe conditions used to completely degrade the carbohydrate fraction for the determination of cell wall carbohydrate composition [25]. Harsher conditions (e.g. higher acid concentration and temperature), while resulting in a substrate that is highly digestible with enzymes to generate fermentable sugars, also result in

above [13, 16-21].

Current paradigms for biofuels production include the production of ethanol by yeast or bacteria from glucose produced from soluble sugars and starch (1st generation ethanol) or from the cellulosic fraction of biomass (2nd generation ethanol). Due to a lack of competition with food production, the latter is typically seen as more sustainable on a long-term basis [9, 10]. An emerging option is the co-production of ethanol and hydrogen via consolidated biopro‐ cessing [11]. In addition to the now little-used anaerobic A-B fermentations discussed above, another scheme for biofuels production from plant biomass involves the anaerobic production of methane by microbial consortia (anaerobic digestion) [12, 13]. The common link for all of these strategies is the exploitation and optimization of natural microbial activity to produce energy-rich molecules for combustion to produce energy. Direct thermochemical conversion of biomass via pyrolysis or gasification is also possible, although these strategies involve a large amount of energy input by heating the biomass to very high temperatures (normally >500°C) and are therefore independent of microbial activity [14].

Regardless of the means by which biofuels are produced by microbial activity from extant plant material, the same essential challenge must be faced: the substrate for biofuels production is the carbohydrate fraction, which must be made available to the microorganisms in order for the biochemical reactions to proceed efficiently. In the case of 1st generation ethanol, soluble sugars and starch are relatively easily converted to glucose that is fermented into ethanol by yeast. Strategies that utilize the non-food portion of crops, however, face a more formidable challenge. The resource from which energy is to be produced consists of three major biopol‐ ymers: cellulose (β(1,4)-linked glucose residues with a degree of polymerization up to ~15,000); hemicellulose (a heterogeneous, short-chained, branched carbohydrate with both 5- and 6 carbon sugars); and lignin (a complex aromatic polymer consisting of nonrepeating covalently linked units of coniferyl, sinapyl, and coumaryl alcohols). These polymers exist together in the plant as a composite, tightly interconnected molecule called lignocellulose [15]. Within lignocellulose, the lignin fraction in particular acts as a barrier to enzyme or microbial penetration, which greatly decreases the yields of fermentable sugars and negatively affects the overall process of energy production from these resources to the extent that it is uneco‐ nomical [5, 16]. To overcome this limitation, some form of pretreatment of the biomass is required for economical and efficient production of biofuels by any of the strategies described above [13, 16-21].

Meeting the world's energy demands requires resources that are abundant and inexpensive to produce. Biomass from forestry and agricultural activities is certainly a candidate, as hundreds of millions of tonnes of agricultural waste from rice, wheat, corn, and other crops are produced worldwide, which could generate billions of litres of ethanol [4]. For ethanol, butanol, methane, and other biofuels to be produced economically, however, requires an integrated approach, with a number of value-added co-products produced in addition to the energy – a "biorefinery" that stands in analogy to petroleum refineries that produce both energy and a wide range of petroleum-based chemicals and products [5-7]. The biorefinery concept is hardly new, as the industrial-scale bacterial fermentation of starch to acetone and butanol (A-B) was developed a century ago. These A-B fermentations were done on an industrial scale in the West during World War I and persisted into the 1950's. They continued in Russia until late in the Soviet era, ultimately using corn cobs and other agricultural residues as input [8]. However, releasing the energy and co-product potential of plant-based material requires energy inputs and processing steps, as discussed below; this hinders the ability of biofuels to compete economically with petroleum resources, which have been exposed to

182 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

millions of years of geological energy input to reach their current biochemical state.

>500°C) and are therefore independent of microbial activity [14].

Current paradigms for biofuels production include the production of ethanol by yeast or bacteria from glucose produced from soluble sugars and starch (1st generation ethanol) or from the cellulosic fraction of biomass (2nd generation ethanol). Due to a lack of competition with food production, the latter is typically seen as more sustainable on a long-term basis [9, 10]. An emerging option is the co-production of ethanol and hydrogen via consolidated biopro‐ cessing [11]. In addition to the now little-used anaerobic A-B fermentations discussed above, another scheme for biofuels production from plant biomass involves the anaerobic production of methane by microbial consortia (anaerobic digestion) [12, 13]. The common link for all of these strategies is the exploitation and optimization of natural microbial activity to produce energy-rich molecules for combustion to produce energy. Direct thermochemical conversion of biomass via pyrolysis or gasification is also possible, although these strategies involve a large amount of energy input by heating the biomass to very high temperatures (normally

Regardless of the means by which biofuels are produced by microbial activity from extant plant material, the same essential challenge must be faced: the substrate for biofuels production is the carbohydrate fraction, which must be made available to the microorganisms in order for the biochemical reactions to proceed efficiently. In the case of 1st generation ethanol, soluble sugars and starch are relatively easily converted to glucose that is fermented into ethanol by yeast. Strategies that utilize the non-food portion of crops, however, face a more formidable challenge. The resource from which energy is to be produced consists of three major biopol‐ ymers: cellulose (β(1,4)-linked glucose residues with a degree of polymerization up to ~15,000); hemicellulose (a heterogeneous, short-chained, branched carbohydrate with both 5- and 6 carbon sugars); and lignin (a complex aromatic polymer consisting of nonrepeating covalently linked units of coniferyl, sinapyl, and coumaryl alcohols). These polymers exist together in the plant as a composite, tightly interconnected molecule called lignocellulose [15]. Within lignocellulose, the lignin fraction in particular acts as a barrier to enzyme or microbial The purpose of this chapter is to review the various pretreatment options available for lignocellulosic biomass, with particular emphasis on agricultural residues and on strategies that exploit the natural metabolic activity of microbes to increase the processability of the biomass. These microbial-based strategies can be effective pretreatments on their own or, more probably, can be used in combination with thermomechanical pretreatments in order to provide a cost-effective means to make lignocellulosic substrates available for conversion to biofuels by microorganisms. The key advantages and disadvantages of this strategy will be presented along with a vision for how microbial pretreatment can be integrated into an economical biorefinery process for biofuels and co-product production.
