**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 positive benefits to the overall economics of biofuels processes.

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 a higher yield of compounds derived from pentoses (furfural), hexoses (5-hydroxymethylfur‐ fural) and lignin (low molecular weight phenolic compounds) that are inhibitory to subsequent fermentation by ethanologenic yeasts [26]. The mathematical concept of combined severity, which combines the various factors that define acid hydrolysis conditions (e.g. temperature, residence time, pH), allows objective comparisons between different conditions that enables the determination of optimal conditions for a given substrate [26]; however, doubts have been raised about its accuracy [17].

biofuel production by fermentation [4]. A successful combination of microwave and chemical pretreatments in a microwave-acid-alkali-hydrogen peroxide sequence resulted in efficient enzymatic saccharification of rice straw [36]. A related pretreatment option that has been exploited to improve the enzymatic digestibility of switchgrass is the use of radio frequency heating in combination with alkali; this treatment has the key advantage of allowing a much higher solids content than conventional heating [37]. Irradiation of biomass can also enhance

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

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A number of other pretreatment options exist, including ammonia fiber explosion (AFEX), liquid hot water, alkalai/wet oxidative pretreament, and others; several recent reviews discuss these processes and their advantages and disadvantages in detail [4, 17-20, 23, 30, 38, 39]. Regardless of the strategy employed, a common feature of any pretreatment option is that energy input is required. Pretreatment is a major part of the overall operating expense and energy efficiency of any biofuels process, and, while essential, typically accounts for over 30% of the costs of biorefinery operation [40, 41]. Strategies to reduce these costs will have a major

Microorganisms have evolved a capacity to modify and access lignocellulosic biomass to meet their metabolic needs. The exploitation of this capacity offers a natural, low-input means for preparing biomass for biofuels processes. Natural modification and degradation of the lignin component in particular can reduce the severity requirements of subsequent thermochemical pretreatment steps. For example, Itoh et al. [42] used a variety of lignin-degrading white-rot fungi to treat wood chips prior to extracting lignin by an organosolv method, and demon‐ strated that improved ethanol yields were obtained from the solid fraction along with a 15% savings in electricity use. Similarly, brown-rot fungal species *Coniophora puteana* and *Postia placenta* have been successfully used to improve glucose yields upon enzymatic saccharifica‐ tion of pine, acting as a complete replacement for thermomechanical pretreatments [43]. While it is clear that it is possible to exploit the metabolic capabilities of microorganisms to facilitate biofuels production, the very wide taxonomic array of microorganisms that modify or degrade lignocellulose presents a tremendous variety of choices for implementing such a strategy. Each

One approach for applying the power of microbial metabolism to the challenges of biofuel production involves ensiling, which is a commonly used means for enhancing the digestibility of forage and other biomass for ruminants [44, 45]. The process of ensiling exploits the capacity of naturally occurring bacteria, mostly Lactobacillaceae, to ferment the sugars within ligno‐ cellulosic residues and produce a substrate that is more easily digested by ruminal microor‐ ganisms. While these bacterial consortia lack the ability to substantially degrade the lignin component, the changes effected on the biomass can improve yields of fermentable sugars

impact on the energy balance and economic sustainability of biorefineries.

methane production by anaerobic digestion [12].

**3. Biological pretreatments**

**3.1. Microbial consortia**

approach carries its own advantages and challenges.

Another highly effective pretreatment strategy is steam explosion, in which biomass is briefly heated to high temperatures (~200°C) under high pressure, then subjected to a rapid pressure drop that renders the biomass more penetrable by enzymes for subsequent hydrolysis [18]. In some cases, steam explosion is enhanced by the addition of an acid catalyst such as sulfuric acid [27]. For lignocellulosic agricultural residues, steam explosion under optimized condi‐ tions has been shown to be an effective pretreatment strategy for enzymatic saccharification [28]. Steam explosion has also been successfully used in combination with other physiochem‐ ical pretreatments such as acid/water impregnation of cereal straws [29]. Both of the latter studies resulted in the release of hemicellulose-derived pentose oligomers into the liquid fraction, and it was suggested that the use of ethanologenic strains capable of converting these pentoses into ethanol would further improve overall process efficiency [28]. Other assessments have suggested that the hemicellulose fraction would be more efficiently converted to other value-added products rather than ethanol using post-treatment enzyme addition or further acid hydrolysis [30].

Organosolv is a process by which the lignin fraction is chemically modified and essentially removed from biomass using high-temperature extraction with alcohols such as methanol or ethanol or other solvents, sometimes with dilute acid (e.g. hydrochloric or sulfuric acid) as a catalyst [17]. While organosolv processes require a solvent recovery step to be economical and efficient, they provide a robust means of generating three streams of potential products: an extracted, modified lignin component, a hemicellulose-enriched aqueous phase, and a residue that is highly enriched in cellulose and an excellent substrate for the production of biofuels by enzymatic saccharification followed by bacterial or yeast fermentation. Organosolv is one of the pretreatment options that results in a fraction containing chemically modified, low molecular weight lignin components. This stream has a good deal of product potential in addition to its possible use as a fuel for combustion to provide energy to the process [7, 31]. While organosolv is particularly suited to very lignin-rich feedstocks such as wood [32], there is increasing interest in using organosolv extractions for agricultural residues such as wheat straw and dedicated biofuels crops [33]. Goh et al. [34] optimized organosolv conditions for empty palm fruit bunch using combined severity calculations, with excellent results and the ability to accurately predict product stream yields.

Microwave pretreatment of biomass is another option that has been reported to improve subsequent enzymatic saccharification of rice straw [35]. Microwaves have the advantage of combining very rapid heating times with a lower energy input than conventional heating strategies. This irradiative pretreatment creates localized hotspots, which open up the lignocellulose composite molecule, thereby facilitating enzyme access for saccharification and biofuel production by fermentation [4]. A successful combination of microwave and chemical pretreatments in a microwave-acid-alkali-hydrogen peroxide sequence resulted in efficient enzymatic saccharification of rice straw [36]. A related pretreatment option that has been exploited to improve the enzymatic digestibility of switchgrass is the use of radio frequency heating in combination with alkali; this treatment has the key advantage of allowing a much higher solids content than conventional heating [37]. Irradiation of biomass can also enhance methane production by anaerobic digestion [12].

A number of other pretreatment options exist, including ammonia fiber explosion (AFEX), liquid hot water, alkalai/wet oxidative pretreament, and others; several recent reviews discuss these processes and their advantages and disadvantages in detail [4, 17-20, 23, 30, 38, 39]. Regardless of the strategy employed, a common feature of any pretreatment option is that energy input is required. Pretreatment is a major part of the overall operating expense and energy efficiency of any biofuels process, and, while essential, typically accounts for over 30% of the costs of biorefinery operation [40, 41]. Strategies to reduce these costs will have a major impact on the energy balance and economic sustainability of biorefineries.
