**4. Conclusions and future outlook**

Pretreatment of lignocellulosic materials with white- or brown-rot fungi can be incorporated into any strategy for the production of biofuels and bioproducts, with significant advantages including decreased energy requirements for subsequent steps, production of fewer fermen‐ tation-inhibiting substances, and the potential for the production of value-added co-product streams [73, 74]. With the wide variety of potential strains and substrates available, and the possibility to create or select new strains with more desirable properties, it seems likely that biological pretreatment can be used on nearly any biomass that is currently produced. One of the most important benefits of biological pretreatment is the resultant reduction in the severity of the subsequent thermomechanical or thermochemical pretreatment step that is required for efficient enzymatic saccharification. While this is a very important benefit, Keller et al. [136] identified six criteria for strains to be selected for biological pretreatment of agricultural waste: little carbohydrate degradation, low costs for nutrients, a reasonable storage time, ability to compete with endogenous microbiota, decreased thermomechanical pretreatment severity, improved yields of glucose upon enzymatic saccharification, and a lack of production of compounds inhibitory to fermenting organisms. These criteria underscore the major limita‐ tions of biological pretreatment, the most important of which are the propensity of the organisms to degrade the carbohydrate component, their inability to establish growth on unsterilized biomass, and the relatively long incubation times that are required. These limitations are related to the ecological niche that these saprophytic fungi fill in nature. That is, they have evolved to access and utilize those plant carbohydrates that are difficult for other microorganisms to access. For this reason, these fungi typically appear at the end of an ecological succession of organisms that degrade decaying wood and are often ill equipped to compete with the faster-growing molds and bacteria that access the more easily degraded plant carbohydrates [137]. While it may be possible using reverse genetics tools and/or strain selection to limit carbohydrate degradation by pretreatment fungi [107], it is likely that such strains will be even less able to compete with endogenous microorganisms; therefore, estab‐ lishment on recently harvested biomass will remain a challenge. Some sort of treatment of the biomass to suppress the growth of endogenous molds prior to inoculation with the pretreat‐ ment fungi will likely be necessary.

modify lignin would provide a means to avoid this drawback of fungal pretreatment. Early studies with strains that were deficient in the production of cellulase met with only moderate success, with substantial degradation of cellulose observed [128, 129]. This is probably attributable to the high degree of redundancy in fungal cellulases, with large numbers of genes contributing to the hydrolytic degradation of cellulose and hemicellulose in various species [15]. More recently, we have applied a strain of *Trametes versicolor* that is unable to produce cellobiose dehydrogenase (CDH) to the pretreatment of canola residue, and found that the strain was proficient in lignin degradation but was unable to catabolize the cellulose [107]. Xylose within the substrate appeared to have been utilized to support the greatly decreased fungal growth compared to the wild-type strain. Furthermore, we found that the application of a fungal cell wall-degrading enzyme cocktail (glucanex; a concentrated supernatant of a SmF culture of *Trichoderma harzianum*) to the fungus-treated biomass resulted in the release of fungal cell wall-associated glucose [107]. Biological pretreatment with *T. versicolor* therefore had the overall effect of converting some of the xylose within the substrate to glucose, which

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

Studies such as these also provide biological data regarding the role of the genes that are downregulated in the mutant strains. This reverse genetics approach is a powerful method for investigating gene function, and in the current genomic era reverse genetics tools can often be applied in the known context of the entire genome of the fungus. Gene silencing by RNA interference (RNAi) is a common method for down-regulating genes in a variety of model systems [130, 131], and the recent demonstration of RNAi mechanisms in the model white-rot fungus *Phanerochaete chrysosporium* [132] suggests that RNAi could be used for targeted downregulation of specific genes in species that are useful for biological pretreatment. The availa‐ bility of convenient gene silencing transformation vectors for ascomycetes such as pSilent [133] and pTroya [134] as well as pHg/pSILBAγ for basidiomycetes [135] will greatly facilitate the investigation of gene function and may also result in the development of modified strains featuring enhanced properties for biological pretreatment of lignocellulosic substrates for

Pretreatment of lignocellulosic materials with white- or brown-rot fungi can be incorporated into any strategy for the production of biofuels and bioproducts, with significant advantages including decreased energy requirements for subsequent steps, production of fewer fermen‐ tation-inhibiting substances, and the potential for the production of value-added co-product streams [73, 74]. With the wide variety of potential strains and substrates available, and the possibility to create or select new strains with more desirable properties, it seems likely that biological pretreatment can be used on nearly any biomass that is currently produced. One of the most important benefits of biological pretreatment is the resultant reduction in the severity of the subsequent thermomechanical or thermochemical pretreatment step that is required for efficient enzymatic saccharification. While this is a very important benefit, Keller et al. [136] identified six criteria for strains to be selected for biological pretreatment of agricultural waste:

is more easily fermented by ethanologenic yeasts.

biofuels production.

**4. Conclusions and future outlook**

The unavoidable expense of the pre-inoculation treatment can be compensated by taking advantage of a potential benefit of biological pretreatment that has received very little attention: wood-degradingfungimaymodifythelignincomponentsufficientlytoprovidepositivebenefits for particle compression of agricultural biomass during densification. Densification (briquet‐ ting or pelleting) of biomass aims to increase the bulk density of agricultural residues far be‐ yondwhatisachievablebybaling,anditisanessentialstepforprovidingbiomasswithsufficient caloric density for efficient transportation [138]. The production of biomass pellets provides a substrate that is suitable for conversion into biofuels through microbial processes or gasifica‐ tion[139,140],orcanbecombusteddirectlytoproduceenergy[141].Awidevarietyofagricultur‐ alfeedstocksissuitableforpelleting[142];however,untreatedbiomassisverydifficulttodensify and, without pretreatment, produces weak, powdery pellets that are expensive to produce and cannotwithstandthephysicalrigorsoftransportation.Ligninactsasanaturalbinderthatprovides strength and durability to biomass pellets, and pretreatment of the biomass is required in order to release lignin fragments during compaction and produce pellets with the desired characteris‐ tics [139, 143]. A number of options are available to prepare biomass for pelleting, with two very promisingmethodsbeingmicrowaveheatingandradiofrequencyheating.Bothofthesemethods provide a number of advantages over conventional heating, particularly regarding treatment times [144-146]. We have found that a very brief microwave treatment of a variety of agricultur‐ al feedstocks suppresses the growth of endogenous molds and bacteria sufficiently for inoculat‐ ed white-rot fungi to establish growth on these substrates. Moreover, canola residue treated with *Trametes versicolor* produces pellets with excellent compaction characteristics and durability (Canam, Town, and Dumonceaux, unpublished). Such pellets would retain the thermochemi‐ calpretreatmentbenefitsaffordedbythefungalpretreatmentintermsofenzymaticsaccharifica‐ tion [107], but would offer vastly increased transportation efficiency in a full-scale biorefinery scenario.

thermal pretreatment of the biomass, but primarily growth suppression of endogenous microbiota. The lightly treated biomass would then be inoculated with a fungal suspension or formulation and transferred via auger to a pile analogous to a silage pile, but with aeration. The inoculated biomass would be incubated at ambient temperatures for several weeks to allow fungal growth. The fully infested biomass would then be milled to an appropriate size using standard equipment. After cooling and/or drying, pellets formed from the milled, pretreated biomass would be suitable for transport to a biorefinery for biofuels/bioproducts manufacture. In the absence of a viable product stream or a biorefinery, biomass pellets could be burned in a high-efficiency oven to exploit their calorific value [139]. Biological pretreatment would provide an array of benefits along this production chain, including decreased milling energy, decreased compression energy requirements, improved densification characteristics and the consequent reduction in transportation cost, decreased severity of thermochemical pretreatments, decreased production of fermentation inhibitors, improved yield of fermenta‐ ble sugars upon enzymatic saccharification, and possibly co-products derived from the more easily extractable lignin phase. All of these benefits would be realized with a fairly minor energy input, and although it is difficult to avoid the long incubation times, SSF can be performed on time scales only slightly longer than the common on-farm practice of ensilage. Biological pretreatments can therefore, in theory, be performed on-farm or nearby, offering significant logistical and technical advantages when incorporated into an overall process for

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

biofuels manufacture.

**Author details**

Thomas Canam1

katoon, Canada

**References**

, Jennifer Town2,3, Kingsley Iroba4

1 Department of Biological Sciences, Eastern Illinois University, Charleston, IL, USA

2 Agriculture and Agri-Food Canada Saskatoon Research Centre, Saskatoon, Canada

3 Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada

4 Department of Chemical and Biological Engineering, University of Saskatchewan, Sas‐

[1] Wolff EW. Greenhouse gases in the Earth system: A palaeoclimate perspective. Philo‐ sophical Transactions of the Royal Society A: Mathematical, Physical and Engineer‐

\*Address all correspondence to: tim.dumonceaux@agr.gc.ca

ing Sciences. 2011;369(1943):2133-47.

, Lope Tabil4

and Tim Dumonceaux2,3\*

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

195

**Figure 1.** A possible scheme for incorporating biological pretreatment into biofuels manufacturing. A. Treatment to suppress the growth of endogenous microorganisms to allow establishment of the inoculated fungal culture. A varie‐ ty of treatments could be utilized, including ultraviolet, microwave, or radio frequency treatment. This scheme is based on the successful biopulping inoculation strategy described by Scott et al. [66] B. Overall scheme for biofuels produc‐ tion including biological pretreatment. (B.5. photo courtesy of Jay Grabiec, Eastern Illinois University).

We can therefore envision a means by which fungal pretreatment might be incorporated into an overall process for producing energy from biomass by a variety of strategies (Figure 1). Biological pretreatment should be included at the earliest stages in order to take maximum advantage of its beneficial effects. Building on the successful bio-pulping model described by Scott et al. [66], chopped biomass would be briefly decontaminated by microwave or radio frequency heating within a conveyor; the objective of this is not sterilization or complete thermal pretreatment of the biomass, but primarily growth suppression of endogenous microbiota. The lightly treated biomass would then be inoculated with a fungal suspension or formulation and transferred via auger to a pile analogous to a silage pile, but with aeration. The inoculated biomass would be incubated at ambient temperatures for several weeks to allow fungal growth. The fully infested biomass would then be milled to an appropriate size using standard equipment. After cooling and/or drying, pellets formed from the milled, pretreated biomass would be suitable for transport to a biorefinery for biofuels/bioproducts manufacture. In the absence of a viable product stream or a biorefinery, biomass pellets could be burned in a high-efficiency oven to exploit their calorific value [139]. Biological pretreatment would provide an array of benefits along this production chain, including decreased milling energy, decreased compression energy requirements, improved densification characteristics and the consequent reduction in transportation cost, decreased severity of thermochemical pretreatments, decreased production of fermentation inhibitors, improved yield of fermenta‐ ble sugars upon enzymatic saccharification, and possibly co-products derived from the more easily extractable lignin phase. All of these benefits would be realized with a fairly minor energy input, and although it is difficult to avoid the long incubation times, SSF can be performed on time scales only slightly longer than the common on-farm practice of ensilage. Biological pretreatments can therefore, in theory, be performed on-farm or nearby, offering significant logistical and technical advantages when incorporated into an overall process for biofuels manufacture.
