The advantages of biological pretreatment include minimum facility cost, low energy requirement and mild environmental conditions. However, for practical application, there are two major disadvantages associ­ated with this process. First, fungi growth consumes hol — ocellulose as an energy source leading to significant carbohydrate loss; second, most biological pretreat­ments are long processes due to slow microbial growth and delignification reaction rates. Since lignin break­down in the biomass would lead to enzyme access to cel­lulose and hemicellulose, selective lignin degradation by white-rot fungi hold some promise for real application in biomass pretreatment if the procedure can be cut shorter and sugar consumption can be controlled to an insignificantly low level. However, not even white-rot fungi can use lignin as a sole carbon and energy source; fungi growth inevitably results in carbohydrate loss (Fan et al., 2012; Sanchez, 2009). Strategies taken to shorten biological pretreatment time and decrease car­bohydrate consumption include (1) selection for natu­rally occurring white-rot fungi that preferentially attack lignin (Ander Eriksson, 1977; Kirk and Moore, 1972; Lee et al., 2007; Muller and Trosch, 1986; Salvachua et al., 2011), (2) selection of cellulase-deficient mutants (Akin et al., 1993; Eriksson et al., 1980; Ruel et al., 1981), or (3) repression of cellulase and hemicellulase expression (Yang et al., 1980). As an example of strain se­lection, among 22 screened Basidiomycetes, mostly the white-rot fungi Pleurotus sp. "florida" preferentially at­tacks lignin in wheat straw to increase cellulose accessi­bility. After 90 days pretreatment with Pleurotus sp. "florida", the resulting biomass can release the same amount of glucose as Avicel, the lignin-free cellulose (Muller and Trosch, 1986). However, pretreatment using this strain is still time consuming.

Furthermore, there are many limitations to the strate­gies for strain improvement. First, carbohydrate con­sumption is needed for microbial growth; therefore, strains can only be selected for increased delignification and decreased sugar loss and not for minimal sugar loss. In addition, decreasing the secretion of carbohydrate hy­drolysis enzymes would lower the reaction rate and lead to even longer pretreatment time. Genetic modification of white-rot fungi to improve the required features may help resolve some of the drawbacks, but the tech­nical process is quite challenging (Fan et al., 2012).

Another way to improve the biological pretreatment process is through optimization of nutrients, tempera­ture, and preprocessing time to reach a balance between maximum sugar release and minimum sugar loss within the shortest possible time. Based on the enzymatic activity profile obtained in a 28-day pretreatment anal­ysis, switchgrass is pretreated with P. chrysosporium for 7 days. The pretreatment of switchgrass led to higher glucan, xylan, and total sugar yields than the unpre — treated sample, suggesting enzyme profile assays may be utilized for initial estimation of pretreatment time in or­der to enhance sugar yields and reduce sugar loss (Maha — laxmi et al., 2010). By monitoring compositional changes during biological pretreatment, a 15-day pretreatment time was selected for the pretreatment of the woody bio­masses Prosopis juliflora and Lantana camara with the white-rot fungus Pycnoporus cinnabarimus (Gupta et al.,

2011) . This 15-day pretreatment resulted in a relatively small weight loss in the pretreated feedstocks with decreased lignin and increased holocellulose contents. Enzymatic hydrolysis of the pretreated biomass led to sugar releases of 389 and 402 mg per gram of dried solid.

Alternatively, as a compromise, preliminary microbial pretreatment of biomass can be used in combination with downstream thermochemical, chemical or other pre­treatment. This procedure would reduce, for example, the amount of acid needed combined with lower temper­ature and shorter time, thus reducing energy and chemi­cal costs. In addition, there would be less biomass degradation and inhibitor production compared to con­ventional thermochemical pretreatment. Preliminary tests showed that after corn stover pretreatment with

P. chrysosporium, the shear forces needed to obtain the same shear rates of 3.2—7 rev/s were reduced 10- to 100-fold, respectively. The digestibility of C. stercoreus — pretreated corn stover showed a three — to fivefold improvement in enzymatic cellulose digestibility (Keller et al., 2003). Sawada et al. reported that combination of fungal pretreatment with less severe steam explosion maximizes enzymatic saccharification of beech wood meal (Sawada et al., 1995). Compared to steam explosion alone, combined pretreatments improve saccharification by 20—100% of the polysaccharide in the wood. However, 17% of the holocellulose was degraded during fungal pretreatment, and there was an unspecified holocellulose loss during steam explosion at optimum 215 °C for

6.5 min (Sawada et al., 1995). Pretreatment of wheat straw with P. juliflora followed by acid hydrolysis led to a reduc­tion in acid load and an increase in sugar release as well as ethanol yield (Kuhar et al., 2008).

Interestingly, a recent study showed that by simply changing the pretreatment sequence, i. e. when the wood Pimus radiata biomass was treated first with steam explosion followed by fungi pretreatment, a 10-fold increase in glucose yield was achieved after enzymatic hydrolysis (Vaidya and Singh, 2012). A combination of selected fungal pretreatment with a mild alkali treat­ment of wheat straw led to a maximum of 69% glucose yield and an ethanol yield of 62% with no inhibitor for­mation during the pretreatment (Salvachua et al., 2011). Also, a combination of the white-rot fungus Lenzites betulina C5617 pretreatment with LHW treatment enhanced the enzymatic hydrolysis of the poplar wood Populus tomentosa led to the highest hemicellulose removal of 92.33%, which was almost two times higher than that of LHW treatment alone and a 2.66-fold increase in glucose yield (Wang et al., 2012).