After the pretreatment step, the bioconversion of lignocellulosic materials includes the biopolymers hydrolysis and the sugar fermentation. These two steps can be performed separately (SHF, separate hydrolysis and fermentation) or simultaneously (SSF, simultaneous saccharification and fermentation). SSF technology is generally considered more advantageous than SHF technology, for several reasons:

• reduced number of the process steps (Koon Ong, 2004)

• reduced end product inhibition because of the rapid conversion of glucose into ethanol by yeast (Viikari et al., 2007)

• reduced contamination by unwanted microorganisms thanks to the presence of ethanol (Elumalai & Thangavelu, 2010).

However, the optimum temperature for the enzymatic hydrolysis is typically higher than that of fermentation. Therefore, in SHF process, the temperature for the enzymatic hydrolysis can be optimized independently from the fermentation temperature, whereas a compromise must be found in SSF process (Olofsson et al., 2008). Another obstacle of the SSF process is the difficulty to carry out continuous fermentation by recirculating and reusing the yeast due to the presence of the solid residues from the hydrolysis.

High solids loadings are usually required to obtain high ethanol levels in the fermentation broths (high gravity fermentation). In particular, solids loadings of pretreated biomass up to 30% (w/w) could be necessary to reach an ethanol concentration of 4-5 wt% that is considered a threshold level for a sustainable distillation process. However, increasing the amount of the solids content in a bioreactor, the hydrolytic performances of the enzymes mixture tends to worsen. In particular, the high initial substrate consistency causes a viscosity increase (Sassner et al., 2006) that is an obstacle toward the homogeneous and effective distribution of the enzymes in the bioreactor. This problem could be partly overcome by using thermostable enzymes. In particular, the hydrolysis could be carried out in two steps: a former step at elevated temperatures with thermostable hydrolytic enzymes producing the liquefaction of biomass (SHF); the latter step, aimed at completing the biomass saccharification, could be carried out at milder temperatures by using the SSF approach (Olofsson et al., 2008).

2. Innovative bioreactor geometries and process strategies

A major requirement in cost-efficient lignocellulosics-to-ethanol process is to employ reactor systems yielding the maximal conversion of the cellulose with the minimal enzyme dosage. As consequence, one of the most important parameter for the design and operation of bioreactors for lignocellulosic conversion is the effective use of the biocatalysts to obtain high specific rates of cellulose conversion (namely the yield of glucose obtained per amount of enzymes). The maximization of the product concentration, i. e. the amount of glucose obtained per liquid volume, is also an important parameter as well as the optimization of the volumetric productivity, in this case the rate of glucose formation per reactor volume. When the hydrolysis is carried out with high dry matter contents, hence high cellulose levels, the product concentration will drive up. For this reason, some recent researches have been finalized into attempting the enzymatic biomass conversion at high-solids loads (Jorgensen et al., 2007; Tolan, 2002). The most important problem of high solid loadings is related to the fact that the viscosity of the reaction mixture is very high and the rheology of the mixture has to be well studied: normal stress might become very significant during bioconversion. In particular, mixing and mass transfer limitations, and, presumably increased inhibition by intermediates come into play. Various fed-batch strategies have been attempted with the scope of supplying the substrate without reaching excessive viscosities and unproductive enzyme binding to the substrate (Rosgaard et al., 2007a; Rudolf et al., 2005).

As said, the currently employed cellulolytic enzyme systems, that include the widely studied T. reesei enzymes, are significantly inhibited by the hydrolysis products cellobiose and glucose. This inhibition retards the overall conversion rate of lignocellulosics-to glucose (Gan et al., 2002; Katz and Reese, 1968). Product inhibition is particularly significant during processing at high substrate loadings mainly because the glucose concentration is higher than that obtained in diluted biomass suspensions. (Kristensen et al., 2009; Rosgaard et al., 2007a). As consequence, both the conversion rate and the glucose yields achievable in batch processing of lignocellulose are reduced (Rosgaard et al., 2007b; Tengborg et al., 2001). General criteria in the bioreactor design and in the selection of the operating conditions could be: use of reactors or reaction regimes that allow a rapid reduction of the glucose concentration; running of the reactions at low to medium substrate concentrations in order to maintain higher conversion rates and hence obtain higher volumetric productivity of the reactor (AndriC et al. 2010, a).

The integration of the bioreactor with a separation unit (reaction-separation hybrids) has shown promising results with product inhibited or equilibrium limited enzyme-catalyzed conversions, because it is possible to remove the products as they are formed (Ahmed et al., 2001; Gan et al., 2002). In this regard, membrane (bio) reactors could be a viable process configuration. Unlike the SSF approach in which the glucose consumption is carried out by the microrganisms simultaneously available in the hydrolyzate, the use of membrane bioreactors would accomplish the same function without any compromise in the reaction temperature. A membrane (bio-) reactor is a multifunction reactor that combines the reaction with a separation, namely in this case product removal by membrane separation, in one integrated unit, i. e. in-situ removal, or alternatively in two or more separate units. The membrane bioreactors hitherto used for the separation in enzymatic processes have been mainly ultra — and nanofiltration (Pinelo et al., 2009). However, the use of this technology is limited by the bank-up of unreacted lignocellulosics (lignin and particularly recalcitrant cellulose) in large — scale and/ or continuous processing (Andric et al. 2010, b). Already in the past, some authors improved the efficiency of the continuous stirred tank bioreactor (CSTR) by incorporating separation membranes in the reactor design. In particular, Henley et al (1980) incorporated an UF membrane (UF) or hollow-fiber cartridge (HFC) into the CSTR-UF and CSTR-HFC system, respectively (Henley et Al., 1980). Ishihara et al. (1991) accomplished a semi-continuous hydrolysis reaction by using a continuously stirred reservoir tank, connected to a suction filter unit for the removal of the lignin-rich residue and an ultra-filtration membrane unit (tubular module), through which the filtrate was pumped in order to separate the hydrolysis products from cellulases. The concentration of the lignocellulosic substrate in the reactor was maintained almost constant by the addition of fresh substrate at appropriate intervals. The filter and ultrafiltration units were operated intermittently, while the enzymes were added at the start, recovered in the UF module, and recycled back into the reactor (Ishihara et al., 1991). More recently, Yang et al. (2006) designed the removal of reducing sugars during the cellulose enzymatic hydrolysis through a system consisting in a tubular reactor, in which the substrate was retained with a porous filter at the bottom and buffer entered at the top through a distributor. The hollow-fiber ultrafiltration module with polysulfone membrane enabled the permeation and the separation of the sugars. To keep the volume constant in the tubular reactor, all the remaining buffer was recycled back from the UF membrane and the make-up buffer was continuously supplied from the reservoir (Yang et al., 2006). In some applications an additional microfiltration unit has exceptionally been used to retain the unconverted lignin — rich solid fraction due to the presence of tightly bound enzymes (Knutsen and Davis, 2004) or has been employed to remove the unconverted substrate from the reactor. These set-ups result in slightly complex process layouts for the hydrolysis.

It is evident that the optimization of the reactor designs will permit to overcome both the rheological and inhibition limit of the bioconversion and maximize the enzymatic conversion. Therefore, the reactor design become strong relevant for large-scale processing of cellulosic biomass (Lynd et al., 2008; Wyman, 2008).

3. Conclusion

In this chapter an overview of the current knowledge on the hydrolysis of lignocellulosics for bioethanol production has been presented. In the last years several important breakthroughs have been made either on the biochemical and technological sides. This is confirmed by several industrial initiatives spread over the world. Among these, in recent days, the first brick of the lignocellulosic bioethanol demo plant (40 kton/y) has been layed in Northern Italy by the Mossi and Ghisolfi Group. Some cooperation agreements were strengthen with Novozymes for improving the efficiency of the hydrolysis step. This event represents an important stage for all the Europe making the production of lignocellulosic ethanol closer to the industrialization and opening the way to new lignocellulosic biorefineries.