SHF may be performed two different ways: separate hydrolysis and separate fermentation (SHSF) and separate hydrolysis and co-fermentation (SHCF). In SHSF, production of cellulolytic enzymes, hydrolysis of pretreated biomass and fermentation are performed in separate vessels. SHSF would allow performing each step at its corresponding optimum conditions. Generally, the production of cellulolytic enzymes using T. reesei is performed at 25-30°C and at pH 4.5-5.5. Hydrolysis of pretreated lignocellulosic biomass is performed in the temperature range of 50-55°C at pH 4.5-5.5. Ethanol fermentation is performed in the temperature range of 30-35°C at pH 5-6. Saccharomyces cerevisiae and Pichia stipitis may be respectively used to ferment both glucose and xylose to ethanol. However, P stipitis requires micro aeration (1 mmol of air per liter per hour) for xylose metabolism (Fig. 5). Iogen, Inc. (based in Canada) uses SHSF for the ethanol production. The optimal process integration would result in higher ethanol productivity.

SHCF is very similar to SHSF in that the hydrolysis of pretreated biomass and fermentation are performed in separate vessels. Unlike SHSF, in SHCF fermentation of different sugars (such as glucose and xylose) is performed in the same vessel. Simultaneous utilization of sugars for ethanol fermentation may be performed using either a single microorganism culture or co-culture of microorganisms. Most of the microorganisms use glucose as a carbon source to produce ethanol and are resistive to xylose uptake. The simultaneous utilization of xylose along with glucose for ethanol production could be approached several different ways.

Adhikari et al. (2009) has reported that the thermotolerant yeast Kluyveromyces sp. IIPE453 MTCC 5314 may consume a wide range of mono — and disaccharide sugars including glucose, xylose, mannose, arabinose simultaneously at temperature range of 40-65°C and pH range of 3.5-5.5 with ethanol productivity 13.8 gl-1h-1 on sugarcane bagasse in continuous

Figure 5. Overview of the xylose metabolic pathway found in yeasts such as Pitchia stipitis including the engineered xylose isomerase (XI) reaction (Pitkanen et al. 2005; Chu and Lee

2007).

Abbreviations: HXT, hexose transporters; Sym, symporter; XR, xylose redutase; XDH, xylitol dehydrogenase; XI, xylose isomerase; XK, xylulokinase; X5P, xylulose-5-phosphate; TKL, transketolase; TAL, transaldolase; S7P, sedoheptulose-7-phosphate; GA3P, glyceraldehyde — 3-phosphate; RPE, L-ribulose-5-phosphate 4-epimerase; Ru5P, L-ribulose 5-phosphate; RKI, ribose-5-phosphate isomerase; R5P, ribose-5-phosphate; E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; GND, 6-phosphogluconate dehydrogenase; 6PG, 6-phospho-D-gluconate; 6PGL, 6-phospho-D-glucono-1,5-lactone; ZWF, glucose-6-phosphate-1-dehydrogenase; G6P, glucose-6-phosphate; HXK, hexokinase; PFK, 6-phosphofructokinase; F6P, fructose — 6-phosphate; F16BP, fructose 1,6-bisphosphate; PGI, glucose-6-phosphate isomerase; FBA, fructose-bisphosphate aldolase; DHAP, dihydroxyacetone phosphate; GPD, glycerol-3- phosphate dehydrogenase; GO3P, glycerol-3-phosphate; GPP, glycerol-3-phosphatase; TPI, triose-phosphate isomerase; TDH, glyceraldehyde-3-phosphate dehydrogenase; BPG, 1,3-bisphosphoglycerate; PEP, phosphoenol pyruvate; PPPh, phosphoenolpyruvate phosphatase; PDC, pyruvate decarboxylase; ADH, alcohol dehydrogenase; ALD, acetaldehyde dehydrogenase; ACS, acetyl-CoA-synthetase; PYC, pyruvate carboxylase; PDB, pyruvate dehydrogenase beta subunit; PCK, phosphoenol pyruvate carboxykinase; AcCoA, acetyl coenzyme A; CIT, citrate synthase; CITR, citrate; ACO, aconitate hydratase; ICTR, isocitrate; AKG, alpha-ketoglutarate; IDP, isocitrate dehydrogenase kinase; KGD, alpha-ketoglutarate decarboxylase; SucCoA, succinyl CoA; LSC, succinyl-CoA ligase; SUC, succinate; SDH, succinate dehydrogenase; FUM, fumarate; FUMH, fumarate hydralase; MAL, malate; MDH, malate dehydrogenase; OAA, oxaloacetate; ICL, isocitrate lyase; GLO, glyoxylate; and, MLS, malate synthase.

fermentation. In addition, Kluyveromyces sp. IIPE453 MTCC 5314 may be recycled up to 20 days in the continuous process at 50°C (Adhikari et al. 2009).

Xylose isomerase (XI) enzyme converts xylose to xylulose that could be easier to use as a carbon source by microorganisms such as yeast S. cerevisiae for ethanol production. Xylose isomerase can be used either separately or along with cellulolytic enzymes. The optimum activity of xylose isomerase is at a pH of 7-8 and at a temperature range of 60-80°C. However, a urease coated xylose isomerase could work under acidic conditions as urease coats a separate inner basic environment from the exterior acidic environment (Rao et al. 2007). Rao (2007) has mentioned that using the urease coated xylose isomerase along with 0.05 M tetrahydroxyborate could convert 86% of xylose into xylulose under acidic condition at temperature 34°C. However, the uptake of xylulose by yeast decreases with increasing ethanol concentration at less than 4% (w/v) in the media (Chiang et al. 1981; Chandrakant and Bisaria 1998).

The use of genetically engineered microorganisms such as Zymomonas mobilis and Escherichia coli may simultaneously consume both glucose and xylose in co-fermentation. The genetically engineered bacteria improve the ethanol yield from 0.39 g g-1 to 0.44-0.52 g g-1 with high productivity up to 0.18-0.96 g l1 h1 (Olsson and Hahn-Hagerdal 1996). The main issues with the genetically engineered strains are stability, reproducibility and regulatory issues with use of biomass byproduct for animal feed.