The enzymatic hydrolysis of algal cell walls and other biopolymers is a promising alternative to energy-consuming mechanical pretreatment and chemical catalytic hydrolysis at high temperature. It has large potential to increase the digestion rate and methane yield. Treatment of WAS by carbohydrases increased the biogas yield by 13% [ 195] . Pretreatment with pancreatic lipases (250 units/mg protein, dose

0. 25 g/L at 25°C for 5.5 h) of slaughterhouse wastewater with pork fat particles resulted in 35% hydrolysis of the neutral fat, but did not significantly increase the fat hydrolysis rate in the anaerobic reactor (sequencing batch type, 25°C) and did not influence the methane yield [244]. The authors suggested that at relatively low temperature (25°C), anaerobic oxidation of LCFA is the rate-limiting step.

Natural hydrolysis pretreatment of green macroalgae in percolators has been extensively studied [175, 245-249]. This method can be viewed as a type of two — step ADP and is discussed in the subsequent reactor design subsection.

The endo-b-1,4-glucanase from Cellulomonas sp. YJ5 hydrolyzed Chlorella sorokiniana cell wall and caused cells lysis after 60-180 min of treatment [250]. Immobilized cellulases hydrolyzed Chlorella cells (reduced sugars yield 62%) and gave a twofold increase in lipids extraction efficiency [251]. Other advantages of enzymatic hydrolysis include an absence of inhibiting by-products and achievement of high selectivity [252]. While this method has a large potential, it is necessary to solve several technological blocks before it can be applied in the biofuel industry.

The major roadblocks are higher cost of enzymes production and their handling, high enzymes to substrate specificity, enormous diversity of algal cell envelope composition and structure.