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Ozonolysis, as a lignocellulosic biomass pretreatment, goes back to 80s; however, in the last years it is becoming widespread again owing to its efficiency and mild operation conditions. Ozone reacts preferably with lignin than carbohydrates, promoting biomass destructuration and delignification, and so the sugar release by enzymatic hydrolysis.The hydrolysate from pretreated biomass has being used as sugars source for secondgeneration fuels production, mainly ethanol, methane and hydrogen. Short-chain carboxylic acids are the main inhibitory compounds generated, being properly removed by water washing. The most common inhibitory compounds reported for other pretreatments, furfural and HMF (5-hydroxymethylfurfural) † , are not found in ozonepretreated hydrolysates. Composition of pretreated biomass and ozone consumption depends on several process parameters: reactor design, moisture content, particle size, pH, reaction time, ozone/air flow and ozone concentration. Additional studies are necessary to clarify process parameters effect and to optimize the process to achieve high yields with economic feasibility.

IntroductionAlgal cell walls separate the inside cell content from the environment to protect the cell against desiccation, pathogens, and predators while still allowing exchange of compounds. Toward application of algae biomass as a sustainable resource, disruption of this cell wall (¼cell disruption) is an essential pretreatment step to maximize product recovery in downstream processes of the algae biorefinery. Also for direct use of algae in feed or food, cell rupture is required to increase the bioavailability of algae constituents. Depending on the cell wall structure, the size, and the shape of algae, cell disruption can be challenging. A variety of cell disruption methods is currently available, and new approaches are being elaborated in parallel. Since downstream processing is responsible for a large part of the operational costs in the whole production chain, cell disruption technologies should be low cost and energy efficient and result preferably in high product quality. This chapter provides information on cell wall types and gives an overview of physical-mechanical and (bio-)chemical cell disruption technologies with attention to development stage, energy efficiency, product quality, costs, emerging approaches, and applicability on large scale. Cell wall types in various groups of microalgae and cyanobacteriaThe cell wall composition and architecture of algae and cyanobacteria are highly variable ranging from tiny membranes to multilayered complex structures. Despite the importance of algal cell wall properties in biotechnology, little structural information is available for most species (Scholz et al., 2014). Based on the complexity of surface structures, four cell types could be distinguished (Barsanti and Gualtieri, 2006;Lee, 2008) (Fig. 6.1). A simple cell membrane (Fig. 6.1, Type 1) is present in short-lived stages (e.g., gametes), chrysophytes, raphidophytes, green algae Dunaliella, and haptophytes Isochrysis. It consists of a lipid bilayer with integrated and peripheral proteins. Sometimes a cap of glycolipids and glycoproteins envelops the outer surface of cell membrane. Cell membranes with additional extracellular material are known in cyanobacteria and many groups of algae, including palmelloid phases. It is the most diverse cell wall type that includes various membrane-associated structures (cell wall, Microalgae-Based Biofuels and Bioproducts. http://dx

Steam explosion (150-200 °C, 5-30 min) was performed on a commercial cellulose presented in two configurations (fiberized and compact sheet) and its effect on their chemical and physical properties was studied, along with the influence of two different preservation methods (acetone drying and freezing) after pretreatment. No degradation compounds were produced during pretreatment, although solid recovery (R) decreased with temperature from 90% to 62%. Similar particle size and surface conditions (increased porosity) were found for both types of pretreated samples despite the extremely different initial configuration. Crystallinity diminished for 150 °C samples, but 200 °C pretreatment promoted recrystallization. Pretreatment also reduced polymerization degree, although enzymatic accessibility did not improve. Both acetone and freezing processes extremely affected cellulose properties. Acetone drying counterbalanced crystallinity and enzymatic accessibility variations of pretreated samples, while decreasing polymerization degree. Freezing dramatically decreased enzymatic accessibility of pretreated samples down to 15.8%.

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Biomass grown in wastewater treatment photobioreactors is a cheap raw material with high contents of carbohydrates, proteins and lipids. This work studies the production of fermentable monosaccharides from three biomasses grown in piggery wastewater (P), domestic wastewater (W) and synthetic medium (S) by applying chemical pretreatment and enzymatic hydrolysis, using a Taguchi design.ANOVA identified temperature, chemical reagent type and chemical reagent concentration as significant operational parameters. However, the biomass concentration, pretreatment time, enzyme dosage and enzymatic hydrolysis time had no remarkable effect.The bacterial content of the biomass had no relevant impact on carbohydrate and protein solubilisation but had a remarkable effect on the degradation of the released carbohydrates (57, 60 and 37% for P, W and S), while also affecting lipid solubilisation. Pretreatment with HCl 2M at 120ºC resulted the optimal conditions, achieving a monosaccharide recovery of 53, 59 and 80% for P, W and S biomasses, respectively. Highlights Temperature was the most influential factor on sugar production from algal biomass.  HCl resulted in higher monosaccharide recovery than NaOH. No effect of enzymatic hydrolysis operational factors on sugar production was found  High carbohydrate solubilisations were achieved from biomasses grown in wastewater. Biomass grown in synthetic medium achieved the highest monosaccharide recovery.